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分子神経生物学分野(1)

Kato T, Abe Y, Hirokawa S, Iwakura Y, Mizuno M, Namba H, Nawa H. Neurobehavioral
Differences Between Mice Receiving Distinct Neuregulin Variants as Neonates; Impact
on Sensitivity to MK-801. Current Molecular Medicine 2015, 15, 222-236

分子神経生物学分野(2)

Sakai M, Watanabe Y, Someya T, Araki K, Shibuya M, Niizato K, Oshima K, Kunii Y,
Yabe H, Matsumoto J, Wada A, Hino M, Hashimoto T, Hishimoto A, Kitamura N, Iritani
S, Shirakawa O, Maeda K, Miyashita A, Niwa S, Takahashi H, Kakita A, Kuwano R ,Nawa H. Assessment of copy number variations in the brain genome of schizophrenia
patients. Sakai et al. Molecular Cytogenetics (2015) 8:46 DOI 10.1186/s13039-0150144-5細胞神経生物学分野(1)

Watanabe-Iida I, Konno K, Akashi K, Abe M, Natsume R, Watanabe M, Sakimura K.
Determination of kainate receptor subunit ratios in mouse brain using novel chimeric
protein standards. J Neurochem. 2015 Oct 8. doi: 10.1111/jnc.13384.

細胞神経生物学分野(2)

細胞神経生物学分野(3)Kakegawa W, Mitakidis N, Miura E, Abe M, Matsuda K, Takeo YH, Kohda K, Motohashi J,
Takahashi A, Nagao S, Muramatsu S, Watanabe M, Sakimura K, Aricescu AR, Yuzaki M.
Anterograde c1ql1 signaling is required in order to determine and maintain a singlewinner climbing fiber in the mouse cerebellum. Neuron. 2015 Jan 21;85(2):316-29. doi:
10.1016/j.neuron.2014.12.020.
Ageta-Ishihara N, Yamazaki M, Konno K, Nakayama H, Abe M, Hashimoto K, Nishioka T,
Kaibuchi K, Hattori S, Miyakawa T, Tanaka K, Huda F, Hirai H, Hashimoto K, Watanabe
M, Sakimura K, Kinoshita M. A CDC42EP4/septin-based perisynaptic glial scaffold
facilitates glutamate clearance. Nat Commun. 2015 Dec 10;6:10090. doi:
10.1038/ncomms10090.

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Watanabe T, Sasaki M, Komagata S, Tsukano H, Hishida R, Kohno T, Baba H, Shibuki K,
Spinal mechanisms underlying potentiation of hindpaw responses observed after
transient hindpaw ischemia in mice. Scientific Reports 5, 11191, 2015.

システム脳生理学分野(2)Tsukano H, Horie M, Bo T, Uchimura A, Hishida R, Kudoh M, Takahashi K, Takebayashi
H, Shibuki K, Delineation of a frequency-organized region isolated from the mouseprimary auditory cortex. J .Neurophysiol. 113, 2900-2920, 2015.

Meguro R, Hishida R, Tsukano H, Yoshitake K, Imamura R, Tohmi M, Kitsukawa T,
Hirabayashi T, Yagi T, Takebayashi H, Shibuki K, Impaired clustered protocadherin-aシステム脳生理学分野(3)(cPcdh-a) leads to aggregated retinogeniculate terminals and impaired visual acuity inmice. J. Neurochem. 133, 66-72, 2015.
Nakashima M, Saitsu H, Tohyama J, Kato M, Shiina M, Takei N, Kitaura H, Shirozu H,
Masuda H, Watanabe K, Ohba C, Tsurusaki Y, Miyake N, Takebayashi H, Ogata K,
病理学分野/デジタル医学分野/
Kameyama S, Kakita A*, Matsumoto N* (*: co-last authors). Somatic mutations in the
脳疾患標本資源解析学分野(1)MTOR gene cause focal cortical dysplasia type IIb. Annals of Neurology. 78(3): 375386, 2015
Ogura R, Tsukamoto Y, Natsumeda M, Isogawa M, Aoki H, Kobayashi T, Yoshida S,
病理学分野/デジタル医学分野/ Okamoto K, Takahashi H, Fujii Y, Kakita A. Immunohistochemical profiles of IDH1,
脳疾患標本資源解析学分野(2) MGMT and P53: practical significance for prognostication of patients with diffuse
gliomas. Neuropathology. 35(4): 324-335, 2015
Kimura T, Kitaura H, Masuda H, Kameyama S, Saito Y, Sugai K, Otsuki T, Nakazawa A,
病理学分野/デジタル医学分野/ Morota N, Yamamoto T, Iida K, Nakagawa M, Mizuno T, Takahashi H, Kakita A.
脳疾患標本資源解析学分野(3) Characteristic expression of p57/Kip2 in balloon cells in focal cortical dysplasia.
Neuropathology. 35(5): 401-409, 2015
分子病態学（客員）分野(1)Tanji K, Odagiri S, Miki Y, Maruyama A, Nikaido Y, Mimura J, Mori F, Warabi E,
Yanagawa T, Ueno S, Itoh K, Wakabayashi K. p62 deficiency enhances α-synuclein
pathology in mice. Brain Pathol 25: 552-564, 2015

分子病態学（客員）分野(2)Nakamura K, Mori F, Kon T, Tanji K, Miki Y, Tomiyama M, Kurotaki H, Toyoshima Y,
Kakita A, Takahashi H, Yamada M, Wakabayashi K. Filamentous aggregations ofphosphorylated α-synuclein in Schwann cells (Schwann cell cytoplasmic inclusions) in
multiple system atrophy. Acta Neuropathol Comm 3: 29, 2015

分子病態学（客員）分野(3)Mori F, Miki Y, Tanji K, Kakita A, Takahashi H, Utsumi J, Sasaki H, Wakabayashi K.
Sortilin-related receptor CNS expressed 2 (SorCS2) localizes in Bunina bodies inamyotrophic lateral sclerosis. Neurosci Lett 608: 6-11, 2015

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Fukuda M, Takao T, Hiraishi T, Aoki H, Ogura R, Sato Y, Fujii Y. Cortico-corticalactivity between the primary and supplementary motor cortex: An intraoperative nearinfrared spectroscopy study. Surg Neurol Int 6: 44, 2015. DOI: 10.4103/21527806.153872.
Ogura R, Tsukamoto Y, Natsumeda M, Isogawa M, Aoki H, Kobayashi T, Yoshida S,
Okamoto K, Takahashi H, Fujii Y, Kakita A. Immunohistochemical profiles of IDH1,
MGMT and P53: practical significance for prognostication of patients with diffuse
gliomas. Neuropathology. 35(4): 324-335, 2015
Nishiyama K, Yoshimura J, Fujii Y. Limitations of neuroendoscopic treatment forpediatric hydrocephalus and considerations from future perspectives. Neurol Med Chir2015 (doi: 10.2176/nmc.ra.2014-0433)
Kanazawa M, Kawamura K, Takahashi T, Miura M, Tanaka Y, Koyama M, Toriyabe M,
Igarashi H, Nakada T, Nishihara M, Nishizawa M, Shimohata T. Multiple therapeutic
effects of progranulin on experimental acute ischaemic stroke. Brain 2015;138:19321948.
Hokari M, Yokoseki A, Arakawa M, Saji E, Yanagawa K, Yanagimura F, Toyoshima Y,
Okamoto K, Ueki S, Hatase T, Ohashi R, Fukuchi T, Akazawa K, Yamada M, Kakita A,
Takahashi H, Nishizawa M, Kawachi I. Clinicopathological features in anterior visual
pathway in neuromyelitis optica. Annals of Neurology 2016;79(4):605-624. doi:
10.1002/ana.24608. PMID: 26836302.

神経内科学分野(3)Ikeda T, Takahashi T, Tsujita M, Kanazawa M, Toriyabe M, Koyama M, Itoh K, Nakada T,
Nishizawa M, Shimohata T. Effects of Alda-1, an Aldehyde Dehydrogenase-2 Agonist,
on Hypoglycemic Neuronal Death. PLOS ONE 2015;10:e0128844.

統合脳機能研究センター(1)Suzuki Y, Nakamura Y, Yamada K, Igarashi H, Kasuga K, Yokoyama Y, Ikeuchi T,
Nishizawa M, Kwee IL, Nakada T. Reduced CSF Water Influx in Alzheimer's Disease
Supporting the β-Amyloid Clearance Hypothesis. PLoS One. 10(5):e0123708, 2015.

統合脳機能研究センター(2)Nakada T. The Molecular Mechanisms of Neural Flow Coupling: A New Concept. J
Neuroimaging. 25(6):861-5, 2015.

統合脳機能研究センター(3)Igarashi H, Suzuki Y, Huber VJ, Ida M, Nakada T. N-acetylaspartate Decrease in AcuteStage of Ischemic Stroke:A Perspective from Experimental and Clinical Studies. Magn
Reson Med Sci. 14(1):13-24, 2014.

遺伝子機能解析学分野/
生命情報工学分野(1)Sato Y, Bernier F, Yamanaka Y, Aoshima K, Oda Y, Ingelsson M, Lannfelt L, Miyashita
A, Kuwano R, Ikeuchi T. Reduced plasma desmosterol-to-cholesterol ratio andlongitudinal cognitive decline in Alzheimer’s disease. Alzheimer’s & Dementia:
Diagnosis, Assessment & Disease Monitoring 1:67-74, 2015

遺伝子機能解析学分野/
生命情報工学分野(2)Kasuga K, Kikuchi M, Tokutake T, Nakaya A, Tezuka T, Tsukie T, Hara N, Miyashita A,
Kuwano R, Ikeuchi T. Systematic review and meta-analysis of Japanese familial
Alzheimer’s disease and FTDP-17. Journal of Human Genetics 60:281-283, 2015

遺伝子機能解析学分野/
生命情報工学分野(3)Yajima R, Tokutake T, Koyama A, Kasuga K, Tezuka T, Nishizawa M, Ikeuchi T. ApoEisoform-dependent cellular uptake of amyloid-b is mediated by lipoprotein receptor
LR11/SorLA. Biochemical Biophysical Research Communications 456:482-488, 2015

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Chiken S, Sato A, Ohta C, Kurokawa M, Arai S, Maeshima J, Sunayama-Morita T,
Sasaoka T, Nambu A. Dopamine D1 receptor-mediated transmission maintains
information flow through the cortico-striato-entopeduncular direct pathway to release
movements. Cerebral Cortex 2015 Dec; Vol.25 No.12:4885-97. doi:Nakamura T, Sato A, Kitsukawa T, Sasaoka T, Yamamori T. Expression pattern ofimmediate early genes in the cerebellum of D1R KO, D2R and wild type KO mice undervarious conditions. Frontiers in Cell and Developmental Biology.Vol.3, Article 38, June2015 doi: 10.3389/fcell.2015.00038
Hayashiji N, Yuasa S, Miyagoe-Suzuki Y, Hara M, Ito N, Hashimoto H, Kusumoto D, Seki
T, Tohyama S, Kodaira M, Kunitomi A, Kashimura S, Takei M, Saito Y, Okata S, EgashiraT, Endo J, Sasaoka T, Takeda S and Fukuda K. G-CSF supports long-term muscle
regeneration in mouse models of muscular dystrophy. Nature Communications 2015
Natsumeda M, Maitani K, Liu Y, Miyahara H, Kaur H, Chu Q, Zhang H, Kahlert U,
Eberhart CG. Targeting Notch Signaling and Autophagy Increases Cytotoxicity in
Glioblastoma Neurospheres. Brain Pathol. 2015 Nov 27. p 1-p11※論文は出版元の転載許可を受けています。

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Current Molecular Medicine 2015, 15, 222-236

Neurobehavioral Differences Between Mice Receiving Distinct
Neuregulin Variants as Neonates; Impact on Sensitivity to MK-801
T. Kato1, Y. Abe1, S. Hirokawa2, Y. Iwakura1, M. Mizuno1, H. Namba1 and H. Nawa*,11

Department of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585,
Japan
2

Department of Molecular Genetics, Niigata University Graduate School of Medical and Dental Sciences,
Niigata 951-8510, Japan
Abstract: Neuregulin-1 (NRG1) is a well-recognized risk gene for schizophrenia and is often implicated in theneurodevelopmental hypothesis of this illness. Alternative splicing and proteolytic processing of the NRG1
gene produce more than 30 structural variants; however, the neuropathological roles of individual variants
remain to be characterized. On the basis of the neurodevelopmental hypothesis of schizophrenia, we
administered eNRG1 (0.1~1.0 μg/g), a core epidermal growth factor-like (EGF) domain common for all splicing
NRG1 variants, to neonatal mice and compared their behavioral performance with mice challenged with a fullmature form of type 1 NRG1 variant. During the neonatal stage, recombinant eNRG1 protein administrated
from the periphery passed the blood-brain barrier and activated its receptor (ErbB4) in the brain. In adults, themice receiving the highest dose exhibited lower locomotor activity and deficits in prepulse inhibition and tonedependent fear learning, although the hearing reduction of the eNRG1-treated mice may explain thesebehavioral deficits. Neonatal eNRG1 treatment also significantly potentiated MK-801-driven locomotor activity
in an eNRG1 dose-dependent manner. In parallel eNRG1 treatment enhanced MK-801-driven c-Fos induction
and decreased immunoreactivity for NMDA receptor subunits in adult brain. In contrast, mice that had beentreated with the same molar dose of a full mature form of type 1 NRG1 as neonates did not exhibit
hypersensitivity to MK-801. However, both animal models exhibited similar hypersensitivity tomethamphetamine. Collectively, our findings suggest that aberrant peripheral NRG1 signals during
neurodevelopment alter later behavioral traits and auditory functions in the NRG1 subtype-dependent manner.

Keywords: Behavior, MK-801, Neuregulin-1, NMDA receptor, hearing, schizophrenia.

INTRODUCTION
Genetic polymorphism of neuregulin-1 (NRG1) issuggested to associate with vulnerability to psychiatricdiseases such as schizophrenia and bipolar disorder[1-3]. The genetic association of NRG1 withschizophrenia was first demonstrated with the
haplotype block called HAPICE that is located in the 5’end of the gene and consists of three single nucleotide
polymorphisms (SNPs) and two microsatellites [1]. Thisinitial finding has been replicated in multiple samples,but not in all populations [1-3]. Alternative splicing or
promoter usage of the NRG1 gene produces multiple
isoforms of NRG1 precursor proteins (type I-VI) [4-6].
The posttranslational processing of the NRG1
precursors further modifies the protein structures ofthese isoforms to alter their biological activity [7].
However, the pathological contribution of each NRG1
isoform is not fully understood. Genetic mutant micethat lack the isoform-specific exon(s) or overexpress a
specific isoform of NRG1 exhibit distinct behavioral
abnormalities relevant to schizophrenia endophenotypes, potentially suggesting the divergence of the*Address correspondence to this author at the Department of
Molecular Neurobiology, Brain Research Institute, Niigata University,
1-757 Asahimachi-dori, Niigata 951-8585, Japan;
E-mail: hnawa@bri.niigata-u.ac.jp15/15 $58.00+.00－ 41 －

isoforms’ functions in vivo [8-12]. These experiments
were often performed by independent researchers anddid not allow us to accurately compare the behavioral
consequencesoftheindividual
isoforms’
manipulations. To test the functional difference of the
NRG1 isoforms, here, we investigated and compared
the behavioral traits of the mice treated with type1
NRG1 (T1-NRG1) and those with its processing
product; an epidermal growth factor (EGF) domain
peptide (eNRG1). These NRG1 derivatives arepotentially induced or recruited in the prenatal andperinatal immune inflammatory processes that are
implicated in schizophrenia etiology [13,14].
We have been testing the neurodevelopmental
hypothesisofschizophrenia,
assessingtheneurobehavioral impact of neonatal exposure to T1NRG1 [15]. In addition to T1-NRG1, here, we
challenged mouse neonates with eNRG1 to compare
the neurobehavioral effects of the two NRG1 variants,
eNRG1 and T1-NRG1. Although there are multiple
isoforms of NRG1 precursor proteins (type I-VI), which
are generated by alternative splicing or promoter usageof the NRG1 gene, but all NRG1 isoforms carry thiscore active domain (i.e., eNRG1) for their receptor
binding [5,6]. Moreover, eNRG1 is naturally produced
by proteolytic processing of T1-NRG1 with a protease,neuropsin, and suggested to exert a distinct action in© 2015 Bentham Science Publishers

Neuregulin-1 Variants and Behavioral Difference

hippocampal synaptic plasticity [7]. We assessed basal
behavioral traits to evaluate the relevancy of eNRG1injected mice to an animal model for schizophrenia,
comparing the previous results from the mice exposed
to T1-NRG1 [15]. As NRG1 signals are known toregulate NMDA receptor expression and function in thebrain [16-19]. Thus, we investigated the sensitivity ofeNRG1-treated mice to the NMDA receptor antagonist
MK-801 as well as the expression of NMDA receptor
subunits in these mice [17,18].

MATERIALS AND METHODS
Ethics Statement
All of the animal experiments described wereapproved by the Animal Use and Care Committee of
Niigata University and performed in accordance withthe guidelines of NIH (USA).
Animals
C57BL/6NCrj mouse dams of late pregnancy werepurchased from Nihon Charles River (Yokohama,Japan) and their pups were used for experiments. Mice
were housed with their dam in polypropylene cages(24L × 17W × 12H cm) in temperature-controlledcolony room maintained under a 12-h light-dark cycle(light on 8:00 h) with free access to food and water. At
postnatal day (PND) 25-28, pups were weaned andseparated into cage (three or four animals per cage).
Both male and female mice were subjected tobehavioral tests during PND 56-70 [15]. Behavioral
tests were performed during the light-cycle.
Reagents
Recombinant epidermal growth factor domain ofhuman NRG 1β1/heregulin 1β1 was purchased from
PeproTech EC (London, UK). This NRG1 peptide issufficient for activation of the ErbB4 receptor tyrosine
kinase. We will refer to it simply as "eNRG1".
Alternatively, we used the full mature form ofrecombinant type-1 NRG1β1 protein (T1-NRG1; MW
25400 Da) [15]. (+)-MK-801 (dizocilpine) was
purchased from Tocris Cookson Ltd (Ellisville, MO,
USA).
NRG1-Treatment and Psychotomimetic ChallengeeNRG1
orT1-NRG1
wasadministeredsubcutaneously (s.c.) to individual litters during
postnatal days PND 2-10 at the nape of the neck at adose of 0.1, 0.3, or 1.0 µg/g body weight (injection
volume 15 µl/g). The experimental design andprocedures were based on our previous studies of the
EGF model for schizophrenia [20]. Control littermates
received a phosphate-buffered saline (PBS; vehicle)
injection of the same volume. MK-801 (0.3 µg/g; 10µl/g) and methamphetamine (1.0 µg/g; 10 µl/g)
dissolved in saline were administered intraperitoneally(i.p.) to adult mice (PND 56-70) [15,21].

Current Molecular Medicine, 2015, Vol. 15, No. 3

223

Immunoprecipitation
Protein extracts were prepared from whole brains ofeNRG1-treated or vehicle-treated control mice on
PND2 [15]. Each brain was homogenized in a RIPA
lysis buffer [50 mM Tris-HCl buffer (pH 7.4) with 1%
TritonX-100, 1% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 150 mM NaCl, 5 mM EDTA, 1
mM Na3VO4, and 1 mM NaF) plus a protease inhibitor
cocktail (Complete Mini; Roche, Mannheim, Germany).
After centrifugation, supernatants were harvested andprotein content was determined using a Micro BCA kit(Pierce Chemical, Rockland, IL, USA). Each brain
lysate (2 mg protein) was then incubated with 2 µg ofanti-ErbB4 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) overnight and mixed with Protein G
Sepharose beads (20 µl; GE Healthcare Bio-Science
AB, Uppsala, Sweden) for 3 h. Sepharose beads were
washed with RIPA buffer, and denatured with 100 µl of
2 × SDS sample buffer (125 mM Tris-HCl pH 6.8, 4%
SDS, 10% 2-mercaptoethanol). The specificity of theanti-ErbB4 antibody was ascertained by immunoblotting of cell lysates containing individual ErbB1-4
proteins (HN, unpublished data). Immunoprecipitates
were then analyzed by gel electrophoresis as
described below.
Immunoblotting
Brain tissues were homogenized in the sample lysis
buffer (62.5 mM Tris-HCl pH 6.8, 2 % SDS, 0.5 % NP40, 5 mM EDTA) plus the protease inhibitor cocktail(Roche). Protein samples (15-30 µg/lane) wereseparated by SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes. Membranes were probed with antibodies directed against
phosphotyrosine (1:1000, Millipore, Bedford, MA,
USA), ErbB4 (1:1000, Santa Cruz Biotechnology).
Alter-natively, immunoblots were probed with
antibodies directed against excitatory and inhibitory
neuronal markers [GluA1 (1:500, Millipore), GluA2/3(1:1000, Millipore), GluA4 (1:1000, Millipore), GluN1(1:1000, Millipore), GluN2A (1:500, Millipore), GluN2B(1: 500, Millipore), GluN2C (1:500, Millipore), GluN2D(1:1000, Santa Cruz Biotechnology), GAD65/67(1:1000, Millipore), parvalbumin (1:3000, Abcam,
Cambridge UK), tyrosine hydroxylase (1:1000,
Millipore) neuregulin-1 (1:300, RandD Systems,
Minneapolis, MN), and β-actin (1:2000, Millipore)].
Immunoreactivity on membranes was detected byperoxidase-conjugated anti-immunoglobulin antibodies
followed by chemiluminescence reaction (ECL kit, GE
Healthcare) and film exposure. The intensity of an
immunoreactive band, whose size matched theauthentic molecular weight, was measured by animage processing software, GENETOOLS (Syngene,
Cambridge, UK).
Auditory Brain-Stem Evoked Response
The lowest level, at which an auditory brain-stem
evoked response (ABR) pattern is recognized, was－ 42 －

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Current Molecular Medicine, 2015, Vol. 15, No. 3

Kato et al.

determined in each mouse [15]. Mice wereanesthetized with an intramuscular injection of
ketamine (75 mg/kg) and xylazine (7.5 mg/kg), andplaced in a close acoustic room [15]. A stainless-steel
electrode was inserted subcutaneously into the vertex(positive pole), retroauricular region (negative pole),
and opposite retroauricular region (background).
Acoustic stimuli, consisting of a tone burst (0.1 ms riseand fall no plateau) at a frequency of 10 kHz, were
presented to each mouse with a sound stimulator(DPS-725, Diamedical System, Tokyo, Japan) andspeaker (PT-R9, Pioneer, Tokyo, Japan) in an open
field. For each time point, 500 responses for eachmouse were recorded and filtered for band widths of
100–3000 Hz. The Neuropac μ (Nihonkoden, Tokyo,
Japan) was used to analyze the ABR response.
c-Fos Immunohistochemistry
Levels of c-Fos expression were assessed bycounting the number of c-Fos-immunoreactive cells[22]. Mice were deeply anesthetized with halothane(Takeda Pharmaceutical Co, Osaka, Japan) 3 h after
the single injection of MK-801 (0.3 µg/g) and perfused
intracardiacally
with
PBS,
followedby4%paraformaldehyde in 0.1 M phosphate-buffer (pH 7.2).
Coronal sections (40 µm) were made by a cryostat andincubated with a blocking solution containing PBS, 5%
nonfat milk and 0.25% TritonX-100 for 2 h andincubated with rabbit anti-c-Fos antibody (1:20000; PC38, Calbiochem, La Jolla, CA, USA) in the blocking
solution 48 h at 4°C, followed by biotinylated anti-rabbit
immunoglobulin antibody (1:200; BA-1000, Vector
Laboratories). Sections were washed and processedwith the avidin-biotinylated horseradish peroxidase
complex (Vector ABC kit Vector Laboratories,
Burlingame, CA, USA). Immunoreactivity wasvisualized with the chromogen 3, 3’- diaminobenzidine.
To quantify the number of c-Fos positive cells in thebrain, we used an all-in-one microscope (BZ-9000,

Keyence, Osaka, Japan) and a BZ-Analyzer(Keyence) to scan the sections, and quantified the cellnumbers from the digital images using NIH Image (ver.
1.61) cell-counting system [23].

Behavioral Testing
Acoustic startle response and prepulse inhibition(PPI) were measured as fully described previously[10,15]. Test paradigm of context- and tone-dependent
fear learning, locomotor measurement, residentintruder assessment [24] and further procedural details
of individual behavioral tests, see Supplementary
Information. Three independent cohorts of mice weremade for each NRG1 subtypes and each doses andassigned to the following three types of experiments;
basal behavioral tests (locomotor assessment, soundstartle test, and fear conditioning in the less stressful
order), the MK-801 experiments, and the residentintruder test (see below).

－ 43 －

Statistical Analysis
Data are presented as mean ± SEM. Computation
was carried out using the SPSS 11.0 for Windows(Tokyo, Japan). Behavioral or immunohistochemical
data were first subjected to the Shapiro-Wilk test (forthe fitting to Gesian distribution) and Levene's test (forthe equality of standard deviations) and then either
one-way analysis of variance (ANOVA) or Student’s ttest. ANOVA adopts NRG1-treatment as a betweensubject factor, NRG1-treatment as a between-subject
factor and time as a within-subject factor, or eNRG1/
T1-NRG1 treatment and drug injection as betweensubject factors. ABR data were subjected to two-way
ANOVA with eNRG1-treatment as a between-subject
factor and frequency of auditory stimulus as a withinsubject factor. Other data stem from experimentalgroups were subjected to Student’s t-test. Some of thedata in the frequency of c-fos, startle responses and
PPI scores had showed biased distributions or
difference in deviations among groups, suggesting therisk of type 1 errors in ANOVA (Supplemental Table
S1). Subsequently, a Tukey HSD test was applied tothe data carrying equal deviations as a post hoc test.
Alternatively, a Scheffe's test was applied to the data
having non-equal deviations to validate the initial
ANOVA assessment. When there was significant
interaction between eNRG1-treatment and drug, data
were analyzed separately to avoid the interaction for
the final analysis. A p value < 0.05 was regarded asstatistically significant.

RESULTSeNRG1 Penetration into the Brain
Since the blood-brain barrier of neonatal mice isimmature, peripherally administered cytokines canreach the brain [15,25]. To confirm that eNRG1 incirculation penetrates the blood-brain barrier andactivates
intracerebral
NRG1
receptors,
wesubcutaneously administered eNRG1 (1.0 µg/g) tomouse pups (PND2) and examined eNRG1
immunoreactivity and the phosphorylation of ErbB4 inthe cerebrum. Immunoblotting revealed that there was
eNRG1 immunoreactivity in the cerebrum of theeNRG1-injeced mice but not in that of saline-injected
controls (Fig. 1A). Peripheral injection of eNRG1 alsosignificantly increased phosphorylation levels of ErbB4[F(4,11) = 11.4, p < 0.01]. The increase persisted
between 1-6 h after the injection with a peak of ErbB4
phosphorylation at 3 h (Fig. 1B, C). Thus, a peripheral
injection of eNRG1 to mouse pups can produce hypersignaling of NRG1/ErbB4 in the neonatal mouse brain.
Exploratory Activity in Novel Environment
We administered various concentrations (vehicle
alone, 0.1, 0.3, and 1.0 µg/g) of eNRG1 repeatedly tomouse neonates. We monitored several parameters ofNeuregulin-1 Variants and Behavioral Difference

Current Molecular Medicine, 2015, Vol. 15, No. 3

mouse development; tooth eruption, eyelid opening,and body weight gain (Table 1). NRG1, which belongs
to the EGF polypeptide family, promotes tooth eruption
and eyelid opening [15,26]. A one-way ANOVA
revealed that repeated eNRG1 injections significantly
stimulated eyelid opening [dose: F(3,56) = 90.31,
p < 0.001] and tooth eruption [dose: F(3,56) = 35.54,

A

Control

(kDa)

eNRG1

Posi

8.5
3.5

eNRG1 Standard

B

IP : ErbB4
eNRG1 (h)

0h

3h

1h

6h

12 h

P- ErbB4
ErbB4

C% of control (%)

400

**

300

***

***

200
100
0

0

1

3

Time (h)612Fig. (1). ErbB4 activation and eNRG1 immunoreactivity in thebrain following peripheral eNRG1 injection. (A) Neonatal mice
(PND2) were subcutaneously challenged with eNRG1 (1.0µg/g) and intracardiacally perfused with PBS 3 h after eNRG1
injection. Brain lysates (200 µg/lane) were subjected to SDS
polyacrylamide electrophoresis with the authentic eNRG1
protein (1 ng; Posi). The presence of eNRG1 protein in thebrain was examined by immunoblotting (n = 4 mice pergroup). (B) Whole brain lysates were prepared 0, 1, 3, 6, and
12 h after subcutaneous injection of eNRG1 (1.0 µg/g) toneonatal mice (PND2). ErbB4 phosphorylation levels were
examined using immunoblotting for phosphotyrosine
combined with ErbB4 immunoprecipitation. (C) Relative
levels of ErbB4 phosphorylation in the brains of eNRG1injected mice are presented (% of 0 time; mean ± SEM, n = 3
per each time points). *p < 0.05 compared with 0 time by
Tukey HSD.

225

226

p < 0.001] in a dose-dependent manner. These results
verified the physiological activity of injected eNRG1 in
vivo. In addition, injections with higher doses of eNRG1(i.e. 0.3 μg/g and 1.0 μg/g), produced a 10-20%
decrease in body weight gain that persisted until
adulthood (Table 1).

Tab

At the young adult stage (PND54-70), we assessed
the locomotor activity of eNRG1-treated mice in a novel
environment. A one-way ANOVA yielded a significant
difference among eNRG1 doses [F(3,36) = 4.6, p <0.01]. Post hoc comparisons revealed that the micetreated with the highest dose (1.0 µg/g) of eNRG1
displayed significantly fewer horizontal movements
compared with the other three groups (p < 0.05) (Fig.
2A), whereas stereotypic behaviors of these mice weresimilar among the groups [dose: F(3,36) = 0.7, p =0.54] (Fig. 2B). There were no significant differences in
the above indices between males and females [gender× dose: F(3,32) = 1.23, p = 0.31 for horizontal
movements; gender × dose: F(3,32) = 0.27, p = 0.85
for stereotypic movements], although stereotypic
movements fundamentally differed between males andfemales [gender: F(3,32) = 1.23, p < 0.01].

0.1

Effects of Neonatal eNRG1-Treatment on Other
Behavioral Traits
Using the mice treated with 1.0 and 0.3 μg/g dosesof eNRG1, we further analyzed other behaviors ofthese animals in adulthood (PND56-70) such as fear
learning ability, sensorimotor gating and social
behavioral traits. The mice treated with the 1.0 μg/g
dose of eNRG1 as neonates exhibited a marked
reduction of PPI [eNRG1: F(1,49) = 61.3, p < 0.001,
repeated ANOVA for 4 prepulses] (Table 2). There
were no significant differences in eNRG1 effects
between males and females [gender × eNRG1: F(1,47)= 2.26, p = 0.14]. These mice also showed a reduction
in tone-dependent learning scores but did not alter
context-dependent learning scores [F(1,39) = 0.004, p= 0.95 for context-dependent learning; F(1,39) = 23.8,
p < 0.001 for tone-dependent learning, both repeated
ANOVA for 5 bins]. There was no significant differencein shock sensitivity [F(1,39) = 0.005, p = 0.95] (Table2). There were no significant differences between
males and females in eNRG1 effects on the above
indices, either [gender × eNRG1; F(1,37) = 1.20, p =0.28 for conditioning: gender × eNRG1; F(1,37) = 0.41,
p = 0.53 for context-dependent learning: gender ×
eNRG1: F(1,37) = 1.87, p = 0.18 for tone-dependent
learning]. Similar behavioral alterations were observed
when we administered the lower dose of eNRG1 (0.3μ/g) to mouse pups (Supplemental Fig. S1). This dose
(0.3 μ/g) was approximately 40 pmol per gram bodyweight and equivalent to the molar dose of T1-NRG1
that had been given to mouse pups in our previous
study [15].
To investigate the impact of neonatal eNRG1treatment on social behaviors in adulthood, we used a
resident-intruder behavioral assay. In this assay, agroup-housed male mouse (intruder) was placed inanother home cage where a resident male mouse had－ 44 －

Do

To

0.3

1.0

Ey

0.1

0.3

1.0

Bo

0.1

0.3

1.0

Bo

0.1

0.3

1.0

eNRG
weig
two-t

bee
trea
com
unp
betw
figh
agg
asc
(i.e.

Effe
Driv

S
rece
anim
sch
eNR
resp
801
env
exp
Afte
a 0.
MKhyp
sed
in b
F(1,

225

226

Current Molecular Medicine, 2015, Vol. 15, No. 3

sults
1 in
RG1
20%
until

Table 1.

ssed
ovel
cant
p <
mice
RG1
ents
(Fig.
were
p =
es in
nder
ontal
0.85
ypic
and

ther

oses
s of
fear
ocial
μg/g
rked
001,
here
ects
,47)
ction
alter
4, p
23.8,
ated
ence
able
ween
bove
p =
0.41,
er ×
dent
rved
(0.3
dose
body
RG1
ious

RG1ed a
y, a
d in
had

Kato et al.

Summary of physical development.

Dose 

Vehicle eNRG1

0.1 μg/g

11.6 ± 0.2

10.4 ± 0.2***

0.3 μg/g

11.6 ± 0.2

10.2 ± 0.2***

1.0 μg/g

10.9 ± 0.2

9.4 ± 0.2***

0.1 μg/g

13.9 ± 0.1

11.5 ± 0.2***

0.3 μg/g

13.7 ± 0.2

10.8 ± 0.1***

1.0 μg/g

13.1 ± 0.2

10.5 ± 0.2***

0.1 μg/g

5.72 ± 0.13

5.52 ± 0.07

0.3 μg/g

6.18 ± 0.20

5.62 ± 0.14*

1.0 μg/g

6.05 ± 0.17

5.08 ± 0.22**

Tooth Eruption (PND)

Eyelid Opening (PND)

Body Growth; P11

Body Growth; P56

Male

FemaleMale

Female0.1 μg/g

24.2 ± 0.6

20.2 ± 0.3

23.8 ± 0.2

19.3 ± 0.3

0.3 μg/g

24.2 ± 0.6

20.3 ± 0.4

21.4 ± 0.3**

18.4 ± 0.5*

1.0 μg/g

19.4 ± 0.3

16.6 ± 0.3

17.6 ± 0.4**

14.3 ± 0.6**

eNRG1 or vehicle was administered (s.c.) daily to neonatal mice during PND2-10. During and after treatment, we monitored eyelid opening, tooth eruption and bodyweight of all mice. Data are expressed as mean ± SEM (n = 10 per group). P-values, compared between eNRG1-treated and vehicle-treated groups by unpaired
two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

been housed alone until the test day (Table 2). eNRG1treated mice exhibited decreased sniffing durationcompared with vehicle-treated controls (p < 0.05,
unpaired t-test), whereas no significant difference
between groups was found with sniffing counts orfighting counts (Table 2). The lower frequency ofaggressive behaviors of the present mice can be
ascribed to the mouse strain used in the present study(i.e., C57BL/6N) [27,28].
Effects of Neonatal eNRG1-Treatment on MK-801Driven-Locomotor Activity
Sensitivity to psychostimulants, such as NMDA
receptor antagonists, have often been evaluated inanimal models to assess their relevancy to aschizophrenia model. To test whether neonataleNRG1-treatment (0.1, 0.3, and 1.0 μg/g) alters acuteresponsiveness to an NMDA receptor antagonist, MK801, in adulthood, mice were first acclimated to a novel
environment to minimize the substantial difference of
exploratory activity and/or motivation between groups.
After one hour acclimation, mice were challenged witha 0.3 μg/g dose of MK-801 (Fig. 3). The given dose of
MK-801 was the minimum dose that can induce
hyperlocomotion in mice [29]. MK-801 markedly increased the locomotor activity of mice for approximately 2 h
in both the eNRG1-treated and control groups [drug:
F(1,36) = 29.7, p < 0.001]. However, the magnitude of－ 45 －

MK-801-induced locomotor activity was significantly
higher in the eNRG1-treated group than in the control
group [eNRG1-treatment: F(1,36) = 5.2, p < 0.05;
eNRG1 × drug: F(1,36) = 6.0, p < 0.05] (Fig. 3A). The
sensitivity appeared to depend on the dose of eNRG1[eNRG1 dose: F(1,36) = 3.2, p < 0.05] (Fig. 3C). Thus,
these results indicate that neonatal exposure to eNRG1enhances behavioral sensitivity to MK-801 in a dosedependent manner. However, there was no significant
difference in MK-801-triggered stereotypic scores
between eNRG1-treated and vehicle-treated control
mice at any dose of eNRG1 [eNRG1 dose: F(3,36) =1.2, p = 0.34] (Fig. 3B, D).
Effects of Neonatal eNRG1-Treatment on c-FosExpression in the Brain
The NMDA antagonist MK-801 increases both
dopamine and glutamate efflux primarily in theforebrain regions [30,31] and induces the gene expression of c-Fos through MK-801-evoked glutamatergic
neurotransmission and subsequent MAP kinase/AP-1
activation [22]. Employing c-Fos expression as amarker of neuronal activity, we examined the effects of
neonatal eNRG1 treatment on MK-801-induced c-Fos
expression and attempted to confirm the above
observation; the higher sensitivity of eNRG1-treatedmice to MK-801. We counted the number of c-Fos
positive cells in the medial prefrontal cortex (mPFC),
nucleus accumbens (NAc), piriform cortex, andNeuregulin-1 Variants and Behavioral Difference

Horizontal locomotor activity(cm, counts/60 min)eNRG1 (0.3 μg/g)
eNRG1 (1.0 μg/g)
1400
1200
1000
800

Table 2.

*

600
400
200
0

18000

Stereotypic counts(counts/60 min)

227

228

compared between control and eNRG1-treated mice inthe saline challenged-groups, however, there was asignificant difference in the basal frequency of c-Fospositive cells in the mPFC (p < 0.001). In the striatum,
we found no detectable increase in c-Fos expression
following MK-801 challenge [drug: F(1,12) = 0.1, p =0.79] (Fig. 4I).

ControleNRG1 (0.1 μg/g)

A

B

Current Molecular Medicine, 2015, Vol. 15, No. 3

15000
12000
9000
6000
3000
0

Fig. (2). Baseline locomotor activity and stereotypic behavior
in a novel environment. (A) Total locomotor activity and (B)stereotypic behavior counts of eNRG1 (0.1, 0.3 and 1.0µg/g)-treated and vehicle-treated control mice for 60 min(n = 10 mice per group). Data is expressed as mean ± SEM.
*p < 0.05 compared with control mice by Tukey HSD.

striatum. MK-801 challenge significantly increased thenumber of c-Fos-positive cells in these regions in bothcontrol and eNRG1-treated mice [drug: F(1,12) =384.1, p < 0.001 for mPFC; F(1,12) = 139, p < 0.001
for NAc; F(1,12) = 69.6, p < 0.001 for piriform cortex].
In the mPFC and NAc, however, we found significant
statistical interactions between MK-801 challenge andeNRG treatment [eNRG1 × drug: F(1,12) = 8.5, p <0.05 for mPFC, F(1,12) = 12.4, p < 0.01 for NAc]. Theinteractions indicate that MK-801-triggered increases in
the number of c-Fos-positive cells were greater in
eNRG1-treated mice than in control mice (Fig. 4A-H).
In contrast, the number of c-Fos positive cells in thepiriform cortex was indistinguishable in control andeNRG1-treated mice following MK-801 challenges[eNRG1-treatment: F(1,12) = 1.4, p = 0.27, eNRG1treatment × drug F(1,12) = 0.6, p = 0.45] (Fig. 4I). As

Summary of behavioral traits.
VehicleeNRG1

Prepulse inhibition (% inhibition)
73dB

20.5 ± 3.2-2.5 ± 3.9***

76dB

38.5 ± 3.5

4.7 ± 4.8***

79dB

47.2 ± 3.3

3.7 ± 5.3***

82dB

57.6 ± 3.2

8.7 ± 4.9***

Shock sensitivity

61.3 ± 3.4

60.0 ± 4.7Context-dependent

66.5 ± 1.5

65.7 ± 2.1

Tone-dependent

66.0 ± 1.3

41.5 ± 3.2***

Sniffing Duration (sec)

178.3 ± 8.9

145.8 ± 8.1*

Sniffing Counts (times)

9.7 ± 0.8

8.1 ± 0.7

Aggression (frequency)

2.28 ± 0.14

3.61 ± 0.23

Learning (% freezing)

Resident-intruder test

Neonatal mice were treated with eNRG1 (1.0 μg/g) or vehicle (control) as
described in the Materials and Methods. In adulthood, PPI levels (%) were
measured with 73, 76, 79 and 82 dB prepulse stimuli (n = 25-26 mice pergroup) and a main pulse of 120 dB. eNRG1-treated and vehicle-treated mice
were also subjected to context-tone-foot shock pairs to evaluate their fear
learning. Mean freezing rates (%) were measured for 150 sec (30 sec/ bin)during conditioning or during the same context exposure and during tone
exposure in the test paradigm (n = 20-21 mice per group). In the residentintruder assay, we measured the total time duration of sniffing behaviors andfrequency of sniffing and fighting behaviors of the resident males over a 10-min
period (n = 18 mice per group). Data are expressed as mean ± SEM. *p < 0.05,*** p < 0.001 by Tukey HSD or Student’s t-test.

NMDA Receptor-Like Immunoreactivity in eNRG1Treated Mice
Alterations in brain expression of NMDA receptors
are often implicated in the behavioral sensitivity to the
NMDA receptor blockers MK-801, phencyclidine, andketamine [24,32]. We aimed to elucidate the molecular
phenotypes underlying the hypersensitivity of eNRG1(1.0 μg/g eNRG1)-treated mice to MK-801. Using
Western blot techniques, we estimated protein levels
for NMDA receptor subunits (GluN1, GluN2A-2D) and
AMPA receptor subunits (GluA1-A4) in the frontal
cortex, hippocampus, and striatum plus NAc (Fig. 5A).
In addition to these receptors, we also examinedmolecular markers for GABAergic and dopaminergic
neurons. Among the receptors examined, there weresignificant decreases in hippocampal levels of GluN2Clike immunoreactivity and frontal cortex levels of
GluN2D-like immunoreactivity in eNRG1-treated micecompared with those of control mice (Fig. 5B). In
addition, there was a decrease in GAD67-like－ 46 －

Fig.
ster
MKmin
con
timi
MKtrea
eNR

imm
perf
com
How
like
The
and
grou

RG1-

tors
the
and
cular
RG1
sing
vels
and
ontal
5A).
ined
ergic
were
N2Cs of
mice
. In
-like

Kato et al.

Control + salineeNRG1 (1.0 μg/g) + saline
Control + MK-801
eNRG1 (1.0 μg/g) + MK-801

A

*****
** **
**
*

6000
5000
4000
3000

5000

Stereotypic counts(counts/10 min)

7000

4000
3000
2000

2000
1000
0

B

1000

###

0

Time (min)Time (min)Control + MK-801
eNRG1 (0.1 μg/g) + MK-801

eNRG1 (0.3 μg/g) + MK-801

eNRG1 (1.0 μg/g) + MK-801

C

#

50000
40000

D
40000

*

Stereotypic counts(counts/120 min)ol) as
were
e per
mice
r fearc/ bin)tone
idents and
0-min
0.05,

Current Molecular Medicine, 2015, Vol. 15, No. 3

Horizontal locomotion activity(cm, counts/10 min)

ce in
as a
Fosum,
sion
p =

228

Horizontal locomotor activity(cm, counts/120 min)

227

30000

30000

20000

20000

10000

10000
0

0

Fig. (3). Hyper-locomotor activity and stereotypic behavior induced by MK-801. (A, C) Horizontal locomotor activity and (B, D)
stereotypic behavior of eNRG1 (0.1, 0.3 and 1.0 µg/g)-treated and vehicle-treated control mice were monitored before and afterMK-801 (0.3 µg/g, i.p.) or saline challenge every 10 min. Prior to MK-801 challenge, mice were acclimated to a test chamber tominimize their exploratory movement. Total locomotor activity and stereotypic counts of eNRG1-treated and vehicle-treatedcontrol mice were calculated and presented (n = 10 mice per group). Data is expressed as mean ± SEM. Arrows mark thetiming of MK-801 challenge. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with vehicle-treated control mice challenged by#
MK-801 and p < 0.05 compared between marked groups by Tukey HSD. Note; There was a significant main effect of eNRG1
treatment on total locomotor activity during the acclimination period of the four groups of mice; F(1, 36) = 19.3, ###p < 0.001 foreNRG1, F(1,36) = 0.46, p = 0.503 for MK-801, F(1,36) = 1.46, p = 0.234 for eNRG1 x MK-801.

immunoreactivity in eNRG1-treated mice. As we
performed multiple comparisons here, we attempted tocompensate the statistics with a Bonferroni’s method.
However, the differences in GluN2C-like and GluN2Dlike immunoreactivites still remained to be significant.
There were no significant differences in other receptors
and other GABAergic/dopaminergic markers between
groups after compensation.

－ 47 －

Distinct Sensitivity of eNRG1-Treated and T1NRG1-Treated Mice to MK-801 But Not toMethamphetamine
Recently, we reported that neonatal exposure to fulllength T1-NRG1 results in behavioral impairments andhypersensitivity to methamphetamine in mice [15]. To
compare the in vivo effects of eNRG1 and T1-NRG1,

Neuregulin-1 Variants and Behavioral Difference

Current Molecular Medicine, 2015, Vol. 15, No. 3

mPFC

229

230

NAc

ControleNRG1AControl

E

F

Saline

B

eNRG1

ac

D

H

G

MK-801

C

ac

ac

ac

The number of cT
c-fos
positive cells
s

I

***
400

###

***

###

300
200

*

Control + salineeNRG1 (1.0 g/g) + saline
Control + MK
MK-801801
eNRG1 (1.0 g/g) + MK-801

###
###

### ###

100
0

mPFC

NAc

Piriform
cortexStriatumFig. (4). Neonatal exposure to eNRG1 enhances c-Fos expression following MK-801-challenge. In adulthood, vehicle-treatedmice (A, C, E, G) and eNRG1-treated mice (B, D, F, H) were subjected to c-Fos immunohistochemistry 2 h after saline (A, B, E,F) or MK-801 (C, D, G, H) challenge. Representative pictures of mPFC (prelimbic cortex; A - D) and NAc (E - H) are shown.
Scale bar, 100 μm. (I) The number of c-Fos-positive cells in the microscopic field (725 × 965 μm) was counted bilaterally using
five to seven sections of mPFC (+1.70 ~ +1.98 mm from Bregma), NAc, and striatum (+1.18 ~ +1.54 mm from Bregma), piriform
cortex (+1.18 ~ +1.98 mm from Bregma), averaged for each mouse, and subjected to statistical analysis (n = 4 mice per group).
###
Values are expressed as mean ± SEM. *p < 0.05, ***p < 0.001 compared between marked groups. p < 0.001 compared withsaline-challenged groups by Scheffe's test.

we measured the sensitivity of T1-NRG1-treated andeNRG1-treated mice to the NMDA receptor antagonist
and methamphetamine (Figs. 6, 7). We prepared bothtypes of mouse models by daily administering the same
molar dose of T1-NRG1 (1.0 μg/g body weight) andeNRG1 (0.3 μg/g body weight) to mouse pups, allowed

them to reach 2 months of age, and challenged with
MK-801 or saline. This dose was approximately 40
pmol per gram body weight and equivalent to the molar
dose of T1-NRG1 that had been given to mouse pups
in our previous study [15].

－ 48 －

Fig.
mar
eNR
the
GluA
(TH
disp
and
sam
GluA
n=

229

ated
B, E,
own.
sing
form
oup).
with

with
40
molar
pups

230

Current Molecular Medicine, 2015, Vol. 15, No. 3

A

Frontal Cortex
Control eNRG

Kato et al.

Striatum+
Hippocampus Nucleus Accumbens
Control eNRG

Control eNRG

GluN1
GluN2A
GluN2B
GluN2C
GluN2D
GluA1

GluA2/3
GluA4
GAD65/67
Parvalbumin
Tyrosine
Hydroxylase
-actinB
GluN1 #
GluN2A
GluN2B # 
GluN2C
GluN2D #GluA1 #GluA2/3
GluA4 #GAD65
GAD67
ParvalbuminTH-actin (n=5-6)(n=9-10) #Frontal Cortex(% of control)

Hippocampus(% of control)

Striatum + Nucleus Accumbens(% of control)93 488  11100  6103  1758  7**121  13107  399  292  384  3*104  892  3101  6104  3105  15111  12110  1750  7**124  1696  780  1192  7116  399  296  680  598  1099  599  599  898  1379  1196  10104  6105  14109  1294  390  896  790  797  998  4

Fig. (5). Influences of neonatal eNRG1 treatment on the immunoreactivity of glutamate receptors, GABAergic and dopaminergic
markers. Protein extracts were prepared from the frontal cortex, hippocampus, and striatum puls NAc of vehicle-treated andeNRG1 (1.0 μg/g)-treated mice (n = 4-5 mice per group) at the adult stage (PND70-80) and subjected to immunoblotting withthe antibodies directed against NMDA receptors (GluN1, GluN2A, GluN2B, GluN2C, GluN2D), AMPA receptors (GluA1,
GluA2/3 and GluA4), GABAergic markers (glutamic acid decarboxylase; GAD65/67 and parvalbumin), a dopaminergic marker(TH; tyrosine hydroxylase), and a loading control (β-actin). (A) Typical signals of two samples in each brain region aredisplayed. (B) The intensity of the immunoreactivity appearing at the authentic size(s) was measured by densitometoric analysis
and subjected to an unpaired two-tailed t-test. *p < 0.05, **p < 0.01 without Bonferroni’s compensation. #) The number ofsamples was increased to 9 or 10 when the values of SEM from 4-5 mice had been larger than 20%; GluN1, GluN2B, GluA1,
GluA4, and β-actin. Relative levels of the markers in eNRG1-treated mice are presented (% of control; mean ± SEM; n = 4-5 or
n = 9-10 for #).

－ 49 －

Neuregulin-1 Variants and Behavioral Difference

**

50000

#

40000
30000
20000
10000
0

ControleNRG1T1 NRG1

231

Saline group

A

MAP group
60000

**

50000

*

40000
30000
20000
10000
0

ControleNRG1T1 NRG1

40000

B

30000
20000
10000
0

ControleNRG1T1 NRG1

Fig. (6). Difference of eNRG1-treated and T1-NRG1-treatedmice in MK-801-driven locomotor activity and stereotypic
behaviors. eNRG1 (0.3 µg/g)-treated, T1-NRG1 (1.0 µg/g)treated and vehicle-treated mice (control) as neonates werechallenged with MK-801 (0.3 µg/g; black boxes) or saline(white boxes) and their horizontal locomotor activity (A) andstereotypic behavior (B) were compared. Total horizontal
activity and stereotypic score of mice were measured for the
120 min period after challenge and presented with the scalesof cm/120 min and counts/120 min, respectively (n = 10 - 23
mice per group). Data is expressed as mean ± SEM. **p <0.01, compared between control and eNRG1-treated mice#challenged with MK-801 and p < 0.05 compared betweeneNRG1-treated mice challenged with MK-801 and T1-NRG1treated mice challenged with MK-801 by Tukey HSD.

An initial 2-way ANOVA on horizontal locomotor
activity, using the subject factors of NRG1 isoform andMK-801, revealed a main effect of MK-801 [F(1,74) =23.57, p < 0.001] and a significant interaction between
NRG1 isoform and MK-801 [F(2,74) = 5.17, p < 0.01].
We then stratified on MK-801 treatment (MK-801challenged and saline-challenged groups; Fig. 6A). In
the saline-challenged control groups, there was no

Stereotypic counts(counts/120 min)Horizontal locomotor activity(cm/120 min)Stereotypic counts(counts/120 min)

MK-801 group

Horizontal locomotor activity(cm/120 min)Saline group

A

B

Current Molecular Medicine, 2015, Vol. 15, No. 3

***

70000
60000
50000

*

40000
30000
20000
10000
0

ControleNRG1T1 NRG1

Fig. (7). Similar enhancement of eNRG1-treated and T1NRG1-treated mice in methamphetamine-driven locomotor
activity and stereotypic behaviors. eNRG1 (0.3 µg/g)-treated,
T1-NRG1 (1.0 µg/g)-treated and vehicle-treated mice(control)as
neonates
werechallenged
withmethamphetamine (MAP; 1.0 μg/g black boxes) or saline(white boxes) and their horizontal locomotor activity (A) andstereotypic behavior (B) were compared. Data is expressed
as mean ± SEM. *p < 0.05, **p < 0.01 between control andeNRG1-treated or T1-NRG1 treated mice challenged with
MAP by Tukey HSD.

difference in basal locomotor activity among T1-NRG1treated, eNRG1-treated and control mice [NRG1
isoform : F(2,37) = 0.70, p = 0.50]. A one-way ANOVA
for the MK-801-challenged group revealed a significant
effect of isoform [F(2,37) = 4.80, p < 0.05]. Post-hoc
analyses suggest that MK-801-driven horizontal
locomotor activity of T1-NRG1-treated mice wasindistinguishable from that of control mice receiving－ 50 －

232

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231

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ated,
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and
with

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ontal
was
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232

Current Molecular Medicine, 2015, Vol. 15, No. 3

Kato et al.

MK-801 whereas eNRG1-treated mice displayed alarger locomotor response to MK-801 than control
mice. A two-way ANOVA for stereotypic movement
revealed no significant effect of NRG1 isoform or nointeraction between NRG1 isoform and MK-801 [NRG1
isoform: F(2,74) = 0.15, p = 0.86; NRG1 isoform × MK801: F(2,74) = 0.99, p = 0.37] (Fig. 6B). The resultsfrom the horizontal activity test revealed a significant
difference in behavioral sensitivity to the NMDA
receptor antagonist between T1-NRG1-treated andeNRG1-treated mice.
We also compared the magnitude of methamphetamine effects on locomotor activity. An initial 2-way
ANOVA on horizontal movement, with between subject
factors of NRG1 isoform and methamphetamine,
revealed a main effect of methamphetamine [F(1,92) =60.2, p < 0.001] and an interaction between NRG1
isoform and methamphetamine [F(2,92) = 5.19, p <0.01]. We then stratified on methamphetamine
treatment (methamphetamine-challenged and salinechallenged groups; Fig. 7A). In the saline-challenged
groups, there were no significant differences amongeNRG1-treated, T1-NRG1-treated, and control groups[F(2,46) = 0.60, p = 0.56]. A one-way ANOVA for themethamphetamine-challenged groups revealed asignificant effect of NRG1 isoform [F(2,46) = 5.68, p <0.01]. Post-hoc analyses detected a significant
difference between eNRG1-treated and control groups
and between T1-NRG1-treated and control groups, but
not between eNRG1-treated and T1-NRG1-treatedgroups. These results suggest that neonatal treatmentswith eNRG1 and T1-NRG1 similarly enhancemethamphetamine-induced horizontal locomotion. We
analyzed the effects of NRG1 isoform on stereotypic
movements in a similar manner (Fig. 7B). There was amain effect of methamphetamine [F(1,92) = 101, p <0.001] and an interaction between NRG1 isoform andmethamphetamine [F(2,92) = 4.74, p < 0.05]. Thus, we
stratified on methamphetamine treatment. In the salinechallenged groups, there were no significant
differences among groups [F(2,46) = 0.0064, p = 0.99].
In the methamphetamine-challenged groups, however,there was a significant effect of NRG1 isoform [F(2,46)= 5.19, p < 0.01]. Post-hoc tests detected a significant
difference between eNRG1-treated and control groups
and between T1-NRG1-treated and control groups, but
not between eNRG1-treated and T1-NRG1-treatedgroups. These analyses suggest that neonatal treatments with eNRG1 and T1-NRG1 similarly enhancemethamphetamine-triggered stereotypic movements.
Auditory Perception of eNRG1-Treated Mice
NRG1 signaling is involved in the survival ofcochlear sensory neurons; thus, we tested soundstartle responses of the mice treated with eNRG1 (0.3
and 1.0 μg/g) to increasing noises and also measured
the auditory stimulus thresholds in different tone
frequencies. In the sound startle response test, thelower dose of eNRG1-treated mice exhibited a marked
increase in startle responses to 110-dB and 120-dB
tones, compared with control mice [eNRG1 × pulse:－ 51 －

F(6,114) = 30.6, p < 0.001] (Fig. 8A). Conversely, theeNRG1-treated mice exhibited marked increases in theauditory stimulus thresholds at all stimulus frequencies
at the ABR test [eNRG1 × stimulus frequencies:
F(5,90) = 11.2, p < 0.001] (Fig. 8B). The mice treatedwith the higher dose of eNRG1 (1.0 μg/g) similarly
exhibited the increases in startle responses and theauditory stimulus thresholds [eNRG1 × pulse: F(6,114)= 21.4, p < 0.001 for startle responses; eNRG1 ×
stimulus frequencies: F(5,90) = 13.4, p < 0.001 forauditory stimulus thresholds] (Fig. 8A, B). The resultsfrom the ABR test suggest deterioration in hearing ofeNRG1-trearted mice. We speculate that, in the soundstartle response test, the saturated levels of tone
stimuli appear to produce higher startle responses inburst-noise-naïve
eNRG1-treated
mice
with
deterioration in hearing.

DISCUSSION
In our preceding study, mice pups were treated with
T1-NRG1 and their behavioral consequences wereanalyzed with respect to the relevancy to an animalmodel for schizophrenia [15]. The T1-NRG1-treatedanimals exhibit various behavioral abnormalities in PPI
and social traits but their gross learning performanceand hearing ability are normal [15]. In addition to theisoform of T1-NRG1, here mouse pups werechallenged with eNRG1, the core domain peptide thatis produced by the neuropsin-dependent processing of
T1-NRG1 [7]. In agreement with the preceding resultsfrom T1-NRG1-treated mice, transient exposure ofeNRG1 during the neonatal stage similarly altered adultbehavioral traits in PPI and resident-intruder test. Even
though the mice were treated with the same molar
dose of NRG1, eNRG1-treated mice exhibited
additional behavioral deficits in tone-dependent fear
learning, sound-startle responses and sensitivity to the
NMDA receptor antagonist MK-801.
We also found that eNRG1-treated mice have ahearing disability. This finding is consistent with theprevious report that ErbB4 signals regulate thedevelopment of hair follicles in the inner ear and thatthe manipulation of NRG1 signals indeed impair
hearing in mice [33,34]. In this context, we can suggestthat the deficits in prepulse inhibition, sound-startle
responses, and tone-dependent learning of eNRG1treated mice are, in part, due to the deterioration inhearing.
The decrease of 1.0 μg/g eNRG1-treated mice in
exploratory movement is also noteworthy. In contrast to
the basal locomotor reduction, these mice rather
exhibited the higher locomotor responses following MK801 challenge. Although the lower basal movement
might reflect the elevation of their anxiety levels, we do
not rule out the possibility that these behavioralfeatures of eNRG1-treated mice might be indirectly
influenced by their deterioration in hearing as well.
In the present study, we additionally prepared theanimal treated with the full soluble type 1 isoform of T1NRG1 at the same molar dose of eNRG1 andNeuregulin-1 Variants and Behavioral DifferenceeNRG1 (0.3 μg/g)

###

***

***

300
200

###

100
0

## ### ###90

200

Control100
80

eNRG1 (0.3 μg/g)

***

60

*** ***

40

***

***

***

20
0

4

8

10

16

20

###

100
0

### ######
95 100 105 110 115 120

90

Pulse (dB)100
80

Test frequency (kHz)

***

60

ControleNRG1 (1.0 μg/g)

***

40

**

***

***

***

20
0

32

###

***

***

300

95 100 105 110 115 120

233

###

ControleNRG1 (1.0 μg/g)

400Pulse (dB)ABR thresholds (dB)

B

Control

400

500

###

Startle amplitude

500

ABR thresholds (dB)Startle amplitudeA

Current Molecular Medicine, 2015, Vol. 15, No. 3

4

8

10

16

20

32

Test frequency (kHz)Fig. (8). Auditory responses of eNRG1-treated mice as adults. (A) Relative amplitudes of startle responses of eNRG1 (left; 0.3µg/g and right; 1.0 µg/g)-treated and vehicle-treated mice (control) were monitored with 90 − 120 dB tones (n = 10 mice per#####group). Data is expressed as mean ± SEM. *** p < 0.001 compared between groups. p < 0.01 and p < 0.001 compared withstartle levels at 90 dB by Tukey HSD. (B) ABR thresholds of eNRG1 (left; 0.3 µg/g and right; 1.0 µg/g)-treated and vehicletreated mice were determined with specific auditory stimuli (4, 8, 10, 16, 20, and 32 kHz) by varying the sound pressure levels(n = 9-11 mice per group). Data is expressed as mean ± SEM. **p < 0.01 and *** p < 0.001 compared between groups byScheffe's test.

compared the sensitivities of eNRG1-treated and T1NRG1-treated mice to two psychotomimetics, MK-801
and methamphetamine. The MK-801-driven locomotor
enhancement was larger in mice treated with eNRG1
as neonates, compared with T1-NRG1-treated mice,even though the given molar concentration of T1-NRG1
was nearly equal to the dose of eNRG1. The
hypersensitivity of the eNRG1-treated mice to MK-801
was also apparent in the magnitude of c-Fos inductionin the brain. Following MK-801 challenge, eNRG1treated mice contained a higher number of c-Fospositive cells in the mPFC as well as in the NAc thancontrol mice receiving MK-801. Both brain regions are

implicated as target regions of MK-801 [35]. In contrast
to the responses to MK-801, there were no significant
difference in methamphetamine-triggered locomotor
enhancement between T1-NRG1-treated and eNRG1treated mice. The behavioral difference of T1-NRG1treated and eNRG1-treated mice might reflect
qualitative differences in in vivo activities of eNRG1and T1-NRG1. Alternatively, the quantitative differencein the efficacy of blood-brain barrier permeability or inmetabolic rates in the brain might underly the
behavioral difference between these NRG1 variants.
However, the magnitude and peak time of ErbB4
phosphoryration appeared to be similar between－ 52 －

234

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233

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Kato et al.

eNRG1 and T1-NRG1 (i.e. both ~3-fold increases threehours after injection of their 1.0 μg/g dose) [15].
Several reports indicate the activity difference
among NRG1 splicing variants. Major splicing variantsof NRG1 (type 1; NDF, type 2; GGF, and type 3;
SMDF) exhibit distinct interactions to individual ErbB14 receptor subtypes, presumably leading to distinct
biological effects in the brain [35,36]. NRG1 isoforms
exhibit qualitative differences due to their specificstructures such as an immunoglobulin-like domain.
This domain, which is found in T1-NRG1 but noteNRG1, carries a heparin binding activity and regulates
tissue distribution and receptor interactions of theligand [7,37,38].
eNRG1 is produced from T1-NRG1 as well as
potentially from the type2 isoform of NRG1 through theneuropsin-dependent proteolytic processing [7]. The
protease neuropsin recognizes and cleaves theimmunoglobulin-like domain of the NRG1 precursors
and liberate their carboxyl terminal domain; eNRG1.
Tamura et al. [7] demonstrate the biological implicationof the neuropsin-dependent NRG1 processing in
hippocampal synapse plasticity. As the molar dose ofeNRG1 was adjusted to that of T1-NRG1 in some ofthe present experiments, their dose difference cannot
illustrate the behavioral differences between T1-NRG1treated and eNRG1-treated mice. Nonetheless, thepresent results demonstrate that the biological actionsof individual NRG1 variants significantly differ in vivo
and result in distinct behavioral consequences.
Corroborating our findings, mice deficient in type 3
NRG1 exhibit distinct behavioral deficits from type 1
NRG1-knockout mice [8,9,11]. However, it remains to
be explored whether the present behavioral differencesof T1-NRG1-treated and eNRG1-treated mice areascribed to the unique activity of the immunoglobulinlike domain or that of the neuropsin-cleaving producteNRG1 [7,38].
To elucidate the molecular underpinnings of thegiven hypersensitivity to the NMDA receptor
antagonist, we assessed the neurochemical influencesof eNRG1 in adulthood, focusing on NMDA-type and
AMPA-typeglutamate
receptors.
There
were
reductions in NMDA receptor-like immunoreactivities(i.e. GluN2C and GluN2D) in the frontal cortex andhippocampus of eNRG1-treated mice, which can be
involved in the hypersensitivity to MK-801. The
specificity of the anti-GluN2D antibody, but not that for
the anti-GluN2C antibody, has been verified with theuse of knockout mice [39,40] Although the molecularnature of the present GluN2C-like immunoreactivity
required further validation, the above arguments are inagreement with the observation that NMDA receptorhypomorphic mutant mice show hypersensitivity to an
NMDA receptor blocker [24,41]. Recently, Belforte etal. (2010) also provide supportive evidence that thepsychomimetric activity of MK-801 is ascribed to adecrease in the NMDA receptor sensitivity ofparvalbumin-positive GABA neurons during postnatal
development [42]. As parvalbumin-positive GABA
neurons are enriched with ErbB4 receptors for NRG1,－ 53 －

the expression of NMDA receptors in this cell
population might be affected by excess eNRG1 in thepresent model [43-46]. However, we do not rule out thepossibility that the eNRG1-driven hypersensitivity to
MK-801 may involve glutamatergic, dopaminergic, or
cholinergic brain circuits as suggested previously [47].
NRG1 is one of the most promising candidate
genes for schizophrenia. Most of the single nucleotide
polymorphisms (SNPs) associating with schizophrenia
are located in the genomic promoter region and intronsof the 5’-side of the NRG1 gene [4,48,49]. Although thefunctional effects of these polymorphisms on genetranscription or alternative splicing of NRG1 isoforms
are largely speculative, postmortem studies indicatethat schizophrenic alterations of NRG1 mRNA levels
are isoform-specific [3,50,51]. In agreement, theindividual SNPs in human NRG1 are differentially
implicated in psychotic symptoms, spatial working
memory capacity, and white matter density andintegrity [52,53]. In addition to schizophrenia, thegenetic and neuropathologic association with NRG1 is
implicated in several other psychiatric disease major
depression, and bipolar disorder as well althoughseveral controversies still remain [54,55]. The
differential expression of NRG1 processing enzymes isreported in schizophrenia and bipolar disorder [56].
Thus differential processing of NRG1 protein may
contribute to the disease specificity as well.
The present results indicate that the composition of
NRG1 isoforms might influence behavioral or cognitive
traits in humans as well. Future studies should
investigate the biological or pathological actions ofindividual NRG1 splicing isoforms underlying the SNPdependent cognitive and anatomical traits in normal
subjects and/or patients with schizophrenia.

ABBREVIATIONSABR= Auditory brain-stem evoked response

ANOVA= Analysis of variance

EGF= Epidermal growth factoreNRG1= An epidermal growth factor domain
peptide of neuregulin-1

mPFC= Medial prefrontal cortex

NAc= Nucleus accumbens

NRG1= Neuregulin-1

PBS= Phosphate-buffered saline

PND= Postnatal day

PPI= Prepulse inhibition

SNP= Single nucleotide polymorphisms

T1-NRG1 = Type1 neuregulin-1

CONFLICT OF INTEREST
The authors confirm that this article content has notconflicts of interest.

Neuregulin-1 Variants and Behavioral Difference

ACKNOWLEDGEMENTS
The authors are grateful to Minoru Tanaka andMichisuke Yokoyama for their assistance with mouse
breeding. This work was supported by MEXT KAKENHI(No.24116010), JSPS KAKENHI (No. 22300107), anda grant for Promotion of Niigata University Research
Projects.

SUPPLEMENTARY MATERIAL

Current Molecular Medicine, 2015, Vol. 15, No. 3
[17]
[18]
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Supplementary material is available on the
publisher’s web site along with the published article.

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Received: November 11, 2014

Revised: December 20, 2014

Accepted: January 17, 2015ction,
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Sakai et al. Molecular Cytogenetics (2015) 8:46
DOI 10.1186/s13039-015-0144-5

Sak

RESEARCH

Open Access

Assessment of copy number variations in
the brain genome of schizophrenia patients
Miwako Sakai1,2, Yuichiro Watanabe2, Toshiyuki Someya2, Kazuaki Araki1, Masako Shibuya2, Kazuhiro Niizato3,
Kenichi Oshima3, Yasuto Kunii4, Hirooki Yabe4, Junya Matsumoto4, Akira Wada4, Mizuki Hino4, Takeshi Hashimoto5,
Akitoyo Hishimoto5, Noboru Kitamura5, Shuji Iritani3,6, Osamu Shirakawa5,7, Kiyoshi Maeda5,8, Akinori Miyashita9,
Shin-ichi Niwa4, Hitoshi Takahashi10, Akiyoshi Kakita10, Ryozo Kuwano9 and Hiroyuki Nawa1*
Abstract
Background: Cytogenomic mutations and chromosomal abnormality are implicated in the neuropathology ofseveral brain diseases. Cell heterogeneity of brain tissues makes their detection and validation difficult, however.
In the present study, we analyzed gene dosage alterations in brain DNA of schizophrenia patients and compared thosewith the copy number variations (CNVs) identified in schizophrenia patients as well as with those in Asian lymphocyte
DNA and attempted to obtain hints at the pathological contribution of cytogenomic instability to schizophrenia.
Results: Brain DNA was extracted from postmortem striatum of schizophrenia patients and control subjects (n = 48each) and subjected to the direct two color microarray analysis that limits technical data variations. Disease-associatedbiases of relative DNA doses were statistically analyzed with Bonferroni’s compensation on the premise of brain cellmosaicism. We found that the relative gene dosage of 85 regions significantly varied among a million of probe sites.
In the candidate CNV regions, 26 regions had no overlaps with the common CNVs found in Asian populations andincluded the genes (i.e., ANTXRL, CHST9, DNM3, NDST3, SDK1, STRC, SKY) that are associated with schizophrenia and/or
other psychiatric diseases. The majority of these candidate CNVs exhibited high statistical probabilities but their signal
differences in gene dosage were less than 1.5-fold. For test evaluation, we rather selected the 10 candidate CNV regionsthat exhibited higher aberration scores or larger global effects and were thus confirmable by PCR. Quantitative PCR
verified the loss of gene dosage at two loci (1p36.21 and 1p13.3) and confirmed the global variation of the copy
number distributions at two loci (11p15.4 and 13q21.1), both indicating the utility of the present strategy. These
test loci, however, exhibited the same somatic CNV patterns in the other brain region.
Conclusions: The present study lists the candidate regions potentially representing cytogenomic CNVs in the brain of
schizophrenia patients, although the significant but modest alterations in their brain genome doses largely remain to
be characterized further.
Keywords: CNV, Caudate, Genome instability, Schizophrenia, Somatic mutation

Background
Copy number variation (CNV) is defined as a deletion or
duplication/multiplication of a genomic fragment spanning more than 1 kb when compared to a reference genome [1–3]. Approximately 37,000 sites of common CNVs
have been identified in the human genome and they occupy 12 % of the entire genome [4, 5]. The genome-wide* Correspondence: hnawa@bri.niigata-u.ac.jp
1
Department of Molecular Neurobiology, Brain Research Institute, Niigata
University, 1-757, Asahimachi-dori, 951-8585 Niigata, Japan
Full list of author information is available at the end of the article

association studies (GWAS) on schizophrenia analyzed
DNA which was isolated from peripheral lymphocytes
and have identified risk CNV sites, some of which are notpresent in the patients’ parents [6–9].
Somatic mosaicism of genome sequences and structures have recently drawn particular attention [10–12].
Nearly 30 % of developing brain cells in human are reported
to harbor aberrant chromosomal compositions [13, 14]. In
addition, there are significant genomic differences in somatic cells between monozygotic twins and among tissues[15–18]. Accordingly, aberrant cytogenomic variations in© 2015 Sakai et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

－ 56 －

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human brain are implicated in neurodegenerative andneurodevelopmental disorders such as Alzheimer’s disease,amyotrophic lateral sclerosis, and Huntington’s diseases[19–25]. It is an open question whether the brain-specificsomatic mutation or CNV might also contribute to the
etiology or neuropathology of schizophrenia [26–28].
To obtain hints at the above question, we prepared
DNA from the brain tissue of 48 schizophrenia patientsand 48 control subjects. Labeling brain DNA samples, we
directly applied those to Agilent 1 M comparative genomic
hybridization (CGH) arrays to measure relative gene doses
without the use of reference genome. This direct comparison through the case–control pairing reduces technicaldata deviations and enhances the statistical power ofdetection [29, 30]. With the potential genomic mosaicism
of heterogeneous brain cell mixtures, we expected that thetarget genome could be diluted with normal DNA fromthe off-target cells and thus assumed non-integer valuesof CNVs in this analysis [31]. Technical limitations of thisapproach are further discussed below.

Results
The striatum contain neural stem cells that proliferate
throughout human life and carries somatic mutation in its
mitochondrial genome [32, 33]. Therefore, we hypothesized that the striatum may be a potential candidate region
that would exhibit somatic mosaicism in brain genome
structures. DNA was extracted from postmortem striatumof patients with chronic schizophrenia (n = 48) and agematched controls who had no history of neuropsychiatricdisorders (n = 48) (Additional file 1: Table S1). Although
there were significant differences in postmortem intervals(PMIs) between groups, there was no detectable difference
in DNA quality (data not shown). All other indices wereindistinguishable between schizophrenia patients and control subjects. A DNA sample was randomly picked fromeach group, paired to a sample in the other group, andsubjected to two-color competitive CGH analysis with
1 M SurePrint G3 Human CGH Microarrays.
We applied the ADM-2 algorithm to the CGH signals of
individual microarray probes (nearly 1 million) and searched
for the primary candidate CNV loci associated with schizophrenia. A flowchart of the present study design is shown in
Additional file 1: Figure S1. We chose1381 chromosomal
loci that exhibited large group differences in gain/loss calls(Selection 1). In each probe site located on the primary
candidate loci, we plotted the distribution of log2 signal
ratios from 48 sets of microarray analyses and tested thenull hypothesis that the mean log2 signal ratios was zero,
indicating that the two groups were indistinguishable(Selection 2). We calculated total probabilities and averaged log2 signal ratios for individual candidate loci andjudged their statistical significance with Bonferroni’s correction. The number of the candidate loci maintaining thestatistical significance through Selection 2 was reduced to
85 (Details in Additional file 1: Table S2).
Positive CNV loci were found in almost all chromosomes except chromosome 17 and 21 (Fig. 1). Individualloci covered 1–746 probe sites (3–2200 kb) and exhibitedaverage log2 ratios of −1.46 to +0.63 (i.e., odds ratio (OR) =0.36–1.55). A majority of the average log2 ratios were between −0.59 to +0.59 (i.e., < 1.5-fold differences) and only
4 loci showed more than 1.5-fold differences in array
CGH signals. A genomic region spanning from 6p22.2
to 6p21.32 contained six CNV loci and included genes
for the major histocompatibility complex that is highly
associated with schizophrenia in GWAS [34]. Amongthe 85 CNV loci in Selection 2, 59 loci were reported
and 26 loci were not reported in the CNV study on
leukocyte DNA samples of Asian populations (Additional
file 1: Table S2) [2].
To validate the authenticity of the present procedure,
we attempted to verify the above genome dosage changesof several candidate loci using quantitative polymerase
chain reaction (qPCR). According to the following two criteria, we selected the test loci whose signal differenceswere larger between groups and could be detectable withthe given accuracy of qPCR; (i) those exhibiting the large
and consistent gain/loss calls across the limited sample
pairs (from Selection 1) and (ii) the loci represented larger
global effects shared in most of the schizophrenia samples(from Selection 2).
In the former category, the gene dosage of Hs03385437(1p13.3), CC70L1J (1p36.21), Hs03318079 (Chr18:q22.1),
Hs04794356 (4q24), Hs05080419 (9q22.2), and Hs07134106(19p12) produced exclusive gain/loss calls in not less thanfour sample pairs. No discrepant calls were detected inany sample pairs. Using the same DNA pairs showing thedifference in the penetrance call (Selection 1), we determined and confirmed the gene dosage of those DNA samples using qPCR. ANOVA detected significant gene dose
differences at two loci (Hs03385437 and CC70L1J) between patient and control groups (Fig. 2).
In this measurement, we used RNaseP gene as an internal
DNA dose control. Measured genome doses of the above
regions appeared not to be integer levels in several control
samples, potentially reflecting the cell mosaicism of the original tissues. We also extracted DNA from the prefrontal
cortex of the same subjects of both groups and compared
the genome doses of the above loci (Hs03385437 andCC70L1J). We calculated the copy number ratio of thepatient’ DNA dosage to that of the control subject’ dosage
and compared these ratios between the brain regions. At
both loci, almost all the copy number ratios were markedly lower than 1.0 except the C26:S34 pair, supporting
our primary observation that the absolute gene dosages ofthese loci were decreased in the schizophrenia samples.
However, copy number ratios did not significantly differ－ 57 －

Fig. 1 Statistically positive loci for log2 signal ratios in 48 sets of CGH microarrays. The signal bias was tested at each probe site by a two-tailed t-test. Bars represent the chromosomal position and lengths
represent the logarithmic value of the probability of rejecting the null hypothesis. As comparisons were repeated 1381 times, we applied the Bonferroni’s correction and decreased a statistical threshold fromp = 0.05 to p = 0.05/1381 (−log p = 4.44). Within a positive candidate CNV locus, neighboring probe sites were merged and their probabilities were cumulated (Additional file 1: Table S2). Note: Data fromchromosomes 17 and 21 are not shown because these did not include any positives

Sakai et al. Molecular Cytogenetics (2015) 8:46
Page 3 of 9

－ 58 －
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Sakai et al. Molecular Cytogenetics (2015) 8:46

Page 4 of 9at the levels of integers but with several exceptions. These
candidate CNVs appear to reflect the gene dosage differences of germinal origin.

Fig. 2 Gene dosage evaluation of the individual positive pairs in CGH
analysis. DNA was prepared from the striatum and prefrontal cortex of thesame subjects whose pairs resulted in the exclusive penetrance calls inarray CGH analysis (Selection 1). DNA was then subjected to qPCR using
the RNase P gene as an internal control in quadruplicate. Moreover, tocompare the gene dosage between the striatum and prefrontal cortex,
ratios of DNA dosages of the schizophrenia patients to those of thecontrol subjects were calculated in each brain region and plotted.
Statistical comparisons of gene dosages or their ratios were conducted by
two-way ANOVA with the subject factors of disease and sample pair,considering technical deviations. *p < 0.05, **p < 0.01, ***p < 0.001

between these brain regions in any of the sample pairs(Fig. 2). At least at these two candidate loci, we failed tofind evidence for a gene dosage difference between thesebrain regions.
In the latter category, Hs0358779 (6p22.1), Hs03265736(7p21.3), Hs03765933 (11p15.4), and Hs03298358 (q21.1)
exhibited higher log2 signal ratios and were thus subjectedto the test evaluation. Gene dosage of these four loci were
determined by qPCR using all the DNA samples in control
and schizophrenia groups (n = 48 each). Differences in genedosages were replicated by qPCR for Hs03765933 andHs03298358 (Fig. 3). In contrast to the data distributions ofFig. 2, almost all the values of the gene doses were located

Discussion
Several recent reports have indicated the neuropathologicalcontribution of somatic CNV or DNA instability of thebrain genome [19–28, 35–40]. In accordance with thesefindings, a small proportion few percent of brain cells are
known to exhibit aneuploidy and carry large CNVs [13,14, 41]. Aneuploidy is detected by fluorescence in situ
hybridization (FISH) and appears to be increased by theonset of Alzheimer’s disease [20, 22]. The aneuploidy ofchromosome 1, 18 and X was also identified in the brainof schizophrenia patients [21, 41]. Despite its advantages, FISH cannot be employed in exploratory investigations, unless the specific genome region of the CNV ofinterest is identified. Since bonafide genome structuresfrom off-target cells could dilute the abnormal genome
DNA population, more sensitive technologies remain to
be developed, which detect low quantities of CNV in heterogeneous cell mixtures of the brain tissue [42, 43]. In thepresent study, we attempted to evaluate the efficacy of the
CGH microarray technique to extract somatic CNVs in
the postmortem brains of schizophrenia patients [42, 43].
With given semi-quantitative nature of the microarray
technique, we applied statistics to the 1 M array CGH results from 48 sample pairs. Using the high density CGH
array and statistical approach, we found 85 candidate CNVloci in the present study; 59 CNV loci are overlapped withthe common CNV regions and the remaining 26 loci are
not reported in peripheral leukocyte-derived DNA of Asianpeople [2, 44]. Of note, the 26 candidate regions encode theseven genes that are associated with or implicated inschizophrenia or other psychiatric diseases; ANTXRL,CHST9, DNM3, NDST3, SDK1, STRC, and SKY (Additional file 1: Table S2). DNM3 in the candidate region of
1q24.3 is disruptively mutated in some of schizophrenia patients [45]. ANTXRL and CHST9 are located in the CNV
regions associated with bipolar disorder and autism [46,47]. NDST3 and STRC are the risk genes for schizophrenia
and hearing impairment that are identified by GWAS, respectively [48, 49]. SDK1 and SKY are the genes whose expression levels are markedly altered in the brain of
schizophrenia patients [50, 51]. Accordingly, the present
listing of the candidate brain CNVs is informative for future
cytogenomic studies on schizophrenia [21, 41].
It was difficult for us to validate most of the abovementioned 85 candidate loci with qPCR analysis with thegiven small signal differences between groups (i.e., less than
1.5-fold). Therefore, we selected the best 10 test loci thatexhibited relatively large and/or wide effects on gene dosage. The six loci were chosen from Selection 1 as putative
rare CNVs, which exhibited exclusive gain/loss calls in the－ 59 －

Sakai et al. Molecular Cytogenetics (2015) 8:46

Page 5 of 9

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Fig. 3 qPCR evaluations of candidate CNV loci from Selection 2. Gene dosages of the candidate loci 6p22.1, 7p21.3, 11p15.4, 13q21.1, and the
RNase P (internal control) were determined by qPCR using all sample DNAs and TaqMan probes (Additional file 1: Table S3). Individual genedosages of 48 patients’ or 48 controls’ DNAs were plotted and compared between groups using the Chi-square and Mann–Whitney U tests

limited number of samples. From Selection 2, the four lociwere chosen as provisional common variants, whichshowed large effects and higher probability levels in theabove parametric analysis. The qPCR analysis confirmed
the schizophrenia-associated gene dosage differences atnearly half of the candidate CNV loci, suggesting the validity of the present strategy.
Unfortunately we had neither stored peripheral tissues
nor information about these CNVs in peripheral DNA of
the same subjects. To estimate the contribution of somatic
CNVs to the present CNV listing, therefore, we were compelled to compare the gene dosages between the two brain
regions or to search for their absence in the databases ofAsian CNVs of leukocyte origin [2.44]. In the test PCR,
however, we could not detect significant differences in genedosages between the striatum and prefrontal cortex, at least,

at these test CNV loci. If somatic CNVs were produced
prior to neuroectodermal differentiation, there should be nodifference between these two neural tissues, suggesting thatthe present comparison between these brain regions was inappropriate. Therefore, a comparative analysis of DNA from
germinal cells of the same subjects will warrant this definitive conclusion [45].
Among the CNV candidate regions in Fig. 1, 26 candidate regions are not reported as the common CNVs of
Asian populations [2, 44]. The majority of theseloci exhibited high statistical significance with the probabilities of less than 10−100, such as 4q35.2, 6p11.2, 7q11–12,
11p15.4–15.5, and 15q11.2. In contrast, their CGH signal
differences between patients and controls were markedly
smaller (OR = 0.988–1.055). As discussed above, thesecandidate CNV loci include the peculiar genes that are－ 60 －

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implicated in schizophrenia [45–51]. These regions, which
exhibited small signal differences, might represent more
promising candidates of somatic CNV sites because thegenome aberration of target cells is presumably diluted in
the brain and should result in smaller ORs. However, suchsmall differences in gene dosage should make the conventional qPCR verification more challenging with the given
technical deviations [52]. To avoid target DNA dilution
with cell mosaicism, single cell qPCR or FISH may be more
beneficial in theory [20–22, 43]. However, it would be difficult to independently perform microdissection of
hundreds of cells and perform single-cell analysis
unless the target cell population is identified with molecular markers and its sensitivity of gene detection ishigh enough. FISH also requires properly fixed andprocessed brain tissues of the same subjects. With thegiven technical difficulties, therefore, we have been unable to verify these small variations.

Conclusion
The present CGH analysis lists the potential candidate
regions of somatic CNVs associated with schizophrenia,
although most of those exhibited the modest but highly
significant alterations in brain genome doses. Future studies aim to develop more elaborate techniques for somatic
genome mosaicism and to verify the schizophreniaassociated cytogenomic instability in the above CNV
candidates [53–56].

University. In brief, the left cerebral hemisphere was fixedin formalin for diagnostic examination and the right hemisphere was frozen at −80 °C. Tissue samples were takenfrom postmortem brains that did not exhibit neurodegenerative abnormalities by conventional pathological staining(data not shown). The striatum (caudate) was identified infrozen coronal slices according to a human brain atlas. All
tissues were collected and stored according to the principles of the Declaration of Helsinki, and tissue use was incompliance with the Human Tissue Act 2004.

DNA extraction

High molecular weight DNA was extracted by the guanidinium − phenol procedure (Gentra Pure Gene Tissue
Kit, Qiagen, Tokyo, Japan) according to the manufacturer’s protocol. Extracted DNA was quantified by spectrophotometry using a Nanodrop ND-2000® (Thermo
Scientific Wilmington, DE, USA). Samples with absorbance ratios of A260/280 ~ 1.80 and A260/230 > 1.90,
respectively, were regarded as sufficiently pure andsuitable for CGH analysis. Some DNA samples were
subjected to 1.0 % agarose gel electrophoresis for quality
control. Evidence of DNA degradation was not detectedin randomly-picked DNA samples from patient or control groups (data not shown).

Comparative genomic hybridization (CGH)

Methods
Ethical approval

The study was approved by Niigata University Medical
Ethics Committee (No. 683). The use of postmortem brain
tissues was authorized by the Matsuzawa Hospital Ethics
Committee, Kobe University Medical Ethics Committee,
Fukushima Medical University Ethics Committee, andNiigata University Medical Ethics Committee. The families of the control and schizophrenia patients provided
written informed consent to allow the use of brain tissues
for pathological investigations.
Brain tissue

Postmortem brain tissue was collected from patients withchronic schizophrenia (30 men, 18 women; mean age,
64.5 ± 12.5 years old) and from age-matched control subjects (30 men, 18 women; mean age, 64.2 ± 12.0 years old),with no history of neuropsychiatric disorders (Additional
file 1: Table S1). The diagnosis of schizophrenia was confirmed by examining the patient’s report according to
DSM-III or DSM-IV categories (American Psychiatric
Association). Postmortem brains of schizophrenia patientswere collected at Matsuzawa Hospital, Kobe University,
Fukushima Medical University and Niigata University,
while those of control subjects were collected at Niigata

Array-based CGH was performed by the manufacturer
Takara Bio Dragon Genomics Center (Seta, Shiga,
Japan). In brief, DNA (2 micro g) was fluorescentlabeled by random priming DNA synthesis in the presence of Cy3-dUTP (control group) or Cy5-dUTP (patient group) (Genomic DNA Enzymatic Labeling Kit;
Agilent Technologies, Hachioji, Tokyo, Japan). DNA labeling efficiency was estimated by spectrophotometry(Nanodrop ND-2000®) measuring optical absorbance at
260 nm for DNA, at 550 nm for Cy5, and at 649 nm forCy3. Cy5- and Cy3-labeled DNAs were randomly paired,
mixed, and hybridized to SurePrint G3 Human CGH
Microarrays (1 M) in the presence of human Cot-1 DNA(Oligo aCGH/ChIP-on-chip Hybridization Kit, Agilent
Technologies). Following hybridization for 24 h, microarray slides were washed according to the manufacturer’s
instructions and immediately scanned on a DNA Microarray Scanner (Agilent Technologies). With the given
limitation of the sample number, we took an advantage ofthe above direct comparison between case and control
samples [57]. This approach allowed us to determine relative ratios of their gene dosages but not their absolutegene dosages. However this procedure decreased data deviations, compared with the CGH analysis utilizing two
microarrays and reference genome DNA [30].

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Quantitative polymerase chain reaction (qPCR)

To validate the results from the microarray experiments,
we performed qPCR using TaqMan probes (Applied
Biosystems, Foster City, CA) as described previously [32].
Gene dosages of the following genomic regions of interest
were measured for the sample pair sets that exhibited theexclusive positive penetrance call with the Aberration
Detection Method 2 (ADM-2) algorithm; CC70L1J (1p13.3),
Hs03385437 (1p36.21), Hs04794356 (4q24), Hs05080419(9q22.2), Hs03318079 (18q21.1), and Hs07134106 (19p12).
Using all the samples, we also determined the gene dosages of the candidate CNV loci that exhibited lower probability scores by the global t-test analysis; Hs03587795(6p22.1), Hs03265736 (7p21.3), Hs03765933 (11p15.4),
and Hs03298358 (13q21.1). DNA sequences of TaqMan
probes and PCR primers are shown in the Additional
file 1. We obtained cycle threshold (CT) values for theregion of interest for each sample with FAM™-labeledprobes, simultaneously monitoring those for RNaseP gene(an internal control) with its VIC®-labeled probe (ABI
PRISM 7900HT Sequence Detection System and SDS v2.3
software, both Applied Biosystems). These CT values of
the target gene and RNaseP gene were obtained for all
the DNA samples. Copy number of the target gene wasestimated from CT values by CopyCaller v1.0 software(Applied Biosystems).
Statistics

The ADM-2 algorithm prompted by Genomic Workbench
software (edition 5.0.14, Agilent Technologies, 2010) wasused to identify individual and common aberrations for 48
microarray data sets. This algorithm identifies all aberrant
intervals with consistently high or low log ratios based on
the statistical score. The algorithm searches for intervals
where a statistical score based on the average qualityweighted log ratio of the sample and reference channels
exceed a user-specified threshold. For the primary screening (Selection 1), we applied the following filtering options
to the human genome assembly hg19 (excluding sex chromosomes): sensitivity threshold = 6, fuzzy zero = On, binsize = 10, and centralization threshold = 6. We then selected the primary candidate loci of somatic CNVs which
exhibited > =4 difference in gain/loss calls in the whole
penetrance summary.
To calculate mean signal OR and the probability of
CNVs between groups, we plotted individual log2 signal
ratios at all the probe sites within the above candidateloci. The Kolmogorov − Smirnov test revealed that log2
signal ratios were judged to fit into the Gaussian distribution at more than 80 % of probe sites. Assuming their
Gaussian distribution, we analyzed their statistical biases
against log2 = 0 (i.e., the null hypothesis of equal signal
intensities between patients and controls) by two tailed
t-test at each probe position. Within a candidate CNV

locus containing multiple probe sites, their log2 signal
ratios were averaged and probabilities were summed andthen subjected to Bonferroni’s correction (Selection 2).
Statistical difference of qPCR results between individual
sample pairs was determined with ANOVA or two tailed
t-test, incorporating technical errors into account. Alternatively, group differences of qPCR results from individual
samples were estimated by the chi-square and Mann–
Whitney U tests. Statistical analyses were performed using
SPSS software (IBM Japan, Tokyo, Japan).

Sak

2.

3.
4.

5.
6.

7.

Additional file
8.
Additional file 1: Figure S1. A flowchart of the study design. Table S1.
Autopsy and clinical information of the subjects used. Table S2. List of
candidate CNV regions and their statistical details. Table S3. Custom
Taqman PCR primers and probes used.
Abbreviations
ADM: Aberration detection method; ANOVA: Analysis of variance;
CGH: Comparative genomic hybridization; CNV: Copy number variation;
CT: Cycle threshold; FISH: Fluorescent in situ hybridization; OR: Odds ratio;
PMI: Postmortem interval; qPCR: Quantitative polymerase chain reaction.
Competing interests
The authors have no conflicts of interest to declare except OS; The author
received honoraria from Otsuka Pharmaceutical; Shionogi; GlaxoSmithKline;
Eli Lilly; and Tanabe Mitsubishi Pharma, and donations for research from the
Otsuka Pharmaceutical Corporation.
Authors’ contributions
TS, RK, and HN designed the experiments and wrote the manuscript; M.
Sakai, YW, KA, M. Shibuya, AM, and HN performed the experiments; KN, KO,
YK, HY, JM, AW, MH, TH, AH NK, SI, OS, KM, SN, HT, and AK collected andexamined postmortem tissues. All authors read and approved the final
manuscript.

9.

10.
11.
12.

13.

14.

15.

16.
17.

Acknowledgments
This work was supported by Grant-in-Aids for Scientific Research on Innovative
Areas (No. 24116010) and for Challenging Exploratory Research (No. 21659273)
and a grant for Promotion of Niigata University Research Projects.
Author details
Department of Molecular Neurobiology, Brain Research Institute, Niigata
University, 1-757, Asahimachi-dori, 951-8585 Niigata, Japan. 2Department of
Psychiatry, Graduate School of Medical and Dental Sciences, Niigata
University, 1-757, Asahimachi-dori, 951-8510 Niigata, Japan. 3Matsuzawa
Hospital, Setagaya-ku, 156-0057 Tokyo, Japan. 4Departments of
Neuropsychiatry, Fukushima Medical University School of Medicine, 960-1295
Fukushima, Japan. 5Division of Psychiatry and Neurology, Kobe University
Graduate School of Medicine, 650-0017 Kobe, Hyogo, Japan. 6Department of
Mental Health, Nagoya University Graduate School of Medicine, 466-8550
Nagoya, Aichi, Japan. 7Department of Neuropsychiatry, Kinki University
Faculty of Medicine, 589-8511 Osaka-Sayama, Osaka, Japan. 8Department ofSocial Rehabilitation, Kobe University School of Medicine, 654-0142 Hyogo,
Japan. 9Department of Molecular Genetics, Brain Research Institute, Niigata
University, 951-8585 Niigata, Japan. 10Pathology and Brain Disease ResearchCenter, Brain Research Institute, Niigata University, 951-8585 Niigata, Japan.
1

18.

19.

20.

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22.

23.

Received: 9 March 2015 Accepted: 12 May 201524.
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JOURNAL OF NEUROCHEMISTRY| 2016 | 136 | 295–305

doi: 10.1111/jnc.13384

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*Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan†CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan‡Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan

Abstract
Kainate-type glutamate receptors (KARs) are tetramericchannels assembled from GluK1-5. GluK1-3 are low-afﬁnity
subunits that form homomeric and heteromeric KARs, while
GluK4 and GluK5 are high-afﬁnity subunits that require coassembly with GluK1-3 for functional expression. Although thesubunit composition is thought to be highly heterogeneous in
the brain, the distribution of KAR subunits at the protein level
and their relative abundance in given regions of the brain
remain largely unknown. In the present study, we titrated Cterminal antibodies to each KAR subunit using chimeric
GluA2-GluK fusion proteins, and measured their relative
abundance in the P2 and post-synaptic density (PSD)
fractions of the adult mouse hippocampus and cerebellum.
Analytical western blots showed that GluK2 and GluK3 werethe major KAR subunits, with additional expression of GluK5 in
the hippocampus and cerebellum. In both regions, GluK4 wasvery low and GluK1 was below the detection threshold. The
relative amount of low-afﬁnity subunits (GluK2 plus GluK3)
was several times higher than that of high-afﬁnity subunits(GluK4 plus GluK5) in both regions. Of note, the highest ratioof high-afﬁnity subunits to low-afﬁnity subunits was found in
the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-afﬁnity subunits
highly accumulate at hippocampal synapses. In comparison,
this ratio was decreased to 0.15 in the cerebellar PSD fraction,suggesting that KARs consisting of low-afﬁnity subunits are
more prevalent in the cerebellum. Therefore, low-afﬁnity
KAR subunits are predominant in the brain, with distinct
subunit combinations between the hippocampus andcerebellum.
Keywords: antibody, GluK2, GluK3, kainate receptor,
quantiﬁcation, western blot.
J. Neurochem. (2016) 136, 295–305.

A kainate type of the ionotropic glutamate receptor family(KAR) is widely expressed in the central nervous system andinvolved in neurotransmission (Lerma 2003, 2006; Pinheiro
and Mulle 2006; Contractor et al. 2011). There are ﬁve
members of KAR subunits, GluK1-GluK5, which signiﬁcantly differ in spatio-temporal patterns of expressions in thebrain (Wisden and Seeburg 1993). KAR subunits aregrouped into low-afﬁnity subunits GluK1-GluK3, and highafﬁnity subunits GluK4 and GluK5 (Bettler et al. 1992).
When expressed in cell lines or Xenopus oocytes, lowafﬁnity subunits can form functional homomeric KAR
channels (Bettler et al. 1990, 1992; Egebjerg et al. 1991),
while high-afﬁnity subunits require any of GluK1-GluK3 toform functional KAR channels (Herb et al. 1992; Sakimura
et al. 1992). KAR subunits are expressed in various neurons,
such as pyramidal neurons, granule cells, and inhibitory
interneurons in the hippocampus (Bahn et al. 1994; Bureau

et al. 1999) and Purkinje cells, granule cells, and inhibitory
interneurons in the cerebellum (Wisden and Seeburg 1993).
KAR-mediated responses are recorded in cerebellar neurons (Renard et al. 1995; Savidge et al. 1999; Bureau et al.
2000), where GluK2 and GluK3 are mainly expressed

Received July 2, 2015; revised manuscript received September 4, 2015;
accepted October 1, 2015.
Address correspondence and reprint requests to Kenji Sakimura,
Department of Cellular Neurobiology, Brain Research Institute, Niigata
University, Niigata 951-8585, Japan. E-mail: sakimura@bri.niigata-u.ac.
jpAbbreviations used: AMPAR, AMPA-type glutamate receptor; DG,
dentate gyrus; DIG, digoxigenin; KAR, kainate-type glutamate receptor;
KO, knockout; NBT/BCIP, nitro-blue tetrazolium and 5-bromo-4chloro-30 -indolyphosphate; NMDAR, NMDA-type glutamate receptor;
PB, phosphate buffer; PFA, paraformaldehyde; PSD, post-synaptic
density; SDS, sodium dodecyl sulfate; TNT, Tris-NaCl-Tween.

© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 65 －

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I. Watanabe-Iida et al.

(Gallyas et al. 2003). KAR-mediated excitatory post-synaptic currents (EPSCs) are detectable at mossy ﬁber-CA3
synapses in the hippocampus, but are lost in GluK4/GluK5double knockout mice (Fernandes et al. 2009), suggestingthat low-afﬁnity subunits alone are insufﬁcient, and coexpression of high-afﬁnity subunits are essential, to form
functional KARs at this hippocampal synapse. Thus, thecomposition of low-afﬁnity and high-afﬁnity KAR subunits
and their functional contribution appear to be differentiallyregulated depending on the brain regions. Despite the wealthof knowledge on expressions at the transcription level(Wisden and Seeburg 1993), the distribution of KAR
subunits at the protein level and their relative abundance ingiven regions of the brain remain largely unknown.
In the present study, we undertook this issue by testing thespeciﬁcity and standardizing the titer of C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion
proteins. With this novel method, we studied the relative
abundance of KAR subunits in the adult mouse hippocampus
and cerebellum, and found that low-afﬁnity subunits are
predominantly expressed in both regions.

Materials and methods
Animal experiments
All animal experiments were carried out in accordance with theguidelines laid down by the animal welfare committees and theethics committees of Niigata University.
Fixation and sections
Under deep pentobarbital anesthesia (100 mg/kg body weight, ip.),
mice were ﬁxed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for chromogenic in situ
hybridization. After excision from the skull, brains were further
post-ﬁxed for 3 days at 20C and cryoprotected with 30% sucrose
in 0.1 M phosphate buffer, and cryosections (30 lm) wereprepared on a cryostat (CM1900; Leica Microsystems, Wetzlar,
Germany).

Production of GluK4-KO mice was previously reported (Akashi
et al. 2009).
Antibody
We produced anti-GluK1 antibody in the guinea pig against Cterminal 36 amino acid (aa) residues of mouse anti-GluK1 (898–934, NM_146072) and anti-GluK3 antibody in the rabbit against Cterminal 17 aa residues of mouse GluK3 (903–919,
NM_001081097). We also used rabbit polyclonal antibodies toanti-GluK2 (Synaptic Systems, Goettingen, Germany), anti-GluK4(Akashi et al. 2009), and anti-GluK5 (Millipore Corporation,
Bedford, MA, USA), which were raised against the C-terminal 55
aa residues of rat GluK2 (844–908, NM_019309), C-terminal 91 aa
of mouse GluK4 (866–896, NM_175481) and C-terminal 21 aa of
rat GluK5 (960–979, NM_031508).
RT-PCR
Total RNA was isolated from whole brains of adult and post-natal
day 3 (P3) mice using TRIzol LS Reagent (Thermo Fisher ScientiﬁcInc., Waltham, MA, USA) following the protocol of the manufacturer. Semiquantitative reverse transcription (RT)-PCR was performed using an RT-PCR kit from Clontech (Takara, Tokyo, Japan).
Brieﬂy, 1 lg of total RNA was reverse transcribed using random
hexamer primers for 1 h at 42°C. The resulting cDNA wasappropriately diluted, and ampliﬁed using the following primers:
GluK1 (exon 10–12), sense, 50 -TCGCTTGCCTAGGAGTCAGT30 ; antisense, 50 -GGGTGAAAAACCACCATATTC-30 ; glyceraldehyde-3-phosphate dehydrogenase, sense, 50 -AGGTCGGTGTGAACGGATTTG-30 ; antisense, 50 -TGTAGACCATGTAGTTGAGGT
CA-30 . Ampliﬁed products were electrophoresed and visualized byethidium bromide staining. The relative expression of GluK1 wasmeasured after scanning the bands with a CS Analyzer ver.3.0(ATTO, Tokyo, Japan).

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In situ hybridization
Mouse cDNA fragments of GluK1 (nucleotides 46–850 bp;
GenBank accession number, NM_146072), GluK2 (42–788,
NM_001111268), GluK3 (24–798, NM_001081097), GluK4(59–889, NM_175481), and GluK5 (11–894, NM_008168) were
subcloned into pBluescript II plasmid vector. Digoxigenin-labeledcRNA probes were transcribed in vitro (Yamasaki et al. 2010).
Fragmentation of riboprobes by alkaline digestion was omitted toincrease the sensitivity and speciﬁcity. The chromogenic in situ
hybridization was carried out as reported previously (Konno et al.
2014).

Construction of chimeric proteins
To standardize the titer of each C-terminal antibody, we generated
standard chimeric proteins consisting of mouse GluA2 subunit (1–833 aa, NM_013540) in the N-terminal side and mouse KAR
subunits (GluK1, 841–934; GluK2, 841–908; GluK3, 842–919;
GluK4, 826–952; GluK5, 825–979) in the C-terminal side
(Fig. 3a). cDNAs for these chimeric proteins were subcloned into
the pEF-BOS vector (Mizushima and Nagata 1990). Plasmid
vectors were transiently transfected into COS-7 cells and/or
HEK293 cells using the Plus Reagent and Lipofectamine Reagent(Invitrogen, Carlsbad, CA, USA) grown in the Dulbecco’s
Modiﬁed Eagle Medium containing 10% fetal bovine serum at
37°C in 5% CO2. 24 h after transfection, cells were lysed at 4°C in
the sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 3.3%
glycerol, 125 mM Tris-HCl, pH 7.4). The extract was centrifuged
at 10 000g for 10–20 min to remove cell debris, and thesupernatant was used for SDS–Polyacrylamide Gel Electrophoresis(SDS-PAGE) experiments.

KAR-knockout mice
GluK2, GluK3, GluK4, and GluK5-knockout (KO) mice weregenerated using ES cell line RENKA derived from C57BL/6N(Mishina and Sakimura 2007). Detailed characterization of GluK2KO, GluK3-KO, and GluK5-KO mice was described in Figure S1.

Preparation of brain samples
C57BL/6N mice were purchased from Charles River Laboratories
Japan (Yokohama, Japan), and were decapitated by cervicaldislocation. Whole brains from P3 and adulthood (7–12 weeks ofage) were homogenized in the homogenate buffer (0.32 M sucrose,

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5 mM EDTA, 5 mM HEPES-NaOH, pH 7.4, complete proteaseinhibitor cocktail tablet; Roche, Mannheim, Germany). Aftermeasuring the total protein concentration by the bicinchoninicacid (BCA) Protein Assay Reagent (Thermoﬁsher Scientiﬁc Inc.),
the whole brain lysates were recovered in the SDS sample buffer
and used for western blot analysis. Fractionated protein samples
were prepared from the hippocampus and cerebellum of adult male
C57BL/6N mice and of adult male GluK2-KO, GluK3-KO,
GluK4-KO, and GluK5-KO mice. Preparation of fractionatedprotein samples and western blot was performed as previously
described (Abe et al. 2004; Fukaya et al. 2006; Yamazaki et al.
2010). Brain tissues were excised and homogenized in thehomogenate buffer and centrifuged at 1000g for 10 min. The
resulting supernatants were then centrifuged at 10 000g for 10 min
to obtain P2 fractions. The pellets were resuspended withhomogenate buffer and aliquoted for P2 fraction, and the remaining
samples were further separated on a sucrose density gradient (0.32,
0.8 and 1.2 M sucrose) by ultracentrifugation at 90 000g for 2 h at
2°C to obtain synaptosomal fractions. Triton X-100 was added tothe aliquot samples to a ﬁnal concentration of 1% and incubated at
2°C for 30 min and centrifuged at 10 000g for 10 min at 2°C toobtain P2 fractions. To prepare PSD fraction, the synaptosomal
fraction was further solubilized with 0.5% Triton X-100 and thencentrifuged at 200 000g for 1 h. The resulting pellet (PSD fraction)
was suspended in 1% SDS, 40 mM Tris-HCl, pH 8.0 and diluted
in SDS sample buffer. Protein sample of each fraction was heatedat 100°C for 5 min in the presence of 2-mercaptoethanol and0.002% Bromophenol blue and subjected to SDS-PAGE forwestern blot.
Western blot
Protein samples were separated by 8% SDS-PAGE under reducing
conditions and electrophoretically transferred to a nitrocellulose
membrane (Amersham, Buckinghamshire, UK). Membranes were
blocked with 5% non-fat milk with Tris-buffered saline-Tween 20(TBS-T) (137 mM NaCl, 0.1% Tween 20, 20 mM Tris-HCl, pH
7.6) for 1 h at 20C. After washing for 30 min with three changes of
TBS-T, membranes were incubated with each primary antibody(1 lg/mL) in TBS-T for 3–4 h at 20C. After three washes with
TBS-T, the membranes were incubated with horseradish peroxidaseconjugated secondary antibody for 1 h at 20C in TBS-T. After
three washes with TBS-T, proteins were visualized with enhanced
chemiluminescence (ECL) (GE Healthcare, Piscataway Township,
NJ, USA and EZ capture MG; ATTO, Tokyo, Japan) using a
medical X-ray ﬁlm (FUJIFILM Corporation, Tokyo, Japan) and aluminescence image analyzer with an electronically cooled chargecoupled device camera (LAS-4000 mini; GE Healthcare).
Quantitative analysis of western blot
Signal intensities of immunoreacted bands were determined bydensitometric measurement using ImageJ software (available fromthe US National Institutes of Health), imageQuant TL (GEHealthcare) and CS Analyzer ver.3.0 (ATTO).

297

Results
GluK1-5 mRNA expressions in the mouse brain
Distinct regional expression patterns of mRNAs for ﬁve GluK
subunits have been reported in the rat brain (Wisden and
Seeburg 1993; Bahn et al. 1994). In this study, we performedin situ hybridization for GluK1-5 mRNAs in the adult mousebrain using Digoxigenin-labeled cRNA probes, with special
interest in the hippocampus and cerebellum (Fig. 1). Expression levels were generally low for GluK1 mRNA, but the
hybridizing signals were selective to interneurons in the
hippocampus and Purkinje cells in the cerebellum (Fig. 1a,f,k
and p). GluK2 mRNA was widely expressed, with intense
signals in the olfactory bulb, striatum, hippocampus, andcerebellum (Fig. 1b). In the hippocampus, GluK2 mRNA wasexpressed in both principal neurons (i.e., pyramidal andgranule cells) and interneurons, which were identiﬁed as cells
forming compact pyramidal cell and granule cell layers orcells dispersed inside and outside the layers respectively. The
intensity of GluK2 mRNA expression was in the order of thedentate gyrus > CA3 > CA1 (Fig. 1g). In the cerebellum,
granule cells expressed GluK2 mRNA intensely (Fig. 1l andq). GluK3 mRNA was moderately expressed in the cerebral
cortex, hippocampus and cerebellum (Fig. 1c). GluK3mRNA was detected in granule cells in the dentate gyrus,interneurons in the hippocampal Ammon’s horn, andinterneurons in the cerebellum; cerebellar interneurons wereidentiﬁed as cells dispersed in the molecular and granularlayers (Fig. 1h,m and r). Expression levels were generally
low for GluK4 mRNA, but intense signals were noted inpyramidal cells in the hippocampal CA3, granule cells in thedentate gyrus, and Purkinje cells in the cerebellum (Fig. 1d,i,n and s). Strong signals for GluK5 mRNA were found in thewhole brain, especially in pyramidal cells in the hippocampal
CA1-CA3, granule cells in the dentate gyrus, and interneurons and granule cells in the cerebellum (Fig. 1e,j,o and t).
These distinct regional and cellular expressions in the adultmouse brain were consistent with those in the adult rat brain(Wisden and Seeburg 1993). We then examined how thedistinct regional expressions of respective GluK subunits
were reﬂected at the protein level.
Specificity of GluK antibodies
We checked the speciﬁcity of GluK2-5 antibodies withwestern blot analysis using hippocampal extracts from wildtype and GluK-defective mutant mice (Fig. 2a). Each of
GluK2-5 antibodies recognized protein bands at expected
molecular weights in the hippocampus of wild-type mice:
GluK2, 102 kDa; GluK3, 104 kDa; GluK4, 107 kDa;
GluK5, 109 kDa. No such bands were detected in the
hippocampus of mutant mice lacking the corresponding
GluK subunits. These results verify the speciﬁcity of these
GluK2-5 antibodies and further indicate their applicability toquantitative western blot analysis.

ies
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Statistical analysis
All data are presented as mean  SE. Groups were compared using
the two-tailed, unpaired Student’s t-test. Statistical signiﬁcance isindicated as follows: ***p < 0.001.

305© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 67 －

I. Watanabe-Iida et al.

298

(a)

(f)

(k)

(p)

(b)

(g)

(l)

(q)

(c)

(h)

(m)

(r)

(d)

(i)

(n)

(s)

(e)

(j)

(o)

(t)

Fig. 1 Expressionofkainate-typeglutamate receptor (KAR) subunit mRNAs
in mouse brain. In situ hybridization withspeciﬁc RNA probes for KAR subunits: (a)GluK1 (b) GluK2 (c) GluK3 (d) GluK4 (e)GluK5, in C57BL/6N mouse whole brain.
High power images of hippocampal andcerebellar layer structures; (f, k, p) GluK1,(g, l, q) GluK2, (h, m, r) GluK3, (i, n, s)
GluK4, (j, o, t) GluK5. CA1-3, CA1-3 regionsof the Ammon’s horn; DG, dentate gyrus;GL, granular layer; ML, molecular layer;
PCL, Purkinje cell layer. Arabic numerals
indicate cerebellar lobules. Scale bars: a–e,
1 mm; f–j, 200 lm; k–o, 100 lm; p–t,
100 lm.

Tit
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Gl
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Fig. 2 Characterization of each anti-GluK
subunit antibody. (a) Hippocampal lysate(30–40 lg) of wild type and GluK2, GluK3,
GluK4, and GluK5-knockout (KO) mice
were examined by western blot analyses
with anti-GluK2 (Synaptic systems), antiGluK3, anti-GluK4 (ours), and anti-GluK5(Millipore) antibodies. Asterisks showcorrect bands of GluK2, GluK3, GluK4,
and GluK5. (b) Characterization of guinea
pig anti-GluK1 C36 antibody withrecombinant GluK1-GluK3 proteins andbrain
P2
fractionspreparedfromcerebellum (Cb.) and striatum (St.),
indicating no cross-reactivity to GluK2 andGluK3. (c) Whole brain lysates (50 lg) fromjuvenile (P3) (WT pool 1, pool 2, pool 3) andadult mice were loaded and detected with
our anti-GluK1 C36 antibody.

Because commercially available GluK1 antibody was
found to cross-react to GluK2 (Figure S2), we produced
GluK1 antibody in the guinea pig. To test the speciﬁcity ofguinea pig GluK1 antibody, we used lysates of COS-7 andHEK293 cells transfected with GluK1-3 cDNAs, and foundselective protein band in GluK1-transfected lysates (Fig. 2b).
However, no protein bands were observed in the extracts
from adult wild-type cerebellum and striatum. Because
transcription levels of GluK1 were transiently increased

during the perinatal period (Bettler et al. 1990; Bahn et al.
1994; Figure S3), we examined brain lysates prepared frompost-natal day 3 (P3) and adult mice. A putative GluK1
protein band just above 100 kDa was faintly detected inbrains at P3, but not in adulthood (Fig. 2c). This indicatesthat the amount of GluK1 proteins is below the detection
threshold in adult brains when using this GluK1 antibody.
Thus, we measured GluK2-5 protein levels in the subsequent
analyses.

© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 68 －

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Determination of KAR subunit ratios

299

Titration of GluK antibodies
We titrated GluK2-5 antibodies using GluA2-KAR chimeric proteins, in which the C-terminal sequence of GluA2(downstream to the M4 domain) was replaced with that of
GluK2-5 (Fig. 3a). Because all of the GluK2-5 antibodies
were raised against the C-terminal regions, the titer of
GluK2-5 antibodies could be standardized by using GluA2
antibody raised against the N-terminal domain (Fig. 3b).
The densitometric measurement of protein bands fordifferent loads of chimeric proteins showed linear doseintensity plots for each of GluK2-5 and GluA2 antibodies(Fig. 3c). Since an extreme difference in the intensities

between chimeric protein bands detected by GluA2 andGluK2-5 antibodies failed to stabilize their titer ratio, weused each of their signal intensities from the same range
and generated standard curves (Fig. 3c). From theseplots, the titer ratios of GluK2-5 antibodies were calculated
to be 8.0, 2.4, 30.5, and 24.6 times that of GluA2
antibody.

Fig. 3 Unique method of GluK subunits determination by western blot.
(a) Scheme of a chimeric protein of GluA2K2-GluA2K5 used forquantiﬁcation analysis whose N-terminal GluA2 was fused with four Cterminal GluK2-5. Each titer was corrected by the titer of anti-GluA2 Nterminal antibody, and used for quantitative analysis of the four GluK
subunits. (b) Chimeric proteins prepared from COS-7 cells or HEK293
cells were used for determining the ratio of antibody titers. Eachantibody binds to chimeric protein depending on each afﬁnity titer, andthis reaction can be seen as western blot band signals. (c) Western

blot for determination of each titer of GluK2-5 antibodies with chimeric
GluA2K2-5 proteins detected by GluA2 antibody and GluK2-5 antibodies. Each number of loaded samples of transfected cell lysates
represents protein amount (lg). Examples of standard curves of
GluA2K2-5 signal intensities detected by anti-GluA2 antibody (blacksquare) and anti-GluK2-5 antibodies (white square) are shown. The
ratios of anti-GluK2-5 antibody titers to anti-GluA2 antibody were
determined by using GluA2K2-5 intensities.

Relative amounts of GluK in hippocampus and cerebellum
Using the titer ratios, we estimated the relative amount of
GluK2-5 proteins in the P2 and PSD fractions prepared fromthe hippocampus and cerebellum (Figs 4 and 5). The

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© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 69 －

300

I. Watanabe-Iida et al.

Fig. 4 Determination of GluK subunits in hippocampus. (a) Analyticalwestern blots of GluK subunits in P2 and post-synaptic density (PSD)
fractions of hippocampus. Graded dilutions of hippocampal P2 and
PSD fractions and each of chimeric GluA2K were subjected toanalytical western blot. In order to simplify the picture, loaded protein
amounts are indicated with relative one (see Table S1). The molecularweights are growing larger, in the order of GluA2 < GluA2K2-

5 < GluK2-5. Arrow heads show correct bands of kainate-type glutamate receptor (KAR) subunits. (b) The ratios of KAR GluK2, GluK3,
GluK4 and GluK5 to GluA2. Ratios of KAR subunits in hippocampal P2
and PSD fractions are shown in the table and the bar graphs. The
ratios of GluK2 (white), GluK3 (gray), GluK4 (stripe), and GluK5 (black)are normalized by the amount of GluA2 in each brain region andfraction. Error bars represent SEM.

amounts of loaded protein samples on western blots werechanged according to the titer of each antibody and subunit
content in each fraction, as summarized in Table S1. We
generated standard curves of chimeric proteins and endogenous proteins (GluA2, GluK2-5) in every experiments(n = 3). A representative example is shown in Figure S4,
where we made four standard curves of GluA2K2 detectedby GluA2 antibody, GluA2K2 detected by GluK2 antibody,
GluA2 in hippocampal PSD fraction, and GluK2 inhippocampal PSD fraction in the same intensity range(< 30000 (intensity), see vertical axis in Figure S4). Using
these standard curves, we titrated their titer ratio anddetermined the ratio of GluK2 to GluA2. The western blot
images (Figs 4a and 5a) are of one of those experiments
determining GluK2-GluK5. We averaged the amounts of
GluK2-5 proteins relative to GluA2 protein in each experiment (GluK2-GluK5, n = 3, respectively), as shown in thegraph and table (Figs 4b and 5b).
In the hippocampus, GluK2, GluK3, GluK4, and GluK5
amounted to 9.2  0.8%, 3.5  1.0%, 0.77  0.30%, and2.0  0.3%, respectively, of GluA2 in the P2 fraction (n = 3
for each), and to 7.0  0.6%, 4.2  0.8%, 0.47  0.09%,
and 3.1  0.2%, respectively, of GluA2 in the PSD fraction(n = 3 for each) (Fig. 4b). In the cerebellum, GluK2, GluK3,
GluK4, and GluK5 amounted to 22  2%, 17  4%,1.9  0.4%, and 2.8  0.5%, respectively, of GluA2 in
the P2 fraction (n = 3 for each), and to 9.9  2.4%,
7.3  1.0%, 1.1  0.1%, and 1.4  0.2%, respectively, of
GluA2 in the PSD fraction (n = 3 for each) (Fig. 5b). These
data show that the composition of GluK2-5 subunits isdifferent depending on the brain regions and biochemical
fractions, with GluK2 being the major component in the
hippocampus and cerebellum.
By setting the relative amount of GluK2 in each fraction as
1.00, we compared the ratio of four GluK subunits among thefour hippocampal and cerebellar fractions (Fig. 6a). In all
fractions, the relative abundance of GluK2-5 subunits was in
the order of GluK2 > GluK3 > GluK5 > GluK4, with relative enrichment of GluK3 in the cerebellar fractions and of
GluK5 in the hippocampal PSD fraction (Fig. 6a). To
simplify the understanding of composition of GluK subunits
in each fraction, we also set GluK2 in the hippocampus P2
fraction as 1.00 and compared the ratio between the P2 and
PSD fractions in the hippocampus and cerebellum (Fig. 6b).
When collectively calculating low-afﬁnity subunits (GluK2
plus GluK3) as 1.00, the ratio of high-afﬁnity subunits© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 70 －

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Determination of KAR subunit ratios

301

Fig. 5 Determination of GluK subunits in cerebellum. (a) Analyticalwestern blots of GluK subunits in P2 and post-synaptic density (PSD)
fractions of cerebellum. Detailed explanation is as shown in Fig. 4. (b)Ratios of kainate-type glutamate receptor subunits in cerebellum P2

and PSD fractions are shown in the table and the bar graphs. The ratiosof GluK2 (white), GluK3 (gray), GluK4 (stripe), and GluK5 (black) arenormalized by the amount of GluA2 in each brain region and fraction.
Error bars represent SEM.

(GluK4 plus GluK5) was higher in the hippocampus (0.22 in
the P2 and 0.32 in the PSD fraction) than in the cerebellum(0.12. and 0.15 respectively). By setting the relative amountin the P2 fraction as 1.00 in each region, each GluK subunit
showed 3–5-fold enrichment in the PSD fraction of the
hippocampus and cerebellum (Table 1).

as demonstrates by using GluK2-5 defective mice as negative
controls, and by linear dose-intensity plots using chimeric
GluA2-KAR protein standards. Here, we have shown that
low-afﬁnity KAR subunits are predominantly expressed in theadult mouse brain, where the relative amount of GluK2 plus
GluK3 was several times higher than that of GluK4 plus
GluK5 in the hippocampus and cerebellum.
Native KARs are thought to be heteromeric receptors
composed of various combinations of subunits. When
expressed in HEK293 cells and Xenopus oocytes, GluK4 and
GluK5 subunits associate with GluK1-3 subunits to form
heteromeric receptors with pharmacological properties distinct
from homomeric receptors consisting of the low-afﬁnity
subunits. Moreover, the amplitude of channel activity of
GluK2/GluK5 heteromeric receptors is several times larger
than that of GluK2 homomeric receptors (Sakimura et al.
1992). Immunoprecipitated analysis has revealed that GluK5
is co-immunoprecipitated with GluK2 in CA3 pyramidal cells(Wenthold et al. 1994). A single molecule imaging of
ﬂuorescence-tagged GluK2 and GluK5 subunits revealed thatthey assemble with a 2 : 2 stoichiometry (Reiner et al. 2012).
Crystal structure analysis has shown that the N-terminal
domains of GluK2 and GluK5 subunits preferentially coassemble as heterodimers rather than as homodimers (Kumar
et al. 2011). In the hippocampus of GluK4/GluK5-double

Discussion

as
he
all
in
elof
To
its
P2
nd
b).
K2
its

In the present study, the relative amount of KAR subunits
was compared by standardizing the titer of KAR subunit
antibodies using chimeric GluA2-GluK fusion proteins.
Based on the titrated antibodies, we examined the relative
amount of each KAR subunit in biochemical fractions
prepared from the adult mouse hippocampus and cerebellum.
This method enabled the assessment of the amount of KAR
subunits relative to GluA2 subunit in the same biochemical
fractions. Compared to standardized immunoblot analysis
using endogenous standard proteins, such as actin, this method
is advantageous in that it is applicable to the comparison ofrelative amounts of multiple subunits using different antibodies. Although its low sensitivity such as inability of GluK1
detection in the adult mouse brain is to be further improved,
the reliability of the present comparison for GluK2-5 subunits
was ensured by speciﬁc detection of GluK2-5 protein bands,

305© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 71 －

302

I. Watanabe-Iida et al.

Ta
(PS

Hip
Hip
Ce
Ce

Th

Fig. 6 Compositionofkainate-typeglutamate receptor (KAR) subunits inhippocampus and cerebellum. (a) Relative
ratios of KAR subunits in hippocampal P2,
post-synaptic density (PSD) and cerebellar
P2, PSD are shown in the table and the bar
graph. The ratios of GluK3 (gray), GluK4(stripe), and GluK5 (black) are normalized
by the amount of GluK2 (white) in the brain
regions respectively. (b) Relative amountsof KAR subunits normalized by the amountof GluK2 in hippocampus P2 fraction are
shown in the table and the bar graph. Lowafﬁnity GluK2 and GluK3 are described in
the left side of position 0, and high afﬁnity
GluK4 and GluK5 are described in the rightside.

knockout mice, detectable KAR-mediated EPSCs are lost at
mossy ﬁber-CA3 synapses (Fernandes et al. 2009). Theseﬁndings indicate that one form of functional KARs in thebrain is heteromeric receptors consisting of high- and lowafﬁnity subunits. GluK4 and GluK5 subunits in hippocampal and cerebellar fractions are likely to represent, mostly if
not all, such heteromeric receptors. The highest GluK5-toGluK2 ratio and its marked increment from the P2 fraction(0.22) to the PSD fraction (0.44) in the hippocampus further
suggest that heteromeric receptors consisting of high- andlow-afﬁnity subunits highly accumulate at hippocampal
synapses.
Another form of functional KARs in the brain is homomeric and heteromeric receptors consisting of only lowafﬁnity KAR subunits. Application of kainate and glutamate
elicits current responses in HEK293 cells expressing GluK1(Q), GluK2(Q/R), GluK1/GluK2, GluK3a, and GluK3b(Sommer et al. 1992; Coussen et al. 2005). GluK1 and
GluK2 subunits co-assemble to generate recombinant receptors with novel functional properties (Cui and Mayer 1999).
Two splice variants of GluK2 subunit (GluK2a and GluK2b)also co-assemble to regulate KAR trafﬁcking and function(Coussen et al. 2005). The ratio of low-afﬁnity (GluK2 plus
GluK3) to high-afﬁnity (GluK4 plus GluK5) was higher inthe cerebellum (8.5 in the P2 fraction and 6.7 in the PSD
fraction) than in the hippocampus (4.6 and 3.1, respectively),suggesting that KARs consisting of low-afﬁnity subunits are
more prevalent in the cerebellum. There have been manystudies on KAR-mediated synaptic transmission focusing on
hippocampus CA3-dentate gyrus synapses, reporting loss of
GluK4 and GluK5 showed no detectable KAR-EPSCs inhippocampus. In this study, it is reasonable to expect that
GluK2 homomer in cerebellum granule cells would not show
KAR-EPSCs. Indeed, cerebellar granule cells in wild typeshowed no KAR-mediated synaptic transmission, but loss of
AMPAR activity increased GluK5 expression and GluK2/5containing KAR-mediated synaptic transmission (Yan et al.
2013). KAR activity is possibly regulated by an increase in
GluK4 or GluK5 expression, when AMPAR activity
decreased.
Expression patterns of ﬁve KAR subunits by in situ
hybridization are quite similar between rat and mouse brains.

© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305－ 72 －

In
va
cer
thu
afﬁ
bra
wh
the
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Determination of KAR subunit ratios

Table 1 Enrichment of GluK subunits in the post-synaptic density(PSD) fraction of the hippocampus and cerebellum

Hippocampus P2
Hippocampus PSD
Cerebellum P2
Cerebellum PSD

GluK2

GluK3

GluK4

GluK5

1
3.57
1
2.99

1
5.56
1
2.83

1
2.84
1
4.00

1
7.24
1
3.46

These values are calculated from Fig. 6b.

hippocampus, concentration levels of both GluK2 andGluK3 are fourfold higher in PSD fractions than in P2
fraction. The proportions of AMPAR and KAR subunits inenriched synaptic membrane fractions in rat cortex withsynaptic puriﬁcation technique (Phillips et al. 2001; Pinheiro
et al. 2003, 2005) showed that GluK2/3 was predominantly
present in PSD fractions (about 70–80%) with much lower
levels of pre-synaptic and non-synaptic synaptosomal protein fractions (about 20–30%) (Feligioni et al. 2006), which
are consistent with our data as mentioned above. On theother hand, GluA1 and GluA2 were not predominantly
present in PSD fraction (40–60%) compared with GluK2/3.
Biochemical distribution and protein solubility of otherglutamate receptors showed that many NMDA-type glutamate receptors were in the insoluble membrane fractions,while AMPARs were in the insoluble membrane fractions
and the soluble fractions (Fukaya et al. 2006; Inamura et al.
2006), suggesting that the distribution of KAR at ultrasynaptic fraction may be different from that of AMPAR. These
data might elucidate the controversy of subcellular localization of KAR including pre- and/or post-synapse, and point toa hidden role of the post-synaptic afferent in the neuron.
Future study will provide an insight into whether lowafﬁnity KAR subunits mediate neurotransmission signal at
post-synaptic sites.

itu
ns.

In both species, GluK5 mRNA is prominently expressed invarious brain regions, including the hippocampus andcerebellum (Wisden and Seeburg 1993; this study). It was
thus unexpected to ﬁnd predominant protein contents of lowafﬁnity KAR subunits over high-afﬁnity ones in the adultbrain. This was particularly prominent in the cerebellum,
where GluK4 and GluK5 mRNAs are highly expressed, but
their protein contents were less than one-tenth of that of
GluK2. This discrepancy raises the possibility that the
predominant low-afﬁnity subunit expression is constructed
mainly at the post-transcriptional level. Considering thathigh-afﬁnity subunits cannot form functional homomeric
KARs (Bettler et al. 1990, 1992; Egebjerg et al. 1991; Herbet al. 1992; Sakimura et al. 1992), homomeric receptors
consisting of high-afﬁnity subunits alone might be unstableand prone to be degraded, even if they are constructed inneurons. In NMDARs, heteromeric subunit conﬁguration isessential for channel function and synaptic expression.
Without GluN2 subunits, GluN1 subunit is unable toaccumulate on the post-synaptic membrane and its turnoverrate is accelerated (Abe et al. 2004). Without GluN1 subunit,
GluN2 subunits are retained in the ER for degradation(McIlhinney et al. 1996; Fukaya et al. 2003). In KAR,
GluK5 also has an ER retention motif and enables recruiting
the membrane when co-assembled with GluK1, GluK2, andGluK3 (Gallyas et al. 2003). Without them, GluK5 was alsoheavily retained in the ER and lost its current in heterologous
cells (Hayes et al. 2003; Ren et al. 2003). We also foundthat the amount of GluK5 is less enriched in PSD fraction of
cerebellum than that of hippocampus (Table 1), indicating
the trafﬁcking of GluK subunit to the post-synaptic plasma
membrane may be differentially regulated in these regions.
Perhaps its turnover rate is maintained depending on thebrain region. Overall, this discrepancy of the amount of
GluK5 at mRNA and protein level remains to be investigated.
In this study, we noticed the importance of low-afﬁnity
subunits as major KAR subunits, although their main roles
have not been understood so far. In our data, GluK2-GluK5
showed positive concentration efﬁciency to PSD fractions
from P2 fractions. The fractionation procedure was
performed as previously described (Abe et al. 2004; Fukaya
et al. 2006; Yamazaki et al. 2010; see method section). In

305© 2015 International Society for Neurochemistry, J. Neurochem. (2016) 136, 295--305

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303

Acknowledgments and conflict of interest
disclosure
We thank C. Nakamoto, H. Azechi and Y. Suzuki for technicalassistance and advice. We also thank M. Kawamura and H. Uchidafor the valuable discussion. This study was supported by CREST
and Grants-in-aid for Scientiﬁc Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan(21300118, 24650195, 24240048 to K.S.). We have no conﬂict ofinterest to declare.
All experiments were conducted in compliance with the ARRIVE
guidelines.

Supporting information
Additional supporting information may be found in the online
version of this article at the publisher's web-site:
Figure S1. (a) To construct a Grik2 targeting vector, a 770 bp
DNA fragment carrying exon 14 of the Grik2 was ampliﬁed by
PCR, and inserted to the targeting vector as described previously(Akashi et al. 2009). In this clone, a DNA fragment of pgk
promoter-driven Neo-poly(A) ﬂanked by two frt sites (Neo cassette)and loxP sequence was located at the site 311 bp upstream of theexon 14, while the other loxP sequence was placed at the site 241 bp
downstream of the exon 14. It contained a 6.83 kb Grik2 at the 50
side and 3.79 kb Grik2 at the 30 side, followed by an MC1 promoterdriven diphtheria toxin (DT) gene. (b) To construct a Grik3 targeting
vector, a 817 bp DNA fragment carrying exon 12 of the Grik3 wasampliﬁed by PCR, and inserted to the SacI sites of the middle entry
clone (pDME-1). In this clone, a DNA fragment of Neo cassette and－ 73 －

304

I. Watanabe-Iida et al.

loxP sequence was located at the site 428 bp upstream of the exon
12, while the other loxP sequence was placed at the site 270 bp
downstream of the exon 12. The 5.07 kb upstream and 5.24 kb
downstream homologous genomic DNA fragments were retrieved
from the BAC clone (RP23-131M1), and then subcloned to 50 entry
clone (pD3UE-2) and 30 entry clone (pD5DE-2) respectively. For
targeting vector assembly, the three entry clones were recombined toa destination vector plasmid (pDEST-DT; containing a cytomegalovirus enhancer/chicken actin (CAG) promoter-driven DT gene byusing a MultiSite Gateway Three-fragment Vector construction Kit(Invitrogen) (Sasaki et al. 2004). (c) To construct a Grik5 targeting
vector, a 770 bp DNA fragment carrying exon 13, 14, and 15 of theGrik5 was ampliﬁed by PCR, and inserted to the targeting vector as
described previously (Akashi et al. 2009). In this clone, a DNA
fragment of Neo cassette and loxP sequence was located at the site
411 bp upstream of the exon 13, while the other loxP sequence wasplaced at the site 96 bp downstream of the exon 15. It contained a
6.53 kb Grik5 at the 50 side and 3.13 kb Grik5 at the 30 side,
followed by an MC1 promoter-driven DT gene. Each targeting
vector introduced into C57BL/6N ES cell line RENKA andrecombinant clones were identiﬁed by Southern blot. To produce
germline chimeras, recombinant ES clones were microinjected into
eight cell stage embryos of CD1 mouse strain. To generate Grik2,
Grik3 and Grik5-null mice, each ﬂowed mouse was crossed with thetelencephalin-Cre mice (Nakamura et al. 2001).
Figure S2. Speciﬁcity of GluK1 antibody (Upstate) was checkedwith western blot analysis of COS-7 cell lysates transfected witheach cDNA of GluK1-GluK3. Cross reactivity of GluK1 antibodies
between GluK1 and GluK2 was tested using the brain extracts,
hippocampal P2 fractions, derived from wild type and GluK2-KO
mice. A detected GluK1 band of the GluK1 antibody in the wild
type disappeared in GluK2-KO mice.
Figure S3. (a) RT-PCR was performed using primers spanningexon 10–12 with whole brain of adult wild type (C57BL/6N),
GluK3ﬂox/ﬂox mice and post-natal day 3 (P3) GluK3ﬂox/ﬂox mice.
Ampliﬁcation of glyceraldehyde-3-phosphate dehydrogenase gene
was used as a positive control for the RT reaction (bottom panel). P3
GluK3ﬂox/ﬂox mice were used instead of wild type mice. (b)GluK1 transcript was decreased in adult mice, compared to P3 mice
(top panel). The expression level of adult mice GluK1 transcript was
normalized by that of P3 mice. Error bars represent SEM. Student’s
t-test was used for statistical analysis.
Figure S4. (a) Determination of GluK2 ratio to GluA2 examinedby chimeric protein and hippocampus PSD sample is shown as an
example of the determination method. Amounts of loaded COS-7
cells lysate expressing GluA2K2 and hippocampal PSD sample areshown as a relative value. (b) Standard curves of GluA2K2 (left) andhippocampus PSD sample (right) were detected by GluA2 antibody(black square and circle) and GluK2 antibody (white square andcircle). The protein ratio of GluK2 to GluA2 in hippocampus PSD
was quantiﬁed by correcting titer ratio of GluK2 antibody to GluA2
antibody. (c) The titer of GluK2 antibody to GluA2 antibody was
obtained from calculation using the slopes of GluA2K2 standard
curves (b, left). The relative ratio of GluK2 protein to GluA2 protein
was also calculated by using the slopes of GluK2 and GluA2
standard curves (b, right) and GluK2 titer ratio mentioned above.
Table S1. The amount (mg) of loaded protein samples ofhippocampus and cerebellum.

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Neuron

ArticleC1q Family Proteins and Synaptic Functions
AnterogradeC1ql1of Signaling
Based on the behavioral phenotypes
ataxic mutant mice Is Required in Order
mGluR1, PKCg, and GluD2, monoinnervation of Purtolacking
Determine
andMaintaina Single-Winnerkinjecells by CFs was thought
to be required
for motor coordination (Chen and Tonegawa, 1997). In contrast, although
Climbing
Fiber in the Mouse Cerebellum
C1ql1-null and PC-Bai3-null Purkinje cells remain innervatedrepresented as mean ± SEM. Statistical analyses were performed using the
Excel statistics 2012 add-in software (Social Survey Research Information),and significant differences were defined as *p < 0.05, **p < 0.01, and ***p <0.001.
ACCESSION NUMBERS

by multiple CF inputs, these mice have normal gaits without

The coordinates and structure factors for the reported crystal structure are

1,2 Nikolaos Mitakidis,3 Eriko Miura,1,2 Manabu Abe,2,4 Keiko Matsuda,1,2 Yukari H. Takeo,1,2
WataruKakegawa,
significant
impairment
in beam or rotor-rod tests (Figure S8; deposited in the Protein Data Bank (PDB) under PDB ID 4d7y.
1,2 Junko Motohashi,1,2 Akiyo Takahashi,1,2 Soichi Nagao,5 Shin-ichi Muramatsu,6Kazuhisa
Kohda,
Movie S1). However,motor
learning, as measured by hOKR,
1,2,*
Masahiko Watanabe,2,7 Kenji Sakimura,2,4 A. Radu Aricescu,3 andMichisuke Yuzaki
SUPPLEMENTAL
INFORMATION

was severely impaired in these mice (Figure 6). Recently,
1Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
mGluR1b-rescue
mice, in which the splice variant of mGluR1
2Core
Research for Evolutional Science and Technology (CREST), Japan Scienceand Technology
Agency Supplemental(JST), Tokyo Experimental
102-0075, Japan
Supplemental
Information includes
Procedures,is expressedin Biology,
mGluR1-nullPurkinjecells, wereshown
to
3Divisionof Structural
Wellcome
Trust Centre
for Human
Genetics,
University
Oxford,OxfordOX3
UK with this article online ateight
figures,ofand
two movies
and
can7BN,be found4Department
display normal
motor
coordination,
thoughtheir Purkinje
of Cellular
Neurobiology,
Braineven
Research
Institute,
Niigata University,
Niigata 951-8585, Japan
http://dx.doi.org/10.1016/j.neuron.2014.12.020.
5Laboratory
cells wereinnervatedbyControl,
multipleRIKEN
CFs.Brain
These
mice Institute,showedSaitama 351-0198, Japanfor Motor
Learning
Science
6Division
impaired
LTD andeyeblink
conditioning,anotherformJapanof AUTHOR CONTRIBUTIONS
of Neurology,
Jichi
Medical University,
Tochigi
329-0498,
7Department of Anatomy, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan
cerebellum-dependent motor learning (Ohtani et al., 2014).
*Correspondence:
myuzaki@a5.keio.jp
Similarly, a single
injection of recombinant Cbln1 restored W.K. designed the project, carried out the electrophysiological and behavioral
works, analyzed the data, and wrote the paper. N.M. and A.R.A. performed
http://dx.doi.org/10.1016/j.neuron.2014.12.020
LTD and eyeblink conditioning in adult Cbln1-null mice without
structural and SPR analyses and designed mutant constructs. E.M. performed
significantly affecting the CF innervation pattern or LTP at the anatomical experiments and the IUE approach. M.A. and K.S. designed
PF-Purkinje cell synapses (Emi et al., 2013). Furthermore, and generated mutant mice. K.M. performed the cell binding assays. Y.H.T.
Watanabeand Kano,2011). Multiple
CFsK.K.,
initially
SUMMARY
GluD2DCT7-rescue mice, in which GluD2 lacking seven C-termi-1996;
designedand prepared
the microRNA
constructs.
A.T.,innervate
J.M., and S.M.
designedand prepared
the recombinantviruses.
S.N. supervised
the hOKR
Purkinjecell soma,
but a single
CF becomesdominant
nal amino acids was expressed in GluD2-null Purkinje cells,a single
M.W. generated
essential antibodies.
designedand superpostsynaptic,
voltage-dependent
Ca2+ M.Y.
Neuronal
are anddynamicallymodified despite
by in aexperiment.
channelactivitydisplayednetworks
impaired LTD
eyeblink conditioning
vised the project and wrote the paper.
manner during postnatal days (P) 3–7 in micemorphologically
PF duringand CFdevelopment
synapses (Kakegawa
selective
synapsenormal
pruningand dependentandHashimoto,2009; Watanabe and Kano, 2011). The
et al., 2008).
These findings
indicateconnections
that LTD, rather
adulthood.
However,how certain
winthan(Kano
ACKNOWLEDGMENTS
of Purkinjecellsandby CFs,likely linked to mo-‘‘single-winner’’ CF translocates to the Purkinje cell dendrites
themonoinnervationcompetition withothersare issubsequently
P9, andthe restofOtsuka
the CFs
remaining
the soma
tor learningis innot
thefully
cerebellum
and indicate
We thank
S. Narumi
and S.
for theirtechnicalon
assistanceandareS. Matmaintained
understood.
Here,that
we C1ql1-Bai3
show aroundsuda andeliminatedK.F. Tanakaby
forP21
their (Hashimoto
useful comments.
We2009).
also thankMiyazaki
et al.,
CF T.
prunsignaling is required for normal CF functions, such as complexeventuallythat C1ql1, a member of the C1q family of proteins,
andK. Konno for
their support
anatomical
experiments,
H.mediatedOkado for lentireportedly
regulatedby onactivitiesin Purkinje
cells
spikes and CF-evoked afterhyperpolarization, which are neces-ing is
is provided by climbing fibers (CFs) and serves as
virus
production,
H.
SakumaandT.
Watanabe(Zeiss)fortheirby parallel-fiber (PF, an axon of a granule cell) inputs throughsupport
me- onsary for LTD.
superresolution microscopy analysis, and the staff at Diamond beamline I04
a crucial
anterograde
to determineareandmain- intabotropic
glutamate receptor 1 (mGluR1), as well as inhibitory
Familymembers of signal
C1ql1 (C1ql2–C1ql4)
expressed
for synchrotron assistance. This work was supported by a grant-in-aid for scitainvarious
the single-winner
CF in
mouse
cerebellum
through(GABA)
receptors(Kano andbrain regions (Iijima
et theal., 2010).
Bai3
and its relatedinputs
entific
researchg-aminobutyricfrom the Ministry ofacid
Education,
Culture,Sports, Sciencethroughout
development
andC1ql1(Kee
speHashimoto,2009;and
WatanabeKano,2011).
Althoughamembers Bai1and Bai2 are
alsoadulthood.
widely expressed
et al.,andTechnologyof Japanthe JapanandSocietyfor thePromotionof Sciencecifically
binds
theimplicatedbrain-specificangiogenesis
live-imaging
indicatedthat translocationof CF syn2004) andhavetobeen
in psychiatricdisorders, suchrecent(W.K.
and M.Y.); thestudy
CREST
from the Japan
Science and Technology
Agency(M.Y.);
the Kanae Foundation(W.K.);
Nakajima Foundation(W.K.);
the Keio
inhibitor
3 (Bai3), andwhich
is disorders
a memberof thecellto dendrites
may provide
a thecompetitive
advantage
to theas schizophrenia
bipolar(Lanoue
et al.,
2013).apses
GijukuMemorial
Fund howfor theAdvancementof strengthEducation and
CFFukuzawa(Carrillo etal., 2013),
a selected
CF isThus, signalingmechanisms similar
to theone mediatedadhesionG-protein-coupled
receptorfamilyand ex- bywinner
Research (W.K.); the Takeda Science Foundation (W.K. and M.Y.); the UK
C1ql1 andin the cerebellum
likely regulate
synaptic compe-ened remains unclear. Furthermore, although brain-derived neupressedon Bai3
postsynaptic
Purkinjecells. C1ql1-Bai3
Medical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD studentrotrophic
factorand insulin-like growth factor are suggested totition andin various
circuits
signalingismaintenancerequired for
motorneuronallearning
but essential
not for for ship (N.M.).
Further support from the Wellcome Trust Core Award 090532/Z/
beinvolvedinstrengthening
the site Fellow.
of synaptic
brain
functions.
Inthehippocampus,
C1ql2
and
C1ql3
are
highly09/Z is acknowledged. A.R.A. isCF
an synapses,
MRC Senior Researchgross motor performance or coordination. Because
expressed in adult dentate gyrus granule cells, and their mossyexpression and action remains largely elusive (Watanabe andrelated family members of C1ql1 and Bai3 are exAccepted:
26, while
2014 CF synapses further undergo ac2011). November
In addition,
fiber afferents make dynamic branched axon-type synapsesKano,pressed in various brain regions, the mechanism
Published: January
21, 2015 or retraction throughout adulthood
expansion
like CFs. Notably, mossy fiber synapses reportedly displaytivity-dependent
described
here likely
appliesto synapse
learning-induced
structural
plasticity
related to formation,
the precision of(Cesa and Strata, 2009), how the winner CF is maintained is
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The cell-adhesion
late Bolliger,
postnatalof inactive
synapses
betweenretinal G
INTRODUCTION
protein-coupled
receptor
BAI3geniculate
is a high-affinity
receptorforC1q-like
proteins.
ganglion
cells
and
the
lateralnucleusinvivo(Stevens
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accor-referred to as the C1q family. Like adiponectin, which is released
Homosynaptic long-term synaptic potentiation of the ‘‘winner’’ climbing fiber
dance with thesetthedown
by the Animal
Resource
Committee
strengthened
by guidelines
increasing
numberof theirsynaptic
con- offrom adipose tissues and regulates glucose and lipid metabolism
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Keio
University.
C1ql1-null,
PC-Bai2-null,
and
PC-Bai3-null
mice
were
genertacts, while weak inputs are physically removed. As a model of in muscle and liver, most C1q family members are secreted andCarrillo, J., Nishiyama, N., and Nishiyama, H. (2013). Dendritic translocation
ated and maintained on C57BL/6N backgrounds. As controls, WT mice wereinvolvedin various signaling pathways (Kishore et al., 2004; Yuactivity-dependent synapse refinementin the central nervous
establishes the winner in cerebellar climbing fiber synapse elimination.
used for C1ql1-null mice and Bai2flox, and Bai3flox mice were used for PCIn 33,
the7641–7653.
cerebellum, Cbln1, a cerebellin family of C1q
system (CNS), postnatal pruning of supernumerary synapses be- zaki,J.2008).
Neurosci.
Bai2-null and PC-Bai3-null mice. For the behavioral analyses, littermateshomologous,isreleased
fromAxonal
granulecells anda crucialtween
climbing
fibers
(CFs,
axons
of
inferior
olivary[IO]
neurons)
were used as controls. Experimental details of mouse generation as well as
Cesa, R., and Strata, P. (2009).
competitionin theplays
synaptic
wiring of thein the formationand maintenance
PF-Purkinje
cell synapandelectrophysiological,
Purkinje cells in the
cerebellum
has been
extensively
studiedana-rolecerebellar
anatomical,
biochemical,
structural,and behavioral
cortex during
development ofandin the mature
cerebellum.
by binding to162,
its 624–632.
postsynaptic receptor, the delta2 glutamate
(Cesa
and
2009;
Kano
and Hashimoto,2009;
Lohof et Dataal., areses Neurosciencelyses
areStrata,
described
in theSupplemental
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Procedures.
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Chen,
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Emi,inhi
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Kano, M
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Kee, H.J
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og1q).
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cial
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C1q-likefamilymembersgenerefinement
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than
P9 were specificallyplasticity,
activity-dependentneuraldevelopment,
and memoryinrons.impaired
Mol. Psychiatry18, 943–950.
(C1ql1–C1ql4)
are also
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Shimono et al., 2010) and are implicated in synapse formation or

Next, we evaluated CF synapse function by assessing CF-

the central
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mechaDavie,
J.T., Clark, in
B.A.,
and Häusser,
M. (2008). neurons
The origin inof thecomplexelimination
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et al., 2011). It is interesting that C1ql1 mRNA is selectively

cells with whole-cell patch-clamp recordings. The number ofLuo, B.H., Springer, T.A., and Takagi, J. (2003). Stabilizing the open conformaDuman, J.G., Tzeng, C.P., Tu, Y.K., Munjal, T., Schwechter, B., Ho, T.S., andand highly expressed in IO neurons (Iijima et al., 2010) during functional CF synapses on single Purkinje cells was estimatedtion of the integrin headpiece with a glycan wedge increases affinity for ligand.
Tolias, K.F. (2013). The adhesion-GPCR BAI1 regulates synaptogenesis bydevelopment and throughout adulthood. Here, we examinedProc.byNatl.
varying
the stimulus intensity because a single CF input
Acad. Sci. USA 100, 2403–2408.
controlling the recruitment of the Par3/Tiam1 polarity complex to synaptic
hypothesisthat C1ql1 provided by CFs may regulate CF syn- has a single threshold for excitation. At P4–P6, both WT andsites.theJ. Neurosci.
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innervatedbyal.a(2010).
similarnumberY., Ito-Ishida,A., cells
Kondo,were
T., Shigemoto,R., etCbln1
isEccles, J.C., Llinás, R., and Sasaki, K. (1966). The excitatory synaptic action of
1F and1G).orgaAmongthat fibres
C1ql1onplays
a crucialcells
roleofinthe
strengthening
maintaining(fromto nine)of CFsreceptor(p = 0.15;
a ligandforthreean orphanglutamated2, aFiguresbidirectional
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SD/mean
CF-EPSCamplitudes;
0.67 ±
3 (Bai3),a cell-adhesion
G Kohda,protein-coupled
receptor
Mikuni,
Uesaka, N.,
Okuno,
Hirai, H., of
Deisseroth,K., Bito,
H., and Kano,
Emi,inhibitor
K., Kakegawa,W., Miura,
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M. (2013).
is a Clql1-nullpostsynaptic(0.65
mediatorof activity-dependent
syn0.05, Arc/Arg3.1
n = 24) and± 0.05,n = 26) Purkinje
cells(GPCR)
that isofconstitutively
expressed
in Purkinjecells. Further(2013).
Reevaluation
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althoughsingle
CF-EPSCs
were elicitedmore,
our usingin vivo
results
demonstratethat
C1ql1–Bai3
signalingapse(pelimination

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P/Q-type
Hashimoto,K., Ichikawa,R., Takechi, H., Inoue, Y., Aiba, A., Sakimura, K.,
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C1ql1 Is Required to Win CF Competition

mice (p < 0.01; Figure 1G). Furthermore, while the amplitude of

Nakayama, H., Miyazaki, T., Kitamura, K., Hashimoto, K., Yanagawa, Y.,
the largest CF-EPSCs steadily increased during development
Obata, K., Sakimura, K., Watanabe, M., and Kano, M. (2012). GABAergic inhiin WT mice, it remained constant after P7–P9 in C1ql1-nullbition regulates developmental synapse elimination in the cerebellum. Neuron
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rents,
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Translocationa ‘‘winner’’ climbing
to the Purkinje
dendrite and subtionof multiple
CFK.,inputs
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et al.,
proteins
in theofwild-type(WT)themouse
Kase,D., Kassai,
H., Nakao,
Hirata, T.,et al. (2014).
The(Hashimoto
synaptic targeting
sequent
elimination‘‘losers’’ fromsoma cerebellum.
in developing Immunohistocerebellum.
its carboxyl-terminal
domaincrucialfor cerebellar
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Neuron63, 106–118.
chemical(IHC) analysis revealed punctate C1ql1 immuno-of mGluR1
2011),bywere
similar between
WTisand
Clql1-null
cerebella(Fig34,These
2702–2712.
ure S2).
results suggest that the winning CFs, which
reactivity
in the M.,
molecularof theK.,cerebellumalong
Hashimoto,K., Tsujita,
Miyazaki, layerT., Kitamura,
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Watanabe,dendrites
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Postsynaptic
P/Qselected during
the initial
functional
stageof PurkinjecellsM.(Figure
The C1ql1-imA.L.,
Mandell,
J.W., andRavichandran,(2007). BAI1
is an
mediates
synaptic
competitionand eliminatypemunopositive
Ca2+ channel in Purkinjepunctacellweremostlylocatedat the edgeof CF ter-Klibanov,by P7,
were
not further
strengthenedin K.S.
the absenceof C1ql1.
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tion in developing cerebellum. Proc. Natl. Acad. Sci. USA 108, 9987–9992.
minals (74% ± 4%, n = 1,195 puncta from 10 slices), which wereengulfment
To examine
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immunopositive for vesicular glutamate transporter 2 (vGluT2).

and determination of the winner CFs, we expressed C1ql1

evident during development (Figures 1D and 1E). In contrast,

existing CF synapses and inducing the elimination of inactive

Rondard, P., Huang, S., Monnier, C., Tu, H., Blanchard, B., Oueslati, N.,
Chandy, N., Hill, R.S., Partlow, J.N., Al-Saffar, M., et al. (2013). Deletions in
Similarly, postembedding immunogold electron microscopy together with enhanced GFP in WT IO neurons at P0–P1 with
Malhaire, F., Li, Y., Trinquet, E., Labesse, G., et al. (2008). Functioning of the
GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in(EM) analysis revealed high levels of C1ql1 immunoreactivity atdimericlentivirus
2A). domain
GFP expression
detected) receptor(Figure
extracellularrevealed bywas
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GABA(Bvectors
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Bednarek,
E.,At
Genoud,C.,Purkinje
Strata, P.,cells
Sacchetti,
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Eur.
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Neurosci. 14,the73–82.
provided by CFs localizes at CF synapses.
CFs overexpressing
C1ql1,
with aconnectivity
dominant CF
and vated
Caroni,by
P.multiple(2011). Learning-related
feedforward
inhibitory
Hirai, H., Pang, Z., Bao, D., Miyazaki, T., Li, L., Miura, E., Parris, J., Rong, Y.,growthrequired formemoryprecision.
Nature473, 514–518.
producingthelargestEPSC ina mannersimilar to that of GFPTo M.,
investigate
the Morgan,
physiologicalof C1ql1,
Watanabe,
Yuzaki, M., andJ.I. (2005).functions
Cbln1 is essential
for syn- we
Selimi,F.,
Cristea,
I.M.,
Heller,E.,
Chait,
B.T.,
and
Heintz,Proteomicthat
only-expressing
Purkinjecells(Figure2C). N.
It (2009).
is interesting
generated
mice
in
which
the
C1ql1
gene
was
disrupted(Figuresaptic integrity and plasticity in the cerebellum. Nat. Neurosci. 8, 1534–1541.
studiesofamplitudes
a single CNSofsynapse
type: the CF-EPSC
parallel fiber/purkinje
cellsynapse.
S1A
and
S1B
available
online).
IHC
andimmunogold
EM
the
the
strongest
and
also
of
the weaker
Holtmaat, A., and Svoboda, K. (2009). Experience-dependent structural synPLoS
Biol. 7, e83.were increased by C1ql1 overexpression in CFs (FigCF-EPSCs
essentiallyno Rev.
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apticanalyses
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Manabe,R., Yamada,
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C1ql1,
at leastT.,when
overexpressed
in IO neurons,ures
1A, 1B,and S1C)M.,inand
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(2010). DistinctTheexpression
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of C1ql1-nullmiceexhibited
normalfoliationand laminatedcould enhance synaptic connectivity between CF and Purkinjeof C1q-likefamily mRNAs
in mousebrain andbiochemical
characterizationofIdentification and characterization of nCLP2, a novel C1q family protein extheircorticalencoded structuresproteins. Eur.(Figure
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31,IHC
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cells,
regardless of the original synaptic strength.
in the central nervous system. J. Biochem. 147, 565–579.
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as a thatneuronal
Neurobiol. 78,
Unexpectedly, at P14–P15, 79% of Purkinje cells had alreadyslices(Figure1C)circuitry
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of vGluT2-positive
Stephenson, J.R., Purcell, R.H., and Hall, R.A. (2014). The BAI subfamily of
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attained
a one-to-one
relationship
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C1ql1
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GPCRs:
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TrendsPharmacol.
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detectionand representationin theolivo-cerebellar
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Motohashi,J.,TheamplitudeofMehalow,
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all131,developmental
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N., Uchigashima,M., dominant
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observed in C1ql1-null cerebella at P9 but became increasingly sults indicate that C1ql1 has dual functions: strengthening
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hibitor
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transporter
1 (vGluT1,
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Recently, affinity chromatography identified C1ql proteins as
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forIsope,
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Bai3,
a C1q
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andfamily proteins:transneuronalGPCR
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and Selimi,
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adhesion-GPCR
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S1F).
TheseTheresults
indicateBAI3,
thata CFCell.(Bolliger
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Neuron85, 316–329,
January
21, 2015ª2015
Elsevier
Inc. Inc.
329317
Neuron85, 316–329,
January
21, 2015ª2015
Elsevier－ 77 －A C1q Family Proteins and Synaptic Functions
B

C

represented as mean ± SEM. Statistical analyses
D were performed using theBased on the behavioral phenotypes of ataxic mutant mice Excel statistics 2012 add-in software (Social Survey Research Information),lacking mGluR1, PKCg, and GluD2, monoinnervation of Pur- and significant differences were defined as *p < 0.05, **p < 0.01, and ***p <0.001.
kinje cells by CFs was thought to be required for motor coordination (Chen and Tonegawa, 1997). In contrast, although ACCESSION NUMBERS
C1ql1-null and PC-Bai3-null Purkinje cells remain innervatedby multiple CF inputs, these mice have normal gaits without The coordinates and structure factors for the reported crystal structure are
significant impairment in beam or rotor-rod tests (Figure S8; deposited in the Protein Data Bank (PDB) under PDB ID 4d7y.
Movie S1). However, motor learning, as measured by hOKR,
was severely impaired in these mice (Figure 6). Recently, SUPPLEMENTAL INFORMATION
E
mGluR1b-rescue mice, in which the splice variant of mGluR1
Supplemental Information includes Supplemental Experimental Procedures,is expressed in mGluR1-null Purkinje cells, were shown toeight figures, and two movies and can be found with this article online at
display normal motor coordination, even though their Purkinje http://dx.doi.org/10.1016/j.neuron.2014.12.020.
cells were innervated by multiple CFs. These mice showed
impaired LTD and eyeblink conditioning, another form of AUTHOR CONTRIBUTIONS
cerebellum-dependent motor learning (Ohtani et al., 2014).
Similarly, a single injection of recombinant Cbln1 restored W.K. designed the project, carried out the electrophysiological and behavioral
works, analyzed the data, andF LTD and eyeblink conditioning in adult Cbln1-nullG
H wrote the paper. N.M. and A.R.A. performed
mice without
structural and SPR analyses and designed mutant constructs. E.M. performed
significantly affecting the CF innervation pattern or LTP at the anatomical experiments and the IUE approach. M.A. and K.S. designed
PF-Purkinje cell synapses (Emi et al., 2013). Furthermore, and generated mutant mice. K.M. performed the cell binding assays. Y.H.T.
GluD2DCT7-rescue mice, in which GluD2 lacking seven C-termi- designed and prepared the microRNA constructs. K.K., A.T., J.M., and S.M.
nal amino acids was expressed in GluD2-null Purkinje cells, designed and prepared the recombinant viruses. S.N. supervised the hOKR
displayed impaired LTD and eyeblink conditioning despite experiment. M.W. generated essential antibodies. M.Y. designed and supermorphologically normal PF and CF synapses (Kakegawa vised the project and wrote the paper.
et al., 2008). These findings indicate that LTD, rather than ACKNOWLEDGMENTS
monoinnervation of Purkinje cells by CFs, is likely linked to motor learning in the cerebellum and indicate that C1ql1-Bai3 We thank S. Narumi and S. Otsuka for their technical assistance and S. Matsignaling is required for normal CF functions, such as complex suda and K.F. Tanaka for their useful comments. We also thank T. Miyazaki
spikes and CF-evoked afterhyperpolarization, which are neces- and K. Konno for their support on anatomical experiments, H. Okado for lentivirus production, H. Sakuma and T. Watanabe (Zeiss) for their support on
Figure
1. for
C1ql1
Is Localized at CF Synapses during Their Formation and Maturation
sary
LTD.
superresolution
microscopy byanalysis,and the arestaffmagnified
at Diamondbeamline(A) IHC images of endogenous C1ql1 in adult WT (left) and C1ql1-null (right) mouse cerebella.
Areas surrounded
white squares
in thelower I04
Family members of C1ql1 (C1ql2–C1ql4) are expressed infor synchrotron assistance. This work was supported by a grant-in-aid for scipanels. Scale bars in upper and lower panels, 20 mm and 3 mm, respectively.
various brain regions (Iijima et al., 2010). Bai3 and its related entific research from the Ministry of Education, Culture, Sports, Science and(B) Postembedding immunogold EM images of endogenous C1ql1 in adult WT mouse
cerebellum. C1ql1 and vGluT2 (a marker for CF terminals) were immembers
widelyexpressed(Kee etcellal.,spine.Technology
Japan goldand particles
the Japan
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Red arrowsofindicate
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Histogramthe verticalof C1ql1-positive
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imagesofvGluT2in adult
WT (left) andC1ql1-nullmousecompecerebella. Dottedlines(W.K.);
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Purkinjesoma,
C1ql1 and Bai3 in the
cerebellum
likelyregulate(right)
synaptic
Medical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD studentrespectively. Scale bar, 20 mm.
tition and maintenance in various neuronal circuits essential for ship (N.M.). Further support from the Wellcome Trust Core Award 090532/Z/(D) Developmental changes in the density of vGluT2 puncta.
brain functions.
In thehippocampus,
C1ql2
and C1ql3
are highly
09/Z is acknowledged.
A.R.A.
is an100%
MRCisSeniorResearch
Fellow.
(E) Developmental
changesin thelocalization of vGluT2
puncta.
The relative
height is represented
as a percentage,
whereat the topof Purkinje
cell distal
expressed
adultdentatecell
gyrus
dendritesand 0% in
is at
the Purkinje
soma.granule cells, and their mossy
Accepted:
November
2014
fiber afferents
makedynamic
branchedaxon-typesynapses(F) CF-EPSC
traces evokedby thepaired-pulse
stimulation(50 ms interstimulusinterval)in various
intensities26,
in P5(top) and adult (bottom) WT (left) and C1ql1Published: January 21, 2015
null (right)
mice. Notably, mossy fiber synapses reportedly display
like CFs.
(G andH) Developmental changes
in thepercentageof the tonumberof CFs innervating
single Purkinje cells (G) and the strongest CF-EPSC amplitudes (H).
learning-induced
structural
plasticity
relatedthe precisionof
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ures
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electrophysiological,
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structural,
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cerebellum.
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3A, 3C,162,
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Purkinje cells attained a one-to-one relationship with CFs (p <Bae, C.S., and Kim, K.K. (2004). Expression of brain-specific angiogenesis inWatanabe, M., and Kano, M. (2011). Climbing fiber synapse elimination in
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FigureKishore,U., Gaboriaud,
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activity
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< 0.001;
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and 3E).
In addition,
thefactor
depthof themutant
Together,these inresults
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nocopied C1ql1-null mice.
nalsV.,wassignificantly
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M. further(2008). Cbln
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aslices)(84%± 1%,= 10 slices) BAI3,
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Neuron85, 316–329,
January
21, 2015ª2015
Elsevier
Inc. Inc.
329319Neuron85, 316–329,
January
21, 2015ª2015
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C1q FamilyAProteins and Synaptic Functions
B
Based on the behavioral phenotypes of ataxic mutant micelacking mGluR1, PKCg, and GluD2, monoinnervation of Purkinje cells by CFs was thought to be required for motor coordination (Chen and Tonegawa, 1997). In contrast, althoughC1ql1-null and PC-Bai3-null Purkinje cells remain innervatedby multiple CF inputs, these mice have normal gaits without
significant impairment in beam or rotor-rod tests (Figure S8;
Movie S1). However, motor learning, as measured by hOKR,
was severely impaired in these mice (Figure 6). Recently,
mGluR1b-rescue mice, in which the splice variant of mGluR1
is expressed in mGluR1-null Purkinje cells, were shown todisplay normal motor coordination, even though their Purkinjecells were innervated by multiple CFs. These mice showed
impaired LTD and eyeblink conditioning, another form of
cerebellum-dependent motor learning (Ohtani et al., 2014).
Similarly, a single injection of recombinant Cbln1 restored
LTD and eyeblink conditioning in adult Cbln1-null mice without
significantly affecting the CF innervation pattern or LTP at
E
PF-PurkinjeDcell synapses (Emi et al., 2013). Furthermore,
GluD2DCT7-rescue mice, in which GluD2 lacking seven C-terminal amino acids was expressed in GluD2-null Purkinje cells,
displayed impaired LTD and eyeblink conditioning despite
morphologically normal PF and CF synapses (Kakegawa
et al., 2008). These findings indicate that LTD, rather thanmonoinnervation of Purkinje cells by CFs, is likely linked to motor learning in the cerebellum and indicate that C1ql1-Bai3
signaling is required for normal CF functions, such as complex
spikes and CF-evoked afterhyperpolarization, which are necessary for LTD.
F
Family members of C1ql1 (C1ql2–C1ql4) are expressed invarious brain regions (Iijima et al., 2010). Bai3 and its related
members Bai1 and Bai2 are also widely expressed (Kee et al.,2004) and have been implicated in psychiatric disorders, suchas schizophrenia and bipolar disorders (Lanoue et al., 2013).
Thus, signaling mechanisms similar to the one mediated by
C1ql1 and Bai3 in the cerebellum likely regulate synaptic competition and maintenance in various neuronal circuits essential for
J In the hippocampus, C1ql2 and C1ql3
K
L
brain functions.
are highly
expressed in adult dentate gyrus granule cells, and their mossy
fiber afferents make dynamic branched axon-type synapses
like CFs. Notably, mossy fiber synapses reportedly display
learning-induced structural plasticity related to the precision oflearning in adult mice (Ruediger et al., 2011). Therefore, futurestudies should assess whether and how C1ql proteins mediate
activity-dependent structural plasticity essential for memory
and learning.

represented as mean
C ± SEM. Statistical analyses were performed using the
Excel statistics 2012 add-in software (Social Survey Research Information),and significant differences were defined as *p < 0.05, **p < 0.01, and ***p <0.001.

All procedures related to animal care and treatment were performed in accordance with the guidelines set down by the Animal Resource Committee of
Keio University. C1ql1-null, PC-Bai2-null, and PC-Bai3-null mice were generated and maintained on C57BL/6N backgrounds. As controls, WT mice wereused for C1ql1-null mice and Bai2flox, and Bai3flox mice were used for PCBai2-null and PC-Bai3-null mice. For the behavioral analyses, littermates
were used as controls. Experimental details of mouse generation as well as
electrophysiological, anatomical, biochemical, structural, and behavioral analyses are described in the Supplemental Experimental Procedures. Data are

The coordinates and structure factors for the reported crystal structure aredeposited in the Protein Data Bank (PDB) under PDB ID 4d7y.

－ 80 －

Duman,
Figu
Tolias, K
regcontrollinsites.cien
J.

larly

SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,eight figures, and two movies and can be found with this article online at
http://dx.doi.org/10.1016/j.neuron.2014.12.020.
AUTHOR CONTRIBUTIONS
W.K. designed the project, carried out the electrophysiological and behavioral
works, analyzed the data, and wrote the paper. N.M. and A.R.A. performed
structural and SPR analyses and designed mutant constructs. E.M. performed
the anatomical experiments and the IUE approach. M.A. and K.S. designed
Gmutant mice. K.M. performed the cell binding assays. Y.H.T.
and generated
designed and prepared the microRNA constructs. K.K., A.T., J.M., and S.M.
designed and prepared the recombinant viruses. S.N. supervised the hOKR
experiment. M.W. generated essential antibodies. M.Y. designed and supervised the project and wrote the paper.
ACKNOWLEDGMENTS
We thank S. Narumi and S. Otsuka for their technical assistance and S. MatI comments. We also thank T. Miyazakisuda and K.F. H
Tanaka for their usefuland K. Konno for their support on anatomical experiments, H. Okado for lentivirus production, H. Sakuma and T. Watanabe (Zeiss) for their support onsuperresolution microscopy analysis, and the staff at Diamond beamline I04
for synchrotron assistance. This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science andTechnology of Japan and the Japan Society for the Promotion of Science(W.K. and M.Y.); the CREST from the Japan Science and Technology Agency(M.Y.); the Kanae Foundation (W.K.); the Nakajima Foundation (W.K.); the Keio
Gijuku Fukuzawa Memorial Fund for the Advancement of Education andResearch (W.K.); the Takeda Science Foundation (W.K. and M.Y.); the UK
Medical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD studentship (N.M.). Further support from the Wellcome Trust Core Award 090532/Z/
09/Z is acknowledged. A.R.A. is an MRC Senior Research Fellow.
Accepted: November 26, 2014
Published: January 21, 2015
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N., and Selimi, F. (2013). The adhesion-GPCR BAI3, a
See also

Yuzaki, M. (2008). Cbln and C1q family proteins: new transneuronal cytokines.
Cell. Mol. Life Sci. 65, 1698–1705.

age)

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21, 2015ª2015
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Inc. Inc.
329321
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January
21, 2015ª2015
Elsevier－ 81 －

and Synaptic Functions
A C1q Family ProteinsB

represented
as mean± SEM. Statistical
performedElimiusing the
Figure4. C1ql1-Bai3Signaling
Triggers analyses
Surplus were
CF Synapse

Excel
statistics
2012 Mice
add-in software (Social Survey Research Information),in Adult
MutantBased on the behavioral phenotypes of ataxic mutant micenationand significant
were definedas *p< 0.05, **p< 0.01,and***p <(A) Cartoon
showingdifferences
the AAV injection
into adultC1ql1-nullmouse
IO forthelacking mGluR1, PKCg, and GluD2, monoinnervation of Pur0.001. of C1ql1. Experiments were performed at 14–21 days postinfection
expression
kinje cells by CFs was thought to be required for motor coordi(dpi).
nation (Chen and Tonegawa, 1997). In contrast, although(B) CF-EPSCs
recorded
from adult C1ql1-null Purkinje cells innervated by CFs
ACCESSIONNUMBERSC1ql1-null and PC-Bai3-null Purkinje cells remain innervatedexpressing WT C1ql1 (C1ql1WT, center), C1ql1GW (right), and empty vectorleft).coordinates and structure factors for the reported crystal structure are
by multiple CF inputs,
these miceDhave normal gaits without(CTL,TheCD) Histograms
showing
theBank
percentageof thePDB
numberof CFs innerdepositedin the Protein
Data(PDB) under
ID 4d7y.
significant impairment in beam or rotor-rod tests (Figure S8;(C andMovie S1). However, motor learning, as measured by hOKR,vating single Purkinje cells (C) and the strongest CF-EPSC amplitudes (D) ineach condition.
was severely impaired in these mice (Figure 6). Recently, SUPPLEMENTAL INFORMATION(E) Diagrams (left) for C1ql1WT, C1ql1GW, and their globular domains (gC1ql1WT
mGluR1b-rescue mice, in which the splice variant of mGluR1and gC1ql1GW, respectively), and the equilibrium SPR data of each C1ql1
Supplemental Information includes Supplemental Experimental Procedures,is expressed in mGluR1-null Purkinje cells, were shown tobinding to eBai3 (right). RU, resonance unit.
eight figures, and two movies and can be found with this article online at
display normal motor coordination, even though their Purkinje(F) Cartoon
representation of the gC1ql1WT (top) and gC1ql1GW (bottom)
http://dx.doi.org/10.1016/j.neuron.2014.12.020.
trimeric
structures.
Individual monomers are separately colored. Glycosylation
cells were innervated by multiple CFs. These mice showed
introducedbymutagenesis (original residues were Q211 and S244) are
E impaired LTD and eyeblink conditioning, another form ofsitesAUTHORCONTRIBUTIONS
indicated by spheres in C1ql1WT. The gC1ql1GW trimeric structure is shown
cerebellum-dependent motor learning (Ohtani et al., 2014).
with W.K.
modeledMan9GlcNAc2
glycans
sphere representation).
designed
the project,carried(atoms
out theinelectrophysiologicaland behavioral
Similarly, a single injection of recombinant Cbln1 restored(G) Cartoon
showing thethe data,
lentivirus
injection
adult
PC-Bai3-nullworks, analyzed
and wrotetheintopaper.
N.M.
and A.R.A.mouse
performed
LTD and eyeblink conditioning in adult Cbln1-null mice withoutcerebellum for the expression of Bai3. Experiments were performed at 14–21
structural and SPR analyses and designed mutant constructs. E.M. performed
significantly affecting the CF innervation pattern or LTP atdpi. the anatomical experiments and the IUE approach. M.A. and K.S. designed
recordedfromadultK.M.
PC-Bai3-nullPurkinje
expressing
PF-Purkinje cell synapses (Emi et al., 2013). Furthermore,(H) CF-EPSCs
and generated
mutantmice.
performed
the cellcells
binding
assays.WT
Y.H.T.
Bai3DCUB
and empty
vectorK.K.,(CTL,left).J.M., and S.M.
(Bai3WT, center),
GluD2DCT7-rescue mice, in which GluD2 lacking seven C-termi-Bai3designed
and preparedthe(right),
microRNA
constructs.
A.T.,
J) Histograms
showing thethe percentage
the number
CFs innervating
and preparedrecombinantofviruses.
S.N.ofsupervised
the hOKR
nal amino acids was expressed in GluD2-null Purkinje cells,(I anddesigned
Fsingle Purkinje cells (I) and the strongest CF-EPSC amplitudes (J) in eachdisplayed impaired LTD and eyeblink conditioning despite experiment. M.W. generated essential antibodies. M.Y. designed and supercondition. the project and wrote the paper.
morphologically normal PF and CF synapses (Kakegawa*p < vised
0.05, ***p < 0.001 by Kruskal-Wallis test followed by Steel post hoc test.
et al., 2008). These findings indicate that LTD, rather thanns, nosignificance. Data are represented as mean ± SEM.
ACKNOWLEDGMENTS
monoinnervation of Purkinje cells by CFs, is likely linked to mo-See also Figures S4 and S6 and Table 1.
tor learning in the cerebellum and indicate that C1ql1-Bai3 We thank S. Narumi and S. Otsuka for their technical assistance and S. Matsignaling is required for normal CF functions, such as complex suda and K.F. Tanaka for their useful comments. We also thank T. Miyazaki
theofanatomical
the strongest
CF-EPSCs
wereand K. Konno
for amplitudes
their support on
experiments,
H. Okado
for lentispikes and CF-evoked afterhyperpolarization, which are neces-Furthermore,
GW and T. Watanabe (Zeiss) for their support on
virus
production,
H.
Sakumaexpressioninadult
C1ql1-nullmicenotincreasedby
C1ql1
sary for LTD.
superresolution microscopy analysis, and the staff at Diamond beamline I04
Family members of C1ql1 (C1ql2–C1ql4) are expressed in(Figures 4B and 4D). These results indicate that the function offor synchrotron assistance. This work was supported by a grant-in-aid for sciin determining a single-winner CF in the adult brain isvarious brain regions (Iijima et al., 2010). Bai3 and its relatedC1ql1
entific research from the Ministry of Education, Culture, Sports, Science andby an interface used for Bai3 binding.
members Bai1 and Bai2 are also widely expressed (Kee et al.,mediatedTechnology of Japan and the Japan Society for the Promotion of Science
Finally,
to M.Y.);
examinewhetherof Bai3in adult PC2004) and have been implicated in psychiatric disorders, such (W.K. andthe CREST
from expression
the Japan Scienceand Technology
AgencyPurkinjecells could
alsotherestoreCFphenotypes,
we Keio(M.Y.); theKanae Foundation(W.K.);
Nakajima
Foundation(W.K.); theas schizophrenia and bipolar disorders (Lanoue et al., 2013).Bai3-null
WT
GijukuFukuzawa
Memorial
Fund fortheAdvancement
lentiviral
vectors
to express
Bai3
or Bai3DCUBofinEducation
Purkinje andThus, signaling mechanisms similar to the one mediated byused
DCUB
Research(W.K.);
the WT
TakedaScienceFoundation(W.K. andM.Y.); the UK
and
Bai3
were
similarly
expressed
cells(Figure4G).
Bai3
C1ql1 and Bai3 in the cerebellum likely regulate synaptic compeMedical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD studentin
dendritic
spinesof
Purkinjecells(FigureS6B),
indicatingthattition and maintenance in various neuronal circuits essential for ship (N.M.). Further support from the Wellcome Trust Core Award 090532/Z/
of C1ql1 was likely to be nonessential for localizing
brain functions. In the hippocampus, C1ql2 and C1ql3 are highlybinding
09/Z is acknowledged. A.R.A. is an MRC Senior Research Fellow.
expressed in adult dentate gyrus granule cells, and their mossyBai3 at postsynaptic sites. Whole-cell patch-clamp recordings
DCUB
that November
Bai3WT, butor empty vector, restored
Accepted:
26, not
2014Bai3
fiber afferents make dynamic branched axon-type synapsesshowed
G like CFs. Notably,H
normal processof 2015
CF synapse elimination in PC-Bai3-nullJanuary 21,
mossy fiber synapses reportedly displaythe Published:learning-induced structural plasticity related to the precision ofmice (p < 0.05; Figures 4H and 4I). Similarly, the amplitudes of
WT
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apseresistanteliminationformin theofdeveloping
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in vivo,
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PC-Bai3Ohtani,Y., Miyata,
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Fukaya,
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Theno
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S1). TheyJ. Neurosci.
2702–2712.
showed34,normal
motor coordination on the rotor-rod test (Figures
Park,S8A
D., Tosello-Trampont,
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Lu, M., Haney, L.B.,
Ma, Z.,and S8B). Nystagmus,
whichM.R.,
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S8E). These results indicate that innervation of Purkinje cells

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Malhaire, F., Li, Y., Trinquet, E., Labesse, G., et al. (2008). Functioning of the
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bellum,we next
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the parallel
fiber/purkinje
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apticreferplasticity
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mouse
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slices eliminapreparedysis M.,of cerebellar
slicesrevealed
thatYuzaki,
the numberof vGluT2-posregulation of synaptic plasticity and cerebellar motor learning by the
Cell 131,
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C1ql1-nulland PC-Bai3-null mice by simultaneously actiitive puncta was significantly reduced in mice expressing miR-tion.from
C-terminal PDZ-binding motif of GluRdelta2. J. Neurosci. 28, 1460–1468.
N., PFs
Uchigashima,T.,current-clamp
Nakao, H., Hirai, H.,vatingand CFsM.,
forMikuni,
2 minT.,atNakazawa,
1 Hz in themode
C1ql1 (110 ± 9, n = 10 slices; Figures 5C and 5D) compared toUesaka,Kano, M., and Hashimoto, K. (2009). Synapse elimination in the central nerAiba,
A.,
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mice expressing miR-SCR (180 ± 6, n = 10 slices; p < 0.001; Fig- (Figure 6E). This protocol robustly induced LTD ofvous system. Curr. Opin. Neurobiol. 19, 154–161.
signaling regulates synapse elimination in the developing mouse brain.
ures 5C and 5D). In addition, the depth of the molecular layer EPSCs (PF-EPSCs) in WT, but not C1ql1-null (p < 0.001; FigScience 344, 1020–1023.
Kee, H.J., Ahn, K.Y., Choi, K.C., Won Song, J., Heo, T., Jung, S., Kim, J.K.,

thickness reached by the most distal CF terminals was reduced

ures 6F and 6G) or PC-Bai3-null (p < 0.001 versus control; Fig-

Bae, C.S., and Kim, K.K. (2004). Expression of brain-specific angiogenesis inWatanabe, M., and Kano, M. (2011). Climbing fiber synapse elimination inures 6F
and 6H), Purkinje cells. Activation of CF input triggers
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10 slices; Figurescerebellar
hibitor(BAI3)
in normal brain
and implications
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and 5E) compared
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complexspikesin WTPurkinje(Ecclesal., 1966).
n = 10p < C.,
0.05;
Figures
5C and5E).
In contrast,
whole-cell
Kishore,U., slices,
Gaboriaud,
Waters,P., Shrive,
A.K.,
Greenhough,
T.J.,
Reid,
Oscillating
Purkinje
neuronactivity
causingcells
involuntary
eyeetmovement
in aTheoccurrenceof thein complex
spike
was d2significantly
reduced inrecordings
fromC1q
adult
cerebellar
revealedmutant
K.B.,patch-clamp
Sim, R.B., and Arlaud,
G.J. (2004).
and tumor
necrosisslices
factor supermouse deficientthe glutamate
receptor
subunit. J. Neurosci.
family:modularityand versatility. Trends
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25,were551–561.
Purkinje cells from C1ql1-null (53 ± 9 in 120 trials, n = 22 versus
thatsimilar percentagesof Purkinje
cells
innervated by sin-24, 2440–2448.
WTM.[118± 2Cblnin 120
trials,n =proteins:22]; p <new
0.001
by Mann-Whitney
gle V.,
CFsin miceexpressing
miR-C1ql1(83%;
Figures
5F and(2008).
and C1q
familytransneuronalcytokines. U
Lanoue,Usardi,A., Sigoillot,
S.M., Talleur,M., Iyer,K., Mariani,J., Isope,
P.,5G)Yuzaki,
Cell.test)
Mol. Life65, 1698–1705.
Vodjdani,G., Heintz, (89%,
N., and pSelimi,
F. (2013).
The
BAI3, aandSci.
PC-Bai3-nullmice (64 ± 8 in 120 trials, n = 24 versus
and miR-SCR= 0.51;Figures
5Fadhesion-GPCR
and 5G). Nevertheless,
Neuron85, 316–329,
January
21, 2015ª2015
Elsevier
Inc. Inc.
329323
Neuron85, 316–329,
January
21, 2015ª2015
Elsevier－ 83 －

and Synaptic Functions
A C1q Family Proteins B
Based on the behavioral phenotypes of ataxic mutant micelacking mGluR1, PKCg, and GluD2, monoinnervation of Purkinje cells by CFs was thought to be required for motor coordination (Chen and Tonegawa, 1997). In contrast, althoughC1ql1-null and PC-Bai3-null Purkinje cells remain innervatedby multiple CF inputs, these mice have normal gaits without
significant impairment in beam or rotor-rod tests (Figure S8;
Movie S1). However, motor learning, as measured by hOKR,
was severely impaired in these mice (Figure 6). Recently,
mGluR1b-rescue mice, in which the splice variant of mGluR1
is expressed in mGluR1-null Purkinje cells, were shown todisplay normal motor coordination, even though their Purkinje
C cells were innervated by multiple CFs. These mice showedD
impaired LTD and eyeblink conditioning, another form of
cerebellum-dependent motor learning (Ohtani et al., 2014).
Similarly, a single injection of recombinant Cbln1 restored
LTD and eyeblink conditioning in adult Cbln1-null mice without
significantly affecting the CF innervation pattern or LTP at
PF-Purkinje cell synapses (Emi et al., 2013). Furthermore,
GluD2DCT7-rescue mice, in which GluD2 lacking seven C-terminal amino acids was expressed in GluD2-null Purkinje cells,
displayed impaired LTD and eyeblink conditioning despite
morphologically normal PF and CF synapses (Kakegawa
et al., 2008). These findings indicate that LTD, rather thanEmonoinnervation of Purkinje cells by CFs, is likely linked to motor learning in the cerebellum and indicate that C1ql1-Bai3
signaling is required for normal CF functions, such as complex
spikes and CF-evoked afterhyperpolarization, which are necessary for LTD.
Family members of C1ql1 (C1ql2–C1ql4) are expressed invarious brain regions (Iijima et al., 2010). Bai3 and its related
members Bai1 and Bai2 are also widely expressed (Kee et al.,2004) and have been implicated in psychiatric disorders, such
F as schizophrenia and bipolar disorders (Lanoue et al., 2013).
Thus, signaling mechanisms similar to the one mediated by
C1ql1 and Bai3 in the cerebellum likely regulate synaptic competition and maintenance in various neuronal circuits essential forbrain functions. In the hippocampus, C1ql2 and C1ql3 are highly
expressed in adult dentate gyrus granule cells, and their mossy
fiber afferents make dynamic branched axon-type synapses
like CFs. Notably, mossy fiber synapses reportedly display
learning-induced structural plasticity related to the precision oflearning in adult mice (Ruediger et al., 2011). Therefore, future
whether and how C1ql proteins mediate
G studies should assess H
activity-dependent structural plasticity essential for memory
and learning.

represented as meanFigure± SEM.5.Statisticalanalyses
were performed
using theC1ql1-Bai3
Interaction
Is Essential
Excel statistics 2012for
add-insoftware (SocialofSurvey
Research
the Maintenance
CF Synapse
in Information),
Adultsand significant differenceswere showing
defined as< 0.05,**p <into
0.01,
and***p <(A) Cartoon
the*pAAV
injection
adult
WT
0.001.
mouse IO for the introduction of miR-C1ql1 or miRSCR. Experiments were performed at 14–21 dpi.
(B) IHC images (left) and the quantitative data (right)
ACCESSION NUMBERS
showing C1ql1 is knocked down by miR-C1ql1
but not
miR-SCRin GFP-expressing
IO are
The coordinates and(right)
structure
factors
for the(left)reported
crystal structure
neurons.
Scale(PDB)bar, 100
mm.PDB ID 4d7y.
deposited in the Protein
Data Bank
under(C) IHC images of vGluT2 in adult WT mouse cerebella with CFs expressing GFP plus miR-SCR (left)
SUPPLEMENTAL INFORMATION
or miR-C1ql1 (right). vGluT2 immunoreactivities arebinarizedin theSupplemental
bottom. Dottedlines and asterisks
Supplemental Information
includesExperimental
Procedures,
represent
the withmolecular
layer online
and ateight figures, and twomovies pial
and surface
can be offound
this article
Purkinje cell soma, respectively. Scale bar, 20 mm.
http://dx.doi.org/10.1016/j.neuron.2014.12.020.
(D and E) Quantification of the density (D) and theheight (E) of vGluT2 puncta on the molecular layer of
AUTHOR CONTRIBUTIONS
each condition.
(F) CF-EPSCs
evokedby various stimulus andintensities
W.K. designed the project,carried out
the electrophysiologicalbehavioral
adult
WT
cerebellar
slicesN.M.
withand
CFsA.R.A.
introducing
works, analyzed the in
data,
and
wrote
the paper.
performed
miR-SCR,
miR-C1ql1
plus E.M.
a resistant
structural and SPR analyses
andmiR-C1ql1,
designed mutant
constructs.
performed
WT) or M.A.
miR-C1ql1
plus
aform ofand
C1ql1(rC1ql1
the anatomical experiments
the IUEapproach.
and K.S.
designed
GW.
rC1ql1and generated mutant
mice. K.M.
performed the cell binding assays. Y.H.T.
(G andHistograms
showing K.K.,
the percentage
theS.M.
designed and preparedthe H)
microRNA
constructs.
A.T., J.M.,ofand
numberof CFs innervating
Purkinje cells
designed and prepared
the recombinantviruses.single
S.N. supervisedthe(G)hOKR
and the strongest
CF-EPSC amplitudes(H) inandeachexperiment. M.W. generated
essential antibodies.
M.Y. designed
supervised the project andcondition.
wrote the paper.
*p < 0.05, ***p < 0.001 by Mann-Whitney U test in (B),(D), and (E). *p < 0.05, ***p < 0.001 by Kruskal-Wallis
ACKNOWLEDGMENTS
test followed by Scheffé post hoc test in (H). ns, nosignificance.
are represented
as mean ±SEM.
We thank S. Narumi and
S. OtsukaData
for theirtechnical assistanceand
S. MatSee
suda and K.F. Tanaka
foralsotheirFigure
usefulS7.
comments. We also thank T. Miyazaki
and K. Konno for their support on anatomical experiments, H. Okado for lentivirus production, H. Sakuma and T. Watanabe (Zeiss) for their support onsuperresolution microscopy analysis, and the staff at Diamond beamline I04
for synchrotron assistance. This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science andTechnology of Japan and the Japan Society for the Promotion of Science(W.K. and M.Y.); the CREST from the Japan Science and Technology Agency(M.Y.); the Kanae Foundation (W.K.); the Nakajima Foundation (W.K.); the Keio
Gijuku Fukuzawa Memorial Fund for the Advancement of Education andResearch (W.K.); the Takeda Science Foundation (W.K. and M.Y.); the UK
Medical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD studentship (N.M.). Further support from the Wellcome Trust Core Award 090532/Z/
09/Z is acknowledged. A.R.A. is an MRC Senior Research Fellow.

All procedures related to animal care and treatment were performed in accordance with the guidelines set down by the Animal Resource Committee of
Keio University. C1ql1-null, PC-Bai2-null, and PC-Bai3-null mice were generated and maintained on C57BL/6N backgrounds. As controls, WT mice wereused for C1ql1-null mice and Bai2flox, and Bai3flox mice were used for PCBai2-null and PC-Bai3-null mice. For the behavioral analyses, littermates
were used as controls. Experimental details of mouse generation as well as
electrophysiological, anatomical, biochemical, structural, and behavioral analyses are described in the Supplemental Experimental Procedures. Data are

Neuron85, 316–329,
January
21, 2015ª2015
Elsevier
324 328
Neuron85, 316–329,
January
21, 2015ª2015
Elsevier
Inc. Inc.

－ 84 －

Davie, J
spike inDuman,
Tolias, K
controllinsites. J.

Eccles, J
climbing
268–296

Emi, K.,(2013). R
ditioning
doi.org/1

HashimoMishina,(2001). R
glutamatduring p

HashimoTransloc
sequent
Neuron 6

Hashimo
H.S., Watype Ca2
tion in deHills, L.B
Chandy,GRID2 lehumans.

Hirai, H.
density i

Hirai, H.,
Watanabaptic inte

Holtmaa
aptic pla

Iijima, T.
of C1q-li
their enc

Accepted: November 26, 2014
Published: January 21, 2015

Ito, M. (2272–303

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represented as mean ± SEM. Statistical analyses were performed using the
C1q Family
WTProteins and Synaptic Functions
Based on the behavioral phenotypes of ataxic mutant mice Excel statistics 2012 add-in software (Social Survey Research Information),
C significant differences were definedDas *p < 0.05, **p < 0.01, and ***p <lacking AmGluR1, PKCg, and GluD2, Bmonoinnervation of Pur- and0.001.
"Maintenance"
Purkinje
kinje cells by CFs was thought to be required for motor coordicell"Maturation"nation (Chen and Functional
Tonegawa, 1997). In contrast, although ACCESSION NUMBERS
differentiation
C1ql1-null and PC-Bai3-nullPurkinje cells remain innervatedby multiple C1ql1
CF inputs, these mice have normal gaits without The coordinates and structure factors for the reported crystal structure are
C1ql1, Bai3
Data Bank (PDB) under PDB ID 4d7y.
significant impairment in beam Translocation
or rotor-rod tests (Figure S8; deposited in the Protein knockdown
Movie S1). However, motor learning, as measured by hOKR,
Bai3
was severely impaired in these mice (Figure 6). Recently, SUPPLEMENTAL INFORMATION
mGluR1b-rescue mice, in which the splice variant of mGluR1
Supplemental
Information includes Supplemental Experimental Procedures,
Dominant
Less dominant"Elimination"is expressed in mGluR1-null Purkinje cells, were shown toeight figures, and two movies and can be found with this article online atCFCFs
display normal motor coordination, even though their
Purkinje
C1ql1- Bai3 http://dx.doi.org/10.1016/j.neuron.2014.12.020.
signalingcells were innervated by multiple CFs. These mice showed
impaired LTD and eyeblink conditioning, another form of AUTHOR CONTRIBUTIONS
cerebellum-dependent motor learning (Ohtani et al., 2014).
PC-Bai3-null
Similarly,C1ql1-nulla single / injectionof recombinant Cbln1 restored W.K. designed the project, carried out the electrophysiological and behavioral
works, analyzed the data, and wrote the paper. N.M. and A.R.A. performed
LTD and eyeblink conditioning in adult Cbln1-null mice without
structuraland SPR analyses and designed
EFG
H mutant constructs. E.M. performed
significantly affecting the CF innervation pattern or LTP at the anatomical experiments and the IUE approach. M.A. and K.S. designed
PF-Purkinje cell synapses (Emi et al., 2013). Furthermore, and generated mutant mice. K.M. performed the cell binding assays. Y.H.T.
Functional
GluD2DCT7-rescue mice,in which GluD2 lacking seven C-termi- designed and prepared the microRNA constructs. K.K., A.T., J.M., and S.M.
differentiation
nal amino acids was expressed in GluD2-null Purkinje cells, designed and prepared the recombinant viruses. S.N. supervised the hOKR
C1ql1,
Bai3 antibodies. M.Y. designed and superTranslocation
essential
displayed impaired LTD and eyeblink conditioning
despite experiment. M.W. generated
expression
Translocation
vised the project and wrote
the paper.
morphologically normal PF and CF synapses (Kakegawa
et al., 2008). These findings indicate that LTD, rather than ACKNOWLEDGMENTS
monoinnervation of Purkinje cells by CFs, is likely linked to motor learning in the cerebellum and indicate that C1ql1-Bai3 We thank S. Narumi and S. Otsuka for their"Elimination"technical assistance and S. Matsignaling is required for normal CF functions, such as complex suda and K.F. Tanaka for their useful comments. We also thank T. Miyazaki
and K. Konno for their support on anatomical experiments, H. Okado for lentispikes
CF-evoked
afterhyperpolarization,
which
are necesFigure
7. A andModel
for C1ql1-Bai3Signaling in Synaptic
Competition
at CF Synapses
virus production, H. Sakuma and T. Watanabe (Zeiss) for their support onsary
fordisparity
LTD. is formed among multiple CFs innervating a single Purkinje cell soma
(A andE) Theby P7 in both WT (A) and C1ql1-null or PC-Bai3-null (E) cerebellum.
superresolution microscopy analysis, and the staff at Diamond beamline I04
Familymembers signalingof C1ql1(C1ql2–C1ql4)
expressed
inCF may trigger a signaling pathway to eliminate less dominant synapses. The(B) Although
the C1ql1-Bai3
promotes
maturation ofare
all CFs,
the winningfor synchrotron assistance. This work was supported by a grant-in-aid for sciwinningCF mayoccupythe postsynaptic
close toBai3
proximaland prevent translocation of somatic synapses formed by less dominant CFs tovariousbrain
regions(Iijima et regional., 2010).
anddendrites
its related
entific research from the Ministry of Education, Culture, Sports, Science andproximaldendrites.
members
Bai1 and Bai2 are also widely expressed (Kee et al., Technology of Japan and the Japan Society for the Promotion of Science(C) CFs remaining on the Purkinje cell soma are eventually eliminated.
2004) and have been implicated in psychiatric disorders, such (W.K. and M.Y.); the CREST from the Japan Science and Technology Agency(D) Knockdown of C1ql1 or Bai3 revealed that the C1ql1-Bai3 signaling is required to maintain the winner CF in adult.
as schizophrenia and bipolar disorders (Lanoue et al., 2013). (M.Y.); the Kanae Foundation (W.K.); the Nakajima Foundation (W.K.); the Keio(F) In the absence of C1ql1-Bai3 signaling, the dominant CF cannot be strengthened enough to prevent the translocation of synapses formed by lessThus,
signaling mechanisms similar to the one mediated by Gijuku Fukuzawa Memorial Fund for the Advancement of Education anddominant CFs.
Research (W.K.); the Takeda Science Foundation (W.K. and M.Y.); the UK
C1ql1
Bai3 inthe cerebellum
likelyregulate
synaptic
compe(G) Weak
CFandsynapsestranslocatedto proximal
dendrites
escapefrom theelimination process at soma.
Medical Research Council (MRC) (A.R.A.); and a Wellcome Trust PhD student(H) Expression
of
C1ql1
or
Bai3
in
adult
C1ql1-nullor
PC-Bai3-nullPurkinje
cells
determines
single-winner CF by promoting the maturation of the dominant CF
tition and maintenance in various neuronal circuits essential for ship the(N.M.). Further support from the Wellcome Trust Core Award 090532/Z/
and eliminating
less dominant
brain functions.
In theCFs.
hippocampus, C1ql2 and C1ql3 are highly 09/Z is acknowledged. A.R.A. is an MRC Senior Research Fellow.
expressed in adult dentate gyrus granule cells, and their mossy
Accepted: November
26, 2014
fiber[120afferents
axon-typesynapsesdevelopment
controlin 120 maketrials, dynamic
n = 30]; pbranched< 0.001 by
Mann-Whitneyby binding
to its postsynaptic receptor Bai3 in
Published:
21, 2015 competition during development islike CFs.
Notably, mossy
fiberspikes
synapsesreportedly
displayPurkinje
U test).
Furthermore,dendritic
reportedly
conveycells.January
CF synapse
additionalsignals by structural
triggeringplasticity
a pause related
in firingtofollowing
the ofdivided into at least three stages: selection of functionally domilearning-induced
the precision
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to a level similar to that in WT mice. By contrast, the amplitude ofof the small GTPase Rac, a powerful regulator of actin cytoskel-

If this is the case, then what is the function of C1ql1-Bai3

CF territories, its deficiency was not sufficient to allow PFs to

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the largest CF-EPSCs remained constant after P7–P9 in C1ql1- etal organization. Indeed, Bai3 was shown to interact with Elmo1
Malhaire, F., Li, Y., Trinquet, E., Labesse, G., et al. (2008). Functioning of the
GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze innull mice (Figure 1H). These findings indicate that C1ql1 does notdimericin vitro(Lanoue
et al., 2013). Further studies are clearly warGABA(
humans. Neurology 81, 1378–1386.
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Cbln1 is essential
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Heller,E., Chait,
B.T., andN. (2009).
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areformed.
These
twoHeintz,
domains
are Proteomic
dynamically
cells,
involves
molecules
as Gaq,phospholi-Selimi,
aptickinje
integrity
and whichplasticity
in the cerebellum.
Nat.such
Neurosci.
8, 1534–1541.
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synapsetype: theparallelfiber/purkinje
cell synapse.
pase
Cb4,
proteinkinaseCg(PKCg)(Kano
andHashimoto,establishedandmaintainedbycompetition
betweenPFs and
Holtmaat, A., and Svoboda, K. (2009). Experience-dependent structural synBiol.(Cesa7, e83. and Strata, 2009; Watanabe and Kano, 2011). For
Uesakaet al., 2014;
and Kano,2011), andPLoSCFs
aptic2009;plasticity
in the mammalianbrain. WatanabeNat. Rev. Neurosci.
10, 647–658.
C., Manabe,T., Fukuda,S., Kawai,
Furutani, Y.,
example,when R.,
PFYamada,
synapses
are reduced
in J.,
Cbln1-null(Hirai
Sema7Aet al.,
2014).
In M.
mice
lackingof theseShimono,
Iijima,T., Miura,(UesakaE., Watanabe,M., and
Yuzaki,(2010).
Distinctone
expression
Tsutsui, K., Ikenaka, K., Hayashizaki, Y., and Sekiguchi, K. (2010).
mGluR1signaling
components,
Purkinjecells remain
et al., 2005) or GluD2-null (Hashimoto et al., 2001) mice, CFs
of C1q-likefamilymRNAs inpathway
mouse brain
and biochemical
characterizationofIdentification and characterization of nCLP2, a novel C1q family protein extheirinnervatedencoded proteins.
Eur. J. Neurosci.
31,in1606–1615.
by multiple
CFs, evenadulthood. Because mGluR1pressed
formin synapses
on distal dendrites of Purkinje cells. Conversely,
the central nervous system. J. Biochem. 147, 565–579.
Ito, M.
Cerebellar
circuitry
as ainputs,
neuronalinnervation
machine. Prog.
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mainlyby PF
ofNeurobiol.
Purkinje78,cells when CF synaptic activity was pharmacologically inhibited or IO
Stephenson, J.R., Purcell, R.H., and Hall, R.A. (2014). The BAI subfamily of
272–303.
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were
lesioned,
CF synapses
were
lostPharmacol.
and PFs formedby multiple CFs in GluD2-null or Cbln1-null mice is thoughtadhesion
GPCRs:
synaptic
regulation
and beyond.
TrendsSci.
Ito, M.
Error detection
and representationin theformation
olivo-cerebellar
systo (2013).
be causedby impaired
PF synapse(Watanabe
synapseson
proximalPurkinje
celldendrites(Cesa
and Strata,
35, 208–215.
tem.andFront.
Neural2011).
CircuitsHowever,
7, 1, http://dx.doi.org/10.3389/fncir.2013.00001.
2009;
and Kano,
PFs invaded
Kano,
unlike in C1ql1-null or PC-Bai3-nullStevens,
B.,Watanabe
Allen, N.J., Vazquez,
L.E., 2011).
Howell, Similarly,
G.R., Christopherson,
K.S., CF2+
Kakegawa,
W.,
Miyazaki,T.,Emi, K.,
Matsuda,K., Motohashi,
territories
in K.D.,
P/Q-type
CaA.K.,
channel-deficient
Purkinje
cells
mice, the numbers andheightsof K.,
theKohda,
CF terminals
are J.,
unaf-Nouri,N., Micheva,
Mehalow,
Huberman, A.D., Stafford,B., et al.
Mishina,M., inKawahara,
Watanabe,mice
M., and
Yuzaki, M. (2008).
The classicalcomplement
cascade mediates
CNS
eliminafected
mGluR1S.,knockout(Hashimoto
et al., Differential
2001). Simi-(2007).
(Miyazaki
et al.,
2004). Notably,
although
CFsynapse
synaptic
inputsregulation of synaptic plasticity and cerebellar motor learning by the
Cell 131,
1164–1178.
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larly, CF-EPSC amplitudes were also unaffected in Sema7Ation.were
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N., Uchigashima,M., Mikuni, T., Nakazawa,T., Nakao,
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S1EH.,and
knocked-down Purkinje cells (Uesaka et al., 2014), indicatingUesaka,Kano, M., and Hashimoto, K. (2009). Synapse elimination in the central nerAiba,
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Watanabe,
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PFs toPurkinjebind toGluD2(Matsudaal., 2010).
Because inGluD2
pressedin all CFs
at P0–P1(FigureIn addition,
whenOscillating
Kishore,U., Gaboriaud,C., Waters,P., Shrive,
A.K., 2D).
Greenhough,T.J., Reid,
neuronactivity
causing etinvoluntary
eye movementa2+clustering
is destabilized
by Ca
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was G.J.
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adult
WT mice,the CF-mutant
K.B.,C1ql1
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and thenumberof 25,
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synapses decreased24, 2440–2448.
2001), reduction of Ca2+ channel activities may be a prerequisite
for M.
PFs
to form
synapses
onproteins:the proximal
dendriticcytokines.
domain of(Figures
5 andS7). Similarly,
theM.,
numberCF terminals(2008).
Cbln andC1q family
new transneuronal
Lanoue,V., Usardi,A., Sigoillot,
S.M., Talleur,Iyer, K., ofMariani,J., Isope, P.,wasYuzaki,
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Vodjdani,G., Heintz,
N., and Selimi,(2013). The adhesion-GPCR
BAI3, a 1Cell.Purkinjecells.
reducedin C1ql1-null
andF.PC-Bai3-null
cerebella (Figures

Neuron85, 316–329,
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21, 2015ª2015
Elsevier
Inc. Inc.
329327
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January
21, 2015ª2015
Elsevier－ 87 －

represented
represented
as meanas mean± SEM.
± SEM.
Statistical
Statisticalanalyses
analyses
werewere
performed
performed
usingusing
the theC1qC1qFamilyFamilyProteins
Proteins
andandSynaptic
Synaptic
Functions
Functions
statistics
statistics
20122012
add-inadd-insoftwaresoftware(Social(SocialSurveySurveyResearchResearch
Information),
Information),
Based
Based
on theon thebehavioral
behavioral
phenotypes
phenotypes
of ataxicof ataxic
mutantmutantmicemiceExcelExceland significant
differences
differences
wereweredefineddefinedas *pas< *p0.05,< 0.05,**p <**p0.01,< 0.01,and ***pand <***p <
lackinglackingmGluR1,mGluR1,PKCg,
PKCg,
andandGluD2,
GluD2,
monoinnervationmonoinnervationof Purof Pur-and significant
0.001.
0.001.
kinjekinjecellscellsby CFsby CFswaswasthought
thought
to be
to required
be required
for motor
for motor
coordicoordination
nation(Chen(Chen
andandTonegawa,
Tonegawa,1997).
1997).
In contrast,
In contrast,
althoughalthoughACCESSIONACCESSIONNUMBERSNUMBERSC1ql1-null
C1ql1-nullandandPC-Bai3-null
PC-Bai3-nullPurkinje
Purkinjecellscells
remainremaininnervatedinnervatedby multiple
by multiple
CF CF
inputs,inputs,these
these
micemice
havehavenormalnormalgaitsgaits
without
withoutThe The
coordinates
coordinates
and andstructure
structure
factors
factors
for thefor reported
the reported
crystalcrystalstructure
structure
are aredepositedin theinProtein
the Protein
DataData
BankBank
(PDB)
(PDB)
under
under
PDBPDB
ID 4d7y.
ID 4d7y.
significant
significant
impairment
impairment
in beamin beamor rotor-rod
or rotor-rod
teststests(Figure(FigureS8; S8;deposited
Movie
Movie
S1).S1).
However,
However,
motor
motor
learning,
learning,
as measured
as measured
by hOKR,by hOKR,SUPPLEMENTAL
INFORMATIONINFORMATION
waswasseverely
severely
impaired
impairedin thesein these
micemice(Figure(Figure6). 6).
Recently,
Recently,SUPPLEMENTAL
mGluR1b-rescue
mGluR1b-rescue
mice,
mice,
in which
in whichthe thesplicesplicevariant
variant
of mGluR1
of mGluR1
Supplemental
Supplemental
Information
Information
includesincludesSupplemental
Supplemental
Experimental
Experimental
Procedures,
Procedures,is expressedis expressedin mGluR1-nullin mGluR1-nullPurkinje
Purkinjecells,
cells,
were
were
shown
shown
to to
eighteight
figures,figures,
and and
two twomoviesmoviesand andcan canbe foundbe foundwith withthis article
this article
onlineonlineat at
display
display
normalnormalmotor
motor
coordination,
coordination,eveneven
thoughthoughtheirtheir
Purkinje
Purkinjehttp://dx.doi.org/10.1016/j.neuron.2014.12.020.
http://dx.doi.org/10.1016/j.neuron.2014.12.020.
cellscells
were
were
innervatedinnervatedby multiple
by multiple
CFs.CFs.
These
These
micemice
showedshowedimpaired
impaired
LTDLTD
andandeyeblink
eyeblink
conditioning,
conditioning,anotheranother
formformof ofAUTHORAUTHORCONTRIBUTIONSCONTRIBUTIONS
cerebellum-dependent
cerebellum-dependent
motor
motor
learning
learning(Ohtani(Ohtaniet al.,
et al.,
2014).
2014).
designeddesigned
the project,
the project,carriedcarriedout theoutelectrophysiologicalthe electrophysiologicaland behavioral
and behavioral
Similarly,
Similarly,
a singlea single
injection
injectionof recombinantof recombinant
Cbln1
Cbln1
restored
restoredW.K.W.K.
works,
works,
analyzed
analyzed
the data,
the data,
and andwrotewrote
the paper.
the paper.
N.M.N.M.
and and
A.R.A.
A.R.A.
performed
performed
LTDLTD
andandeyeblink
eyeblink
conditioning
conditioningin adult
in adult
Cbln1-null
Cbln1-nullmicemice
without
without
structural
structuraland SPRand SPR
analyses
analysesand designed
and designed
mutantmutantconstructs.
constructs.
E.M.E.M.
performed
performed
significantly
significantly
affectingaffectingthe theCF CF
innervation
innervation
patternpatternor LTPor LTPat atthe anatomical
the anatomical
experiments
experiments
and andthe IUEthe approach.
IUE approach.
M.A.M.A.
and and
K.S. K.S.
designeddesigned
PF-Purkinje
PF-Purkinje
cell cell
synapses
synapses(Emi(Emiet al.,
et al.,
2013).
2013).
Furthermore,
Furthermore,and generated
and generated
mutantmutantmice.mice.
K.M.K.M.
performed
performed
the cellthe binding
cell binding
assays.
assays.
Y.H.T.
Y.H.T.
DCT7DCT7
GluD2
GluD2
-rescue-rescuemice,
mice,
in which
in which
GluD2
GluD2
lackinglackingseven
seven
C-termiC-termi-designed
designedand preparedand prepared
the microRNA
the microRNA
constructs.
constructs.
K.K.,K.K.,
A.T.,A.T.,
J.M.,J.M.,
and S.M.
and S.M.
designedand andpreparedprepared
the recombinant
the recombinantviruses.
viruses.
S.N. S.N.
supervised
supervised
the hOKR
the hOKR
nal nal
amino
amino
acids
acids
waswasexpressed
expressed
in GluD2-nullin GluD2-nullPurkinje
Purkinjecells,
cells,designed
experiment.
M.W.M.W.
generated
generatedessential
essential
antibodies.
antibodies.
M.Y.M.Y.
designeddesignedand superand superdisplayed
displayed
impaired
impaired
LTDLTD
andandeyeblink
eyeblink
conditioning
conditioning
despitedespiteexperiment.
the project
the project
and wrote
and wrote
the paper.
the paper.
morphologicallymorphologicallynormalnormalPF PF
andandCF CF
synapses
synapses(Kakegawa(Kakegawavisedvised
et al.,
et al.,
2008).
2008).
These
These
findings
findings
indicate
indicate
thatthat
LTD,LTD,ratherratherthanthanACKNOWLEDGMENTS
ACKNOWLEDGMENTS
monoinnervationmonoinnervationof Purkinjeof Purkinjecellscellsby CFs,by CFs,is likely
is likely
linkedlinkedto moto motor tor
learning
learning
in thein thecerebellum
cerebellum
andandindicate
indicate
thatthatC1ql1-Bai3
C1ql1-Bai3We thankWe thank
S. Narumi
S. Narumi
and S.
andOtsukaS. Otsuka
for their
for theirtechnical
technical
assistanceassistanceand S.
andMatS. Matand K.F.
and K.F.
TanakaTanakafor their
for their
usefulusefulcomments.
comments.
We also
We alsothankthankT. Miyazaki
T. Miyazaki
signaling
signaling
is required
is required
for normal
for normal
CF functions,
CF functions,suchsuch
as complex
as complexsudasuda
and
Konno
K. Konno
for their
for their
supportsupporton anatomical
on anatomical
experiments,
experiments,
H. Okado
H. Okado
for lentifor lentispikes
spikesandandCF-evoked
CF-evoked
afterhyperpolarization,
afterhyperpolarization,
which
which
are arenecesneces-and K.
virus
virus
production,
production,
H.
SakumaH.
Sakumaandand
T.
WatanabeT.
Watanabe(Zeiss)(Zeiss)
for
their
for
their
supportsupporton onsarysaryfor LTD.
for LTD.
superresolution
superresolution
microscopy
microscopy
analysis,analysis,and theandstaff
the staff
at Diamondat Diamondbeamlinebeamline
I04 I04
FamilyFamilymembers
membersof C1ql1
of C1ql1(C1ql2–C1ql4)(C1ql2–C1ql4)
are areexpressed
expressed
in infor synchrotron
for synchrotron
assistance.
assistance.
This This
workwork
was wassupported
supportedby a bygrant-in-aid
a grant-in-aid
for scifor scivarious
variousbrain
brain
regionsregions(Iijima(Iijima
et al.,
et al.,
2010).
2010).
Bai3Bai3
andandits related
its relatedentificentific
research
research
fromfrom
the Ministry
the Ministryof Education,of Education,
Culture,Culture,Sports,Sports,Science
Scienceand andmembers
members
Bai1Bai1
andandBai2Bai2
are arealsoalso
widelywidelyexpressed
expressed(Kee(Keeet al.,
et al.,Technology
Technologyof Japanof Japanand andthe Japan
the Japan
SocietySocietyfor thefor Promotion
the Promotionof Scienceof Science
2004)
2004)
andandhavehavebeen
been
implicatedimplicatedin psychiatricin psychiatricdisorders,disorders,suchsuch(W.K.(W.K.
and M.Y.);and M.Y.);
the CREST
the CREST
fromfrom
the Japan
the Japan
Science
Scienceand Technology
and Technology
AgencyAgency(M.Y.);
the Kanae
the Kanae
Foundation
Foundation(W.K.);(W.K.);
the Nakajima
the Nakajima
Foundation
Foundation(W.K.);(W.K.);
the Keio
the Keioas schizophrenia
as schizophrenia
andandbipolarbipolardisordersdisorders(Lanoue(Lanoue
et al.,
et al.,
2013).
2013).(M.Y.);
GijukuFukuzawa
Fukuzawa
Memorial
Memorial
FundFundfor thefor Advancement
the Advancementof Educationof Education
and andThus,
Thus,
signaling
signalingmechanisms
mechanisms
similarsimilarto theto theoneonemediated
mediatedby byGijuku
ResearchResearch(W.K.);(W.K.);
the Takeda
the Takeda
Science
ScienceFoundation
Foundation(W.K.(W.K.
and andM.Y.);M.Y.);
the UK
the UK
C1ql1
C1ql1
andandBai3Bai3in thein cerebellum
the cerebellum
likelylikelyregulate
regulate
synaptic
synaptic
compecompeMedical
MedicalResearchResearch
Council
Council(MRC)(MRC)(A.R.A.);(A.R.A.);and aandWellcomea Wellcome
TrustTrust
PhD PhD
studentstudenttition
titionandandmaintenancemaintenancein various
in various
neuronal
neuronal
circuits
circuits
essential
essential
for forship ship
(N.M.).
(N.M.).
Further
Further
supportsupportfromfrom
the Wellcome
the Wellcome
TrustTrust
CoreCore
Award
Award
090532/Z/
090532/Z/
brain
brain
functions.
functions.
In theIn hippocampus,
the hippocampus,
C1ql2
C1ql2
andand
C1ql3
C1ql3
are highly
are highly09/Z09/Z
is acknowledged.
is acknowledged.
A.R.A.
A.R.A.
is anisMRC
an MRC
SeniorSeniorResearchResearch
Fellow.
Fellow.
expressed
expressed
in adult
in adult
dentatedentategyrus
gyrus
granulegranulecells,
cells,
andandtheirtheir
mossy
mossy
Accepted:
NovemberNovember26, 2014
26, 2014
fiberfiber
afferents
afferents
make
makedynamic
dynamic
branched
branchedaxon-typeaxon-typesynapses
synapsesAccepted:
Published:
January
January
21, 2015
21, 2015
like like
CFs.CFs.
Notably,
Notably,
mossy
mossy
fiberfiber
synapses
synapsesreportedly
reportedly
display
displayPublished:learning-induced
learning-induced
structural
structural
plasticity
plasticity
relatedrelatedto theto theprecision
precisionof of
REFERENCESREFERENCES
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EXPERIMENTAL
EXPERIMENTAL
PROCEDURES
PROCEDURES

Bosman,Bosman,L.W.,L.W.,
Takechi,Takechi,H., Hartmann,
H., Hartmann,J., Eilers,J., Eilers,J., andJ., Konnerth,and Konnerth,A. (2008).
A. (2008).
All procedures
All procedures
relatedrelatedto animal
to animal
care careand treatment
and treatment
werewere
performed
performed
in accorin accorHomosynaptic
Homosynaptic
long-term
long-term
synaptic
synaptic
potentiation
potentiationof theof‘‘winner’’the ‘‘winner’’
climbing
climbing
fiber fiber
dance
dance
with withthe guidelines
the guidelines
set down
set down
by theby Animal
the Animal
Resource
Resource
Committee
Committee
of ofsynapsesynapsein developingin developing
Purkinje
Purkinjecells.cells.
J. Neurosci.
J. Neurosci.
28, 798–807.
28, 798–807.
Keio Keio
University.
University.
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the central
ner- ner-Aiba,Aiba,
A., Watanabe,A., Watanabe,M., M.,
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Kano,
Kano,
M. (2014).
M. (2014).
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semaphorin
semaphorin
vousvous
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19, 154–161.
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signaling
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regulates
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synapsesynapseelimination
eliminationin thein thedeveloping
developing
mouse
mouse
brain.
brain.
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1020–1023.
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H.J.,H.J.,
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K.Y.,K.Y.,
Choi,Choi,
K.C.,K.C.,
WonWonSong,
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T., Jung,
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Expressionof brain-specificof brain-specificangiogenesis
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Climbing
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fiberfiber
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3 (BAI3)
in normal
in normal
brainbrain
and andimplications
implications
for BAI3
for BAI3
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in ischemia-inducedcerebellar
cerebellar
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Oscillating
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involuntary
involuntary
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and andArlaud,Arlaud,G.J. G.J.
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January
21, 2015ª2015
Elsevier－ 89 －

AR

G

ARTICLE
Received 19 Sep 2015 | Accepted 30 Oct 2015 | Published 10 Dec 2015

DOI: 10.1038/ncomms10090

OPEN

A CDC42EP4/septin-based perisynaptic glial
scaffold facilitates glutamate clearance
Natsumi Ageta-Ishihara1, Maya Yamazaki2, Kohtarou Konno3, Hisako Nakayama4, Manabu Abe2, Kenji
Hashimoto5, Tomoki Nishioka6, Kozo Kaibuchi6, Satoko Hattori7, Tsuyoshi Miyakawa7,8, Kohichi Tanaka9,
Fathul Huda10, Hirokazu Hirai10, Kouichi Hashimoto4, Masahiko Watanabe3, Kenji Sakimura2& Makoto Kinoshita1

The small GTPase-effector proteins CDC42EP1-5/BORG1–5 interact reciprocally with CDC42
or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively
expressed in Bergmann glia and localizes beneath speciﬁc membrane domains enwrapping
dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers,
which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4  /  mice,
GLAST is dissociated from septins and is delocalized away from the parallel ﬁbre-Purkinje cell
synapses. The excitatory postsynaptic current exhibits a protracted decay time constant,reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (gDGG)
and excessive baseline inward current in response to a subthreshold dose of a nonselective
inhibitor of the glutamate transporters/EAAT1–5 (DL-TBOA). Insufﬁcient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects,
which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septinbased glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efﬁciencyof glutamate-buffering and clearance.

1 Division of Biological Sciences, Department of Molecular Biology, Nagoya University Graduate School of Science, Nagoya 464-8602, Japan. 2 Department of
Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan. 3 Department of Anatomy, Hokkaido University Graduate Schoolof Medicine, Sapporo 060-8638, Japan. 4 Department of Neurophysiology, Graduate School of Biomedical and Health Sciences, Hiroshima University,
Hiroshima 734-8551, Japan. 5 Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba 260-8670, Japan. 6 Department of
Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya 466-8560, Japan. 7 Division of Systems Medical Science, Institute forComprehensive Medical Science, Fujita Health University, Toyoake 470-1192, Japan. 8 Center for Genetic Analysis of Behavior, National Institute forPhysiological Sciences, Okazaki 444-8585, Japan. 9 Laboratory of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University,
Tokyo 113-8510, Japan. 10 Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511,
Japan. Correspondence and requests for materials should be addressed to M.K. (email: kinoshita.makoto@c.mbox.nagoya-u.ac.jp).

NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 90 －

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

lutamate transporters (Excitatory Amino Acid Transporter 1–5/EAAT1–5) are membrane-bound solute
carrier proteins that terminate glutamatergic neurotransmission and maintain the glutamate homeostasis by the
symport of extracellular glutamate and Na þ /H þ into the glial
and neuronal cytoplasm1. The extracellular glutamate
concentration surges to 160–250 mM near perisynaptic
domains of Bergmann glia in the cerebellum, which is
rapidly buffered and cleared by binding to and reuptake
through EAATs2,3. When the glutamate-buffering/clearance
capacity falls short of glutamate-release activities, glutamate
pervades the extracellular space. Excessive external glutamate
entails protracted activation of glutamate receptors in thenearest synaptic and extrasynaptic membranes, and in
the neighbouring synapses, and of glial responses. In the
cerebellar molecular layer, GLAST/EAAT1/SLC1A3 is highly
concentrated along perisynaptic Bergmann glial membranes4
and plays a major role in the clearance of glutamate released
from the parallel ﬁbres (PFs, from granule cells in the
cerebellar granule cell layer) and climbing ﬁbres (CFs, fromneurons in the inferior olivary nuclei) to Purkinje cells (PCs),
while GLT-1/EAAT2, the major glial transporter in theforebrain, and neuronal EAAT4 play relatively minor roles5.
Genetic loss of GLAST results in anomalous CF–PC
innervations and defective motor coordination/learning6,
attesting its central role in Bergmann glia-mediatedglutamate homeostasis in the cerebellum.
Despite the developmental and physiological signiﬁcance of
GLAST, and the linkage to human episodic ataxia andschizophrenia7–9, little is known about the post-translational
regulation. Physical interactions with beta-III spectrin/GTRAP41
and ARHGEF11/GTRAP48 were reported to facilitate theanchorage and activity of EAAT4 (ref. 10). However,physiological signiﬁcance of these ﬁndings from heterologous
cells, and whether these proteins modulate the localization
and/or activity of GLAST, remains unknown. Previous studies
demonstrated a physical interaction between GLAST and
septins (a family of polymerizing GTPases that constitute themembrane skeleton) in vitro and in heterologous cells, and their
partial co-localization in Bergmann glia11,12. However,hypothetical septin dependence of the perisynaptic targeting
and activity of GLAST has never been directly tested in vivo,
partly because of the redundancy among the septin family (seeDiscussion).
Another line of biochemical and cell biological studies showedthat a family of CRIB-domain proteins CDC42EPs/BORGs binds
to septin hetero-oligomers or CDC42 (a signalling small GTPasethat controls cytoskeleton and cell morphogenesis) in a mutually
exclusive manner. The major determinant of the binding
preference of CDC42EPs is the status of CDC42-boundnucleotide; GTP-CDC424septins4GDP-CDC42 (refs 13–15).
However, again, in vivo relevance of the hypothetical CDC42CDC42EP-septin pathway remains unclear.
Given the above background, genetic deletion of the dominant
CDC42EP species in Bergmann glia is a rational approach toaddress the two open issues: physiological roles of the CDC42CDC42EP-septin pathway and of the septin–GLAST interactionin the brain. We ﬁnd robust co-expression and co-localization of
CDC42EP4 and septins in Bergmann glia, generate CDC42EP4null mice and conduct biochemical, ﬁne morphological,
electrophysiological, pharmacological and behavioural analyses.
The unique and systematic approach reveals the requirementof CDC42EP4 in Bergmann glia for the septin-mediatedperisynaptic localization of GLAST, and for the efﬁcientbuffering and clearance of glutamate from around synapses
towards PCs.
2

Results
Selective expression of CDC42EP4 in Bergmann glia. We
conducted immunoblot (IB) for the expression proﬁling of the
CDC42EP4 protein in the whole brain, two brain subregions(cerebellum and hippocampus) and seven non-neural tissues
from adult C57BL/6N mice (Fig. 1a). The major band of B39
kDa, which ﬁts the calculated molecular mass of 37,980, was themost abundant in the cerebellum (Fig. 1b and Supplementary
Fig. 15) as predicted from public gene expression databases
including National Center for Biotechnology Information.
Fluorescence in situ hybridization (FISH) for Cdc42ep4 mRNA
highlighted the PC layer in the cerebellum (Fig. 1c, left), which isconsistent with the data in the Allen Mouse Brain Atlas#71723875 (Allen Institute for Brain Science). Double-label FISH
showed complementary distribution of mRNAs for CDC42EP4
and calbindin (PC marker) and overlap between mRNAs for
CDC42EP4 and GLAST (Bergmann glia marker; Fig. 1c, right),
demonstrating highly Bergmann glia-selective expression of theCdc42ep4 gene.
Clustering of CDC42EP4 in perisynaptic glial processes. Consistently, double-label immunoﬂuorescence (IF) for CDC42EP4
and calbindin, respectively, highlighted Bergmann glia and PCs ina mutually exclusive manner (Fig. 1d). At a higher magniﬁcation
with a lower gain level, CDC42EP4 immunoreactivity appeared as‘hotspots’, which were interspersed along PC dendrites andtightly apposed to dendritic spines (Fig. 1d, right). CDC42EP4
was far more concentrated in the processes of Bergmann glia thanin the cell bodies (Fig. 1e).
To analyse the subcellular distribution of CDC42EP4 at higher
resolutions, we conducted silver-enhanced immunoelectron
microscopy. Bergmann glial processes thoroughly ensheatheddendritic spines of PCs, except for synaptic contact sites withaxon terminals. Gold particles for CDC42EP4 were commonly
found as submembranous clusters in Bergmann glial processes
that surrounded dendritic spines, particularly around the spine
neck (Fig. 1f). Quantitative analysis demonstrated a gradient of
CDC42EP4 from the base (neck) to the apex (head) of spines(Fig. 1g). These data indicate that CDC42EP4 in Bergmann glia is
localized beneath speciﬁc membrane domains that are facing
dendritic spines of PCs. The characteristic distribution of
CDC42EP4 is reminiscent of those of septin subunits, SEPT4/
H5 (ref. 16), SEPT7/hCDC10 (ref. 16) and SEPT2/Nedd5 (ref.
11), whose physiological role has been unknown.
Generation of Cdc42ep4-ﬂoxed and -null mice. To explore
physiological roles of Cdc42ep4 using a reverse genetic approach,
we generated a line of C57BL/6N mice harbouring a ﬂoxed allele,
which was subsequently converted to a null allele by crossing withanother line that ubiquitously expresses Cre recombinase(Fig. 2a–c). Analyses of Cdc42ep4 � / � offspring demonstrated
the total absence of CDC42EP4 in IB and IF (Fig. 2d,e andSupplementary Fig. 15). For the following analyses, weconsistently compared Cdc42ep4 � / � (knockout; KO) andCdc42ep4ﬂ/ﬂ (wild type; WT) male littermates generated from
Cdc42ep4ﬂ/ � (heterozygous) parents. There was no difference bygenotype in the gross appearance, body weight and fertility(Supplementary Fig. 1).
Normal architecture of the Cdc42ep4 � / � cerebellum. We
conducted IF with speciﬁc markers for the four major neuronaland glial components (PFs, CFs, PCs and Bergmann glia), which
did not show obvious anomaly in the cerebellar cortex of KO
mice (Figs 3a and 5b). Transmission electron microscopy (TEM)
images showed no recognizable ultrastructural defects in the

NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 91 －ARTICLE

CDC42EP1

CRIB

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Liver > Placenta
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214

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Whole brain
Cerebellum
Hippocampus
Heart
Lung
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SpleenIntestine
KidneyOvary

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Leukocytes > Uterus

254

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Cerebellum > Lung

349
150

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Figure 1 | Bergmann glia-selective expression and unique perisynaptic localization of CDC42EP4. (a) The CDC42EP/BORG family and gene expression
pattern in the mouse. Each CDC42EP contains a set of basic-CRIB-BD domains. Anti-CDC42EP4 antibody was raised against a region between the CRIB
and BD3 (septin-binding) domains. The numbers denote amino-acid residues. CDC42EP1/2/3/4/5, respectively, corresponds to BORG5/1/2/4/3.
(b) Lysates from adult mouse tissues (50 mg total protein per lane) were immunoblotted for CDC42EP4 and were reprobed for b-actin as a loading control.
The major B39-kDa band was most abundant in the cerebellum. (c) FISH for Cdc42ep4 mRNA in the adult mouse cerebellum. (Left) Labels for Cdc42ep4(green) highlighted the PC layer in a parasagittal section. (Top) Double-label FISH for mRNAs for Cdc42ep4 and a PC marker calbindin (red). Cdc42ep4
mRNA was excluded from PCs (*). (Bottom) Double-label FISH for Cdc42ep4 and Glast (red) mRNAs, and TOTO-3 stain for DNA (blue). The two mRNAsignals overlapped in all (n ¼ 115) Bergmann glial cells identiﬁed in a representative section. Scale bars, 1 mm and 20 mm. (d) (Left and centre) Double-label
IF for CDC42EP4 (red) and calbindin (green) in the cerebellar cortex. The diffuse granular signals for CDC42EP4 distributed throughout the molecular layer
and in the PC layer, which were excluded from PCs and the granule cell layer. (Right) At a higher magniﬁcation, CDC42EP4-positive puncta wereinterspersed and aligned along PC dendrites. Scale bars, 20 and 5 mm. (e) Double-label IF for CDC42EP4 (red) and a Bergmann glial marker Phgdh (green).
The limited overlap in Bergmann glial cell bodies (arrowheads) indicated selective localization of CDC42EP4 in glial processes. Scale bar, 5 mm. (f) (Left)
Immunoelectron microscopy image for CDC42EP4 in the molecular layer. Gold particles for CDC42EP4 were found as submembranous clusters in terminal
processes of Bergmann glia (tinted), each surrounding a dendritic spine (Sp) of a PC. PF, parallel ﬁbre terminal. Scale bar, 200 nm. (Right) Quantiﬁcation ofglia-selective localization of CDC42EP4. Data represented as mean±s.e.m. (g) Histogram showing a gradient of CDC42EP4 signals relative to thegeometry of dendritic spines of PCs; higher in regions facing spine base (arrows in f) than in regions facing the spine head.

molecular layer (Fig. 3b), including the lengths of the postsynaptic density (PSD) in PF–PC synapses (Fig. 3c). Four major
proteins at glutamatergic synapses, GluA1, GluA2, GluA4 andPSD-95, did not show quantitative differences by genotype(Fig. 3d and Supplementary Fig. 15). These data indicate that
CDC42EP4 is dispensable for the morphological development of

the major neuronal and glial components, and synapse architecture in the cerebellar cortex.
Septin oligomers as the major binding partners of CDC42EP4.
For unbiased identiﬁcation of physiological binding partners of
CDC42EP4 in Bergmann glia, we conducted proteomic analysis

NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 92 －

3

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Cdc42ep4 genome(Cdc42ep4+)
Targeting vector

S

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Figure 2 | Generation of Cdc42ep4 � / � mice and morphology of the major neuronal components. (a) The KO strategy of the Cdc42ep4 gene. A
schematic diagram showing the wild-type (Cdc42ep4 þ ), ﬂoxed (Cdc42ep4ﬂ) and null (Cdc42ep4 � ) alleles, and the targeting vector. Note that Cremediated loxP recombination leaves no coding exon. The restriction sites for Sac I (S), EcoR V (E) and Hinc II (H), three probes (grey bars) used forSouthern blot analysis and three PCR primer sites are indicated. Signs; coding region (grey box), untranslated region (open box), loxP (black triangle), frt(open half-circle), neomycin resistance cassette (Neo), diphtheria toxin A-chain cassette (DT). (b) Southern blot analysis. Genomic DNAs puriﬁed from
WT and the chimera (Cdc42ep4 þ / þ ;Cdc42ep4ﬂ/ þ ) mice were digested with the restriction enzymes and hybridized with the probes as indicated.
The band patterns, as seen in preceding Southern blot analysis of ES cell clones, reconﬁrmed successful homologous recombination of the clone. See
Methods for details. (c) PCR genotyping. Two sets of primers discriminated genomic DNAs from Cdc42ep4 þ / þ , Cdc42ep4ﬂ/ﬂ (WT), Cdc42ep4ﬂ/ � andCdc42ep4 � / � (KO) mice. (d) Expression and extractability of CDC42EP4 in the adult mouse cerebellum. The pellet/supernatant assay showed that
CDC42EP4 is partitioned mostly to the detergent-extractable, supernatant (s) fraction but not to the inextractable, pellet (p) fraction. CDC42EP4 wasabsent from KO tissues. a-Tubulin was used as a loading control. (e) IF for CDC42EP4 on parasagittal brain sections of adult male littermate WT and KO
mice. The molecular layer of the WT cerebellum (Cb) was intensely labelled for CDC42EP4, which was absent from the KO brain. The faint, diffuse labellingof the entire brain is attributed to astrocytes. These results are consistent with the immunoblot data (Figs 1b and 2d) and warrant the speciﬁcity(high signal-to-noise ratio) of the antibody. Scale bar, 1 mm. UTR, untranslated repeat.

sion
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of cerebellar lysates. Immunoafﬁnity chromatography for
CDC42EP4 followed by mass spectrometry identiﬁed CDC42EP4,nine septin subunits (SEPT2/3/4/5/6/7/8/10/11) and a few othercytoskeletal proteins including Myosin-10 (nonmuscle myosin
heavy chain IIB) and a-II/b-II spectrins (Table 1). The minimal
background noises in the three negative control experiments (forexample, anti-CDC42EP4 IgG captured no peptides from KO
samples) reconﬁrmed the absence of CDC42EP4 in the KO
cerebellum and the antibody speciﬁcity. Intriguingly, however,
CDC42 and Tc10/RhoQ (another small GTPase that can bind to
CDC42EPs in vitro13) were not detected (Table 1 andSupplementary Fig. 13). Thus, the major physiological binding
partners of CDC42EP4 in the adult mouse cerebellum are notsmall GTPases, but hetero-oligomers of septins.
Physiological network of CDC42EP4, septins and GLAST.
Previous studies with recombinant proteins separately demonstrated direct interactions between CDC42EP5/BORG3 and septin
hetero-oligomers (for example, SEPT6/7 and SEPT2/6/7, but not
individual subunits)14,15, and between SEPT2 and GLAST11.
However, higher-order molecular network composed of
4

CDC42EPs, septin hetero-oligomers and GLAST has never beentested. We addressed this with their co-immunoprecipitation (co-IP)
from cerebellar lysate (Fig. 4a and Supplementary Figs 13–15), anddouble-label IF that showed their partial overlap in Bergmann glia
(Fig. 4e). These data, along with the previous studies and our
proteomic and IF ﬁndings, indicate physiological molecular networkthat contains CDC42EP4, septin hetero-oligomers and a subset
of GLAST.
Loss of CDC42EP4 diminishes septin–GLAST interaction. To
validate the signiﬁcance of CDC42EP4 in the molecular network,
we compared the status with or without CDC42EP4. Pellet/
supernatant assay of cerebellar lysates showed that the amountand solubility of SEPT7, SEPT4 and GLAST were unaffected by
the loss of CDC42EP4 (Fig. 4b–d and Supplementary Fig. 15).
Their distribution patterns assessed using IF showed no recognizable difference by genotype (Fig. 5b). Intriguingly, however, aco-IP/IB assay revealed a signiﬁcant reduction (D50%) of GLAST
that was pulled down with SEPT4 (a Bergmann glia-selective
septin subunit) from the Cdc42ep4 � / � cerebellum, when theinteractions with two other major septin subunits in Bergmann

NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 93 －ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Cdc42ep4fI/fI (WT)

Cdc42ep4–/– (KO)

AR

Tab

Ide
ML

CD
SEP
SEP
SEP
SEP
SEP
a-I
SEP
My
SEP
SEP
SEP
b-I

PCL

VGluT1/Car8

ML

PCL

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Cdc42ep4fI/fI (WT)

Cdc42ep4–/– (KO)

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lysa
num

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PC

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PF

scar
part
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sign
sign
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con
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syn

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PC

PC

PF

WT KO

NS

60
40

WT
KO

20
0

0

0.16 0.32 0.48 0.64 0.80
PSD length (μm)

WT KO

GluA1

GluA2

GluA4

kDa
100

PSD-95

PSD-95

PSD-95

100

2

2

2
NS
1
0

WT KO

NS
1
0

WT KO

GluA4
/PSD-95

80

PC

WT KO

GluA2
/PSD-95

100

GluA1/PSD-95

Cumulative frequency (%)

PF

NS
1
0

WT KO

Figure 3 | Morphological analysis of the neuronal and glial components in Cdc42ep4ﬂ/ﬂ and Cdc42ep4 � / � cerebellar cortices. (a) Double-label IF of
WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel ﬁbre (that is, granule cell) marker VGluT1 (top, green) or a climbing ﬁbre
marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal componentsof KO-derived samples. Scale bar, 20 mm. (b) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructuraldifference was found between the genotypes. PF, parallel ﬁbre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted.
Scale bar, 200 nm. (c) Cumulative histogram of PSD length of the PF–PC synapses, showing no signiﬁcant difference between the genotypes (n ¼ 92
synapses from two littermates for each genotype, NS, P40.05 by Kolmogorov–Smirnov test). (d) Quantitative immunoblot of WT and KO cerebellar PSD
fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no signiﬁcant quantitative difference
by genotype (n ¼ 3, NS, P40.05 by t-test).

glia (SEPT2 and SEPT7) were unaffected (Fig. 5a andSupplementary Fig. 15). The signiﬁcant dissociation of GLAST
from septin hetero-oligomers by the loss of CDC42EP4 indicatesa role for CDC42EP4 as a stabilizer and/or an adapter for the
association between GLAST and septin hetero-oligomers.
GLAST delocalizes away from perisynapse without CDC42EP4.
Previous ﬂuorescence recovery after photobleaching assay showedthat septin depletion via RNA interference augmented diffusional
mobility of green ﬂuorescent protein-tagged GLAST on theplasma membrane, while septin ﬁlament stabilization gave theopposite effect, indicating a role for septins as submembranous
scaffold and/or diffusion barrier for GLAST12. To assess whether
the dissociation from septins by the loss of CDC42EP4 could alter
the distribution of GLAST, we conducted quantitative mappingof GLAST in Bergmann glia by postembedding immunoelectron microscopy technique (Fig. 5c–e). As expected, gold
particles for GLAST were distributed along Bergmann glia
membrane enwrapping pre- and postsynaptic elementsof PF–PC synapses, whereas neuronal membranes were

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5

Cdc
To e
from
mic
trop
cere
mul
exci
tage
(n ¼
larg
dua
the
betw
CD
PC
(tha
wer
indi
are
G
extr
GLA
in K
PFdela
mol
faci
the
stud
(AM
syna
6

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Table 1 | Proteomic analysis for the binding partners of CDC42EP4 in the cerebellum.
Identiﬁed proteins (69)

Peptide number
KO IgG

CDC42EP4
SEPT7
SEPT4
SEPT11
SEPT5
SEPT2a-II spectrin
SEPT3
Myosin-10
SEPT8
SEPT10SEPT6b-II spectrin

KO ep4

Sequence coverage (%)

WT IgGWT ep4
20
21
19
14
12
11
18
7
10
6
5
5
4

1
1

8
2

1

1

KO IgG

KO ep4WT IgG
2.1
1.9

4.7
1.3

0.81

0.8

WT ep4
63
45
43
34
42
47
9.1
22
6.7
24
16
26
2.8

List of proteins puriﬁed from WT cerebellar lysate with CDC42EP4-immunoafﬁnity column and identiﬁed LC-MS/MS analysis (rightmost columns). The numbers denote the count of peptides assigned toeach protein (left) and the sequence coverage (right). The blank columns are zero. The speciﬁcity of the counts is corroborated by few false-positive counts in the controls (three left columns, that is, KOlysate captured with nonimmune IgG or anti-CDC42EP4 antibody, and WT lysate captured with nonimmune IgG). Another co-IP method used for the immunoblot analyses detected GLAST (peptide
number ¼ 2, sequence coverage ¼ 6.45), but not CDC42. Co-IP, Co-immunoprecipitation; KO, knockout; WT, wild type.

scarcely labelled. The distribution and density of immunogold
particles were comparable between WT and KO mice (Fig. 5d).
Intriguingly, however, the distance from perisynaptic GLAST
signals to the nearest PSD edge (arrowheads, Fig. 5c) was
signiﬁcantly longer in KO mice than that in WT mice (Fig. 5e).
Together, the biochemical and ﬁne morphometric dataconsistently indicate that CDC42EP4 in Bergmann glia isrequired for GLAST to interact with septins and to localize
along perisynaptic membrane domains enwrapping PF–PC
synapses.

IF of
ﬁbre
ents

nted.
92
PSD
ence

the
ous
ther
alter
ping
nogold
glia
ents
were
5

Cdc42ep4 � / � mice exhibit insufﬁcient glutamate clearance.
To explore whether the anomalies of GLAST (that is, dissociationfrom septins and delocalization from synapses) in Cdc42ep4 � / �
mice affect glutamatergic neurotransmissions, we assessed electrophysiological properties of the major glutamatergic synapses incerebellar slices. We ﬁrst examined the CF–PC synapse by stimulating CFs in the granule cell layer and recording the evoked
excitatory postsynaptic currents (EPSCs) with a whole-cell voltage clamp conﬁguration (Fig. 6a). Most PCs in 7-week-old WT(n ¼ 16/18, 89%) and KO (n ¼ 17/21, 81%) mice elicited a single
large EPSC in an all-or-none manner as stimulus intensity gradually increased (Fig. 6a, left). The number of steps, which reﬂects
the number of CFs innervating a given PC17,18, was comparable
between WT and KO mice (Fig. 6a, right), indicating that
CDC42EP4 is dispensable for the postnatal establishment of CF–
PC monoinnervation. The major parameters of CF-EPSC kinetics(that is, amplitude, 10–90% rise time and decay time constant)were also comparable (Supplementary Table 1). These data
indicate that properties of the CF-mediated synaptic transmission
are normal in young adult KO mice.
Given the major role for GLAST in glutamate clearance fromextracellular space around PF–PC synapses5, the delocalization of
GLAST away from PSD (Fig. 5e) might affect PF–EPSC kinetics
in KO mice. To test this, we measured the decay time constant of
PF-EPSC, which is known to protract as glutamate reuptake
delays19–22. The PF-EPSCs elicited by the stimulation in themolecular layer of KO mice showed normal paired-pulse
facilitation (Fig. 6b and Supplementary Table 1), indicating thatthe presynaptic functions were largely unaffected. As previous
studies showed that desensitization of the AMPA receptors(AMPARs) masks the effects of GLAST insufﬁciency on PF–PC
synapses20, we also employed cyclothiazide (CTZ) to block6

desensitization of AMPARs. While bath application of CTZ
prolonged the decay time constant of PF-EPSCs in both
genotypes, it was signiﬁcantly longer in KO than in WT(P ¼ 0.010, Fig. 6c). These data suggest a recognizable delay inthe glutamate clearance from PF–PC synapses in KO mice.
To corroborate the above ﬁndings, we compared glutamate
transients in PF-PC synapses using a low-afﬁnity competitive
inhibitor of the AMPARs, g-D-glutamylglycine (gDGG).
gDGG is a rapidly unbinding competitive antagonist that isreadily displaced from AMPARs by released glutamate23.
Therefore, the degree of inhibition of the EPSC roughly
reﬂects the relative size and kinetics of the glutamate transientin the synaptic cleft. Bath application of 1 mM gDGG inhibited
the PF-EPSC amplitude to B50% of the basal level in WT, while
only to B80% in KO, suggesting that the size of glutamate
transient at PF–PC synapses was signiﬁcantly higher in KO
than in WT (WT versus KO, 51.5±2.2/79.1±4.2%, n ¼ 7,
Po0.001, Fig. 6d).
The above ﬁndings, in conjunction with the molecular-levelanomalies, suggest that PF–PC synapses in KO mice havehotspots around active zones with subnormal glutamate-buffering
capacity. Since the density of AMPARs is the highest in
postsynaptic active zones, we hypothesized that KO mice aresensitive to subthreshold doses of glutamate clearance inhibitors.
To test this, we applied 50 mM DL-TBOA, which nonselectively
inhibits glutamate transporters (EAAT1–5) but only partially as
the half-maximal inhibitory concentration for EAAT1/GLAST is
70 mM. The treatment with the low-dose DL-TBOA in addition to
CTZ elicited a signiﬁcantly larger baseline inward current in KO
PCs (WT versus KO, 310±26/864±185 pA, n ¼ 11/12, Po0.001,
Fig. 6e, trace 3). The aberrant baseline inward current of KO mice
was abolished by additional application of an AMPAR antagonist,
NBQX (from 895±260 to 367±84 pA, n ¼ 7, P ¼ 0.016, Fig. 6e,trace 4). Thus, the large baseline inward current in KO PCs isgenerated by AMPARs exposed to excessive external glutamate.
A previous study showed that 200 mM DL-TBOA augmented
AMPAR-mediated inward current of PCs in WT mice19, while
KO PCs were sensitive enough to react to 50 mM DL-TBOA.
These results further support the compromised glutamatebuffering/clearance in KO mice, which may be due to thedelocalization of GLAST from PF–PC synapse active zones(Fig. 5e), and/or their dissociation from the perisynaptic clusters
of septins and CDC42EP4 (Fig. 5a).

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SEPT7

50

SEPT2

50

GAPDH

37

Sup

GLAST
/GAPDH

37

GLAST

SEPT7

EP4
0
1

NS

1
0
2

NS

1
0

KO

Sup

Pellet

1
0

WT

KO

Sup

WT

KO

SEPT4/α-Tubulin

SEPT7/α-Tubulin

NS

Pellet
kDa

GLAST
50
α-Tubulin

50

50

2
NS

NS

1

0

KO WT KOlgG EP4 EP4WT KO WT KO

37
α-Tubulin

NS

WT
lgG

50

50

2

***
***

kDa

SEPT4

α-Tubulin

***

Sup

WT KO WT KO

kDa
50

***
***

0

WT

*

*

***

0
1

NS

*

*

*

0
1

1
0
2

**

SEPT4

NS

0
2

PelletWT KO WT KO

0
1

1

***
***

GLAST/α-Tubulin

50
37

0
2

***

SEPT7

SEPT4

***

1

AR

lP:αEP4

1

SEPT2

25
75

Input

2

GLAST

37

SEPT4
/GAPDH

CDC42
EP4

SEPT7
/GAPDH

kDa
50

SEPT2
/GADH

KO

EP4
WT

lP:αEP4
lgG
WT
KO

KO

WT

Input

EP4
/GAPDH

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

WT

Pellet

KO

Sup

WT

KO

2

NS

NS

1

0

Pellet

WT

KO

Sup

WT

KO

Pellet

GLAST
CDC42EP4

GLAST
CDC42EP4

CDC42EP4

CDC42EP4

Figure 4 | Biochemical analysis of binding partners of CDC42EP4 in Cdc42ep4ﬂ/ﬂ and Cdc42ep4 � / � cerebella. (a) Co-IP/IB assay of CDC42EP4 withrepresentative septin subunits and GLAST from WT and KO cerebellar lysates. (Input) IB for SEPT4, SEPT7, SEPT2 and GLAST, respectively, detected aquadruplet of 54, 52, 48 and 44 kDa, a doublet of 51 and 48 kDa, a single 42 kDa band and a broad 55 kDa band in the cerebellar lysate. (IP) AntiCDC42EP4 antibody pulled down SEPT4, SEPT7, SEPT2 and GLAST only from WT cerebellar lysate. The graphs show densitometric quantiﬁcation of theyield (n ¼ 3, ***Po0.001, **Po0.01, *Po0.05, NS, P40.05 by one-way ANOVA with post hoc Tukey test). (Note: the extraction condition including thelysis buffer composition was optimized to detect GLAST, which was distinct from the one used mainly for the proteomic analysis (Table 1). See Methods.)(b–d) Pellet/supernatant assay results on the quantity and extractability of SEPT7, SEPT4 and GLAST in WT and KO cerebella. There was no signiﬁcantdifference in their amount and partitioning by genotype (n ¼ 3, NS, P40.05 by t-test). The same membranes were reprobed for a-tubulin as a loading
control, which was used for normalization. (e) Double-label IF for GLAST (green) and CDC42EP4 (red) in WT cerebellar cortex showing their partial
co-localization in Bergmann glial processes. Scale bars, 20 and 5 mm.
NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 96 －

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Figu
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amo
sept
(b)
reco
and
distr
glia
467
8 pa
edge
syna
by K
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IP:αSEPT4

0.5
0

WT

KO

1
0.5
0

*
IP:αSEPT4
GLAST/SEPT4

37
50

1

1.5

WT KO

GLAST/Car8

1
0.5
0

WT KO

ML

Cdc42ep4–/– (KO)

50

SEPT2
GLAST

Cdc42ep4 f/fl (WT)

SEPT7

1.5

IP:αSEPT4
SEPT2/SEPT4

50

SEPT4

NS

NS

kDa
IP:αSEPT4
SEPT7/SEPT4

WT
KO
WT
KO

lgG SEPT4

PCL

Cdc42ep4 –/– (KO)Cdc42ep4 f/fl (WT)

PF

PF

PC

PC

Cumulative frequency (%)

Density of immunogold(particles per μm)

NS
25

WT

20

KO

15
10
5

NS

0
Glialmembrane

with
ed a
f the
g the
ods.)
cant
ding
al

7

Postsynapticmembrane

100
80
60
40

***
WT

20
0

KO
0

0.1

0.2

0.30

Distance fromnearest PSD edge (μm)

Figure 5 | Loss of CDC42EP4 dissociates the GLAST–septin interaction and delocalizes GLAST away from synapses. (a) Quantitative co-IP/IB assay
between SEPT4 and SEPT7, SEPT2 or GLAST in WT and KO cerebellar lysates. While the interaction among the septin subunits did not differ, the relative
amount of GLAST pulled down with SEPT4 from KO lysate was signiﬁcantly less than that from WT (n ¼ 3, *Po0.05, NS, P40.05 by t-test), indicating that
septin–GLAST interaction depends on CDC42EP4. See Fig. 4a–d for the comparable amount and solubility of these proteins in WT and KO cerebella.
(b) Double-label IF for GLAST (green) and a Purkinje cell marker Car8 (red) in WT and KO cerebellar cortices. Genetic loss of CDC42EP4 caused norecognizable difference in the distribution of GLAST up to the resolution. Scale bars, 20 and 1 mm. (c) Immunoelectron microscopy images for GLAST in WT
and KO molecular layers. PF, parallel ﬁbre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. The pattern of GLAST
distribution appears comparable to that of a previous study4. Scale bar, 200 nm. (d) Quantitative analysis of immunoelectron microscopy data. Bergmannglia selectivity and labelling density of GLAST were comparable between WT and KO mice: Bergmann glia; 22.4±1.3/21.1±1.2 particles per mm (n ¼ 519/
467 particles from two littermate pairs, NS, P40.05 by Mann–Whitney U-test). Postsynaptic membrane; 0.50±0.21/0.68±0.24 particles per mm (n ¼ 5/
8 particles, P ¼ 0.68 by Mann–Whitney U-test; cf. Figs 4d and 5a–c). (e) Cumulative histogram of the distance of GLAST measured from the nearest PSD
edge (for example, arrowheads in c). A signiﬁcant right shift of the curve for KO mice demonstrates delocalization of GLAST away from PSDs of PF–PC
synapses (median, WT ¼ 0.27 mm, KO ¼ 0.31 mm from the nearest edge of PSD; n ¼ 22/19 synapses from two littermates for each genotype, ***Po0.001
by Kolmogorov–Smirnov test).
8

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WT
KO

Cdc42ep4–/– (KO)

40

KO

20

WT
KO

Decay time constant (ms)

KO
10

1.4

WT
KO

1.2
Baseline inward
current (nA)

2

3

4

KO

WT

ControlγDGG
Wash out

CTZ

Glu
Gln
Gly
GA
L-S
D-S
5-H
5-H
NE
MH
HV
DA
DO

0.4

100

*

Control

Ne

0.8

WT

**

WT
1

1.2

0

20

0

CTZ

1.6

1
2
Number of steps

30

Tab
tra
and

NS

2.0

60

WT

Control

WT

80

0

KO

NS

Paired pulse ratio

Number of cells (%)

100

Amplitude (% of control)

Cdc42ep4 fl/fl (WT)

KO

***

80
60

The
tissu

40
20
0

WT

KO

*
3

1.0
0.8
***
2

0.6
0.4

* 4

***

1

0.2
1

2

0
CTZ
TBOA
NBQX

3

–
–
–

+
–
–

+
+
–

+
+
–

+
+
+

Figure 6 | Cdc42ep4 � / � mice exhibit insufﬁcient glutamate-buffering/clearance capacity. (a; Left) Sample traces of CF-EPSCs. Two to three traces
were superimposed. Scale bars, 10 ms and 500 pA. (Right) Summary histogram showing the number of CF-EPSC steps (n ¼ 21/18, P ¼ 0.513 by Mann–
Whitney U-test). Holding potential was � 10 mV in a and � 70 mV in b–e. (b; Left) PF-EPSCs in response to paired stimuli at 50 ms intervals. Scale bars,
10 ms and 100 pA. (Right) Summary graph of the paired-pulse ratio (n ¼ 22/19, P ¼ 0.824 by Mann–Whitney U-test). (c; Left) Sample traces of PF-EPSCs
with (grey) or without (black) 100 mM CTZ in a WT and a KO mice. Scale bars, 25 ms and 50 pA. (Right) The decay time constant of PF-EPSCs. Although
CTZ prolonged the decay time constant both in WT and KO (n ¼ 11/17, WT, from 9.3±0.8 to 17.9±1.8 ms, ***Po0.001; KO, from 11.6±0.8 to
22.6±1.3 ms; ***Po0.001), the effect was signiﬁcantly larger in KO than in WT (**P ¼ 0.006 by two-way ANOVA with post hoc Tukey test), which resulted
in more protracted postsynaptic response in KO PCs (*P ¼ 0.010). (d) (Left) PF-EPSCs before (black), in the presence of 1 mM gDGG (grey), and after
washout (dashed). Scale bars, 10 ms and 200 pA. (Right) Summary histogram showing the effects of gDGG. KO PF-EPSCs were signiﬁcantly more
insensitive to gDGG than WT ones (n ¼ 7/7, ***Po0.001 by Mann–Whitney U-test). (e; Left) Sample traces of PF-EPSCs in a WT and a KO mice in control
ACSF (1), in the presence of CTZ (2), CTZ plus 50 mM TBOA (3), CTZ, TBOA plus 10 mM NBQX (4). Grey and dashed lines, respectively, indicate the zero
offset level and baseline holding current level in the control ACSF. Scale bars, 200 ms and 100 pA. (Right) The summary graph showing the holding current.
The effects of CTZ plus TBOA was signiﬁcantly larger on KO than on WT (n ¼ 11/12, ***Po0.001 by two-way repeated measures ANOVA with post hoc
Tukey test). (Two right plots) Additional application of NBQX signiﬁcantly diminished the inward current by TBOA (n ¼ 7, *P ¼ 0.016 by Wilcoxon signedrank test).

In addition, the content of glutamate, glutamine and relatedamino acids and monoamines in cerebellar tissues from WT and
KO mice was comparable, as assessed using high-performanceliquid chromatography (Table 2). Thus, the total amount of theglutamate/glutamine shuttle components, and other major
neurotransmitters and metabolites, are unaffected by the loss of
CDC42EP4.
Impaired motor coordination/learning in Cdc42ep4 � / � mice.
As a means of unbiased screening for neural dysfunctions, acohort of WT and KO littermates were subjected to a systematicbattery of behavioural tests. In 14 behavioural test paradigms
including the rotating rod (rota-rod) test, WT and KO mice
performed almost comparably (Table 3 and Supplementary
Figs 1–12). Given the robust compensatory potential of the
motor control circuitry, and the molecular and electrophysiological endophenotype of KO mice, we assessed theirmotor coordination and motor learning with the balance beam
test, which is superior to the rota-rod test in detection sensitivity24. While the moving speed of 12-week-old KO mice on
the initial trial (the intercept) was normal, the slopes of theirlearning curves were signiﬁcantly ﬂatter than those of WT mice

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AR

9

(n ¼
diam
coh
con
and
T
the
man
CDC
EAA
DLnot
pha
ent
rota
hyp
in v
clea

Dis
Prev
(CD
man
CD
in
ang
acto
end
mai
for
mor
phy
prim
mR
Atla
Cdc
cere
G
SEP
ano
mic
SEP
10

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Table 2 | The bulk content of representative neurotransmitters and metabolites in the cerebella of Cdc42ep4ﬂ/ﬂ
and Cdc42ep4 � / � littermate mice.
Neurotransmitters and metabolites (per mg tissue)
Glu (nmol)
Gln (nmol)
Gly (nmol)
GABA (nmol)
L-Ser (nmol)
D-Ser (nmol)
5-HT (ng)5-HIAA (ng)NE (ng)
MHPG (ng)HVA (ng)
DA (ng)
DOPAC (ng)WT
8.07±0.521
5.54±0.459
0.66±0.065
1.43±0.102
0.40±0.028
0.003±0.0001
0.16±0.021
0.09±0.01
0.35±0.037
0.12±0.062
0.005±0.003
0.004±0.001
0.006±0.002

KO7.97±0.253
5.25±0.128
0.81±0.13
1.48±0.133
0.40±0.028
0.003±0.0000
0.16±0.013
0.11±0.007
0.39±0.013
0.07±0.0006
0.008±0.002
0.005±0.003
0.008±0.004

The amount of glutamate, glutamine and other substances extracted from WT and KO cerebellar
tissues (n ¼ 3, 3). There was no signiﬁcant difference. KO, knockout; WT, wild type.

ces
ann–
bars,
PSCs
ough

ulted
after

ntrol
zero
rent.
hoc
ned-

gms
mice
tary
the
troheir
eam
itivonheir
mice
9(n ¼ 13/13, P ¼ 0.0003 and 0.0004, respectively, for the beam
diameters of 28 and 11 mm; Fig. 7a). In a follow-up of the samecohort at 24 weeks of age, the learning curve of KO mice wasconsistently lower than that of WT mice (n ¼ 12/12, P ¼ 0.018
and 0.014; Fig. 7a).
To test whether the persistent underperformance is caused by
the insufﬁcient glutamate-buffering/clearance capacity in Bergmann glia, and not by the loss of extracerebellar astrocytic
CDC42EP4 (Fig. 2e), we attempted the partial inhibition of
EAATs with DL-TBOA. Direct subpial injection of 100 mM
DL-TBOA with CTZ above lobule VI of the cerebellar cortex did
not affect the motor performance of WT mice, whereas thepharmacological decompensation elicited signiﬁcant and transient motor incoordination in KO mice, as was obvious now in therota-rod test (n ¼ 5/4, P ¼ 0.0011, Fig. 7b). The remarkablehypersensitivity to the subthreshold DL-TBOA recapitulated
in vivo has corroborated the insufﬁcient glutamate-buffering/clearance capacity in KO mice.
Discussion
Previous studies indicated that CDC42EPs bind to small GTPases(CDC42, RhoQ/Tc10) and septins in a mutually exclusive
manner13,14, and that interaction of CDC42EP1/BORG5 with
CDC42 and atypical protein kinase C is required for cell motility
in early mouse embryo25. A recent study revealed defective
angiogenesis in Cdc42ep1 � / � mice, which is due partly to septin/
actomyosin dysregulation that affects migration of vascularendothelial cells26. In contrast, CDC42EP4 in Bergmann glia is
mainly in complex with septins and GLAST, and is dispensablefor developmental cell migration and post-developmental
morphological integrity. The distinct binding partners andphysiological roles of CDC42EPs may reﬂect their distinct
primary structure and/or expression pattern. Since Cdc42ep1
mRNA is also expressed in Bergmann glia (Allen Mouse Brain
Atlas #68342384), Cdc42ep1 � / � mice and Cdc42ep1 � / � ;Cdc42ep4 � / � double-mutant mice would exhibit intriguing
cerebellar phenotypes.
Genetic loss of a vital septin subunit (that is, SEPT7, SEPT9 or
SEPT11) causes embryonic lethality27–29, whereas obvious brain
anomaly or motor incoordination has never been reported inmice that lack one or two other septin subunits (that is, SEPT3,
SEPT4, SEPT5, SEPT6, SEPT3 and SEPT5, or SEPT4 and SEPT6),
10

despite their abundance in the cerebellum and other brain
regions30–34. In contrast, GLAST � / � mice exhibit protracted
glutamate transient, multiple CF–PC innervations and failure in
the rota-rod test6,21.
The present study demonstrated that Cdc42ep4 � / � micedevelop CF–PC innervations with largely normal wiring patternand electrophysiological properties (Figs 3a and 6a), and pass therotating rod (rota-rod) test (Table 3). However, pharmacologicaldecompensation with inhibitors of AMPAR, AMPAR desensitization or EAATs consistently indicated their insufﬁcient
glutamate-buffering/clearance capacity in electrophysiologicalanalyses (Fig. 6c–e), and caused failure in the rota-rod test
(Fig. 7b). Thus, the severity of cerebellar phenotypes is ranked
GLAST � / � 4Cdc42ep4 � / � 4septin-null mice, which would
justify an interpretation of the phenotype of Cdc42ep4 � / � miceas a mild insufﬁciency of GLAST function, rather than as a partial
loss of septin function. Since Sept4 � / � mice and Bergmann gliaselective Sept7 � / � mice exhibit subnormal motor coordination
only in infancy (Kinoshita, Ageta-Ishihara et al., unpublished),
the pharmacological decompensation technique may help
differentiate these and other septin mutant mice from WT
littermates.
Glutamate released from PF boutons/terminals not only
depolarizes PCs via AMPARs; however, the spillover fraction
evokes Ca2 þ responses in the perisynaptic compartments(‘microdomains’) of lamellar Bergmann glial processes mainly
via mGluR1 (refs 35,36). A subdomain in such a compartment
tightly enwraps a PF–PC synapse, where GLAST outnumbersother EAATs4,37. While EAAT4 is localized to PC spines for slow
glutamate clearance20, GLAST is concentrated along perisynaptic
processes of Bergmann glia and contributes to the fast andefﬁcient limitation/termination of glutamate neurotransmission
and spillover1,5,20. However, the molecular basis to target andconcentrate GLAST to the proximity of synaptic gap remains
unclear.
Physical and functional interactions of the cytoplasmic tail of
GLAST with submembranous septins have been consistently
demonstrated in vitro, in tissue culture cell cortex and Bergmannglial processes11,12. The interaction with submembranous septins
has been hypothesized to provide membrane-bound GLAST
trimers with scaffolds, limit their lateral diffusion and/or augment
their transport activity, which collectively contribute toperisynaptic concentration of glutamate clearance activity.
However, the hypothesis has never been tested in vivo partly
because of the aforementioned redundancy problem in septin
reverse genetics.
This study demonstrated that CDC42EP4 clusters beneath
Bergmann glial membrane subdomains enwrapping dendritic
spines where septins and GLAST abound4,11,16. In the absence of
CDC42EP4, GLAST is dissociated from septins (Fig. 5a) anddelocalized away from synapses (Fig. 5e). These ﬁndings support
the above hypothesis of septins as scaffold/barrier for GLAST,
and may further implicate subtle alterations in tripartite synapse
geometry, for example, retraction of glial components fromsynaptic clefts, with or without secondary remodelling of
neuronal components.
The distribution of GLAST visualized with IF and immunoelectron microscopy appears more diffuse along Bergmann glial
membranes than that of CDC42EP4 (Figs 1f, 4e and 5c), whichcorresponds to the IP/IB data that only a subset of GLAST ispulled down with CDC42EP4 and/or septins. These data suggest
other membrane-proximal proteins that contribute to perisynaptic localization of GLAST. One such candidate is NHERF1(Na þ /H þ -exchange regulatory factor-1) whose PDZ domaininteracts with the cytoplasmic tail of GLAST12,38. So far, however,neither GLAST-related phenotype for Nherf1 � / � mice39 nor

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Table 3 | Systematic behavioural test results of Cdc42ep4ﬂ/ﬂ and Cdc42ep4 � / � littermate mice.

Behavioural despair
Latency to immobility
Spatial memory
Time spent around target hole
Motor coordination/learning Moving speed

Alteration from wild-type
Cdc42ep4 � / �-

Relatedﬁgures*1

2

Moving speed (cm s–1)

Indices measured(inexhaustive)
General health andGeneral health
Body weightneurological screening
Rectal temperature
Grip strength
Hanging persistenceLight/dark transition test
Exploratory activity
Distance travelled in the lightchamberLight avoidanceDistance travelled in the darkchamber
Latency to the ﬁrst entry to thelight chamber
Time stayed in the lightchamberNumber of transitions between
chambers
Open ﬁeld test
Exploratory activity
Distance travelled
Avoidance from open space Centre time
Anxiety-like behaviour
Vertical activity
Stereotypical movements
Elevated plus maze test
Exploratory activity
Distance travelled
Height avoidance
Entries into open arms
Number of entries
Time stayed on open arms
Acoustic startle response
Startle reﬂex to loudness
Amplitude of body motion
Prepulse inhibition (PPI) test
Sensorimotor gating
Decrement of startle amplitude
Porsolt forced swim test
Despair-like behaviour
Latency to immobility
Home cage monitoring
Diurnal cycle of locomotor
Activity level (distanceactivity
travelled)
Social behaviour
Mean number of particles
Social interaction test (one-chamber, stranger Social behaviour, anxiety-like Distance travelledpair)behaviour
Number of contacts
Total duration of active
contacts
Mean contact duration
Total duration of contacts
Social interaction test (three chambers, one Social behaviour, anxiety-like Time spent with novel stranger
to two caged strangers
behaviour
Distance travelled
Rota-rod test
Motor coordination/learning Latency to fall
Tail suspension test
Barnes maze test
Balance beam test

Mental/physical activities

3

-

5a
5b
6
7

-

8

4

Latency to fall (s)Tests- (k with CTZ þ TBOA)
k

9

Fig. 7b,10
11
12
Fig. 7aComparative table of the results of systematic behavioural tests with WT and KO littermate mice (C57BL/6, male, n ¼ 13-8/13-9 (the dropouts are due to failed data acquisition or spontaneous deaths)).
Symbols (KO phenotype relative to WT): -, no signiﬁcant difference; m/k, statistically signiﬁcant increase/decrease (Po0.05). *See Figs. 7a,b and Supplementary Figs 1–12 for details.

expression and localization NHERF1 in Bergmann glia has beenreported. Thus, the unique phenotype of Cdc42ep4 � / � miceprovides the ﬁrst in vivo evidence for the signiﬁcance of
perisynaptic concentration of GLAST by submembranous
scaffold and/or diffusion barrier.
GLAST abounds near PF–PC synapses and the buffering/clearance capacity far surpasses the level of glutamate release,which confers remarkable tolerance to EAAT inhibitors. Ourelectrophysiological analysis with pharmacological interventions
revealed insufﬁcient glutamate-buffering/clearance capacity inCdc42ep4 � / � PF–PC synapses. The decay time constant of
EPSC, which reﬂects the retention of extracellular glutamate, wasmildly protracted on inhibition of AMPAR desensitization withCTZ (Fig. 6c). Additional inhibition of EAATs with subthreshold
DL-TBOA signiﬁcantly augmented AMPAR-mediated inward
current only in the Cdc42ep4 � / � cerebellum (Fig. 6d,e). Further,a low-afﬁnity glutamate analogue gDGG was less effective in theinhibition of EPSC (Fig. 6d). These data concordantly indicate

higher extracellular glutamate level in the mutant. Given theproperties of EAATs and their distribution around glutamatergic
synapses to PCs5,20,37, and unaltered expression of the major
AMPAR subunits in the Cdc42ep4 � / � cerebellum (Fig. 3d), thephenotype is attributed principally, if not all, to the insufﬁciency
of GLAST function. As the total amounts of glutamate and
GLAST are unaltered in the Cdc42ep4 � / � cerebellum, thedelocalization of GLAST from perisynaptic glial processes(Fig. 5e), with or without altered transporter activity due to thedissociation from CDC42EP/septins, appears to be the major
cause of the glutamate intolerance.
Overall, the unique phenotype of Cdc42ep4 � / � mice provided
the ﬁrst in vivo evidence for submembranous molecular networkthat is required for the perisynaptic concentration and efﬁciency
of GLAST. This study illuminated the spatial regulation of
GLAST by the CDC42EP4/septin-based scaffold beneath perisynaptic glial membranes, which ensures optimal buffering/clearanceof extracellular glutamate from around synapses. Since CDC42EPs

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11

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

φ = 28 / 11 mm
0.5 m

WT
KO

L=1m

20

10

10

0
Trial

0
Trial

2

3

Latency to fall (s)φ = 28 mm

7a

s)).

the
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EPs
11

4

Day

φ = 11 mm

1

2

6-week-old
4h
6h
Post-injection
Post-injection

P = 0.551

P = 0.0011

300

P = 0.014

3

φ = 28 mm

24 h
Pre-injection

P = 0.957

4
φ = 11 mm

24 h
Post-injection
P = 0.518

200
100
0
Trial 1
Day

7b,

30

20

Day 1

P = 0.018

1
2
3
4
5
6
7
8
9
10
11
12

30

24-week-old

P = 0.0004

1
2
3
4
5
6
7
8
9
10
11
12

Moving speed (cm s–1)

12-week-old
P = 0.0003

WT
KO
2

3
1

4

5

6

7

8

9 10 11 122

13 14 15 16
3

Figure 7 | Motor coordination and motor learning defects in Cdc42ep4 � / �
mice. (a; Top) Set-up for the balance beam test. The height (0.5 m above
the ﬂoor) and illumination (100 lux) motivate mice to escape by traversing
along a horizontal rod (1 m � 28 or 11 mm) into a dark box. (Bottom) The
learning curves of a cohort of WT and KO mice measured at 3 and 6 monthsof age. The moving speeds are plotted for seven trials with a 28-mm rod
and subsequent ﬁve trials with an 11-mm rod over 4 days. The motor
coordination defects in KO mice remained uncompensated up to 6 monthsof age (n ¼ 13/13 and 12/12, P ¼ 0.0003, 0.0004, 0.018, 0.014 by two-way
repeated measures ANOVA). (b) The learning curves of 6-week-old WTand KO littermate mice assessed by the rota-rod test before and after direct
cerebellar cortical injection of CTZ plus DL-TBOA (100 mM each per 10 ml).
The local inhibition of EAATs with the subthreshold dose of DL-TBOA
elicited a signiﬁcant motor coordination defects transiently (around 4 hpost-injection) and only in KO mice (n ¼ 5/4, P ¼ 0.551, 0.0011, 0.957, 0.518
by two-way repeated measures ANOVA). The aberrant hypersensitivity of KO
mice indicates their glutamate clearance deﬁcit that is adaptively compensated.

and septins, as with GLAST, are implicated in human neurologicaland psychiatric disorders including schizophrenia by unbiased
proteomic and genome-wide association studies8,9,40–43, whether
relevant mechanisms underlie the pathophysiology in other brain
regions is to be tested in future studies.
Methods

Animal experiments. Animal experiments had been approved by the institutional
review committees and conducted in accordance with the regulations for the careand use of animals at Nagoya University, Niigata University, Hokkaido University,
Hiroshima University, Fujita Health University and Gunma University.
We consistently compared male littermates raised in the same cages unlessotherwise noted.
Antibodies. We raised rabbit polyclonal antibodies against seven antigens andconsistently used one (#2) against a recombinant polypeptide Arg58–Met100 from12

CDC42EP4 that gave the best signal/noise ratio in IB and IF analyses. The
speciﬁcity was warranted by the absence of the signals in Cdc42ep4 � / � tissues(Fig. 2d,e). We used antibodies for septins, SEPT2 (1:2,000), SEPT4 (1:3,000) and
SEPT7 (1:4,000) as previously described33,44, GLAST45, 3-phosphoglycerate
dehydrogenase (Phgdh)46, calbindin47, carbonic anhydrase 8 (Car8)48, VGluT1,2 (ref. 49) and commercial antibodies for GluR1, 2 (Alomone Labs, AGC-004,
1:200, AGC-005, 1:150), GLAST (Frontier Institute, Rb-Af660, 1:2,000), PSD-95(Cell Signaling, 3450, 1:1,000), CDC42 (Santa Cruz, L0809, 1:100), b-actin (Sigma,
A5441, 1:5,000) and a-Tubulin (Sigma, T9026, 1:10,000). For secondary antibodies,
we used Alexa 488-, Cy3- or horseradish peroxidase-conjugated IgGs fromrabbit, goat or guinea pig (Jackson ImmunoResearch, 706-545-148, 711-165-152,
111-035-003) diluted at 1:200–1,000. Histidine-tagged recombinant CDC42(Cytoskeleton) was used to quantify endogenous CDC42.
Biochemical fractionation and IB analysis. Each tissue was dissected, weighed
and homogenized by sonication in 3 ml g � 1 of buffer A (10 mM Tris-HCl at pH
7.6, 0.15 M NaCl, 1% Triton X-100, protease inhibitors). The supernatant after
centrifugation at 20,400g at 4 C for 0.5 h was labelled as soluble fraction. The pellet
was dissolved with sonication in 1 ml g � 1 buffer B (3% SDS, 5% 2-mercaptoethanol), which was termed pellet fraction. The samples were resolved by
10%PAGE (SDS-PAGE), transferred to reinforced nitrocellulose or PVDF membranes and subjected to IB analysis using a blocking buffer containing 5% skim
milk in TBST (0.1 M Tris-HCl at pH 7.4, 0.15 M NaCl, 0.05% Tween 20). Chemiluminescence detection and densitometry were performed with horseradish
peroxidase-conjugated secondary antibodies, ECL-Plus reagent (PerkinElmer),
TrueBlot (Rockland) and an image analyser LAS-3000mini with MultiGauge
software (Fuji). Uncropped version of each image is shown in Supplementary
Fig. 15.
Immunoprecipitation. For IP/IB experiments, whole cerebella were lysed withbuffer A’ (0.2 M Tris-HCl at pH 7.35, 0.3 M NaCl, 0.2% SDS, 0.2% Triton X-100,
0.2% sodium deoxycholate, 20 mM 2-mercaptoethanol and protease inhibitors),
centrifuged and the supernatant was mixed with protein A Sepharose CL-4B(GE Healthcare) or Afﬁ-Prep Protein A Support (Bio-Rad) that had been preincubated with anti-CDC42EP4, anti-SEPT4 or nonimmune rabbit IgG (as a negative
control). After washing the beads with the same buffer, bound proteins were lysed
for IB. For IP/MS experiments, we used buffer C (20 mM Tris-HCl at pH 8.0,
0.15 M NaCl, 1 mM EDTA, 1% NP-40 and protease inhibitors) with more stringent
wash condition50.
Mass spectrometry and peptide mass ﬁngerprinting. After IP, bead-boundproteins were eluted with solution (7 M guanidine, 50 mM Tris-HCl at pH 8.0),
reduced with 5 mM dithiothreitol for 0.5 h, alkylated with 10 mM iodoacetamide
for 1 h in the dark, demineralized and concentrated by methanol/chloroform
precipitation and digested (0.5 mg trypsin in 1.2 M urea and 50 mM Tris-HCl
at pH 8.5).
Mass spectrometric analysis (LC-MS/MS) was carried out as previously
described50. Brieﬂy, we used an LTQ Orbitrap XL mass spectrometer (Thermo
Scientiﬁc) connected to an HTC-PAL autosampler and a Paradigm MS4 HPLC
with a C18 reversed-phase column and ADVANCE Plug and Play Nano Source(Michrom Bioresources). The reversed-phase chromatography condition was a
linear gradient (0 min, 5% B; 70 min, 100% B) of solvent A (2% CH3CN with 0.1%
triﬂuoroacetic acid (TFA)) and solvent B (98% CH3CN with 0.1% TFA) at a ﬂow
rate of 500 nl min � 1. The mass spectrometer was operated in the data-dependent
MS2 mode. Peak lists were generated and calibrated by using the Mascot software(Matrix Science), and validation of the MS/MS-based peptide and protein
identiﬁcations was made with the Scaffold programme (ver. 3, Proteome Software).
The peptide probability by Mascot was used for the Scaffold Local FDR algorithm.
Peptide identiﬁcation was approved by the presence of at least two peptides thatexceeded the probability of 95%. Proteins that contained similar peptides and could
not be differentiated by MS/MS data alone were grouped to satisfy the principle ofparsimony.
Measurement of neurotransmitters and metabolites. We used previously
described methods for the extraction and measurement of amino acids, amines andmetabolites51–54. Brieﬂy, cerebellar hemispheres dissected on ice were frozen inliquid N2 and stored at � 80 C until extraction. For measurement of amino acids,
each sample was weighed, homogenized in 1.5 ml CH3OH on ice, centrifuged at
3,000g for 6 min at 4 C and 20 ml of the supernatant was dried by evaporation
at 40 C. The residue was dissolved with 20 ml water, 20 ml 0.1 M borate buffer at pH
8.0 and 60 ml 50 mM 4-ﬂuoro-7-nitro-2,1,3-benzoxadiazole (NBD-F; Tokyo Kasei
Kogyo) in CH3CN, reacted at 60 C for 2 min and stopped by adding 100 ml 0.1%
TFA and 10% CH3CN in water. Total D-/L-serine levels were measured using acolumn-switching HPLC system (SCL-10A VP, Shimadzu)51. Glutamate,glutamine, glycine and GABA were measured using an HPLC system (LC-20AT,
Shimadzu)52. Fluorescence emission at 530 nm was measured with an excitation at
470 nm. For monoamines and their metabolites, tissue samples were homogenizedin 0.2 M HClO4 containing 0.1 mM EDTA and 100 mg l � 1 isoproterenol (internal
standard), and centrifuged at 20,000g for 15 min at 4 C. Supernatants were ﬁltered

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30 HEPES at pH 7.3, adjusted with CsOH). To block inhibitory synaptictransmission, 10 mM bicuculline was consistently supplemented to the normal
ACSF. All the drugs were obtained from Tocris Bioscience. Stimulation pipettes(B5 mm tip diameter) ﬁlled with the normal ACSF were used to apply squarepulses for focal stimulation (duration, 0.1 ms; amplitude, 0–90 V). PFs were
stimulated in the molecular layer. CFs were stimulated in the granular layer aroundthe PC soma under investigation. The stimulation was given at 0.2 Hz (Fig. 6a,b) or
0.05 Hz (Fig. 6c–e). The number of CFs innervating the recorded PC was estimatedby the number of discrete CF-EPSC steps in response to a gradual rise in thestimulus intensity. The stimulation pipette was systematically moved around each
PC soma. Ionic currents were recorded with a patch clamp ampliﬁer (EPC10,
HEKA Elektronik). The pipette access resistance was compensated by 70–80%.
Online data acquisition and ofﬂine data analysis were conducted with software,
PATCHMASTER and FITMASTER (HEKA).

Neur
forel
by pu
a spr
meas
wire
meas

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

through 0.45-mm pore membranes (Millex-LH, Millipore). We used a reversedphase column (SC-5ODS) and an electrochemical detector (ECD-300) in an HPLC
system (EP-300, DG-300 and EPC-300, all from Eicom) with the mobile phase
containing 0.1 M acetate–citric acid buffer at pH 3.5, 16% CH3OH, 5 mg l � 1 EDTA
and 0.19 g l � 1 sodium octyl sulfate.
Tissue preparation for histochemistry. Under deep pentobarbital anaesthesia(0.1 mg per g of body weight), mice were ﬁxed transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.2 for IF and immunoelectron
microscopy, or with 4% paraformaldehyde/0.1% glutaraldehyde in PB for TEM.
After excision from the skull, brains for immunohistochemistry were immersed in
the same ﬁxative for 2 h. In IF for CDC42EP4, ﬁxed brains were sliced at 50 mm
with a microslicer (VT1000S, Leica) and subjected to free-ﬂoating incubation, while
4-mm-thick parafﬁn sections with a sliding microtome (SM1000R, Leica) were used
for Car8, GLAST, VGluT1 and VGluT2. Brains for FISH were removed under deep
pentobarbital anaesthesia and immediately frozen in powdered dry ice, cut at
20 mm with the cryostat and mounted on silane-coated glass slides.
FISH. Mouse cDNA fragments for Cdc42ep4 (nucleotide 331–1,118, GenBank
BC003857), calbindin (37–1,071, BC016421), GLAST (1,620–2,576, BC066154)
and Phgdh (1–1,799, BC110673) were subcloned into the pBluescript II plasmid
vector. Digoxigenin (DIG)- or ﬂuorescein-labelled cRNA probes were transcribedin vitro55.
Sections were processed as follows: acetylation with 0.25% acetic anhydride in
0.1 M triethanolamine-HCl at pH 8.0 for 10 min and prehybridization for 1 h inbuffer A (50% formamide, 50 mM Tris-HCl at pH 7.5, 0.6 M NaCl, 0.02% Ficoll,
0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 200 mg l � 1 tRNA,
1 mM EDTA and 10% dextran sulfate). Hybridization with cRNA probes was
performed in buffer A at 63.5 C for 12 h, and then sections were washed at 61 Cwith 5 � standard sodium citrate (SSC) for 0.5 h, 4 � SSC containing 50%
formamide for 0.7 h, 2 � SSC containing 50% formamide for 0.7 h and 0.1 � SSC
for 0.5 h. Sections were incubated successively in NTE buffer (0.5 M NaCl, 0.01 M
Tris-HCl at pH 7.5, 5 mM EDTA), 20 mM iodoacetamide in NTE buffer and TNT
buffer (0.1 M Tris-HCl at pH 7.5, 0.15 M NaCl), each for 0.3 h at room temperature.
For double-labelling FISH, sections were reacted with peroxidase-conjugatedanti-ﬂuorescein antibody (Invitrogen, A21253, 1:1,500) and incubated with the
FITC-TSA plus ampliﬁcation kit (PerkinElmer). After inactivation of residual
peroxidase activities with 3% H2O2, the sections were incubated successively with
DIG-labelled cRNA probe, the peroxidase-conjugated anti-DIG IgG (Roche, 11 207
733 910, 1:1,000) and the Cy3-TSA Plus reagents (PerkinElmer), and thencounterstained with TOTO-3 (Invitrogen, T3604, ﬁnal 0.2 mM) in PBS.
Immunohistochemistry. For immunohistochemistry, 4-mm-thick parafﬁn sections
were incubated with 10% normal donkey serum for 20 min, a mixture of primary
antibodies (1 mg l � 1) overnight and a mixture of Alexa 488- or Cy3-labelled
species-speciﬁc secondary antibodies for 2 h at a dilution of 1:200. Fluorescenceimaging was conducted using scanning laser confocal microscopes IX81/FV1000(Olympus) with a � 40 objective lens (numerical aperture (NA) 1.0) and LSM-780(Zeiss) with � 40 (NA 1.1) and � 63 (NA 1.4) objective lenses, and wide-ﬁeld
microscopes (BZ-9000, Keyence or BX-60, Olympus) with � 40 and � 20 objective
lenses (NA 1.3, Nikon or NA 0.7, Olympus).
Electron microscopy. Conventional electron microscopy, pre-embedding silverenhanced immunoelectron microscopy and postembedding immunogold electron
microscopy were conducted as described previously56,57. Brieﬂy, pre-embedding
immunoelectron microscopy was conducted by incubation in blocking solution forgoat gold conjugates (Aurion, Electron Microscopy Sciences) for 0.5 h, and thenwith CDC42EP4 antibody (1 mg l � 1) diluted with 1% BSA and 0.004% saponin in
PBS overnight. Anti-rabbit IgG linked to 1.4 nm gold particles (1:100, Nanogold
labelling reagents, Nanoprobes, #2003) were incubated for 2 h, and gold particles
were intensiﬁed using a silver enhancement kit (R-Gent SE-EM, Aurion). In postembedding immunogold, 80-nm-thick sections were cut with an ultramicrotome(Ultracut, Leica), incubated overnight with rabbit GLAST antibody (20 mg l � 1)
and then with colloidal gold (10 nm)-conjugated anti-rabbit or anti-guinea pig IgG(British Biocell, EM.GAR10, 1:100) for 2 h. The samples were mounted on grids
and stained with 2% uranyl acetate for 20 min, and observed with a TEM (H-7100,
Hitachi).
Electrophysiology. We prepared 250-mm-thick cerebellar slices as described
previously58,59. In brief, 5–7-week-old mice deeply anaesthetized with CO2 were
decapitated. The cerebella were quickly cut parasagittally with a microslicer(VT1200S, Leica) in a chilled cutting solution ((in mM) 120 choline-Cl, 3 KCl, 1.25
NaHPO4, 28 NaHCO3, 8 MgCl2 and 25 glucose), incubated in normal ACSF
(artiﬁcial cerebrospinal ﬂuid; 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4,
26 NaHCO3 and 20 glucose, bubbled with 95% O2 and 5% CO2) at 36 C for 0.5 h
and then kept at room temperature. All recordings were performed at 32–34 C.
Whole-cell recordings were made from visually identiﬁed PCs under an uprightmicroscope (BX51WI, Olympus). Pipette solution contained (55 CsCl, 10 Cs
D-gluconate, 20 TEA-Cl, 20 BAPTA, 4 MgCl2, 4 ATP, 0.4 GTP, 5 QX314-Cl andGeneration of Cdc42ep4-ﬂoxed and null mice. The targeting strategy is illustrated in Fig. 2a. We constructed the targeting vector using the MultiSite Gateway
Three-Fragment Vector Construction kit (Invitrogen) and BAC Subcloning Kit(Gene Bridges) with some modiﬁcations. Brieﬂy, a DNA fragment carrying theloxP sequence and pgk-promoter-driven neomycin-resistance (Neo) cassette
ﬂanked by two Flp recognition target sites was inserted in reverse orientation
directly downstream of the sole coding exon of Cdc42ep4 isolated from a C57BL/6
mouse genomic BAC clone RP23-366J17. The other loxP site was inserted into theintron directly upstream of the coding exon. The resulting targeting vector, which
consisted of 50 - and 30 -homology arms, the ﬂoxed coding region with the Neo
cassette and diphtheria toxin gene (DT) for negative selection, was linearized andelectroporated into the C57BL/6N mouse embryonic stem (ES) cell line, RENKA60.
Genomic DNA from neomycin/G418-resistant ES cell clones were analysed with
Southern blot analysis, and positive clones with proper homologous recombination
were microinjected to host embryos as described previously61. The null allele(Cdc42ep4 � ) was created by crossing heterozygous ﬂoxed (Cdc42ep4ﬂ/ þ ) micewith CAG-Cre transgenic mice62. For Southern blot analysis, genomic DNA wasdigested with Sac I, EcoR V and Hinc II, and hybridized with 50 , 30 and neo probes,
respectively. The predicted fragment sizes for the WT and ﬂoxed alleles for eachprobe, 13/11.3, 16.4/12.7 and N.A./16.1 (kb), respectively, were conﬁrmed. We
thereafter conducted genotyping using PCR with the following primers: (a) 50 TGCTTCAGTACCTTCGGAC-30 , (b) 50 -TTCGAGTTCACAGAGCTGGA-30 and(c) 50 -TCATAGAGAAGGTGGCAGC-30 to distinguish the WT, ﬂoxed and nullalleles by 490-bp (with primers a þ b), 580-bp (a þ b) and 480-bp (a þ c) products,
respectively. After conﬁrming germline transmission and Mendelian inheritance of
the Cdc42ep4ﬂ and Cdc42ep4 � alleles, the lines have been deposited to RIKEN
Bioresource Center (RBRC04894 and RBRC09539, respectively).
Balance beam and rota-rod tests. All systematic behavioural tests were conducted on male littermates (10–38 weeks old) as described previously33,63. In thebalance beam test, the motor coordination was quantitatively assessed by theperformance on the ﬁrst trial, the moving speed and the numbers of pauses andslips along a 1-m-long rod (diameter, 28 mm). After six trials on Days 1–3, ﬁve
trials with a thinner rod (diameter, 11 mm) were conducted on Days 3–4. The
increment in the moving speed and decrements in the numbers of pauses and slips
were quantiﬁed as indices of motor learning. In the rota-rod test, we measured thelatency to fall from a rotating rod (diameter, 30 mm) that was accelerated from 4 to
40 r.p.m. over 5 min per trial. For pharmacological experiment (see below), eachmouse was subjected to four sessions (four trials per session) at 24 h before
injection, and 4, 6 and 24 h after injection.
Direct cerebellar cortical injection. We applied a method established for viraldelivery64 with minor modiﬁcations. Brieﬂy, 6-week-old mice (WT, 4F þ 1M; KO,
3F þ 1M) anaesthetized with ketamine (100 mg kg � 1) and xylazine (16 mg kg � 1)
were mounted in a stereotactic frame, and the occipital bone was drilled at 5 mm
caudal to the bregma. A blunt-ended Hamilton syringe tip (33 G) attached to a
micropump and a controller (UltramicroPump II, Micro4; World Precision
Instrument) was placed beneath the pia mater above lobule VI. The mixture of
CTZ and DL-threo-b-benzyloxyaspartic acid (DL-TBOA; 100 mM each in 10 ml) was
injected at a rate of 333 nl min � 1. After suturing the scalp, and recovery fromanaesthesia on a warm pad, the mice were returned to home cages.
Systematic behavioural analysis. We applied our standard protocols63,65,66 to acohort of male littermates (n ¼ 13, 13) reared in the same cages: behavioural testing
was conducted between 9 a.m. and 6 p.m. except for the continuous home cage
monitoring. Each apparatus was cleaned with sodium hypochlorite solution tominimize odour after use. We conducted tests in the following order: general healthand neurological screening (including body weight and temperature
measurements, grip strength test, righting test, whisker touch test and ear twitch
reﬂexes, wire hang test), light/dark transition test, open ﬁeld test, elevated plus
maze test, one-chamber social interaction test, rota-rod test, three-chambersociability and preference for social novelty test, prepulse inhibition test of acoustic
startle response, Porsolt forced swim test, Barnes maze test, tail suspension test andlong-term monitoring of locomotion and social interaction in home cage. Intervals
between tests were 424 h.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

Neuromuscular strength tests. Neuromuscular strength was assessed with theforelimb grip strength test and wire hang test. Forelimb grip strength was measured
by pulling a mouse in the tail while its forepaws hung on to a wire grid attached toa spring balance. The tensile force (N) when the mouse released the grid wasmeasured three times, and the greatest value was analysed. In the wire hang test, awire mesh with a mouse on top was slowly inverted and the latency to fall wasmeasured.
Light/dark transition test. The apparatus had a pair of differentially illuminated(390 versus 2 lux) chambers (21  41  25 cm) connected with a door in the
middle. Each mouse was released in the dark chamber, and image data wereacquired from the top with a CCD (charge-coupled device) camera for 10 min. The
latency until the ﬁrst entry into the light chamber, the time spent in each chamber,the number of transitions and the total distance travelled were automaticallymeasured using ImageLD software (see Image analysis).
Open ﬁeld test. Voluntary locomotor activity was measured in an open ﬁeld test.
Each mouse was placed in the centre of the open ﬁeld apparatus (40  40  30 cm;
Accuscan Instruments) illuminated at 100 lux. The following indices were monitored for 120 min: total distance travelled, time spent in the centre area of20  20 cm, number of rearing and beam-breaks were automatically measured bycounting interruptions of infrared beams.
Elevated plus maze test. The apparatus had two open arms (25  5 cm, with
3-mm-high plastic ledges) and two closed arms (25  5 cm, with 15-cm-hightransparent walls) interconnected via a central crossing (5  5 cm), which was set at
55 cm height and illuminated at 100 lux. The numbers of entries into, and the timespent in the open and enclosed arms, were recorded for 10 min. Image data wereacquired from the top with a CCD camera, and the number of entries into and thetime spent in the open/closed arms, and total distance travelled, were measured
automatically using the ImageEP software (see Image analysis).
Acoustic startle response and prepulse inhibition test. A mouse restrained in acylinder was placed in the chamber of a startle reﬂex measurement system (O’Hara& Co.) with 70-dB background white noise. After 10 min, the mouse’s startle
response to a startle stimulus (110 or 120 dB white noise for 40 ms) was measured
by a motion sensor for 140 ms. A test session was a random sequence of four trials
each with a prepulse stimulus (74 or 78 dB white noise for 20 ms that preceded thestartle stimulus by 100 ms) and two without. Six blocks of six trials were presented
in a pseudorandom order with the average intertrial interval of 15 s.
Porsolt forced swim test. Each mouse was released in 7.5-cm-deep water at 23 Cin an acrylic cylinder (10 cm in diameter), and the duration of the motion forevacuation was measured up to 10 min automatically using the ImageTS/PS software (see Image analysis).
Social interaction and voluntary activity in the home cage. The position of eachmouse housed alone in a cage was monitored from the top continuously for a week.
The distance travelled along the diurnal cycle was measured automatically using
the ImageHA software (see Image analysis). Two mice of the same genotype that
had been separately reared were housed together in a home cage and their twodimensional (2D) images from the top were captured at 1 fps for a week. Their
physical contact and separation were represented, respectively, as one and twoparticles, and their locomotor activity was quantiﬁed by the differentials of pixels
between successive frames by using the ImageHA software (see Image analysis).
One-chamber social interaction test. The positions of two mice placed in a novel
chamber (40  40  30 cm) were monitored from the top at three frame s  1. Theirhorizontal distance travelled and the number of contacts were measured automatically using the ImageSI software (see Image analysis).
Three-chamber test for sociability and social novelty. The apparatus for
Crawley’s test had three chambers (20  40  22 cm) separated by two transparent
partitions each with an opening (5  3 cm) and a lid with an infrared CCD camera.
A male mouse (9-week old, C57BL/6J, termed Stranger 1) that had no prior contact
with the subject mice was enclosed in a cylinder cage (9 cm in diameter, set in theleft chamber) that allowed nose contacts. Each subject mouse was released in the
middle chamber and allowed to explore for 10 min, while the time spent in eachchamber and within 5 cm from each cage was measured automatically using theImageCSI software (see Image analysis). Subsequently, another unfamiliar mouse(Stranger 2) was placed in another cylinder cage (in the right chamber) andmonitored likewise for another 10 min.
Rota-rod test. For the initial screening, each mouse was subjected to nine trials
over 3 days. See Balance beam and rota-rod tests for details.
14

Tail suspension test. The movement of each mouse suspended by the tail at aheight of 30 cm was recorded for 10 min and analysed by using the ImageTS/PS
software (see Image analysis).
Barnes maze test. The apparatus consisted of a blight (800 lux), white circular
platform (diameter, 1 m) with 12 holes along the perimeter, 0.75 m above the ﬂoor,
and a dark escape box (containing cage bedding) set under one of the holes(‘target’). Following habituation sessions on the day before, each mouse was consistently trained to learn a randomly assigned target position (1–3 trials per day  6days). The platform was turned 90 per day to minimize the inﬂuence from local
cues. The distance travelled, time spent and number of errors from the centre to thetarget hole were measured from time-lapse images with a video-tracking software,
ImageBM. One day (24 h) after the last training session, the ﬁrst probe test was
carried out without escape box to exclude local cue-dependent navigation, whenmice were allowed to explore for 3 min and the time spent around each hole wasmeasured. The probe test was followed by a training session with the target. To
assess long-term spatial memory retention, the mice were again subjected to theprobe test 1 month later.
Image analysis. The application programmes for behavioural data acquisition andanalysis (ImageLD, EP, SI, CSI, TS/PS, BM, HA) were created on the ImageJ(http://rsb.info.nih.gov/ij/). ImageLD and EP are freely available from http://
www.mouse-phenotype.org/software.html.
Statistical analysis. Quantitative data are represented as mean±s.e.m. Prism 4.0(GraphPad Software), Stat View (SAS Institute) or Sigma Plot (SYSTAT), Excel(Microsoft) were used for statistical analyses. Behavioural data were analysed byanalysis of variance (ANOVA; one-way and two-way repeated measures), unlessotherwise noted. For the other data, t-test, Mann–Whitney U-test, Wilcoxon
signed-rank test (two rightmost plots in Fig. 6e) and two-way (Fig. 6c) or two-way
repeated measures ANOVA (Fig. 6e) followed by Tukey’s post hoc comparisonswere applied. For behavioural tests, either one-way ANOVA or two-way repeatedmeasures ANOVA was applied for statistical analyses unless otherwise noted.

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Acknowledgements
We thank C. Oshima (Nagoya University) and M. Yanagisawa (Tokyo Medical and
Dental University) for the technical help, T. Miyazaki (Osaka University) for CAG-Cretransgenic mice and M. Itakura (Kitasato University) for GluA4 antibody, and theMochida Memorial Foundation and Hori Science Foundation for ﬁnancial aids. This
work was supported in part by Grant-in-Aid for Scientiﬁc Research, Grant-in-Aid forYoung Investigator, Grants-in-Aid for Scientiﬁc Research on Innovative Areas ‘Synapse
pathology’, ‘Micropsychopathology’, ‘Neural Diversity and Neocortical Organization’,‘Glial Assembly’ and ‘Adaptive Circuit Shift’, Technical Supports for Fluorescent Probes
and Imaging, for Behavioural Analysis and for Antibody Production from Comprehensive Brain Science Network, and the Strategic Research Program for Brain Sciences(Integrated Research on Neuropsychiatric disorders) each from the MEXT of Japan.

NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 104 －

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms10090

M.Y., M.A. and K.S. established the Cdc42ep4ﬂ line; N.A.-I., K.Ko. and M.W. conducted thebiochemical and morphological analyses; H.N. and Ko.H. conducted electrophysiologicalanalyses, Ke.H. supervised transmitter measurement; T.N. and K.Ka. conducted mass
spectrometric analysis, K.T. provided reagents and critical suggestions for GLAST
experiments; S.H. and T.M. supervised the behavioural tests; F.H. and H.H. designed andconducted drug injection surgery; and M.K. designed the study and wrote the manuscriptwith W.M. and Ko.H. All authors read and approved the ﬁnal manuscript.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.

Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Ageta-Ishihara, N. et al. A CDC42EP4/septin-based perisynaptic
glial scaffold facilitates glutamate clearance. Nat. Commun. 6:10090 doi: 10.1038/
ncomms10090 (2015).
This work is licensed under a Creative Commons Attribution 4.0
International License. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicated otherwise
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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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NATURE COMMUNICATIONS | 6:10090 | DOI: 10.1038/ncomms10090 | www.nature.com/naturecommunications－ 105 －

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Received: 18 March 2015
Accepted: 23 April 2015
Published: 13 July 2015

wwwSpinal mechanisms underlying
potentiation of hindpaw responses
observed after transient hindpaw
ischemia in mice
Tatsunori Watanabe1,2, Mika Sasaki2, Seiji Komagata1, Hiroaki Tsukano1, Ryuichi Hishida1,
Tatsuro Kohno2, Hiroshi Baba2 & Katsuei Shibuki1
Transient ischemia produces postischemic tingling sensation. Ischemia also produces nerve
conduction block that may modulate spinal neural circuits. In the present study, reduced mechanicalthresholds for hindpaw-withdrawal reflex were found in mice after transient hindpaw ischemia,
which was produced by a high pressure applied around the hindpaw for 30 min. The reduction in thethreshold was blocked by spinal application of LY354740, a specific agonist of group II metabotropic
glutamate receptors. Neural activities in the spinal cord and the primary somatosensory cortex(S1) were investigated using activity-dependent changes in endogenous fluorescence derived frommitochondrial flavoproteins. Ischemic treatment induced potentiation of the ipsilateral spinal andcontralateral S1 responses to hindpaw stimulation. Both types of potentiation were blocked byspinal application of LY354740. The contralateral S1 responses, abolished by lesioning the ipsilateral
dorsal column, reappeared after ischemic treatment, indicating that postischemic tingling sensationreflects a sensory modality shift from tactile sensation to nociception in the spinal cord. Changes inneural responses were investigated during ischemic treatment in the contralateral spinal cord andthe ipsilateral S1. Potentiation already appeared during ischemic treatment for 30 min. The present
findings suggest that the postischemic potentiation shares spinal mechanisms, at least in part, withneuropathic pain.

The mechanism of postischemic tingling sensation has been attributed to enhanced excitability of peripheral nerves during recovery from ischemic conduction block1,2. However, nerve conduction block is
known to quickly induce plasticity in the central nervous system in experimental animals3 and humans4–6.
Cessation of low frequency spontaneous firing of Aβ tactile afferents after partial denervation produces
potentiation of neural responses in the primary somatosensory cortex (S1) elicited via the remaining
nerves7. Reduction in the mechanical thresholds for paw-withdrawal reflex is observed at the same time7.
This reduction in mechanical thresholds can be inhibited by spinal application of LY3547407, a specificantagonist of group II metabotropic glutamate receptors (mGluRs)8. If similar spinal mechanisms arealso responsible for postischemic tingling sensation, this should be abolished by spinal application of
LY354740. In the present study, we reproduced postischemic mechanical allodynia in mice, and tested
roles of spinal mechanisms using this model.
Neuropathic pain is produced by nerve injury in animal models9–11, and the resulting pain is usually
evaluated using behavioral tests more than 24 h after the injury to allow the recovery of animals fromsurgical injury. Therefore, the findings obtained from behavioral tests are affected by complex cascades
1

Department of Neurophysiology, Brain Research Institute, Niigata University,1-757 Asahi-machi, Chuo-ku,
Niigata 951-8585, Japan. 2Department of Anesthesiology, School of Medicine, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata 951-8510, Japan. Correspondence and requests for materials should be addressed to K.S.
(email: shibuki@bri.niigata-u.ac.jp)

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a1,
Figure 1. Reduced mechanical thresholds for hindpaw-withdrawal reflex. (a) Application of high pressure(250 mm Hg) to the left thigh. (b) Mechanical thresholds for left hindpaw-withdrawal reflex before and after
ischemic or sham treatment applied to the left thigh. Mean ± SEM are shown. (c) Mechanical thresholds for
hindpaw-withdrawal reflex after hindpaw ischemia in mice with spinal application of LY354740 (10 nM, 5 μ l)or saline alone. (d) Comparison of the thresholds for the left hindpaw-withdrawal reflex at 2 h after ischemicor sham treatment, with or without spinal application of LY354740. In the ischemia group, data were mixed
in mice with or without spinal application of saline. In the sham-treated group, data were mixed in micewith sham treatment to the left thigh or to the right thigh.

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1of inflammation and gene expression during the recovery period12–14. However, postischemic mechanical allodynia is induced very quickly after transient hindpaw ischemia. Thus, if neural changes reflecting postischemic tingling sensation are found, these changes may be useful for investigating the neuralmechanisms that trigger neuropathic pain. Activity-dependent flavoprotein fluorescence signals reflect
aerobic energy metabolism in mitochondria15, and are useful for investigating fine neural activities andplasticity16–18. Flavoprotein signals are resistant to and spontaneously recover from photobleaching19, andquantitatively reflect neural activities20,21. Spinal activities elicited by peripheral stimulation have beenvisualized using flavoprotein fluorescence signals22. An initial phase of neuropathic pain after partial denervation is observed as long-lasting potentiation of flavoprotein fluorescence signals in S17. Furthermore,a modality shift from tactile sensation to nociception can be visualized as reappearance of S1 responses
to tactile stimulation in mice with ipsilateral dorsal column lesioning7. It is because the tactile sensationis mediated via the ipsilateral dorsal column to the contralateral S123, while nociception is mediated via
the contralateral spinothalamic tract24. In the present study, we confirmed that such a modality shift fromtactile sensation to nociception was produced within 30 min after ischemic treatment.

Results

Reduced mechanical thresholds for paw-withdrawal reflex after ischemic treatment. Because

unpleasant postischemic tingling sensation is exacerbated by external mechanical forces, we measured
the thresholds for hindpaw-withdrawal reflex using von Frey filaments25. The thresholds before hindpaw
ischemia were 0.57 ± 0.10 g (mean ± SEM, n = 6). Mice were transiently anesthetized with 1% isoflurane,
and a pressure of 250 mmHg was applied for 30 min to a rubber cuff set around the thigh (Fig. 1a). At
30 min after the ischemic treatment was finished, the mechanical thresholds were significantly reduced
compared with the corresponding data in sham-treated mice with no pressure application (P < 0.01,
Fig. 1b). The thresholds reached the minimal values of 0.07 ± 0.02 g (n = 6) at 2 h after the ischemic treatment (Fig. 1b), and were clearly and significantly smaller than the corresponding values in sham-treatedmice (P < 0.005). Isoflurane anesthesia alone produced no reduction in the threshold in sham-treatedmice (Fig. 1b).
Next, we tested whether the reduction in the thresholds was sensitive to spinal application of 10 nM
LY354740 (Fig. 1c), which can block the initial phase of neuropathic pain after partial denervation7.

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Figure 2. Potentiation of the spinal responses after hindpaw ischemia. (a) Example of ipsilateralspinal responses elicited by vibratory stimulation applied to the left hindpaw at T13 and L1 level (upper
panels). The left-most panels are original fluorescence images, and others are pseudocolor images of
response magnitudes in Δ F/F0 recorded at the time before and after the stimulus onset shown on each
image. Another example of ipsilateral spinal responses elicited by vibratory stimulation applied to the left
hindpaw at T12 and T13 level (lower panels). (b) Example of spinal responses recorded in the same mouse
before, during, and 30–120 min after ischemic treatment applied to the left thigh for 30 min. The response
amplitudes were measured in the square window of 100 × 25 pixels shown in the second panel. (c) Relative
amplitudes of spinal responses during and after ischemic treatment. The amplitudes were normalized bythose recorded before hindpaw ischemia. (d) Example of spinal responses recorded before and at 60 min
after sham treatment. (e) Example of spinal responses recorded before and at 60 min after hindpaw ischemia
in a mouse with spinal application of 10 nM LY354740. (f) Comparison of the normalized response
amplitudes in mice at 60 min after hindpaw ischemia, sham treatment, or hindpaw ischemia with spinal
application of 10 nM LY354740.

When the ischemic treatment was applied to the mice that received spinal application of LY354740,
the thresholds at 2 h after hindpaw ischemia were 0.63 ± 0.12 g (n = 6). In contrast, the thresholds were0.12 ± 0.06 g (n = 6) in mice that received spinal application of saline alone (Fig. 1c). The difference
at 2 h after hindpaw ischemia or sham-treatment was statistically significant (P < 0.01). We confirmedthat spinal application of LY354740 alone had no apparent effect on the mechanical thresholds forpaw-withdrawal reflex (Fig. 1d). Interestingly, a slight but significant reduction in the threshold wasobserved in the right hindpaw (Supplementary Fig. S1), suggesting that transient hindpaw ischemia
might also have some effects on the contralateral spinal cord.

Flavoprotein fluorescence responses in the spinal cord. Because the behavioral test data suggested the presence of some spinal plasticity, we recorded ipsilateral spinal responses elicited by vibratory hindpaw stimulation using flavoprotein fluorescence imaging. Hindpaw stimulation produced anincrease in fluorescence on the dorsal surface of the ipsilateral spinal cord corresponding to the dorsal
horn at the T13 and L1 level (Fig. 2a). Because minimal response was found at T12, the fluorescencechanges were attributed mainly to localized activities in the dorsal horn but not to ascending afferent
activities mediated via the dorsal column. The responses started at 0.2 s after the onset of the stimulation,
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and peaked at approximately 1% in Δ F/F0 at 0.6–0.8 s after the stimulus onset. Although these properties were similar to those recorded in cortical areas7,26, the decay was slower than that of the corticalresponses, as the hemodynamic responses were not marked in the spinal cord (Supplementary Figs S2).
The spinal responses to vibratory hindpaw stimulation were abolished by a pressure of 250 mmHg
applied to the thigh, indicating that ischemic conduction block was successfully produced at 250 mmHg
(Fig. 2b). The responses reappeared within 30 min after hindpaw ischemia, and the magnitude of the
responses estimated at 30 min after hindpaw ischemia was slightly but significantly potentiated (P < 0.03,
Fig. 2b,c). At 1 h after hindpaw ischemia, the response amplitudes reached maximal values, and werepotentiated to 138 ± 11% (n = 7) compared with those before hindpaw ischemia (Fig. 2c). No clear
potentiation was observed in sham-treated mice, and the difference between the two groups was statistically significant (P < 0.002, Fig. 2d,f).
Next, we applied 10 nM LY354740 on the surface of the spinal cord during imaging experiments.
Application of LY354740 had no immediate effect on the spinal responses to vibratory hindpaw stimulation (Fig. 2e). However, the spinal responses were not clearly potentiated after hindpaw ischemia, andthe relative amplitudes at 1 h after hindpaw ischemia were significantly smaller than those in mice withno LY354740 (P < 0.003, Fig. 2e,f). These data suggest that transient hindpaw ischemia produced spinal
potentiation, and the reduction in mechanical thresholds for paw-withdrawal reflex, likely via similar
spinal mechanisms sensitive to LY354740.
The spinal responses occasionally appeared in separated segments (for example, Fig. 2a), which mightreflect the presence of functional units. Therefore, we tested the fine somatotopic maps in the spinal
responses elicited by vibratory stimulation applied to each toe. The spinal responses to stimulation ofeach toe appeared in small areas that were different each other (Supplementary Fig. S2). The responsive areas were arranged almost linearly (Supplementary Figs S2). These results were compatible with aprevious morphological study on spinal distribution of afferent fibers from each digit27. Therefore, theapparent segments in the spinal responses likely reflected strong stimulation of the skin convexes by thebrush used for hindpaw stimulation.

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emia

Flavoprotein fluorescence responses in S1. Flavoprotein fluorescence responses elicited by hindpaw stimulation appeared in the contralateral S1 (Fig. 3a). The initial time course of S1 responses elicitedby hindpaw stimulation was similar to that of spinal responses, while hemodynamic responses obscured
the later phase of the responses (Supplementary Figs S3). Although neural responses in S1 are arranged
according to somatotopic maps over a large scale26,28,29, the responses elicited by stimulation of each toe
were not clearly separated, and no fine somatotopic map was found (Supplementary Figs S3). When apressure of 250 mmHg was applied to the thigh, S1 responses were almost completely abolished (Fig. 3b).
However, the responses reappeared and were potentiated after hindpaw ischemia (Fig. 3b,c). The potentiation was maintained up to 3 h after hindpaw ischemia. The relative amplitudes at 1 h after hindpaw
ischemia (165 ± 18%, n = 13) were significantly larger than those in sham-treated mice (99 ± 4%, n = 8,
P < 0.003, Fig. 3d,g). Potentiation of S1 responses after ischemic treatment was almost completely abolished by spinal application of 10 nM LY354740 (Fig. 3e,f). The relative amplitudes of S1 responses in micewith spinal application with LY354740 were 105 ± 6% (n = 9) at 1 h after hindpaw ischemia, and were significantly smaller than those in mice with spinal application of saline alone (150 ± 16%, n = 8, P < 0.007,
Fig. 3g). Therefore, the suppression of S1 potentiation is likely attributable to the pharmacological effects
of LY354740, but not to non-specific spinal injury caused by the drug application.
Modality shift from tactile sensation to nociception induced by hindpaw ischemia. Potentiation

4740,
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in S1 after ischemic treatment may be produced by a modality shift from tactile sensation to nociception, as observed after partial denervation7. When we disrupted the ipsilateral dorsal column at the
T11 level (Fig. 4a), S1 responses elicited by hindpaw stimulation were abolished (Fig. 4b). However, S1
responses reappeared at 30 and 60 min after ischemic treatment applied to the hindpaw (Fig. 4b). As
no such response was observed in sham-treated mice (Fig. 4c), the reappearing S1 responses cannot be
attributed to spontaneous recovery from the injury caused by dorsal column lesioning. The difference in
S1 responses in the operated mice was statistically significant at 30 and 60 min after hindpaw ischemia
or sham treatment (P < 0.01 for both, Fig. 4d). The reappearing S1 responses suggest that the modality
shift from tactile sensation to nociception was induced within 30 min after ischemic treatment.

Potentiation in the contralateral spinal cord and the ipsilateral S1 during hindpaw
ischemia. Changes in neural responses were not observable during ischemic treatment in the ipsilat-eral spinal cord or in the contralateral S1 because of conduction block of the peripheral nerves. However,
we found that the spinal responses elicited by vibratory stimulation applied to the hindpaw contralateral to the ischemic treatment were significantly potentiated during ischemic treatment (Fig. 5a,b). The
potentiation was maintained for at least 60 min after hindpaw ischemia. Cortical responses in S1 ipsilateral to the ischemic treatment were similarly potentiated during and after hindpaw ischemia (Fig. 5c,d),indicating that spinal and cortical potentiation had already been initiated during ischemic treatment.
Comparison of the potentiation at 60 min after hindpaw ischemia in the spinal cord (ipsilateral:
138 ± 11%, n = 7; contralateral: 140 ± 6%, n = 5) and S1 (contralateral: 165 ± 18%, n = 13; ipsilateral;
171 ± 14%, n = 14) revealed that the neural changes in the contralateral spinal cord and the ipsilateral

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Figure 3. Potentiation of the S1 responses after ischemic treatment. (a) Example of contralateral S1
responses elicited by vibratory stimulation applied to the left hindpaw. (b) Example of S1 responses recordedin the same mouse before, during, and 30–180 min after ischemia applied to the left thigh for 30 min. The
response amplitudes were measured in the square window of 60 × 60 pixels shown in the second panel.
(c) Relative amplitudes of S1 responses during and after hindpaw ischemia. (d) Example of S1 responses
recorded before and at 60 min after sham treatment. (e) Example of S1 responses recorded before andat 60 min after hindpaw ischemia in a mouse with spinal application of saline alone. (f) Example of S1
responses recorded before and at 60 min after hindpaw ischemia in a mouse with spinal application of 10 nM
LY354740. (g) Comparison of the normalized response amplitudes in mice at 60 min after hindpaw ischemia,
sham treatment, hindpaw ischemia with spinal application of saline alone, or hindpaw ischemia with spinal
application of 10 nM LY354740.

S1 were comparable to those in the opposite sides. These results suggest that reduction in the mechanicalthresholds of the right hindpaw-withdrawal reflex could be observed after ischemic treatment applied
to the left thigh. In accordance with these results, a slight but significant reduction in the mechanicalthreshold (P < 0.02) was observed in the right hindpaw withdrawal reflex at 3 h after ischemia applied
to the left hindpaw. However, S1 responses to forepaw stimulation were not clearly affected by ischemia
applied to the hindpaw (Supplementary Figs S4).

Discussion

Postischemic mechanical allodynia produced by spinal mechanisms in mice. In the present
study, we found that mechanical thresholds for hindpaw-withdrawal reflex were reduced after ischemic
treatment. Previous studies also reported that hyperalgesia30 or mechanical allodynia31 is observed after
hindpaw ischemia in animal experiments. However, it is difficult to differentiate between tingling sensation (or dysesthesia) and hyperalgesia or mechanical allodynia using behavioral tests, because all of these
symptoms produce reduced mechanical thresholds. The reduced mechanical thresholds after ischemic
treatment in the present study can be regarded as dysesthesia, because tingling sensation rather than painis frequently experienced by human subjects after reversible hindpaw ischemia. Usually, postischemic
dysesthesia due to hyperexcitability of peripheral nerves continues for approximately 5 min after ischemic
treatment1, while use of a tourniquet to prevent bleeding for more than 2 h can produce long-lasting
dysesthesia32.
The reduced mechanical thresholds were accompanied by potentiation in the spinal cord and S1.
These three changes after ischemic treatment were abolished by spinal application of LY354740. Spinal
and S1 responses to the right hindpaw stimulation were also potentiated during ischemia applied to
the left hindpaw. Although long lasting-ischemia and reperfusion produce peroxides and resulting
cytokine-mediated adverse pathophysiological effects on peripheral tissues33,34, potentiation in the5

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Figure 4. Sensory modality shift induced by ischemic treatment. (a) Lesion in the left dorsal column
at T11 level. (b) Example of the right S1 responses modified by the left column lesioning and ischemic
treatment applied to the left thigh. (c) Example of the right S1 responses modified by the left column
lessoning and sham treatment applied to the left thigh. (d) Response amplitudes immediately after dorsal
column lesioning, and at 30 min and 60 min after ischemic or sham treatment.

orded
The

s

contralateral spinal cord or in the ipsilateral S1 was observed before reperfusion was started, and S1
responses to forepaw stimulation were not affected by ischemia applied to the hindpaw. Taken together,
these data suggest that postischemic tingling sensation was produced by localized spinal mechanisms in
our mouse model.

Spinal neural circuits and mechanisms producing postischemic plasticity.

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5Neural imaging isuseful for investigating mechanisms underlying postischemic tingling sensation. We used endogenous
fluorescence signals derived from the mitochondrial flavoproteins15. Because flavoproteins are endogenous proteins of the electron transfer system in the mitochondria, it is very unlikely that flavoproteinswork as a calcium chelator and have some artificial effects on the calcium dynamics, which play essential
roles in induction of synaptic plasticity. We have successfully recorded various types of neural plasticity using this imaging method16,17,20,35. Flavoprotein fluorescence responses are mainly derived fromsynaptically-driven neuronal activity21, and no clear fluorescence response was found from the ipsilateral
dorsal column that mediates tactile sensory information during hindpaw stimulation. Therefore, the spinal flavoprotein fluorescence responses were presumably derived from dorsal parts of the dorsal horn,which is composed of lamina I–VI36. Of these, tactile inputs mediated by Aβ fibers are terminated mainly
in lamina III–V. Lamina II, or the substantial gelatinosa, has a critical role in neuropathic pain37,38, andreceives nociceptive inputs mediated mainly by C fibers39. Group II mGluRs are located in the substantial
gelatinosa40. Taken together, the spinal fluorescence responses elicited by tactile stimuli to the hindpaw
are likely derived from neuronal activity in lamina III–V, and the potentiation observed after ischemic
treatment likely reflects the recruitment of neuronal activity in the substantial gelatinosa.
An initial phase of neuropathic pain after partial denervation is abolished by spinal application of
LY3547407, and this compound and similar group II mGluR agonists alleviate neuropathic pain8,41.
Because the potentiation in the present study was also susceptible to spinal application of LY354740,
these two types of potentiation may share similar mechanisms, at least in part, with neuropathic pain
(Fig. 6a). Activation of postsynaptic group II mGluRs reduces presynaptic transmitter release42, produces
membrane hyperpolarization by opening inward rectifier K+ channels43,44, and modulates spontaneous
Ca2+ spikes45. Therefore, failure of group II mGluR activation during conduction block of peripheral
nerve activities may increase the excitability and intracellular Ca2+ concentration of postsynaptic neurons. These changes induce down-regulation of the neuron-specific KCl cotransporter (KCC2) in dorsal
horn neurons and a resulting reduced neuronal Cl– gradient46–48. Nociceptive spinal neurons are dynamically regulated by inhibition from various sources49,50, and modulation of the Cl– gradient can explain
the increased responsiveness of spinothalamic tract neurons to innocuous mechanical stimuli in animals
with neuropathic pain48,51. The spinal potentiation is induced not only in the ipsilateral site that fails toreceive basal afferent firing, but also in the contralateral site or nearby site after partial nerve cutting.
Because stimulation of different skin areas produces separate spinal responses (for example, Fig. 3), thepotentiation in nearby spinal sites may have been induced by diffusible mediators produced in spinal neurons that failed to receive basal afferent firing (Fig. 6b). It is suggested that hindpaw ischemia produced

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Figure 5. Responses to peripheral stimulation contralateral to hindpaw ischemia. (a) Example of spinal
responses to left hindpaw stimulation before, during, and at 30 min and 60 min after ischemic treatment
applied to the right hindpaw. (b) Relative amplitudes of the responses to left hindpaw stimulation before,
during, and at 30 min and 60 min after ischemic or sham treatment applied to the right hindpaw. Responses
were normalized by those recorded before ischemic or sham treatment. (c) Example of S1 responses to left
hindpaw stimulation before, during, and after ischemic treatment applied to the right hindpaw. (d) Relative
amplitudes of the responses to left hindpaw stimulation.

some diffusible mediators that produced not only potentiation of contralateral hindpaw responses but
also contralateral mechanical allodynia. Diffusible mediators may also play a role in potentiation of spinal responses ipsilateral to ischemic treatment. In accordance with this concept, neuropathic pain wasreported to be facilitated by a number of diffusible mediators such as nitric oxide52 and ATP/cytokines53.
The mechanical allodynia observed after hindpaw ischemia seemed to be more clearly observed in micecompared with similar phenomena in humans. Furthermore, humans do not clearly exhibit tingling sensation contralateral to the ischemic side or after partial nerve cutting. This difference may be attributed tothe small size of spinal circuits in mice, in which diffusible messengers such as nitric oxide can be very
effective. Another important consideration is that the potentiation observed during ischemic treatmentin the present study could not be induced by repetitive activity in peripheral nerves, another condition
known to induce spinal potentiation54,55.

Postischemic changes as an experimental model for investigating neuropathic
pain. Neuropathic pain is usually induced by peripheral nerve injury in animal models9–11. It is pro-

duced by a complex cascade of inflammation and gene expression, and not only neurons but also glial
cells in the spinal cord have important roles in neuropathic pain12–14. We have reported that an initial
phase of neuropathic pain is observed at a few hours after partial denervation7. However, postischemic
tingling sensation and plasticity, which share similar spinal mechanisms with neuropathic pain, wereobserved at 30 min after reversible hindpaw ischemia. Furthermore, potentiation of spinal responses
contralateral to the ischemic side appeared within 30 min of ischemic treatment. Regardless of the species difference, the very early onset of neural plasticity during reversible hindpaw ischemia or transient functional deafferentation in mice make them useful as an experimental model for observing and7

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Figure 6. Mechanisms underlying postischemic plasticity. (a) Sensory modality shift from tactile totingling sensation at the spinal cord level. (b) Schematic drawings of spinal potentiation ipsilateral andcontralateral to ischemic treatment. Expected spinal potentiation induced after partial nerve cutting may also
be induced by similar mechanisms.

investigating the detailed cellular and molecular cascades that trigger human neuropathic pain. However,further studies on neuronal and glial activities in the spinal cord are required for establishing a new
preclinical model of early neuropathic pain based on the present studies.

Materials and methods

The experiments in the present study were approved by the ethics committee of animal experiments in
Niigata University (approved number: 233-4), and were carried out in accordance with the approved
guidelines. Male C57BL/6 mice between 7 and 10 weeks old, purchased from Charles River Japan (Tokyo,
Japan), were used in the present study.

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Estimation of mechanical thresholds for hindpaw-withdrawal reflex. The mechanical thresholds for hindpaw-withdrawal reflex were measured using von Frey filaments25. The forces producedby von Frey filaments were between 0.008 and 1.4 g (respective sizes: between 1.65 and 4.17). Mice
were separately placed into a transparent plastic box with a mesh floor, and accustomed to the state for
30 min. The thresholds were determined from the minimal force at which hindpaw withdrawal reflex
was induced more than twice in eight trials. Transient ischemia was applied to the left hindpaw of micelightly anesthetized with 1% isoflurane. Urethane anesthesia (1.65 g/kg, i.p.) was also used for imaging
experiments. A small rubber cuff was set around the left thigh, and covered with a hard plastic tube
(Fig. 1a). Air pressure at 250 mmHg was applied for 30 min to the tubing connected to the cuff using
a mercury manometer. The pressure was directed to the thigh, because inflation of the rubber cuff waslimited by the hard plastic tube. We confirmed that this treatment was sufficient to produce transientischemia of the hindpaw, as neural responses elicited by vibratory stimulation to the hindpaw were
blocked by this manipulation (for example, Figs 2c,4b). No apparent impairment except reduced threshold of paw-withdrawal reflex was found in the left leg after the mice recovered from isoflurane anesthesia.
Imaging experiments.

The surgical procedures were performed as described previously7. Mice wereanesthetized with urethane (1.65 g/kg, i.p.), and a tracheotomy was performed for facilitating spontaneous respiration. During the experiments, body temperature was monitored using a rectal probe andmaintained at 38 °C using a silicon rubber heater. These surgical operations were usually finished within
60 min. Recordings were started at 30 min after the surgical operations. Additional doses of urethane(0.1–0.2 g/kg, s.c.) were administered when necessary. When spinal responses to hindpaw stimulation
were investigated, the vertebral arch was removed at the T13 and L1 level, and the dorsal surface of the
spinal cord with the intact dura mater was exposed. The surface was cleaned with saline, and covered
with 2% agarose to prevent spinal movement. The surface of the agarose gel was covered with a mixture of petroleum jelly and liquid paraffin to prevent drying. The spinal cord was fixed under a microscope using a clamp (STS-A; Narishige, Tokyo, Japan). Spontaneous respiration was maintained during
the imaging experiment, because the movement of the spinal cord caused by respiration was minimal.

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wwwFlavoprotein fluorescence imaging was performed as described previously7. Endogenous green fluorescence (λ = 500–550 nm) was recorded in blue light (λ = 450–490 nm). Images (128 × 168 pixels) of the
spinal cord or S1 were recorded at 9 frames/s using a cooled charge coupled device camera (ORCA-R2;
Hamamatsu Photonics, Hamamatsu, Japan). The camera was attached to a binocular epifluorescencemicroscope (M165 FC; Leica Microsystems, Wetzlar, Germany) with a 75-W xenon light source and a
1× objective lens. Serial images were taken in recording sessions repeated at 50 s intervals. Brush vibration (amplitude: 0.2 mm; frequency: 50 Hz) was applied for 600 ms to the sole of the hindpaw using amechanical stimulator (DPS-290; Dia Medical, Tokyo, Japan). When fine somatotopic maps were investigated in the spinal cord or S1, brush vibration was applied to each toe. Fluorescence changes elicitedby the stimulation were averaged over 24 trials. Because it took approximately 20 min to obtain datafrom 24 trials, the recording time of the averaged data was defined as the middle point of the recording period. Spatial averaging in 5 × 5 pixels and temporal averaging in three consecutive frames wereused for smoothing and improving the image quality. The images were normalized, pixel by pixel, withrespect to a reference image, which was obtained by averaging five images taken immediately before thestimulation. In the figures, parts of the normalized images are shown in a pseudocolor scale representing
the fractional fluorescence changes (Δ F/F0). The response amplitude was evaluated as values of Δ F/F0
in a square window of 100 × 25 pixels or 3.84 × 0.96 mm. The location of the window was adjusted tomaximize the response amplitude in Δ F/F0. After the recordings, mice were euthanized with an overdoseof pentobarbital (300 mg/kg, i.p.).
For investigating the S1 responses to hindpaw stimulation, the disinfected head skin was incised,
and the skull over the right S1 was exposed. The surface of the skull was cleaned with sterile saline, anda small piece of metal was attached to the skull with dental acrylic resin (Super Bond; Sun Medical,
Shiga, Japan) to fix the head under a microscope. The surface of the skull was covered with a mixture of
petroleum jelly and liquid paraffin to keep the skull transparent. The response amplitude was evaluatedas values of Δ F/F0 in a square window of 60 × 60 pixels or 1.55 × 1.55 mm.

Dorsal column lesioning. The left dorsal column was disrupted using an ultrasonic cutter (NE87;
NSK, Kanuma, Japan) at the T11 level, as described previously7. To verify the lesion, the spinal cord
was isolated after the recording experiments and fixed with 10% paraformaldehyde. Serial spinal cord
sections of 100-μ m thickness were cut using a microslicer (PRO-7; Dosaka, Kyoto, Japan), and the translucent images were observed.
Spinal application of LY354740 to the spinal cord. In behavioral experiments, LY354740 (10 nM,

5 μ l), obtained from Santa Cruz Biotechnology (Santa Cruz, USA), was applied to the spinal cord withintrathecal injection before hindpaw ischemia. In imaging experiments of the spinal responses, epiduralapplication and infiltration of 10 nM LY354740 for at least 30 min was performed before the epidural
surface was covered with 2% agarose. Epidural application and infiltration of saline alone was naturally performed in other imaging experiments of the spinal responses. In imaging experiments of S1
responses, the epidural application and infiltration of 10 nM LY354740 or saline alone was performed at
the T13–L1 level.

Statistics.

Statistical significance in data was analyzed using StatView software (SAS Institute Inc.,
Cary, USA). Unpaired data obtained from different mice were evaluated by the Mann Whitney U-test.
Paired data obtained from the same mice were evaluated by the Wilcoxon signed rank test. Only P values
less than 0.05 are shown.

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4. Tinazzi, M. et al. Transient deafferentation in humans induces rapid modulation of primary sensory cortex not associated with
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Acknowledgements

We thank S. Maruyama and A. Matsushima for their technical assistance. This work was supported by
a Grant-in-Aid for Scientific Research (No. 22115011) and Core Research for Evolutional Science andTechnology program (CREST) from the Japanese Government.

J Neu
First

De
aud

Author Contributions

T.W., M.S, S.K., and K.S. designed the experiments; T.W., M.S. and H.T. conducted experiments; T.W.,
H.T., R.H., T.K., H.B. and K.S. analyzed the data; T.K. and H.B. provided materials; T.W., and K.S. wrote
the manuscript.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Watanabe, T. et al. Spinal mechanisms underlying potentiation of hindpaw
responses observed after transient hindpaw ischemia in mice. Sci. Rep. 5, 11191; doi: 10.1038/
srep11191 (2015).
This work is licensed under a Creative Commons Attribution 4.0 International License. The
images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under theCreative Commons license, users will need to obtain permission from the license holder to reproduce
the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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J Neurophysiol 113: 2900 –2920, 2015.
First published February 18, 2015; doi:10.1152/jn.00932.2014.

Delineation of a frequency-organized region isolated from the mouse primary
auditory cortex
X Hiroaki Tsukano,1 Masao Horie,2 Takeshi Bo,1 Arikuni Uchimura,3 Ryuichi Hishida,1Masaharu Kudoh,4 Kuniyuki Takahashi,5 Hirohide Takebayashi,2 and Katsuei Shibuki1

T.W.,
wrote

1

Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan; 2Division of Neurobiology andAnatomy, Graduate School of Medicine and Dental Sciences, Niigata University, Niigata, Japan; 3KOKORO-Biology Group,
Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; 4Departmentof Physiology, Teikyo University School of Medicine, Tokyo, Japan; and 5Department of Otolaryngology, Graduate School of
Medicine and Dental Sciences, Niigata University, Niigata, Japan
Submitted 21 November 2014; accepted in ﬁnal form 17 February 2015

w

. The
Comr theoduce

Tsukano H, Horie M, Bo T, Uchimura A, Hishida R, Kudoh M,
Takahashi K, Takebayashi H, Shibuki K. Delineation of a frequencyorganized region isolated from the mouse primary auditory cortex. J
Neurophysiol 113: 2900 –2920, 2015. First published February 18,
2015; doi:10.1152/jn.00932.2014.—The primary auditory cortex (AI)is the representative recipient of information from the ears in the
mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging,that two distinct areas of the AI, located ventrally and dorsally, areactivated by high-frequency tones, whereas only one area is activatedby low-frequency tones. Here, we show that the dorsal high-frequency
area is an independent region that is separated from the rest of the AI.
We could visualize the two distinct high-frequency areas using ﬂavoprotein ﬂuorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Speciﬁcally, the ratio of
SMI-32-positive pyramidal neurons to nonpyramidal neurons waslarger in the dorsal high-frequency area than the rest of the AI. We
named this new region the dorsomedial ﬁeld (DM). Retrograde tracing
showed that neurons projecting to the DM were localized in the rostralpart of the ventral division of the medial geniculate body with adistinct frequency organization, where few neurons projected to the
AI. Furthermore, the responses of the DM to ultrasonic courtshipsongs presented by males were signiﬁcantly greater in females than inmales; in contrast, there was no sex difference in response to artiﬁcial
pure tones. Our ﬁndings offer a basic outline on the processing of
ultrasonic vocal information on the basis of the precisely subdivided,
multiple frequency-organized auditory cortex map in mice.
auditory cortex; mapping; multiple frequency organization; courtshipsong; mice
THE PRIMARY SENSORY CORTEX of the mammalian brain is widely
known to receive the ﬁrst thalamic inputs that convey peripheral sensory information, such as hearing and vision. In theauditory cortex, the primary auditory cortex (AI) is the mainrecipient of information from the ears into the cortex andtransfers this information to higher-order auditory corticalregions (Kaas and Hackett 2000). The AI is important in termsof being both the receiver and the relay point in hierarchicalauditory processing and has been studied using various animals, including mice.

Address for reprint requests and other correspondence: H. Tsukano, Dept. of
Neurophysiology, Brain Research Institute, Niigata Univ., 1-757 Asahimachidori, Chuo-ku, Niigata 951-8585, Japan (e-mail: tsukano-nii@umin.ac.jp).

11

2900

The mouse is widely used in auditory physiological researchbecause of its merits as an animal model in auditory corticalstudies involving two-photon Ca2 imaging (Bandyopadhyay
et al. 2010; Bathellier et al. 2012; Honma et al. 2013; Issa et al.
2014; Rothschild et al. 2010), voltage-sensitive imaging (Sawatari et al. 2011; Takahashi et al. 2006), anatomical studies(Barkat et al. 2011; Hackett et al. 2011; Hofstetter and Ehret
1992; Horie et al. 2013; Llano and Sherman 2008; Oviedo et al.
2010), genetic manipulation (Barkat et al. 2011; Rotschafer
and Razak 2013; Xiong et al. 2012), and behavioral analysis(Tsukano et al. 2011). Accordingly, the attainment of precise
knowledge of the AI in mice is essential. However, the delineation of the map of the mouse auditory cortex is under debate,
as maps obtained by electrophysiology have been amended inrecent studies using optical imaging.
The classical mouse auditory cortex map was drawn using
unit recording. The anterior auditory ﬁeld (AAF) and AI withclear frequency gradients are placed at the center as the core
and are surrounded by the belt region, considered the higherorder region, including the secondary auditory ﬁeld (AII), theultrasonic ﬁeld (UF), and the dorsoposterior ﬁeld (DP)(Stiebler et al. 1997). The AAF and AI have frequencyorganized arrangements covering the frequencies up to 40 kHz;neurons with characteristic frequency over 50 kHz are localized in the UF (Fig. 1A). A subsequent study indicated that a
distinct UF region does not exist, with the AAF and AI having
the full range of frequency organization, processing sounds
from 4 kHz up to 64 kHz (Guo et al. 2012). The term “UF” hasthus been redeﬁned as subparts within the AAF and AI thatprocess high-frequency sounds (Fig. 1B). Recently, however,Issa et al. (2014) have reported elegantly that the AI is divided
into two rostral areas that process high-frequency tones, despite only one caudal area being activated by low-frequency
tones. This leads to the generation of forked, dual-frequency
gradients inside of the AI (Issa et al. 2014). The larger dorsal
branch of the fork-shaped frequency gradients travels toward
the high-frequency area of the AI, referred to as the UF, asreported by Guo et al. (2012), whereas the smaller ventral
division has an axis of the frequency organization toward the
AII (Fig. 1C) (Issa et al. 2014). This new map was obtainedusing optical imaging. Moreover, they clearly revealed that thedirection of the frequency organization of the AAF was present
and is directed from the dorsorostral to ventrocaudal direction.
Their study used transgenic mice, in which the calcium-

Licensed under Creative Commons Attribution CC-BY 3.0: © the American Physiological Society. ISSN 0022-3077.

－ 117 －

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NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A Map of the auditory cortex

B Map of the auditory cortexDPDP

revealed by Stiebler et al. (1997)AI

revealed by Guo et al. (2012)

UF

UF

AI

AAF

AAF
AII

AII

C Map of the auditory cortex

revealed in Issa et al. (2014)

D Map of the auditory cortex

revealed in the present study.

UF

DP

AI

DM
DA

AI
AAF

AAF

AII

AII

Low

High

Dorsal2901

Fig. 1. Schematic maps of the mouse auditory cortex inprevious studies and the present study. A: schematic map of
the auditory cortex in Stiebler et al. (1997). Frequency
gradients of the anterior auditory ﬁeld (AAF) and primary
auditory cortex (AI) included neurons with characteristicfrequencies up to 40 kHz, and neurons with characteristicfrequencies higher than 50 kHz are located in the distinct
ultrasonic ﬁeld (UF). B: map of the auditory cortex in Guo
et al. (2012). The AAF and AI had ultrasonic bands higher
than 50 kHz. The UF was interpreted to be high-frequency
subparts in the high-frequency area of the AAF and AI but
not a distinct region. A and B: these maps were drawn
according to the results obtained by unit recording. C: mapof the auditory cortex in Issa et al. (2014), showing 2
streams of frequency gradients inside of the AI. The major
gradient runs toward the dorsal part, including the UF area,
whereas the minor gradient runs toward the secondary
auditory ﬁeld (AII). The direction of the frequency organization of the AAF is drawn along the ventrocaudal axis,
consistent with the data reported by Horie et al. (2013). D:
map of the auditory cortex reported in the present study.
Newly delineated, distinct region dorsomedial ﬁeld (DM)was isolated from the AI high-frequency area. The directionof the frequency organization of the AI is the one travelingtoward AII. The direction of the frequency organization of
the AAF matches that reported in the previous studies(Horie et al. 2013; Issa et al. 2014). The region that Stiebler
et al. (1997) deﬁned as UF [referred to as dorsoanterior ﬁeld(DA)] responds to slow-frequency modulation (FM) components, regardless of tonal frequency range. C and D: thesemaps were drawn according to the results obtained by
optical imaging. DP, dorsoposterior ﬁeld.

Rostralsensitive protein GCaMP3 was initially expressed uniformly,
offering much better spatial resolution regarding the auditory
cortical surface than that achieved by unit recording. Principally, however, previous anatomical studies reported that frequency-organized maps in the AAF and AI reﬂect distinct
topological projections from frequency-organized maps in the
medial and lateral parts, respectively, of the ventral division of
the medial geniculate body (MGv; MGB) in mice as well(Horie et al. 2013; Takemoto et al. 2014). Moreover, various
mammals have multiple frequency-organized regions with distinct, unique frequency direction (Higgins et al. 2010; Kalatsky
et al. 2005; Kanold et al. 2014); spectral, temporal, and spatial
sensitivities (Bizley et al. 2009; Imaizumi et al. 2004; Polley etal. 2007); and thalamocortical projections (Lee et al. 2004;
Storace et al. 2010, 2012) without redundancy. The mouse AI,
therefore, would be exceptional if the fork-shaped frequency
gradients existed inside of a single region.
In this study, we tried to clarify whether the frequency
gradient inside of the AI proceeds in a straight line or shows aforked shape. For this purpose, we used a combination of
ﬂavoprotein autoﬂuorescence imaging and anatomical techniques. Flavoproteins are endogenous ﬂuorescent proteins inmitochondria, and ﬂuorescence imaging of these proteinshas been used to map precisely the auditory cortex (Honma
et al. 2013; Horie et al. 2013; Kubota et al. 2008; Ohshima
et al. 2010; Takahashi et al. 2006), visual cortex (Andermann et al. 2011; Tohmi et al. 2009, 2014; Yoshitake et al.
2013), somatosensory cortex (Komagata et al. 2011), andinsular cortex (Gogolla et al. 2014). With the use of thistechnique, we reproduced the ﬁnding that the mouse AI is
divided into two areas in response to high-frequency tones(Issa et al. 2014; Tsukano et al. 2013a). Additionally, we used
SMI-32 immunolabeling, which has been used to partition
various cortical regions (Boire et al. 2005; Budinger et al.
2000; Mellott et al. 2010; Ouda et al. 2012; Paxinos et al. 2009;
Rothschild et al. 2010; van der Gucht et al 2001; Wong andKaas 2009) to investigate the cytoarchitecture in the two AI
high-frequency areas. We found that the dorsal part of the AI
high-frequency area is a different region from the rest of theAI, including the low-frequency area and the ventral highfrequency area, and the posterior frequency-organized region,
which is referred to as the AI, is restricted to the low-frequency
area and the ventral high-frequency area that has been considered as a supplemental AI area in the study by Issa et al. (2014)(Fig. 1D). Existence of multiple frequency organizations in themouse auditory cortex may unify the auditory cortical maps ofmice and other mammals.
METHODS

Animals. The Committee for Animal Care at Niigata University
approved the experimental protocols used in this study. We used 5- to
7-wk-old C57BL/6 mice (n  197; Charles River Japan, Yokohama,Japan), 7- to 9-wk-old Balb/c mice (n  3; Charles River Japan), and
7- to 9-wk-old CBA/CaJ mice (n  3; The Jackson Laboratory, Bar
Harbor, ME). The animals were housed in cages with ad libitum
access to food pellets and water and were kept on a 12-h light/darkcycle. Male and female mice used in the experiments (see Fig. 14)
were 6-wk-old C57BL/6 mice, produced in our institute, and they
were not housed with mice of the opposite sex after weaning at 3 wk
old.
In vivo ﬂavoprotein ﬂuorescence imaging. In vivo ﬂavoprotein
ﬂuorescence imaging was performed, as described in our previous
studies (Takahashi et al. 2006; Tsukano et al. 2013b). Mice wereJ Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 118 －

2902

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NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

deeply anesthetized with urethane (1.7 g/kg ip; Wako, Osaka, Japan),and their rectal temperatures were maintained at 37°C. After local
anesthesia using bupivacaine, the skin and temporal muscle over theright auditory cortex were incised. A piece of metal was attached tothe skull with dental resin, and the head was ﬁxed by screwing themetal piece onto a manipulator. The skull over the auditory cortex was
removed in mice used for some experiments (see Figs. 2B, 3, 4, 6, 8,
12, and 13) to compare response patterns of the auditory cortex or
further tracer-injection experiments. Transcranial imaging was performed in the other experiments. The right auditory cortex was
observed unless otherwise noted. The exposed surface of the intact
skull was covered with liquid parafﬁn (Wako) to keep the skull
transparent in transcranial imaging. Cortical images (128  168 pixels
after binning) of endogenous green ﬂuorescence (  500 –550 nm) inblue light (  470 – 490 nm) were recorded using a cooled chargecoupled device (CCD) camera system (AQUACOSMOS with
ORCA-R2 camera; Hamamatsu Photonics, Hamamatsu, Japan). Images were taken at 9.7 Hz [54 Hz in some experiments (see Figs. 11
and 14, C–E)]. Images were averaged over 20 trials unless otherwise
noted. Spatial averaging of 5  5 pixels was applied. Images were
calculated as ﬂuorescence change (F)/baseline intensity (F0), whereF  F  F0. The F0 was obtained by averaging the intensity values
during the prestimulus period (500 ms). The response amplitude
was evaluated as F/F0 in a circle window with a diameter of 20
pixels. When the frequency organization was evaluated (see Figs. 5
and 7), a circle window with a diameter of 15 pixels was chosen to
give the largest response amplitude, and the location of the center
pixel was considered to be the frequency-speciﬁc response peak.
In vivo two-photon calcium imaging. Calcium imaging was performed using a two-photon microscope (TCS SP5 MP; Leica Microsystems, Wetzlar, Germany) with a hybrid detector (HyD; Leica
Microsystems) and a Ti-Sapphire mode-locked femto second laser(Chameleon Vision; Coherent, Santa Clara, CA), as described in ourprevious studies (Honma et al. 2013; Tohmi et al. 2014; Yoshitake etal. 2013). Calcium-sensitive dye was prepared by dissolving Fura-2
AM (Invitrogen Life Technologies, Boston, MA) in 20% (w/v)
Pluronic F-127 in DMSO (Invitrogen Life Technologies) and diluted
with Ringer solution containing sulforhodamine 101 (SR-101; Invitrogen Life Technologies). After anesthetic induction with urethane(1.9 g/kg ip), craniotomy, and localization using ﬂavoprotein ﬂuorescence imaging, a solution of calcium-sensitive dye was pressure
injected (5–20 kPa) for 5–10 min into layer II/III using glass pipettes(tip diameter: 2– 4 m). Astrocytes were distinguished from neurons
using SR-101. After injection, the pipette was withdrawn, and thecraniotomy was covered with 2% agarose (1-B; Sigma-Aldrich, St.
Louis, MO) and a thin cover glass (thickness 0.15 mm; Matsunami,
Osaka, Japan), which was ﬁxed to the skull with dental cement (Sun
Medical, Shiga, Japan). Excitation wavelength for Fura-2 was 800
nm, and that for SR-101 was 900 –950 nm. Images (256  256 pixels)
were recorded at 3.7 Hz in a 260  260-m region.
The data were realigned using AQUACOSMOS and MATLAB
software (MathWorks, Natick, MA). Data from ﬁve to six trials of thesame stimulation were averaged. Size-matched regions of interest(ROIs) were chosen. Data were calculated as F/F0, where F  F0 
F. The F0 was obtained by averaging the intensity values during theprestimulus period (3 s). The response of each neuron was deﬁned
as the maximum value in poststimulus observation windows (5 s).
Neurons were deﬁned as responsive when the response peak was 4
SD above the baselines during the prestimulus period to minimize
errors by baseline ﬂuctuations. The best frequency (BF) of a neuron
was deﬁned as the frequency to which the neuron had the greatest
response. We examined the correlation between frequency and location and computed a regression line. The direction of the frequency
organization was deﬁned so as to make the correlation coefﬁcient aslarge as possible. Then, we examined the residuals as a measure ofdeviation from the frequency organization. The bandwidth of tuning
curves was deﬁned as the logarithmic ratio of minimum and maxi-

mum frequencies that resulted in a response 75% of the peak
amplitude.
Auditory stimuli. Tones were made by a computer using a customwritten LabVIEW program (National Instruments, Austin, TX) at asampling rate of 500 kHz. Courtship songs of a male mouse, emitted
when he was placed with a familiar female mouse in estrous in thesame cage, were recorded using the recording software AvisoftRECORDER (Avisoft Bioacoustics, Glienicke, Germany) at a sampling frequency of 250 kHz, with a microphone (CM16/CMPA;
Avisoft Bioacoustics) and a preampliﬁer (UltraSoundGate 116; Avisoft Bioacoustics). Sounds were low-pass ﬁltered at 150 kHz (3624;
NF, Kanagawa, Japan). Pure tones at frequencies of 5– 80 kHz were
amplitude modulated by 20 Hz sine wave. A speaker for 5– 40 kHz(SRS-3050A; Stax, Saitama, Japan) or 50 – 80 kHz (ES105A; Murata,
Kyoto, Japan) was set 10 cm in front of the mice. Sound intensity wascalibrated using the microphone (types 4135 and 2669; Brüel & Kjær,Nærum, Denmark) and the sound level meter (type 2610; Brüel &Kjær). The sound intensity was 60 dB sound pressure level (SPL) for
ﬂavoprotein ﬂuorescence imaging and 80 dB SPL for two-photon
imaging. The sound duration was 500 ms with a rise/fall time of 10
ms. The desired sound spectrum was determined using a digitalspectrum analyzer (R9211A; Advantest, Tokyo, Japan) or the customwritten LabVIEW program. When the UF and DP (Stiebler et al.
1997) were activated speciﬁcally, frequency modulation (FM) direction-reversal stimulation (24 kHz/s) was used (Honma et al. 2013).
The sound intensity was set to 60 dB SPL, and the band frequency was
between 5 and 11 kHz.
Acoustic exposure. For acoustic exposure experiments (see Figs. 9
and 10), home cages were placed in the sound-shielded chamber andexposed to a 5- or 35-kHz sound stimulus through a speaker (SRS3050A; Stax) placed above the cage. The exposure sound consisted ofan amplitude-modulated tone with a carrier frequency of 5 or 35 kHz
and modulation frequency of 20 Hz. Duration of the tones was 500
ms, and a rise/fall time was 10 ms. The sound intensity was adjusted
to 70 dB SPL at the ﬂoor of the cage. This tonal stimulus was repeated
at 1 Hz throughout the exposing periods (P7–P35). Mice of normalgroups were reared in the normal cages. Mice of quiet groups werereared in the chamber but not exposed to any tones. Flavoprotein
ﬂuorescence imaging was performed within 1 wk after exposure
was ﬁnished. When a circular window was put on the response of
the dorsomedial ﬁeld (DM) to evaluate response amplitudes, awindow was put to make the response amplitude maximum, kept
22 pixels apart dorsal to the AI response peak, according to thedata (see Fig. 2E).
Retrograde tracer experiments. To visualize neurons in the MGB
projecting to each cortical region, a neural tracer was injected into thecenter of each region identiﬁed by ﬂavoprotein ﬂuorescence imaging(Horie et al. 2013). A glass capillary (tip diameter 20 –30 m) ﬁlledwith tracer solution and a platinum wire was introduced into the centerof the subregion of the right auditory cortex to 500 m below thesurface. Alexa Fluor 488- or 555-conjugated cholera toxin subunit B(CTB; Molecular Probes, Eugene, OR) was used in injections (seeFig. 13). Fluorescein and Texas Red (Molecular Probes) were used insome animals, but the results were the same. Fluorescent CTB
solution (0.5% in phosphate buffer) was injected iontophoretically bya 5-A pulse current (5 s on; 5 s off) for 15 min. In some experiments(see Figs. 8 and 12), biotinylated dextran amine (BDA; molecularweight 3,000; Molecular Probes) was injected iontophoretically by a
5-A pulse current (7 s on; 7 s off) for 15 min. Survival of 3 days for
ﬂuorescent CTB or 7 days for BDA was ensured until perfusion. After
anesthetizing mice deeply with pentobarbital (1.0 g/kg ip), the brains
were dissected and immersed in 4% paraformaldehyde overnight, anda consecutive series of 40-m-thick coronal or horizontal sections
was cut using a sliding cryotome. To observe ﬂuorescent tracers,
sections were mounted on glass slides and covered with Fluoromount(Cosmo Bio, Tokyo, Japan).

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NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

To visualize BDA, sections were rinsed initially in 20 mM PBS andincubated in PBS containing 3% hydrogen peroxide and 0.1% Triton
X-100 for 15 min at room temperature. After rinsing in 20 mM PBS
containing 0.1% Triton X-100 (PBST), the sections were incubatedfor 40 min in 20 mM PBST containing avidin-biotin peroxidase
complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA).
Sections were rinsed in 20 mM PBS, and BDA was visualized in asolution comprising 0.05% diaminobenzidine tetrahydrochloride and0.003% hydrogen peroxide in 50 mM Tris-HCl buffer (pH 7.4) for 20
min. All sections were ﬁnally, thoroughly rinsed in 50 mM Tris-HCl
buffer and mounted onto gelatin-coated slides. Adjacent sections werecounterstained using 0.1% cresyl violet (Chroma Gesellschaft, Kongen, Germany). After the mounted sections had dried, they were
dehydrated in a graded ethanol series, cleared in xylene, and coverslipped using the covering reagent Bioleit (Okenshoji, Tokyo, Japan).
The borders of the MGB subdivisions were delineated, according to
the SMI-32 immunolabeling pattern (Honma et al. 2013; Horie et al.
2013; LeDoux et al. 1985, 1987) and an atlas (Paxinos 2003; Paxinos
and Franklin 2001; Paxinos et al. 2009). The immunohistochemistryof SMI-32 reacts with a nonphosphorylated epitope in neuroﬁlament
M and H (NNF). Sections were rinsed and incubated in PBST
containing 3% hydrogen peroxide, as described for BDA visualization. After rinsing in 20 mM PBS, the sections were incubatedovernight at room temperature with the monoclonal antibody (MAb)
SMI-32 (1:2,000; Covance Research Products, Berkeley, CA) (Sternberger and Sternberger 1983), diluted with 20 mM PBS containing
0.5% skim milk. Sections were then incubated in anti-mouse IgG(1:100; MBL, Nagoya, Japan) at room temperature for 2 h. The
sections were rinsed in 20 mM PBS, and the immunoreactions werevisualized in a Tris-HCl buffer containing 0.05% diaminobenzidine
tetrahydrochloride and 0.003% hydrogen peroxide for 5 min at room
temperature. After visualization, the sections were coverslipped. All
sections were observed under the light microscope (Eclipse Ni; Nikon,
Tokyo, Japan) and a CCD camera (DS-Fi2; Nikon). The drawings andimages were prepared using CorelDRAW (E Frontier, Tokyo, Japan),Illustrator (Adobe Systems, San Jose, CA), and Photoshop (Adobe
Systems) software.
AAV injection to investigate interhemispheric connectivity. To
conﬁrm that the corresponding subareas have interhemispheric connections with each other via the corpus callosum, adeno-associatedvirus (AAV) vector for GCaMP3 expression (Penn Vector Core,
Philadelphia, PA) was injected into an area of the right AI after
functional identiﬁcation, which stains the axonal branches reaching
the contralateral auditory cortex (see Fig. 3). A glass capillary (tipdiameter: 20 –30 m) ﬁlled with AAV solution (500 L) was introduced to a depth of 500 m from the surface and slowly pressure
injected for 30 min by a custom-made pump. After injection, the holein the skull was covered with 2% agarose (1-B; Sigma-Aldrich), andthe skin was sutured. Mice were recovered from anesthesia in theirhome cages. Two weeks after the injection, the left auditory cortex
was observed using an epiﬂuorescence microscope [excitation (Ex),470 – 490 nm; emission (Em), 500 –550 nm] and a two-photon microscope (Ex, 900 nm; Em, 500 –550 nm).
Statistics. The Mann-Whitney U-test or Wilcoxon signed-rank test
was used to evaluate differences between unpaired or paired data fromtwo groups, respectively. When data from three groups were compared, Tukey-Kramer test was used as a post hoc test after ANOVA.
The correlation coefﬁcients and the P values were calculated using
Spearman rank correlation test. When the difference between two
slopes was evaluated, the slope test was performed. The 2 test wasused to evaluate the difference between two percentages. These
tests were performed using SPSS (IBM, Tokyo, Japan) or MATLAB software. All of the data in the text and graphs are presented
as means  SE.

2903

2904

RESULTS

Finding the new, distinct region identiﬁed from the AI. Prior
imaging studies have identiﬁed distinct auditory cortical regions based on response magnitudes to tones and the topographic organization of tone-frequency responses (cochleotopy) (Harel et al. 2000; Horie et al. 2013; Kubota et al. 2008;
Sawatari et al. 2011; Takahashi et al. 2006). In this study, we
try to conﬁrm frequency organizations by demonstrating distinct response magnitude peaks and mirror-reversed frequency
organization in the AAF and AI using ﬂavoprotein ﬂuorescence imaging. In addition, we try to observe ﬂavoprotein
ﬂuorescence responses precisely within a cortical region, typically deﬁned as the AI in mice, according to the Paxinos
anatomic atlas (Paxinos and Franklin 2001). First, we observed
neural responses in the right auditory cortex of C57BL/6 micein a 5- to 70-kHz range after craniotomy (Fig. 2A). The
frequency-organized map of the AI was arranged in a caudalrostral direction. Although only one area was activated in
response to a 5-kHz tone, the AI responses to a 40-kHz tone
were observed in two different areas, which were dorsoventrally separated from each other, as reported previously (Fig.
2B) (Issa et al. 2014; Tsukano et al. 2013a). We also conﬁrmedthat the AI high-frequency area showed two different response
peaks when stimulated by high-frequency tones using transcranial ﬂavoprotein ﬂuorescence imaging (Fig. 2C). Signiﬁcantly separated double peaks in F/F0 were found by positioning serial ROIs across both dorsal and ventral AI highfrequency areas (Fig. 2E; P  0.01 between ROI 6 vs. 11; P 
0.05 between ROI 11 vs. 20). These data conﬁrmed thepresence of apparently separated AI high-frequency areas inmice. The regions overlapped to the places where Stiebler et al.
(1997) drew the UF and DP in their auditory cortical map,
hereby referred to as Stiebler’s UF and DP in this report, wereconﬁrmed by several studies (Honma et al. 2013; Joachimsthaler et al. 2014; Stiebler et al. 1997), and both areas are known
to be activated by directional changes of slow FM sounds(Honma et al. 2013; Tsukano et al. 2013b). Moreover, corticalresponses to ultrasonic sounds are not speciﬁc enough to divide
the broad area into speciﬁc subareas. Therefore, we veriﬁed
whether the dorsal high-frequency area of the AI overlaps
Stiebler’s UF or DP using FM directional changes. The dorsal
area of the AI mapped between Stiebler’s UF and DP (Fig.
2D). To evaluate these data quantitatively, we positioned ROIs
across Stiebler’s UF and DP, and the same ROIs were placed
onto the image of the responses to a 35-kHz tone obtained fromthe same mouse (Fig. 2F). The response peaks of the dorsal
area of the AI did not overlap Stiebler’s UF or DP (Fig. 2F;P  0.05 at ROI 9; P  0.05 at ROI 21; P  0.05 at ROI 31).
The observation that the AI was divided into two areas in the
response to high-frequency tones was also made in the left
auditory cortex (data not shown). We visualized the auditory
cortex in CBA/CaJ and Balb/c mice as well. The structures of
responses of the AAF, AI, and AII were the same among threestrains, and the AI was divided into two areas in response tohigh-frequency tones (data not shown). These results, alongwith prior studies, suggest that intrinsic response peaks identifyfunctionally distinct regions in the auditory cortex (Kalatsky et
al. 2005).
It remains unclear how auditory cortical regions, deﬁned
according to sound-response magnitudes, align with traditional

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 120 －

Fig. 2
media
obtainwerecauda(a) an
the A
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Wilco
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ﬁned
onal

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A

B

bregmaVC

5 kHz

40 kHzSuperimposed

SC

Cb AC

MCA

OB

rf

Dorsal

C

AI

AI

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AAF

AII

AII

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AI

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-0.8%

b
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40 kHz

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1 mm

D

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35 kHz tone

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40 ROIs

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2904

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2903

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ROIs on image of 35 kHz

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40

Fig. 2. Two distinct responses to high-frequency tones in the AI. A: schematic drawing of the mouse auditory cortex. Auditory cortex (AC); cerebellum (Cb);medial cerebral artery (MCA); olfactory bulb (OB); rhinal ﬁssure (rf); somatosensory cortex (SC); visual cortex (VC). B: neural responses to a 5- or 40-kHz tone
obtained by ﬂavoprotein ﬂuorescence imaging in a mouse with craniotomy. The AI in response to a 40-kHz tone area was divided into 2 parts. Three images
were obtained from the same mouse. Scale bar, 1 mm. C: neural responses to 5–70 kHz tones revealed by transcranial ﬂavoprotein ﬂuorescence imaging. The
caudal AI low-frequency area was clearly activated. When 20 –70 kHz tones were presented, the AI high-frequency area was divided into 2 parts: the dorsal part(a) and the ventral part (b), indicated by white arrows. Four images were obtained from the same mouse. D: positional relationship between the dorsal part of
the AI high-frequency area and Stiebler’s UF and DP. Stiebler’s UF and DP were activated by slow FM directional changes (Honma et al. 2013). Three images
were obtained from the same mouse. E: quantitative analysis of response slopes by a 35-kHz tone. Regions of interest (ROIs) were placed across the 2 highest
peaks in the AI high-frequency areas, and the signiﬁcant double peaks in ﬂuorescence change (F)/baseline intensity (F0) were drawn (*P  0.05; **P  0.01;
Wilcoxon signed-rank test, 10 mice). Each value was normalized as percent of the peak value at ROI 6, which was put on the response peak of the dorsal AI
high-frequency area and used as a landmark to register across individuals. Sites a and b are equivalent to those shown in C. F: quantitative analysis of response
slopes on the response in the experiment shown in D. ROIs were placed across the peaks in Stiebler’s UF and DP, and the same ROIs were placed onto the imageof the responses to a 35-kHz tone obtained from the same mouse (inset). The red line was obtained from the response to a 35-kHz tone, and the blue line wasobtained from the response to FM directional changes in the same mouse. The response peak derived from the dorsal AI high-frequency area was placed in the
middle of Stiebler’s UF and DP (*P  0.05; Wilcoxon signed-rank test, 6 mice). Each value was normalized as percent of the peak value at ROI 30, which was
put on the response peak of Stiebler’s UF and used as a landmark to register across individuals. Transcranial imaging was performed in experiments C–F.
J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 121 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

cytoarchitectonic partitions. To address this problem, we investigated the cytoarchitectural pattern of the AI low-frequency area, the ventral AI high-frequency area, and the dorsal
AI high-frequency area (see Fig. 4A) after identifying theregions by ﬂavoprotein optical imaging. We characterizedthese areas histologically by immunolabeling NNF using MAb
SMI-32, which is widely used to partition and identify various
cortical and brain regions, including the auditory cortex(Budinger et al. 2000; Mellott et al. 2010; Ouda et al. 2012;
Rothschild et al. 2010), MGB (Honma et al. 2013; Horie et al.
2013; Paxinos et al. 2009), visual cortex (Boire et al. 2005; vander Gucht et al 2001; Wong and Kaas 2009), and other regions.
To perform this analysis, it is necessary to determine the lowand high-frequency areas of the AI on slice sections. However,mouse monoclonal SMI-32 immunolabeling using mouse IgG
as a secondary antibody gave high-background staining aftermarking the identiﬁed area with injection of ﬂuorescent beads,
dye, or ink. Therefore, we injected BDA into an identiﬁed areaof the AI in the right hemisphere in vivo and then observed
NNF cytoarchitecture in the left AI on slice sections guided byaxonal branches stained with BDA projecting from neurons in
the right AI. It is widely known that the corresponding areas
have interhemispheric projections via the corpus callosum inmammalian brains (Budinger et al. 2000; Oviedo et al. 2010;
Rouiller et al. 1991; Xiong et al. 2012), as shown in the schemaof Fig. 3A. We conﬁrmed this fact in the present study as well(Fig. 3). After we identiﬁed the precise location of the subregions in the right auditory cortex using ﬂavoprotein ﬂuorescence imaging, we injected AAV-GCaMP3 solution into theAI 5-kHz area (Fig. 3, B–D). This technique visualizes theaxon terminals originating from the injected subarea in vivo
(Glickfeld et al. 2013; Oh et al. 2014). Two weeks after
injection of AAV-GCaMP3 into the AI 5-kHz area in the righthemisphere, the ﬂuorescent axon terminals were observed in
the contralateral left AI 5-kHz area (Fig. 3E). GCaMP3positive axon terminals were visualized using a two-photon
microscopy in the place where an intense signal was observedin Fig. 3E (Fig. 3F), whereas no GCaMP3-positive terminals
were observed outside of the AI (Fig. 3G). The large calcium
responses to a 5-kHz tone derived from GCaMP3-positive
terminals were observed in the left AI 5-kHz area (Fig. 3H),
and the responses of the AAF and AII were close to thoseobserved in a naïve mouse (Fig. 3D). These data conﬁrm that
it is possible to observe the NNF pattern of the identiﬁed areain the contralateral auditory cortex on slice sections, guided byaxon terminals, stained by BDA injected into the right auditory
cortex.
The NNF immunolabeling pattern appeared much denser inthe dorsal part than in the ventral part of the AI high-frequency
area or the AI low-frequency area, whereas the weak NNF
immunolabeling pattern appeared in the latter two areas (Fig. 4B).
Moreover, the dorsal part of the AI high-frequency area appeared to have the particular laminar pattern of immunolabeling. To analyze quantitatively the difference in NNF immunolabeling patterns, we ﬁrst counted the number of NNF-positive
neurons in each area. However, labeled neurons were notsigniﬁcantly different in number among the three areas (Fig.
4C). NNF immunolabeling clearly distinguished pyramidal
from nonpyramidal neurons, the two most common neuron
types. Pyramidal neurons had NNF-positive apical dendrites,
reaching the superﬁcial layer, and a densely labeled large soma

2905

(Fig. 4D). Conversely, nonpyramidal neurons had few NNFpositive apical and basal dendrites, and their soma was labeled
weakly. Therefore, the difference in the ratios of NNF-positive
pyramidal neurons to nonpyramidal neurons could account for
the apparent density difference with the NNF immunolabeling
patterns. In layers II, III, and IV, the ratio of NNF-positive
pyramidal/nonpyramidal neurons did not differ noticeably, andnonpyramidal neurons were dominant in all three areas (Fig.
4E). However, the deeper layers V and VI in the dorsal AI
high-frequency area were almost fully occupied by NNFpositive pyramidal neurons. The ratio of NNF-positive pyramidal/nonpyramidal neurons was signiﬁcantly higher than thatobserved in the other two areas [Fig. 4F; P  0.05 (area a vs.
c); P  0.05 (area b vs. c)] when compared in layers V, VI, and
II–VI, whereas there was no signiﬁcant difference between
areas a and b in any layer. Thus the presence of numerous
NNF-positive pyramidal neurons in the deeper layers could
explain the dense NNF immunolabeling pattern in the dorsal
AI high-frequency area, since these neurons have thick NNFpositive basal dendrites and apical dendrites that reach superﬁcial layers. These data (Figs. 2 and 4) strongly suggest that thedorsal AI high-frequency area, located between Stiebler’s UF
and DP, is likely a newly deﬁned region with a different
cytoarchitectural pattern. We tentatively refer to this new area
as the DM (Fig. 1D).
Frequency organizations of the AI and DM. We next reevaluated the precise frequency organizations of the auditory
cortex based on the ﬁnding that the dorsal AI high-frequency
area is a newly deﬁned region. We investigated frequency
gradients by plotting the response peaks using ﬂavoprotein
ﬂuorescence imaging described in our previous study (Fig. 5)(Honma et al. 2013). The posterior frequency-organized region
labeled as the “AI,” has a low-to-high (5– 80 kHz) tonefrequency response axis directed from the dorsocaudal to theventrorostral temporal cortex (Fig. 5, A and B). The frequency
gradient of the AI ran in a mild clockwise direction. The
corresponding frequency gradient for the newly identiﬁed DM
was directed from ventrocaudal to dorsorostral anatomic axis
(Fig. 5, A and C). Similar maps were obtained using othersound-intensity levels (for example, at 40 dB SPL; data notshown). The slopes of the AI and DM frequency gradientswere not signiﬁcantly different (P  0.9) using comparison ofregression coefﬁcients. Both the AI and DM have high-frequency bands inside of their frequency gradients. The ratios of
the response amplitudes of the DM and AI were plotted against
the stimulus frequency to investigate whether the response
property was similar in these two regions (Fig. 5, D and E).
Although the AI is responsive to ultrasonic sounds, the DM ismore responsive to ultrasonic sounds over 40 kHz than is the
AI (Fig. 5E). These data suggest that the DM and AI havedistinct frequency gradient directions for tone-response topographies and that the DM responds more vigorously to highfrequency sounds than the AI.
Differences in robustness of frequency gradients between
regions have been conﬁrmed using two-photon imaging (Issa et
al. 2014). We have shown that the DM and AI have a robust,
frequency-organized structure using ﬂavoprotein ﬂuorescenceimaging. To evaluate the extent of heterogeneity in the frequency organization at the high-resolution scale, we performedin vivo two-photon imaging to determine the properties ofneurons in the supragranular layers in the AI and DM (Fig. 6),

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 122 －

2906

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2906

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A

ipsi subareas

Flavoprotein
fluorescenceimaging&
AAV-GCaMP3
injection to the AI

Fig. 3. Interhemispheric projection between
the auditory cortices. A: schematic drawing
showing that a neuron in the subarea in theauditory cortex projects to the equivalent
contralateral subarea. B–D: injection site ofadeno-associated virus (AAV)-GCaMP3 in
the right auditory cortex. The areal borders
were drawn according to the ﬂavoprotein
ﬂuorescence response to a 5-kHz tone (C). C
and D: the images shown are the same except
for the color range. , injection site. E:
GCaMP3-positive axon terminals in the AI
5-kHz area in the left auditory cortex observed using a stereoscopic microscope. The
top 10% pixels in intensity are colored green.
F: GCaMP3-positive axon terminals visualized using a 2-photon microscopy in theplace where an intense signal was observedin E. G: no GCaMP3-positive terminals
found outside of the AI 5-kHz area. H: thelarge calcium responses to a 5-kHz tone
derived from GCaMP3-positive terminals in
the left AI. This response was obtained using
an epiﬂuorescent microscope under the same
experimental condition as that to perform usual
ﬂavoprotein ﬂuorescence imaging. Only the AI
showed a much larger response in H due to
GCaMP-stained terminals. Flavoprotein ﬂuorescence responses, which were small compared with GCaMP signals, were observed in
the 5-kHz area in the AAF and AII. Scale bars,
1 mm (B); 10 m (F and G).

Barea A

contra subareas

area Barea B’area A’

E

Right

AAF

AI

Left

AI

AAF
AII

AII
C

Flavoprotein
fluorescenceimaging&
Two-photon
imaging

Flavoprotein imaging

Two-photon imaging

F

G

AAF

AI
AII
-2.5%

D

+2.5%

Flavoprotein imaging

AI

H

AAF

AII

AII
-4.5%

+4.5%

DorsalRostralsince the regional characteristic obtained by low-resolution
imaging reﬂects an ensemble of the single neuronal properties
in layers II/III (Andermann et al. 2011; Marshel et al. 2011;
Tohmi et al. 2014). After identifying the precise location of the
AI or DM using ﬂavoprotein ﬂuorescence imaging (Fig. 6A),
we injected Fura-2 solution to observe the calcium responses ineach neuron (Fig. 6B). The frequency at which the response
amplitude reached the maximum in a given neuron was deﬁned
as the BF (Fig. 6C). Although the overall frequency gradientsin the DM and AI had the same directions as those deﬁned
using ﬂavoprotein ﬂuorescence imaging, the frequency gradient in the DM neurons was less variable than that in the AI(Fig. 6, D and E). The degree of the frequency organization of

AI

AAF

-4.5%
Rostral+4.5%
Dorsalthe microscopic structures was evaluated using residuals between each neuron and the regression line. The value ofresiduals was greater in the AI than in the DM (Fig. 6F; P 
0.001), implying that neuronal responses in layers II/III of the
AI were more complex than those in the DM.
The DM was less sensitive to low-frequency tones than the
AI in ﬂavoprotein ﬂuorescence imaging (Fig. 5E). This was
explained by the neuronal distribution revealed by two-photon
imaging; there were few neurons in the DM with BF 20 kHz(Fig. 6, G and H). In contrast, the bandwidth of the neurons in
the DM and AI was similar (Fig. 6I). A tendency that bandwidth values obtained in two-photon studies, including thisstudy, are smaller than those in electrophysiological study

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 123 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A(site “a”)

(site “b”)

I

c

II

b

III
VI
V

Low
High

(site “c”)

I
II

I

III

II

VI
V

III
VI
V

IV

IV

IV

DorsalRostralLateralF

Ratio ofPyramidal / Non-pyramidal
Ratio ofPyramidal / Non-pyramidal

E

Number of
NNF-positive neurons

DorsalC

site “a”

30

site “b”

D NNF+

NNF+
Pyramidal Non-pyramidalsite “c”

20
10
0

II

III

IV

V

VI Layer

2

2

2

1

1

1

0

2908

DorsalB Low frequency area Ventral
high frequency area high frequency area

Flavoprotein
imaging

AI
a

2907

a

b

Layer II

c

*

10
8

0

8
6

4

4

2
1
0

2
1
0

b

Layer Vcb

Layer III

c

*

10

6

a

a

0

a

b

Layer IV

Fig. 4. Histological properties of the new region
different from the AI in SMI-32 immunolabeling. A:
functional location of the AI obtained using ﬂavoprotein ﬂuorescence imaging. Scale bar, 500 m. B:
sample images of SMI-32 immunolabeling patterns.
We observed nonphosphorylated neuroﬁlament(NNF) at sites a, b, and c, indicated in A. Scale bar,100 m. C: number of NNF-positive neurons in eacharea of the AI. Layers were identiﬁed based on Nissl
staining in an adjacent section. D: typical images ofan NNF-positive pyramidal and nonpyramidal neuron. E: ratio of the number of NNF-positive pyramidal/nonpyramidal neurons in the superﬁcial layers(II, III, and IV). F: ratio of the number of NNFpositive pyramidal/nonpyramidal neurons in the deep
layers (V and VI) and all of the layers (II–VI). *P 
0.05, Tukey-Kramer’s test. Site a, n  6; site b, n 
6; site c, n  7. NNF and biotinylated dextran amine(BDA) were visualized in the adjacent tissue
sections.

cFig. 5revea
2 freq
AI an
tein
20-kH
origin
anima
m.
respo
60 kH
and 8
inset:
red, 5quenc
scalesconne
0.001
in the
frequ
0.001
was 1
again
(right
each
Spear
10 kH
kHz,
n3

*

3
2
1

a

b

Layer VI

c

0

a

b

c

All layers (II – VI)(Guo et al. 2012) might be attributed to the property of a
calcium-sensitive dye in detecting spikes.
Frequency organizations of the AAF. We also found thepresence of the precise frequency organization in the AAF(Fig. 7). The neural response to a 5-kHz tone was placed at themost rostral part of the AAF. When mice heard tones at 5– 80
kHz, neural responses to higher tones shifted to the ventrocaudal direction. No clear response was found near Stiebler’s UF
indicated by the white arrow (Fig. 7A, inset). The plotting of
response peaks elicited by tones at 5– 80 kHz also conﬁrmed
the presence of the precise octave-based frequency organization in the AAF (Fig. 7, B and C), as reported previously (Horieet al. 2013; Issa et al. 2014). Therefore, the low-high andhigh-low mirror-imaged frequency organizations between the

AAF and AI, reported in other species, were preserved in mice
as well.
There is a discrepancy in the direction of the frequency
organization of the AAF between studies using optical imaging(Fig. 7) (Honma et al. 2013; Issa et al. 2014) and studies using
electrophysiology (Guo et al. 2012). The latest mapping study
using electrophysiology considered the whole area, which wascomposed of the AAF, Stiebler’s UF, and the DM in this study
as a single region of the AAF, as shown by the schema in Fig. 8A(Guo et al. 2012), and the direction of the frequency organization of the AAF was drawn dorsocaudally (Fig. 1C). Therefore, we investigated NNF immunolabeling patterns and anatomical properties in the AAF, Stiebler’s UF, and the DM (Fig.
8, B–E) to conﬁrm our physiological mapping shown in Fig. 7.

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 124 －

First
UF
imm
than
diffe
the
NNF
of N
muc
show
labe
and

mice

ency
ging
sing
tudy
was
tudy
. 8A
aniereanaFig.
g. 7.

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A

DM
DP

Stiebler’s UF

5 kHz
10 kHz
20 kHz
30 kHz40 kHz
50 kHz60 kHz70 kHz80 kHzAI

DorsalRostral

B

600

600

450

450

300

300

150

150

0

0

-150

Frequency (linear) (kHz)

D

400
300
200
100

5 10 20 40 80

Response in AI

Response in DM
1.6

1.4

1.4

1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0

5 10 20 30 40 50 60 70 80

Frequency (kHz)-100

Frequency (Octave) (kHz)1.6

First, NNF immunolabeling patterns of the AAF and Stiebler’s
UF were totally different (Fig. 8C); apparently, the grossimmunolabeling pattern of Stiebler’s UF was much weaker
than that of the AAF (Fig. 8C). Although there was nodifference in number of total NNF-positive neurons between
the AAF and Stiebler’s UF (Fig. 8D), Stiebler’s UF lacked
NNF-positive neurons in the layer VI (Fig. 8E), and the densityof NNF immunolabeling within somas and dendrites per se wasmuch weaker in Stiebler’s UF, especially in the layer III,
shown in Fig. 8C. Furthermore, the density of NNF immunolabeling in the DM was denser than that in the AAF (Figs. 4B
and 8C), which is because the DM contained more than twice

500

0

-150

5 10 20 30 40 50 60 70 80

C

10

20

40

80

Frequency ( Octave ) (kHz)

E
1.6
1.4

DM / AI

Fig. 5. Frequency organizations of the AI and DM
revealed by ﬂavoprotein ﬂuorescence imaging. A:
2 frequency gradients for 5– 80 kHz sounds in the
AI and newly identiﬁed DM revealed by ﬂavoprotein ﬂuorescence imaging. The location of the
20-kHz response peak in the AI was used as theorigin of coordinates on which data from several
animals were superimposed. Black scale bar, 200
m. See color-coding key on right. Top inset:
response peak shifts in DM. Blue, 40 kHz; green,
60 kHz; red, 80 kHz; yellow, overlap between 60
and 80 kHz. White scale bar, 100 m. Bottom
inset: blue, 5 kHz; green, 10 kHz; yellow, 20 kHz;red, 50 kHz. White scale bar, 100 m. B: a frequency gradient of the linear (left) and the log
scales (right) in the AI. The horizontal axis is a line
connecting points of 5 and 80 kHz (r  0.85; P 
0.001, Spearman’s rank correlation test). The slope
in the octave axis was 1.01 octave/125 m. C: afrequency gradient in the DM (r  0.69; P 
0.001, Spearman’s rank correlation test). The slope
was 1.17 octave/125 m. D: response amplitudes
against each frequency in the AI (left) and DM(right). E: ratio of amplitudes of the DM to AI byeach stimulus frequency (r  0.63, P  0.001,
Spearman’s rank correlation test). 5 kHz, n  15;10 kHz, n  5; 20 kHz, n  15; 30 kHz, n  8; 40
kHz, n  6; 50 kHz, n  7; 60 kHz, n  8; 70 kHz,n  3; 80 kHz, n  3.

Distance (μm)

egion
g. A:
ﬂavom. B:
erns.
ment
e bar,
each
Nissl
es of
neuramiayers
NNFdeep
*P 
,n
mine
issue

2908

Δ F/F 0 (%)

2907

1.2
1.0
0.8
0.6

5 10 20 30 40 50 60 70 80

Frequency (kHz)0.4

5 10 20 30 40 50 60 70 80

Frequency (kHz)as many NNF-positive neurons as did the AAF and Stiebler’s
UF (Fig. 8D). These data indicate that the area that was deﬁned
as the AAF in the past study (Guo et al. 2012) (Fig. 8A)
includes three subregions with a distinct immunolabeling
pattern.
The Stiebler’s UF is more responsive to slow FM stimuli
than to tones and does not have any frequency-organizedstructure (Honma et al. 2013; Stiebler et al. 1997), suggestingthat the Stiebler’s UF belongs to the belt region that receives
thalamic inputs from the dorsal division of the MGB (MGd)(Winer and Schreiner 2011). Therefore, we investigated fromwhich division of the MGB the Stiebler’s UF receives dense

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 125 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

2-photon imaging

C5

10 20 30 40 50 60 70 80

AAF

1

0
DorsalRostral
(kHz)
80
70
60
50
40
30
20
10
5

80

Frequency (kHz)

DM

AI

DM

H

40

2910

***

50

25

0

AI

***

58%

DM

16%

20
10

AI

5

E AI

G

***

(kHz)

AII

D

F

Frequency (kHz)

DM
AI

B

DM

BF < 20 kHz
BF ≥ 20 kHz

Frequency (kHz)

80

I

40
20
10
5

0.4

BW (Octave)

Flavoprotein imaging

Residual

A

2909

0.2

0

AI

DM

Fig. 6. Frequency organizations of the AI and DM revealed by 2-photon imaging. A: identiﬁcation of subregions in the auditory cortex using ﬂavoprotein
ﬂuorescence imaging. The image was obtained using a 35-kHz tone. Scale bar, 1 mm. B: example of staining with Fura-2 and sulforhodamine 101 in the auditory
cortex. Scale bar, 50 m. C: sample traces of calcium response in a neuron with a best frequency (BF) of 60 kHz. Horizontal scale, 3 s; vertical scale, 2%;
triangles, timing when a tone was given. A series of sounds at 80 dB sound pressure level was presented to a mouse. D: distribution of neurons in the DM. Data
were obtained from 3 animals, and images were superimposed on the location of a 5-kHz response peak in the AI as the origin of coordinates obtained using
ﬂavoprotein ﬂuorescence imaging. Color of each plot represents its BF. A signiﬁcant, progressive increase in frequency was observed (P  0.05, Spearman’srank correlation test; 163 neurons). Scale bar, 200 m. E: distribution of neurons in the AI. Data were obtained from 4 animals and reconstructed. A signiﬁcant,progressive increase in frequency was observed (P  0.0001, Spearman’s rank correlation test; 598 neurons). Scale bar, 200 m. F: comparison of the degreeof the frequency organization between the AI and DM. The extent of disorder was evaluated using the distances of each neuron from the regression line (***P  0.001,
Mann-Whitney U-test). G: averages of BFs (***P  0.001, Mann-Whitney U-test). H: percentages of neurons with BF  10 kHz (***P  0.0001, 2 test). I:
comparison of neuron bandwidths (BW) between the AI and DM, deﬁned as the logarithmic ratio of minimum and maximum frequencies that resulted in a
response 75% (P  0.2, Mann-Whitney U-test) of the peak amplitude.

projections (Fig. 8F). We injected BDA solution into the
Stiebler’s UF as a retrograde tracer after identifying the region,according to the response to FM reversal stimuli (Fig. 2D), andevaluated neuronal distribution using coronal sections between
2.8 and 3.4 mm posterior to the bregma. The MGv and MGd
were partitioned according to the SMI-32 immunolabeling
patterns (Honma et al. 2013; Horie et al. 2013; LeDoux et al.
1985, 1987) and an atlas (Paxinos 2003; Paxinos and Franklin
2001; Paxinos et al. 2009). Whereas 64.6% neurons of BDAstained neurons were placed in the MGv, as much as 35.4%
neurons were placed in the MGd (Fig. 8F). These data areclearly consistent with the results reported by Hofstetter andEhret (1992), showing that the Stiebler’s UF received robust
thalamic projections from both MGd and MGv. In contrast, asfor the region referred to as the AAF in the present study, 90%of the thalamic inputs originated from the MGv, and only 10%
originated from the MGd (Fig. 8F), which is consistent withthe fact that the AAF is one of the lemniscal core regions.
Furthermore, we injected BDA solution into the DM to clarify
the origin of the thalamic input. The DM presumably receives
thalamic information from the MGv but not from the MGd,because the DM has the clear frequency organization shown in
Fig. 5. As expected, it was revealed that as much as 97%
neurons projecting to the DM were localized in the MGv, andremaining 3% neurons were located in the MGd (Fig. 8F).
Overall, these data indicate that the AAF, Stiebler’s UF, andDM are not combined into a single region but three different
regions with distinct histological and anatomical properties.
Hence, these data support our physiological mapping thatreveals that the frequency gradient of the AAF travels ventrocaudally (Fig. 7).
Comparison of the effects of acoustic exposures between the
AI and DM. The number of cortical neurons with a particularcharacteristic frequency increases after animals are passively
exposed for a long time to the tonal stimuli at the same
frequency (de Villers-Sidani et al. 2007; Nakahara et al. 2004;
Zhang et al. 2001). This plastic change can be observed, as
response amplitude increases when observed by ﬂavoprotein
ﬂuorescence imaging (Takahashi et al. 2006; Tohmi et al.
2009). The potentiation induced by the passive acoustic exposure exhibits regional and tonal speciﬁcity. Whereas the AAF
is relatively insensitive to the passive acoustic exposure (Taka-J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 126 －

Fig. 7(rightto 5–data i
kHz,

hash
the
2004
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2910

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A

5 kHz

superimposed

30 kHz

DM

AI
AII
-1.0%

B

AAF

AI

5 kHz area
overlapped area

AAF

AAF

30 kHz area

AII
+1.0%

-1.5%

+1.5%

DorsalRostral

1 mm

C

AI

400

AAF

5 kHz
10 kHz
20 kHz
30 kHz40 kHz
50 kHz60 kHz70 kHz80 kHz300

Distance (μm)

2909

200
100
0
-100

200 μm5

10 20 40 80

Frequency (Octave) (kHz)Fig. 7. Frequency organization of the AAF and AI. A: images of responses to a 5 (left)- and 30 (middle)-kHz tone and a superimposed image of these 2 images(right). The white arrow indicates the region deﬁned as the high-frequency area of AAF in Guo et al. (2012). B: frequency-organized maps of the AI and AAF
to 5– 80 kHz sounds revealed by ﬂavoprotein ﬂuorescence imaging. The response peak of AI to 20 kHz is used as the origin of coordinates to superimpose thedata in several animals. C: frequency gradient of the AAF. The horizontal axis is a line connecting points of 5 and 80 kHz (5 kHz, n  15; 10 kHz, n  5; 20
kHz, n  15; 30 kHz, n  8; 40 kHz, n  6; 50 kHz, n  7; 60 kHz, n  8; 70 kHz, n  3; 80 kHz, n  3; P  0.001).

hashi et al. 2006), the AI responses are clearly potentiated by
the exposure (de Villers-Sidani et al. 2007; Nakahara et al.
2004; Zhang et al. 2001). We tested whether the plastic change
to acoustic exposure was observed in the DM, which is expected to have different properties from those in the AI. We
observed that when mice were reared under 5 kHz tone
exposure from P7 to P35 (Fig. 9A), signiﬁcant potentiation ofneural responses to a 5-kHz tone occurred only in the AI(F/F0 in normal environment, 0.68  0.06%, n  10; quiet
environment, 0.64  0.05%, n  6; exposed, 0.87  0.04%,
n  14), and such potentiation was not found in the AAF or
AII (Fig. 9, B and C). In the AAF, AI, AII, and DM, theresponses to a 35-kHz tone were not clearly potentiated after
exposure to a 5-kHz tone because of the frequency speciﬁcity
in the potentiation (Fig. 10A), as reported previously (Takahashi et al. 2006). When mice were exposed to a 35-kHz tone,
signiﬁcant potentiation was observed only in the AI (normal
environment, 0.95  0.08%, n  10; quiet environment, 0.95 
0.05%, n  6; exposed, 1.09  0.03%, n  14), whereas nopotentiation was observed in the DM as in the AAF or AII(Fig. 9, D and E). Although the size of responsive areas after
the plasticity occurred seemed to be expanded in an experience-dependent manner, the location of the AI peak was
not shifted by the plasticity. Therefore, it is unlikely that the
AI and DM have merged by the plasticity. The neural
responses were not potentiated in all three areas to a 5-kHz
tone (Fig. 10B). Flavoprotein ﬂuorescence imaging showedthat responses of the DM to a 5-kHz tone were very weakand diffused and did not show robust, clear peaks in re-sponse to a 5-kHz tone (Fig. 2B). Moreover, single neuronalanalysis showed that there were few low-frequency neurons
in the DM (Fig. 6). Therefore, reliable analysis was notpossible regarding DM responses to a 5-kHz tone, and weused only a 35-kHz tone to evaluate the effect of acoustic
exposures. Overall, the resistance to plasticity of the DM
indicates that the DM is a functionally distinct region fromthe AI.
Thalamocortical projections from the MGB to the DM.
Auditory cortical regions have different latencies to auditory
stimuli across species. In previous studies, neurons in the AAF
were activated with shorter latencies than those in the AI(Kubota et al. 2008; Linden et al. 2003; Sawatari et al. 2011).
We veriﬁed the latency of the neural response to a 35-kHz tonein the DM using ﬂavoprotein ﬂuorescence imaging (Fig. 11).
The latency was evaluated as the time until the ﬂuorescencesignal reached 25% or 50% maximum of the peak. The latencyof the AAF response to a 35-kHz tone was shorter than that of
the AI, as we described previously (Fig. 11A) (Kubota et al.
2008), which is consistent with the results obtained by voltagesensitive dye imaging (Sawatari et al. 2011). The latency of the
DM was also shorter than that of the AI and comparable withthat in the AAF (Fig. 11A). The latencies to reach 25%
maximum were 122.2  5.4 ms in the AAF, 120.2  4.5 ms
in the DM, 142.0  4.7 ms in the AI, and 146.0  5.8 ms in
the AII (n  18, each; Fig. 11B). The latencies to reach 50%
maximum were 185  4.7 ms in the AAF, 189.3  5.6 ms in
the DM, 212.5  6.5 ms in the AI, and 215.6  7.5 ms in the
AII (n  18, each; Fig. 11B). The latencies to reach 25% and

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 127 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

B

A
DP

Low

SUF

AI

AAF
AII

DorsalRostral

Number of NNF-positive neurons

*

60
40
20
0

AAF SUF DM

Number of NNF-positive neurons

E
80

DM
SUF
AAF

AII

Past delineation of AAF

D

Stiebler’s UF

DP

DM

AI

AAF

Layers II - V

I

II

II

III

III

IV
V

IV
V

VI

VI

8

15

6

10

4

5

2

AAF SUF

DorsalF

Layer VI

20

0

I

5 kHz
35 kHzFM reversal stim0*

BDA-positive neurons (%)

High

C

AAF SUF

50% maximum had the same tendency. The latencies of the
AAF and DM were signiﬁcantly shorter than those of the AI
and AII, and there were no signiﬁcant differences between the
AAF and DM or the AI and AII (Fig. 11C). These data also
support the fact that the DM receives thalamocortical projections directly from the MGv but not via the AAF or AI.
The mouse MGv is composed of several compartments that
topographically project to their corresponding subregions in theauditory cortex (Horie et al. 2013; Takemoto et al. 2014). The
region that projects to the AAF or AI is localized in the middle
part of the MGv. On these bases, we tried to identify the region
that projected to the DM. First, we injected BDA into the AI or
DM after identiﬁcation, and we evaluated the locations ofneurons that project to the AI or DM using coronal sections(Fig. 12). Neurons projecting to the DM were found in theventral half of the MGv, and those projecting to the ventral AI
high-frequency area were observed in a neighboring region
located at the same ventrodorsal level (Fig. 12). However, wefound that neurons projecting to the DM were located rostral tothose projecting to the AI as a whole. Therefore, we preparedhorizontal sections to evaluate the relative rostrocaudal location of neurons projecting to the DM and AI (Fig. 13). We
injected Alexa Fluor-conjugated CTB into the ventral AI highfrequency area or the DM, identiﬁed using a tone at 40 kHz(Fig. 13, A and B). In horizontal sections of the MGv, theneurons projecting to the DM were located more rostrally than
the neurons projecting to the AI (Fig. 13, C–F). The relative
location of the neuronal population projecting to the DM wasmeasured in reference to the averaged coordinates of neurons
projecting to the AI (Fig. 13J). The neurons projecting to the
DM were located signiﬁcantly more rostral and more medialcompared with those projecting to the AI (P  0.001, for both).

MGv
MGd
100

Lateral***** ***

75
50
25
0

2911

Fig. 8. Difference in histological and anatomical properties among the AAF,
Stiebler’s UF, and DM. A: schematic drawing of the auditory cortical map. The area
enclosed by the blue oval was the deﬁnitionof the AAF proposed by Guo et al. (2012).
SUF, Stiebler’s UF. B: auditory cortical map
revealed by ﬂavoprotein ﬂuorescence responses. The area enclosed by the white
rectangle is equivalent to that in the blue oval
in A. Scale bar, 500 m. C: sample images of
SMI-32 immunolabeling patterns in the AAF
and Stiebler’s UF. Scale bar, 100 m. D:
number of NNF-positive neurons in theAAF, Stiebler’s UF, and DM. *P  0.05,
Tukey-Kramer’s test; AAF, n  5; Stiebler’sUF, n  4; DM, n  7. E: number of
NNF-positive neurons in the layer VI and theothers of the AAF and Stiebler’s UF. *P 
0.05, Mann-Whitney U-test; AAF, n  5;Stiebler’s UF, n  4. F: percentage of neuronal distribution in the medial geniculate
body [MGB; the ventral division of MGB(MGv) or the dorsal division of MGB(MGd)] by their cortical target. **P  0.01;***P  0.000001, 2 test after Bonferroni
correction; to AAF, n  150 neurons from 3
mice; to Stiebler’s UF, n  144 neurons
from 6 mice; to DM, n  316 neurons from
7 mice.

AAF SUF DM

Next, we tested whether the frequency organization in the
DM reﬂects the distinct frequency organization in a single
region within the MGv or partly overlapped regions projecting
to the AI and DM, as suggested by the fork-shaped, frequencyorganized map proposed by Issa et al. (2014) (Fig. 1C). We
injected ﬂuorescent CTB into the low- and high-frequency
regions of the DM. Results clearly indicated that the frequency
organization in the MGv projecting to the DM was structured
along a single latero-medial axis within a single compartmentof the MGv (Fig. 13, G–I). The quantitative distribution map
showed a signiﬁcant place shift in location between the areas
projecting to the low- and high-frequency areas of the DM(Fig. 13K; P  0.001). These data indicate that neurons
projecting to the DM are localized in the rostral part of the
MGv with a distinct frequency organization along the lateromedial axis, and no fewer than four compartments were independent from each other within the MGv (Fig. 13L).
Vocalization processing in the auditory cortex. Although thefrequency gradient in the AI also includes ultrasonic bands, theresponses of the DM to ultrasonic tones were larger than thoseof the AI (Fig. 5E). Therefore, we veriﬁed the possibility thatthe DM may be involved in ultrasonic courtship songs produced by male mice for females (Fig. 14A). Although thiscourtship vocalization produced by male mice is one of therepresentative ultrasonic, communication-related sounds(Asaba et al. 2014; Hammerschmidt et al. 2009; Holy and Guo2005), as well as isolation calls produced by pups (Ehret andHaack 1982; Thornton et al. 2005; Uematsu et al. 2007), no
research has been performed about central processing of courtship songs, although several studies about pup isolation calls
have been reported (Galindo-Leon et al. 2009). The courtshipsongs produced by male mice include characteristic features,

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 128 －

2912

Fig. 9
DM.
forme
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5-kH
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NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A P0

P7

P35
Tone exposure(5 kHz or 35 kHz)

P41

Flavoprotein
imaging

B Mice exposed to a 5 kHz tone
Normal

Quiet

AAF

AI

-1.0%

5 kHz Exposed
AAF

AI

AAF

AI

+1.0% -1.0%

1 mm

AII

AII

AII

+1.0% -1.0%+1.0%

DorsalRostralFig. 9. Plasticity after acoustic exposure in the AI and
DM. A: schedule of experiments. Imaging was performed within 1 wk after cessation of acoustic exposure. P, period. B and C: response amplitudes to a
5-kHz tone in mice exposed to a 5-kHz tone (*P 
0.05, Mann-Whitney U-test; normal, n  10; quiet, n 
6; exposed, n  14). Mice rearing in a normal home
cage (Normal), in the chamber without presentation of
any tones (Quiet), or in the chamber with acoustic
exposure (Exposed). D and E: response amplitudes to a
35-kHz tone in mice exposed to a 35-kHz tone (*P 
0.05, Mann-Whitney U-test; normal, n  10; quiet, n 
6; exposed, n  14).

C

ΔF/F 0 (%)

anaAAF,drawareanition012).
map
e rewhiteoval
ges of
AAF
m. D:
the
0.05,
bler’s
er of
d the
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 5;
neuulate
MGB
MGB
0.01;
rroni
om 3
urons
from

2912

Responses to a 5 kHz tone

Normal

AII

AAF

AI

1.0

1.0

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

0.2

0.2

0

0

0

*

*

Quiet

Exposed1.0
0.8

D Mice exposed to a 35 kHz tone
Normal

Quiet

AAF

AI
-1.5%

E

AII

35 kHz Exposed
DM

DM

DM

ΔF/F 0 (%)

2911

AI

+1.5% -1.5%

Responses to a 35 kHz tone

AAF

AI

1.2

1.2

1.0

*

1 mm

AII

AII

+1.5% -1.5%

AAF

AI

AAF

Normal

+1.5%

Quiet

AII

DM

1.2

1.2

1.0

1.0

1.0

0.8

0.8

0.8

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

0.2

0.2

0.2

0

0

0

0

such as a high-frequency band over 50 kHz, fast FM, pitch
jumps, and intermittent rhythms (Fischer and Hammerschmidt
2011; Lahvis et al. 2011). The AAF, AI, AII, and DM in both
sexes were clearly activated by stimulation with songs produced by males (Fig. 14B). Interestingly, the temporal order ofthe rising phase of neural responses to a male’s courtship song
was different from that of an artiﬁcial tone (Fig. 14, C and D).
The DM was activated ﬁrst, followed by simultaneous activation of the AAF and AI, and the AII was activated last. The
latency to reach 25% maximum was 143  6.2 ms in the DM,
170  9.4 ms in the AAF, 181  7.4 ms in the AI, and 208 
13.7 ms in the AII (Fig. 14E), and the latency to reach 50%
maximum was 243  6.5 ms in the DM, 279  8.7 ms in the*

ExposedAAF, 291  9.3 ms in the AI, and 340  11.9 ms in the AII(n  20 in total; male, n  7; female, n  13; Fig. 14E). As
for the latency, there were no differences between male andfemale mice in all of the regions. These data indicate that
vocalization is similarly processed in the DM of both male andfemale mice regarding the latency of the DM responses to amale’s courtship song. In contrast, the amplitudes of the response to a male’s song were signiﬁcantly larger in females
than in males in the DM alone (Fig. 14F; male, 1.61  0.10%,n  15; female, 2.0  0.19%, n  16; P  0.05). The response
amplitudes in the AAF, AI, and AII were slightly larger infemales than in males, although there were no signiﬁcantdifferences between the sexes (AAF, P  0.6; AI, P  0.8;

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 129 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

A

Mice exposed to 5 kHz
Responses to 35 kHz
AAF

ΔF/F 0 (%)

1.2

AI

1.2

AII

1.2

1.0

1.0

1.0

0.8

0.8

0.8

0.8

0.6

0.6

0.6

0.6

0.4

0.4

0.4

0.4

0.2

0.2

0.2

0.2

0

0

0

0

Fig. 10. . Frequency speciﬁcity in plasticity after
acoustic exposure. A: the response amplitudes to a
35-kHz tone in mice exposed to a 5-kHz tone. There
was no signiﬁcant difference between normal andexposed or quiet and exposed mice. Normal, n  10;quiet, n  6; exposed, n  14. B: the response
amplitudes to a 5-kHz tone in mice exposed to
35-kHz. There was no signiﬁcant difference between
normal and exposed or quiet and exposed mice.
Normal, n  10; quiet, n  6; exposed, n  14.

Exposed

Quiet

Normal

DM

1.2

1.0

B Mice exposed to 35 kHz
Responses to 5 kHz
AAF

ΔF/F 0 (%)

1.0

AI

1.0

AII

1.0

0.8

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.4

0.2

0.2

0.2

0

0

0

AII, P  0.5). The slight difference in the AAF, AI, and AII
might be attributed to the signiﬁcant difference in the DM, as
it was reported that neural responses in a downstream region
with a longer latency are affected by neural responses in an
upstream region with a shorter latency in the auditory cortex(Kubota et al. 2008). As expected from this idea, there was no

A

AAF

AI

DM

AI

2914

Exposed

Quiet

Normal2913

difference between the sexes in response to a 70-kHz artiﬁcial
tone (Fig. 14G). These data imply that ultrasonic courtshipsongs produced by males, mediated via a distinct rostral compartment of the MGv, reach the DM ﬁrst and are processed
further through pathways from the DM to other cortical regionsin mice.

AAF

Fig. 1
DM i
parce
media
the a
ionto
BDA
quenc
(left).
also larrow
Scaleof theneuro
Neuroof the
AI hi
Whitn
156 n

DM

DISC

25% Max
500 msB******

Latency (ms)

160

220

150

210

140

200

130

*** ***

120
110

***
***

230

AAF

DM

AI

AII

*** **

190
180
170

AAF

25% Max

C
AI

1 DM

AI

AII

50% Max
(ms)
150

2
AII 2

DM

Fig. 11. Responses in the DM and AAF faster than those in the
AI and AII. A: temporal proﬁles of F/F0 in the AAF, AI, andDM in response to a 35-kHz tone. Frame rate was set at 54 Hz.
Horizontal black bars indicate the stimulus period. B: times
required to reach 25% (left) and 50% (right) of the maximal
F/F0 after the stimulus onset in the AAF, DM, AI, and AII(***P  0.001, **P  0.01, 1-way repeated-measures
ANOVA; n  18 each). C: schema of latencies reaching 25%
maximum by color. The numbers indicate the order of
response.

Dorsal1

AAF

Rostral120
J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 130 －

In
in th
imag
and
re-d
be a
soun
Joac
1997
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2014
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n the
, and
4 Hz.
times
ximal
d AII
sures
25%
er of

A

B

SMI-32

Injection to AI highInjection to DM

LP
MGd

Fig. 12. Distribution of MGv neurons projecting to the AI or
DM in coronal sections. A: regional subdivisions of the MGB
parcellated using SMI-32. LP, lateral posterior nucleus; MGm,
medial division of MGB. B: neurons in the MGB projecting to
the auditory cortex in coronal sections. BDA was injectediontophoretically into the AI high-frequency area or the DM.
BDA-positive MGB neurons projecting to the AI high-frequency area are located in the ventrolateral part in the MGv(left). BDA-positive MGB neurons projecting to the DM arealso located in the ventrolateral part in the MGv (right). White
arrows indicate where BDA-positive neurons are assembled.
Scale bar, 200 m. C and D: quantitative analysis. The centerof the MGB was deﬁned as (0, 0). The relative location of eachneuron was measured, and the coordinates were averaged (D).
Neurons projecting to the DM were localized in the lower halfof the MGv and at the same level as the region projecting to theAI high-frequency region dorsoventrally (n.s., P  0.9, MannWhitney U-test; 131 neurons projecting to the AI in 7 mice;
156 neurons projecting to the DM in another 7 mice).

MGv

MGv

MGv
200 μ mDorsalLateralC

D

Distance (μm)
centercenter40

60

dorsal ( μ m)
neuron A(a1, a2)

40
60

n.s.

80
100
120

center (0, 0)

80 100 120
lateral

20

MGv

a2

20

ventralﬁcial
shipomssedions

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEXDistance (μm)

after
to a
There
and
 10;
ponse
ed to
ween
mice.
4.

2914

MGm

2913

To AI highTo DM

a1 lateral( μ m)DISCUSSION

In the present study, we observed the regional characteristics
in the mouse auditory cortex using ﬂavoprotein ﬂuorescenceimaging, two-photon calcium imaging, immunohistochemistry,
and tracer experiments. The regional borders of the AI werere-delineated by isolating the DM, which has been reported to
be a part of the AI, specialized for processing high-frequency
sound signals in mice (Guo et al. 2012; Issa et al. 2014;
Joachimsthaler et al. 2014; Sawatari et al. 2011; Stiebler et al.
1997). In the present study, we identiﬁed the DM as the ﬁfth
frequency-organized region in the cortex, in addition to thethree frequency-organized regions—the AAF, AI, and AII—in
the auditory cortex (Issa et al. 2014; Kubota et al. 2008) and theinsular auditory ﬁeld (IAF) in the insular cortex (Gogolla et al.
2014; Sawatari et al. 2011; Takemoto et al. 2014). We demonstrated that the frequency gradient of the AI ran from thedorsocaudal to ventrorostral direction, whereas the frequency
gradient in the DM ran from the ventrocaudal to dorsorostral
direction. The direction of the frequency organization of the
AAF was from dorsorostral to ventrocaudal, consistent withrecent reports (Horie et al. 2013; Issa et al. 2014; Kubota et al.
2008). The DM responses to a courtship song by males weresigniﬁcantly larger in female mice, which is the ﬁrst evidence
to suggest that the gender-speciﬁc biological importance ofcourtship songs might be reﬂected in the properties of themouse auditory system.

Technical merits of using ﬂavoprotein ﬂuorescence imaging
to investigate cortical functions in mice. There were several
technical advantages in the present study to localize the newly
found DM region. First, we used ﬂavoprotein autoﬂuorescenceimaging to visualize the mouse auditory cortex. Flavoprotein
ﬂuorescence imaging gave us very similar results to thoseobtained in GCaMP3-expressing mice (Issa et al. 2014) regarding the regional borders between the DM and surrounding
regions. This method detects neural activity based on activitydependent oxygen metabolism; therefore, there is no need tostain neurons using dye solutions, thus allowing uniform observability on the cortical surface (Llano et al. 2009; Shibuki etal. 2003, 2006) and allowing the delineation of regional borders, as in our previous studies (Honma et al. 2013; Kubota etal. 2008; Ohshima et al. 2010). Because of the merits of
intrinsic ﬂuorescence imaging, we were able to ﬁnd a small
trough in neuronal activity between the AI and DM (Fig. 2), as
seen in GCaMP3-expressing mice (Issa et al. 2014). A criticalrequirement for delineating small cortical regions is the uniform distribution of ﬂuorophores in the brain, which was basedon endogenous ﬂavoproteins or homogenously expressed
GCaMP3. In contrast, unit recording requires the insertion ofan electrode into the cortex, and therefore, the recording sites
must be set at some intervals. Therefore, the regional border,
such as that between the AAF and AI, must be deﬁned as aresult of the assumption that the border corresponds to thefrequency-gradient reversal line based on unit recording de-J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 131 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

D

A

R

DM
AI

B

2915

R C

L

L

2916

R

MGB

AAF

LGB

AC

AII

D AI

Hi p pocampus

E DM

F Merge
MGm
MGv

G DM low

K

***

200
100

***

0
0

100

200

300

Distance (μm)

Distance (μm)

J

I Merge

H DM high

L

***

200
100

Dorsal

***

0
0

400

100

200

Distance (μm)

Distance (μm)

To AI
To DM

To DM low
To DM highRostralLateral Low

High

To AAFTo AI
To DM
To IAF

Fig. 13. Distribution of MGv neurons projecting to the DM in horizontal sections. A: injection sites in the AI and DM. Image of the brain surface and merged images
obtained through a green or a red ﬁlter. The dotted lines indicate the outlines of responses to a 40-kHz tone. Scale bar, 1 mm. D, dorsal; R, rostral. B: injection site after
a horizontal slice was prepared. The dotted lines indicate the outlines of the auditory cortex. Inset: the same image observed under bright-ﬁeld microscopy. Scale bars,
500 m. L, lateral. C: bright-ﬁeld view of the MGB in a horizontal slice. The dotted rectangle represents the window containing images of D–F. Scale bar, 500 m.
LGB, lateral geniculate body. D–F: neurons projecting to the AI ventral high-frequency area and DM (F). The arrowheads indicate the lateral edge of the MGv as shownin C. Scale bar, 100 m. G–I: neurons projecting to the low- and high-frequency areas of the DM (I). Scale bar, 100 m. J: the relative coordinates of neurons projecting
to the DM vs. the AI. The average of the coordinates of neurons projecting to the AI was set as the origin. In 6 mice, 71 neurons projecting to the AI and 103 neurons
projecting to the DM were found. ***P  0.001, Mann-Whitney U-test. K: the relative coordinates of neurons projecting to a low-frequency (25 kHz) area vs.
high-frequency (60 kHz) area in the DM. The average of the coordinates of neurons projecting to a low-frequency area was set as the origin. In 3 mice, 33 neurons
projecting to the low-frequency area of the DM and 28 neurons projecting to the high-frequency area were found. ***P  0.001, Mann-Whitney U-test. L: 3-dimensional
schematic view of the relative locations of 4 compartments within MGv. The schema of the compartment projecting to the insular auditory ﬁeld (IAF), AAF, or AI wascompensated, according to the elegant study by Takemoto et al. (2014) and, in part, the study by Horie et al. (2013).

Fig. 1(top).
The d
which
peaks
(n 
maxim
25%
***P
in av
a 70-

scribed in previous studies. Thus it is no wonder that the small
trough between the DM and the ventral AI high-frequency area
was not found in previous studies using unit recording. The
DM is sensitive to ultrasonic sounds over 40 kHz, and the

al. 2
thes
A
varia

response of the AI is somewhat weaker than that of the DM.
These factors might have led to the conclusion that a single
frequency gradient extended from the AI low-frequency area to
the DM in previous electrophysiological experiments (Guo et

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 132 －

DM.
ngle
ea to
o et

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEXA

B Response to male song

Frequency (kHz)

120

DM

20

1s

120

AI

AAF

AII
20

Time

-1.2%

50 ms

+1.2%

AI

2

AI

DM

25% Max

AAF

2

AII 3

AII

Rostral

(ms)
210

1 DM

AAF

Dorsal

1 mm

D

C

140

500 msDM AAF

25% Max

Latency (ms)

240

AII

DM AAF*** *** ***
n.s.
***
***
n.s.
AI
***
*
AII
*** *** *50% Max

AAF

200

2.0

160

*** *** ***
n.s.
***
***
n.s.
AI
***
***
AII
*** *** ***

Male

260

DM

AAF

AI

220

AII

*

0.6
0.5
0.4

1.2

0.3

0.8

0.2

0.4

0.1

DM

AAF

AI

Female
DM

G

1.6

0

AII

AAF

300

Responses to male song
2.4

AI

DM

280

120

F

AI

DM

AII

0

Response amplitude (%)

E

Δ of amplitude (%)

mages
after
bars,
m.
hown
ectingurons
ea vs.
urons
sional
I was

2916

Response amplitude (%)

2915

AAF

AI

AII

Responses to 70 kHz
1.2
1.0

Male

Female

AAF

AI

0.8
0.6
0.4
0.2
0

DM

AII

Fig. 14. Auditory responses to courtship male songs. A: recorded spectrogram of a courtship song produced by a male C57BL/6 mouse when approaching a female(top). The red box indicates the segment used in this study as a stimulus (bottom). B: the typical image of the response in the auditory cortex to the male’s song.
The dorsal anterior “UF” region identiﬁed by Stiebler et al. (1997) was not activated, probably because this region is responsive to slow FM sweeps, 24 kHz/s,
which were not included in the male’s songs. The DM was identiﬁed based on the responses to a 70-kHz tone, as performed in the previous sections. The response
peaks of the DM to a 70-kHz tone and a courtship song did not shift signiﬁcantly. C: temporal proﬁles of F/F0 in each region in response to the male’s song(n  20). Images were obtained at 54 Hz. D: schema showing the latency of responses to reach 25% maximum. E: the time required to reach 25% (left) or 50%
maximum (right); male, n  7; female, n  13. Insets: statistical signiﬁcance of pair-wise comparisons between regions with respect to latency required to reach
25% maximum (left) and 50% maximum (right) of the response peak to a male’s song. The sexes were mixed, because there was no difference between them.
***P  0.001, *P  0.05, 1-way repeated-measures ANOVA; n  20. F: response amplitudes to a male’s song (left axis). The blue plots indicate the differencein averaged amplitudes between females and males (right axis); male, n  15; female, n  16. *P  0.05, Mann-Whitney U-test. G: response amplitudes to
a 70-kHz ultrasonic tone; male, n  15; female, n  16.

al. 2012) or that the strong frequency gradient traveled acrossthese two different regions (Issa et al. 2014).
Another beneﬁt of ﬂavoprotein ﬂuorescence imaging is thatvariability of the signal amplitudes in F/F0 is very small ineach mouse, especially when transcranial imaging is performed. Because the skull of a mouse is transparent, andﬂavoproteins exist originally in neurons, we can observe thesame pattern of responses transcranially without craniotomy,

J Neurophysiol • doi:10.1152/jn.00932.2014 • www.jn.org－ 133 －

NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEX

which might confound the results. Moreover, as ﬂavoproteinsare an endogenous protein in the mitochondrial electron transport chain, it is very unlikely that ﬂavoproteins work as a
calcium chelator and have some artiﬁcial effects on the calcium
dynamics, which is essential for the induction of corticalplasticity (Zucker 1999).
Finally, we were able to combine ﬂavoprotein ﬂuorescenceimaging with anatomical techniques using ﬂuorescent traces,
because the ﬂavoprotein signals were much weaker than thosefrom the ﬂuorescent tracers. The combination of ﬂavoprotein
ﬂuorescence imaging and microinjection of a retrograde ﬂuorescent tracer could reveal multiple compartments in the MGv.
Because the MGv is located deep within the brain, it is difﬁcult
to perform precise MGv experiments without anatomical studies. Direct optical imaging to observe responses in the MGv
has not been reported. Although functional MRI (fMRI) imaging can be used to observe activities deep within the brain,
the spatial resolution is too low to ﬁnd multiple compartmentsin the mouse MGv. Without the ﬁne spatial resolution achievedby anatomical studies, the precise multicompartments with adistinct frequency gradient (Fig. 13L) could not be visualized,
because the general concept of one frequency organization in
one region is also applicable in mice. The combination of
optical imaging and localized tracer injection into functionallyidentiﬁed, small cortical regions is expected to be greatly
advantageous for detecting ﬁne topological organization inother sensory cortices other than the auditory cortex.
New deﬁnition of regions included in the mouse auditory
cortex. Our ﬁndings suggest that the term UF might not beappropriate. Stiebler et al. (1997) deﬁned the UF as a distinct,
frequency-unorganized region, where neurons have a characteristic frequency over 50 kHz (Fig. 1A). Then, UF as a distinct
region was denied, and the term UF has also been used toindicate the high-frequency area of the AAF and AI (Fig. 1B)(Guo et al. 2012) or only the dorsal AI high-frequency area
(Fig. 1C) (Issa et al. 2014). However, optical imaging revealed
that frequency direction of the AAF runs ventrocaudally (Issa
et al. 2014) and that the AII has the distinct frequency organization (Issa et al. 2014; Kubota et al. 2008). By delineating
the DM, we have identiﬁed the four distinct frequency-organized regions of the AAF, AI, DM, and AII, in total, each ofwhich has its own high-frequency area (up to 80 kHz) corresponding to the UF. Optical imaging also revealed that a vacantregion insensitive to pure tones containing vocalization-speciﬁc neurons is located between the AAF and AI dorsal
high-frequency area (Issa et al. 2014), and we have alreadyreported that the region between the AAF and DM is responsive to slow FM components, regardless of tonal frequency
range (Fig. 1D) (Honma et al. 2013; Tsukano et al. 2013b).
These facts indicate that the distinct FM-sensitive region exists
in this area in the mouse auditory cortex, similar to thatobserved in bats (Suga and Jen 1976). Actually, there is apossibility that Stiebler et al. (1997) might perform recordings
without distinguishing between the DM and frequency-unorganized, FM-sensitive region. They mentioned in their paperthat neurons observed in the rostral part of the UF were FM
sensitive but not tone sensitive (Stiebler et al. 1997). The
confusion of two different regions might obscure a frequencyorganized structure and lead to the misconception that UF was
not frequency organized (Stiebler et al. 1997). Overall, we
propose to subdivide the dorsal part of the auditory cortex

2917

precisely and to assign new, anatomical names: the dorsoanterior ﬁeld (DA) to the FM-sensitive region just dorsal to the
AAF and the DM to the region newly identiﬁed in the present
study (Fig. 1D), in addition to the classical DP named previously (Stiebler et al. 1997).
Multiple frequency organizations have been found in various
mammals. By identifying the DM with a distinct frequency
gradient using ﬂavoprotein ﬂuorescence imaging, multiple frequency organizations were revealed in the mouse auditory
cortex in the present study. In primates, frequency-organizedstructures exist in the belt region besides the core region. Withthe use of fMRI, it was revealed that the caudal-medial (CM)ﬁeld in macaques, which belongs to the belt, has stronger
frequency gradient than do ﬁelds in the core, and other beltﬁelds also have a frequency gradient (Petkov et al. 2006).
Moreover, multiple frequency-organized structures were also
found in rodents, such as guinea pigs (Nishimura et al. 2007),
ferrets (Bizley and King 2009), chinchillas (Harel et al. 2000),
and rats (Higgins et al. 2010; Kalatsky et al. 2005) using
optical imaging. Especially the auditory cortical maps revealedin rats (Higgins et al. 2010; Kalatsky et al. 2005) are quite
similar to those we revealed in mice. Several imaging studies
have revealed that the AII is frequency organized in mice (Issa
et al. 2014; Kubota et al. 2008). The IAF in the mouse insularcortex anterior to the auditory cortex is also frequency organized (Sawatari et al. 2011; Takemoto et al. 2014). Hence, atleast ﬁve frequency-organized regions exist inside and near theauditory cortex in mice. Each frequency-organized region
receives distinct thalamic information from equivalent compartments with a distinct frequency organization in the MGv(Fig. 13L) (Horie et al. 2013; Takemoto et al. 2014), and thesound responses are presumably conveyed through the courseof the hierarchical, inter-regional processing in the auditory
cortex (Fig. 14) (Kaas and Hackett 2000; Kubota et al. 2005).
The basic concept of “core and belt” prevails in the auditory
cortex across species (Winer and Schreiner 2011). The coreregion contains the AAF and AI, which receive dense projections from the frequency-organized MGv and process lemniscal information from the cochlea. The belt region surrounding
the core receives nonlemniscal information from the frequency-unorganized MGd across species basically (Winer andSchreiner 2011). Although the DM in mice is located dorsal to
the AAF and AI and should be considered as the belt (Fig. 1D),
the DM has a clear frequency gradient and receives topographic thalamic inputs directly from the MGv (Fig. 13). In
rats, the ventral auditory ﬁeld, which is frequency organizedand placed ventral to the AAF and AI, also receives dense
thalamic projections from the caudal part of the MGv (Storace
et al. 2010). These ﬁndings suggest that the belt regions inrodents receive frequency-organized, lemniscal-thalamic information directly. In contrast, the belt ﬁelds with a frequency
organization, including the CM, receive thalamic inputs fromthe frequency-unorganized MGd in marmosets (de la Mothe etal. 2012), suggesting that the frequency representation in thebelt may derive from the core after intracortical interactions inprimates. Although these present and prior observations suggest that rodents and primates have multiple frequency-organized belt auditory ﬁelds, it remains to be seen to what degree
these ﬁelds are functionally homologous or analogous between
rodents and primates.

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Many animal species use ultrasonic frequencies for navigation or communication with others. Actually, rodents communicate with each other using ultrasonic frequency vocalization
over 50 kHz. Ultrasonic songs produced by male mice induce
exploratory behaviors in female mice (Hammerschmidt et al.
2009; Holy and Guo 2005), and isolated pups attempt tocommunicate with their mother by producing isolation calls(Ehret 2005; Hahn and Schanz 2005). Therefore, it is criticalfor mice to receive and process ultrasonic sounds. The DM has
fewer neurons with low BFs and responds well to ultrasonicsounds compared with the AI (Figs. 5, D and E, and 6).
Moreover, strong plasticity is not induced in the DM byacoustic exposure (Fig. 9). The degree of variability in thefrequency organization in cellular resolution recording is lessmarked in the DM than in the AI (Fig. 6). These data imply thatthe DM is not dynamically regulated by learning; instead, theDM is dedicated for processing some congenitally importantsounds, such as courtship chirps, as shown in Fig. 14.
The DM may play a role of recipient and relay point forprocessing of courtship songs. The thalamocortical auditory
pathway comprises parallel pathways, and each region in theauditory cortex receives a topological projection from thecorresponding subregion in the MGv (Horie et al. 2013; Takemoto et al. 2014). The DM receives dense projections directlyfrom the rostral part of the MGv (Fig. 13) and is the ﬁrst region
to be activated in both sexes by a male courtship song. These
data imply that the MGv determines the region of the auditory
cortex to which song information will be sent. A characteristic
waveform and frequency included in voices emitted under aspeciﬁc condition (Hammerschmidt et al. 2009; Holy and Guo
2005; Insel et al. 1986) may selectively activate gating channels in the thalamus (Schiff et al. 2013) to choose a speciﬁcrecipient in the auditory cortex. Furthermore, there was asigniﬁcant difference in response amplitudes of a male courtship song in the DM between sexes (Fig. 14F), which may
reﬂect the results of previous behavioral studies that female
mice respond speciﬁcally to male courtship songs (Hammerschmidt et al. 2009). The auditory cortex is required for fear
conditioning using complex sounds (Letzkus et al. 2011).
Therefore, subregions of the auditory cortex may themselves
have roles for evaluating the biological meanings attached tonatural sounds.

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NEWLY DELINEATED REGION IN MOUSE AUDITORY CORTEXACKNOWLEDGMENTS
The authors thank S. Maruyama for technical assistance and A. Matsushima
for mouse breeding and maintenance.
GRANTS
Support for this study was provided by the Japan Society for the Promotionof Science KAKENHI (Grant Number 24700320; to H. Tsukano).
DISCLOSURESThe authors declare no competing interests.
AUTHOR CONTRIBUTIONS
Author contributions: H. Tsukano, R.H., M.K., K.T., H. Takebayashi, andK.S. conception and design of research; H. Tsukano, M.H., T.B., and A.U.
performed experiments; H. Tsukano, M.H., T.B., and A.U. analyzed data; H.
Tsukano, M.H., and K.S. interpreted results of experiments; H. Tsukano
prepared ﬁgures; H. Tsukano drafted manuscript; H. Tsukano, M.K., H.

Takebayashi, and K.S. edited and revised manuscript; H. Tsukano, M.H., T.B.,
R.H., M.K., K.T., H. Takebayashi, and K.S. approved ﬁnal version of manuscript.

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JOURNAL OF NEUROCHEMISTRY| 2015 | 133 | 66–72

doi: 10.1111/jnc.13053

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*Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata
University, Niigata, JapanM†Department of Neurophysiology, Brain Research Institute, Niigata University, Niigata, Japan

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‡Japan Science and Technology Agency-Core Research for Evolutional Science and Technology(CREST), Osaka University, Suita, Japan§KOKORO-Biology Group, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

Abstract
Clustered protocadherins (cPcdhs) comprising cPcdh-a, -b,
and -c, encode a large family of cadherin-like cell-adhesion
molecules speciﬁc to neurons. Impairment of cPcdh-a results inabnormal neuronal projection patterns in speciﬁc brain areas.
To elucidate the role of cPcdh-a in retinogeniculate projections,
we investigated the morphological patterns of retinogeniculateterminals in the lateral geniculate (LG) nucleus of mice with
impaired cPcdh-a. We found huge aggregated retinogeniculateterminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in theolivary pretectal nucleus and the ventral LG nucleus. These

aggregated terminals appeared between P10 and P14, justbefore eye opening and at the beginning of the reﬁnement
stage of the retinogeniculate projections. Reduced visual acuity
was observed in adult mice with impaired cPcdh-a, whereas theorientation selectivity and direction selectivity of neurons in the
primary visual cortex were apparently normal. These ﬁndings
suggest that cPcdh-a is required for adequate spacing of
retinogeniculate projections, which may be essential for normal
development of visual acuity.
Keywords: aggregated terminals, lateral geniculate nucleus,
protocadherin, retinal terminals, visual acuity.
J. Neurochem. (2015) 133, 66–72.

Clustered protocadherins (cPcdhs) are the largest subgroup of
the cadherin superfamily of neuronal adhesion molecules(Kohmura et al. 1998; Junghans et al. 2005; Shapiro et al.
2007; Yagi 2012). Mammalian cPcdh genes consist of threefamilies, cPcdh-a, -b, and -c, each of which has a clusteredstructure in a small genome locus on a single chromosome (Wu2005). In normal mice, olfactory sensory neurons expressing
the same odorant receptor project their axons into a speciﬁc
glomerulus in the main olfactory bulb (Mori and Sakano2011). However, these projection patterns are abnormal in
cPcdh-a knockout (KO) mice, in which the constant region of
cPcdh-a is deleted (Hasegawa et al. 2008). Furthermore,abnormal aggregation of serotonergic axons is observed in
cPcdh-a KO mice (Katori et al. 2009), suggesting that cPcdha is important for the normal development of axonal projection
patterns derived from speciﬁc types of neurons.

In the visual system of mice, cPcdh-a is expressed in the
optic nerve ﬁbers, in the lateral geniculate nucleus (LG), andin the primary visual cortex (V1) for 2 weeks after birth(Morishita et al. 2004). This period before eye opening isvery important for eye-speciﬁc segregation of the retinogeniculate ﬁbers (Jaubert-Miazza et al. 2005; Guido 2008), the66

Received November 22, 2014; revised manuscript received January 16,
2015; accepted January 26, 2015.
Correspondence and present address: Reiko Meguro, School of Health
Sciences, Faculty of Medicine, Niigata University, 2-746 Asahi-machi,
Chuo-ku, Niigata 951-8518, Japan. E-mail: meguror@clg.niigata-u.ac.jp
Abbreviations used: BDA, biotinylated dextran amine; cPcdh, clustered protocadherin; CTb, cholera toxin b subunit; DAB, diaminobenzidine; IGL, intergeniculate leaﬂet; LG, lateral geniculate nucleus; LGd,dorsal LG; LGv, ventral LG; OPT, olivary pretectal nucleus; PBS,
phosphate-buffered saline; V1, primary visual cortex.

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72－ 138 －

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Aggregated LGd terminals in cPcdh-a KO mice053

reﬁnement of retinogeniculate synapses (Chen and Regehr
2000; Hooks and Chen 2006, 2008; Liu and Chen 2008), andthe maturation of inhibitory neurons (Golding et al. 2014) inthe dorsal LG (LGd). In the present study, we thus
investigated the morphology of retinogeniculate terminals
in cPcdh-a KO mice and found abnormally aggregatedterminals mainly in LGd. These aggregated terminals foundin adults were also observed at P14 but not until P10. The
abnormal aggregation can result in impaired transmission ofvisual information through LGd. As expected, we foundsigniﬁcantly deteriorated visual acuity in cPcdh-a KO mice,
which was determined using a behavioral test (Prusky et al.
2000), whereas the orientation tuning of V1 neurons wasapparently normal in cPcdh-a KO mice.

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Materials and methods
Animal experiments
We used male and female mice of cPcdh-a-KO (Hasegawa et al.
2008) and C57BL/6J strain obtained from Charles River Japan(Tokyo, Japan). All experiments were performed in accordance withthe guide for the Care and Use of Laboratory Animals of the Science
Council of Japan, and were approved by the Animal ExperimentCommittee of Niigata University.
Neural tracing experiments
Five wild type (WT) mice and ﬁve cPcdh-a KO mice of aged 8–
10 weeks were anesthetized intraperitoneally with a mixture of
ketamine (300 mg/kg; Daiichi-Sankyo, Tokyo, Japan) and xylazine(30 mg/mL; Bayer, Leverkusen, Germany). The cholera toxin b
subunit (CTb, 5 ng in 1 lL of saline; List Biological Laboratories,
Campbell, CA, USA), an anterograde neural tracer, was injected
into the left eye using a glass pipette. Five days after the injection,
the mice were anesthetized with an overdose of pentobarbital(100 mg/kg, i.p.) and perfused with phosphate-buffered saline,
followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH7.2). For the developmental study, two WT and two cPcdh-a KOmice at each of P10, P14, and P19 were tested in a similar mannerwith a minimum survival time of 48 h for CTb transportation (Wuet al. 2000). The transported CTb in LG was visualized as
described previously (Horie et al. 2013). Brieﬂy, 50 lm coronal
sections, prepared using a cryostat, were incubated with a goatpolyclonal anti-CTb antibody (1 : 8000 dilution; List Biological
Laboratories) overnight, followed by incubation with a biotinylated
anti-goat IgG antibody (1 : 200 dilution; Jackson, West Grove,
PA, USA) for 2 h. The sections were further incubated with anavidin–biotin complex (1 : 100 dilution; Vector Laboratories,
Burlingame, CA, USA) and the CTb immunoreactivity wasvisualized with oxidized diaminobenzidine. The reacted sections
were mounted on gelatin-coated glass slides and air-dried. Some
serial CTb-labeled sections were counter-stained with cresyl violet.
Sections were observed using a microscope (BX-50; Olympus,
Tokyo, Japan) and images were acquired using a CCD camera(CS-600; Olympus). The terminal size measurement was takenusing a program based on Matlab (Mathworks, Natick, MA, USA).
To visualize the corticogeniculate ﬁbers in two of the each WT
and cPcdh-a mice, biotinylated dextran amine (BDA-3000;

67

Invitrogen, Carlsbad, CA, USA) was injected into V1, as described
previously (Horie et al. 2013; Tohmi et al. 2014).
Two-photon calcium imaging of V1 neurons
Orientation selectivity and direction selectivity in V1 neurons were
tested using two-photon calcium imaging in ﬁve WT and ﬁve
cPcdh-a KO mice. The two-photon calcium imaging was performed
as described previously (Honma et al. 2013; Yoshitake et al. 2013;
Tohmi et al. 2014). Brieﬂy, V1 was identiﬁed using ﬂavoprotein
ﬂuorescence imaging in mice anesthetized with urethane (1.75 g/kg,
i.p.). After removing the skull above V1, a glass pipette ﬁlled withthe cocktail of fura-2 AM (Invitrogen) and sulforhodamine 101 (SR101; Invitrogen) was inserted at the center of V1 and was advanced
into the supragranular layers, down to a depth of 200–300 lm fromthe surface. The cocktail was injected using a pressure of 4–15 kPa
for 5–10 min, so that the cells in the area located 200–300 mm fromthe tip of the pipette were labeled with fura-2. Astrocytes labeledwith SR-101 were excluded from further analysis. After the mice
were set under a two-photon microscope (TCS SP5 MP; Leica
Microsystems, Wetzlar, Germany), grating patterns moving in eight
directions (from 0° to 315° in 45° steps) were administered to themice and V1 neuronal responses were analyzed, as described
previously (Honma et al. 2013; Yoshitake et al. 2013).
Behavioral assessment of visual acuity
Visual acuity of mice was measured via a visual water maze task
developed by Prusky and coworkers (Prusky et al. 2000; Prusky andDouglas 2004). Ten WT and nine cPcdh-a KO mice (aged 7–
12 weeks) were trained and tested in the visual water maze task in
32 trials every day to assess their visual acuity. During the training
phase, they were trained to choose a sine-wave vertical grating
pattern at a low spatial frequency (0.14 cycles/degree) from ahomogeneous gray pattern with the same averaged luminous
intensity. In the subsequent testing phase, the limit of theirdiscriminatory ability was assessed at spatial frequencies higher
than 0.14 cycles/degree. A preliminary grating threshold wasestablished at which the mice achieved 70% correct choices. The
spatial frequencies around the threshold were retested and theaverage performance was calculated to plot a frequency-of-seeing
curve. The point at 70% accuracy was determined from the curve
and was deﬁned as the visual acuity.
Statistical tests
The Kormogorv–Smirnov test was used to evaluate the differencesin the cumulative distributions of terminal area sizes between WT
and cPcdh-a mice. For group comparisons, the Mann–Whitney U
test was used.

Results
Abnormal aggregation of retinogeniculate terminals in
cPcdh-a KO mice
The gross anatomical features of the thalamus and the tectum
were checked in WT (Fig. 1a) and cPcdh-a KO (Fig. 1b) mice
aged 8–10 weeks. The distribution of CTb-positive terminals
originating from the contralateral eye was apparently similar
between the two types of mice. At a higher magniﬁcation,© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72－ 139 －

68

R. Meguro et al.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

apparently normal terminals in the LGd of cPcdh-a KO mice.
Such abnormal terminals were also observed in the ipsilateralretinogeniculate projection, within the other side of LGd.
Some aggregated terminals were also found in the intergeniculate leaﬂet, whereas few were observed in the olivary
pretectal nucleus and in the ventral LG (LGv) in cPcdh-a KO
and WT mice (data not shown). As a control, we tested the
distribution pattern of corticogeniculate terminals, which
represent another main input into the LGd. BDA-positive
terminals in LGd, labeled after BDA injection into V1, were
not aggregated, and were apparently similar between a WT
(Fig. 1g) and a cPcdh-a (Fig. 1h) mouse.

Fig. 1 Retinogeniculate axon terminals in cPcdh-a KO mice. (a and b)
Gross distribution patterns of cholera toxin b subunit (CTb)-positive
brown terminals derived from the contralateral eye in olivary pretectal
nucleus (OPT), dorsal LG (LGd), intergeniculate leaﬂet (IGL; arrowhead), and ventral LG (LGv) of a WT (a) and a cPcdh-a KO (b) mouse,
aged 8–10 weeks. Sections were processed by Nissl staining. (c andd) Higher magniﬁcation of LGd shown in a (c) and b (d). Many
aggregated terminals (arrows) were scattered in the LGd of cPcdh-a
KO mice (d). (e and f) Other images of CTb-positive terminals at ahigher magniﬁcation in a WT (e) and a cPcdh-a (f) mouse. Thesesections were not counter-stained. (g and h) Corticogeniculate terminals in LGd (arrowheads) labeled after biotinylated dextran amine(BDA) injection into the primary visual cortex (V1) of a WT (g) and acPcdh-a KO (h) mouse. No aggregated terminals were found. Scalebars, 1 mm in (a and b), 20 lm in (c and d), and 10 lm in (e–h).

however, many aggregated terminals were found in LGd of
cPcdh-a KO mice (Fig. 1d and f) but not in WT mice (Fig. 1cand e). Some aggregated terminals (arrows in Fig. 1d) werecomparable in size to the soma of Nissl-stained LGd neurons.
The abnormally aggregated terminals were intermingled with

Quantitative analysis of isolated retinogeniculate terminals
in cPcdh-a-KO mice
We arbitrarily categorized the aggregated retinogeniculateterminals into three types on the basis of their approximate
sizes: large (≥ 100 lm2), medium (< 100 and > 40 lm2),
and small (≤ 40 lm2) types. The large type was distributed
all over the LGd in cPcdh-a KO mice, except the area
receiving CTb-negative terminals from the ipsilateral eye(Fig. 2b). In WT mice, only a few terminals of the large typeappeared near the lateral surface of the LGd (Fig. 2a). The
medium terminals were observed throughout the LGd in bothtypes of mice (Fig. 2a and b), and the small terminals were aswell (data not shown). A quantitative analysis of size
distribution in the isolated terminals with or without
aggregation revealed a signiﬁcant difference between WT
and cPcdh-a KO mice (p < 0.001, Fig. 2c). The mean size ofthe isolated terminals was 13.1 lm2 in WT mice and27.7 lm2 in cPcdh-a KO mice. This difference was statistically signiﬁcant (p < 0.005, Fig. 2d). Fine terminals(< 13.1 lm2) occupied 71% of isolated terminals in WT
mice and 56% of those in cPcdh-a KO mice. The density ofﬁne terminals in cPcdh-a KO mice was 62.5% of thatobserved in WT mice; this difference was also statisticallysigniﬁcant (p < 0.005, Fig. 2e).
Appearance of the aggregated retinogeniculate terminals at
P14 in cPcdh-a KO mice
We examined the post-natal development of retinogeniculateterminals in cPcdh-a KO mice. Gross distribution patterns
were similar between WT and cPcdh-a KO mice at all stages
after P10, including the normal completion of eye-speciﬁc
segregation patterns up to P10 (data not shown). In WT mice,the retinogeniculate terminals did not change much in shapeand size between P10 and P19, whereas their densities werereduced between P10 and P19 (Fig. 3a, c, and e). In cPcdh-a
mice, the aggregated retinogeniculate terminals, which were
not present at P10, were observed at P14, at the day of eye
opening (Fig. 3b and d). Dark rearing had no effect on theseresults (data not shown), suggesting that visual experience is
not required for aggregation. At P19, the aggregatedterminals increased in size and were as large as those© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72－ 140 －

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p < 0.005
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WT
cPcdh - KOn=5
n=5Fig. 2 Quantitative analysis of isolated retinogeniculate terminals. (aand b) Typical schematic distribution of retinal terminals at the center
level of dorsal LG (LGd), including the area that receives inputs fromthe ipsilateral eye. Aggregated terminals were arbitrarily categorized
into large (≥ 100 lm2), medium (< 100 and > 40 lm2), and small(≤ 40 lm2) types. Only the large and medium types are shown. The
areas surrounded by a dotted line received inputs from the ipsilateraleye. (c) Cumulative distribution of area sizes. The isolated aggregatedterminal size measurement was taken on the binarized digital imagesof cholera toxin b subunit (CTb)-stained sections. (d) Sizes of isolated
retinogeniculate terminals. (e) Number of ﬁne type isolated retinogeniculate terminals (< 13.1 lm2) per mm2 in LGd sections. In theanalyses shown in (c–e), the areas that received inputs from theipsilateral eye were omitted.

Fig. 3 Ontogenetic observation of retinogeniculate terminals. (a, c,and e) Retinogeniculate terminals at P10 (a), P14 (c), and P19 (e) in
WT mice. (b, d, and f) Retinogeniculate terminals at P10 (b), P14 (d),and P19 (f) in cPcdh-a KO mice. Scale bars, 20 lm in (a–f).

KO mice (Fig. 4a and b), as reported previously (Yoshitake
et al. 2013). We further tested the visual acuity of mice using abehavioral test (Prusky et al. 2000), and compared the results
between WT and cPcdh-a KO mice (Fig. 4c). WT and cPcdha KO mice similarly performed low-frequency tasks at 0.14
cycles/degree during the training phase. The visual acuity of
WT mice [0.56  0.01 cycles/degree (mean  SEM),
n = 10] was comparable to the values reported previously(Prusky et al. 2000; Prusky and Douglas 2004). However, thevisual acuity of cPcdh-a KO mice (0.41  0.01 cycles/
degree, n = 9) was signiﬁcantly deteriorated compared withthat observed in WT mice (p < 0.0003; Fig. 4d and e).

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Deteriorated visual acuity in cPcdh-a KO mice
The presence of the aggregated retinogeniculate terminals
suggests abnormal visual functions in cPcdh-a KO mice.
Therefore, we performed two-photon calcium imaging of theV1 neurons. However, the orientation selectivity and direction
selectivity were apparently similar between WT and cPcdh-a-72© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72

observed in adult mice aged 8–10 weeks (Fig. 3f). The mean
size of the isolated terminals were 11.72, 12.58, 14.63 lm2
in two WT mice and 16.57, 20.63, 25.39 lm2 in two cPcdha KO mice, at P10, P14, and P19, respectively.

Discussion
Mechanisms underlying terminal aggregation in cPcdh-a KOmice
In the present study, we found abnormal terminal aggregation in the LGd of cPcdh-a KO mice. Impairment of cPcdh-a
results in abnormal projection patterns from olfactory
sensory neurons expressing a given odorant receptor onto a－ 141 －

R. Meguro et al.

70

WT

(a)

cPcdh - KO

10%
F/F 0
10 s

(c)

WT

100

cPcdh - KO
100

50

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0.3 0.4 0.5 0.6
Cycles/degreep < 0.0003

0.5
0.4
0.3
WT cPcdh -KO(n = 10) (n = 9)

Fig. 4 Reduced visual acuity in cPcdh-a KO mice. (a) Sample tracesof neuronal calcium response to moving grating patterns (from 0° to
315° in 45° steps) in a WT mouse (left) and a cPcdh-a KO mouse(right). (b) Cumulative distributions of the orientation selectivity index(OSI) of 481 neurons in WT mice and 423 neurons in cPcdh-a KO mice(left). The distribution of the direction selectivity index (DSI) of 197
neurons in WT mice and 164 neurons in cPcdh-a KO mice is also
shown (right). DSI was estimated in neurons with an OSI > 0.4. (c)Schematic drawing of the Prusky water maze used to estimate visualacuity. (d) Examples of the results obtained for a WT and a cPcdh-a
KO mouse. (e) Visual acuity in 10 WT and nine cPcdh-a KO mice.

single glomerulus in the olfactory bulb (Hasegawa et al.
2008). Abnormal aggregation of serotonergic ﬁbers is also
found in cPcdh-a mice (Katori et al. 2009). The corticocortical pathways connecting the primary somatosensory cortices in both hemispheres are also impaired in cPcdh-a KOmice (Yamashita et al. 2012). These ﬁndings indicate thatcPcdh-a is required for normal development of axonal

projection patterns derived from speciﬁc types of neurons.
Although cPcdh-a is expressed in most neurons, impairment
of cPcdh-a has dominant effects exclusively in speciﬁc typesof neurons or synapses. The retinal ﬁber innervating areas,such as LGd, LGv, and the superior colliculus, also express
cPcdh-a, and almost all the optic tract ﬁbers contain cPcdh-a
protein during the early post-natal stage, with abnormality
appearing in the LGv and the superior colliculus. Theseﬁndings suggest that other cPcdhs or cell-adhesion molecules
might replace cPcdh-a in other neurons or synapses.
Distinct subsets of the cPcdh cluster are expressed
differentially in individual neurons, and enormous cellsurface diversity may result from their combinatorial
expression (Kohmura et al. 1998; Wang et al. 2002a; Esumi
et al. 2005; Kaneko et al. 2006). The cell surface diversity
attributed to cPcdhs assists the formation of speciﬁc synapses
between neurons expressing similar sets of cPcdhs (Shapiro
and Colman 1999; Phillips et al. 2003; Yagi 2012). Alternatively, cPcdh-c supposedly plays a role in self-avoidance
between neurites that express the same sets of cPcdh-c(Lefebvre et al. 2012; Zipursky and Grueber 2013). The
abnormal terminal aggregation observed in the LGd of
cPcdh-a KO mice can be explained by either of the following
mechanisms: it may be attributed to non-speciﬁc synapse
formation between a single LGd neuron and many retinal
ganglion cells, or the failure of avoidance between retinogeniculate terminals, which avoid one another in WT mice.
In cPcdh-a KO mice, terminal aggregation was notobserved at P10, and appeared just before normal eye
opening at P14; thus, cPcdh-a is likely to play an importantrole in the reﬁnement process of retinogeniculate terminals
between P10 and P14 (Chen and Regehr 2000; Hooks andChen 2006, 2008; Liu and Chen 2008). The slight mean size
difference of retinogeniculate terminals already appeared at
P10, the starting point of the reﬁnement process with lots ofexcess terminals to be eliminated. cPcdh-a might have someeffects on the size of immature terminals. In contrast, cPcdhc is required for neuronal survival and for preparing
appropriate synapses in the early development of the spinal
cord (Wang et al. 2002b; Prasad et al. 2008; Prasad andWeiner 2011; Chen et al. 2012). Moreover, cPcdh-c-deleted
mutant mice lack voluntary movements and reﬂexes and die
shortly after birth (Wang et al. 2002b; Chen et al. 2012).
Therefore, cPcdh-a and cPcdh-c may play distinct roles in
the neural circuit formation at different developmental stages.
Relationship between terminal aggregation and impaired
visual acuity in cPcdh-a KO mice
In the present study, we observed aggregation of retinogeniculate terminals and impaired visual acuity in cPcdh-a KO
mice. However, the relationship between the two phenomena
remains unclear, because there could be other abnormalities
in retinal circuits, in which cPcdh-c plays an important role
(Lefebvre et al. 2012), or in circuits higher than LGd, such

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72－ 142 －

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as geniculocortical projections, intracortical circuits in V1 orhigher visual areas. To date, orientation selectivity, direction
selectivity, and ocular dominance plasticity in V1 areapparently normal in cPcdh-a KO mice (Fig. 4 and Yoshitake et al. 2013). The estimation of visual acuity required
visual discrimination learning, in which no apparent difference was found between WT and cPcdh-a KO mice when agrating pattern of 0.14 cycles/degree was used. However, V1
plasticity induced by spatial mismatches between whisker
and visual inputs is impaired in cPcdh-a KO mice, suggesting the presence of some abnormalities in higher areas ofthese mice (Yoshitake et al. 2013). The possibility of retinal
circuit and geniculocortical circuit abnormality also needs tobe considered. However, an important ﬁnding in the present
study is that the magnitude of impaired visual acuity in
cPcdh-a KO mice was comparable to that of the aggregatedretinothalamic terminals. We investigated visual acuity in thepresent study, since the aggregated retinogeniculate terminals
had suggested impaired visual acuity in cPcdh-a KO mice.
However, we cannot exclude the possibility that otherparameters such as the size of receptive ﬁeld are abnormal
in cPcdh-a KO mice.
Normal retinogeniculate synapses are highly efﬁcient insignal transmission (Budisantoso et al. 2012), and aggregation of terminals is unlikely to further assist signal transmission. Instead, non-speciﬁc mixing of the information
conveyed by each retinal ganglion axon at the aggregatedterminals probably impairs signal transmission. Therefore,
the ﬁne spatial information conveyed through LGd may be
mediated mainly via ﬁne terminals (< 13.1 lm2). The
density of ﬁne terminals in LGd was reduced to 62.5% in
cPcdh-a KO mice compared with WT mice. Assuming that
retinogeniculate terminals with spatial information were
distributed in the grid, the visual acuity discriminating two
vertical (or horizontal) lines are inversely proportional to thegrid intervals, or proportional to the square root of theterminal density. When the visual acuity determined using
grating patterns would be proportional to the square root of
the ﬁne-terminal density, it would deteriorate to 79% in
cPcdh-a KO mice. Because the visual acuity observed in WT
mice was 0.56 cycles/degree, the expected visual acuity in
cPcdh-a KO mice could be 0.44 cycles/degree. This expectedvalue is very close to 0.41 cycles/degree, which was thevalue obtained in behavioral experiments. This simpliﬁed
argument suggests that the aggregation of retinogeniculateterminals in cPcdh-a KO mice explains a considerable part ofthe deteriorated visual acuity in cPcdh-a KO mice, although
the possibility that other abnormalities may contribute to theobserved visual impairment cannot be excluded (Katori et al.
2009; Lefebvre et al. 2012; Yoshitake et al. 2013). Therefore, morphological and functional studies on the retinogeniculate synapses in mice with genetically manipulated cPcdhs
may be useful for elucidating the functions of cPcdhs in the
future.

-72© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72

ns.
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71

Acknowledgments and conflict of interest
disclosureThis work was supported by JSPS KAKENHI Grant Number
23659091 and JST-CREST. The authors declare no competing
ﬁnancial interests.
All experiments were conducted in compliance with the ARRIVE
guidelines.

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© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 66--72－ 144 －RESEARCH ARTICLE

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Somatic Mutations in the MTOR Gene
Cause Focal Cortical Dysplasia Type IIb
Mitsuko Nakashima, MD, PhD,1a Hirotomo Saitsu, MD, PhD,1a
Nobuyuki Takei, PhD,2 Jun Tohyama, MD, PhD,3 Mitsuhiro Kato, MD, PhD,4

R.
nal

the
79–

Hiroki Kitaura, DDS, PhD,5 Masaaki Shiina, MD, PhD,6 Hiroshi Shirozu, MD, PhD,7
Hiroshi Masuda, MD,7 Keisuke Watanabe, PhD,8 Chihiro Ohba, MD, PhD,1

the
ble
ce.

Yoshinori Tsurusaki, PhD,1 Noriko Miyake, MD, PhD,1 Yingjun Zheng, MD, PhD,5
Tatsuhiro Sato, PhD,9 Hirohide Takebayashi, MD, PhD,8cell

Kazuhiro Ogata, MD, PhD,6 Shigeki Kameyama, MD, PhD,7
Akiyoshi Kakita, MD, PhD,5b and Naomichi Matsumoto, MD, PhD1b

S.,
n of
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T.
ded
4.
elf-

Objective: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architecturalabnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb.
Methods: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD typeIIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep
sequencing of the entire gene’s coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase
chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluatedby immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments.
Results: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb
showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the directtarget of mTOR kinase.
Interpretation: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating
somatic mutations in MTOR cause FCD type IIb.
ANN NEUROL 2015;78:375–386

F

ocal cortical dysplasia (FCD) is a cortical malformation frequently associated with drug-resistant epilepsy
that requires surgical treatment.1,2 FCD can be classified

into three types (I, II, and III) based on its clinicopathological features.3 Type I refers to abnormalities in corticalarchitecture without cellular anomalies,3 whereas type III

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24444
Received Oct 8, 2014, and in revised form May 26, 2015. Accepted for publication May 26, 2015.
aThese authors contributed equally to this work.
b

These are co-last authors.

Address correspondence to Dr Naomichi Matsumoto, Department of Human Genetics, Yokohama City University Graduate School of Medicine, 3–9
Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: naomat@yokohama-cu.ac.jp
From the 1Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2Department of Molecular
Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan; 3Department of Child Neurology, Nishi-Niigata Chuo National Hospital,
Niigata, Japan; 4Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan; 5Department of Pathology, Brain Research Institute,
University of Niigata, Niigata, Japan; 6Department of Biochemistry, Yokohama City University Graduate School of Medicine, Yokohama, Japan; and7
Department of Functional Neurosurgery, Epilepsy Center, Nishi-Niigata Chuo National Hospital, Niigata, Japan; 8Division of Neurobiology andAnatomy, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan; and 9Division of Biochemistry, School of Pharmaceutical
Sciences, Kitasato University, Tokyo, Japan.
Additional Supporting Information can be found in the online version of this article.

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C 2015 American Neurological AssociationV375－ 145 －

ANNALS of Neurology

involves cortical lamination abnormalities associated witha principal lesion, which are usually adjacent and affectthe same cortical area or lobes.3 FCD type II is characterized by severe architectural abnormalities and dysmorphic neurons and can be further subdivided into types
IIb (with balloon cells) and IIa (without balloon cells).3,4
It has been suggested that FCDs are likely to arise
from somatic mutations in cells in the developing brain,
but that the molecular basis of the three types of FCD
can differ.2,4 Several studies have demonstrated that thephosphatidylinositol 3-kinase (PI3K)/AKT3/mechanistic
target of rapamycin (mTOR)–signaling pathway is activated in FCD type IIb.5–11 This pathway regulates cellproliferation, metabolism, autophagy, and apoptosis indeveloping cerebral cells. Furthermore, some cortical malformations are demonstrated to possess somatic mutations in genes of the PI3K/AKT3/mTOR-signaling
pathway.12–19 In this study, we analyzed paired brain
lesions and blood leukocytes (or saliva) from individuals
with FCDs who underwent surgery to evaluate the possible involvement of somatic mutations in the disease.

Subjects and Methods
Study Subjects and Samples
Thirteen individuals with FCD type IIb, 5 with type IIa, and 6
with type I were investigated in this study. All participants
underwent preoperative clinical evaluations, including seizure
charts, electroencephalography, and neuroimaging with magnetic resonance imaging (MRI), followed by surgical treatment.
Subjects or their families provided us with written informed
consent for participation in this study. The institutional review
boards of Yokohama City University, Nishi-Niigata Chuo
National Hospital, Yamagata University, and the University of
Niigata approved this study. Surgically operated brain tissues(lesions) and peripheral blood leukocytes (or saliva) (normal tissues) were obtained from all participants. Fresh surgical specimens were immediately cut into 5-mm blocks in the operating
room, and some of the blocks were fixed with 20% bufferedformalin and embedded in paraffin wax. Serial 4-lm-thick sections were then cut and stained with hematoxylin-eosin orKl€uver-Barrera stain. Histopathological diagnosis was performed
according to the recent classification system.3 As controls for
immunohistochemistry (IHC) and Western blotting analyses,
we selected 6 subjects (ID/age [years]/sex 5 15473/18/M,
15732/38/F, 15294/26/M, 16080/43/M, 16312/12/M, and
16337/12/M) who had suffered from localized neocortical seizures for years and underwent surgery to treat epilepsy. In allcases, histopathological evaluation of resected brain lesions
revealed none of the obvious abnormalities in cortical layeringthat are required for a diagnosis of FCD type I.3 These cases
could thus be regarded as mild malformations of corticaldevelopment.3

DNA Preparation
Fresh frozen brain tissues were dissolved in sodium dodecyl sulfate (SDS)/sodium chloride–based lysis solution with proteinase
K and incubated at 558C for 1 hour, then 378C overnight.
Genomic DNA was extracted from peripheral blood leukocytes
and saliva using QuickGene-610L (Fujifilm, Tokyo, Japan) andOragene (DNA Genotek Inc., Kanata, Ontario, Canada),
respectively, according to the manufacturer’s instructions.

Somatic Variant Calling Using Paired Whole
Exome Sequencing Data
Paired brain lesions and blood leukocytes (or saliva) from nine
individuals with FCD Type IIb were analyzed by whole-exome
sequencing (WES). DNA was captured using a SureSelectHuman All Exon V5 Kit (Agilent Technologies, Santa Clara,
CA) and sequenced on an Illumina HiSeq 2500 (Illumina, San
Diego, CA) with 101-bp paired-end reads. Read bases below
the Phred quality score of 20 were trimmed from the 30 end ofreads. These cleaned reads were aligned to the human reference
genome sequence (UCSC hg19, NCBI build 37) using Novoalign (Novocraft Technologies, Petaling Jaya, Malaysia). Paired
WES data (from brain and blood/saliva) were analyzed by
MuTect20 and VarScan 221 to detect de novo somatic singlenucleotide variants (SNVs) in brain tissues. For MuTect, we
carefully examined both filter-passed and -rejected variants toavoid false-negative results, considering the cellular complexity
in brain tissues. For Varscan 2, minimum read depth forsomatic variant calls in normal and tumor samples was set to12. Exclusion criteria were: (1) variants on mutant alleles with
<2 reads in the brain and �2 reads in leukocytes (or saliva);(2) variants registered in dbSNP 137, except for clinicallyassociated single-nucleotide polymorphisms (flagged); (3) variants registered in 6,500 exomes of the National Heart, Lung,
and Blood Institute Exome Sequencing Project Exome Variant
Sever and the 1000 Genomes database22,23; (4) variants
observed in our 575 in-house control exomes; and (5) synonymous variants. Called variants were annotated using ANNOVAR software.24

Validation of Candidate Somatic Variants
Candidate somatic variants extracted from WES data were validated by polymerase chain reaction (PCR)-based deep sequencing using specific primers. Sequencing libraries were preparedusing the SureSelect XT Library Prep Kit (Agilent Technologies) and sequenced on a MiSeq (Illumina) with 150-bp pairedend reads. Trimming and alignment of reads were performed as
described above. Allele counting was performed using Integrative Genomics Viewer software (IGV).25

Somatic Mutation Screening of MTOR
A total of 16 primer sets covering the entire coding region andintron-exon boundaries of MTOR (MIM 601231; RefSeq accession number: NM_004958.3) were used to amplify DNA frombrain lesions and blood leukocytes (or saliva). Sequence libraries
were prepared with the Nextera DNA Sample Preparation Kit(Illumina). An Agilent DNA 1000 Kit (Agilent Technologies)

376

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Nakashima et al: Somatic MTOR mutations and focal cortical dysplasia

ulse
ht.
es
nd
a),

and KAPA Library Quantification Kits (Kapa Biosystems, Wilmington, MA) were used to check size distribution and DNA
concentration of the libraries, respectively. Libraries weresequenced on an MiSeq (Illumina). Trimming and alignment ofreads were performed as described above. Variants were calledby MuTect. All mutations were visually reviewed by IGV.

Droplet Digital PCR

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Somatic MTOR mutations were confirmed by droplet digital
PCR (ddPCR). Custom locked nucleic acid probes for eachwild-type (WT) and mutant allele- and region-specific primers
were purchased from Integrated DNA Technology (Coralville,
IA). A 20-ll assay mix containing 50ng of genomic DNA,ddPCR Supermix for Probes (No dUTP; Bio-Rad, Hercules,
CA), 250nM of WT and mutant probes, and 900nM of PCRprimers were emulsified into 20,000 droplets using a QX100
Droplet Generator (Bio-Rad). Target-specific primers andprobes are shown in Supporting Table 1. In preliminary experiments, appropriate Tm values and thresholds were examined foreach WT and mutant allele using cloned DNAs to minimize
background. These experiments revealed that virtually no falsepositive droplets were observed in the optimized experimental
conditions. We tested six paired samples with MTOR mutations
using mutation-specific primers and probes sets. PCR was performed using the following cycles: 10 minutes at 958C; 40
cycles of 30 seconds at 948C and 2 minutes at 598C
(c.6644C>A,
c.6644C>T)or
538C(c.4376C>A,
c.4379T>C); and 10 minutes at 988C. Droplet reading anddata analysis were performed using QX200 Droplet Reader andQuantaSoft software (Bio-Rad), respectively.

Statistical Analysis of Correlation Between
MTOR Mutations and Clinical Features
We assessed the relevance between MTOR mutations and clinical features, including seizure onset age, seizure frequency, neurological findings, IQ at pre- or postsurgery, and lesion volume,
by Wilcoxon rank-sum or Fisher exact test (p < 0.05 was considered as significant). Lesion volume in each subject was calculated from MRI proton density weighted images (PDWIs)
using OsiriX MD (Pixmeo, Geneva, Switzerland). In brief, themargin of high-intensity signals in PDWIs, which indicates
FCD, obtained just before surgery was delineated manually as aregion of interest (ROI) area in both axial and coronal views,
and the ROI area in each slice was calculated. Then, the volume of the lesion was automatically calculated based on eacharea and slice thickness. In subjects 14434, 17424, and 11683,
lesion volume was manually calculated. All analyses were performed using R software (version 3.1.0; R Foundation for Statistical Computing, Vienna, Austria).

Immunohistochemistry
Paraffin-embedded sections were immunostained with rabbit
polyclonal antibody against the ribosomal protein, phospho-S6(Ser235/236; diluted 1:1,000; Cell Signaling Technologies,
Danvers, MA), as previously described.26

FIGURE 1: Flow chart of somatic mutation analysis in subjects with focal cortical dysplasia (FCD). The flow of analysis
in this study is outlined. ddPCR 5 droplet digital polymerase
chain reaction; PCR 5 polymerase chain reaction.

Structural Analysis of mTOR
FoldX software (version 3.0b3) was used to calculate freeenergy changes after mutation using the mTOR crystal structure (amino acids [aa] 1,376–2,549)/mLST8 complex (Protein
Data Bank [PDB] 4JSN).27 The calculation was repeated threetimes, and the resultant data are presented as an average value
with a standard deviation.

Western Blotting Analysis
Fresh-frozen tissues of surgical specimens taken from the 5 subjects with MTOR mutations (ID: 16964, 17424, 11683,
14434, and 16578) and the six controls (described above).
Fresh-frozen tissues of a patient with an MTOR mutation (ID:15622) were not available for Western blotting. These werelysed with SDS sample buffer containing protease (Complete;Roche Diagnostics, Mannheim, Germany) and phosphataseinhibitor cocktails (PhosSTOP; Roche Diagnostics). Protein
concentration was determined using BCA protein assay reagent(Thermo Fisher Scientific, Waltham, MA). Equal amounts ofprotein (40lg/lane for phospho and total S6 and 2lg/lane foractin) were separated by SDS polyacrylamide gel electrophoresis(PAGE) and analyzed by Western blotting with rabbit polyclonal antibodies against total S6 (Cell Signaling Technologies)
and phospho-S6 (Ser235/236), and with a mouse monoclonal
antibody against actin (Merck Millipore, Billerica, MA).

September 2015377

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ANNALS of Neurology

TABLE 1. Summary of Whole-Exome Sequencing in Individuals with FCD Type IIb (n 5 9)

Subject

Mean Depthb(Brain/Blood)%_bases_above_
203(Brain/Blood)

Program

De novo
SNV Call(Filter Passed)

CommonVariants in
Two Programs

Candidate Gene(s)Common in Two orMore Cases

11683

193.7/178.4

95.0/94.3

MuTect422 (13)

13

UNC79

Varscan 2

35

MuTect446 (6)

Varscan 2

74

MuTect428 (10)

Varscan 2

28

MuTect318 (10)

Varscan 2

148

MuTect357 (12)

Varscan 2

232

MuTect348 (8)

Varscan 2

79

MuTect376 (9)

Varscan 2

706

MuTect460 (10)

Varscan 2

43

MuTect319 (6)

Varscan 2

68

a

15035
15622
16129
16578

17562
14434

15381
16325

192.9/194.5
146.3/121.3
109.2/67.37
117.8/143.2

158.2/166.2
154.2/259.4

127.5/108.6
131.0/141.2

94.9/94.7
95.6/94.6
93.8/88.4
94.5/95.4

95.8/95.8
92.8/95.5

94.8/93.7
93.7/94.0

25
11

NME5

30

NME5

56

NBPF8, UNC79,
MTOR, KAT6B

20

NBPF8

102

MTOR, KAT6B,
CES4A

23
22

NBPF8, CES4A

a

DNA from saliva instead of blood was used.
Against protein coding sequences of RefSeq genes.
FCD 5 focal cortical dysplasia; SNV 5 single-nucleotide variant.
b

Human WT MTOR complementary DNA (cDNA;
NM_004958.3) and mutant MTOR cDNA (p.Ala1459Asp,
p.Leu1460Pro, p.Ser2215Phe and p.Ser2215Tyr) were cloned in
pEF-BOS-FLAG vector.28 Human EIF4EBP1 (NM_004095.3)
was introduced into pCMV-FLAG.29 HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum and penicillin/streptomycin at 378C in
an atmosphere of 5% CO2. HEK293T cells were cotransfectedwith FLAG-4EBP and WT or mutant MTOR plasmids using
Fugene6 (Roche Diagnostics), according to the manufacturer’s
instructions. To assess the activity of MTOR mutants, cells werestarved for serum and amino acids in Hank’s balanced salt solution for 1 hour. Cells were then lysed and incubated with antiDYKDDDDK antibody beads (WAKO, Osaka, Japan) at 48Cfor 4 hours. Immunoprecipitates were analyzed by SDS-PAGE
and Western blotting with anti-phospho-4EBP (Thr37/46; Cell
Signaling Technologies) or anti-DYKDDDDK (WAKO), as previously described.30

Results
Somatic Mutation Screening and Validation
A flow chart of our analysis is illustrated in Figure 1.
Using MuTect20 and VarScan 2,21 we detected a numberof possible somatic variants in WES data of nine paired
samples (Table 1). To quickly narrow down candidate
variants, we first searched for those variants commonly
detected by the two programs, then focused on candidate
genes whose variants were found in two or more cases(Table 1). Among six candidate genes, MTOR is of interest. It encodes the serine/threonine kinase mTOR, a key
regulator of the PI3K/AKT3/mTOR-signaling pathway,31,32 and an MTOR somatic mutation has beenreported in hemimegalencephaly.13 Considering the possible involvement of the mTOR pathway in FCD, weinvestigated variants in candidate genes involved in themTOR pathway (Supporting Table 2). We only detected378

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IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

IIb

11683

15035a

15622

16129

16578

17562

14434

15381

16325

15524

16964

17424a18437

September 2015

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ND

ND

ND

ND

c.1820C>T(p.Ala607Val)—

c.6644C>T(p.Ser2215Phe)—c.4379T>C(p.Leu1460Pro)

c.1897T>C(p.Ser633Pro)

c.4376C>A(p.Ala1459Asp)—

c.6644C>A(p.Ser2215Tyr)

Variant in
MTOR by
MuTect

ND

ND

ND

ND

2/59
(3.28)—

6/314(1.88)—

4/199(1.97)

2/67(2.90)

3/221(1.34)—

4/357(1.11)

Mut/Ref
Counts
(%b)

ND

ND

ND

ND

0/59
(0.00)—

0/502(0.00)—

0/174(0.00)

0/97(0.00)

0/187(0.00)—

0/318(0.00)

Mut/Ref
Counts
(%b)

Blood

ND

ND

ND

ND

False

—

True

—

True

False

True

—

True

Mutation

ND

ND

ND

ND

20/11,990(0.17)—

160/6,876(2.27)—

623/28,382(2.15)

24/26,447(0.09)

406/28,605(1.40)—

406/28,719(1.39)

Mut/Ref
Counts
(%b)

Brain

ND

ND

ND

ND

74/42,140(0.17)—

1/17,069(0.01)—

52/103,377(0.05)

29/35,285(0.08)

18/38,815(0.05)—

9/31,396(0.03)

Mut/Ref
Counts(%b)

Blood2,003/1,9375

1,483/4,723

2,606/6,393

3,168/5,350

1,224/1,422

2478/2,215

1,148/3,373

2,198/2,851

3,195/15,181

2,546/2,611

860/3,017

1,619/1,189

1,968/1,335

Mean depthd(Brain/Blood)—c.6644C>A(p.Ser2215Tyr)c.4379T>C(p.Leu1460Pro)

—

—

—

c.6644C>T(p.Ser2215Phe)—c.4379T>C(p.Leu1460Pro)—

c.4376C>A(p.Ala1459Asp)—

c.6644C>A(p.Ser2215Tyr)

Mutation—

74/721(9.31)

111/2,167(4.87)———

24/747(3.11)—

150/9,303(1.59)—

33/1,970(1.65)—

38/2,426(1.54)

Mut/Ref
Counts(%b)

Brain

MTOR-Targeted Deep Sequencingcb

DNA from saliva instead of blood was used.
Percent of mutant allele was calculated by mutant allele reads/(mutant allele reads 1 reference allele reads).
c
Variants were called by MuTect, and reads were manually counted by Integrative Genomics Viewer software (IGV).
d
Against protein coding sequences of MTOR.
FCD 5 focal cortical dysplasia; Mut 5 mutant allele; N.D. 5 whole-exome sequencing and variant-targeted sequencing were not performed; Ref 5 reference allele.

a

Type

Subject

Brain

Whole-Exome Sequencing

Variant-Targeted Deep Sequencingc(Validation)

TABLE 2. Summary of Deep Sequencing of MTOR in Individuals With FCD—

3/3,665(0.08)

4/8,255(0.05)———

0/2,556(0.00)—

26/42,551(0.06)—

6/10,394(0.06)—

0/1,242(0.00)

Mut/Ref
Counts(%b)

Blood

Nakashima et al: Somatic MTOR mutations and focal cortical dysplasia

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ANNALS of Neurology

somatic MTOR mutations were verified in 6 individuals
with FCD type IIb (6 of 13 [46%]; Fig 2A; Table 2).
No candidate mutations were identified in individuals
with FCD type I or IIa (Supporting Table 3). Mutant
allele frequencies in brain lesions were very low (range,
1.54–9.31%; average, 3.67%) but were clearly higher
than those in blood leukocytes or saliva (all <0.1%;
Table 2), excluding possible PCR or sequencing errors.
Furthermore, we verified the mutant allele frequencies by
ddPCR. Fractional abundance in each sample was nearlyconsistent with mutant allele frequencies from the deep

FIGURE 2: Somatic MTOR mutations in individuals with focal
cortical dysplasia (FCD) type IIb. (A) Schematic of the mammalian target of rapamycin (mTOR) protein with HEAT repeats,
FAT domain, kinase domain comprising N- and C-lobes, FRB
domain, and FATC domain. All four identified missense mutations occurred at evolutionarily conserved amino acids in the
FAT domain (n 5 2; Ala1459 and Leu1460) or N-lobe of thekinase domain (n 5 2; Ser2215). Multiple amino acid sequences of mTOR proteins were aligned with tools available on the
CLUSTALW website (http://www.genome.jp/tools/clustalw/).
(B) Percentage of mutant alleles of MTOR in blood leukocytes
and brain lesions in FCD type IIb. All samples showed bothresults analyzed by targeted amplicon deep sequencing(NGS) and ddPCR. ddPCR 5 droplet digital polymerase chain
reaction; NGS 5 next-generation sequencing.

variants in six mTOR-related genes (MTOR, AKT3, TSC1,
TSC2, PIK3, and PTEN) by MuTect and subsequently
examined by targeted deep sequencing. Four MTOR mutations were validated: c.4376C>A (p.Ala1459Asp);
c.4379T>C (p.Leu1460Pro); c.6644C>A (p.Ser2215Tyr);and c.6644C>T (p.Ser2215Phe; Table 2). No variants inother genes were validated because they were absent in targeted deep sequencing, suggesting that they were false positive. We also examined germline or somatic mutations incandidate genes involved in the mTOR pathway (Supporting
Table 2) using nonbrain Varscan 2 data, but we could notdetect any pathogenic variants in these genes.
We then screened the entire MTOR coding region
using deep sequencing of PCR amplicons from 13 individuals with FCD type IIb, including the 9 analyzed by
WES. In addition, to explore possible involvement ofsomatic MTOR mutations in other types of FCD, wealso screened 5 subjects with FCD type IIa and 6 with
type I. We identified two recurrent mutations(c.4379T>C and c.6644C>A) in an additional two individuals with FCD type IIb. A total of four lesion-specific

FI
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FIGURE 3: Brain magnetic resonance imaging (MRI) and histopathological features of subjects with somatic MTOR
mutations. Coronal or axial brain MRI of subjects withsomatic MTOR mutations. T2-weighted (A, D, G, J, M, P)
and proton-density–weighted (B, E, H, K, N, Q) images
show abnormal configurations of cerebral gyri and high signal, respectively. Yellow arrowheads indicate focal corticaldysplasia (FCD) lesions. Photomicrographs of hematoxylineosin–stained histology sections (C, F, I, L, O, R) demonstrating characteristic dysplastic features with dysmorphic
neurons (arrowheads) and balloon cells (arrows) in surgical
specimens taken from 6 individuals with somatic MTOR
mutations. Bar 5 20lm.

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FIGURE 4: Expression of phospho-S6 in focal cortical dysplasia (FCD) type IIb. Immunohistochemical features (A–D) and Western blotting (E) for phospho-S6 in brain tissues taken from subjects with FCD type IIb and controls. (A–C) Many dysmorphic
neurons and balloon cells in an FCD type IIb brain section with an MTOR mutation (A: 16578, B: 15622; Table 2 and Supporting Table 3) and without a mutation (C: 15381; Table 2 and Supporting Table 3) showed intense immunoreactivity to phosphoS6. (D) Neurons in the control (#16080) showed no or very faint reactivity. Bar 5 120lm. (E) Expression of phospho-S6 (Ser235/236), total S6 protein, and actin are depicted in the upper, middle, and lower panel, respectively. Western blotting showed alarger amount of phosphorylated S6 in brain tissues of FCD type IIb (lanes 1–5: 16964, 17424, 11683, 14434, and 16578,
respectively), compared to controls (lanes 1–6: 15473, 15732, 15294, 16080, 16312, and 16337, respectively). Equal amounts
of protein (40lg/lane for phospho and total S6 and 2lg/lane for actin) were applied.

sOR
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OR

3

sequencing data (Fig 2B). Fractional abundance in blood
was extremely low or zero (0–0.034%; Fig 2B), being
equivalent to no-template control. Low-prevalence mosaic
rates for all mutations were reproducible by ddPCR.
Correlation Between MTOR Mutations andClinical Features
Correlation of MTOR mutations with clinical phenotypes
was statistically analyzed. Clinical features of the 13
individuals with FCD type IIb are described in detail (Sup-porting Table 4). Brain MRI showed severe architecturalabnormalities in 6 individuals with somatic MTOR mutations (Fig 3), as well as in 7 without MTOR mutations (datanot shown). We found no statistical difference for any clinical feature between mutation-positive and -negative groups.
Phosphorylation of Ribosomal Protein S6 in
FCD Samples
At primary histopathological examination, all specimens
with FCD type IIb (Table 2 and Supporting Table 4)

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T

FIGURE 5: Mammalian target of rapamycin (mTOR) signaling activation by MTOR mutations. Structural consideration of thefour mutations. (A) Crystal structure of mTOR (residues 1,376–2,549) in complex with mLST8, ATPcS, and magnesium ions (Protein Data Bank [PDB] code 4JSP). FAT domain, N- and C-lobes of the kinase domain, and the FRB domain in the N-lobe are colored blue, magenta, cyan, and light blue, respectively. Nonhydrolyzed ATP analog, ATPcS, and magnesium ions are shown asgray sticks and orange balls, respectively. Residues at mutation sites are shown as van der Waals spheres in red. The secondary
structure assignment is according to published nomenclature. (B and C) Close-up of regions around the mutation sites from thefront and back. Side chains of some residues near the mutation sites are represented as transparent van der Waals spheres.
(D) Free energy change upon amino acid mutations, estimated from calculations using FoldX software. (E) HEK293T cells weretransfected with FLAG-MTOR or pcDNA3.1-MTOR hyperactive mutant and FLAG-4EBP plasmids. Cell lysates were immunoprecipitated with anti-Flag antibody beads and then probed with anti-phospho-4EBP (Thr36/47; left, upper panel), anti-Flag for
mTOR (left, middle panel), and 4EBP (left, lower panel). Three separate experiments were performed. Transfection efficacy
was almost the same in each transfection. Note that all the mutants found in this study led to significant activation of themTOR pathway, and the mutants are comparable with the hyperactive mutant (right panel). n 5 3 for WT and mutants.
*p < 0.0001 by analysis of variance. Western blotting were quantified by densitometric analysis using ImageJ software (NIH,
Bethesda, MD). Bars represent means 6 standard error of the mean. ATP 5 adenosine triphosphate; FCD 5 focal corticaldysplasia.

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TABLE 3. Prediction of Somatic MTOR Mutation Pathogenicity

Subject

Mutation

SIFT

PolyPhen2

Mutation Taster

11683 and 17424

c.6644C>A (p.Ser2215Tyr)0

Possibly damaging (0.876)

Disease-causing (0.999)

15622

c.4376C>A (p.Ala1459Asp)

0

Benign (0.133)

Disease-causing (0.999)

16578 and 16964

c.4379T>C (p.Leu1460Pro)

0

Benign (0.352)

Disease-causing (0.999)

14434

c.6644C>T (p.Ser2215Phe)

0

Possibly damaging (0.824)

Disease-causing (0.999)

SIFT (http://sift.jcvi.org/): scores of less than 0.05% indicate substitutions that are predicted as intolerant. PolyPhen-2 (http://
genetics.bwh.harvard.edu/pph2/): HumVar scores are evaluated as 0.000 (most probably benign) to 0.999 (most probably damaging). Mutation Taster (http://www.mutationtaster.org/): rapid evaluation of DNA sequence alterations. Alterations are classified as
disease-causing or polymorphisms.

he
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eor
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he
ts.
H,
al

3

showed numerous dysmorphic neurons and balloon cells
regardless of the presence or absence of mutations (Fig
3C, F, I, L, O, R). We then examined the activation of
the mTOR pathway in FCD type I, IIa, and IIb using
IHC for phospho-S6, which is a downstream phosphorylated protein in the mTOR pathway. In FCD type IIb
specimens with or without MTOR mutations, almost all
dysmorphic neurons and balloon cells were distributed inboth the cortex and subcortical white matter and showed
intense reactivity for phospho-S6 (Fig 4A–C). A small
proportion of normal-looking pyramidal neurons distributed in the cortex also showed a variable intensity ofreactivity for phospho-S6. On the other hand, neurons
showed no or faint reactivity for phospho-S6 in all control specimens (Fig 4D). We also confirmed the expression level of phospho-S6 by Western blotting of proteinsextracted from lesion-specific brain sections. The ratio ofphosphorylation (the ratio of intensities of phospho-S6/
total-S6) in each FCD type IIb individual with an
MTOR mutation (range, 0.72–1.24; average 5 1.17) wasmuch higher than that in the control specimens (range,
0.22–0.63; average 5 0.51; Fig 4E). Note that the lowest
ratio (0.72) in FCD type IIb was still greater than thehighest ratio (0.63) in the control. These results indicated that mTOR signaling was increased in FCD typeIIb cases of MTOR mutations.
MTOR Mutations Activate the mTOR Pathway
mTOR (aa 1,376–2,549) consists of Huntingtin, elongation factor 3, a subunit of protein phosphatase 2A, andTOR1 (HEAT) repeats, a FRAP, ATM, and TRAP
(FAT) domain, a kinase domain comprising N- and Clobes that form a catalytic cleft, a FKBP12/rapamycin
binding (FRB) domain, and a FRAP, ATM, TRRAP, andC-terminal (FATC) domain (Figs 2A and 5A).31 The
neighboring p.Leu1460Pro and p.Ala1459Asp mutations
are located in the FAT domain, whereas p.Ser2215Tyrand p.Ser2215Phe are at the same residue within the

kinase domain (Figs 2A and 5A). All altered amino acids
are evolutionarily well conserved (Fig 2A), and the mutations were predicted to be deleterious by at least two
online programs (Table 3). To evaluate the effects of missense mutations on mTOR function from a structural
viewpoint, we mapped their location on the mTOR crystal structure (aa 1,376–2,549) in complex with mLST8,
ATPcS, and magnesium ions.31 The FRB domain in the
N-lobe and helix 9b in the C-lobe hinder substrate accessto the active site, negatively regulating mTOR kinase
activity.33–35 Because residues at sites Ala1459 andLeu1460 are located in an N-terminal helix of the FAT
domain and are involved in a hydrophobic core (Fig 5A,
C), mutations here would destabilize the local structureof the FAT domain and impair its interaction with thekinase domain. This could release it from the restrictionof active site accessibility, thereby hyperactivating mTOR
kinase activity as reported previously.33 Ser2215 islocated in helix 3b, which is adjacent to the structural
frame involving closely located helices 3, 9, and 9b (Fig
5A, B). Thus, mutations of Ser2215 to Tyr and Phe arelikely to destabilize the structural frame and affect helix
9b conformation, restricting active site accessibility, andleading to mTOR kinase hyperactivation. Thus, all four
somatic MTOR mutations were predicted to induce
mTOR pathway hyperactivation. The free energy change
calculated by FoldX software revealed a remarkableincrease after all mutations, supporting our structural
predictions (Fig 5D). Moreover, the MTOR mutations
found in FCD type IIb were analyzed in cultured cells(in vitro). Plasmids carrying WT and mutant MTORs(p.Ala1459Asp, p.Leu1460Pro, p.Ser2215Phe, andp.Ser2215Tyr) were transfected into HEK293T cells
together with FLAG-4EBP, the best-known substrate for
mTOR complex 1 (mTORC1). Mutant MTOR-transfected cells exhibited significantly strong phosphorylationof 4EBP, compared to WT MTOR-transfected cells, indicating that all MTOR mutants are constitutively active

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FIGURE 6: Schematic presentation of the mammalian targetof rapamycin (mTOR)-signaling pathway. Upstream regulators of mTOR complex 1 (mTORC1), after stimulation withgrowth factors, are PI3K, Akt, TSC1/2, and Rheb. PTEN suppresses Akt activity. Another regulatory pathway that is
activated by amino acids is Rags/Ragulator signaling.
Gator1 suppresses mTORC1 activity. Activated mTORC1
directly phosphorylates 4EBP, p70S6K, and ULK1. Focal
cortical dysplasia is caused by gain-of-function mutations(red arrows) or loss-of-function mutations (blue arrows) inmTOR-related genes. Black arrows and cross-end bars indicate activation of phosphorylation and inhibition of phosphorylation, respectively.

(Fig 5E). Signal intensities of all MTOR mutants werecomparable with that of the plasmid carrying a hyperactive mutation of rat mTOR, which was used as a positive
control.34

Discussion
We found that the low-prevalence somatic MTOR mutations in 6 of 13 individuals with FCD type IIb (46%;
Table 2 and Supporting Table 4). While this article wasbeing reviewed, Lim et al also reported that somatic
MTOR mutations caused FCD type IIb.36 These results
corroborated that low-prevalence somatic MTOR mutation is one of the genetic causes in FCD type IIb. However, there may be mutations in up- or downstream
genes in the mTOR cascade in the other subjects having
no MTOR mutation (Fig 6). Indeed, somatic mutations
in PTEN and germline mutations in DEPDC5, encoding
a component of Gator1, were reported in FCD type
IIb.16–18,37 PTEN is one of the tumor-suppressor genes,
encoding plasma membrane lipid phosphatase, whichantagonizes PI3K-Akt signaling.38 Gator1 has GTPaseactivating activity for RagA/B and thus suppresses
mTORC1 activity in static and low-amino-acid conditions.39 Therefore, loss of function in PTEN and Gator1

may cause mTOR activation. Similarly, mutations inother genes involved in the mTOR cascade are involvedin tuberous sclerosis (with mutations in TSC1 andTSC2) and hemimegalencephaly (with mutations in
AKT3, DEPDC5, MTOR, PIK3CA, and PTEN), whichshare histopathological similarities.12–17,19,37 These findings strongly suggest that unidentified mutations inrelated molecules of the mTOR pathway may possibly be
the pathogenesis of FCD type IIb.
Somatic MTOR mutations in FCD type IIb are
likely to cause hyperactivation of the mTOR-signaling
pathway, which is involved in growth, migration, and thematuration of neurons and glial cells.8 All individuals
with MTOR mutations showed distinctive balloon cells
in their brain tissues (Fig 3 C, F, I, L, O, R). Such cells
are also observed in tuberous sclerosis and hemimegalencephaly, in which the mTOR-signaling pathway is activated,2,8,10,13,32 raising the possibility that they aremarkers of aberrant mTOR signaling. Our IHC andWestern blotting results clearly demonstrated high levelsof phospho-S6, the well-known hallmark of mTOR cascade activation, in FCD type IIb lesions with MTOR
mutations, specifically in dysmorphic neurons and balloon cells (Fig 4A, B, E). We failed to detect apparent
differences on IHC expression of phospho-S6 between
FCD type IIb with and without mutations (Fig 4A–C).
In both cases, a large proportion of the abnormal cells(dysmorphic neurons and balloon cells) were phosphoS6-positive. Furthermore, we found no clinical differences between FCD type IIb individuals with or without
MTOR mutations. These findings supported the hypothesis that MTOR-mutation–negative subjects may havemutations in other molecular members of the pathway,
which lead to net activation of the mTOR signaling.19
We also investigated MTOR mutations in limited
numbers of brain samples with FCD type I and IIa (sixin type I and five in type IIa) and found no associatedvariants (Supporting Table 3). IHC examination of FCD
type IIa revealed that many dysmorphic neurons showed
phospho-S6-immunoreactivity as those noted in FCD
type IIb (data not shown). In our series of FCD type IIa,
both the number of dysmorphic neurons and intensitiesof phospho-S6-immunopositivity differed markedly
among cases. Interestingly, Lim et al also reported
somatic MTOR mutations in FCD type IIa.36 Therefore,
a further study with a large number of FCD type IIa
cases would be necessary for better understanding of themolecular profiles and precise pathomechanisms underlying FCD type IIa. In control neocortical tissues, wefound no dysmorphic neurons, balloon cells, or apparent
cytoarchitectural abnormalities without any trace of aberrant mTOR signaling (Fig 4D, E). Consistent with this,

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we observed no apparent phospho-S6 immunoreactivity
in FCD type I (data not shown).mTOR activity is controlled by various extracellular cues, such as nutrients andgrowth factors in the brain.8 In a culture system, all of
the mutants found in this study were constitutively active(Fig 5E), regardless of any extracellular stimuli. Therefore, these somatic mutations could induce dysregulationof growth of neurons and glia, or presumably of theirprogenitors during brain development. A large numberof somatic mutations in cancer cells are clustered in the
FAT domains or the kinase domains, and most activate
the mTOR-signaling pathway.6,33 The mTOR protein
can form two distinct complexes—mTORC1 andmTOR complex 2 (mTORC2)—and involvement ofthese different complexes results in divergence of the signaling pathway into two distinct channels. mTORC1 iscomposed of mTOR, mLST8, and Raptor and phosphorylates the downstream proteins, S6K1 and 4EBP, which
regulate cell growth, proliferation, ribosome biogenesis,
and autophagy (Fig 6).32,40 mTORC2 is composed ofmTOR, mLST8, Rictor, and mSin1 and phosphorylates
Akt, PKC, and SGK, which are involved in cell cycle,
cell survival, and actin organization and morphology.32,40,41 Rapamycin, an allosteric inhibitor of mTOR,
binds to mTOR with FKBP12 and inhibits kinase activity of mTORC1. mTORC1 is known to play a pivotal
role in cerebral cortical development, though little is
known about mTORC2.8,9 The mutations, p.Ser2215Tyrand p.Leu1460Pro, lead to the constitutive activation ofmTORC1 signaling, but not that of mTORC2, andboth mutations are sensitive to rapamycin.6,42 Thesefindings imply that mTOR inhibitors would be able toalleviate intractable epilepsy in FCD type IIb.

K. Takabe, and S. Nigorikawa for their excellent technical assistance. The plasmids of pCMV-FLAG and hyperactive mTOR mutant were kindly gifted by Dr K.
Yonezawa and Dr T. Maeda, respectively.

Authorship
M.K., A.K., and N.Ma. conceived the study. M.N.,
H.Sa., and N.Ma. designed and performed analyses.
M.N., Y.T., N.Mi., and H.Sa. performed exome sequencing. M.N. and C.O. performed targeted ampliconsequencing. M.N. performed statistical analysis andddPCR. M.S. and K.O. performed protein structural analyses. J.T., M.K., H.Sh., H.M., and S.K. evaluated patientsand provided samples. H.K, K.W., H.T., Y.Z., and A.K.
processed tissue samples and performed pathological evaluation. N.T. and T.S. validated mTORC1 signaling. M.N.,
H.Sa., N.T., A.K., and N.Ma. wrote the manuscript.
M.K., A.K., and N.Ma. obtained research funding.

Potential Conflicts of Interest
Nothing to report.

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This work was supported by grants from the Ministry of
Health, Labor and Welfare of Japan; a Grant-in-Aid for
Scientific Research (A), (B), and (C); a Grant-in-Aid for
Exploratory Research from the Japan Society for the Promotion of Science; the Takeda Science Foundation; thefund for Creation of Innovation Centers for Advanced
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Thorvaldsdottir H, Robinson JT, Mesirov JP. Integrative Genomics
Viewer (IGV): high-performance genomics data visualization andexploration. Brief Bioinform 2013;14:178–192.

41.

Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes,
only one of which is rapamycin sensitive, have distinct roles in cell
growth control. Mol Cell 2002;10:457–468.

26.

Miyahara H, Natsumeda M, Shiga A, et al. Suppressed expressionof autophagosomal protein LC3 in cortical tubers of tuberous
sclerosis complex. Brain Pathol 2013;23:254–262.

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Grabiner BC, Nardi V, Birsoy K, et al. A diverse array of cancerassociated MTOR mutations are hyperactivating and can predict
rapamycin sensitivity. Cancer Discov 2014;4:554–563.

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Neuropathology 2015; 35, 324–335

doi:10.1111/neup.12196

Or i g i n a l A r t i c l e

Immunohistochemical profiles of IDH1, MGMTand P53: Practical significance for prognosticationof patients with diffuse gliomas
Ryosuke Ogura,2 Yoshihiro Tsukamoto,2 Manabu Natsumeda,1 Mizuho Isogawa,1 Hiroshi Aoki,1Tsutomu Kobayashi,1 Seiichi Yoshida,1 Kouichiro Okamoto,1 Hitoshi Takahashi,2 Yukihiko Fujii1
and Akiyoshi Kakita2
Departments of 1Neurosurgery and 2Pathology, Brain Research Institute, University of Niigata, Niigata, Japan

Genetic and epigenetic status, including mutations ofisocitrate dehydrogenase (IDH) and TP53 and methylationof O6-methylguanine-DNA methyltransferase (MGMT),
are associated with the development of various types ofglioma and are useful for prognostication. Here, using routinely available histology sections from 312 patients withdiffuse gliomas, we performed immunohistochemistry using
antibodies specific for IDH1 mutation, MGMT methylation
status, and aberrant p53 expression to evaluate the possible
prognostic significance of these features. With regard to
overall survival (OS), univariate analysis indicated that an
IDH1-positive profile in patients with glioblastoma (GBM),
anaplastic astrocytoma (AA), anaplastic oligoastrocytoma
and oligodendroglioma, or a MGMT-negative profile inpatients with GBM and AA were significantly associatedwith a favorable outcome. Multivariate analysis revealed
that both profiles were independent factors influencing
prognosis. The OS of patients with IDH1-positive/MGMTnegative profiles was significantly longer than that ofpatients with negative/negative and negative/positive profiles. A p53 profile was not an independent prognostic
factor. However, for GBM/AA patients with IDH1negative/MGMT-negative profiles, p53 overexpression was
significantly associated with an unfavorable outcome. Thus,
the immunohistochemical profiles of IDH1 and MGMT areof considerable significance in gliomas, and a combinationof IDH1, MGMT and p53 profiles may be useful for prognostication of GBM/AA.

Correspondence: Akiyoshi Kakita, MD, PhD, Department of
Pathology, Brain Research Institute, University of Niigata, 1-757
Asahimachi, Chuo-ku, Niigata 951-8585, Japan. Email: kakita@
bri.niigata-u.ac.jp
Received 21 October 2014; revised 21 December 2014 and accepted
22 December 2014; published online 6 May 2015.

Key words: glioma, IDH1, immunohistochemistry, MGMT,p53.

INTRODUCTION
Glioma is the most common type of primary brain tumor.
Recent molecular studies of various histopathologicaltypes of glioma1 have revealed several genetic pathways
critical for the development of astrocytic and oligodendroglial tumors.2,3 For prognostication of patients withdiffuse gliomas, it has been shown that the molecular profiles of isocitrate dehydrogenase 1/2 (IDH1/2) mutation,
O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, 1p19q co-deletion, and glioma CpG
island methylator phenotype (G-CIMP) are possible
factors, whereas the impact of TP53 and EGFR mutation
has remained unclear.4 Moreover, a recent genetic andepigenetic study has revealed that a combination of IDH1
mutation and MGMT methylation status outperforms
information about either IDH1 or MGMT alone in predicting the survival of patients with glioblastoma (GBM).5
Thus, in clinical practice, it is important to have information on these molecular profiles. However, identification ofmolecular profiles by DNA sequencing requires intensive
laboratory work by trained personnel using specialized
equipment, and therefore the methodology is not always
available for daily clinical use at every medical center.
On the other hand, immunohistochemistry is a techniquethat can be applied to routinely available histology sections.
For detection of IDH1 mutations, an IDH1-R132H
mutation-specific antibody is available, and the results showconcordance with DNA sequencing without any crossreactivity with the wild-type or other mutant IDH1
proteins.6,7 Moreover, immunohistochemistry with a monoclonal antibody MT3.1 against MGMT protein has been

© 2015 Japanese Society of Neuropathology
3－ 157 －

325

IDH1/MGMT/p53 immunoprofiles and glioma prognosis
Table 1 Clinical profiles of patients in each group
Group

Grade IV

Grade IIINo. of patientsAge (years)
MedianRange
Age (years), n (%)<50
50–59 years
60–69 years≧70
Gender, n (%)
Male
Female
KPS, n (%)
100
70–90
<70
Surgery, n (%)
Total/Subtotal
PartialStereo biopsy
Others
Initial treatment, n (%)
None
RT alone
CRT
Others

165

61

40

19

27

64
18−85

58
24–89

59
22–81

38
24–67

45
25–74

25 (15.1)
26 (15.8)
60 (36.4)
54 (32.7)

23 (37.7)
10 (16.4)
7 (11.5)
21 (34.4)

8 (20)
11 (27.5)
10 (25.0)
11 (27.5)

12 (63.2)
5 (26.3)
2 (10.5)
015 (55.6)
4 (14.8)
6 (22.2)2 (7.4)93 (56.3)
72 (43.6)

33 (54.1)
28 (45.9)

26 (65)
14 (35)9 (47.4)
10 (52.6)

14 (51.9)
13 (48.1)

4 (2.4)95 (57.6)
66 (40.0)

11 (18.0)
32 (52.5)
18 (29.5)

4 (10.0)
19 (47.5)
17 (42.5)

12 (63.2)
7 (36.8)09 (33.3)
17 (63.0)
1 (3.7)72 (43.6)
53 (32.1)
38 (23.0)
2 (1.2)

12 (19.6)
27 (44.3)
19 (31.2)
3 (4.9)21 (52.5)
19 (47.5)
00

12 (63.2)
7 (36.8)
00

17 (63.0)
9 (33.3)
1 (3.7)0

8 (4.8)61 (37.0)
95 (57.6)
1 (0.6)2 (3.3)21 (34.4)
36 (59.0)
2 (3.3)0
8 (20.0)32 (80.0)012 (63.2)
7 (36.8)
00

11 (40.7)
16 (59.3)
0
0

GBM

AA

Grade II
AOA/AODDA

OA/OD

GBM, glioblastoma; AA, anaplastic astrocytoma; AOA, anaplastic oligoastrocytoma; AOD, anaplastic oligodendroglioma; DA, diffuse astrocytoma; OA, oligoastrocyotma; OD, oligodendroglioma; KPS, Karnofsky performance status; RT, radiotherapy; CRT, chemoradiotherapy.

employed to assess in vivo MGMT methylation status, and asignificant negative association of MGMT with survival has
been detected in patients with WHO grades II and IV
astrocytomas.8 Furthermore, immunohistochemical detection of overall p53 protein expression may be an independent indicator of shorter survival in patients with astrocytic
tumors, even though overexpression of p53 is not always
associated with point mutations in the TP53 gene.9 In thepresent study, we performed immunohistochemistry using
antibodies specific for IDH1 mutation, MGMT status, andaberrant p53 expression in order to evaluate the possible
prognostic significance of these features and their combinations in patients with diffuse gliomas.

MATERIALS AND METHODSPatients
We reviewed the medical records of 312 consecutive
patients (175 male, 137 female: age at surgery, mean 1
SD = 58.4 1 15.6 years) who were admitted to the University of Niigata Hospital, Japan, between 2000 and 2013, andwere diagnosed pathologically as having diffuse gliomas,
including diffuse astrocytoma (DA), oligodendroglioma(OD), oligoastrocytoma (OA), anaplastic astrocytoma(AA), anaplastic oligodendroglioma (AOD), anaplastic
oligoastrocytoma (AOA) and GBM. The study wasapproved by the hospital Ethics Committee, and written
informed consent to use resected tissues for research purposes was obtained from all patients. First, in accordancewith the histopathological diagnosis of diffuse gliomas,1 thepatients were divided into five groups: GBM, AA, AOA/
AOD, DA and OA/OD. Table 1 summarizes the clinicalprofiles of the patients in each of these histologicallydefined groups.

Histology and immunohistochemistry
The surgical specimens were fixed with 20% buffered formalin and embedded in paraffin. Histopathological examination was performed on 4-μm-thick sections stained with
HE. The paraffin-embedded sections were processed
for immunohistochemistry using methods previously
described.10 Primary monoclonal antibodies against thefollowing antigens were used: human IDH1 R132H (clone
H09; Dianova, Hamburg, Germany; dilution 1:100),
MGMT (clone MT3.1; Chemicon International, Temecula,
CA, USA; 1:50), and human p53 (clone DO-7; Dako,
Glostrup, Denmark; 1:50). Immunohistochemical features were evaluated by two persons (RO and YT)© 2015 Japanese Society of Neuropathology－ 158 －

326

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325

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R Ogura et al.

independently without information on the patients’ profiles or histological grades, and then were confirmed by apathologist (AK).

IDH1 mutations: direct DNA sequencing andimmunohistochemical evaluation
Genomic DNA was extracted from paraffin-embedded
sections, as described previously,11 and PCR amplification
was performed using primer sets (forward: 5′-CGGTCTTCAGAGAAGCCATT-3′; and reverse: 5′-TTCATACCTTGCTTAATGGGTGT-3′) for exon 4 of the IDH1 genearound the R132 residue. The PCR products were thensequenced on a 3130xl Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA) with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) inaccordance with the manufacturer’s instructions.
IDH1 immunoreactivity was judged as “positive” whena large proportion of tumor cells showed strong cytoplasmic reactivity (Fig. 1A), whereas it was regarded as “negative” if weak or no reactivity was observed (Fig. 1B).
Labeling of macrophages was ignored.
To compare the results of both direct DNA sequencing
and immunohistochemistry, we preliminarily applied bothmethods to specimens taken from 37 patients with GBM.
All four patients with an IDH1-immunopositive profile
showed an identical heterozygous mutation in IDH1
(CGT→CAT) (Fig. 1C) resulting in an Arg→His (R132H)
amino acid substitution. The other 33 patients withan IDH1-immunonegative profile showed the wild-type
IDH1 gene. Furthermore, direct DNA sequencing using
samples taken from areas with sparsely infiltrating tumor
cells failed to detect the mutation, although in one patientwith anaplastic astrocytoma immunohistochemistry demonstrated a small number of positive cells in this type ofarea (Fig. 1C). Thus, application of immunohistochemistryfor detection of IDH1 R132H mutation was shown to bereliable and of practical use.

Evaluation of MGMT and p53
MGMT immunoreactivity was evaluated in representative
areas of the tumors showing the characteristic features
defining their histological grades.1 Individual tumor nuclei
were regarded as positive when they showed an intensity
similar to that of vascular endothelial cells used as an internal positive control (Fig. 1D,E). In accordance with aprevious report,12 we determined percentages of MGMTpositive tumor cells in individual cases by continuously
counting over 400 nuclei observed in high-magnification
views. We carefully excluded possible positive staining of
non-neoplastic cells, including oligodendrocytes and infiltrating inflammatory cells.13,14 For individual cases, when
the proportion of labeled nuclei/all tumor nuclei was 30%

or more, the case was regarded as “positive”, whereas acase was considered “negative” when the proportion wasless than 30% (Fig. 1D,E). Indeed, there was a clear difference between the numbers of “negative” and “positive”
cases (Fig. 1F), which enabled us to judge with confidence.
Immunonegativity for MGMT may represent the
methylation status of the MGMT gene, and is positively
linked to the response of a tumor to chemotherapy.15
Evaluation of p53 expression was performed in a semiquantitative manner by continuously counting over 1000
tumor cells in the areas of highest expression. According to
the percentage of tumor cells showing reactivity, we distinguished four groups: many (>50%), several (10–50%), rare(<10%), or no cells positive (Fig. 1G–J).16 In accordancewith previous reports,9,17 we considered cases in the manyand several groups as “positive” and those in the rare andnegative groups as “negative”.

Statistical analysis
Data analysis was carried out using the SPSS ver17.0
software package (SPSS Inc., Chicago, IL, USA) and
GraphPad Prism 5.0 statistical software (http://graphpadprism.software.informer.com/5.0/). Overall survival (OS)and progression-free survival (PFS) were calculated fromthe time of surgery until death, disease progression, or last
follow-up according to the Kaplan-Meier method with logrank test for comparison between groups. Univariate andmultivariate analysis with the Cox proportional hazards
model was used to assess prognostic factors. Results are
expressed as relative risk with the 95% confidence interval(95% CI).

RESULTS
Significance of immunohistochemical profiles of
IDH1, MGMT and p53
Tables 2 and 3 summarize the immunohistochemical profiles and their prognostic values in terms of OS and PFS for
patients included in each of the histologically definedgroups. Figure 2 shows the Kaplan-Meier curves of the OS
rate and median OS (mOS) for patients with grades III/IV
malignant astrocytomas (AA/GBM).
The numbers of patients with IDH1-positive profiles
were significantly higher and lower than those of patientswith IDH1-negative profiles for grade II (DA, P = 0.003;
OA/OD, P = 0.004) and grades III (AA, P = 0.007)/IV(GBM, P < 0.001) gliomas, respectively, and there were no
significant differences in the numbers of patients with bothprofiles in the AOA/AOD group (P = 0.114). The numbersof patients with MGMT-negative profiles in the oligodendroglial tumor groups (OA/OD, P = 0.001; AOA/AOD,© 2015 Japanese Society of Neuropathology－ 159 －

327

IDH1/MGMT/p53 immunoprofiles and glioma prognosis

328

Tab

toch

Gro

GB
N
m
m
AA
N
m
m
AO
N
m
m
DA
N
m
m
OA
N
m
m

P
m
astr
olig

P<
wit
pat
(DA
alm
pos
app
gra
54%
pos
pat
P=

Fig. 1 Evaluation of isocitrate dehydrogenase 1 (IDH1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 status.
Immunohistochemistry for IDH1 (A, B), MGMT (D, E), and p53 (G–J). Examples of IDH1 positivity (A), IDH1 negativity (B), MGMT
positivity (D), and MGMT negativity (E). Arrowheads in D and E indicate endothelial cells used as an internal positive control. Examplesof p53 many (G), several (H), rare (I), and no (J) cells positive. (C) Demonstration of IDH1 immunohistochemistry and direct DNA
sequencing of IDH1 in a patient with anaplastic astrocytoma. In the tumor center (left panels), many IDH1-positive cells and heterozygous
mutation of IDH1 R132H (arrow) are evident, whereas in the infiltrating area (right panels), scattered IDH1-positive cells but nomutations are demonstrated. (F) A histogram showing the proportion of the MGMT-labeled nuclei/all tumor nuclei for all 165 cases ofglioblastoma. A clear numerical distinction is evident between less than 30% (regarded as “negative”) and 30% and more (“positive”).
Scale bars = 50 μm.

© 2015 Japanese Society of Neuropathology－ 160 －

cat
wit
tive
AA
MG
P<
GB
out
IDH
MG
ent
(Ta
OA
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327

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”).

328

R Ogura et al.

Table 2 Prognostic value of mOS and mPFS of patients included in each histologically defined group, in accordance with immunohistochemical profiles of IDH1, MGMT and p53 of the gliomas
Group
GBM
No. of patients (%)
mOS (months)
mPFS (months)
AA
No. of patients (%)
mOS (months)
mPFS (months)
AOA/AOD
No. of patients (%)
mOS (months)
mPFS (months)
DA
No. of patients (%)
mOS (months)
mPFS (months)
OA/OD
No. of patients (%)
mOS (months)
mPFS (months)

Total

IDH1

MGMT

p53

Positive

Negative

P value

Negative

Positive

P value

Negative

P value

165
14
5

6 (4)
66
59

159 (96)14
4<0.001
0.006
0.005

89 (54)18
7

76 (46)12
3

0.312<0.001<0.00162 (38)
13
5

0.001
0.921
0.671

61
22
5

20 (33)46
22

41 (67)
15
4

0.007
0.013
0.002

33 (54)30
11

28 (46)
15
3

0.522
0.009
<0.00128 (46)
18
6

0.522
0.967
0.847

40
80
24

15 (38)NRNR

25 (62)
25
10

0.114
0.025
0.001

32 (80)8028

8 (20)
21
7<0.001
0.206
0.160

19 (47)
25
20

0.752
0.522
0.470

19
NR
NR

16 (84)NRNR

3 (16)
ND
51

0.003
–
–

11 (58)NR44

8 (42)
ND
NR

0.491
–
–

14 (74)
ND
51

0.039
–
–

27
NR
NR

22 (81)NRNR

5 (19)
40
19

0.004
0.100
0.080

21 (78)NRNR

6 (22)
NR
19

0.001
0.381
0.142

23 (85)NR
NR<0.0010.021
0.031

P-values were calculated by the log-rank test and Chi-square test.
mOS, median overall survival; mPFS, median progression free survival; NR, not reached; ND, not determined. GBM, glioblastoma;AA, anaplastic
astrocytoma; AOA, anaplastic oligoastrocytoma; AOD, anaplastic oligodendroglioma; DA, diffuse astrocytoma; OA, oligoastrocyotma; OD,
oligodendroglioma

P < 0.001) were significantly higher than those of patientswith MGMT-positive profiles, whereas the numbers ofpatients with either profile in the astrocytic tumor groups(DA, P = 0.491; AA, P = 0.522; GBM, P = 0.312) were
almost equal. The proportion of patients with a p53positive profile in each histologically defined group
appeared to increase in accordance with malignancy
grading (OA/OD 15%, DA 26%, AOA/AOD 53%, AA
54% and GBM 62%). The number of patients with p53positive profiles was significantly higher than that ofpatients with p53-negative profiles for grade IV (GBM,
P = 0.001) only.
With regard to OS and PFS, univariate analysis indicated that either an IDH1-positive profile for patientswith GBM (OS and PFS: P = 0.014 and P = 0.015, respectively), AA (P = 0.020 and P = 0.004) (Fig. 2A for GBM/
AA) and AOA/AOD (P = 0.035 and P = 0.003), or a
MGMT-negative profile for patients with GBM (bothP < 0.001) and AA (P = 0.016 and P = 0.006) (Fig. 2B for
GBM/AA) was significantly associated with a favorable
outcome. Multivariate analysis revealed that both an
IDH1-positive profile (P = 0.031 and P = 0.013) and a
MGMT-negative profile (both P < 0.001) were independent factors affecting prognosis for patients with GBM(Table 3). For patients with grade II gliomas (DA andOA/OD), the prognostic value of IDH1 and MGMT
status was less significant.

On the basis of the IDH1 and MGMT profiles, a total of
226 patients with grades III/IV malignant astrocytomas(165 and 61 patients with GBM and AA, respectively) weredivided into four groups. The mOS of patients with
IDH1-positive/MGMT-negative (n = 18), positive/positive(n = 8), negative/negative (n = 104), and negative/positive(n = 96) profiles was 56, 22, 17 and 12 months, respectively(Fig. 2C). The mOS of patients with positive/negative
profiles was significantly longer than that of patientswith negative/negative (P = 0.002) and negative/positive(P < 0.001) profiles. Similarly, the mOS of patients withpositive/positive and negative/negative profiles was significantly longer (P = 0.008 and P < 0.001, respectively) thanthat of patients with a negative/positive profile. Thus, acombination of both IDH1 and MGMT profiles was able toaccurately predict outcome in patients with malignant
atrocytomas.
On the other hand, a p53 positive/negative profile was
not an independent factor affecting prognosis for patientswith malignant astrocytomas (Table 3) when we defined
the many and several groups (p53 >10%) (Fig. 2D) or the
many group only (p53 >50%) (Fig. 2E) as “positive”. Next,
we examined the effects of combinations of the IDH1,
MGMT and p53 profiles (Fig. 2F–I). For patients with
IDH1-positive profiles (Fig. 2F,G), neither p53 >10%
(Fig. 2F) nor p53 >50% (Fig. 2G) were a prognostic factor.
Similarly, for patients with IDH1-negative profiles© 2015 Japanese Society of Neuropathology－ 161 －

IDH1/MGMT/p53 immunoprofiles and glioma prognosis

329

330

astr
(Fi
poo
(n =
mO
out

Table 3 Prognostic univariate and multivariate Cox regression analyses of patients included in each histologically defined group
Univariate
GBM
IDH1
MGMT
p53
KPS
Age
Total/subtotal
CRT
AA
IDH1
MGMT
p53
KPS
Age
Total/subtotal
CRT
AOA/AOD
IDH1
MGMT
p53
KPS
Age
Total/subtotal
CRT
DA
IDH1
MGMT
p53
Age
Total/subtotal
RT
OA/OD
IDH1
MGMT
p53
Age
Total/subtotal
RT

OS
HR

95% CI

P-valueHR

95% CI

P-valuePos vs. neg
Neg vs. pos
Pos vs. neg≧70 vs. <70≧65 vs. <65
Yes vs. no
Yes vs. no

0.230
0.463
1.014
0.614
1.632
0.530
0.446

0.071–0.742
0.323–0.665
0.711–1.445
0.431–0.876
1.142–2.332
0.367–0.765
0.313–0.638

0.014<0.001
0.940
0.007
0.007
0.001
<0.001

0.228
0.503
1.073
0.717
1.226
0.605
0.585

0.069–0.751
0.347–0.729
0.740–1.556
0.493–1.043
0.848–1.774
0.420–0.872
0.400–0.854

0.015<0.001
0.709
0.082
0.278
0.007
0.005

Pos vs. neg
Neg vs. pos
Pos vs. neg≧70 vs. <70≧60 vs. <60
Yes vs. no
Yes vs. no

0.459
0.473
1.066
0.334
3.120
0.382
0.565

0.239–0.884
0.257–0.872
0.583–1.951
0.169–0.660
1.651–5.898
0.173–0.844
0.305–1.046

0.020
0.016
0.835
0.002
<0.001
0.017
0.069

0.373
0.416
0.987
0.413
1.964
0.370
0.883

0.189–0.736
0.221–0.780
0.530–1.839
0.204–0.835
1.001–3.850
0.160–0.858
0.461–1.692

0.004
0.006
0.968
0.014
0.050
0.021
0.707

Pos vs. neg
Neg vs. pos
Pos vs. neg≧70 vs. <70≧60 vs. <60
Yes vs. no
Yes vs. no

0.291
0.521
0.742
0.339
2.067
0.260
0.560

0.092–0.916
0.184–1.474
0.294–1.874
0.122–0.945
0.777–5.501
0.092–0.737
0.198–1.578

0.035
0.219
0.528
0.039
0.146
0.011
0.272

0.238
0.529
0.760
0.394
3.267
0.399
0.836

0.093–0.611
0.212–1.320
0.356–1.622
0.173–0.896
1.407–7.584
0.184–0.866
0.333–2.096

0.003
0.172
0.477
0.026
0.006
0.020
0.702

Pos vs. neg
Neg vs. pos
Pos vs. neg≧45 vs. <45
Yes vs. no
Yes vs. no

NR
NR
6.329
2.171
0.812
NR

1.014
2.276
0.645
0.400
0.714
1.290

0.117–8.774
0.384–13.497
0.075–5.544
0.046–3.451
0.140–3.643
0.255–6.525

0.990
0.365
0.690
0.405
0.685
0.758

Pos vs. neg
Neg vs. pos
Pos vs. neg≧45 vs. <45
Yes vs. no
Yes vs. no

0.246
0.458
6.407
1.504
0.345
1.829

0.306
0.399
4.155
2.299
0.321
3.236

0.075–1.239
0.112–1.420
1.025–16.839
0.547–9.657
0.076–1.348
0.397–26.349

0.097
0.156
0.046
0.255
0.121
0.272

Multivariate
GBM
IDH1
MGMT
KPS
Age
Total/subtotal
CRT

PFS

0.569–70.412
0.131–35.996
0.066–9.975

0.133
0.588
0.871

0.040–1.498
0.076–2.746
1.052–39.020
0.251–9.006
0.058–2.068
0.204–16.389

0.128
0.393
0.044
0.655
0.244
0.589

OS

Pos vs. neg
Neg vs. pos≧70 vs. <70≧65 vs. <65
Yes vs. no
Yes vs. no

PFS

HR

95% CI

P-valueHR

95% CI

P-value

0.265
0.457
0.694
1.207
0.582
0.580

0.079–0.886
0.315–0.663
0.481–1.002
0.814–1.791
0.402–0.841
0.390–0.864

0.031<0.001
0.051
0.349
0.004
0.007

0.209
0.483
0.667
0.982
0.618
0.675

0.061–0.715
0.329–0.710
0.454–0.980
0.657–1.469
0.427–0.895
0.446–1.023

0.013<0.001
0.039
0.931
0.011
0.064

P values were calculated by Cox proportional hazard models.
CI, confidence interval; HR, hazard ratio; neg, negative; pos, positive; GBM, glioblastoma; AA, anaplastic astrocytoma; AOA, anaplastic
oligoastrocytoma; AOD, anaplastic oligodendroglioma; DA, diffuse astrocytoma; OA, oligoastrocyotma; OD, oligodendroglioma; KPS, Karnofsky
performance status; RT, radiotherapy; CRT, chemoradiotherapy(Fig. 2H,I), p53 >10% (Fig. 2H) was not a prognostic
factor, and a MGMT-negative profile appeared to operate
as a favorable factor. However, the OS of patients with p53>50% who were MGMT-negative (n = 28) was significantly
shorter than that of patients with p53 250%/MGMT-

negative profiles (n = 76, P = 0.044) (Fig. 2I). Thus, forpatients with IDH1-negative/MGMT-negative profiles, p53>50% was associated with a poor outcome.
Based on combinations of the IDH1, MGMT andp53 profiles, three groups of patients with malignant© 2015 Japanese Society of Neuropathology－ 162 －

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astrocytomas were distinguishable in terms of outcome(Figs 2J and 3). Group C (n = 124) showed significantly thepoorest outcome (mOS = 12 m) relative to Group B(n = 76, mOS = 18 m, P < 0.001) and Group A (n = 18,mOS = 56 m, P < 0.001). Group B also showed a poorer
outcome than Group A (P = 0.005).

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Clinical factors and prognosis
For patients with malignant astrocytomas, several clinicalcharacteristics, including Karnofsky performance statusscore, age, total/subtotal removal of the tumor, and useof chemoradiotherapy (CRT: yes vs. no, n = 131 vs. 95;
Fig. 2K) showed a significant relationship with OS(Table 3). Multivariate analysis indicated that total/
subtotal removal of the tumor and use of CRT were independent factors significantly associated with a favorable
outcome (Table 3).
For patients with malignant astrocytomas who received
CRT (n = 131; Fig. 2L), the mOS of those with a MGMTnegative profile (n = 77, 24 months) was significantly(P < 0.001) longer than that of patients with a MGMTpositive profile (n = 54, 15 months).

DISCUSSION
Recent molecular analyses of glioma tissue have revealed
that several profiles, including IDH1/2 mutation, TP53
mutation, TERT promoter mutation, ATRX mutation,
1p/19q co-deletion, and MGMT methylation status, areassociated with the development of various histologicaltypes of glioma. In the present study, we performed
immunohistochemistry to evaluate subsets of thesemolecular profiles using routinely available histology sections (Fig. 1). This revealed that the significance of the
profiles differed in accordance with the histopathologicalclassification of the gliomas, and that combination of the
profiles was of prognostic value (Tables 2 and 3, and Figs 2
and 3).
Patients with IDH1-mutated malignant glioma may
survive longer than those with wild-type IDH1 glioma.18–20
Recently, a genetic and epigenetic study has indicated a
possible mechanism underlying this phenomenon, in which
IDH1 mutations induce the CpG island methylator

phenotype, a factor associated with biologically benign
behavior of the glioma cells.21 Therefore, in clinical practice, detection of IDH1 mutations is important, and for thispurpose direct DNA sequencing is frequently employed.
However, tissue samples from diffuse gliomas are usuallyan admixture of tumor cells and pre-existing brain parenchyma; therefore the sensitivity of this method for detection of a mutant sequence within wild-type sequences
depends on the ratio of the two tissue components, and isestimated to be 20% or more.22 Pyrosequencing analysis isa much more sensitive method, capable of detecting aminimum of 5% mutant alleles.22,23 Moreover, the use of
BEAMing (beads, emulsion, amplification, magnetics)
PCR and droplet digital PCR can detect a mutant allele ina tissue sample with a sensitivity of 0.01%.24 Thus, advancesin methodology have improved the sensitivity of mutation
detection; on the other hand, these techniques require specific equipment and skill, and as a consequence they aresometimes unavailable in a standard clinical laboratory.
Immunohistochemistry is a technique that can be performed simultaneously with practical histopathologicaldiagnosis of tumors. This method has apparently highsensitivity for detection of IDH1 mutations.7 Consistent
with this, our preliminary attempt to perform bothimmunohistochemistry and direct DNA sequencing on
tissues showing glioma infiltration showed that mutation
was detectable by immunohistochemistry only (Fig. 1C).
Immunohistochemistry with the IDH1-R132H antibody
is highly specific for detecting the most common mutationin the gene.7 The sensitivity of this method is estimated tobe 94%,25 but other IDH1 mutations, including R132C,
R132S, R132G, R132L, R132V and R132P,6,7,20,26–30 or IDH2
mutations31 are not recognized by this antibody. Therefore,
even though such IDH1/2 mutations are rare in practice,for IDH1-immunonegative cases, direct DNA sequencing
for IDH1/2 may provide more detailed information on thegenes.32–34
The frequencies of patients with IDH1-immunopositive
profiles in the present study (Table 2) were similar to thosereported previously, where direct DNA sequencing identified IDH1 mutations in 70%-80% of diffuse gliomas,
including DA (72.51%), OD (77.81%), OA (81.43%), AA(48.71%), AOA (66.40%), AOD (32.79%) and primary

stic
sky

or
53

nd
ant

▶

Fig. 2 Kaplan-Meier curves. Overall survival rate (OS) for patients with grades III/IV malignant astrocytomas (anaplastic astrocytoma
and glioblastoma) (A–J). Median OS (months: m) for patients with (A) isocitrate dehydrogenase 1 (IDH1), (B) O6-methylguanine-DNA
methyltransferase (MGMT), (C) the IDH1/MGMT combination, (D) p53 (>10%: many and several groups, or 210%), (E) p53 (>50%:
many groups only, or 250%), (F) in patients with IDH1 positivity, combination of p53 (>/210%)/MGMT, (G) in patients with IDH1
positivity, combination of p53 (>/250%)/MGMT, (H) in patients with IDH1 negativity, combination of p53 (>/210%)/MGMT, and (I) in
patients with IDH1 negativity, combination of p53 (>/250%)-immunopositive (pos) or negative (neg) profiles are described. (J) OS for
patients in Groups A, B and C. The groups were defined as depicted in Figure 3. (K) OS for patients with (yes) or without (no)chemoradiotherapy (CRT). (L) For the patients with CRT, a MGMT-negative profile was significantly associated with a favorable
outcome. P-values were calculated by the log-rank test.

© 2015 Japanese Society of Neuropathology－ 163 －

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R Ogura et al.

Fig. 3 Schematic representation of Groups A, B and C distinguishable on the basis of combinations of the isocitrate dehydrogenase 1 (IDH1), O6-methylguanine-DNA methyltransferase(MGMT) and p53 immunoprofiles. The median overall survival(mOSs) of the three groups differ significantly. Eight patients with
IDH1-positive and MGMT-positive profiles were excluded. Notethat for the patients with IDH1-negative and MGMT-negative
profiles, the p53 profile (> or 250%) was a useful factor for
prognostication.

GBM (4.43%).31 In grade II gliomas, it is still unclear
whether IDH1 mutations influence outcome.35–38 In thepresent study, IDH1 immunoprofiles showed no significant
association with the outcome of patients with grade II
gliomas (Tables 2 and 3). On the other hand, for patients
who were diagnosed histopathologically as having grade
III or IV glioma, IDH1 positivity was a factor significantly
predictive of a favorable outcome (Tables 2 and 3).
At present, MGMT status is recognized to be a prognostic factor and a powerful predictor of the response totemozolomide in patients with GBM.15 Based on several
clinical studies involving patients with GBM, it has beenshown that MGMT promoter methylation assessed bymethylation-specific PCR or pyrosequencing is a favorable
prognostic factor.12,39,40 MGMT status can be regulated byseveral mechanisms, including histone modification41–43 andpost-transcriptional regulation with miRNA,44 and for thisreason MGMT status is not always identical at the DNA,RNA and protein levels. For example, it has been shownthat mRNA expression is transcriptionally regulated,

resulting in a low level in approximately 20% of patients, inwhom the MGMT promoter is unmethylated.45,46 Moreover, it has been demonstrated that variation in MGMT
promoter methylation can occur within the same tumor
after treatment.47 Furthermore, a previous study employing
multivariate analysis has indicated that expression of
MGMT protein, rather than MGMT status, was correlatedwith the survival of patients with GBM.48 Thus, immunohistochemical evaluation of MGMT protein status inglioma tissue may provide useful prognostic information.
Recent molecular studies have provided more detailsof the complex mechanisms that determine MGMT
methylation status. For example, it seems plausible that
IDH mutations can lead to MGMT methylation, as gliomas
with IDH mutation show a reduction of α-ketoglutarate
and accumulation of 2-hydroxyglutarate, resulting in
genome-wide histone and DNA methylation.49 Moreover,TP53 mutation may also influence MGMT methylation
status, in association with TNF-α-induced NF-κB activation and chronic inflammation, which can induce DNA
methylation.50–52 Thus, IDH1 and TP53 mutations may alter
MGMT methylation status.
There are some technical issues concerning MGMTimmunohistochemistry, including intra- and inter-observer
variability in the assessment of immunoreactivity,8,12,53
the need for exclusion of non-neoplastic cells,13 variability
among antigen retrieval methods, the antibodies
employed, and the cutoff values adopted.53 According toour criteria for judgment of MGMT positivity or negativity(Fig. 1D–F), we found that a MGMT-negative profile wasassociated with a favorable outcome for patients withmalignant atrocytomas (Tables 2 and 3, and Fig. 2B). Consistent with this evidence, a recent study of both protein
expression and DNA methylation found that patients withhigh protein expression had a poor outcome, even though
the promoter was methylated,41 and that patients with low
protein expression and a methylated promoter had afavorable outcome.54,55
We therefore evaluated the association between CRT
and MGMT profiles and survival in patients with malignant astrocytomas, and found that a MGMT-negative
profile predicted a favorable outcome for patients who
received CRT (Fig. 2L), suggesting that glioma cells withlow MGMT expression may be much more sensitive to
CRT. The proportions of patients with MGMT-negative
profiles in the oligodendroglial (OA/OD and AOA/AOD)
groups were high (Table 2). This seems to reflect the factthat MGMT promoter methylation is frequently evident inoligodendroglial tumors.56
It has been recognized that the p53 pathway plays a rolein the pathogenesis of gliomas. However, at present it
remains controversial whether TP53 mutation can be
regarded as a true prognostic factor. Immunoreactivity of© 2015 Japanese Society of Neuropathology－ 165 －

IDH1/MGMT/p53 immunoprofiles and glioma prognosis333

p53 may be associated with TP53 mutation, LOH of TP53,
and aberrant expression of various p53-regulatory genes,
including MDM2 and p14ARF.1,3,57–59 In the present study, as
the grade of malignancy increased, the proportion ofpatients with p53-positive profiles tended to increase;
however, expression of p53 in patients with any tumor
grade showed no significant association with outcome(Tables 2 and 3).
In the present study, we found that a combination ofboth IDH1 and MGMT profiles would be useful for prognostication (Fig. 2C). Therefore, after diagnosis of GBM
and AA, on the basis of immunohistochemistry for both
IDH1 and MGMT, it would be possible to estimate themOS of the patients. Moreover, if patients were found tohave a MGMT-negative profile, CRT could be considered.
Consistent with this, a recent molecular study has revealed
that the combination of IDH1 mutation and MGMT
methylation status outperforms information on either
IDH1 or MGMT alone for predicting the survival ofpatients with GBM: patients with IDH1 mutations/MGMT
methylation survived longer, whereas those with noIDH1 mutations/no MGMT methylation showed shorter
survival.5
Furthermore, we found that p53 overexpression inpatients with IDH1-negative and MGMT-negative profiles
could be a useful prognostic factor (Fig. 2I). Therefore,
based on a combination of the IDH1, MGMT and p53
profiles, three groups were distinguishable, and their mOSs
differed significantly (Figs 2J and 3). Clinicopathologically,
these findings appear to have potential importance forpredicting the outcome of patients with malignant
astrocytomas.
In conclusion, the immunohistochemical profiles of
IDH1, MGMT and p53 are of significant importance forprognostication in patients with malignant astrocytomas. A
combination of IDH1 positivity and MGMT negativity
may indicate a favorable prognosis, whereas a combinationof IDH1 negativity and MGMT positivity may be unfavorable. For patients with IDH1 negativity and MGMT negativity, the p53 profile may have a crucial prognostic impact,
overexpression conferring an unfavorable prognosis. If
information on these profiles were immediately available
following histopathological diagnosis, it would be usefulfor prompt and accurate decision-making with regard totreatment.

and a grant (24-7) for Nervous and Mental Disorders from
the Ministry of Health, Labor and Welfare, Japan.

ACKNOWLEDGMENTS
We thank Professor Kouhei Akazawa, Department ofMedical Informatics, University of Niigata, for statisticalanalysis. This work was supported by a Grant-in-Aid for
Scientific Research (no. 25250008) from the Ministry of
Education, Culture, Sports, Science, and Technology, Japan,

334

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Neuropathology 2015; 35, 401–409

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doi:10.1111/neup.12199

Characteristic expression of p57/Kip2 in balloon
cells in focal cortical dysplasia
Tadashi Kimura,1,7 Hiroki Kitaura,1 Hiroshi Masuda,2 Shigeki Kameyama,2 Yuko Saito,3Kenji Sugai,3 Taisuke Otsuki,3 Atsuko Nakazawa,4 Nobuhito Morota,4 Takamichi Yamamoto,5Kouji Iida,6 Masanori Nakagawa,7 Toshiki Mizuno,7 Hitoshi Takahashi1 and Akiyoshi Kakita1
1
Department of Pathology, Brain Research Institute, University of Niigata, 2Department of Functional Neurosurgery,Epilepsy Center, Nishi-Niigata Chuo National Hospital, Niigata, 3National Center of Neurology and Psychiatry, 4National
Medical Center for Children and Mothers, National Center for Child Health and Development, Tokyo, 5Comprehensive
Epilepsy Center, Seirei Hamamatsu General Hospital, Hamamatsu, 6Department of Neurosurgery, University ofHiroshima, Hiroshima and 7Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural
University of Medicine, Kyoto, Japan

Balloon cells are a pathognomonic cellular feature ofvarious cortical malformations, including focal cortical dysplasia type IIb (FCD IIb), cortical tubers of tuberous sclerosis (TSC) and hemimegalencephaly (HME). In thepresent study, we investigated the immunohistochemical
expression of p57/Kip2, a member of the Cip/Kip family of
cyclin-dependent kinase inhibitory proteins, in balloon cells
in surgical specimens taken from 26, 17 and six patients withFCD IIb, TSC and HME, respectively. Characteristic dotlike reactivity with a faint, intense, reticular and process-likepattern was confined to the proximal portion of the cytoplasmic processes of the cells. Immunoelectron microscopy
revealed the p57/Kip2 reactivity on intermediate filamentsin the proximal portion of the processes. The immunohistochemical profile appeared similar to that of CD34; however,a double immunofluorescence study demonstrated that nocells showed reactivity for both p57/Kip2 and CD34. The
frequencies of the p57/Kip2-positive cells in FCD IIb and
HME were significantly higher than those in TSC, suggesting that the balloon cells may be heterogeneous. Thesefindings suggest some functional significance of the protein

Correspondence: Akiyoshi Kakita, MD, PhD, Department of
Pathology, Brain Research Institute, University of Niigata, 1-757
Asahimachi, Chuo-ku, Niigata 951-8585, Japan. Email: kakita@
bri.niigata-u.ac.jp
Sources of financial support: This work was supported by a Grantin-Aid for Scientific Research (no. 25250008) and Exploratory
Research (no. 25640027) from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan, and a grant (24-7) for Nervousand Mental Disorders and a Health Labour Sciences Research Grant(H26-052) from the Ministry of Health, Labor and Welfare, Japan.
Received 22 December 2014; revised 18 January 2015 and accepted
19 January 2015; published online 7 May 2015.

on the cytoplasmic processes of balloon cells and appear
consistent with the notion that the cells are abnormallydifferentiated progenitor cells.
Key words: balloon cell, focal cortical dysplasia,
hemimegalencephaly, p57/Kip2, tuberous sclerosis.

INTRODUCTION
Balloon cells are a pathognomonic cellular feature ofvarious cortical malformations, including focal cortical dysplasia type IIb (FCD IIb),1 cortical tubers of tuberous sclerosis (TSC)2 and hemimegalencephaly (HME).3 Patientswith these malformations often suffer intractable epilepsy
requiring surgical resection of the lesions. The origin andidentity of these cells are uncertain. However, a large
number of studies have indicated that subsets of balloon
cells express various proteins characteristic of glial,
neuronal and stem cell/progenitor cells, including GFAP,
vimentin, nestin,4,5 neurofilament, neuronal specific nuclear
protein (NeuN),microtubule associated protein 2 (MAP2),6class III beta-tubulin (TuJ1),7 glutamate receptor,8 CD133,
CD34,9 and minichomosome maintenance complex component 2 (Mcm2).10 With regard to the origin of balloon cells,
an in vitro study of isolated balloon cells taken from patientswith FCD IIb and TSC has indicated that such cells areabnormal stem cells that contribute to the pathogenesis ofthese disorders.11 Furthermore, an immunohistochemical
study using a panel of several antibodies specific for certain
progenitor cell types, including brain lipid-binding protein(BLBP), Otxl, GFAP-δ and Pax6, has indicated that balloon
cells are derived from radial glial progenitor cells.5 Anothermorphological and electrophysiological study of FCD IIb© 2015 Japanese Society of Neuropathology－ 169 －

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T Kimura et al.

has also proposed that balloon cells represent radial glial
cells that have failed to undergo postnatal degeneration.12
Moreover, an immunohistochemical study examining theexpression of cell cycle proteins, including nonphosphorylated retinoblastoma protein, cdk4 and p53, inballoon cells in FCD IIb has hypothesized that these cells
are the remnants of early cortical cells, which have failed toundergo differentiation or elimination during development
and have undergone cell cycle arrest.13 Thus, dysregulationof cell cycle proteins may be a pivotal mechanism underlying the dysplastic features of balloon cells, including abnormal cell fate determination, cell immaturity, and subsequentabnormal morphology.
In the present study, we examined the immunohistochemical expression of p57/Kip2 and p27/Kip1, membersof the Cip/Kip family of cyclin-dependent kinase (CDK)inhibitory proteins, in balloon cells in surgical specimens of
FCD IIb, TSC and HME taken from patients with epilepsy.
Recent biological studies have provided evidence thatthese proteins play important roles in cell cycle regulation,
proliferation, differentiation and migration of various
types of cancer cells14 and of cortical precursors in thedeveloping brain.15 We found that various proportions ofballoon cells in the malformed brain tissue showed reactivity for p57/Kip2, but not for p27/Kip1.

MATERIALS AND METHODS
Subjects
The study was approved by the various institutional ethics
committees, and all patients provided consent to use theirresected tissues for research purposes. We examined surgical specimens taken from 26 patients with FCD IIb(14 male, 12 female: 15 right, 11 left: age at resection
mean 1 SD = 16.4 1 12.9 years), 17 patients with TSC (ninemale, eight female: seven right, 10 left: age 6.29 1 7.6 years)
and six patients with HME (three male, three female: fourright, two left: age 1.00 1 1.2 years), who had undergone
resection of neocortical tissue because of intractable epilepsy (Table 1). Five of the patients with TSC were confirmed as harboring mutations in the TSC1 or TSC2 gene,as previously reported 16 (Table 1). The surgical specimens
were cut into slices and fixed with phosphate-buffered 20%
formalin and embedded in paraffin wax. Serial 4-μm-thicksections were then cut, and subjected to HE and KB staining. Diagnoses of FCD IIb,1 cortical tubers of TSC2 andHME,3 were made on the basis of histopathological features, clinical information, images and prognosis.
The term “balloon cells” is used for the abnormal cells
observed in FCD IIb and HME, whereas “giant cells” is theterm sometimes applied for those in cortical tubers. Thesecells are morphologically indistinguishable. For clarity, theterm “balloon cells” is used here for all cell populations.2,11

Immunohistochemistry, cell counting andstatistical analysis
The paraffin-embedded sections were processed by theavidin-biotin-peroxidase complex (ABC) method with a
Vectastain ABC kit (Vector, Burlingame, CA, USA). As
the primary antibodies, we used a rabbit polyclonal antibody against p57/Kip2 (Abcam, Cambridge, MA, USA;
1:200), and mouse monoclonal antibodies against p27/Kip1(clone 57, BD Biosciences Pharmingen, Franklin Lakes, NJ,
USA; 1:200) and CD34 (clone Qbend 10, Immunotech,Monrovia, CA, USA; 1:200). Pretreatment was performedby heat/autoclaving for 10 min at 121°C in 10 mmol/L
sodium citrate buffer.
For cell counting, a single p57/Kip2-immunostained
section from each case was used. The sections wereobserved under a light microscope (BX53, Olympus,
Tokyo, Japan) and images were captured by a digital
camera (DP73, Olympus). Balloon cells were identified as
bizarre cells with abundant cytoplasm and eccentric nuclei(Fig. 1A). The numbers of the p57/Kip2-positive and-negative balloon cells in the images were counted manually using the “Count tool” of the Adobe Photoshop software package.
Statistical significance of differences in the frequency of
p57/Kip2-positive balloon cells in any of the FCD IIb, TSC
or HME cases was analyzed with nonparametric KruskalWallis test and Mann-Whitney U-test at a significance levelof P < 0.01. The results are shown as mean 1 SD. Data
analysis was carried out using the IBM SPSS statistics
version 20 software package (IBM SPSS Inc., Chicago, IL,
USA).

Double immunofluorescence
A double-labeling immunofluorescence study was performed on several sections from eight patients with FCD
IIb using the rabbit polyclonal anti-p57/Kip2 antibody(1:100) and the mouse monoclonal anti-CD34 antibody(1:100). The secondary antibodies used were Alexa Fluor
555 goat anti-mouse IgG and Alexa Fluor 488 goat antirabbit IgG (both Molecular Probes, Eugene, OR, USA;
1:1000). Slides were treated with Autofluorescence Eliminator Reagent (Millipore, Bellerica, MA, USA), and thenmounted with glass coverslips using VECTAshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI)
nuclear stain (Vector Laboratories, Burlingame, CA,
USA). A laser-scanning confocal microscope (Carl
Zeiss LSM-700, Jena, Germany) was used to visualize
immunoreactivity.
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p57/Kip2 in balloon cells
Table 1 Clinical and pathological profiles of the patients with FCD, TSC and HME
Case ID

Sex

Age
(y)

Onset
(y)

LocationSide

Pathology

Number ofballoon cells

Number of
p57/Kip2
positive cells

Percentages

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32**
33
34
35
36
37
38
39*
40**
41
42*
43**
44
45
46
47
48
49

F
F
M
M
M
M
F
F
M
F
F
M
M
M
M
F
F
F
M
F
F
M
M
M
F
M
F
F
M
M
M
F
F
M
M
F
F
F
F
M
M
M
M
M
M
F
M
F
M

2
2
3
7
8
8
8
9
10
10
10
11
11
12
12
12
17
18
20
20
23
26
34
38
41
54
0
0
1
2
2
2
2
3
3
3
4
5
9
11
11
24
25
0
0
0
1
2
3

1
–
0
6
5
7
4
5
1
3
0
9
5
6
5
0
7
5
10
4
7
6
10
1
5
3
0
0
–
0
0
0
0
1
2
0
2
0
0
0
–
10
6
0
0
0
0
0
0

T
P
F
F
F
F
F
P
F
P
F
O
P
F
F
T
F
P
O
F
P
P
F
M
P
F
F
F
F
F
F
T
T
F
F
T
P
P
F
F
F
F
F
P
F
F
F
F
T

R
L
L
R
R
L
L
R
R
R
L
R
L
R
L
R
L
R
L
R
R
R
R
L
R
L
L
R
R
L
R
L
L
R
L
L
L
L
L
R
L
R
R
L
L
R
R
R
R

FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
FCD IIb
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
TSC
HME
HME
HME
HME
HME
HME

95
69
78
51
120
65
163
13
19
85
154
21
73
33
63
157
66
81
50
58
84
109
162
42
78
31
340
140
70
71
18
81
117
37
52
43
37
14
10
9
24
18
30
23
94
58
140
31
150

67
50
39
50
97
42
80
11
12
64
102
16
62
5
31
73
50
66
26
39
70
94
95
36
40
21
1
0
18
2
6
14
1
22
4
0
1
0
1
1
5
15
4
4
82
30
7
18
91

70.53
72.46
50.00
98.04
80.83
64.62
49.08
84.62
63.16
75.29
66.23
76.19
84.93
15.15
49.21
46.50
75.76
81.48
52.00
67.24
83.33
86.24
58.64
85.71
51.28
67.74
0.29
0.00
25.71
2.82
33.33
17.28
0.85
59.46
7.69
0.00
2.70
0.00
10.00
11.11
20.83
83.33
13.33
17.39
87.23
51.72
5.00
58.06
60.67

F, female; M, male; F, frontal; T, temporal; P, parietal; O, occipital; R, right; L, left; FCD, focal cortical dysplasia; TSC, tuberous sclerosis; HME,
hemimegalencephaly; y, year(s). Mutations in * TSC1 and ** TSC2 genes.

Immunoelectron microscopy
The ultrastructural localization of p57/Kip2 was examinedusing surgical specimens taken from five patients with FCD
IIb, employing the post-embedding method previously
described.17 Small tissue blocks were prepared fromformalin-fixed tissue, and washed with PBS. The tissue
blocks were then washed with gradually increasing concentrations of dimethylformamide, and embedded in LR
White resin (London Resin Company, Berkshire, UK).
Ultrathin sections were cut, incubated with the anti-p57/
Kip2 antibody (1:25) for 36 h, and reacted with 15-nm gold

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T Kimura et al.

Fig. 1 p57/Kip2 immunohistochemical features. Balloon cells in focal cortical dysplasia (FCD) type IIb (A-F), tuberous sclerosis (TSC)(G), and hemimegalencephaly (HME) (H), and dysmorphic neurons in FCD type IIb (I, J). (A, B) Low-power magnification views of whitematter of a sample demonstrating several balloon cells with abundant cytoplasm and eccentric nuclei (A), and dot-like immunoreactivity
around the cytoplasm of the balloon cells (B). (C-F) Higher-magnification views of balloon cells showing faint (C), intense (D), reticular(E) and process-like (F) fashion. (G) No balloon cells with immunoreactivity are seen. (H) Many balloon cells showing similar
immunoreactivity. (J) No dysmorphic neurons with immunoreactivity are seen. (A, I) Hematoxylin and eosin stain. (B-H, J)
Immunostained and then counterstained with hematoxylin. Images of A and B, and those of I and J are taken from serial sections.
Bars = 50 μm for A, B, G and H, and 20 μm for C-F, I and J.

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p57/Kip2 in balloon cells
colloidal particle-conjugated anti-rabbit IgG (British
BioCell, Cardiff, UK, 1:30). The sections were then stainedwith lead citrate, and examined with a Hitachi H-7700
digital electron microscope at 75 kV.

and short cytoplasmic processes with abundant filaments(Fig. 3A, C), as previously reported.18,19 Immunogold labeling for p57/Kip2 was observed mainly on the filaments at
the peripheral cytoplasm and proximal portion of the processes (Fig. 3B, D, E).

RESULTSDISCUSSION

p57/Kip2 immunohistochemistry
In FCD IIb, balloon cells showed characteristic p57/Kip2
immunoreactivity (Fig. 1B) in the form of dot-like profiles
around the abundant cytoplasm, showing faint (Fig. 1C),
intense (Fig. 1D), reticular (Fig. 1E) and process-like(Fig. 1F) patterns. These profiles appeared to be confinedto the cytoplasmic membrane and proximal processes of
the cells. No apparent immunoreactivity was evident within
the perikaryon proper. Balloon cells with faint or intense
p57/Kip2 immunoreactivity were found predominantly
within the white matter, whereas those with the reticularand process-like patterns were scattered in deeper layers of
the cortex and at its boundary with the white matter. Interestingly, a large proportion of balloon cells observed in
TSC were p57/Kip2-negative (Fig. 1G). On the other hand,balloon cells in HME (Fig. 1H) showed immunoreactivity
similar to that in FCD IIb.
The median frequencies of the p57/Kip2-positive
balloon cells were 69.1% in FCD IIb (range, 51.8 to 81.9),
54.9% in HME (range, 17.4 to 60.7), and 10.0% in TSC(range, 0.9 to 20.8) (Table 1). The frequencies in FCD IIb
and HME were higher than those in TSC (χ2 (2,n = 49) = 22.9, P < 0.001).A post-hoc test revealed a significant difference between FCD IIb and TSC (P < 0.001), but
not between HME and TSC.
We found no morphological differences between
balloon cells with p57/Kip2 immunoreactivity and those
without the immunoreactivity. No dysmorphic neurons(Fig. 1I) or other neurons with normal morphology showed
reactivity for p57/Kip2 (Fig. 1J).
With regard to p27/Kip1, no labeling of balloon cells or
dysmorphic neurons was evident in FCD IIb, TSC or HME(data not shown).

Double immunofluorescence
As the expression pattern of p57/Kip2 in balloon cells(Fig. 1B) appeared similar to that of CD34,9 we performed
double immunofluorescence for detection of both p57/
Kip2 and CD34. Surprisingly, we encountered no balloon
cells that were immunoreactive with both of the antibodies
used (Fig. 2).

Electron microscopy
Ultrastructurally, the balloon cells exhibited abundantcytoplasm containing numerous intermediate filaments,

In the present study, for the first time, we have demonstrated the expression of p57/Kip2 in balloon cells(Fig. 1). The characteristic immunohistochemical features(Figs 1,3) suggest some significant role of the protein in theprocesses. Furthermore, we have shown that in comparison
with balloon cells in FCD IIb and HME, those in TSC
express p57/Kip2 much more rarely (Table 1), indicatingthat the roles of the protein in balloon cells may differ
somewhat between FCD IIb/HME and TSC.
The CDK inhibitors of the Cip/Kip family, including
p57/Kip2 and p27/Kip1, are known to regulate the cell
cycle. Recent understanding of the roles of the family
members in development of the CNS has been expanding, and it is now known to be involved in multiple
aspects of neurogenesis through cell cycle arrest, specificcell-type differentiation and neuronal migration.15,20,21
Recently, it has been revealed that the members of thisfamily differentially regulate neuroglial fate determination depending on environmental signals and developmental stage.22 Balloon cells are considered to be theconsequence of aberrant differentiation of radial glial
cells, a type of progenitor cell, to neurons and glia.5
Therefore, expression of p57/Kip2 in balloon cells may be
associated with abnormal differentiation of the progenitor cells. It seems unlikely that the p57/Kip2 profiles
observed in the present study reflected aberrant
neurogenesis or neuronal migration, because neurons
with normal or dysplastic morphologies were distributed
irregularly within the cortex and white matter of FCD
IIb/TSC/HME, but they showed no p57/Kip2
immunoreactivity.
It has been hypothesized that balloon cells may bearrested in cell cycle G1, based on the findings that theyfrequently express G1 markers in their nuclei.13 Concerning the cell cycle, a biological study demonstrated that
p57/Kip2 was strongly expressed in association with thelate G1 phase of developing cells of the CNS.20 Therefore,
the p57/Kip2 selected expression in the balloon cells in thepresent study appears compatible with the notion that they
are arrested in G1. However, we could not consider close
association between the p57/Kip2 expression and the cell
cycle of the balloon cells, because G1 associated proteins
usually localize within the nucleus, but p57/Kip2 wasobserved at the cytoplasmic processes, rather than the
nucleus, of the balloon cells.

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T Kimura et al.

Fig. 2 Double immunofluorescence. Immunofluorescence with the p57/Kip2 (green, A, D) and CD34 (red, B, E) signals to demonstrate
the spatial relationship of the two antigens. (C, F) Merged image of both signals. Nuclei are labeled with 4,6-diamidino-2-phenylindole(DAPI) (blue). High-power views demonstrating a single balloon cell with two nuclei (A-C) and lower-power views showing two cells(arrow and arrowhead) (D-F) in FCD type IIb. Note the cells show either signal. No co-localized signals are observed. Bars = 10 μm for
A-C, and 20 μm for D-F.

TSC is an autosomal-dominant disease resulting frommutations of either TSC1 or TSC2, encoding hamartin andtuberin, respectively. The TSC gene products form a functional heterodimer, which negatively regulates the activities
of mammalian target of rapamycin (mTOR),23 a serine/
threonine kinase known to be a major effector of cell
growth. In the CNS, activation of mTOR signaling may
cause abnormal development of neurons and glia.24 On theother hand, recent genetic studies have revealed somatic
mutations in the PIK3CA, AKT3 and MTOR genes in brain
tissue of patients with HME, indicating aberrant activationof mTOR signaling.25,26 The molecular genetic mechanisms
underlying FCD IIb have remained uncertain. FCD IIb,
TSC and HME often share a number of histopathologicalfeatures, including disruption of cortical cytoarchitecture,dysmorphic neurons and balloon cells. On the other hand,based on our experience of routine histopathological diagnoses of these developmental disorders, we have formed an
impression that calcification and astrocytosis are much

more remarkable in TSC than in FCD IIb and HME. Moreover, the frequency of dysmorphic neurons appears to bemuch higher in HME than in FCD IIb and TSC. In a subsetof patients with HME, balloon cells were not observed.
Furthermore, a previous biochemical analysis demonstrated somewhat differential Pi3K-pathway activation in
FCD IIb and TSC.27 Another electrophysiological study
demonstrated that FCD IIb and TSC display differences inthe topography of abnormal cells, excitatory and inhibitory
synaptic network properties, and GABA(A) receptor
sensitibity.28 Accordingly, certain differences in thepathomechanisms of these disorders may be associated withdifferences in the histological picture and expression of
p57/Kip2 in balloon cells (Table 1). In the present study, we
found no clear association between genetic profiles and thefrequencies of p57/Kip2-positive balloon cells in patientswith TSC. For example, the frequency in two patients with
TSC1 mutations (cases #39 and #42 in Table 1) were considerably differed.
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p57/Kip2 in balloon cells

Fig. 3 Ultrastructural localization of p57/Kip2. (A) Low-power view of a balloon cell showing eccentric nucleus, abundant cytoplasm withfilaments and cell organelle, and short cytoplasmic processes. (B) A higher magnification view of the short processes. The area indicatedby a square in A. Note the immunoreactivity in the proximal portion of the cytoplasmic processes (arrows). (C-E) Another example ofballoon cell with abundant intermediate filaments. (D, E) Higher magnification views of the areas indicated by square 1 rotated by 45° andsquare 2 in C, respectively, showing the immunoreactivity on the intermediate filaments. Bars = 10 μm for A, 500 nm for B, D and E, and5 μm for C.

CD34, a stem cell/progenitor cell marker, is well known
to be expressed in balloon cells in FCD IIb,TSC and HME,
where the immunolabeled cells are exclusively locatedwithin the deep white matter or close to the gray matterboundary,but not in the cortex.9 In the present study,we also
detected several CD34-labeled balloon cells; however,double-labeling immunofluorescence revealed distinct
populations of balloon cells showing immunoreactivity for
either CD34 or p57/Kip2 (Fig. 2). Even though these populations were located closely to each other within the whitematter, we found no balloon cells that expressed both proteins (Fig. 2). CD34 is known to be expressed during neuraltube formation.29 Moreover, it has been reported that
CD34-labeled progenitor-like cells are sometimes observedin ganglioglioma and other epilepsy-associated brain

tumors taken from patients with epilepsy.30 However, thesignificance of CD34, and also p57/Kip2, in balloon cells hasremained uncertain. It seems unlikely that certain cellularmechanisms would induce some sort of conflict in theexpression of CD34 and p57/Kip2. Therefore, we speculatethat the available evidence points to heterogeneity of thecells.
We were able to observe the ultrastructural localizationof p57/Kip2 on intermediate filaments, presumably mainly
vimentin filaments and possibly glial filaments as reported
previously,19 in the proximal parts of the cytoplasmic processes (Fig. 3). This ultrastructural localization appeared tobe compatible with the immunohistochemical picture(Fig. 1). In the perikarya of balloon cells, the intermediatefilaments are loosely arranged and intermingled with© 2015 Japanese Society of Neuropathology－ 175 －

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T Kimura et al.

intracellular organelles, whereas in the proximal processes,
the filaments show a tendency to be much more densely
packed and aligned in the direction of extension of theprocesses (Fig. 3D, E). Up to now, the significance of p57/
Kip2 localization in balloon cells has been uncertain.
However, recent biochemical studies have provided evidence for a critical role of p57/Kip2 in the regulation ofactin cytoskeletal dynamics, and thus migration ability, incancer cells.31 Therefore, there is a possibility that p57/Kip2
may be involved in the intermediate filament assembly thatis necessary for extension of the cytoplasmic processes ofballoon cells.
In conclusion, we have reported aberrant expression of
p57/Kip2 in balloon cells in various types of brain malformation. Our observations appear to be consistent with thenotion that these cells represent abnormal differentiation ofprogenitor cells. Characteristically, the immunoreactivity
appears to be localized in the proximal portion of cytoplasmic processes, implying that the protein has functional significance in the processes. The significant difference in thefrequency of immunoreactive cells between FCD IIb and
TSC suggests that the function of the protein may differ
between these malformations.

ACKNOWLEDGMENTS
We thank Prof. Kouhei Akazawa, Department of Medical
Informatics, University of Niigata, for statistical analysis.

DISCLAIMER
The authors report no conflicts of interest concerning thematerials or methods used in this study or the findingspresented.

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Brain Pathology ISSN 1015-6305

RESEARCH ARTICLE

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p62 Deficiency Enhances α-Synuclein Pathology in Mice
Kunikazu Tanji1*; Saori Odagiri2*; Yasuo Miki1; Atsushi Maruyama3; Yoshikazu Nikaido4;
Junsei Mimura3; Fumiaki Mori1; Eiji Warabi5; Toru Yanagawa5; Shinya Ueno4; Ken Itoh3;
Koichi Wakabayashi1
Department of Neuropathology, 3 Department of Stress Response Science, 4 Department of Neurophysiology, Hirosaki University Graduate
School of Medicine, Hirosaki, 2 Department of Neuroanatomy, Cell Biology and Histology, 5 Majors of Medical Sciences, University of Tsukuba,
Tsukuba, Japan.
1

Keywords
α-synuclein, Lewy body disease,
p62/Sequestsome 1/SQSTM1, Parkinson’s
disease, proteolysis, stress.
Corresponding author:
Kunikazu Tanji, PhD, Department of
Neuropathology, Hirosaki University
Graduate School of Medicine, 5
Zaifu-cho, Hirosaki 036-8562, Japan(E-mail: kunikazu@cc.hirosaki-u.ac.jp)
Received 16 June 2014
Accepted 02 October 2014
Published Online Article Accepted 10 October
2014* These authors contributed equally to themanuscript.

Th

Abstract
In Lewy body disease (LBD) such as dementia with LBs and Parkinson’s disease, several
lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly
involved with intracellular proteolysis and is a component of ubiquitin-positive inclusions
in various neurodegenerative disorders. However, it is not clear whether p62 deficiency
affects inclusion formation and abnormal protein accumulation. To answer this question,
we used a mouse model of LBD that lacks p62, and found that LB-like inclusions wereobserved in transgenic mice that overexpressed α-synuclein (Tg mice) with or without thep62 protein. p62 deficiency enhanced α-synuclein pathology with regard to the number ofinclusions and staining intensity compared with Tg mice that expressed p62. To further
investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we
assessed the mRNA and protein levels of several molecules, and found that the neighbor of
the brca1 gene (NBr1 ), which is functionally and structurally similar to p62, is increased in
Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and
NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative
modulation of α-synuclein aggregation.

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doi:10.1111/bpa.12214

INTRODUCTION
Lewy body disease (LBD) including dementia with LBs and Parkinson’s disease (PD) is pathologically characterized by the presence of intracellular inclusions called LBs. α-Synuclein has been
identified as a component of LBs (41), and the duplication andtriplication of the α-synuclein gene are found in both sporadic andearly onset forms of PD (40). Mutations (A30P and A53T) in the
α-synuclein gene are linked to autosomal dominant forms of PD(20, 31). Originally, α-synuclein is a proteinase K (PK)-soluble
protein that localizes at presynaptic terminals; however,
α-synuclein becomes resistant to PK and widely deposited
throughout the brain of patients with LBD (19, 42). These findings
suggest that α-synuclein is significantly involved in the pathogenesis of both familial and sporadic cases of LBD.
α-Synuclein is physiologically processed by two intracellulardegradation systems, including the ubiquitin–proteasome andautophagy–lysosome systems. In case of α-synuclein overload,
the autophagy–lysosome system, including chaperone-mediatedautophagy, predominantly aids in the degradation of excess
α-synuclein (6, 22, 46). Thus, it is possible that dysfunction of
intracellular degradation system results in the up-regulation of

α-synuclein expression and contributes to abnormal protein accumulation. Indeed, several lysosomal-related genes were identified
as a causative mutation in familial PD, including leucine-richrepeat kinase 2 and adenosine-3-phosphate 13A2. Furthermore,
PD has been genetically linked to rare lysosomal storage diseases,
including Gaucher’s disease (25) and Sanfilippo syndrome (47).
p62/SQSTM1/sequestosome 1 (referred to as p62) is a multifunctional protein that is strongly associated with the intracellulardegradation system. P62 knockout (KO) mice exhibit matureonset obesity, insulin and leptin resistance (37). Pathologically,
loss of p62 results in the accumulation of hyperphosphorylated tau
and insoluble K63-linked polyubiquitin chains (33, 48). p62 contains a ubiquitin-associated (UBA) domain at the C-terminus thatenables its interaction with ubiquitinated and misfolded proteins.
Additionally, p62 possesses a Phox and Bem1p (PB1) domain at
the N-terminus and a LC3 interacting region, suggesting that p62
is able to interact with proteasome components and autophagosomal membranes (29, 38). Thus, it has been suggested that p62
can efficiently degrade ubiquitinated and misfolded proteinsthrough the proteasome and autophagy–lysosome systems. It has
been reported that p62 is an inducible protein that easily aggregates under several pathological conditions, such as oxidative
1

Brain Pathology •• (2014) ••–••© 2014 International Society of Neuropathology－ 178 －

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The influence of p62 on α-synuclein in mice

Tanji et alstress and neurodegeneration (1, 11, 27). Accordingly, dysfunctionof the intracellular degradation systems induces p62 aggregationin vivo (2, 16). Furthermore, loss of p62 suppressed ubiquitinpositive inclusions in neurons of brain-specific autophagydeficient mice (17). Additionally, ubiquitin- and p62-positive
protein aggregates were abrogated in Atg8 and p62 double-mutant
flies (26). These findings suggest that p62 may be responsible forthe formation of cytoplasmic inclusions and abnormal protein
accumulation.
In this study, we used transgenic (Tg) mice overexpressing
α-synuclein with a A53T mutation as a model for LBD. We
crossed the Tg mice with p62 KO mice to examine the involvementof p62 in abnormal α-synuclein pathology. Immunohistochemical
analyses showed that p62 deficiency enhanced α-synuclein pathology, as shown by an increase in inclusion number and staining
intensity. We assessed several genes and proteins related to stressresponse and proteolysis. These data revealed that the expressionof neighbor of brca1 gene (NBR1), which is a functional homologue to p62, was increased in p62-deficient mice.

Figure 1. Characterization of p62 protein deficiency in an animal modelof Lewy body disease. A. Breeding strategies to generate p62 deficiency in α-synuclein transgenic (Tg) mice. Initially, heterozygous
α-synuclein Tg and homozygous p62-knockout (KO) mice were crossed.
Next, littermates and heterozygous p62-deficient mice were mated togenerate Tg mice without p62 (Tg/KO), of which four groups were usedin this study (black circles). B. Immunoblot analysis confirmed thatα-synuclein was overexpressed in Tg and Tg/KO mice and that p62

MATERIALS AND METHODS
Animals and experimental design
α-Synuclein Tg mice have been widely used as an animal modelfor LBD (7, 14, 21, 23, 24, 34, 36, 45). To create this LBD modelin a p62-deficient background, we used mice overexpressing
human α-synuclein with the A53T mutation under the prion promoter (Jackson Laboratories, Bar Harbor, ME, USA) (7) and p62
KO mice with exon 1–4 deleted as previously described (17). The
p62 KO mice lacked abnormal tau pathology. α-Synuclein Tg and
p62 KO mice were backcrossed with C57BL/6J mice for at least 10
generations. First, heterozygous α-synuclein Tg mice were bredwith p62 KO mice to generate α-synuclein+/−/p62+/− mice. Second,
α-synuclein+/−/p62+/− mice were inbred to generate wild type,p62 KO, α-synuclein+/−/p62+/+ and α-synuclein+/−/p62−/− mice
(Figure 1A). Hereafter, wild-type, p62 KO, α-synuclein+/−/p62+/+
and α-synuclein+/−/p62−/− mice are simply referred to as WT, KO,
Tg and Tg/KO mice, respectively. All comparisons were madesignals were diminished in KO and Tg/KO mice (9 weeks of age, n = 6
per group). The molecular mass is indicated on the left side of the panel.
β-Actin was used as a loading control. C. A quantitative analysis showsthat human α-synuclein is expressed in Tg and Tg/KO mice and that p62
is absent in KO and Tg/KO mice. The values of Tg mice are defined as
100%. D. The weight changes of Tg (black circle) and Tg/KO mice (greycircle) are shown (mean ± standard deviation, n = 6–8 per group).

2

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The influence of p62 on α-synuclein in miceTh

synthesis kit (Takara Bio Inc., Otsu, Japan). An aliquot of cDNA
was used for gene expression analysis with the SYBR® Premix Ex
Taq™ II (Perfect Real Time) (Takara Bio Inc.) and CFX RealTime PCR Detection System (Bio-Rad, Hercules, CA, USA) using
the following primer sets: heme oxygenase-1 (Ho-1) (5′-CCA
GCA ACA AAG TGC AAG ATT C-3′; 5′-TCA CAT GGC ATAAAG CCC TAC AG-3′), Nqo1 (5′-GTC ATT CTC TGG CCA ATT
CAG AGT-3′; 5′-TTC CAG GAT TTG AAT TCG GG-3′),glutamate-cysteine ligase catalytic subunit (Gclc) (5′-AAA ATG
CGG AGG CAT CAA-3′; 5′-ATA TGC TGC AGG CTT GGA
AT-3′), p62 (5′-AGC TGC CTT GTA CCC ACA TC-3′; 5′-CAG
AGA AGC CCA TGG ACA G-3′), Cyclophilin A (5-ATG CTGGAC CCA ACA CAA AT-3′; 5-TCT TTC ACT TTG CCA AAC
ACC-3′), Keap1 (5′-CAC AGC AGC GTG GAG AGA-3′; 5′-CAA
CAT TGG CGC GAC TAG A-3′), Lamp1 (5′-CCT ACG AGA
CTG CGA ATG GT-3′; 5′-CCA CAA GAA CTG CCA TTT TTC3′), Cathepsin D (5′-CCC TCC ATT CAT TGC AAG ATA C-3′; 5′TGC TGG ACT TGT CAC TGT TGT-3′), transcription factor EB(Tf EB) (5′-GAG CTG GGA ATG CTG ATC C-3′; 5′-GGG ACT
TCT GCA GGT CCT T-3′); Rab7l1 (5′-GCT GCA GCT CTG
GGA TAT TG-3′; 5′-TAG TAG AGT CGT GTC ATG GAT GTG3′) and Nbr1 (5′-TCA ACA GGA CTC GCA AAC AG-3′; 5′-ATG
CTG CTC CCA TTG TGG-3′). Cyclophilin A was used fornormalization.

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Tanji et al

among littermates to minimize confounding effects by different
genetic backgrounds. Mice were housed with a light/dark cycle of
12 h and were given food and water ad libitum. The experimental
protocol was approved by the Institutional Animal Care and Use
Committee at the Hirosaki University Graduate School of Medicine in Japan. Tg mice were genotyped using real-time polymerase
chain reaction (PCR) analysis (forward primer, 5′-TGT AGG CTC
CAA AAC CAA GG-3′; reverse primer, 5′-TAT GCC TGT GGA
TCC TGA CA-3′), and verified by backcrossing. Conventional
PCR was used for p62 genotyping (primer pair for wild type,forward, 5′-CTT ACG GGT CCT TTT CCC AAC-3′; reverse,
5′-TCC TCC TTG CCC AGA AGA TAG-3′; primer for p62 KO,
forward; 5′-CTG CAT GTC TTC TCC CAT GAC-3′; reverse,
5′-TAG ATA CCT AGG TGA GCT CTG-3′). Mice weretranscardially perfused with phosphate-buffered saline. The brain
was removed, and the right hemisphere was fixed with 4% paraformaldehyde for 48 h. After dehydrating through a graded ethanol
series, the right hemisphere was embedded in paraffin and cut into
4-μm thick sections. The left hemisphere was frozen at −80°C for
subsequent biochemical analyses.

Antibodies and immunohistochemistry
Rabbit antibodies against Keap1 (ProteinTech Group, Inc.,
Chicago, IL, USA), p62 (MBL, Nagoya, Japan), NBR1 (Sigma, St.
Louis, MO, USA and Santa Cruz Biotechnology, Santa Cruz, CA,USA), NAD(P)H quinone oxidoreductase 1 (NQO1) (Sigma), LC3(Sigma and MBL), ubiquitin (DAKO, Glostrup, Denmark),UBQLN1 (Lifespan Biosciences, Seattle, WA, USA),phosphorylated α-synuclein (Abcam, Cambridge, UK) and β-actin(Sigma) were used in this study. Mouse antibodies against p62(BD Biosciences, Franklin Lakes, NJ, USA), SNAP25 (Chemicon,
Temecula, CA, USA), synaptophysin (DAKO), human
α-synuclein (LB509; Zymed, South San Francisco, CA, USA),
human and mouse α-synucleins (4D6; GeneTex, Irvine, CA, USA)and phosphorylated α-synuclein (pSyn#64; Wako, Osaka, Japan)
were also used.
The sections were dehydrated and pretreated with heat retrieval
using an autoclave for 10 minutes in 10 mM citrate buffer (pH 6.0)
for rabbit anti-Keap1 and anti-NBR1 antibodies. The sections werethen subjected to immunohistochemical processing using theavidin-biotin-peroxidase complex method with diaminobenzidine(Sigma). In addition, the sections were counterstained withhematoxylin. For the staining of presynaptic PK-resistantα-synuclein, sections were pretreated with PK (Gibco BRL,
Gaithersburg, MD, USA; 50 μg/mL) in a PK buffer containing
10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Nonidet-P40 at
37°C for 5 minutes. The total number of inclusions immunostainedwith anti-phosphorylated α-synuclein was quantified in contiguous sections. Immunohistochemical studies were performed at 9
weeks of age (n = 6 per group).

Quantitative reverse transcription-polymerase
chain reaction (qRT-PCR)
Total RNA was extracted from the right hemisphere of the brain
using the RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany)at 9 weeks of age (n = 3 per group). cDNA was synthesized from
1 μg of total RNA using the PrimeScript® II first-strand cDNA

Immunoblot analysis
Western blot analysis was performed as previously described (43).
For total cell lysate, we used a lysis buffer with 4% sodium dodecyl
sulfate (SDS; 75 mM Tris-HCl, pH 6.8, 4% SDS, 25% glycerol,
5% β-mercaptoethanol) and passed sample through 21 gauge
needle attached on a 1 mL syringe. For an experiment using
insoluble sample of detergent, samples were weighted and lysedwith 10-fold volume of Tris-based buffer (pH 7.4) containing 0.1%
Triton X-100 on ice. After homogenization with a pestle 20 times,
they were passed 10 times through 21 gauge needle attached on a
1 mL syringe. Lysates were incubated for 5 minutes on ice, andcentrifuged at 12 000 × g for 10 minutes. Supernatant was used asa soluble fraction. The pellets were resuspended with 8 M urea andsonicated (insoluble fraction). Signal detection was performed
according to the protocol provided with the ECL or ECL prime
detection systems (Amersham Pharmacia Biotech, Piscataway, NJ,
USA). We performed each immunoblot analysis a minimum of
three times, and all data were quantified and collected.

Animal behavioral testing
The Morris water maze
Spatial learning was assessed in a round tank of water (0.95 m indiameter) at 30°C. An escape platform (10 cm in diameter) was
placed 1 cm below the water surface. A camera (Primetech Engineering Corp., Tokyo, Japan) was mounted above the maze andattached to a computer running the Smart software (Primetech
Engineering Corp.). The training paradigm for the hidden platformversion of the Morris water maze consisted of two trials per day forfive consecutive days. The time taken to reach the platform(latency to escape) was recorded for each trial. The time limit was
120 s, and the intertrial interval was 1 h. If the animal could not
3Brain Pathology •• (2014) ••–••© 2014 International Society of Neuropathology－ 180 －

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Forced swim test
Immobility time was analyzed using a forced swim test. Animals
were individually placed in a transparent acrylic cylindrical beaker(height: 25 cm, diameter: 18 cm) containing 4600 mL of clear
water at 25 ± 1°C for 6 minutes. A mouse was judged to be immobile when it remained passively floating in the water for more than
2 s. Immobility time was quantified using a Forced Swim Scan
software (Clever Sys Inc., Reston, VA, USA).

Quantitative analysis and statistical analysis
A semi-quantitative analysis of protein levels was performed using
the ImageJ software provided by the NIH. All data were represented as the mean + standard deviation. The statistical significance was evaluated using one-way analysis of variance (ANOVA)
with Bonferroni’s post hoc test to analyze four genotypes andStudent’s t-test to analyze two genotypes. A probability value ofless than 0.05 (P < 0.05) was considered to be significant.

RESULTS
Characterization of α-synuclein Tg mice withor without p62
To test the possibility that p62 is responsible for the formation ofcytoplasmic inclusions and abnormal protein accumulation, we
generated mice that overexpressed human α-synuclein (Tg) on a
p62-deficient background (Figure 1A). First, we crossed Tg micewith p62 KO mice. Next, littermates with or without p62 and/or
human α-synuclein were selected by genotyping and crossed togenerate Tg mice lacking p62. Consequently, littermates with orwithout endogenous p62 and/or human α-synuclein expression
were born at the expected Mendelian ratio. For our studies, weused WT, KO, Tg and Tg/KO mice.
We confirmed that α-synuclein was robustly expressed in thebrains of Tg mice and Tg/KO mice (Figure 1B). We used a human
α-synuclein-specific antibody, LB509, to confirm that human
α-synuclein expression was present only in Tg and Tg/KO mice.
There were no differences in the endogenous and human
α-synuclein levels between the Tg and Tg/KO mice. We also
confirmed that p62 protein levels were diminished in the brains of
KO and Tg/KO mice. Interestingly, the amount of p62 was slightly
higher in Tg mice than it was in WT mice (Figure 1C). An increaseof p62 was also supported by immunohistochemical studies thatshowed an increase in p62 immunoreactivity in Tg mice comparedwith WT mice (Figure 2A). α-Synuclein expression was mainly
observed in the presynapses in the brains of WT and p62 KO mice;
however, additional staining of α-synuclein was observed in thecytoplasm and presynapses in the brains of Tg and Tg/KO mice
(Figure 2B). Consistent with previous papers (28, 37), KO mice
exhibited mature-onset obesity. As they aged, Tg/KO mice had aheavier average body weight than did Tg mice (Figure 1D). The

majority of Tg mice remained healthy until at least 70 weeks ofage. Tg and Tg/KO mice were behaviorally indistinguishable anddisplayed lower food intake and activity at the end stage of thedisease.

Tg/KO mice exhibit an increase inphosphorylated α-synuclein staining andinclusion number compared with Tg mice
Similar to the human pathological conditions, there are two typesof abnormal α-synuclein in the brains of Tg mice (7, 42), including
phosphorylated α-synuclein (P-syn) and PK-resistant α-synuclein(PK-syn). Immunohistochemical analyses showed that P-syn isobserved in both Tg/KO and Tg mice (Figure 3A). We compared
the number of P-syn-positive inclusions in the thalamus of Tg and
Tg/KO mice. Quantitative data indicated that the number of inclusions was higher in Tg/KO mice compared with Tg mice
(Figure 3B). Furthermore, the intensity of P-syn staining wasincreased in the hippocampus and cerebral cortex of Tg/KO micecompared with Tg mice (Figure 3C). Unlike human pathologicalconditions, p62 was not localized in the cytoplasmic inclusions
in the brains of Tg mice. Immunohistochemical studies
demonstrated that PK treatment abolished normal α-synuclein
immunoreactivity, and PK-syn was found in the presynapses of thebrain of both Tg and Tg/KO mice (Figure 3D). Western blot analysis verified that P-syn signal intensity was higher in Tg/KO than Tg
mice using two kinds of antibodies against P-syn (Figure 3E).
Furthermore, we fractionated samples of Tg and Tg/KO mice bybuffer with 0.1% Triton X-100 detergent, and found that insoluble
P-syn level was increased in Tg/KO compared with Tg mice
(Figure 3F). Thus, p62 deficiency modulates α-synuclein pathology with regard to P-syn staining intensity, the number of P-syn
inclusions and solubility.

Behavioral tests revealed a longer immobility
time for p62-deficient mice
Given the presynaptic aggregation of PK-syn in the hippocampusof Tg and Tg/KO mice, we sought to determine whether memory
function was also affected in these mice. We performed the Morris
water maze test using mice at a younger age (9 weeks old) to
exclude differences in body weight. The average weight was comparable between Tg and Tg/KO mice (21.1 g in Tg, 22.0 g in
Tg/KO) at 9 weeks of age. During the training phase of the Morris
water maze test, WT and Tg mice showed a gradual decrease inescape latency over time; however, KO and Tg/KO mice exhibited
longer escape latencies (Supporting Information Video Clip S1and S2). When the platform was removed, 80% of WT mice and
70% of Tg mice found the platform location. In contrast, less than
50% of KO and Tg/KO mice found the platform location. KO and
Tg/KO mice took a longer time to reach to the platform location(Figure 4A) and spent less time in the target quadrant (Figure 4B)
than did WT mice. The lower rate of platform crossing in the KO
mice was due to their immobility (Figure 4C,D), which is consistent with previous results showing that KO mice exhibited immobility during training and probe trials (33). During only the firstminute of a forced swim test, KO mice showed a significantly
increased immobility that lasted longer than 2 s when comparedwith WT mice (Figure 4E,F). Thereafter, the time course for4

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Th

A

B

Figure 2. p62 and α-synuclein staining inwild-type (WT), knockout (KO), Tg and Tg/KO
mice. A. Immunohistochemical analysis showsthat p62 immunoreactivity is observed in WT and
Tg mice but not in KO mice (9 weeks of age,n = 6 per group). Bar = 20 μm. B. Human andmouse α-synuclein is strongly expressed in thepresynapse and cytoplasm of cortical neurons in
Tg and Tg/KO mice. Bar = 10 μm.

floating behavior (the percentage of immobility) was similar
between groups. There was no significant difference between
Tg/KO and WT mice. These results suggest that p62 plays a role inmaintaining neurological functions, such as stress responses andmotivation to escape.

Increased levels of the functional homologue,
NBR1, in p62 KO and Tg/KO mice
To analyze the molecular mechanisms associated with the loss of
p62 on Tg mice, we performed quantitative RT-PCR analysis using

primers for genes related to the stress response and proteolysis(Figure 5). Consistent with the genotype results, the p62 mRNAlevel was diminished in KO and Tg/KO mice. Keap1 is a binding
partner of p62 and functions as a sensor for noxious stimuli suchas oxidants and electrophiles. The mRNA level of Keap1 appeared
to be different between the four groups; however, the data were notstatistically significant (P = 0.069). Previous papers have reported
that autophagy-deficient mice display a higher expression ofdetoxifying enzymes, such as Ho-1, Nqo1 and Gclc (18). There
were no differences in the mRNA levels of these enzymes among
the four groups. Recent evidence indicates that α-synuclein
5Brain Pathology •• (2014) ••–••© 2014 International Society of Neuropathology－ 182 －

Fig
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Tanji et al

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Figure 3. The effect of p62 deficiency on abnormal α-synuclein expression. A. Cytoplasmic inclusions are positive for phosphorylated
α-synuclein (P-syn) in the thalamus of Tg and Tg/knockout (KO) mice.
Bar = 20 μm. B. A quantitative analysis shows that the number of cytoplasmic inclusions is significantly increased in Tg/KO mice when compared with Tg mice (9 weeks of age, n = 6 per group). The groups differedsignificantly [analysis of variance (ANOVA), F(3, 11) = 160.81, P < 0.01].
C. P-syn staining is observed in the neurons of the cerebral cortex andhippocampus in Tg and Tg/KO mice. An increased staining intensity isobserved in Tg/KO mice compared with Tg mice. Bar = 500 μm. D. No

obvious differences in proteinase K-resistant α-synuclein (PK-syn) arefound between Tg and Tg/KO mice. Bar = 250 μm. E. P-syn level issignificantly increased in Tg/KO mice compared with Tg mice. Ratio of
P-syn to β-actin was calculated, and the values of Tg mice are defined as
1.0. The groups differed significantly [ANOVA, F(3, 11) = 147.1, P < 0.01].
F. Triton X-100 soluble and insoluble samples were prepared from Tg and
Tg/KO mice (9 weeks of age, n = 2 per Tg and Tg/KO groups). Insoluble
P-syn level is increased in Tg/KO mice compared with Tg mice. P-synlevels were normalized by total synuclein, and the values of Tg mice were
defined as 1.0 in a soluble or insoluble sample.

overexpression causes dynamic changes in the autophagy–lysosomal system. Therefore, we assessed levels of Tf EB, a majortranscriptional regulator for this system (39), lysosomal enzymes(Lamp1 and cathepsin D), molecules responsible for membranetrafficking (Rab7l1) and selective autophagy markers (Nbr1).
Among these genes, only the Nbr1 mRNA levels were significantly
different (P < 0.01) between the four groups. Consistent with thisresult, the NBR1 protein levels were significantly increased inmice lacking p62 compared with mice with p62 (P < 0.05)(Figure 6A,B). Additionally, Keap1 protein levels were also significantly different among the four groups at the protein level.
There were no alterations in NQO1, synaptic proteins andproteolysis-related molecules, such as ubiquitin and LC3, which
are essential to autophagosomal formation (13). Based on the

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The influence of p62 on α-synuclein in mice

Tanji et alTh

Figure 3. Continued.

increased NBR1 levels in mice lacking p62, we compared the
distribution patterns of p62 and NBR1 in the mouse brain. Interestingly, immunoblotting showed that p62 and NBR1 are similarly
distributed in distinct regions of the mouse brain (Figure 7A,B).
NBR1 was mainly localized in the cytoplasm of neurons, and its
intensity was higher in Tg/KO than in Tg mice (Figure 7C). These
data are consistent with the qRT-PCR and immunoblotting
analyses.

DISCUSSION
p62 is an inducible protein that easily aggregates under pathological conditions, such as oxidative stress and disrupted proteolysis,
and it is localized in cytoplasmic inclusions in LBD and otherneurodegenerative diseases, suggesting that p62 contributes toinclusion formation. Moreover, p62- and ubiquitin-positive inclusions in the neurons of brain-specific Atg7-deficient mice disappear with the loss of p62 (17). Based on these findings, we initiallypredicted that p62 deficiency would lead to a decrease in thenumber of inclusions in Tg mice that overexpressed α-synuclein.
However, our data suggest that p62 deficiency results in an exaggeration of α-synuclein pathology with regard to P-syn staining
intensity and inclusion number. Consistent with our findings,
Doi et al demonstrated that a loss of p62 exacerbated neuropathological outcomes (5) in a mouse model of spinal and bulbar
muscular atrophy, which is one of polyglutamine diseases. Our

pathological data showed that the number of P-syn-positive inclusion increased by 1.5-fold in Tg/KO mice compared with Tg mice.
Consistently, this was supported by Western blot analyses showingthat P-syn level was higher in Tg/KO mice than Tg mice using two
kinds of antibodies against P-syn. Considering that increased
P-syn is mainly resistant to detergent of Triton X-100, it is possiblethat biochemical property of α-synuclein is altered and leads tomore aggregation in Tg/KO mice. Although it remains controversial whether the formation of cytoplasmic inclusions exerts a beneficial or toxic effect on cells, our findings strengthen the idea thatp62 can modulate α-synuclein aggregation and the pathogenesis of
diseases.
Consistent with previous results (28, 37), a p62 deficiency
resulted in mature-onset obesity in mice. Recent evidence indicates that hyperphagia is the primary cause of obesity in p62deficient mice due to the disruption of leptin signaling (9).
Accordingly, p62 is highly expressed in hypothalamic neurons,
including proopiomelanocortin (POMC) neurons in the arcuate
nucleus (3, 9) that are responsible for the control of appetite andenergy intake. Interestingly, lack of autophagic activity in POMC
neurons caused higher post-weaning body weight and p62/
ubiquitin aggregation (4, 32). Furthermore, leptin signaling is also
disrupted in these mice. This may have broad implications for thepathophysiology of p62 KO mice. Because p62 helps shuttle
insoluble and ubiquitinated proteins into autophagosomes, disruption of autophagic flux or loss of p62 gives rise to the accumulation
7

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Tanji et al

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Figure 4. p62-deficient mice exhibit longer escape latencies due tolower activity. A. The probe trial was completed after 5 days of hidden
platform training in the Morris water maze. Wild type (WT, n = 10), p62
knockout (KO, n = 11), α-synuclein Tg (Tg, n = 9) and α-synuclein micelacking p62 (Tg/KO, n = 9) were tested at 9 weeks of age. KO mice takelonger to reach the platform location. The groups differed significantly[analysis of variance (ANOVA), F(3, 39) = 4.53, P < 0.01]. B. The percentage of time spent in the target quadrant (black) during a 60 s probe trialof the Morris water maze test. KO mice spend less time in the target

quadrant. C. The immobility time of the Morris water maze. Longer
immobility times are evident in KO mice. D. Representative path tracings are shown. Light pink indicates the position of the platform. E. A
forced swim test was performed at 9–10 weeks of age (n = 9–11 pergroup) and shows a significant difference in immobility latency, with KOmice lasting longer than 2 s and WT mice remaining mobile for the first
1 minute. F. KO mice exhibit higher immobility times for the first 6
minutes. The groups differed significantly [ANOVA, F(3, 39) = 2.14,
P < 0.05]. *P < 0.05.

8

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The influence of p62 on α-synuclein in mice

Tanji et al

Figure 5. The effect of p62 deficiency on several kinds of genes. The
mRNA levels of genes related to proteolysis and oxidative stress in thebrains of WT, KO, Tg and TG/KO mice were determined at 9 weeks ofage (n = 3 per group). mRNA was measured by quantitative reverse
transcription-polymerase chain reaction (qRT-PCR) using the right hemisphere of the brain. Data are normalized by the Cyclophilin A mRNAlevel in each sample, and the average and standard deviation was
calculated. qRT-PCR reveals that there is no significant difference in the

Keap1, glutamate-cysteine ligase catalytic subunit (Gclc), NAD(P)Hquinone oxidoreductase 1 (Nqo1), heme oxygenase-1 (Ho-1), Lamp1,
Cathepsin D, Tf EB and Rab7l1 levels among the four groups. In contrast, the mRNA level of p62 (P < 0.01) and Nbr1 (P < 0.05) are significantly different. The groups differed significantly [analysis of variance,
F(3, 11) = 226.86, P < 0.01 in p62 mRNA, F(3, 11) = 14.15, P < 0.01 in
Nbr1 mRNA]. The WT values are defined as 100%. *P < 0.05,**P < 0.01.

9Brain Pathology •• (2014) ••–••© 2014 International Society of Neuropathology－ 186 －

Th

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9

The influence of p62 on α-synuclein in mice

Tanji et al

Figure 6. The effect of p62 deficiency on molecules related toproteolysis, oxidative stress and the synapse. A. Expression of Keap1
and NBR1 is significantly increased in Tg/KO mice compared with Tg
mice. NAD(P)H quinone oxidoreductase 1 (NQO1), LC3, ubiquitin,synaptophysin and SNAP25 levels are not significantly different
between the four groups (9 weeks of age, n = 6 per group). B. A

quantitative analysis indicates that the Keap1 and NBR1 levels are
significantly increased in p62-deficient mice compared with mice with
p62. The Tg values are defined as 100%. *P < 0.05, **P < 0.01. The
groups differed significantly [analysis of variance, F(3, 11) = 7.44,
P = 0.011 in Keap1, F(3, 11) = 4.27, P = 0.045 in NBR1].

10

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The influence of p62 on α-synuclein in miceThof p62 target molecules. Accordingly, we revealed that P-syn levelis increased in Triton X-100 insoluble fraction of Tg/KO micecompared with Tg mice. Thus, p62 dysfunction observed in
autophagy-deficient POMC neurons or p62 KO mice might also
affect intercellular environment through disturbance of p62
binding partners or substrates. One of p62 binding partners is
known to be dopamine receptor (15). Because dopamine is widely
involved in physiological conditions such as mood, cognition andmotor control, it is possible that p62 modulates dopamine system,and p62 dysfunction may cause pathogenesis of PD.
Our immunoblot results confirmed that the hypothalamus is oneof the regions with the highest p62 expression level. The hypothalamus is known to regulate various physiological functions,particularly the hypothalamus-pituitary-adrenal axis, which coordinates emotional, neuroendocrine and autonomic inputs in
response to stress. Regarding behavioral abnormalities, we could
not distinguish Tg mice from Tg/KO mice; however, p62-deficient
mice exhibited less activity and depression-like behavior in the
Morris water maze and forced swim test. This is consistent withprevious results (33). It is conceivable that p62 deficiency affects
the hypothalamus-pituitary-adrenal axis, leading to behavioral
abnormalities in response to stress. The immobility rate of Tg/KOmice was comparable with that of normal control mice. Considering previous reports that mice overexpressing α-synuclein are
hyperactive (8, 30, 44), we speculate that the degree of immobility
in Tg/KO mice is recovered because of the hyperactivity of Tg
mice. Taken together, p62 plays an important role in modulating
multiple physiological responses, including nutritional, oxidative
and water stressors.
We screened multiple protein and mRNA levels to study themolecular mechanisms associated with the loss of p62 in Tg
mice. We found that NBR1 was significantly increased in Tg/KOmice compared with Tg mice at both the mRNA and protein
levels. p62 and NBR1 contain an N-terminal PB1 domain, anintermediate LC3 binding region, and a C-terminal UBA
domain, and they function as cargo adapters for the autophagic
degradation of ubiquitinated substrates (10, 12, 29). Intriguingly,
our immunoblotting results suggest that these molecules are
similarly distributed in distinct regions of the mouse brain. This
spatial pattern and functional similarity raise the possibility that
NBR1 levels can be up-regulated to compensate for the loss ofp62 protein. Therefore, the functional redundancy of NBR1 may
mask the anticipated abnormalities of p62-deficient mice.
In conclusion, we have provided evidence that p62 is unnecessary for the formation of inclusions in an animal model thatoverexpresses α-synuclein. In addition, p62 deficiency enhanced
α-synuclein pathology based on the number of inclusions andstaining intensity of P-syn. In support of this finding, it is likelythat p62 indirectly helps sequester abnormal molecules through its
own oligomerization (35). Further analyses at the molecular level
suggest that NBR1 plays a compensatory role for p62 in the central
nervous system. NBR1 and p62 double KO mice would be a useful
tool to test this hypothesis.

(K
the
sit
the
He
Re
of
K.
as

Tanji et al

Figure 7. The spatial patterns of p62 and NBR1 in the mouse brain. A.
Equal amounts of homogenates from the indicated regions wereanalyzed by immunoblotting (12 weeks of age, n = 2 in wild-type mice).
Antibodies against NBR1 (upper) or p62 (bottom) were used to detect
endogenous proteins. NBR1 is mainly expressed in the olfactory bulb,
temporal and occipital cortices, striatum, thalamus and hypothalamus.
The highest expression of p62 is observed in the olfactory bulb,
striatum, temporal and occipital cortices, hippocampus, thalamus, hypothalamus and medulla oblongata. β-Actin is used as a loading control. B.
Distribution patterns of NBR1 and p62 in the sagittal section of micebrains. NBR1 or p62 levels are normalized by β-actin. The circles represent the size of the expression level. C. NBR1 immunostaining in Tg and
Tg/KO mice (9 weeks of age, n = 6 per group). NBR1 immunoreactivity
is mainly detected in neurons of the thalamus of both Tg and Tg/KO
mice. Note the increased intensity of NBR1 immunoreactivity in Tg/KOmice compared with Tg mice. Bar = 30 μm.

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ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI grant numbers
26430050 (K.T.), 26860655 (Y.M.), 26430049 (F.M.), 24300131
11

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12

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The influence of p62 on α-synuclein in mice

vel
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(K.W.); Priority Research Grant for Young Scientists designated by
the president of Hirosaki University (K.T., J.M.); Hirosaki University Institutional Research Grant (K.I., K.W.); grants-in-aid fromthe Research Committee for Ataxic Disease from the Ministry of
Health, Labor and Welfare, Japan (K.W.); and an Intramural
Research Grant (24-5) for Neurological and Psychiatric Disordersof NCNP (K.W.). The authors wish to express their gratitude to
K. Saruta, T. Kon, M. Nakata and A. Ono for their technicalassistance.

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Tanji et al

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TS, Kemppinen A et al (2012) Genetic and pathological links
between Parkinson’s disease and the lysosomal disorder Sanfilippo
syndrome. Mov Disord 27:312–315.
48. Wooten MW, Geetha T, Babu JR, Seibenhener ML, Peng J, Cox N
et al (2008) Essential role of sequestosome 1/p62 in regulating
accumulation of Lys63-ubiquitinated proteins. J Biol Chem
283:6783–6789.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Video Clip S1. The Morris water maze test. A wild-type mouse
successfully reaches the platform within 30 s on the fourth day of
training course.
Video Clip S2. The Morris water maze test. A p62-knockout
mouse does not take an action within 60 s, and end up failing on
the fourth day of training course.

Na
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13

Brain Pathology •• (2014) ••–••© 2014 International Society of Neuropathology－ 190 －

cesNakamura et al. Acta Neuropathologica Communications (2015) 3:29
DOI 10.1186/s40478-015-0208-0

RESEARCH

Open Access

Filamentous aggregations of phosphorylated
α-synuclein in Schwann cells (Schwann cell
cytoplasmic inclusions) in multiple system
atrophy
Keiko Nakamura1,2, Fumiaki Mori1*, Tomoya Kon3, Kunikazu Tanji1, Yasuo Miki1, Masahiko Tomiyama3,
Hidekachi Kurotaki4, Yasuko Toyoshima5, Akiyoshi Kakita6, Hitoshi Takahashi5, Masahito Yamada2
and Koichi Wakabayashi1

o

N

ne

se
of

out
on

13

Abstract
Background: The histological hallmark of multiple system atrophy (MSA) is the presence of filamentous aggregationsof phosphorylated α-synuclein in oligodendrocytes, referred to as glial cytoplasmic inclusions (GCIs). Although GCIs canoccur widely in the central nervous system, accumulation of phosphorylated α-synuclein in Schwann cells has not beenreported in MSA. We immunohistochemically examined the cranial and spinal nerves, peripheral ganglia and visceralautonomic nervous system of patients with MSA (n = 14) and control subjects (n = 20).
Results: In MSA, accumulation of phosphorylated α-synuclein was found in the cytoplasm of Schwann cells. These
Schwann cell cytoplasmic inclusions (SCCIs) were also immunopositive for ubiquitin and p62. SCCIs were found in 12 of 14
patients with MSA (85.7 %). They were most frequent in the anterior nerve of the sacral cord and, to a lesser extent, inthe cranial nerves (oculomotor, glossopharyngeal-vagus and hypoglossal nerves), and spinal and sympathetic ganglia.
SCCIs were rarely found in the visceral organs. Immunoelectron microscopy demonstrated that the SCCIs consisted ofabnormal filaments, 15–20 nm in diameter. No such inclusions were found in controls.
Conclusion: The present findings indicate that Schwann cells are also involved in the disease process of MSA.
Keywords: α-synuclein, Multiple system atrophy, Peripheral nerve, Schwann cell, Schwann cell cytoplasmic inclusion,
Ultrastructure

Introduction
Multiple system atrophy (MSA) is an adult-onset neurodegenerative disorder manifested clinically as a combination of parkinsonism, cerebellar ataxia and autonomic
dysfunction. MSA is now divided into two clinicalsubtypes: MSA with predominant parkinsonian features(MSA-P) and MSA with predominant cerebellar dysfunction (MSA-C) [1]. MSA is characterized pathologically by
any combination of coexisting olivopontocerebellar atrophy, striatonigral degeneration and preganglionic* Correspondence: neuropal@hirosaki-u.ac.jp
1
Department of Neuropathology, Institute of Brain Science, Hirosaki
University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562,
Japan
Full list of author information is available at the end of the article

autonomic lesions [2]. The histological hallmark of MSA
is widespread glial cytoplasmic inclusions (GCIs) in thecentral nervous system [3–6]. These GCIs can be visualized by silver staining such as the Gallyas-Braak method[3], and ultrastructurally they consist of granuleassociated filaments 20–30 nm in diameter [3, 4, 7]. The
major component of GCIs is α-synuclein [8], which is
phosphorylated at Serine 129 [9] and ubiquitinated [10].
Although primary oligodendroglial pathology is themain feature of MSA [11–13], accumulation of phosphorylated α-synuclein is also consistently found in theneuronal cytoplasm, processes and nuclei [14]. Similar
neuronal inclusions are found less frequently in the
peripheral sympathetic ganglia [13, 15].

© 2015 Nakamura et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

－ 191 －

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

Although immunoreactivity of non-phosphorylated
α-synuclein has been reported in normal and neoplastic Schwann cells in the peripheral nervous system of
humans [16], accumulation of phosphorylated α-synucleinin Schwann cells of patients with MSA has not been
described hitherto. Here we immunohistochemicallyexamined the cranial and spinal nerves, peripheral ganglia
and visceral autonomic nervous system of patientswith MSA using antibodies against phosphorylated
α-synuclein, and report for the first time that
Schwann cells in these patients are also affected by filamentous aggregations of phosphorylated α-synuclein.

Materials and methods
Subjects

Thirty-four autopsy cases were included in this study. Fourteen of the patients (age 49–79 years, average = 64.6 years)
had a clinical history of MSA, which was confirmed at autopsy by the presence of numerous GCIs (Table 1). All of
the MSA cases lacked Lewy body pathology. The clinical and neuropathological features of early MSA (cases
2 and 12) have been reported previously [17, 18].
Twenty patients used as controls (age 40–84 years,
average = 70.0 years) were clinically and histopathologically free of neurodegenerative disease. This study
was approved by the Institutional Ethics Committee ofHirosaki University Graduate School of Medicine.
Immunohistochemistry

Immunohistochemical analysis was carried out using
formalin-fixed, paraffin-embedded, 4-μm-thick sections
from the midbrain, upper pons, medulla oblongata,spinal cord (cervical, thoracic, lumbar and sacral segments), and dorsal root and paravertebral sympathetic
ganglia. Oculomotor and trigeminal nerves were examined at the level of the midbrain and upper pons,
respectively. Glossopharyngeal and vagus nerves were
examined at the level of the dorsal vagal nucleus. Since
it was difficult to differentiate glossopharyngeal nervefrom vagus nerve on the sections, these two nerves were
described as a whole. Hypoglossal nerves were examinedat the level of the gracile nucleus. Paraffin sections werealso cut from block samples of the esophagus, stomach,small intestine, colon, heart, lung, thyroid, liver,pancreas, kidney, adrenal gland and urinary bladder. The
sections were subjected to immunohistochemical processing using the avidin-biotin-peroxidase complex
method with diaminobenzidine as the chromogen. The
primary antibodies used were mouse monoclonal antibodies against phosphorylated α-synuclein (#64; Wako,
Osaka, Japan; 1:5,000), aggregated α-synuclein (5G4; EMD
Millipore, Temecula, CA, USA; 1:1,000) [19] and ubiquitin(1B3; MBL, Nagoya, Japan; 1:2,000), rabbit monoclonal
antibody against phosphorylated α-synuclein (EP1536Y;

Page 2 of 9

Abcam, Cambridge, UK; 1:5,000), and rabbit polyclonal
antibody against p62 (MBL; 1:1,000). #64 is a monoclonal
antibody against a synthetic peptide corresponding toamino acid residues 124–134 of human α-synuclein witha phosphorylated Serine 129 residue. EP1536Y is also a
monoclonal antibody against a synthetic peptide corresponding to residues surrounding phosphorylated Serine
129 of human α-synuclein.
In addition to routine immunohistochemical techniques, selected sections from the spinal cord of MSA patients were first stained using the modified Gallyas-Braakmethod [20]. The spinal nerve roots were observed undera ×40 objective lens. After removing the cover glasses
from the slides using xylene, the specimens were decolorized in alcohol, then immunostained with antiphosphorylated α-synuclein (Wako; 1:5,000). The spinal
nerve roots were then observed again under a ×40
objective lens.
Semiquantitative assessment of inclusions in Schwann
cells was performed in each region by anti-phosphorylated
α-synuclein immunolabeling. The numbers of inclusions
were estimated as: −, none; +, 1 to 5 inclusions; ++, >5
inclusions.
Double immunostaining

To characterize the inclusion-bearing cells, anti-S-100
was used as a marker of Schwann cells [21], anti-tubulin
polymerization promoting protein (TPPP)/p25α as amarker of oligodendroglia [22], and anti-phosphorylated
neurofilament as a marker of axons [23]. TPPP/p25α isalso known to be a component of GCIs in MSA [24].
Double immunofluorescence analysis was also performed to detect overlapping expression of phosphorylated α-synuclein and ubiquitin. Paraffin sections fromthe spinal cord of patients with MSA (n = 3) were processed for double-label immunofluorescence. Deparaffinized sections were blocked with donkey serum andthen incubated overnight at 4 °C with a mixture of themonoclonal anti-phosphorylated α-synuclein (Wako;
1:500) and polyclonal anti-S-100 (DAKO, Tokyo, Japan;
1:500), anti-TPPP/p25α (Sigma-Aldrich Japan, Tokyo,
Japan; 1:500) or anti-ubiquitin (DAKO; 1:200), or a
mixture of the mouse monoclonal anti-phosphorylated
neurofilament (SMI31; Cosmo Bio, Tokyo, Japan; 1:500)
and rabbit monoclonal anti-phosphorylated α-synuclein(Abcam; 1:500). The sections were then rinsed and incubated for 1 h at 38 °C with anti-rabbit IgG tagged with
Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA; 1:200)
and anti-mouse IgG tagged with Alexa Fluor 594 (Invitrogen; 1:200), or anti-rabbit IgG tagged with Alexa
Fluor 594 (Invitrogen; 1:200) and anti-mouse IgG taggedwith Alexa Fluor 488 (Invitrogen; 1:200). The sections
were examined using an Olympus BX63 fluorescencemicroscope (Olympus, Tokyo, Japan).

－ 192 －

Na

00
lin
a
ed
is
4].
erryomocfind
he
ko;
an;
yo,
a
ed
00)
ein
uth
00)
vixa
ed
ns
ce

M

M

65

65

66

66

69

71

75

79

7

9

10

－ 193 －

11

12

13

14

4

8

<1

8

13

9

14

7

3

4.5

7

7

1

7

SDS

progressive
supranuclear
palsy

neurologicallynormal

SDS

MSA-C

SDS

MSA-C

MSA-C

MSA-P

MSA-P

MSA-P

SDS

spinocerebellar
degeneration

MSA-P

Clinicaldiagnosis

unsteady gait

gait disturbance

MSA-P

MSA-C

none

snoringsensory disturbanceUDgait disturbance

gait disturbance

gait disturbancetremorUD

UD, constipation,
impotence

ataxialimping

Initial symptoms

MSA-C

MSA-C

MSA-C

MSA-P

MSA-P

MSA-C

MSA-P

MSA-P

MSA-P

MSA-C

MSA-C

MSA-P

Pathologicaldiagnosis

OH, orthostatic hypotension; UD, urogenital dysfunction; SDS, Shy-Drager syndrome; +, present;−, absent

M

M

F

M

M

M

F

F

M

8

61

4

M

61

58

3

F

63

57

2

F

Diseaseduration (years)

+

−

+

−

−

+

+
−

−

+

+

+

+

+

+

+

−

+

+

+

+

+

−

+

+

+

+

+
+

−
+

+
−
+

+

−

+

+

+

+

+

+

+

+

+

−

+

UD

+

+

+

−
+

−

−

+
−

OH

Cerebellar
signs

Parkinsonian
signs

Symptoms

−

−

−

−

−

−

−

+
−

−

−

−

−

−

−
−

−

−
+

+

−

−

−

−

−
−

−

−

+

−
−

−

−

Impotence−+

Dyshidrosis

+

−

+

+

+

−

+

Constipation

hpaak
der
ses
oltinal
40

5

49

1

Gender

nal
nal
to
ith
a
rene

6

Age atdeath (years)

nn
ed
ns
>5

Case
No.

Table 1 Summary of clinical findings of patients with multiple system atrophy (MSA)f9
Nakamura et al. Acta Neuropathologica Communications (2015) 3:29
Page 3 of 9

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

Immunoelectron microscopy

The anterior spinal nerve roots from a case of MSA(case 1) were processed for immunoelectron microscopy. Fifty-micrometer-thick vibratome sections were
cut from the formalin-fixed tissue. The sections were incubated with a rabbit monoclonal anti-phosphorylated
α-synuclein antibody (Abcam; 1:500), followed by incubation with a biotinylated secondary anti-rabbit IgG(Vector, Burlingame, CA, USA; 1:200) and avidinbiotin-peroxidase complex (Vector; 1:200), and thereaction was developed with diaminobenzidine. The
immunolabeled sections were post-fixed in 1 % glutaraldehyde and 1 % osmium tetroxide, dehydrated in ethanol, and then embedded in Poly/Bed 812 resin(Polysciences, Inc., Warrington, PA, USA). Ultrathin
sections were cut and viewed with a JEOL1230 electron
microscope (JEOL Ltd., Tokyo, Japan).

Results
Morphology and immunohistochemical features

Immunostaining with anti-phosphorylated and antiaggregated α-synuclein antibodies as well as the modified
Gallyas-Braak method demonstrated widespread occurrence of GCIs throughout the brain and spinal cord of patients with MSA, but not in control subjects. The
immunostaining with two monoclonal anti-phosphorylated
α-synuclein antibodies and a monoclonal anti-aggregatedα-synuclein antibody revealed Schwann cell cytoplasmic inclusions (SCCIs) in the cranial and spinal nerves, peripheral
ganglia and visceral autonomic nervous system of MSA patients (Fig. 1a–q). They appeared crescent-shaped, coil-like,or cigar-shaped (Fig. 1d–f). The SCCIs enveloped the axons(Fig. 1g) and extended their processes from the cytoplasm
to the axons (Fig. 1h, i). Similar inclusions were detectedwith anti-ubiquitin and anti-p62 antibodies (Fig. 1r, s). The
inclusions could not be visualized with hematoxylin andeosin, Klüver-Barrera or Bodian’s method. GCIs appearedargyrophilic with the modified Gallyas-Braak method,whereas SCCIs were stained only weakly or partially (Fig. 1 t,u). No such inclusions were found in controls.
To further characterize the inclusion-bearing cells, antiS-100 was used as a Schwann cell marker, anti-TPPP/
p25α as an oligodendroglia marker, and phosphorylated
neurofilament as an axon marker. Double immunofluorescence analysis revealed co-localization of phosphorylated
α-synuclein and S-100 (Fig. 2a–c), but not TPPP/p25α(Fig. 2d–f) or phosphorylated neurofilament (Fig. 2g–i), in
the inclusions. Phosphorylated α-synuclein and ubiquitin
were also co-localized in the inclusions (Fig. 2 j–l).
Immunoelectron microscopyPre-embedding immunoelectron microscopy demonstrated
phosphorylated α-synuclein-immunoreactive structures in
the cytoplasm of Schwann cells (Fig. 3a). The SCCIs

Page 4 of 9Naconsisted of randomly arranged, loosely packed, granulecoated fibrils, approximately 15–20 nm in diameter(Fig. 3b). Immunodeposition was also detected in the outerand inner loops of the myelinated axons, where fibril formation was not apparent (Fig. 3c).
Distribution and incidence

The distribution and semiquantitative assessment of
SCCIs in patients with MSA are summarized in Table 2.
SCCIs were present in the cranial nerves (oculomotor,
glossopharyngeal-vagus and hypoglossal nerves) and thespinal nerve roots. In the spinal nerve roots, SCCIs were
found in the anterior nerves at the levels of the cervical,
thoracic, lumbar and sacral segments, as well as in theposterior nerves in all the segments, except at the cervicallevel. They were also seen in the dorsal root and sympathetic ganglia and visceral autonomic nervous system.
SCCIs were found in 12 of 14 patients with MSA(85.7 %). They were most frequent in the anterior nervesof the sacral cord (69.2 %) and tended to be more frequent in the anterior than in the posterior nerves at eachlevel. In one case of MSA (case 1), we examined theproximal and distal portions of the sacral nerve roots,
and found that SCCIs were more numerous in the proximal than in the distal portion. In the cranial nerves, theinclusions were more frequent in the glossopharyngealvagus nerves (46.2 %) than in the oculomotor (28.6 %)and hypoglossal (9.1 %) nerves. SCCIs were found in
66.7 % and 33.3 % of the dorsal root and sympathetic
ganglia, respectively. A small number of SCCIs were also
found in the visceral organs in 2 of 14 patients withMSA (14.3 %): the subserosal nerves of the stomach in
one patient (case 1) and the adrenal gland and urinary
bladder in the other (case 12). There appeared to be no
relationship between the frequency of SCCIs and the disease duration or clinical phenotype (MSA-C vs MSA-P) of
patients with MSA.
Several neuronal cytoplasmic inclusions were found in
the dorsal root ganglia in 2 of 9 MSA patients (cases 5
and 13) (Fig. 1 v, w). No such inclusions were found in
the sympathetic ganglia or visceral organs.

Discussion
In the present study, we have demonstrated for the firsttime that phosphorylated α-synuclein accumulates in thecytoplasm of Schwann cells in patients with MSA. These
SCCIs were also immunopositive for aggregated α-synuclein,
ubiquitin and p62, a ubiquitin- proteasome system-related
protein. Thus, the immunohistochemical profile of SCCIs is
similar to that of GCIs [3, 7, 9, 19, 25]. Ultrastructurally,
SCCIs were composed of randomly arranged, loosely packed,
granule-coated fibrils, approximately 15–20 nm in diameter. Both GCIs and neuronal cytoplasmic inclusions also
consisted of granule-coated fibrils, approximately 20–－ 194 －

F
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th
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a
c
E

f9

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

Page 5 of 9

leter
ter
or-

of
2.
or,
he
ere
al,
he
cal
pa-

SA
ves
rech
he
ts,
oxhe
al%)
in
tic
so
th
in
ary
no
isof
in
5
in

rst
he
ese
in,
ed
is
ly,
ed,
mso
0–

Fig. 1 Schwann cell (a–u) and neuronal (v, w) cytoplasmic inclusions stained with anti-phosphorylated α-synuclein (a, b, d–q, u–w), anti-aggregatedα-synuclein (c), anti-ubiquitin (r), anti-p62 (s) and the Gallyas-Braak method (t). a–i Schwann cell cytoplasmic inclusions (SCCIs) (arrowheads) in theanterior spinal nerve roots. SCCIs displaying crescent-shaped (d), coil-like (e), or cigar-shaped morphology (f). SCCIs enwrapping the axons (g). SCCIs
extending their processes to the axons (h, i). j–l SCCIs in the oculomotor (j), glossopharyngeal-vagus (k) and hypoglossal (l) nerves. m and n SCCIs in
the dorsal root (m) and sympathetic (n) ganglia. o–q SCCIs in the stomach (o), adrenal grand (p) and urinary bladder (q). r and s SCCIs showing
immunopositivity for ubiquitin (r) and p62 (s). t and u Sequential staining of the same sections of the spinal nerve with Gallyas-Braak (t) andanti-phosphorylated α-synuclein (u). SCCIs (arrowheads) are only weakly or partially stained with the Gallyas-Braak method. v and w Neuronalcytoplasmic inclusions in the dorsal root ganglia. Immunostaining with anti-phosphorylated α-synuclein antibodies (#64 for a, e, g–q, u–w; andEP1536Y for b, d, f). Bars = 50 μm in a–c; 10 μm in d–w－ 195 －

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

Page 6 of 9

Na

Ta

Cas

1
2
3
4
5
6
7
8
9
10
11
12

Fig. 2 Double immunofluorescence staining of Schwann cell
cytoplasmic inclusions. Co-localization of phosphorylated α-synuclein(p-α-Syn) and S-100 (a–c), but not TPPP/p25α (d–f) or phosphorylated
neurofilament (p-NF) (g–i), in the inclusions. p-α-Syn and ubiquitin(UBQ) are also co-localized in the inclusions (j–l). P-α-Syn (a, d, g, j)appears red, S-100 (b), TPPP/p25α (e), p-NF (h) and UBQ (k) appear
green, and overlap of S-100 or UBQ and p-α-Syn (c, l) appears
yellow. Bars = 10 μm

30 nm in diameter [3, 4, 7, 26–28]. These findings indicatethat Schwann cells are also involved in the disease processof MSA.
SCCIs were found in 12 of 14 patients with MSA(85.7 %) in the present study. GCIs were consistently
found in the brainstem and spinal cord in all of the
MSA patients. By contrast, SCCIs were not observed in
the cranial or spinal nerves in three patients (cases 2, 12
and 14), two of whom had early MSA [17, 18]. Thesefindings suggest that the occurrence of GCIs precedesthat of SCCIs in MSA.
Recently, expression of human α-synuclein has beenreported in Schwann cells ensheathing the nerve fibersof the urinary bladder in a transgenic mouse model of
MSA showing oligodendroglial overexpression of human
α-synuclein under the control of the proteolipid protein
promoter [29]. Urodynamic analysis revealed a less efficient and unstable urinary bladder in this MSA mousemodel. In human MSA, widespread occurrence of GCIs
in the central nervous system is a cardinal pathologicalFig. 3 Immunoelectron microscopy of Schwann cell cytoplasmic
inclusions in the spinal nerve roots. a Phosphorylated α-synucleinimmunoreactive structures in the cytoplasm of Schwann cells.
b A higher-magnification view of the area indicated by the blackasterisk in (a). The inclusion showing granule-coated fibrillary structures,
about 15–20 nm in diameter. Anti-phosphorylated α-synuclein
antibody labels filamentous and granular structures. c A highermagnification view of the area indicated by the white asterisk in(a). Phosphorylated α-synuclein-immunoreactive structures areevident in the outer (black arrowheads) and inner loops (whitearrowheads) of Schwann cells. M, myelin; Ax, axon. Bars = 1 μm

feature [3–6]. Moreover, neuronal cytoplasmic andnuclear inclusions have been observed in the inferiorolivary and pontine nuclei, substantia nigra, putamen
and cerebral cortex in patients with MSA [14, 28]. Filamentous aggregates of α-synuclein are also found in
neurons in the sympathetic ganglia [14, 15]. In thepresent study, we further demonstrated that accumulation of phosphorylated α-synuclein occurs in the neuronal cytoplasm in the dorsal root ganglia. Sural nerve
biopsy from patients with MSA shows a 23 % reductionof unmyelinated fibers (sensory afferent fibers and postganglionic sympathetic fibers) [30]. Mild degeneration ofcardiac sympathetic nerves can occur in MSA [31].
Thus, MSA is a glio-neuronal α-synucleinopathy involving the central and peripheral nervous systems.
It is noteworthy that SCCIs tend to be more frequent in
the peripheral nerves associated with autonomic function, i.e. glossopharyngeal-vagus nerves, and anterior
spinal nerves of the thoracic and sacral cord. The vagus
nerve is a mixed cranial nerve containing axons of branchiomeric motor neurons, parasympathetic preganglionic fibers, visceral afferent fibers, and somatic sensory
afferent fibers. The glossopharyngeal nerve is related
closely to the vagus nerve, sharing common medullary－ 196 －

13
14

Per

DRG
SCC

nu
Th
the
the
lum
the
mi
my
co
SC
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ary

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

Page 7 of 9

Table 2 Distribution and frequency of Schwann cell cytoplasmic inclusions (SCCIs) in patients with multiple system atrophy
Case No.

Cranial nerves

Spinal nerves
C

III

V

IX/X

XII

A

DRG

T
P

A

L
P

A

SG

Visceral organs

S
P

A

P

1

NE

−

+

+

NE

NE

+

+

++

−

++

+

NE

NE

+ (stomach)

2

−

−

−

−

−

−

−

−

−

−

−

−

−

−

−

3

−

NE

+

−

+

−

−

−

+

−

+

+

+

−

−

4

−

−

+

NE

NE

NE

+

−

+

−

+

−

NE

NE

−

5

+

NE

−

−

−

−

+

−

+

−

+

−

−

+

−

6

NE

NE

+

−

+

−

−

−

−

−

+

−

+

−

−

7

NE

NE

−

−

+

−

−

−

−

−

+

−

+

−

−

8

++

NE

+

NE

−

−

−

−

−

−

−

+

+

−

−

9

NE

−

+

NE

+

−

+

+

−

−

+

+

NE

NE

−

10

NE

NE

−

−

+

−

+

−

−

−

+

−

+

−

−

11

−

NE

−

−

−

−

−

−

−

+

+

−

−

+

−

12

−

−

−

−

−

−

−

−

−

−

−

−

NE

NE

+ (adrenal, urinary bladder)

13

NE

NE

−

−

−

−

+

−

−

−

−

−

+

+

−

14

NE

−

NE

−

−

−

−

−

−

−

NE

NE

NE

NE

−

Percent positive for inclusions

28.6

0

46.2

9.1

41.7

0

42.9

14.3

28.6

7.1

69.2

30.8

66.7

33.3

14.3

DRG, dorsal root ganglia; SG, sympathetic ganglia; C, cervical; T, thoracic; L, lumbar; S, sacral; A, anterior; P, posterior. Semiquantitive assessment:−, none; +, 1 to 5
SCCIs per area; ++, more than 5 SCCIs per area; NE, not examined

nuclei and having similar functional components [32].
The sympathetic ganglia receive preganglionic fibers fromthe intermediolateral nucleus of the spinal cord throughthe anterior roots of all the thoracic and the upper two
lumber nerves [32]. The sacral preganglionic parasympathetic fibers exit from the sacral cord and go to the terminal ganglia of the pelvic plexuses, as well as to themyenteric and submucosal plexuses of the descending
colon and rectum [32]. The widespread occurrence of
SCCIs, at least in part, may play a role for the manifestationof a variety of autonomic symptoms in MSA.
Using the modified Gallyas-Braak method, GCIs werepositive whereas SCCIs were stained only weakly or partially. Ultrastructurally, the constituent filaments of SCCIs(approximately 15–20 nm) appeared thinner than thoseof GCIs (approximately 20–30 nm) [3, 4, 7]. Phosphorylated α-synuclein-immunoreactive filamentous inclusions are also found in oligodendrocytes and astrocytes
in the brains of patients with Parkinson’s disease and dementia with Lewy bodies [33–35] and are argyrophilicwith the modified Gallyas-Braak method [36], suggestingthat the process of α-synuclein aggregation in glial cells
may differ somewhat between the central and peripheral
nervous systems.
Cranial nerves are composed of myelinated and unmyelinated fibers in various proportions [37]. The nerve
fibers of the anterior spinal nerve roots projecting to theautonomic ganglia are myelinated [38]. Both myelinatedand unmyelinated fibers in the peripheral nervous

system are enveloped with Schwann cells. Although thenumber of samples was small, our immunoelectron
microscopy examination demonstrated that inclusionbearing Schwann cells, at least in part, ensheath themyelinated fibers. Considering that postganglionic sympathetic nerve fibers are unmyelinated [39] and a small
number of SCCIs were observed in the visceral autonomic
nervous system in MSA, SCCI formation may also occur
in Schwann cells ensheathing the unmyelinated fibers.
Moreover, immunodeposition was also found in theouter and inner loops of Schwann cells. In the central
nervous system, constituent filaments of GCIs are notevident in the outer or inner loops of oligodendrocytes
in MSA [7]. By contrast, tau- and Gallyas-positive filamentous structures are found in the outer and inner
loops of oligodendrocytes in progressive supranuclear
palsy and corticobasal degeneration [39–41]. These findings suggest that phosphorylated α-synuclein pathology
develops both in the perikarya and distal processes of
Schwann cells, whereas the perikarya is chiefly involvedin oligodendrocytes in MSA.
It is unclear how aggregated α-synuclein in the cytoplasm of Schwann cells interacts with the axon, myelin
and Schwann cell itself. Both oligodendrocytes and
Schwann cells are essential for axonal function andintegrity. These enwrapping glia support axonal growthand myelination by transfer of metabolic substrates andsecretion of neurotrophic factors [42]. Glial cell linederived neurotrophic factor (GDNF) is one of the－ 197 －

Nakamura et al. Acta Neuropathologica Communications (2015) 3:29

neurotrophic factors produced by oligodendrocytes [43]and Schwann cells [44]. The level of GDNF is significantly decreased in the frontal white matter and cerebellum of human MSA patients and in the brain of a MSA
mouse model overexpressing human α-synuclein under
the control of the myelin basic protein promoter [45].
Intraventricular infusion of GDNF improves behavioral
deficits and ameliorates the neurodegenerative pathology
in this MSA mouse model [45]. GDNF induces Schwann
cell migration and axonal regeneration in the peripheral
nervous system [46] and also prevents atrophy of facial
motoneurons following axotomy [47]. Liver kinase B1(LKB1) is also a crucial regulator of the major metabolic
pathway in Schwann cells, which are central to axonal
stability [48]. Deletion of LKB1 leads to energy depletion, mitochondrial dysfunction, abnormalities of lipid
homeostasis and increased lactate release in Schwann
cells [48]. The loss of viability in human neuroblastoma
cells overexpressing wild-type α-synuclein is associatedwith reduced activation of intracellular energy sensors,
including LKB1 [49]. α-Synuclein-overexpressing rat
primary neurons also display lower LKB1 activity [49].
Based on the above findings, it is likely that overexpression of α-synuclein in Schwann cells impairs the activityof neurotrophic factors, leading to axonal destabilizationin peripheral nerves.
The origin of α-synuclein in SCCIs is uncertain. Immunoreactivity of non-phosphorylated α-synuclein has been reported in normal and neoplastic Schwann cells in the
peripheral nervous system of humans [16]. Therefore, it ispossible to consider that overexpression of α-synuclein in
Schwann cells would cause SCCI formation. As anotherpossible mechanism, neuron-to-neuron transmission of αsynuclein fibrils through anterograde axonal transport has
been demonstrated in primary cortical mouse neurons
in vitro [50]. The fact that SCCIs tended to appear more
frequently in the proximal than in the distal spinal
nerve roots is appropriate for anterograde transport of
α-synuclein. α-Synuclein in SCCIs could be derived fromneurons. Future studies will be necessary to clarify theorigin of α-synuclein in MSA Schwann cells.

Conclusion
In conclusion, we have provided for the first time evidence that filamentous aggregation of phosphorylated αsynuclein occurs in Schwann cells in patients with MSA.
Similar inclusions are also observed in the oligodendrocytes and neurons of the central nervous system as wellas in neurons of the peripheral ganglia. Both central andperipheral mechanisms may contribute to the neurodegeneration in MSA.
Competing interests
The authors declare that they have no competing interests.

Page 8 of 9

Na

Authors’ contributions
KN, FM and KW designed the study and analyzed data. KN, FM, KT, HK, YT,
AK, HT and KW performed the pathological observations and evaluations. TK,
YM and MT performed the clinical evaluations. FM, MY and KW supervised
the whole process of the study. KN, FM and KW wrote the manuscript. All
authors read and approved the final manuscript.

11.

Acknowledgements
The authors wish to express their gratitude to M. Nakata and A. Ono fortechnical assistance.

14.

Funding
This work was supported by JSPS KAKENHI Grant Numbers 26430049 (F.M.),
26430050 (K.T.), 26860655 (Y.M.) and 24300131 (K.W.), a Grant for Hirosaki
University Institutional Research (K.W.), the Collaborative Research Project(2014–2508) of the Brain Research Institute, Niigata University (F.M.), theResearch Committee for Ataxic Disease (K.W.) from the Ministry of Health,Labour and Welfare, Japan, and an Intramural Research Grant (24–5) for
Neurological and Psychiatric Disorders of NCNP (K.W.).

15.

Author details
1
Department of Neuropathology, Institute of Brain Science, Hirosaki
University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562,
Japan. 2Department of Neurology and Neurobiology of Ageing, Kanazawa
University Graduate School of Medical Science, Kanazawa, Japan.
3
Department of Neurology, Aomori Prefectural Central Hospital, Aomori,Japan. 4Department of Pathology, Aomori Prefectural Central Hospital,
Aomori, Japan. 5Department of Pathology, Brain Research Institute, Universityof Niigata, Niigata, Japan. 6Department of Pathological Neuroscience, Center
for Bioresource-based Researches, Brain Research Institute, University of
Niigata, Niigata, Japan.

18.

12.
13.

16.

17.

19.

20.

21.
22.

Received: 26 March 2015 Accepted: 30 April 2015

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Neuroscience Letters 608 (2015) 6–11

Contents lists available at ScienceDirect

Neuroscience Lettersjournal homepage: www.elsevier.com/locate/neulet

patienprevalantibo[11]. Bulum [
folding
port ofprevioResearch paper

Sortilin-related receptor CNS expressed 2 (SorCS2) is localized to
Bunina bodies in amyotrophic lateral sclerosis
Fumiaki Mori a,∗ , Yasuo Miki a , Kunikazu Tanji a , Akiyoshi Kakita b , Hitoshi Takahashi c ,Jun Utsumi d , Hidenao Sasaki d , Koichi Wakabayashi aaDepartment of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan
Department of Pathological Neuroscience, Center for Bioresource-based Researches, Brain Research Institute, University of Niigata, Niigata, Japan
Department of Pathology, Brain Research Institute, University of Niigata, Niigata, Japand
Department of Neurology, Hokkaido University Graduate School of Medicine, Sapporo, Japanbch i g h l i g h t s• Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative disease.
• ALS-speciﬁc Bunina bodies are immunoreactive for SorCS2, a member of vacuolar protein sorting 10 family proteins (VPS10Ps).
• Immunoreativity for VPS10Ps, sortilin and SorLA, is decreased in anterior horn cells of ALS patients.
• VPS10Ps may be involved in the pathomechanisms of ALS.

a r t i c l e

i n f oArticle history:
Received 18 June 2015
Received in revised form 7 September 2015
Accepted 24 September 2015
Available online 28 September 2015
Keywords:
Amyotrophic lateral sclerosis
Bunina bodies
Immunohistochemistry
Sortilin-related receptor CNS expressed 2
Vacuolar protein sorting 10 family proteinsa b s t r a c t
Sortilin-related receptor CNS expressed 2 (SorCS2) is one of the vacuolar protein sorting 10 family proteins(VPS10Ps) that have pleiotropic roles in protein trafﬁcking and intracellular and intercellular signaling.
Bunina bodies (BBs) are speciﬁcally detected in the lower motor neurons in patients with amyotrophic
lateral sclerosis (ALS). BBs are immunolabeled with antibodies against cystatin C, transferrin and peripherin and are considered to originate from the endoplasmic reticulum, which is part of the protein sorting
pathway. The present study investigated whether VPS10Ps are involved in the formation of BBs in ALS.
We immunohistochemically examined the spinal cord from patients with ALS and control subjects using
antibodies against VPS10Ps (sortilin, SorLA, SorCS1, SorCS2 and SorCS3). In normal controls, antibodies
against VPS10Ps immunolabeled the cytoplasm of anterior horn cells in a ﬁne granular pattern. In ALS,
almost all BBs (95.1%) were strongly immunopositive for SorCS2, and immunoreativity for sortilin and
SorLA was decreased in anterior horn cells. These ﬁndings suggest that VPS10Ps may be involved in the
disease process of ALS.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Sortilin-related receptor CNS expressed 2 (SorCS2) is a type I
transmembrane glycoprotein receptor that belongs to the mammalian vacuolar protein sorting 10 protein (VPS10P) family [6,19].
VPS10Ps include sortilin, sorting protein-related receptor with Atype repeats (SorLA), SorCS1, SorCS2, and SorCS3 proteins. All
SorCS proteins are expressed predominantly in the brain, especially∗ Corresponding author at: Department of Neuropathology, Institute of Brain
Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki
036-8562, Japan. Fax: +81 172 39 5132.
E-mail address: neuropal@hirosaki-u.ac.jp (F. Mori).

during development [6,8,19]. Interactions have been documented
between VPS10Ps and retromer coat complex (VPS35 and VPS26), a
key component of the intracellular trafﬁcking apparatus that sorts
cargo from the early endosome to the trans-Golgi network [9]. In
recent years, it has been shown that a number of neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s
disease and frontotemporal lobar degeneration (FTLD) are characterized by the misprocessing and missorting of intracellular
proteins (amyloid precursor protein, tau, ˛-synuclein, TDP-43, FUS)
within endosomal–lysosomal pathways. Dysfunction within thesepathways is proposed to be a major contributing factor to diseaseprogression [9].
Bunina bodies (BBs) are small round eosinophilic inclusions,
1–5 m in diameter, observed in the lower motor neurons ofFig. 1. L
immunoand SorC

http://dx.doi.org/10.1016/j.neulet.2015.09.030
0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

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F. Mori et al. / Neuroscience Letters 608 (2015) 6–11

patients with amyotrophic lateral sclerosis (ALS), one of the mostprevalent TDP-43 proteinopathies. BBs are immunolabeled with
antibodies against cystatin C [16], transferrin [10] and peripherin[11]. BBs are considered to originate from the endoplasmic reticulum [21,23], which serves many general functions, including thefolding of protein molecules in sacs called cisternae and the transport of synthesized proteins in vesicles to the Golgi apparatus. Our
previous immunoelectron microscopy observations indicating that7cystatin C is localized to the vesicular membranous structures of
BBs [13] prompted us to examine whether VPS10Ps are involved in
the formation of BBs. Therefore, we immunohistochemically investigated the expression of VPS10Ps in spinal cord samples frompatients with ALS and control subjects. Here we report that SorCS2
immunoreactivity is detectable in BBs in anterior horn cells (AHCs)
and that immunoreativity for sortilin and SorLA is decreased in
AHCs of ALS patients in comparison to controls.

roteins
naling.
trophic
periphsortingin ALS.
s using
ibodies
In ALS,lin andd in theserved.

mentedS26), at sorts[9]. In
degennson’scharellular3, FUS)n theseiseaseusions,ons ofFig. 1. Light micrographs of the anterior horn of the lumbar cord of normal controls (a,c,e,g,i) and patients with amyotrophic lateral sclerosis (ALS) (b,d,f,h,j), showing
immunoreactivity for sortilin (a,b), sorting protein-related receptor with A-type repeats (SorLA) (c,d), sortilin-related receptor CNS expressed 1 (SorCS1) (e,f), SorCS2 (g,h),and SorCS3 (i,j). In ALS, immunoreactivity for sortilin and SorLA is decreased in anterior horn cells (b,d). Bars = 50 m.

－ 201 －

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F. Mori et al. / Neuroscience Letters 608 (2015) 6–11

sections from multiple cortical and subcortical regions were stainedwith hematoxylin and eosin (H&E) or by the Klüver–Barrera
method. We also examined the sections immunohistochemicallyusing anti-TDP-43 antibody (10782-1-AP; ProteinTec Group, Inc.,
Chicago, IL, USA; 1:4000). This study was approved by the Institutional Ethics Committee of Hirosaki University Graduate School of
Medicine.

2.2. ImmunohistochemistryFig. 2. Semiquantiﬁcation of immunoreactivity for sortilin (a), SorLA (b), SorCS1 (c),SorCS2 (d), and SorCS3 (e) of anterior horn cells in normal controls and ALS patients.
The proportions of neurons with intense/moderate (++, black column), weak (+, graycolumn) or no immunostaining (−, white column) relative to the total number ofneurons in the anterior horn are shown. Values are expressed as mean ± standard
error of the mean. Correlations at p < 0.05 were considered signiﬁcant.

Sheep polyclonal antibody against SorCS2 (AF4238; R&D Systems, Inc., Minneapolis, MN, USA; 1:40) and rabbit polyclonal
antibodies against sortilin (HPA006889; Atlas Antibodies AB, Stockholm, Sweden; 1:50), SorLA (ab16642; Abcam, Cambridge, UK;1:50), SorCS1 (23002-1-A1; ProteinTec Group, Inc.; 1:50) and
SorCS3 (HPA049097; Atlas Antibodies AB; 1:500) were used in thisstudy.
We identiﬁed BBs using H&E-stained sections. BBs were deﬁned
as small eosinophilic perikaryal inclusions, 1–5 m in diameter,
which were sometimes clustered. Serial sections (4 m thick)were cut from parafﬁn blocks of the 4th lumbar segment in allcases and were ﬁrst stained with H&E. Digital images of the anterior horn on both sides were captured by a virtual slide system(VS110-S1; Olympus, Tokyo, Japan). AHCs were deﬁned as Nissl
body-containing cells in Rexed VIII and IX, whose somal diameters were greater than 37 m [12]. AHCs with or without BBs wereobserved under a ×40 objective lens, measured by a virtual slide
system (×600 on a display) and numbered on enlarged prints (×80).
When the same neuronal cell body was recognized on several contiguous sections, the same number was marked on the neuron. Thisapproach ensured we avoided missing tiny BBs. After removing thecover glass from the slides in xylene, the specimens were decolorized in alcohol, subjected to heat retrieval using an autoclave for10 min in 10 mM citrate buffer (pH 6.0), and immunostained withsheep antibody against SorCS2 (1:40) using a Vectastain ABC kit(Vector, Burlingame, CA, USA). Diaminobenzidine was used as thechromogen. The sections were counterstained with hematoxylin.
Digital images of the anterior horn on both sides were capturedby the virtual slide system again. The SorCS2 immunoreactivity of
BBs was conﬁrmed under a ×40 objective lens in each neuron on adisplay.
Selected sections were double-immunolabeled with sheep polyclonal anti-SorCS2 (1:20) and rabbit polyclonal anti-cystatin C(A0541; DakoCytomation, Glostrup, Denmark; 1:500) antibodies.
The secondary antibodies were Alexa Fluor 488 donkey anti-sheep
IgG (A-11015; Invitrogen, Carlsbad, CA, USA; 1:200) and Alexa
Fluor 594 donkey anti-rabbit IgG (A-21207; Invitrogen; 1:200).
The sections were examined with an Olympus Provis ﬂuorescencemicroscope (Olympus, Tokyo, Japan).

Fig. 3. Cand c sh2.4. St

Calc
Publishformedmean ±
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3. ResIn n
moder
granulantibowas unSorLA
was nocytoplabetwee2.3. Semi-quantitative analysis
2. Materials and methods
2.1. Subjects
Twelve patients with sporadic ALS (aged 53–77 years, average
65.8 years) were studied. The diagnosis was conﬁrmed both clinically and histopathologically. BBs were found in the AHCs in 8 of
the 12 cases, and TDP-43-positive inclusions were evident in all
the cases. We also examined ﬁve neurologically normal individuals(aged 53–84 years, average 67.8 years). For routine histological examination, 4-m-thick, formalin-ﬁxed, parafﬁn-embedded

The numbers of neurons immunoreactive for sortilin, SorLA,
SorCS1, SorCS2 and SorCS3 in control subjects and patients with ALS
were assessed using a semi-quantitative rating scale: −, unstained;+, weakly stained; ++, moderately or intensely stained. The staining intensity was ﬁrst graded as − or ++, and any intensity that wasneither − nor ++ was deﬁned as +. Three authors (FM, YM, KW),
experienced neuropathologists, rated immunostained sections of
ALS and control cases under blinded fashion. The proportions of sortilin, SorLA, SorCS1, SorCS2 and SorCS3-positive neurons relative tothe total number of neurons were calculated in each case.

－ 202 －

Fig. 4. D
SorCS2 a

F. Mori et al. / Neuroscience Letters 608 (2015) 6–11

9

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pturedvity ofn on ap polyatin C
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scenceSorLA,th ALS
ained;
stainat was, KW),
ons ofof sortive to

Fig. 3. Contiguous sections stained with anti-SorCS2 antibody (a,b) and hematoxylin and eosin (c,d). Higher-magniﬁcation view of the area indicated by the asterisks in aand c showing Bunina bodies (arrowheads) in the lumbar anterior horn of a patient with ALS (b,d). Bars in a and c = 50 m. Bars in b and d = 10 m.

2.4. Statistical analysis
Calculations were performed using Statcel software (OMS
Publishing, Tokorozawa, Japan). Statistical comparison was performed with two-way factorial ANOVA. Values were expressed asmean ± standard error of the mean. Correlations at p < 0.05 wereconsidered signiﬁcant. Good interrater reliability was achieved bythree observers using the method, because the data from each rater
showed the same signiﬁcant result.

3. Results
In normal controls, antibodies against sortilin, SorLA and SorCS3
moderately immunolabeled the cytoplasm of AHCs in a diffuse
granular pattern (Fig. 1a,c,i). With anti-SorCS1 and anti-SorCS2
antibodies, the cytoplasm of AHCs was barely immunolabeled, or
was unstained (Fig. 1e,g). In ALS, immunoreactivity for sortilin and
SorLA was decreased in the majority of AHCs (Fig. 1b,d). There
was no apparent difference in staining intensity of the neuronalcytoplasm immunolabeled with anti-SorCS1, -SorCS2 or -SorCS3
between ALS patients and controls (Fig. 1f,h,j).

Semi-quantitative analysis showed that 20.2% of AHCs weremoderately or intensely immunolabeled with anti-sortilin antibody and that 60.5% were weakly immunolabeled in normal
controls (Fig. 2a, left). In ALS, 31.2% of AHCs showed weak
immunoreactivity and 68.8% were unstained (Fig. 2a, right). Similarly, 27.0% of AHCs were moderately or intensely immunolabeledwith anti-SorLA antibody and 57.7% were weakly immunolabeled
in normal controls (Fig. 2b, left). In ALS patients, 15.6% of AHCs
showed weak immunoreactivity and the rest were unstained(Fig. 2b, right). The differences in the staining intensity of AHCs
immunolabeled with anti-sortilin or anti-SorLA between ALS
patients and controls were statistically signiﬁcant (p < 0.01). There
were no signiﬁcant differences in immunoreactivity for SorCS1,
SorCS2 and SorCS3 between ALS patients and controls (Fig. 2c–e).
Although the cytoplasm of AHCs showed no or only weak
immunoreactivity for SorCS2, BBs were intensely immunolabeledwith anti-SorCS2 antibody (Fig. 3). Sequential staining of thesame sections with H&E and anti-SorCS2 antibody revealed that
almost all BBs were immunopositive for SorCS2 (90.9–100%, average 95.1%). No SorCS2-positive inclusions were observed in 4
cases of ALS, in which no BBs had been detected. No signiﬁcantFig. 4. Double-labeling immunoﬂuorescence demonstrating co-localization of SorCS2 and cystatin C in Bunina bodies in spinal anterior horn cells of a patient with ALS.
SorCS2 appears green (a) and cystatin C appears red (b). Overlap of SorCS2 and cystatin C appears yellow (c). Bars = 10 m.

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F. Mori et al. / Neuroscience Letters 608 (2015) 6–11

immunoreactivity was detectable in sections from controls and ALS
cases treated with phosphate-buffered saline solution or by preincubating the antibody (AF4238) with the corresponding synthetic
protein (4238-SR; R&D Systems, Inc.) (data not shown).
Double-label immunoﬂuorescence revealed co-localization of
SorCS2 and cystatin C in BBs (Fig. 4). No signiﬁcant immunoreactivity was detectable in negative reaction control sections incubatedwithout the primary antibodies.

4. Discussion
A number of genome-wide association studies and biochemical
studies have identiﬁed members of the VPS10Ps (sortilin, SorLA,
SorCS1, SorCS2 and SorCS3) that regulate endosomal sorting as risk
factors for neurodegenerative diseases [9]. In the present study,
we demonstrated that immunoreativity for sortilin and SorLA wassigniﬁcantly decreased in AHCs from patients with ALS and that BBs
were immunoreactive for SorCS2, indicating that sortilin, SorLA andSorCS2 are linked to the pathomechanism of ALS.
Sortilin functions as a trafﬁcking receptor for both Trk receptors
and brain-derived neurotrophic factor through the secretory pathway. Progranulin is targeted to the endosomal–lysosomal pathway
through a sortilin-dependent mechanism [9]. Haploinsufﬁciency
of progranulin is a common genetic cause of FTLD with TDP-43
aggregates. Progranulin levels were sharply increased upon TDP43 reduction and splicing of sortilin was altered [17]. TDP-43 is amajor constituent of inclusions in motor and non-motor neurons
in ALS and FTLD [1,14,22]. Decreased immunoreativity for sortilin
in AHCs of ALS might be associated with altered splicing of sortilin.
SorLA was the ﬁrst member of the family of VPS10 proteins
to be genetically linked with late-onset AD [20], and SorLA mutations have now also been suggested as causes of familial AD [18].
Subsequent in vitro and in vivo studies have shown that SorLA
is indeed required for endosome to TGN trafﬁcking of amyloid
precursor protein [4,24]. Disruption of the VPS26 binding motif
within the SorLA cytoplasmic tail results in increased localization of amyloid precursor protein to endosomal compartments andincreased amyloidogenic processing of amyloid precursor protein
to produce -amyloid [4]. -Amyloid has been reported to trigger
ALS-associated TDP-43 pathology in AD models [7]. These ﬁndings
suggest that decreased immunoreativity for SorLA in AHCs of ALS
might contribute to the pathomechanism of ALS.
We further demonstrated that BBs are immunoreactive for
SorCS2, a member of the VPS10Ps which play various roles in membrane trafﬁcking [9]. Our ﬁndings indicate that SorCS2 is anotherimmunohistochemical marker for BBs in addition to cystatin C [16],which is a member of a super family of protease inhibitors, transferrin, which is an iron-binding plasma protein [10], and peripherin,
which is a type III intermediate ﬁlament protein [11]. These threeproteins show moderate to strong positive reactions in the cytoplasm in almost all neurons without BBs, suggesting that they might
be incorporated into BBs. On the other hand, SorCS2 protein appears
to be less detectable in the cytoplasm of AHCs without BBs, suggesting a different role of SorCS2 in the formation of BBs. SorCS2
is a member of the VPS10Ps, which have pleiotropic roles in protein trafﬁcking and intra- and intercellular signaling in neuronaland non-neuronal cells [9]. Ultrastructurally, BBs are considered tooriginate from the endoplasmic reticulum [21,23], and cystatin C
is localized to the vesicular structures of BBs [13]. These ﬁndings
indicate that deposition of SorCS2 in BBs results from dysfunctionof protein trafﬁcking and intra- and intercellular signaling, leading
to incorporation or mislocalization of cystatin C [16], transferrin[10] and peripherin [11] into BBs.
Sortilin is a sorting receptor that directs target proteins, such as
growth factors, signaling receptors, and enzymes, to their destined

location in secretory or endocytic compartments of cells [2]. However, sortilin is not unique, and just one of ﬁve structurally related
VPS10Ps including SorCS2 [25]. Emerging evidence suggests that
VPS10Ps functionally cooperate, adding another level of complexity to the molecular network in which sortilin acts [15]. SorCS2 hasbeen identiﬁed as a receptor that binds to pro-nerve growth factor,
thereby mediating acute collapse of growth cones of hippocampal
neurons [3]. SorCS2 has also been identiﬁed as a proneurotrophin
receptor, mediating both trophic and apoptotic signals in conjunction with p75NTR [5]. These ﬁndings indicate that SorCS2 isinvolved in regressive changes of neuronal cells. In the present
study, we have demonstrated that BBs, a speciﬁc hallmark of ALS,
are strongly immunopositive for SorCS2. These ﬁndings indicate
that, in ALS, SorCS2-positive BBs represent dysfunctional sorting ofcertain cellular proteins to their destined location in AHCs.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Numbers
26430049 (F.M.), 26430050 (K.T.), 26860655 (Y.M.) and 24300131(K.W.), a Grant for Hirosaki University Institutional Research(K.W.), the Collaborative Research Project (2015-2508) of the Brain
Research Institute, Niigata University (F.M.), and Grants-in Aid fromthe Research Committee for Ataxic Disease, the Ministry of Health,Labour and Welfare, Japan (H.S., K.W.). The authors wish to express
their gratitude to M. Nakata and A. Ono for their technical assistance.
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Surgical Neurology International

OPEN ACCESSFor entire Editorial Board visit :http://www.surgicalneurologyint.com

Editor:James I. Ausman, MD, PhD
University of California, Los
Angeles, CA, USA

Case Report

Cortico-cortical activity between the primary and supplementary
motor cortex: An intraoperative near-infrared spectroscopy study
Masafumi Fukuda, Tetsuro Takao, Tetsuya Hiraishi, Hiroshi Aoki, Ryosuke Ogura,
Yosuke Sato, Yukihiko Fujii
Department of Neurosurgery, Brain Research Institute, University of Niigata, Niigata-City, Japan
E-mail: *Masafumi Fukuda - mfuku529@bri.niigata-u.ac.jp; Tetsuro Takao - takao@bri.niigata-u.ac.jp; Tetsuya Hiraishi - tetsuya_hiraishi@me.com;
Hiroshi Aoki - aoki1123@bri.niigata-u.ac.jp; Ryosuke Ogura - oguryou@bri.niigata-u.ac.jp;Yosuke Sato - yosuke0420@hotmail.com;
Yukihiko Fujii - yfujii@bri.niigata-u.ac.jp*Corresponding author
Received: 18 August 14

Accepted: 02 January 15

Published: 24 March 15

This article may be cited as:
Fukuda M, Takao T, Hiraishi T, Aoki H, Ogura R, Sato Y, et al. Cortico-cortical activity between the primary and supplementary motor cortex: An intraoperative near-infrared
spectroscopy study. Surg Neurol Int 2015;6:44.
Available FREE in open access from: http://www.surgicalneurologyint.com/text.asp?2015/6/1/44/153872
Copyright © 2015 Fukuda M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract
Background: The supplementary motor area (SMA) makes multiple reciprocal
connections to many areas of the cerebral cortices, such as the primary motor
cortex (PMC), anterior cingulate cortex, and various regions in the parietal
somatosensory cortex. In patients with SMA seizures, epileptic discharges from the
SMA rapidly propagate to the PMC. We sought to determine whether near-infrared
spectroscopy (NIRS) is able to intraoperatively display hemodynamic changes inepileptic network activities between the SMA and the PMC.
Case Descriptions: In a 60-year-old male with SMA seizures, we intraoperatively
delivered a 500 Hz, 5-train stimulation to the medial cortical surface andmeasured the resulting hemodynamic changes in the PMC by calculating theoxyhemoglobin (HbO2) and deoxyhemoglobin (HbR) concentration changes
during stimulation. No hemodynamic changes in the lateral cortex were observed
during stimulation of the medial surface corresponding to the foot motor areas. In
contrast, both HbO2 and HbR increased in the lateral cortex corresponding to thehand motor areas when the seizure onset zone was stimulated. In the premotor
cortex and the lateral cortex corresponding to the trunk motor areas, hemodynamicchanges showed a pattern of increased HbO2 with decreased HbR.
Conclusions: This is the first reported study using intraoperative NIRS tocharacterize the epileptic network activities between the SMA and PMC. Our
intraoperative NIRS procedure may thus be useful in monitoring the activities ofcortico-cortical neural pathways such as the language system.

Supplementary motor area (SMA) seizures are short induration and characterized by abrupt, bilateral, tonic

Access this article
onlineWebsite:www.surgicalneurologyint.com
DOI:
10.4103/2152-7806.153872
Quick Response Code:posturing of the extremities, and vocalization without
loss of consciousness.[6] Epileptic discharges fromthe SMA can rapidly propagate through the primary
motor cortex (PMC), anterior cingulate cortex (ACC),－ 206 －

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Key Words: Cortico-cortical activity, epilepsy, hemodynamic change, near-infrared
spectroscopy, primary motor cortex, supplementary motor area

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Surgical Neurology International 2015, 6:44

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and various parietal somatosensory areas, which form
multiple reciprocal connections to the SMA.[2,4] Better
understanding of the mechanisms of SMA propagation canbe achieved through improved methods to probe cortical
network activities associated with epileptic discharges
from the SMA. For example, an ictal single photon
emission computed tomography study indicated that
seizure-associated hyperperfusion areas did not localize
within the SMA, but rather spread to adjacent corticalregions ipsilateral to epileptic foci, such as the ACC and
PMC.[1] Recently, we demonstrated using simultaneoustranscranial near-infrared spectroscopy (NIRS) andelectrocorticography (ECoG) recordings in a patientwith SMA seizures that increased cerebral blood flow wasobserved from the epileptic discharges in the ipsilateral
SMA and spread to the ipsilateral premotor cortex and
PMC as well as the contralateral hemisphere.[7]In a patient with SMA seizure, we used a novel
4-probe device attached to the brain surface to conduct
intraoperative NIRS from four probes to show increased
blood flow in the PMC elicited by stimulation to the SMA.
This is the first report to determine that intraoperative
NIRS can reveal the cortico-cortical activities between
the SMA and PMC. Moreover, by placing probes on thebrain surface, we were able to obtain greater resolution
than with transcranial NIRS methods. Our technique willthus allow improved mapping of cortico-cortical network
activities intraoperatively.

CASE REPORT
A 60-year-old male presented with seizures a few months
before admission to our hospital. His seizures werecharacterized by tonic posturing in the left extremities
and occurred 3–4 times monthly. T2-weighted magnetic
resonance (MR) imaging revealed a high intensity lesionin the medial surface of the right frontal lobe. The lesion
was not enhanced by Gd on the T1-weighted MR images,
and was suspected to be a low-grade glioma. In order toconfirm the relationship between the lesion and the PMC
corresponding to the lower extremities, subdural gridelectrodes were placed to cover the lateral and medialsurfaces adjacent to the PMC. Video-ECoG monitoring
demonstrated seizure onset at the right medial surface
corresponding to the SMA. The seizure activities rapidly
propagated from the SMA to the lateral cortex, including
the PMC.
Cortical electrical stimulation was performed forfunctional cortical mapping. A repetitive square wave withelectrical currents of alternating polarity, a pulse widthof 0.2 ms, and a frequency of 50 Hz were delivered for
5 s (Nihon Koden, Corporation, Japan). Two neighboring
electrodes, with an intensity of 2–5 mA, were stimulated
in a bipolar manner. Positive motor and sensory areas
were identified by positive motor response (i.e. muscle

twitch) and subjective sensory sensation, respectively.
To define the precise location of each electrode on
the surface of the brain, subdural electrodes extracted
from computed tomography (CT) images wereco-registered to three-dimensional volume-rendered MR
images (3.0 T) using image-analysis software (Zed-View,
LEXI, Inc., Japan). The results of this functional corticalmapping are depicted in Figure 1. Cortical stimulation to
the anterior and posterior areas of the seizure onset zone
induced habitual seizures.
Before a partial resection of the lesion for pathology,
intraoperative NIRS recording was performed upon
stimulation of the placed subdural electrodes apparatusof the medial cortical surface. Constant current
stimuli, consisting of five rectangular pulses with 2-ms
interstimulus intervals, were generated and recordedwith a Neuropac (Nihon Koden, Corporation, Japan).
The cathode was positioned at Fz. Motor-evoked
potentials were recorded from the abductor pollicis brevis
and abductor halluces brevis muscles through paired
stainless-steel needle electrodes inserted subdermally.
The band-pass filter was set to a range of 5–3000 Hz.
The applied stimuli were adjusted to the supra-thresholdintensity.
For NIRS monitoring, we developed a novel device
comprising of four recording probes spaced 1.5 cm
apart and equipped with fixable spatula retractors at
the tip of each probe to enable attachment to the brain
surface [Figure 2a and b]. The four probes were set tocover the lateral cortex, including the PMC [Figure 2c].
NIRS was carried out with a 695/830 nm spectrometerequipped to our novel monitoring device (ETG-7100;
HITACHI Medical, Japan). Emitting light intensity
was adjusted to 1 mW (approximately one-fourth

Figure 1: Results of video-electrocorticography (ECoG) monitoring
and functional cortical mapping. 3D brain surface images showing
recording electrodes (pale blue) and the brain tumor (green).The
seizure onset zone (square) was confirmed to reside in the medialsurface. The sites in which stimulation induced habitual seizures(closed square) were noted anterior and posterior to the seizure
onset zone. Before partial tumor resection, both the foot motor
area and seizure onset zone were stimulated for intraoperative
NIRS study (stars). UE: Upper extremities, LE: Lower extremities,
CS: Central sulcus－ 207 －

Surgical Neurology International 2015, 6:44

of scalp NIRS). The oxyhemoglobin (HbO2) anddeoxyhemoglobin(HbR)
concentration
changescorresponding to medial surface stimulation in eachsession were calculated for each segment, which consistedof a 30 s intervals comprising of a prestimulation
block (10 s), a stimulation block, and a poststimulation
block (20 s). Each session was repeated five times. The
present study was approved by the Institutional Review
Board Committee at the Niigata University School of
Medicine, and informed consent was obtained from thepatients (IRB#1559).
In the results, no hemodynamic changes wereobserved during stimulation of the medial surface
corresponding to the foot motor areas at the intensityof 20 mA [Figure 3A]. In contrast, both HbO2 and HbR
increased in the lateral cortex corresponding to the hand
motor areas when the seizure onset zone was stimulated
at an intensity of 16 mA [b and c in Figure 3B]. In thelateral cortex corresponding to the trunk motor areas andthe premotor cortex, hemodynamic changes showed apattern of increased HbO2 with decreased HbR [a and din Figure 3B].

DISCUSSION
To our knowledge, this is the first report using
intraoperative NIRS during cortical stimulation todemonstrate cortico-cortical activity between the SMA
and PMC. We observed that stimulation of the footmotor area elicited no detectable hemodynamic responses
in the lateral cortex, whereas stimulation to the seizure
onset zone elicited hemodynamic responses at all four
probes despite the stimulation intensity decreasing from
20 to 16 mA. These results likely reflected the epileptic
network activities between the SMA and PMC.

http://www.surgicalneurologyint.com/content/6/1/44

Surg

It should be noted that we used high frequency
stimulation (5-trains, 500 Hz) in the present study,
because we were concerned that 50 Hz-stimulation to theseizure onset zone during surgery and functional mapping
might induce seizures. Previously, we characterized
hemodynamic connectivity in the language system ina patient with temporal lobe epilepsy.[8] In this study,
50 Hz-stimulation in the left superior temporal gyrus via
subdural electrode contacts gave rise to hemodynamic
increases in the inferior frontal gyrus, indicating a strong
connection to the stimulation site. In a cortico-corticalevoked potential study,[5] stimulation of the foot motor
cortex elicited responses in the pre- and postcentral
gyrus. Thus, 50 Hz-stimulation of the foot motor areas,rather than 500 Hz employed in our present experiments,
might induce hemodynamic changes in the lateral cortex
as well.

ass
lev
con
sup

In this study, high frequency stimulation caused thehemodynamic changes characterized by increase in both
HbO2 and HbR in the lateral cortex corresponding to thehand motor areas. This phenomenon was also observedin a study of the language system of the frontal andtemporal cortex using simultaneous NIRS and ECoG
recordings during cortical stimulation.[8] In contrast, inthe areas corresponding to the trunk motor areas andpremotor cortex, hemodynamic changes showed a patternof increased HbO2 with decreased HbR as is typicalof task-evoked hemodynamic changes. In a previous
NIRS study carried out simultaneously with corticalstimulation via subdural electrodes,[3] 50 Hz-stimulation
produced significant increases in both HbO2 and HbR
at the stimulation site and surrounding areas, while
5 Hz-stimulation produced a localized increase in HbO2
and a decrease in HbR. An increase in HbO2 with an
A

B

a

b
a
c

d

b

c

Figure 2: (a) Our novel device for the intracranial setting of fourNIRS probes.The inter-probe distance was 1.5 cm. (b) Four probes
were equipped to the NIRS devices. (c) During surgery, the novel
device and its four probes were wrapped by a sterilized cover.The
device was fixed by spatula retractors at the tip of each probe forattachment to the brain surface.The probes were placed to cover
the lateral cortex including the primary motor cortex

Figure 3: Time course changes of HbO2 and HbR in the lateralcortex. The left picture shows the relationship between thesites of each of the four probes (circle) and subdural electrodes.
Recording sites of NIRS were a, b, c, and d. CS: Central sulcus. (A)No hemodynamic changes were noted in any sites, when the footmotor area was stimulated at the intensity of 20 mA. (B) BothHbO2 and HbR increased in the hand motor areas when the seizure
onset zone was stimulated at an intensity of 16 mA (b and c). In thetrunk motor areas (a) and the premotor cortex (d), hemodynamicchanges showed a pattern of increased HbO2 with decreased HbR－ 208 －

Th
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1/44

Surgical Neurology International 2015, 6:44

ncy
udy,
the
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udy,
via
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ong
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otor
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associated increase in HbR is thus indicative of higher
levels of neural activity and indicates that oxygen
consumption exceeds oxygen delivery via the blood
supply.

the
oth
the
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and
CoG
, in
and
tern
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HbR
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HbR

http://www.surgicalneurologyint.com/content/6/1/44

These findings also suggest that stronger stimulations
to the brain surface may likely induce increases in both
HbO2 and HbR not only in stimulation sites but also
in remote areas that make strong neuronal connections
to the stimulation sites. This implies that areas withrobust connections areas are prone to seizure spread,even if remote, and that their connectivity will bereflected in the NIRS data. For instance, in the present
study, hand motor cortex exhibited increases in both
HbO2 and HbR upon stimulation of the seizure onset
zone. Oxygen consumption exceeded the rate of oxygen
delivery, because the two areas had robust connections
that lay within one of the epileptic networks. However,in the trunk motor and premotor cortex, which wereconceivably not located within main epileptic networks
and weakly connected to the seizure onset zone, therate of oxygen consumption did not exceed the rate ofdelivery. Further studies are needed to clarify and extend
the implications and relevance of such hemodynamicchanges in resolving epileptic networks and more weakly
connected surrounding areas.
In this study, we employed intraoperative NIRS fromfour probes to demonstrate that abnormal connectivity
between regions of cortex characterizes epileptic
network activities. Our results highlight the capabilityof intraoperative NIRS to provide us with useful
information about the dynamics of cortico-corticalactivity at high resolution and without artefacts due
to scalp blood flow. This approach can thus expand on
previous studies using NIRS scalp recordings, such as
our description of hemodynamic connectivity between
the superior temporal and inferior frontal cortex in
the language system. The additional ability to makesimultaneous electrical recording would enhance the levelof information obtained through this approach. Recently,
a thin flexible probe, with a probe head of 5.6 × 10 mm
and the total thickness of 0.7 mm, was developed
for simultaneous recording of NIRS and ECoG,[9] byintegrating near-infrared light-emitting diodes andphotodiodes for NIRS measurement placed on the brain
surface and ECoG electrodes. In their initial experimental
study using this novel device, these researchers reported
hemodynamic changes associated with both focal brain
cooling and hyperventilation. If such small devices

were made widely available and applied in patients
undergoing neurosurgery, it could be used to further our
understanding of cortico-cortical projections and expand
on intraoperative NRIS studies.[8]CONCLUSIONSIn a patient with SMA seizures, we employedintraoperative NIRS to demonstrate hemodynamicchanges between the SMA, corresponding to the seizure
onset zone, and the lateral PMC, corresponding to theseizure propagation areas. This is the first report to reportthat NIRS can reveal cortico-cortical activities from thebrain surface intraoperatively. In the future, intraoperative
NIRS will be useful in monitoring cortico-corticalactivities such as in the language system and othercortical processes.

ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant Number
25462246. The authors thank Akihiko Suzuki, Motohiro Soma,
and Kiyoe Nonaka for their technical support.

REFERENCES
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Fukuda M, Masuda H, Honma J, Fujimoto A, Kameyama S,Tanaka R. Ictal SPECTin supplementary motor area seizures. Neurol Res 2006;28:845-8.
Hatanaka N, Tokuno H, Hamada I, Inase M, Ito Y, Imanishi M, et al.
Thalamocortical and intracortical connections of monkey cingulate motor
areas. J Comp Neurol 2003;462:121-38.
Hoshino T, Sakatani K, Katayama Y, Fujiwara N, Murata Y, Kobayashi K, et al.
Application of multichannel near-infrared spectroscopic topography tophysiological monitoring of the cortex during cortical mapping: Technicalcase report. Surg Neurol 2005;64:272-5.
Luppino G, Matelli M, Camarda R, Rizzolatti G. Corticocortical connectionsof area F3 (SMA-proper) and area F6 (pre-SMA) in the macaque monkey.
J Comp Neurol 1993;338:114-40.
Matsumoto R, Nair DR, LaPresto E, Bingaman W, Shibasaki H, Luders HO.
Functional connectivity in human cortical motor system: A cortico-corticalevoked potential study. Brain 2007;130:181-97.
Morris HH 3rd, Dinner DS, Luders H, Wyllie E, Kramer R. Supplementary
motor seizures: Clinical and electroencephalographic findings. Neurology
1988;38:1075-82.
Sato Y, Fukuda M, Oishi M, Shirasawa A, Fujii Y. Ictal near-infrared spectroscopy
and electrocorticography study of supplementary motor area seizures.
J Biomed Opt 2013;18:76022.
Sato Y, Oishi M, Fukuda M, Fujii Y. Hemodynamic and electrophysiologicalconnectivity in the language system: Simultaneous near-infrared spectroscopy
and electrocorticography recordings during cortical stimulation. Brain Lang2012;123:64-7.
Yamakawa T, Inoue T, He Y, Fujii M, Suzuki M, Niwayama M. Development of
an implantable flexible probe for simultaneous near‑infrared spectroscopy
and electrocorticography. IEEE Trans Biomed Eng 2014;61:388-95.

R EVIEW A RTICLE

doi: 10.2176/nmc.ra.2014-0433
Online July 28, 2015

Neurol Med Chir (Tokyo) 55, 611–616, 2015

Limitations of Neuroendoscopic Treatment for PediatricHydrocephalus and Considerations from
Future Perspectives
Kenichi NISHIYAMA,1 Junichi YOSHIMURA,1 and Yukihiko FUJII1
1

Brain Research Institute, University of Niigata, Niigata, Niigata

Abstract
Neuroendoscopy has become common in the field of pediatric neurosurgery. As an alternative procedure
to cerebrospinal fluid shunt, endoscopic third ventriculostomy has been the routine surgical treatment
for obstructive hydrocephalus. However, the indication is still debatable in infantile periods. The predictors of late failure and how to manage are still unknown. Recently, the remarkable results of endoscopic
choroid plexus coagulation in combination with third ventriculostomy, reported from experiences inAfrica, present puzzling complexity. The current data on the role of neuroendoscopic surgery for pediatric hydrocephalus is reported with discussion of its limitations and future perspectives, in this review.
Key words:

hydrocephalus, neuroeodoscopy, cerebrospinal fluid, pediatric, shunt

Introduction

ETV, late failure can occur and may lead to rapid
deterioration. The aim of this report is to review
the current data on the use of neuroendoscopy inchildren and to discuss the limitations and future
perspectives regarding this procedure.

The cerebrospinal fluid (CSF) shunt has long been
the classic treatment for pediatric hydrocephalus. It
can resolve nearly all forms of hydrocephalus, regardless of the etiology. It is a simple procedure that
can aid in having a relatively normal life. However,its failure rate is significant. The reported rate ofshunt malfunction in the first year of placement isapproximately 30%, and about 10% per year thereafter.
The risk of infection is between 5% and 10%. In
addition, the high rates of shunt complication suchas slit ventricle syndrome are unacceptable by current
standards. Children with shunts are dependent on
surgical maintenance throughout their lives.1–8) Therefore, the advent of neuroendoscopy was received
with enthusiasm. Endoscopic third ventriculostomy(ETV) has been a routine surgical practice for the
past two decades and provides an alternative to
the CSF shunt. It is a straightforward procedurefor diversion of the CSF and does not require
placing devices in children’s bodies. The utility
and safety of ETV have been proven for obstructive hydrocephalus that occur secondary to aqueductal stenosis. However, for other indications, itis necessary to examine the anatomy and etiology,
as well as the patient’s age. During follow-up after

Patient Selection and ETV in Infants
ETV has two main purposes: to restore CSF communication between the ventricle and subarachnoid
space and to reduce transmantle pulsatile stressby increasing compliance of the ventricular wall.9)
Since the 1990s, patients with aqueductal stenosis
have been considered ideal candidate for ETV.10,11)
ETV quietly developed into a common procedure,
without any prospective randomized trials comparing
its efficacy to that of the CSF shunt. In spite of strict
patient selection, the overall ETV success rate does
not exceed 80%.12–16) However, as neuroendoscopic
technology has evolved and pediatric neurosurgeons
have gained experience with the procedure, theindications for ETV have broadened. For example,
patients with hydrocephalus who had a previously failed shunt have become ETV candidates.
The success rate of ETV for shunt malfunction isnotable, around 80%.17–20) In recent years, the use of
ETV to treat many pathological conditions has been
debated. These include myelomeningocele, Chiari
type 1 malformation, Dandy-Walker malformation,and previous meningitis or hemorrhage.14,19,21,22)

Received December 11, 2014; Accepted January 22, 2015

611

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K. Nishiyama et al.

In infants, the benefits conferred by ETV may beconsiderable due to the high complication rate of
CSF shunts. However, there are two concerns withthe use of ETV in infants. The first is safety of theprocedure, including the possibility of damage to theventricular and cisternal structures during surgery,CSF leakage, or infection soon after surgery and late
closure of the stoma with rapid deterioration. The
second concern is that an infant successfully treatedwith ETV may be transformed from active hydrocephalus to an arrested type. These infants often
have larger ventricles than children treated with CSF
shunts. No study has attempted to correlate the larger
ventricle size with any measurements of psychomotor
development. According to reports from a multitudeof international studies, the shunt independence rate
ranges from 25% to 89%.23–28) Two-thirds of the reportssuggested that success rate is dependent on age ofthe infant at the time of ETV.29–34) To increase ETV
success rates, greater accuracy of appropriate patientswould be advised. However, because of the high rateof shunt failure and complication, ETV is sometimes
preferred as a first-line treatment. Further, the rangeof what is defined as failure in this age group is very
wide. A multicenter prospective randomized study
on infants up to 2 years of age with no flow at thelevel of the aqueduct, named the International Infant
Hydrocephalus Study (IIHS) is now under way. This
study focuses primarily on the neurodevelopmental
outcomes associated with different treatment paradigms
at 5 years, and includes a comprehensive assessmentof relevant risks and benefits.35)

Failed ETV and Its Management
ETV failure is a possible event. Although most failuresfrom ETV occur in the early period, within a few days
to 2 weeks following the procedure, late failure after
many months may lead to rapid deterioration andeven sudden death. A rapid increase in intracranialpressure caused by late obstruction of the stoma istypically regarded as the mechanism of failure.36–42)
Early failure is attributed to the incorrect surgical
technique or different criteria in the selection ofpatients. However, the predictors of late failure arestill unknown. Therefore, patients who have undergone
successful ETV should be followed on an ongoingbasis. Neurosurgeons should encourage patients andtheir parents to return as soon as possible if any
adverse symptoms develop, because these may havesevere consequences.43) Setting the CSF reservoir
concurrently with ETV is one option for the treatment of emergencies, even though it means implantation of a foreign material. In Japan, a follow-up
magnetic resonance imaging (MRI) examination

including sagittal T2-weighted images, cine-MRI,or constructive interference in steady state (CISS)is often scheduled to detect the CSF flow acrossthe stoma. However, there is currently no evidence
regarding whether the patients with no flow on MRI
following ETV may be at a greater risk to develop
clinical symptoms. For these patients, a repeat ETV
may be performed immediately rather than close
observation. In the literature, repeat ETV has a goodsuccess rate.32,44–49) Therefore, this is one option forpatients with a failed ETV, and it provides a meansof even avoiding the CSF shunt.

ETV vs. Aqueductoplasty
Endoscopic aqueductoplasty (EAP) is a means
to restore the physiological CSF dynamics. This
procedure provides an alternative to ETV, because
it avoids the risk of severe arterial bleeding. EAP
has been performed in cases with membranous orshort segmental occlusion of the sylvian aqueduct.
However, the long-term results of EAP have notbeen as successful as one would expect.50–54) EAP
has been shown to fail frequently. Schroeder et al.
reported a re-closure rate of 50%, and proposed that
one contributing factor to re-closure could be lower
aqueductal CSF flow through the stoma than that
following ETV.55,56) In addition, aqueductoplasty is
generally considered a riskier procedure due to thehigher risk of injuring midbrain structures. It may
lead to neurological deficits such as oculomotor or
trochlear nerve palsy, Parinaud’s syndrome, andperiaqueductal syndrome. Therefore, ETV, which
has higher long-term success rates and less risk,
would be a better alternative for membranous, shortsegmental, and even tumor-related occlusion of the
aqueduct.55,57) However, the condition of isolated fourth
ventricle (IFV) is an exception. Almost all patientswith IFV have a medical history of hydrocephalus
within first year of life, mostly post-infectious or
post-hemorrhagic. Further, following shunt placement, they often experience complicated overdrainage
with aqueductal stenosis. Aqueductoplasty could
be a means of establishing CSF communication
to the formerly isolated ventricular compartment;
however, a stent is mandatory to keep this pathway
open. EAP with a stent could be one choice in theendoscopic treatment of IFV.50,53,55,57,58)

ETV vs. CSF Shunt in Children
As described above, ETV has been widely applied forpediatric hydrocephalus as an alternative to the CSF
shunt mainly in an attempt to avoid foreign body
implantation and to better simulate physiological

Neurol Med Chir (Tokyo) 55, August, 2015

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Neuroendoscopy for Pediatric Hydrocephalus

CSF dynamics. However, the results of CSF shunt
have rarely been compared to ETV. According to thepediatric study by de Ribaupierre et al., the failure
rate of ETV was 26% and that of ventriculoperitoneal(VP) shunt was 42% at 5 years follow-up. In a reviewof the literature, the same trend was seen in otherstudies.59) Kulkarni et al. reported that the relativerisk of ETV failure is initially higher than that for
the CSF shunt. However, the risk became progressively lower at approximately 3 months following
the procedure, and was approximately half therisk of shunt failure at 2 years.60) The decrease inventricle size is usually smaller and happens more
slowly after ETV compared to shunt. Pediatricneurosurgeons are sometimes concerned about therelationship between these neuroimaging changes
and neuropsychological outcomes. Hirsch reviewedthat the postoperative intelligence quotient (IQ) was
not significantly different in 70 patients with shunts
vs. 44 who underwent percutaneous third ventriculostomy.61) Other reports also have found similar
results.62,63) Recently, Kulkarni et al. reported thatat 1 year following surgery, the quality of life and
IQ scores were not significantly different between
an ETV and a VP shunt group.64,65)

Challenging Procedure of ETV + CPC
There has been a resurgence in the technique ofchoroid plexus coagulation (CPC) in combination
with ETV ever since this procedure was performed
for African children with hydrocephalus of various
etiologies in 2005.66–73) Warf and colleagues highlighted that hydrocephalus with shunt dependency
is inadvisable in developing countries because oflimited access to medical centers in the event ofshunt malfunction. In 2005, Warf and colleagues
reported the results of a combined ETV + CPC trial
performed mainly in infant with post-infectioushydrocephalus and in those with myelomeningocele. A
total of 266 patients underwent ETV + CPC, whereas
ETV alone was performed in 284 patients. The
results demonstrated that the ETV + CPC increased
the success rate in infants from 47% to 66%.66) In
2008, they reported the long-term results of ETV +CPC in hydrocephalus with myelomeningocele. The
intention-to-treat analysis showed a shunt independence rate of 76% in 338 infants, which was
higher than the results of ETV alone reported in theliterature.22,32,67) Further analyses regarding ETV +CPC performed in patients with encephalocele,
Dandy-Walker complex, and congenital aqueductal
stenosis have been conducted using their database.70–72)
The success rates achieved were between 70% and80%. However, all pediatric neurosurgeons should613

be cautious about the meaning of “success.” In thesereports, success usually referred to controlling raised
intracranial pressure and avoiding an extra-cranial CSF
shunt. Cognitive function was not the main outcome
index. In addition, all these results have been reported
from Africa. In the coming years, the challenge will
be to see whether these results from Africa can beextrapolated to developed countries.74) The main
advantage of ETV + CPC was avoidance of a shuntin patients who were difficult to follow-up. In Africa,
geographic and socioeconomic constraints reasonsfor poor follow-up. The low possibility of cognitive
salvage or the high rate of shunt complications, suchas holoprosencephaly or hydroanencephaly, may befurther reasons in developed nations. Technically,
Warf and colleagues used a steerable endoscope via
a single burr hole, sometimes in combination withseptostomy, and coagulated the bilateral choroid
plexus from the foramen of Monro to the entranceof the temporal horn.66,73) This procedure appeared tobe safe in some cases of severely dilated ventricles.
For Japanese neuroendoscopic surgeons, the flexible endoscope is more familiar than it is in other
European nations. Nevertheless, almost all pediatricneurosurgeons still believe that the use of ETV + CPC
in pediatric hydrocephalus needs further evaluation.

Conclusion
It is difficult to determine the best strategy for CSF
diversion in pediatric hydrocephalus. ETV is oneof the alternative procedures to the CSF shunt inconditions of obstructive hydrocephalus such as
aqueductal stenosis. However, the preferred indications in infants have not been fully determined. The
mechanisms and predictors of late ETV failure thatmight cause rapid deterioration are not still known.
Recently, other clinical dilemmas, such as the useof ETV + CPC have arisen. Definition of the optimal
indications, perfection of endoscopic techniques,developing strategies for follow-up, and considering socioeconomic constraints are required frompediatric neurosurgeons when selecting a surgicalstrategy in pediatric hydrocephalus.

Conflicts of Interest Disclosure
The authors declare that they have no conflicts ofinterest.

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Address reprint requests to: Kenichi Nishiyama, MD, PhD,
Brain Research Institute, University of Niigata, 1-757
Asahimachi, Chuo-ku, Niigata, Niigata 951-8585,
Japan.
e-mail: nishiken@d4.dion.ne.jp

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Multiple therapeutic effects of progranulin on
experimental acute ischaemic stroke

1 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
2 Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
3 Department of Centre for Integrated Human Brain Science, Brain Research Institute, Niigata University, Niigata, Japan

Received September 19, 2014. Revised December 27, 2014. Accepted January 27, 2015. Advance Access publication April 2, 2015
 The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com－ 216 －

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thrombo-embolic model with delayed tissue plasminogen activator treatment. Intravenously administered recombinant progranulin
reduced cerebral infarct and oedema, suppressed haemorrhagic transformation, and improved motor outcomes (P = 0.007, 0.038,
0.007 and 0.004, respectively). In conclusion, progranulin may be a novel therapeutic target that provides vascular protection, antineuroinﬂammation, and neuroprotection related in part to vascular endothelial growth factor, interleukin 10, and TAR DNAbinding protein-43, respectively.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948| 1933

Correspondence to: Dr Takayoshi Shimohata,
Department of Neurology,
Brain Research Institute,
Niigata University,
1-757 Asahimachi-dori,
Chuoku, Niigata,
Niigata 951-8585,
JapanE-mail: t-shimo@bri.niigata-u.ac.jp

Keywords: progranulin; cerebral ischaemia; VEGF; IL10; TDP-43
Abbreviations: FTLD-TDP = TAR DNA binding protein positive frontotemporal lobar degeneration; OGD = oxygen glucose deprivation; TDP-43 = TAR DNA-binding protein 43; TGF-b = transforming growth factor beta; TNF- = tumor necrosis factor alpha;tPA = tissue plasminogen activator

Introduction－ 217 －

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Progranulin (PGRN) is a secreted N-linked glycoprotein
growth factor implicated in tissue regeneration, wound repair, and inﬂammation (He et al., 2003a). Several in vitro
studies have revealed that PGRN is classiﬁed into two isoforms according to its glycosylation status, the glycosylated
immature isoform (58–68 kDa) and the fully glycosylated
mature secretory isoform (88 kDa) (Shankaran et al.,2008; Naphade et al., 2010; Alquézar et al., 2014).
In the CNS, PGRN is considered to play a crucial role inmaintaining physiological functions and in the survival ofneurons for the following reasons: ﬁrst, primary corticalneurons derived from Pgrn knockout mice showed reduced
neuronal survival (Kleinberger et al., 2010); second, PGRN
has been shown to exhibit neurotrophic activity in vitro(Van Damme et al., 2008) and in vivo (Laird et al.,2010); and third, mutations in the Pgrn gene caused ubiquitin- and TAR DNA binding protein-positive frontotemporal lobar degeneration (FTLD-TDP), a progressive
neurodegenerative disorder with an autosomal dominant
inheritance pattern (Baker et al., 2006; Cruts et al., 2006).
Several studies raise the possibility that PGRN might beinvolved in ischaemic brain injury. First, PGRN might playa vascular protective role against focal cerebral ischaemia
via suppression of blood–brain barrier disruption. Recent
studies have demonstrated that intraventricular administration of recombinant PGRN could suppress cerebral oedemain a mouse transient focal cerebral ischaemia (suture
model) (Egashira et al., 2013), and that Pgrn knockout
mice may prompt post-ischaemic blood–brain barrier disruption (Jackman et al., 2013).
Second, PGRN has been shown to be involved in neuroinﬂammation after ischaemic brain injury. Previous studies have demonstrated that PGRN was induced in
activated microglia after spinal cord injury (Naphadeet al., 2010) and traumatic brain injury (Tanaka et al.,2013), indicating that the induction of PGRN reﬂects
microglial responses. Similarly, in ischaemic brain injury,
microglia have been shown to mediate expansion of theinfarct via inﬂammation in the acute phase (Mabuchi
et al., 2000; Yenari et al., 2010), while it might also contribute to endogenous anti-inﬂammatory protection in thesubacute phase (Faustino et al., 2011). In this context,
recent studies suggested that PGRN might suppress secretion of pro-inﬂammatory cytokines and recruitment of neutrophils associated with focal cerebral ischaemia (Tao et al.,2012; Egashira et al., 2013). However, a recent study
showed that PGRN was not involved in neuroinﬂammation
related to acute focal cerebral ischaemia (Jackman et al.,2013). Therefore, the role of PGRN in neuroinﬂammation
after ischaemic brain injury is controversial.
Third, PGRN might exhibit protective effects on neuronalcells after acute focal cerebral ischaemia based on analyses
using transgenic mice expressing PGRN (Tao et al., 2012)
and Pgrn knockout mice (Jackman et al., 2013), although
the mechanism of neuroprotection remains unknown. We
previously demonstrated that ischaemic neuronal injury
might be caused in part by cleavage and cytoplasmic redistribution of TARDBP (previously known as TDP-43), a key
protein in FTLD-TDP and amyotrophic lateral sclerosis(Kanazawa et al., 2011b). Because PGRN can suppress
the cleavage of TARDBP via inhibiting caspase 3 (Zhang
et al., 2007; Kanazawa et al., 2011b), it raises the possibility that PGRN may prevent ischaemic neuronal injury
via preservation of TARDBP functions.
Based on the abovementioned ﬁndings, we speculated
that PGRN could exert a positive therapeutic effect via
regulation of blood–brain barrier function, suppression of
inﬂammation, and neuroprotection against acute focal cerebral ischaemia. Here, we investigated temporal changes in
PGRN after focal cerebral ischaemia, including its cellular
expression, glycosylation status, and sources of secretion.
We also examined the role of PGRN in neuroinﬂammation
after ischaemic brain injury and the mechanisms of neurovascular protective effects of PGRN. Moreover, we used a
rat autologous thrombo-embolic model (Okubo et al.,2007), which shows cerebral oedema and haemorrhagictransformation when tissue plasminogen activator (tPA) isadministered beyond the therapeutic time window

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Materials and methods

Focal cerebral ischaemia

The sample size was calculated before performing the experiments. We calculated the sample size needed to detect the difference in the amount of cerebral haemorrhage or cerebral
oedema volume between the tPA with the recombinant PGRN
group and tPA with the control group with 80% power( = 0.05; one-sided analysis). Mice and rats were randomly
assigned to various experimental groups, and analyses were performed by an investigator blinded to genotype and treatment.

Immunoblotting
The rats that survived for 10 min, 4 h, 10 h, 18 h, 24 h, and
72 h after reperfusion were euthanized with an overdose of
halothane, followed by intracardiac perfusion with cold
normal saline. We deﬁned ischaemic penumbra as the region
that was rescued by reperfusion, and the ischaemic core as thegenelinkedfrontotemporaldegenerationubiquitinregion
that towas
not rescued lobar
by reperfusionas withdescribed
previimmunoreactive
inclusions
progranulin
production
and secreously (Memezawa
et al.,reduce
1992;
Kanazawa
et al., 2011b).
The
tion.
J BiolChemcorresponding
2008; 283: 1744–53.
corticaltissuesto the ischaemic core or penumShimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D,
bra were dissected for immunoblotting as described
Steinberg GK. Suppression of deltaPKC activation after focal cere(Kanazawa et al., 2011b). Cortical tissues from the shambral ischemia contributes to the protective effect of hypothermia. J
operatedrats
were
also2007;
dissected
as controls. For whole-cell
Cereb
Blood
Flow
Metab
27: 1463–75.
extracts,
proteinsfrombraintissues
extracted J. as
Songsrirote K, Li Z, Ashford D, Bateman A,wereThomas-Oates
described previously(Shimohataal., 2007). methods
The proteins
Development
and applicationof massetspectrometricfor(50 mg)wereofsubjected
to tris-glycine
sodiumdodecyl sulphatetheanalysisprogranulin
N-glycosylation.
J Proteomics
2010;
73:
1479–90.
polyacrylamide
gel electrophoresis (SDS-PAGE).
Swanson
RA, whole-cell
Morton MT,
Tsao-Wu
G, Savalos
C, inFor theextracts
in vitro,
cells RA,
wereDavidson
harvested
Sharp
FR.lysis
A semiautomatedmethod formeasuring braininfarct
cold cell
buffer (Cell Signaling
Technology).
Conditioned
volume.
J Cereb
Blood Flow
1990; 10:(30
290–3.
media werecollected
and Metab
concentrated) using Amicon
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
Ultra-0.5 tubes (Millipore). Proteins (5 mg) in the samples were
et al. Neuritic beading induced by activated microglia is an early
separated by tris-glycine SDS-PAGE and were probed withfeature of neuronal dysfunction toward neuronal death by inhibition
primary
antibodies(Supplementary1), followedby aof
mitochondrial
respiration
and axonalTabletransport.
J Biol Chem
secondaryhorseradish
peroxidase-conjugatedantibody
2005;
280: 10444–54.
(1:10 Y,000).
Signals T,
were
detected K,
by Nishihara
an enhanced
chemiluminTanaka
Matsuwaki
Yamanouchi
M. Exacerbatedescence (Millipore)
andrelated
semiquantiﬁedby microglia
densitometry= 5).
inﬂammatory
responses
to activatedafter (ntraumatic
brain injury
in stripped
progranulin-deﬁcient
Neuroscience
The membranes
wereand probed mice.
with ananti-b-actin
2013;
8–19.
or an250:
anti-transferrin
antibody to conﬁrm even loading of
TaoJ, Ji F, Wang F, Liu B, Zhu Y. Neuroprotective effects of proproteins.
granulin in ischemic mice. Brain Res 2012; 1436: 130–6.
Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, et al. Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance neuronal survival. J Cell
Biol 2008; 181: 37–41.
Yenari MA, Kauppinen TM, Swanson RA. Microglial activation in
Rats and mice euthanized with an overdose inhalation of halostroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
or intraperitoneal
Yinthane
F, Banerjee
R, Thomas injection
B, Zhou ofP, xylazine
Qian L, chloral
Jia T, hydrate
et al.
were sacriﬁcedby transcardialwithsalineExaggerated
inﬂammation,
impaired perfusion
host defense,and normal
neuropatholfollowedby perfusion withogy
in progranulin-deﬁcientmice. cold
J Exp 4%
Med paraformaldehyde
2010; 207: 117–28. in
0.1 M A,phosphate-bufferedsaline(PBS;T,pH
7.4) ath ofandYoshidaYoshida S, Khalil AK,
Ishibashi
Inomata
H. 24
Role
NF-kappaB-mediatedinterleukin-8
expression
in intraocular
neovas- as
72 h after reperfusion.
We stained
free-ﬂoating
sections
cularization.
Invest Ophthalmol
Vis Sci
39: 1097–106.
described previously(Kanazawa
et 1998;
al., 2011b)
and mounted
Zausinger
S, Hungerhuber
Baethmann
A, ReulenInformation
H, Schmid- ofwith Vectashield
DAPI E,(VectorLaboratories).
Elsaesser R. Neurological impairment in rats after transient middle
the primary antibodies is described in Supplementary Table 1.
cerebral artery occlusion: a comparative study under various treatThe sections were examined under a confocal laser-scanningment paradigms. Brain Res 2000; 863: 94–105.
microscope(LSM510
Zeiss). P,The
Zhang
YJ, Xu YF,
Cook C, META;
Gendron CarlTF, Roettges
Linkresults
CD, et wereal.
conﬁrmedin threeindependent
samples.
Aberrantcleavageof TDP-43
enhances
aggregation and cellular toxicity. Proc Natl Acad Sci USA 2009; 106: 7607–12.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
Progranulin mediates caspase-dependent cleavage of TAR DNA
binding
J Neurosci
2007;mice
27: 10530–4.
Deeply protein-43.
anaesthetizedrats andwere perfused intracarZhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D,
dially with cold 4% paraformaldehyde at 24 h and 72 h after
Zhang R, et al. Correlation of VEGF and angiopoietin expression
reperfusion. The brains were removed and embedded inwith disruption of blood-brain barrier and angiogenesis after focal
parafﬁnischemia.
wax. Serial
sections(4-mmMetabthick)2002;
were22:cut
from thecerebral
J Cereb
Blood Flow
379–92.

Immunofluorescence staining andconfocal microscopy

Immunohistochemistryparafﬁn blocks and stained using antibodies (Supplementary
Table 1). The immunoreactive products were visualized using
the ABC Vectastain kit (Vector Laboratories) and diaminobenzidine as the chromogen. The results were conﬁrmed in three
independent samples.
To measure immunoreactivity, we used ImageJ software
1.37r (National Institutes of Health, Bethesda, MD, USA) as
described elsewhere (Krishnamurthy et al., 2009). Brieﬂy,
images of the stained sections were visualized, examinedblindly, and seven randomly chosen non-overlapping highpower ﬁelds ( 400) covering the ischaemic cortex were analysed for both wild-type and Pgrn knockout mice (n = 21). For
each image ﬁeld, the total number of pixels was quantiﬁed ona grey scale (0–255). The levels of immunoreactivities wereexpressed as the average density (mean pixel value in animage ﬁeld  SEM) for each experimental group.

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Kleinberger
G, Wils
H, Ponsaerts
Joris G, Timmermans
JP, Van(Kanazawa
et al.,
2011a; P,
Kawamura
et al., 2014)
andBroeckhoven C, et al. Increased caspase activation and decreased
demonstrated for the ﬁrst time that intravenous administraTDP-43 solubility in progranulin knockout cortical cultures. Jtion of recombinant
PGRN showed therapeutic effects on
Neurochem
2010; 115: 735–47.
the volumesRG,
of cerebral
infarct
andD,oedema,
Krishnamurthy
Senut MC,Zemke
Min J, haemorrhagic
Frenkel MB,
Greenberg
EJ, et al.
acid, a pentacyclic triterpene fromtransformation,andAsiatic
prognosis.
Centella asiatica, is neuroprotective in a mouse model of focal
cerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
Bosch L, Van Damme P, et al. Progranulin is neurotrophic in vivo
and protects against a mutant TDP-43 induced axonopathy. PLoS
This2010;
study5:was
carried out in strict accordance with the recOne
e13368.
ommendations
from
Guide forthe Careand UseLiau
LM, Lallone RL,
Seitz theRS, Buznikov
A, Gregg
JP, Kornblum
HI, ofLaboratory
Animals
the National
Institutes of
Health.
The
etal. Identiﬁcationof ofa humanglioma-associatedgrowthfactorgene,
granulin,differential byimmuno-absorption.
Res
protocolwasusingapproved
the Niigata Cancer
University
2000;
60: 1353–60.
Administrative
Panel on Laboratory Animal Care. All surgeries
MabuchiT, Kitagawa
K, inhalation
Ohtsuki T,
K, Yagita
Y,
were performed
underandKuwabara
intraperitoneal
anaestheYanagihara
T, et al. Contribution
microglia/macrophages
to exsia and according
to the ofARRIVE(Animal Research:pansion of infarction and response of oligodendrocytes after focal
Reporting of In Vivo Experiments) guidelines (Kilkenny
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
et al., 2010).
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged byreperfusion following middle cerebral artery occlusion in rats. Stroke
1992; 23: 552–9.
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
Responses
endothelial
cell and astrocyte
Transient of
acutefocal cerebral
ischaemiamatrix-integrin
was inducedreceptors
using thetoischemia mimic
those observed
the neurovascularrats
unit.weighing
Strokeintraluminalﬁlament
in male inSprague-Dawley
2008;
39: g191–7.
300–370
and wild-type (C57BL/6) and Pgrn knockout mice
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,(C57BL/6 background) weighing 22–28 g (Kanazawa et al.,
Kuret J. Progranulin expression is upregulated after spinal contusion
2011b).
Brieﬂy, anaesthesia was administered via inhalation of
in mice. Acta Neuropathol 2010; 119: 123–33.
1.5% S,halothane
mixture of T,
70%
nitrous oxideand 30%
Okubo
Igarashi in
H, a Kanamatsu
Hasegawa
D, OrimaH,
oxygen for
andextended
with intraperitoneal
10 mg/kg
Katayama
Y. rats
FK-506the therapeutic injection
time windowforxylazine and
300 mg/kg
chloralhydratefor mice.
A nylonthrombolysis
withoutincreasing
the riskof hemorrhagictransformmonoﬁlament,witha diameterof 0.148
mm 1143:
for rats
andation
in an embolic
rat strokemodel. Brain
Res 2007;
221–7.
Osada
T,mm
Gu YH,
Tsubota
HawkinsThe
BT, tip
SpatzofM,the0.074
for Kanazawa
mice, wasM,used
for Y,
suturing.
etal. Interendothelial
claudin-5
expression
depends
on cerebral
nylon
monoﬁlament was
roundedwith heat.
An 11-mm
distalendothelial
cell-matrix
adhesionby 0.350b(1)-integrins.
J Cereb
Bloodforsuture segment
was coatedwithmm diametersiliconFlow Metab 2011; 31: 1972–85.
rats, and a 4-mm distal suture segment was coated withÖzdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
0.185 mm diameter silicon for mice. Middle cerebral artery ocby an intraluminal ﬁlament may cause disturbances in the hippoclusionblood
was performed
insertionof ofthe ofsutureexternal
campalﬂow due toby
anomalies
circleWillisvia
andanﬁlament
carotid arterythe internal
carotid artery. After 90 min of
thickness.
Brain into
Res 1999;
822: 260–4.
ischaemia, thewithdrawn
restoreﬂow.
Tangkeangsirisin
W,suture
Serrero was
G. PC-cellderived to
growthfactorblood(PCDGF/
Body coretemperaturemonitored
withanda rectalprobe
was mainGP88)
stimulates
migration,
invasiveness
VEGF
expressioninbreastcells.Carcinogenesis
25: 1587–92.
a heatingpad. To exclude thetainedcancer
at 37.0
0.5 C, using 2004;
Toh
H, CaoM, Daniels ofE, circle
BatemanExpression
the growtheffectsof anomaliesof A.
Willison theofischaemic
size
factorprogranulin
in endothelial
cells
inﬂuences
growth and patterns
develand oedema
in Pgrnknockout
mice,
we investigatedopmentblood
vessels:
a novelmouse model.
PLoS knockout
One 2013; mice8:of circleof ofWillisin bothwild-typeand Pgrn
e64989.
using transcardial perfusion with carbon black suspended in
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
saline (n 5 7) (Supplementary Fig. 1; Özdemir et al., 1999).
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948

Measurement of the volume of thecerebral infarct and oedema
At 24 h after reperfusion, the rats and mice were given highly
concentrated halothane and deeply anaesthetized. After transcardial perfusion with saline, the brains were extracted. The brains
were cut into 3-mm slices and stained with 2% 2, 3, 5-triphenyltetrazolium chloride solution (#264310; BD, Franklin Lakes).
After staining, the slices were photographed with a scanner(CanoScan LiDE 50; Canon Inc), and cerebral infarct volume
and cerebral oedema volume were measured with ImageJ software 1.37r (National Institutes of Health, Bethesda, MD, USA)according to the method described by Swanson et al. (1990).
These values were expressed as a proportion of the cerebral
hemisphere occupied. The analyses were performed by an investigator blinded to the genotype and treatment.

Neurological evaluations

Pgrn knockout mice
Pgrn knockout mice were produced by breeding heterozygous
pairs in the Graduate School of Agricultural and Life Sciences,at the University of Tokyo, and genotyped using previously
described protocols (Kayasuga et al., 2007). Additionally,
Pgrn knockout mice purchased from RIKEN BioResource
Centre (Tsukuba, Japan) were bred and maintained in the
Department of Comparative and Experimental Medicine,
Brain Research Institute, Niigata University. We used only
homozygous pairs to obtain Pgrn knockout pups. The mice
were maintained under controlled light (lights on, 05:00–
19:00), temperature (23  1 C), and humidity (55  10%)and given free access to food and water.

C6 cell culture and analysis ofglycosylation
Rat C6 cells were grown in Opti-MEM (Gibco/Invitrogen)with 10% foetal bovine serum (FBS) and 1% penicillinstreptomycin. Cells were grown in Opti-MEM without FBS
24 h before the experiments. Cells were harvested in cell lysis
buffer (Cell Signaling Technology) containing 1% TritonTM X100 and inhibitors for proteases and phosphatases (SigmaAldrich). After centrifugation at 14 000 rpm for 5 min (4 C),the supernatants were collected. Additionally, conditioned
media were also obtained for immunoblotting. To analyse glycosylation of PGRN, cell lysates and media were digested at
37 C with peptide-N-glycosidase F (PNGase F; New England
Biolabs) according to manufacturer’s instructions, in triplicate.

Primary cell cultures
Primary murine neuronal cells were prepared from the neocortices of embryonic Day 17 C57Bl/6 mouse embryos (Takeuchi
et al., 2005). Brieﬂy, cortical fragments were dissociated into
single cells in a dissociation solution (Sumitomo Bakelite) andresuspended in nerve culture medium (Sumitomo Bakelite).
Neurons were plated onto poly-D-lysine-coated (P7405,
Sigma-Aldrich) six-well dishes (10  104 cells per well). The
purity of these neuronal cultures was 95% as determined by
NeuN-speciﬁc immunostaining (Takeuchi et al., 2005).
Primary murine microglia and astrocytes were obtained aspreviously described (Milner et al., 2008). Primary mixed glial
cultures were established from the forebrains of postnatal
C57Bl/6 mice by dissociating isolated cerebral cortices inpapain and then growing the resulting cell suspension for 10
days in Dulbecco’s modiﬁed Eagle medium (DMEM) supplemented with 10% FBS. After 10 days, the ﬂasks were shaken
for 15 min to remove the loosely attached microglia. The purityof these microglial cultures was 99% as determined by Mac-1
immunoreactivity in ﬂow cytometry (Milner et al., 2008). For
astrocytes, the ﬂasks were then shaken overnight to remove themicroglia and oligodendrocyte precursors. The remaining monolayer was determined as 495% astrocytes by glial ﬁbrillary
acidic protein (GFAP) immunoreactivity (Milner et al., 2008).
To investigate the effect of microglia and astrocytes on secretion of vascular endothelial growth factor (VEGF) fromastrocyte after oxygen glucose deprivation (OGD), we cultured
astrocytes with microglial conditioned media. Brieﬂy, after we
performed OGD using primary murine microglia, we collected
the microglial low-glucose conditioned media. Then, we
removed the astrocytic high-glucose conditioned media andwashed thoroughly with PBS. We ﬁnally exchanged the PBS
for the collected microglial low-glucose conditioned media.
After overnight incubation under OGD condition, we measured the level of VEGF in the conditioned media using
VEGF Quantikine ELISA Kit (RRV00, R&D Systems), according to the manufacturer’s instructions (n = 6–7). IL10
levels in the conditioned media from microglia were also determined by a sandwich ELISA using the IL10 Quantikine
ELISA Kit (M1000B, R&D Systems), according to the manufacturer’s instructions (n = 5–6).

Oxygen–glucose deprivation
The standardized conditions for OGD have been described indetail elsewhere (Milner et al., 2008). Brieﬂy, before inductionof OGD, serum-containing media was removed from the cell
cultures by washing twice thoroughly with PBS before adding
serum-free high glucose medium (4.5 mg/l, DMEM containing
4 mM L-glutamine, penicillin, and streptomycin, supplemented
with N1 medium) or low-glucose medium (1.0 mg/l, supplemented with DMEM). The cultures containing low-glucose
medium were placed in a hypoxia chamber (BillupsRothenburg), which was ﬂushed with a mixture of 95% N2
and 5% CO2 for 1 h, and then closed for 18 h or 24 h. Oxygen
levels decreased to 0.1–0.4% at 4 h and was maintained
throughout the experiment (del Zoppo et al., 2012).
To determine the effects of PGRN under OGD, we added
full-length recombinant mouse PGRN produced from HEK293
cells (AG-40A-0080, AdipoGen) into conditioned media. The
recombinant PGRN is N-glycosylated, as determined by a

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Neurological evaluations were conducted at 24 h after reperfusion with a 6-point neurological scale (Zausinger et al., 2000).
Speciﬁcally, grade 5 indicated no neurological ﬁndings, grade 4
indicated an inability to move forward with the foot on theaffected side, grade 3 indicated weak resistance to a force that
was applied from the side on a level surface, grade 2 indicatedturning to the affected side when pulled from behind on a levelsurface, grade 1 indicated spontaneously turning to theaffected side, and grade 0 indicated an inability to move
spontaneously or death.

| 1935

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Cell counting protocols

Reverse transcription polymerase
chain reaction

CGAA -3 ), TGF-b (forward, 5 - TGGAGCAACATGTGGAACTC -30 ; reverse, 50 - CAGCAGCCGGTTACCAAG -30 ),IL-10 (forward, 50 - CTAACCGACTCCTTAATGCAG -30 ;reverse, 50 - TTAAAATCACTCTTCACCT -30 ), and actin(forward, 50 - CATCCGTAAAGACCTCTATGCCAAC -30 ; reverse, 50 - ATGGAGCCACCGATCCACA -30 ). The PCR reactions were carried out as follows: 35 cycles for ampliﬁcation at
98� C for 10 s, 55� C for 5 s, and 72� C for 5 s. The genes ofinterest were normalized to actin (n = 5–7).

Quantitative real-time PCR
Primary microglia were collected as pellets after centrifugation
at 13 000 rpm for 10 min. Total RNA was isolated from samples using Nucleospin RNA XS (U0902A, Takara Bio), andﬁrst-strand cDNA was synthesized from total RNA bygenelinked Ace
to frontotemporaldegeneration
with ubiquitinReverTra(TOYOBO). lobar
Quantitative
RT-PCR
was perimmunoreactive
reduce progranulin
production
secrePremix ExTaq
on anda TP-850
formed with inclusions
SYBR Greention.
J Biol Chem
283: 1744–53.
Real-TimePCR 2008;
Detectionsystem (Takara Bio). We designed
Shimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D, 0
primer pairs for IL10 as follows: (forward, 5 Steinberg GK. Suppression of deltaPKC activation
after focal cere- 0
reverse,5ATTTGAATTCCCTGGGTGAGAAG-30 ;bral ischemia contributes to the protective effectof hypothermia. J
0).
PCR
conditions
CACAGGGGAGAAATCGATGACA-3
Cereb Blood Flow Metab
2007;
27:
1463–75.
�
for initialdenaturing,followed by J.40
were 30 sK, atLi 95
Songsrirote
Z, CAshford
D, Bateman
A, Thomas-Oates��C and 30
at 60
C. Expression
levelscycles of 5 sandat 95
Development
applicationof smass
spectrometricmethods
for ofthe analysis
target gene
relative to N-glycosylation.
housekeeping enzyme(TakaraBio)
theof progranulin
J Proteomics
2010;
73:
1479–90.
weredetermined using the CT method (n = 3–4).
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring braininfarct
volume. J Cereb Blood Flow Metab 1990; 10: 290–3.
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
et al. Neuritic beading induced by activated microglia is an earlyfeatureof neuronal
dysfunction
towardneuronal
death
inhibition
Cerebralischaemia
was createdusing
a modelof byfocal
embolicofmitochondrial
respiration
and axonaltransport.
Biol Chemischaemia,
in whichthe animals
had their
middle Jcerebral
artery2005;
280: 10444–54.
occludedby autologous thrombi (Okubo et al., 2007;
Tanaka Y, Matsuwaki T, Yamanouchi K, Nishihara M. Exacerbated
Kanazawa et al., 2011a). tPA was administered intravenouslyinﬂammatory responses related to activated microglia after trauin the brain
form injury
of alteplase(Mitsubishi Tanabe
PharmaCo.) at a
matic
in progranulin-deﬁcientmice.
Neuroscience
dose ofmg/kg per animal at 4 h after cerebral ischaemia,
2013;
250:108–19.
becausepreviouslythat this condition
TaoJ, Ji F,we
Wang
F, Liu B,demonstrated
Zhu Y. Neuroprotective
effects of caused
procerebral inoedema,haemorrhagictransformation,
granulinischemic mice.
Brain Res 2012;
1436: 130–6.and severe
Van
Damme P,(Kanazawa
Van HoeckeetA,al.,Lambrechts
Vanacker P,
E,prognosis
2011a; D,
Kawamura
et Bogaert
al., 2014).
vanJ, et al. Progranulinfunctionsas blooda neurotrophic
factor
We Swieten
demonstrated
that cerebral
corticalﬂow decreased
toneuritebaseline
outgrowthandischaemia
enhance neuronal
J Cell4 h
byregulate
40% from
after
and tPAsurvival.
treatmentBiol
181: 37–41.
after2008;
ischaemia
improved it by 470% from baseline in thisYenari MA, Kauppinen TM, Swanson RA. Microglial activation inthrombo-emobolic model (Kanazawa et al., 2011a).
stroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
Immediately before tPA administration, 100 mg of FLAGYin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
tagged recombinant
PGRN
or control
protein
R5G10Exaggerated
inﬂammation,
impaired
host defense,and (IgG;
neuropathol048,inOEM
Concepts) wasmice.
administereda bolusthrough aogy
progranulin-deﬁcient
J Exp Med as
2010;
207: 117–28.
catheterthe inguinalvein(n Ishibashi= 6–7). The
volumesH.ofRole
the cereYoshida
A, inYoshidaS, KhalilAK,T, Inomata
ofbral infarct and oedema,
cerebralhaemorrhage,neurological
NF-kappaB-mediatedinterleukin-8
expression
in intraocular
neovascularization.
Ophthalmol
ViswereSci 1998;
39: 1097–106.
evaluations,Invest
and mortality
ratio
examined.
Cerebral infarct
Zausinger
S, Hungerhuber
Baethmann A,
Reulen H, Schmidand oedema
and cerebralE,haemorrhage
measurements
were perElsaesser
R. aNeurologicalin rats
after transient middle
formed in
randomizedimpairment
and blindedmanner.
cerebral artery occlusion: a comparative study under various treatment paradigms. Brain Res 2000; 863: 94–105.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxAll data
wereAcadpresented
mean106:� SEM.
Differences in theicity.
Proc NatlSci USAas2009;
7607–12.
parameters
wereanalysedusing
one-wayor R,two-way
Zhang
YJ, Xu YF,
DickeyCA, Buratti
E, Baralle
F, Bailey
et al.
Progranulin
mediates by
caspase-dependentcleavageof TARDNA
ANOVA, followed
Bonferroni’s post
hoc test
or unpaired
binding
protein-43.analyses
J Neurosci
2007;
27: 10530–4.
t-test. Statistical
wereperformed
using SPSS (ver.21;
Zhang
L, Tsang
W, Soltanian-Zadeh
Morris withD,
SPSS ZG,
Inc.).Zhang
Differencesin thefrequencies wereH,assessedZhang
et al. Correlationof VEGF and angiopoietin
Fisher’sR, exact
test or Mann-Whitney’s
U-test. Allexpression
tests werewith disruption of blood-brain barrier and angiogenesis after focal
considered statistically signiﬁcant at a P-value 5 0.05.
cerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

Thrombolytic treatment with tissue
plasminogen activator and PGRN

Statistical analyses

Results
Post-ischaemic changes of PGRN
expression in neuronal cells,
microglia and endothelial cells
in rat cerebral cortex
To investigate the expression and localization of PGRN in
the non-ischaemic cortex of Sprague-Dawley rats, we performed immunoﬂuorescence staining using an anti-PGRN
antibody. At ﬁrst we tested speciﬁcity of the anti-PGRN
antibody by immunoblotting and immunohistochemistry.

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Kleinberger
G,assay
Wils H,
Ponsaerts P, Joris
G, Timmermans
Van
PNGase F(manufacturer’s
datasheet).
Because JP,
a FLAGBroeckhoven
C, et toal. theIncreased
caspaseofactivationand decreased
tag was attachedC-terminus
the recombinant
PGRN
TDP-43in ofprogranulin
cortical
cultures.
J
protein, solubility
localization
exogenousknockout
recombinant
PGRN
proteinNeurochem 2010; 115: 735–47.
was determined by immunohistochemical staining with an
Krishnamurthy RG, Senut MC, Zemke D, Min J, Frenkel MB,
anti-FLAG antibody (Supplementary Table 1).
Greenberg EJ, et al. Asiatic acid, a pentacyclic triterpene fromThe survival
in culture exposed
to OGD
deterCentella
asiatica, ofis cells
neuroprotective
in a mousemodel wasof focalminedbylactatedehydrogenase(LDH)
releaseintotheculturecerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
supernatant
using aA, kit
LairdAS, Van Hoecke
De assay
Muynckin L,six-well
Timmersformat
M, Van(RochedenDiagnostics),
accordingto Progranulin
the manufacturer’s
instructions.
Bosch
L, Van Damme
P, et al.
is neurotrophic
in vivoanda mutant
inducedwere
axonopathy.
PLoSbyThe protects
relativeagainst
assessmentsof TDP-43
cytotoxicity
normalizedOne
2010; 5: with
e13368.
comparison
100% cell damage (n = 5). The survival of
LiauLM, Lallone
Seitzculture
RS, Buznikov
A,to
Gregg
JP,wasKornblumHI,neuronal
cells RL,in theexposedOGD
determinedetal.
Identiﬁcation
of
a
human
glioma-associatedgrowthfactorusing the propidium iodide method in which 33 mg/ml propigene, granulin, using differential immuno-absorption. Cancer Res
dium iodide solution (Sigma) was added to each well, followed
2000; 60: 1353–60.
by incubation for 5 min (Osada et al., 2011).
Mabuchi T, Kitagawa K, Ohtsuki T, Kuwabara K, Yagita Y,
Yanagihara T, et al. Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
Memezawa
H, Smith
ML, Siesjö of
BK.propidium
Penumbraliodide-positive
tissues salvaged neurby
To determine
the frequency
reperfusion
following
middlecerebral wearteryocclusionsevenin rats.
Strokeonal cells afterOGD
conditions,
examinedrandomly
1992;
552–9.
chosen23:non-overlapping
high-power ﬁelds ( � 630) of primary
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
neuronal cells incubated under OGD conditions for 24 h
Responses of endothelial cell and astrocyte matrix-integrin receptors(n = 21) (Kanazawa et al., 2011b). To determine the frequency
to ischemia mimic those observed in the neurovascular unit. Strokeof microtubule-associatedprotein 2 (MAP2)-positive neurons
2008;
39: 191–7.
with cytoplasmic
redistributionof TARDBP,
randomly
NaphadeSB, Kigerl KA,
Jakeman LB,
Kostyk SK, seven
Popovich
PG,
chosennon-overlapping
high-powerﬁeldsafter( �spinal
630) ofprimary
Kuret
J. Progranulin
expression
is upregulated
contusion
neuronal
cellsNeuropathol
under normoxicor OGD
conditions during 24 hinmice. Acta
2010; 119:
123–33.
Okubo
Igarashi (nH,= Kanamatsu
T, Hasegawa
D, Orima
were S,examined
7) (Kanazawa
et al., 2011b).
We H,
also
Katayama
Y. theFK-506extendedfrequencies
the therapeutictime window
determinedappearanceof microglia
and forneuthrombolysis
withoutincreasing
theinriskof hemorrhagictransformtrophils in theischaemic
tissuewild-typeand Pgrn
knockout
ation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
mice by examining seven randomly chosen non-overlapping
Osada T, Gu YH, Kanazawa M, Tsubota Y, Hawkins BT, Spatz M,
high-power ﬁelds ( � 400) (n = 21). These analyses were conet al. Interendothelial claudin-5 expression depends on cerebral
ducted blinded
to theadhesion
sample byinformation.
endothelial
cell-matrixb(1)-integrins. J Cereb Blood
Flow Metab 2011; 31: 1972–85.
Özdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
by an intraluminal ﬁlament may cause disturbances in the hippocampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
thickness. Brain Res 1999; 822: 260–4.
Primary microglia
wereG.collected
as pellets
after
centrifugation
Tangkeangsirisin
W, Serrero
PC-cell derived
growthfactor(PCDGF/at 13 000
rpm for10 min.invasivenessTotal RNA
isolatedfrom inthe
GP88)stimulates
migration,
andwas
VEGF
expressionbreastcancercells.
Carcinogenesis
2004;XS
25:(U0902A,
1587–92. Takara Bio),RNA
samplesusing
NucleoSpin
Toh
H,the
Cao
M, DanielscDNA
E, Bateman
A. Expression
the growthandﬁrst-strand
was synthesized
withofReverTra
Acefactorprogranulin
in endothelial
inﬂuences
growthand devel(FSQ-101,
TOYOBO).
PCR cells
reactions
wereperformed
withopmentof blood
vessels:
a novelmouse model.
PLoS
One
2013;

Max
DNA
Polymerase(TakaraBio)andthe 8:folPrimeSTAR
e64989.
0lowing primers: TNF- (forward, 5 - AAGAGGCACT
Shankaran SS, Capell A,
Hruscha AT, Fellerer K, Neumann M,
CCCCCAAAAG -30 ; reverse, 50 - GCTACAGGCTTGTCACT
Schmid B,0 et al. Missense mutationsin the progranulin
0

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948

Temporal changes in PGRN expression and its glycosylated status in rat
cerebral cortex after acute focal
cerebral ischaemia
To investigate the effects of acute focal ischaemia on theexpression and glycosylation status of PGRN, we next performed immunoblotting using whole-cell extracts of the ischaemic cortex of Sprague-Dawley rats. In the ischaemic
core, the level of 88 kDa PGRN gradually decreased
after reperfusion, although it recovered at 72 h after
reperfusion (Fig. 3A and B). On the other hand, the levelof 58–68 kDa PGRN did not change until 18 h, but
increased at 24 h and markedly increased at 72 h after
reperfusion (Fig. 3A and B). In the ischaemic penumbra,
the 88-kDa PGRN decreased slightly, but not signiﬁcantly, after reperfusion, while the 58–68 kDa PGRN moderately increased (Fig. 3A and B).
To conﬁrm whether these two bands indicated secretoryisoforms of PGRN, an in vitro study was performed. Cell
lysates and conditioned media from rat C6 cells were subjected to immunoblotting, as this tumor cell line produces
PGRN during growth (Liau et al., 2000). We identiﬁed a
low molecular-weight band (58–68 kDa) in the cell lysates
and a high molecular-weight band (88 kDa) in the conditioned media (Fig. 3C). In addition, after digestion with
PNGase F, which cleaves all N-linked oligosaccharides,

the mobility of both bands from rat C6 cells changed to
58 kDa. A similar result was observed from an experimentusing rat non-ischaemic brains (Fig. 3D). Therefore, it isconsidered that the high molecular-weight band(88 kDa) was the secretory form of the fully glycosylated
isoform of PGRN and that the low molecular-weight band
(58–68 kDa) was the pre-secretory form of the glycosylated
immature isoform of PGRN (Fig. 3D).

Production and secretion of the two
isoforms of PGRN after OGD
To determine the potential cellular sources of the glycosylated immature isoform of 58–68 kDa PGRN, whichmarkedly increased after reperfusion in ischaemic rats,
immunoblot analyses of whole-cell extracts and conditioned media from primary murine neurons or microglia
were performed. After 18 h incubation under OGD,
both neurons and microglia were not dead as determinedby the lack of change in LDH release from the cells (Fig.
4A). Under normoxic conditions, 58–68 kDa PGRN was
observed in the neuronal cell extracts similar to C6 cell(Fig. 4B), and the 88-kDa PGRN was observed in bothneuronal and microglial conditioned media (Fig. 4C). We
found that, after 18 h incubation under OGD, neurons
produced 88 kDa and 58–68 PGRN and secreted
88 kDa PGRN, and the microglia produced 88 kDa
PGRN, and secreted both 88 kDa and 58–68 kDa
PGRN (Fig. 4C). Therefore, the glycosylated immature isoform of 58–68 kDa PGRN was secreted only frommicroglia.

Volumes of cerebral infarct andoedema, and outcome after
ischaemia in Pgrn knockout mice
To investigate the roles of PGRN in acute focal cerebral
ischaemia, we compared volumes of cerebral infarct andoedema between Pgrn knockout mice and wild-type mice,and evaluated the outcome by motor scale after middle cerebral artery occlusion. Before the comparison, we evaluatedvascular anomaly of the circle of Willis (Özdemir et al.,1999) of Pgrn knockout mice, and found no signiﬁcant difference in vascular structure between wild-type and Pgrn
knockout mice (Supplementary Fig. 2). There was also no
signiﬁcant difference in infarct size at 24 h after reperfusion
between the two groups (P = 0.12) (Fig. 5A). However, cerebral oedema in the Pgrn knockout mice was larger than thatin the wild-type mice at 24 h after reperfusion (P = 0.048)(Fig. 5B). In addition, motor scale at 24 h after reperfusionin Pgrn knockout mice was worse than that in the wild-typemice (P = 0.02) (Fig. 5C). There were no signiﬁcant differences in cerebral oedema and motor scale at 72 h after reperfusion between the two groups (P = 0.95 and 0.75,
respectively).

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The cell lysates and brain tissue from Pgrn knockout mice
did not show immunoreactivity against the antibody(Supplementary Fig. 1). Confocal microscopic studies revealed that the immunoreactivity for PGRN was detected
only in cortical neurons (Fig. 1). PGRN was detected
in a punctate pattern within neuronal cytoplasm, anddouble immunostaining revealed that PGRN was colocalized with marker proteins for the endoplasmic reticulum, Golgi apparatus, and lysosome [ERp57 (nowknown as PDIA3), Golgi-58k, and LAMP1, respectively].
We did not identify microglia or vessels that expressed
PGRN.
We next investigated the effect of acute focal ischaemia
on the expression and localization of PGRN using ischaemic Sprague-Dawley rats. Immunohistochemical analyses
revealed that PGRN was not observed in the neurons
from the ischaemic core (Fig. 2A), but observed in surviving neurons from the ischaemic penumbra (Fig. 2A). The
expression pattern of PGRN in the surviving neurons fromthe ischaemic penumbra is diffused and strong, which isdifferent from that of the non-ischaemic neurons(Fig. 2A). PGRN-positive microglia were increased at 24 h
and markedly increased at 72 h after reperfusion, especiallyon the border of the ischaemic core and penumbra on theischaemic core side (Fig. 2B). In addition, PGRN expression
was observed in the von Willebrand factor-positive
endothelial cells in the ischaemic penumbra at 24 h after
reperfusion (Fig. 2C).

| 1937

1948
1938 | | BRAIN
BRAIN
2015:
2015:
138;
138;
1932–1948
1932–1948

gene linked to frontotemporal lobar degeneration with ubiquitinimmunoreactive inclusions reduce progranulin production and secretion. J Biol Chem 2008; 283: 1744–53.
Shimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D,
Steinberg GK. Suppression of deltaPKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia. J
Cereb Blood Flow Metab 2007; 27: 1463–75.
Songsrirote K, Li Z, Ashford D, Bateman A, Thomas-Oates J.
Development and application of mass spectrometric methods forthe analysis of progranulin N-glycosylation. J Proteomics 2010;
73: 1479–90.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring braininfarct
volume. J Cereb Blood Flow Metab 1990; 10: 290–3.
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
et al. Neuritic beading induced by activated microglia is an earlyfeature of neuronal dysfunction toward neuronal death by inhibitionof mitochondrial respiration and axonal transport. J Biol Chem
2005; 280: 10444–54.
Tanaka Y, Matsuwaki T, Yamanouchi K, Nishihara M. Exacerbatedinﬂammatory responses related to activated microglia after traumatic brain injury in progranulin-deﬁcient mice. Neuroscience
2013; 250: 8–19.
Tao J, Ji F, Wang F, Liu B, Zhu Y. Neuroprotective effects of progranulin in ischemic mice. Brain Res 2012; 1436: 130–6.
Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, et al. Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance neuronal survival. J Cell
Biol 2008; 181: 37–41.
Yenari MA, Kauppinen TM, Swanson RA. Microglial activation in
stroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
Exaggerated inﬂammation, impaired host defense, and neuropathology in progranulin-deﬁcient mice. J Exp Med 2010; 207: 117–28.
Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H. Role of
NF-kappaB-mediated interleukin-8 expression in intraocular neovascularization. Invest Ophthalmol Vis Sci 1998; 39: 1097–106.
Zausinger S, Hungerhuber E, Baethmann A, Reulen H, SchmidElsaesser R. Neurological impairment in rats after transient middle
cerebral artery occlusion: a comparative study under various treatment paradigms. Brain Res 2000; 863: 94–105.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA 2009; 106: 7607–12.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
Progranulin mediates caspase-dependent cleavage of TAR DNA
binding protein-43. J Neurosci 2007; 27: 10530–4.
Zhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D,
Zhang R, et al. Correlation of VEGF and angiopoietin expression
with disruption of blood-brain barrier and angiogenesis after focal
cerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

Figure 1 Analyses of PGRN expression in neuronal cells and microglia from rat non-ischaemic cerebral cortex. Microtubuleassociated protein 2 (MAP2), endoplasmic reticulum stress protein 57 (ERp57, now known as PDIA3), Golgi-58k, lysosomal associated membraneprotein 1 (LAMP-1), CD68/ED1 (green) / PGRN (red) / 4’,6’-diamidino-2-phenylindole (DAPI; blue) triple labelling in the non-ischaemic cortices
examined by confocal microscopy. MAP2, ERp57, Golgi-58k, LAMP1, and CD68/ED1 are markers for neuronal cells, endoplasmic reticulum, Golgi
apparatus, lysosome, and microglia, respectively. Scale bars = 10 mm.

－ 222 －

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Kleinberger G, Wils H, Ponsaerts P, Joris G, Timmermans JP, Van
Broeckhoven C, et al. Increased caspase activation and decreased
TDP-43 solubility in progranulin knockout cortical cultures. J
Neurochem 2010; 115: 735–47.
Krishnamurthy RG, Senut MC, Zemke D, Min J, Frenkel MB,
Greenberg EJ, et al. Asiatic acid, a pentacyclic triterpene fromCentella asiatica, is neuroprotective in a mouse model of focal
cerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
Bosch L, Van Damme P, et al. Progranulin is neurotrophic in vivo
and protects against a mutant TDP-43 induced axonopathy. PLoS
One 2010; 5: e13368.
Liau LM, Lallone RL, Seitz RS, Buznikov A, Gregg JP, Kornblum HI,
et al. Identiﬁcation of a human glioma-associated growth factor
gene, granulin, using differential immuno-absorption. Cancer Res
2000; 60: 1353–60.
Mabuchi T, Kitagawa K, Ohtsuki T, Kuwabara K, Yagita Y,
Yanagihara T, et al. Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged byreperfusion following middle cerebral artery occlusion in rats. Stroke
1992; 23: 552–9.
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
Responses of endothelial cell and astrocyte matrix-integrin receptors
to ischemia mimic those observed in the neurovascular unit. Stroke
2008; 39: 191–7.
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,
Kuret J. Progranulin expression is upregulated after spinal contusion
in mice. Acta Neuropathol 2010; 119: 123–33.
Okubo S, Igarashi H, Kanamatsu T, Hasegawa D, Orima H,
Katayama Y. FK-506 extended the therapeutic time window for
thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
Osada T, Gu YH, Kanazawa M, Tsubota Y, Hawkins BT, Spatz M,
et al. Interendothelial claudin-5 expression depends on cerebral
endothelial cell-matrix adhesion by b(1)-integrins. J Cereb Blood
Flow Metab 2011; 31: 1972–85.
Özdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
by an intraluminal ﬁlament may cause disturbances in the hippocampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
thickness. Brain Res 1999; 822: 260–4.
Tangkeangsirisin W, Serrero G. PC-cell derived growth factor (PCDGF/
GP88) stimulates migration, invasiveness and VEGF expression inbreast cancer cells. Carcinogenesis 2004; 25: 1587–92.
Toh H, Cao M, Daniels E, Bateman A. Expression of the growth
factor progranulin in endothelial cells inﬂuences growth and development of blood vessels: a novel mouse model. PLoS One 2013; 8:
e64989.
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948| 1939Recruitment of neutrophils andmicroglia after ischaemia in Pgrn
knockout mice

VEGF expression in Pgrn knockout
mice after ischaemia
We next investigated the possibility that cerebral oedema in
Pgrn knockout mice was caused by VEGF expression, because VEGF is a potent angiogenic factor that regulates
vascular permeability after focal cerebral ischaemia(Zhang et al., 2002). Additionally, a recent study, inwhich PDGF (platelet-derived growth factor) was used as
an experimental agent to induce blood–brain barrier leakage, did not determine the VEGF protein level after ischaemia (Jackman et al., 2013). Although VEGF expression
was undetectable in brains of sham-operated wild-type
and Pgrn knockout mice, VEGF expression was observedin the peri-infarct area from brains of ischaemic wild-type
and Pgrn knockout mice 24 and 72 h after reperfusion. At
72 h after reperfusion, the expression was much more
prominent in Pgrn knockout mice than in wild-type mice
(P 5 0.001) (Fig. 5D). VEGF expression was observed
Figure 2 Continued

Figure 2 Post-ischaemic changes in PGRN expression inneuronal cells, microglia and endothelial cells in the rat
cerebral cortex. (A) The absence of PGRN expression in the－ 223 －

ischaemic core and increased PGRN expression in the ischaemic
penumbra at 24 h after reperfusion. MAP2, LAMP1 (green) / PGRN
(red) / DAPI (blue) triple labelling in the ischaemic core and penumbra examined by confocal microscopy. Scale bars = 10 mm. (B)Markedly increased PGRN-positive microglia at 72 h after reperfusion. CD68/ED1 (green) / PGRN (red) / DAPI (blue) triple labellingin the sham-operated or ischaemic cortex (ischaemic core) examined by confocal microscopy. Red scale bar = 10 mm. White scalebars = 20 mm. (C) PGRN expression in endothelial cells and astrocytic end-feet at 24 h after reperfusion. von Willebrand factor(vWF), glial fibrillary acidic protein (GFAP, green) / PGRN (red) /DAPI (blue) triple labelling in the sham-operated or ischaemic
penumbra examined by confocal microscopy. von Willebrand factor
is a marker for endothelial cells, and GFAP is a marker for astrocytes. Scale bars = 10 mm.

Downloaded from http://brain.oxfordjournals.org/ by guest on May 4, 2016

Because a recent study reported that recombinant PGRN
treatment suppressed neutrophil recruitment into ischaemic
mouse brain (Egashira et al., 2013), we investigatedwhether cerebral oedema in Pgrn knockout mice is associated with neutrophil recruitment. Immunohistochemistry
using an anti-myeloperoxidase antibody revealed that there
were no signiﬁcant differences in the numbers of myeloperoxidase-positive neutrophils between the ischaemic wildtype and Pgrn knockout mice at 24 h and 72 h after reperfusion (Supplementary Fig. 3). We also found that there
were no signiﬁcant differences in the numbers of IBA1
(now known as AIF1)-positive microglia in the border ofthe ischaemic core and penumbra between the wild-type
and Pgrn knockout mice at 24 h and 72 h after reperfusion(Supplementary Fig. 4).

19481940 | | BRAIN
BRAIN
2015:
2015:
138;
138;
1932–1948
1932–1948

gene linked to frontotemporal lobar degeneration with ubiquitinimmunoreactive inclusions reduce progranulin production and secretion. J Biol Chem 2008; 283: 1744–53.
Shimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D,
Steinberg GK. Suppression of deltaPKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia. J
Cereb Blood Flow Metab 2007; 27: 1463–75.
Songsrirote K, Li Z, Ashford D, Bateman A, Thomas-Oates J.
Development and application of mass spectrometric methods forthe analysis of progranulin N-glycosylation. J Proteomics 2010;
73: 1479–90.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring braininfarct
volume. J Cereb Blood Flow Metab 1990; 10: 290–3.
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
et al. Neuritic beading induced by activated microglia is an earlyfeature of neuronal dysfunction toward neuronal death by inhibitionof mitochondrial respiration and axonal transport. J Biol Chem
2005; 280: 10444–54.
Tanaka Y, Matsuwaki T, Yamanouchi K, Nishihara M. Exacerbatedinﬂammatory responses related to activated microglia after traumatic brain injury in progranulin-deﬁcient mice. Neuroscience
2013; 250: 8–19.
Tao J, Ji F, Wang F, Liu B, Zhu Y. Neuroprotective effects of progranulin in ischemic mice. Brain Res 2012; 1436: 130–6.
Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, et al. Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance neuronal survival. J Cell
Biol 2008; 181: 37–41.
Yenari MA, Kauppinen TM, Swanson RA. Microglial activation in
stroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
Exaggerated inﬂammation, impaired host defense, and neuropathology in progranulin-deﬁcient mice. J Exp Med 2010; 207: 117–28.
Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H. Role of
NF-kappaB-mediated interleukin-8 expression in intraocular neovascularization. Invest Ophthalmol Vis Sci 1998; 39: 1097–106.
Zausinger S, Hungerhuber E, Baethmann A, Reulen H, SchmidElsaesser R. Neurological impairment in rats after transient middle
cerebral artery occlusion: a comparative study under various treatment paradigms. Brain Res 2000; 863: 94–105.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA 2009; 106: 7607–12.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
Progranulin mediates caspase-dependent cleavage of TAR DNA
binding protein-43. J Neurosci 2007; 27: 10530–4.
Zhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D,
Zhang R, et al. Correlation of VEGF and angiopoietin expression
with disruption of blood-brain barrier and angiogenesis after focal
cerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

Figure 3 Temporal changes in PGRN expression in the rat cerebral cortex after focal cerebral ischaemia. (A) Representative
PGRN protein bands of the rat cerebral cortex from the ischaemic core (left) and ischaemic penumbra (right). Two isoforms (88 kDa and 58–
68 kDa) were observed. b-actin confirmed equal loading of proteins. (B) Optical densitometry analyses for 88 kDa (left) and 58–68 kDa isoforms(right) of PGRN. *P 5 0.05, **P 5 0.01, ##P 5 0.01 versus sham. Data represent relative optical densities of ischaemic brain samples compared tothose of sham-operated samples. (C) Difference in glycosylation status in PGRN between cell lysates and conditioned media of rat C6 cells
analysed by immunoblotting. Note that PGRN shows two isoforms (88 kDa and 58–68 kDa), both of which change to 58 kDa after digestion
with peptide-N-glycosidase F (PNGase F). All findings were confirmed in triplicate. (D) Difference in glycosylation status in PGRN from the rat
non-ischaemic cortex analysed by immunoblotting. Similar to rat C6 cells, PGRN shows two isoforms (88 kDa and 58–68 kDa), both of whichchange to 58 kDa after digestion with PNGase F. All findings were confirmed in triplicate.

mainly in astrocytes and endothelial cells (but not in microglia) within ischaemic mice (Supplementary Fig. 5).
To conﬁrm the increased level of VEGF in ischaemic
Pgrn knockout mice, we performed an in vitro study. We

ﬁrst compared VEGF secretion after OGD from microglia
and astrocytes between wild-type and Pgrn knockout mice
(Fig. 5E). The levels of VEGF from microglia of Pgrn
knockout mice were higher than those of wild-type mice－ 224 －

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Kleinberger G, Wils H, Ponsaerts P, Joris G, Timmermans JP, Van
Broeckhoven C, et al. Increased caspase activation and decreased
TDP-43 solubility in progranulin knockout cortical cultures. J
Neurochem 2010; 115: 735–47.
Krishnamurthy RG, Senut MC, Zemke D, Min J, Frenkel MB,
Greenberg EJ, et al. Asiatic acid, a pentacyclic triterpene fromCentella asiatica, is neuroprotective in a mouse model of focal
cerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
Bosch L, Van Damme P, et al. Progranulin is neurotrophic in vivo
and protects against a mutant TDP-43 induced axonopathy. PLoS
One 2010; 5: e13368.
Liau LM, Lallone RL, Seitz RS, Buznikov A, Gregg JP, Kornblum HI,
et al. Identiﬁcation of a human glioma-associated growth factor
gene, granulin, using differential immuno-absorption. Cancer Res
2000; 60: 1353–60.
Mabuchi T, Kitagawa K, Ohtsuki T, Kuwabara K, Yagita Y,
Yanagihara T, et al. Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged byreperfusion following middle cerebral artery occlusion in rats. Stroke
1992; 23: 552–9.
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
Responses of endothelial cell and astrocyte matrix-integrin receptors
to ischemia mimic those observed in the neurovascular unit. Stroke
2008; 39: 191–7.
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,
Kuret J. Progranulin expression is upregulated after spinal contusion
in mice. Acta Neuropathol 2010; 119: 123–33.
Okubo S, Igarashi H, Kanamatsu T, Hasegawa D, Orima H,
Katayama Y. FK-506 extended the therapeutic time window for
thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
Osada T, Gu YH, Kanazawa M, Tsubota Y, Hawkins BT, Spatz M,
et al. Interendothelial claudin-5 expression depends on cerebral
endothelial cell-matrix adhesion by b(1)-integrins. J Cereb Blood
Flow Metab 2011; 31: 1972–85.
Özdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
by an intraluminal ﬁlament may cause disturbances in the hippocampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
thickness. Brain Res 1999; 822: 260–4.
Tangkeangsirisin W, Serrero G. PC-cell derived growth factor (PCDGF/
GP88) stimulates migration, invasiveness and VEGF expression inbreast cancer cells. Carcinogenesis 2004; 25: 1587–92.
Toh H, Cao M, Daniels E, Bateman A. Expression of the growth
factor progranulin in endothelial cells inﬂuences growth and development of blood vessels: a novel mouse model. PLoS One 2013; 8:
e64989.
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948| 1941(P 5 0.001), although the levels of VEGF from astrocytes
were not different between wild-type and Pgrn knockout
mice (P = 1.00). To investigate the effect of microglia on
VEGF secretion from astrocytes, microglial condition
media of wild-type and Pgrn knockout mice were added
to the astrocytes of wild-type and Pgrn knockout mice,
respectively. Interestingly, the levels of VEGF from theastrocytes with microglial conditioned media of knockout
mice was higher than that of wild-type under OGD(P 5 0.001). In addition, the levels of VEGF from astrocytes with microglial conditioned media of knockout mice
was higher than that from microglia alone (P = 0.013) orastrocytes alone (P = 0.014) of Pgrn knockout mice (Fig.
5E). These results suggest that lack of PGRN or unknown
humoral factors from ischaemic microglia of Pgrn knockout mice might synergically promote VEGF secretion fromthe astrocytes.

Figure 4 Effects of OGD on PGRN secretion from murine
primary cultured neuronal and microglial cells. (A)Cytotoxicity evaluated by lactate dehydrogenase assay between
normoxia and OGD in neuronal cells and microglia (n = 5). (B andC) Representative PGRN protein bands in immunoblotting fromwhole-cell extracts (B) and conditioned media (C) of murine primary cultured cells (neurons and microglia) subjected to normoxia(norm) or OGD. After 18 h incubation under OGD condition, 58–
68 kDa and 88 kDa PGRN in the neuronal cell extracts as well as
88 kDa PGRN in the neuronal conditioned media increased
compared to those under normoxic condition (P = 0.001 and 0.011,
respectively). The 88 kDa PGRN in the microglial conditioned
media and the 58–68 kDa PGRN in the microglial cell extracts also
increased compared to those under normoxic conditions(P 5 0.001 and 0.0098, respectively). Additionally, the 58–68 kDa
PGRN in the microglial conditioned media markedly increased(P 5 0.001). b-actin and transferrin confirmed equal loading ofproteins. Optical densitometry analyses for PGRN are also shown.
Data represent relative optical densities of each sample compared
to microglial samples from OGD (n = 5). *P 5 0.05. **P 5 0.01.

To investigate the effect of recombinant PGRN on VEGF
expression in vitro, we compared VEGF levels in astrocyteswith microglial conditioned media for wild-type mice treated with vehicle controls and recombinant PGRN. The levelof VEGF in astrocytes cultured in microglial conditioned
media was decreased by recombinant PGRN (10 mg/ml)
compared to vehicle treatment (23.0  4.8% reduction;
P = 0.046, n = 3–6). We previously reported that delayedtPA treatment 4 h after ischaemia promoted expression of
VEGF in vivo (Kanazawa et al., 2011a). We investigatedthe effect of recombinant PGRN on VEGF expression using
this thrombo-embolic model. For this purpose we administrated 100 mg recombinant PGRN or vehicle along withtPA treatment 4 h after ischaemia. The co-administrationof recombinant PGRN and tPA decreased the expressionof VEGF compared to co-administration of vehicle andtPA (P = 0.046) (Supplementary Fig. 6).

Comparison of cytokine responseof microglia between wild-type andPgrn knockout mice after OGD
To investigate the effect of PGRN on neuroinﬂammation
after acute ischaemia, we compared mRNA levels of several
cytokines in microglia from wild-type and Pgrn knockout
mice, under normoxic and OGD conditions, because thebalance between pro-inﬂammatory and anti-inﬂammatory
microglial phenotypes is considered to inﬂuence expansionof cerebral infarct (Hu et al., 2012). We found that there
were no differences in the mRNA levels of pro-inﬂammatory cytokines (TNF- and TGF-b) between wild-type andPgrn knockout mice, while the mRNA level of anti-inﬂammatory cytokine IL10 in the Pgrn knockout microglia waslower than that in the wild-type microglia after OGD－ 225 －

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The effects of VEGF expression
by recombinant PGRN treatment
after ischaemia in vitro and in vivo

19481942 | | BRAIN
BRAIN
2015:
2015:
138;
138;
1932–1948
1932–1948

gene linked to frontotemporal lobar degeneration with ubiquitinimmunoreactive inclusions reduce progranulin production and secretion. J Biol Chem 2008; 283: 1744–53.
Shimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D,
Steinberg GK. Suppression of deltaPKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia. J
Cereb Blood Flow Metab 2007; 27: 1463–75.
Songsrirote K, Li Z, Ashford D, Bateman A, Thomas-Oates J.
Development and application of mass spectrometric methods forthe analysis of progranulin N-glycosylation. J Proteomics 2010;
73: 1479–90.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring braininfarct
volume. J Cereb Blood Flow Metab 1990; 10: 290–3.
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
et al. Neuritic beading induced by activated microglia is an earlyfeature of neuronal dysfunction toward neuronal death by inhibitionof mitochondrial respiration and axonal transport. J Biol Chem
2005; 280: 10444–54.
Tanaka Y, Matsuwaki T, Yamanouchi K, Nishihara M. Exacerbatedinﬂammatory responses related to activated microglia after traumatic brain injury in progranulin-deﬁcient mice. Neuroscience
2013; 250: 8–19.
Tao J, Ji F, Wang F, Liu B, Zhu Y. Neuroprotective effects of progranulin in ischemic mice. Brain Res 2012; 1436: 130–6.
Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, et al. Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance neuronal survival. J Cell
Biol 2008; 181: 37–41.
Yenari MA, Kauppinen TM, Swanson RA. Microglial activation in
stroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
Exaggerated inﬂammation, impaired host defense, and neuropathology in progranulin-deﬁcient mice. J Exp Med 2010; 207: 117–28.
Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H. Role of
NF-kappaB-mediated interleukin-8 expression in intraocular neovascularization. Invest Ophthalmol Vis Sci 1998; 39: 1097–106.
Zausinger S, Hungerhuber E, Baethmann A, Reulen H, SchmidElsaesser R. Neurological impairment in rats after transient middle
cerebral artery occlusion: a comparative study under various treatment paradigms. Brain Res 2000; 863: 94–105.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA 2009; 106: 7607–12.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
Progranulin mediates caspase-dependent cleavage of TAR DNA
binding protein-43. J Neurosci 2007; 27: 10530–4.
Zhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D,
Zhang R, et al. Correlation of VEGF and angiopoietin expression
with disruption of blood-brain barrier and angiogenesis after focal
cerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

Figure 5 Comparison of ischaemic outcomes and VEGF expression between wild-type and Pgrn knockout mice. These panels
show volumes of the cerebral infarct (A) and cerebral oedema (B) at 24 h and 72 h after reperfusion (all, n 5 9), and outcome evaluated by a
6-point neurological scale score (C) at 24 h after reperfusion [wild-type (WT) n = 29, knockout (KO) n = 22]. Volumes of cerebral infarct andoedema are expressed as proportions on the ischaemic side of the cerebral hemisphere. *P 5 0.05, **P 5 0.01. (D) VEGF expression in the shamoperated and ischaemic brains (at 24 h and 72 h after reperfusion) from the wild-type and Pgrn knockout mice. The results were confirmed intriplicate. A secondary-only antibody control confirms that extracellular stainings of VEGF after ischaemia were not non-specific (data not shown).
At 72 h after reperfusion, the expression of VEGF was higher in ischaemic brains of Pgrn knockout mice than in those of wild-type mice(**P 5 0.001) (n = 21). Scale bars = 200 mm. (E) The levels of secretory VEGF from microglia, astrocytes, and astrocytes with microglial
conditioned media of wild-type and Pgrn knockout mice after OGD (n = 6–7). *P = 0.01, **P 5 0.01.

－ 226 －

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Kleinberger G, Wils H, Ponsaerts P, Joris G, Timmermans JP, Van
Broeckhoven C, et al. Increased caspase activation and decreased
TDP-43 solubility in progranulin knockout cortical cultures. J
Neurochem 2010; 115: 735–47.
Krishnamurthy RG, Senut MC, Zemke D, Min J, Frenkel MB,
Greenberg EJ, et al. Asiatic acid, a pentacyclic triterpene fromCentella asiatica, is neuroprotective in a mouse model of focal
cerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
Bosch L, Van Damme P, et al. Progranulin is neurotrophic in vivo
and protects against a mutant TDP-43 induced axonopathy. PLoS
One 2010; 5: e13368.
Liau LM, Lallone RL, Seitz RS, Buznikov A, Gregg JP, Kornblum HI,
et al. Identiﬁcation of a human glioma-associated growth factor
gene, granulin, using differential immuno-absorption. Cancer Res
2000; 60: 1353–60.
Mabuchi T, Kitagawa K, Ohtsuki T, Kuwabara K, Yagita Y,
Yanagihara T, et al. Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged byreperfusion following middle cerebral artery occlusion in rats. Stroke
1992; 23: 552–9.
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
Responses of endothelial cell and astrocyte matrix-integrin receptors
to ischemia mimic those observed in the neurovascular unit. Stroke
2008; 39: 191–7.
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,
Kuret J. Progranulin expression is upregulated after spinal contusion
in mice. Acta Neuropathol 2010; 119: 123–33.
Okubo S, Igarashi H, Kanamatsu T, Hasegawa D, Orima H,
Katayama Y. FK-506 extended the therapeutic time window for
thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
Osada T, Gu YH, Kanazawa M, Tsubota Y, Hawkins BT, Spatz M,
et al. Interendothelial claudin-5 expression depends on cerebral
endothelial cell-matrix adhesion by b(1)-integrins. J Cereb Blood
Flow Metab 2011; 31: 1972–85.
Özdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
by an intraluminal ﬁlament may cause disturbances in the hippocampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
thickness. Brain Res 1999; 822: 260–4.
Tangkeangsirisin W, Serrero G. PC-cell derived growth factor (PCDGF/
GP88) stimulates migration, invasiveness and VEGF expression inbreast cancer cells. Carcinogenesis 2004; 25: 1587–92.
Toh H, Cao M, Daniels E, Bateman A. Expression of the growth
factor progranulin in endothelial cells inﬂuences growth and development of blood vessels: a novel mouse model. PLoS One 2013; 8:
e64989.
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948| 1943(P = 0.037) (Fig. 6A and B). We also found that Il10mRNA levels in the Pgrn knockout microglia were lower
than that in the wild-type microglia after OGD by real-time
PCR (P = 0.001) (Fig. 6C). The level of Il10 after 18 h incubation under OGD from microglial conditioned media ofPgrn knockout mice (10.3  3.1 pg/ml) was lower than thatfrom microglial conditioned media of wild-type mice
(37.4  12.2 pg/ml) (P = 0.040).
We also investigated the expression of TNF-, TGF-b,
and IL10 in brains from ischaemic wild-type andPgrn knockout mice 24 and 72 h after reperfusion.
Although the expressions of TNF- and TGF-b in theischaemic brains were not different between wild-type andPgrn knockout mice (P = 0.262 and P = 0.228, respectively)(Supplementary Fig. 7A and B), IL10 expression was lower
in the ischaemic brains of Pgrn knockout mice than inthose of wild-type mice (P 5 0.001) (Supplementary Fig.
7C). IL10 was expressed mainly in the extracellularparenchyma.

Effects of PGRN on neuronal cell
death and cytoplasmic redistributionof TARDBP
We investigated the neuroprotective effect of PGRN on
murine primary neuronal cells under OGD condition.
Twenty-four hours incubation under OGD promoted neuronal cell death as evaluated by a lactate dehydrogenase
assay (Fig. 7A), and the percentage of propidium iodidepositive neuronal cells after 24-h incubation under OGD
was 8.4  3.7% (n = 21) (Supplementary Fig. 8). The attached neuronal cells that showed cytoplasmic redistribution of TARDBP under the OGD conditions wereconﬁrmed to be living as they were propidium iodide-negative (Fig. 7B). However, we found that the recombinant
PGRN (5 mg/ml) suppressed the neuronal cell death
compared to the control (P = 0.045) (Fig. 7C).
Immunocytochemistry conﬁrmed that these surviving
neurons incorporated the exogenously administered－ 227 －

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Figure 6 Comparison of cytokine response of microglia between wild-type and Pgrn knockout mice after OGD. (A)Representative cytokine mRNA bands from primary microglial cells of wild-type (WT) and Pgrn knockout (KO) under normoxic and OGD
conditions. (B) Optical densitometry analyses for inflammatory cytokines such as TNF- and transforming growth factor-b (TGF-b), and antiinflammatory cytokines such as IL-10 (n = 5–7). Data represent relative optical densities of respective samples compared to samples from wildtype mice under normoxic conditions by one-way ANOVA. (C) Data represent relative Il10 mRNA levels compared to samples from wild-typemice under normoxic conditions (n = 3–4). *P 5 0.05, **P 5 0.01.

1948
1944 | | BRAIN
BRAIN
2015:
2015:
138;
138;
1932–1948
1932–1948

gene linked to frontotemporal lobar degeneration with ubiquitinimmunoreactive inclusions reduce progranulin production and secretion. J Biol Chem 2008; 283: 1744–53.
Shimohata T, Zhao H, Sung JH, Sun G, Mochly-Rosen D,
Steinberg GK. Suppression of deltaPKC activation after focal cerebral ischemia contributes to the protective effect of hypothermia. J
Cereb Blood Flow Metab 2007; 27: 1463–75.
Songsrirote K, Li Z, Ashford D, Bateman A, Thomas-Oates J.
Development and application of mass spectrometric methods forthe analysis of progranulin N-glycosylation. J Proteomics 2010;
73: 1479–90.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C,
Sharp FR. A semiautomated method for measuring braininfarct
volume. J Cereb Blood Flow Metab 1990; 10: 290–3.
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
et al. Neuritic beading induced by activated microglia is an earlyfeature of neuronal dysfunction toward neuronal death by inhibitionof mitochondrial respiration and axonal transport. J Biol Chem
2005; 280: 10444–54.
Tanaka Y, Matsuwaki T, Yamanouchi K, Nishihara M. Exacerbatedinﬂammatory responses related to activated microglia after traumatic brain injury in progranulin-deﬁcient mice. Neuroscience
2013; 250: 8–19.
Tao J, Ji F, Wang F, Liu B, Zhu Y. Neuroprotective effects of progranulin in ischemic mice. Brain Res 2012; 1436: 130–6.
Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E,
van Swieten J, et al. Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance neuronal survival. J Cell
Biol 2008; 181: 37–41.
Yenari MA, Kauppinen TM, Swanson RA. Microglial activation in
stroke: therapeutic targets. Neurotherapeutics 2010; 7: 378–91.
Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
Exaggerated inﬂammation, impaired host defense, and neuropathology in progranulin-deﬁcient mice. J Exp Med 2010; 207: 117–28.
Yoshida A, Yoshida S, Khalil AK, Ishibashi T, Inomata H. Role of
NF-kappaB-mediated interleukin-8 expression in intraocular neovascularization. Invest Ophthalmol Vis Sci 1998; 39: 1097–106.
Zausinger S, Hungerhuber E, Baethmann A, Reulen H, SchmidElsaesser R. Neurological impairment in rats after transient middle
cerebral artery occlusion: a comparative study under various treatment paradigms. Brain Res 2000; 863: 94–105.
Zhang YJ, Xu YF, Cook C, Gendron TF, Roettges P, Link CD, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA 2009; 106: 7607–12.
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
Progranulin mediates caspase-dependent cleavage of TAR DNA
binding protein-43. J Neurosci 2007; 27: 10530–4.
Zhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D,
Zhang R, et al. Correlation of VEGF and angiopoietin expression
with disruption of blood-brain barrier and angiogenesis after focal
cerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

Figure 7 Suppression of neuronal cell death and cytoplasmic redistribution of TARDBP (TDP-43) under OGD conditions byrecombinant PGRN. (A) Cytotoxicity of neuronal cells between normoxic and OGD conditions at 18 h and 24 h (n = 5). **P 5 0.01. (B)Confocal microscopic observation of TDP-43 (now known as TARDBP, green)/propidium iodide (PI) (red)/DAPI (blue)-triple labelling in neuronalcells under OGD conditions. Magnification is double in the small inset. Cytoplasmic redistribution of TDP-43 was observed in surviving neurons
under the OGD conditions. These neuronal cells were propidium iodide-negative. Scale bar = 50 mm. (C) Recombinant PGRN (rPGRN) alleviatedcytotoxicity at 24 h under OGD conditions (n = 6). *P 5 0.05. (D) MAP2 (green)/FLAG (red) / DAPI (blue) triple labeling in neurons between
normoxic and OGD conditions by confocal microscopy. FLAG-tagged recombinant PGRN was co-localized in surviving neurons under OGD
condition (arrow). Scale bars = 20 mm. (E) MAP2 (green)/TDP-43 (red)/DAPI (blue) triple labelling in neurons between normoxic and OGD
conditions with or without recombinant PGRN treatment by confocal microscopy. Arrow indicates cytoplasmic redistribution of TARDBP at 24 hunder OGD conditions. Scale bars = 20 mm. (F) The frequencies cytoplasmic redistribution of TARDBP in MAP2-positive neuronal cells between
normoxic and OGD conditions. recombinant PGRN blocked cytoplasmic redistribution of TARDBP at 24 h under OGD conditions. *P 5 0.05,##
P 5 0.01 versus normoxic condition (n = 7).

－ 228 －

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Kleinberger G, Wils H, Ponsaerts P, Joris G, Timmermans JP, Van
Broeckhoven C, et al. Increased caspase activation and decreased
TDP-43 solubility in progranulin knockout cortical cultures. J
Neurochem 2010; 115: 735–47.
Krishnamurthy RG, Senut MC, Zemke D, Min J, Frenkel MB,
Greenberg EJ, et al. Asiatic acid, a pentacyclic triterpene fromCentella asiatica, is neuroprotective in a mouse model of focal
cerebral ischemia. J Neurosci Res 2009; 87: 2541–50.
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
Bosch L, Van Damme P, et al. Progranulin is neurotrophic in vivo
and protects against a mutant TDP-43 induced axonopathy. PLoS
One 2010; 5: e13368.
Liau LM, Lallone RL, Seitz RS, Buznikov A, Gregg JP, Kornblum HI,
et al. Identiﬁcation of a human glioma-associated growth factor
gene, granulin, using differential immuno-absorption. Cancer Res
2000; 60: 1353–60.
Mabuchi T, Kitagawa K, Ohtsuki T, Kuwabara K, Yagita Y,
Yanagihara T, et al. Contribution of microglia/macrophages to expansion of infarction and response of oligodendrocytes after focal
cerebral ischemia in rats. Stroke 2000; 31: 1735–43.
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged byreperfusion following middle cerebral artery occlusion in rats. Stroke
1992; 23: 552–9.
Milner R, Hung S, Wang X, Berg GI, Spatz M, del Zoppo GJ.
Responses of endothelial cell and astrocyte matrix-integrin receptors
to ischemia mimic those observed in the neurovascular unit. Stroke
2008; 39: 191–7.
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,
Kuret J. Progranulin expression is upregulated after spinal contusion
in mice. Acta Neuropathol 2010; 119: 123–33.
Okubo S, Igarashi H, Kanamatsu T, Hasegawa D, Orima H,
Katayama Y. FK-506 extended the therapeutic time window for
thrombolysis without increasing the risk of hemorrhagic transformation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
Osada T, Gu YH, Kanazawa M, Tsubota Y, Hawkins BT, Spatz M,
et al. Interendothelial claudin-5 expression depends on cerebral
endothelial cell-matrix adhesion by b(1)-integrins. J Cereb Blood
Flow Metab 2011; 31: 1972–85.
Özdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
by an intraluminal ﬁlament may cause disturbances in the hippocampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
thickness. Brain Res 1999; 822: 260–4.
Tangkeangsirisin W, Serrero G. PC-cell derived growth factor (PCDGF/
GP88) stimulates migration, invasiveness and VEGF expression inbreast cancer cells. Carcinogenesis 2004; 25: 1587–92.
Toh H, Cao M, Daniels E, Bateman A. Expression of the growth
factor progranulin in endothelial cells inﬂuences growth and development of blood vessels: a novel mouse model. PLoS One 2013; 8:
e64989.
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948| 1945FLAG-tagged recombinant PGRN (Fig. 7D). We also foundthat the neuronal cells, which incorporated the exogenous
FLAG-tagged recombinant PGRN, showed a decrease in
the cytoplasmic redistribution of TARDBP (Fig. 7E): thefrequency of cytoplasmic redistribution of TARDBP in theneurons treated with recombinant PGRN under OGD waslower than that treated with the vehicle control(48.0  35.4% versus 86.9  10.4%; P = 0.010) (Fig. 7F).

oedema and haemorrhagic transformation when tPA is administered beyond the therapeutic time window (4 h)(Kanazawa et al., 2011a; Kawamura et al., 2014). The
group treated with tPA and intravenous administration ofrecombinant PGRN at 4 h after reperfusion showed therapeutic effects on the cerebral infarct volume (Fig. 8A),
oedema volume (Fig. 8B), haemorrhage (Fig. 8C), motor
scale (Fig. 8D), and mortality ratio (Fig. 8E) compared to
the group treated with tPA and control (P = 0.007, 0.038,
0.007, 0.004 and 0.026, respectively).

Therapeutic effects of recombinant
PGRN against delayed tPA treatment
for acute focal cerebral ischaemia

Discussion

Finally, we investigated therapeutic effects of recombinant
PGRN using a rat autologous thrombo-embolic model(Okubo et al., 2007), because this model shows cerebral

We demonstrated for the ﬁrst time the dynamic changes in
PGRN in vivo in the neurons, microglia, and endothelial
cells after ischaemia, including decreased level of PGRN

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Figure 8 The improvements in ischaemic outcomes after delayed tPA treatment with recombinant PGRN. Quantification of
volumes of cerebral infarct (A) and oedema (B), and haemoglobin concentrations of the lysates from the ischaemic hemisphere (C) at 24 h after
reperfusion. Black bars: the tPA 4 h group treated with control protein; grey bars: the tPA 4 h group treated with recombinant PGRN (rPGRN)(n = 5). Six-point neurological scale (D) and mortality ratio (E) of the tPA 4 h group treated with control protein (n = 7) or recombinant PGRN
(n = 6). *P 5 0.05, **P 5 0.01.

1948
1946 ||BRAIN
BRAIN
2015:
2015:
138;
138;
1932–1948
1932–1948

knockout mice than in wild-type mice after focal cerebral
ischaemia. In addition, Pgrn knockout microglia itself secreted VEGF and the conditioned media from Pgrn knockout microglia synergically stimulated VEGF secretion from
Pgrn knockout astrocytes after OGD. Although we could
not identify the speciﬁc molecules that stimulate VEGF expression, a recent study demonstrated that PGRN might
inhibit cerebral oedema via NF-B (Egashira et al., 2013),
which activates VEGF transcription (Yoshida et al., 1998).
PGRN may regulate vascular permeability by inhibiting
microglial production of molecules that activate NF-BVEGF signalling pathway.
In breast cancer, high levels of PGRN result in increased
VEGF production (Tangkeangsirisin et al., 2004), while inthis study of ischaemia, depletion of PGRN increased

genelinkedto frontotemporallobar degeneration
VEGF
expression.
Furthermore,
we showed withclearubiquitinincreases
immunoreactive inclusions reduce progranulin production and secrein PGRN expression in vessels of the penumbra (Fig. 2C),
tion. J Biol Chem 2008; 283: 1744–53.
which isT,similarin peripheral
Shimohata
Zhao toH, whatSung hasJH, been
Sun reported
G, Mochly-Rosen
D,
wounds(He
etal.,2003b).
BecauseelevatedlevelsSteinberg GK. Suppression of deltaPKC activation after focal
cere- ofbral
ischemia
contributes
to the protective
effect effectof hypothermia.
J
PGRN
have
a signiﬁcantbiologicalon vessel
Cereb
Blood
Metab
27: 1463–75.
growththatFlow
mightbe2007;
independentof VEGF (Toh et al.,Songsrirote
K, Liof Z,
D, Pgrn
Bateman
A, Thomas-Oates
J.
2013), sometheAshford
effects ofknockout
on penumbral
Development and application of mass spectrometric methods forblood
vesselsmight
bemediated
directly
by
PGRN
loss
the analysis of progranulin N-glycosylation. J Proteomics 2010;
ratherthan via increased VEGF expression (Jackman
73:
1479–90.
Swanson
RA, Morton
MT,studies
Tsao-Wu
SavalostoRA,DavidsonC,
et al., 2013).
FurtherareG,
neededclarify
the mechSharp
A semiautomatedmethod formeasuringbraininfarctanismFR.
underlying
PGRN-mediatedvascularpermeability.
volume.
J Cereb
Bloodmay
Flow suppress
Metab 1990;
10: 290–3.
Second,PGRN
neuroinﬂammation
after
Takeuchi H, Mizuno T, Zhang G, Wang J, Kawanokuchi J, Kuno R,
acute
focal
cerebralischaemiavia
theanti-inﬂammatory
et al. Neuritic beading induced by activated microglia is an earlycytokine,
IL10. dysfunction
We demonstrated
thatdeaththe bynumberfeatureof neuronal
toward neuronal
inhibition ofofmitochondrial respiration
and axonaltransport. J Biol Chem
PGRN-positive
microglia(CD68/ED1-positive
cells)
2005;
280: 10444–54.
increasedin the ischaemic core of Sprague-Dawley rats,
TanakaY, Matsuwaki
T, Yamanouchi
K, Nishihara
M. Pgrn
Exacerbated
and that
the primarycultured-microglia
fromknockinﬂammatory responses related to activated microglia after trauout
mice
showedadecreaseinthelevels
of
IL10
under
matic brain injury in progranulin-deﬁcient mice. Neuroscience
OGD,250:although
2013;
8–19. levels of inﬂammatory cytokines such as
TNF-changed. Consistentwith
TaoJ, Ji F, and
WangTGF-b
F, Liu B,were
Zhu not
Y. Neuroprotective
effects of progranulin
in ischemic
mice. Brain
Res 2012;
1436:that
130–6.
this ﬁnding,previous
studiesreported
the level of
Van
Damme
Vancells
HoeckeA, Lambrechts
D, Vanacker P, Bogaert
E,
IL10
in P,
glial
from
PGRN-overexpressing
transgenic
van Swieten J, et al. Progranulin functions as a neurotrophic factor
mice
wasneurite
higherthan and
thatenhancefrom neuronalwild-typemiceJ Cell
(Taotoregulateoutgrowthsurvival.
et al.,and that the macrophage/microglia from
Biol
2008;2012),
181: 37–41.
Yenari
Kauppinen
TM,released
Swanson less
RA. IL10
Microglial
in
PgrnMA,knockoutmice
thanactivationfrom wildstroke:
therapeutic
Neurotherapeutics
7: 378–91.
type mice
whentargets.
exposedto bacterial2010;
lipopolysaccharide
Yin F, Banerjee R, Thomas B, Zhou P, Qian L, Jia T, et al.
(Yin et al., 2010). However, ﬁndings on pro-inﬂammatory
Exaggerated inﬂammation, impaired host defense, and neuropatholcytokines
are controversial and inconclusive. It has been
ogy in progranulin-deﬁcient mice. J Exp Med 2010; 207: 117–28.
demonstrated
levelsAK,
of Ishibashi
other pro-inﬂammatory
YoshidaA, Yoshidathat
S, KhalilT, Inomata H. Rolecytoof
in glialin cells
from neovasPGRNNF-kappaB-mediatedexpression
intraocularkines (IL1B, IL6 interleukin-8
and TNF-)cularization.
Invest transgenic
Ophthalmol Vis
Sci 1998;
1097–106.
mice
were 39:lower
than thoseoverexpressing
Zausinger
S,
Hungerhuber
E,
BaethmannA,
ReulenH, thatSchmidfrom wild-type mice (Tao et al., 2012), andthey
Elsaesser R. Neurological impairment in rats after transient middle
wereincreasedinthemacrophage/microglia
from
Pgrn
cerebral artery occlusion: a comparative study under various treatknockout
miceBraincomparedwild-type
ment
paradigms.
Res 2000;to863:
94–105. mice when mice
exposedtoYF,
bacterial
et CD,
al., 2010).
Zhang
YJ, XuCook C,lipopolysaccharide
Gendron TF, Roettges(YinP, Link
et al.
Aberrantcleavageof TDP-43
aggregation and cellular
toxIn contrast,
a veryrecentenhances
study demonstrated
that mRNA
icity.
Proc
Natl
Acad
Sci
USA
2009;
106:
7607–12.
levels of Il1b, Il6, TNF- (now known as Tnf), and TGF-b
Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, et al.
(now
known as Tgfb1) (Il10 not examined) after ischaemic
Progranulin mediates caspase-dependent cleavage of TAR DNA
brain injury
wereJ Neurosci
not associatedwithPgrn knockout conbinding
protein-43.
2007; 27:
10530–4.
ditionZG,as Zhang
seen inresults(Jackman et al.,2013). D,We
Zhang
L, ourTsang
W, Soltanian-Zadeh
H, Morris
Zhang
R, etbased
al. Correlationof VEGF andexpression
consider,on our ﬁndings,
thatangiopoietin
PGRN may
suppresswith
disruption of blood-brain
barrier
after focal
neuroinﬂammation
via IL10
not andby angiogenesis
inhibiting pro-inﬂamcerebral ischemia. J Cereb Blood Flow Metab 2002; 22: 379–92.

matory cytokines.
Third, we conﬁrmed that PGRN might play a neuroprotective role against acute focal cerebral ischaemia in vitro
and in vivo. Neuronal PGRN decreased in the ischaemic
core where neuronal death occurred, and increased in theischaemic penumbra where neuronal survival occurred after
acute focal cerebral ischaemia. In vitro studies demonstrated that the neuronal cells and microglia produced
and secreted PGRN after ischaemia, and that exogenous
recombinant PGRN could suppress neuronal cell deathunder OGD. Although PGRN has been shown to haveneurotrophic effects, the exact mechanism remains unknown (Van Damme et al., 2008; Laird et al., 2010). We
consider that the neurotrophic effects of PGRN might be
explained in part by the inhibition of abnormal cytoplasmic－ 230 －

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Kleinberger
G, in
Wils
H, Ponsaerts
Jorisischaemic
G, Timmermans
JP, Vanexpression
neurons
withinP, thecore, increased
Broeckhoven C, et al. Increased caspase activation and decreased
level of PGRN expression in the surviving neurons, as well
TDP-43 solubility in progranulin knockout cortical cultures. J
as inductionof 115:
PGRN
expression in microglia and endoNeurochem
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thelial cells RG,
in theischaemic
penumbra.
anaKrishnamurthy
Senut
MC, Zemke
D, MinImmunoblot
J, Frenkel MB,
lyses demonstrated
that acid,the afully
glycosylated
Greenberg
EJ, et al. Asiatic
pentacyclic
triterpene mature
fromCentella
is neuroprotective
in a mouse
model
focal
secretoryasiatica,
isoform(88 kDa) decreased,
while
theofglycosycerebral
ischemia.
J
Neurosci
Res
2009;
87:
2541–50.
lated immature isoform (58–68 kDa) markedly increased
Laird AS, Van Hoecke A, De Muynck L, Timmers M, Van den
after reperfusion.
in vitroisexperiments
Bosch
L, Van DammeInP,addition,
et al. Progranulin
neurotrophic revealedin vivothattheglycosylated
matureisoformsecretedPLoS
fromand protects against a mutant TDP-43 inducedwas
axonopathy.
neurons
microglia, and the glycosylated immature isoOne
2010;and5: e13368.
LiauLM,was
Lallone
RL, Seitz
Buznikov
A, Gregg
JP, Kornblum
form
secreted
onlyRS,frommicroglia.
Consistent
withHI,
thisetal.
Identiﬁcation
of
a
human
glioma-associatedgrowthfactor aﬁnding, immunohistochemical analyses demonstrated
gene, granulin, using differential immuno-absorption. Cancer Res
marked increase in PGRN-positive microglia at 72 h after
2000; 60: 1353–60.
reperfusion
in ischaemic
rats. T,These
ﬁndingsK, suggest
that
MabuchiT, Kitagawa
K, Ohtsuki
Kuwabara
Yagita Y,
the marked
in the glycosylated
immature isoform
Yanagihara
T, increase
et al. Contributionof microglia/macrophages
to expansionof infarction
and responseof oligodendrocytes
focalbyof 58–68
kDa PGRN
after reperfusion
was aftercausedcerebralischemia in rats. Stroke 2000; 31: 1735–43.
microglia.
Memezawa H, Smith ML, Siesjö BK. Penumbral tissues salvaged by
We nextfollowing
demonstrated
that artery
PGRN
has multiple
therareperfusion
middle cerebral
occlusionin rats. Stroke
peutic23:effects
1992;
552–9. against ischaemic brain injury. First, wefoundR,that
PGRN
could
blood–brain
barrierGJ.
disMilner
HungS, Wang
X, attenuate
Berg GI, Spatz
M, del Zoppo
Responses
of endothelial
cell andcerebral
astrocyte ischaemia.
matrix-integrin
ruption after
acute focal
Wereceptors
demontoischemiain theneurovascular
unit. Stroke
stratedformimic
the thoseﬁrst observed
time thatPGRN
is expressedin the2008; 39: 191–7.
endothelial
cells
in
the
ischaemic
penumbra.
A
previous
Naphade SB, Kigerl KA, Jakeman LB, Kostyk SK, Popovich PG,
reportJ. Progranulin
has shownexpressionthat PGRN
is inducedin thecontusion
capillary
Kuret
is upregulated
after spinal
endotheliumof wound 2010;
granulation
tissue and promotes theinmice. Acta Neuropathol
119: 123–33.
Okubo
S, andIgarashiH, Kanamatsu
Hasegawa
D, Orima H,
mitosismigrationof adultT,dermal
microvascular
cells
Katayama Y. FK-506 extended the therapeutic time window for(He et al., 2003b), although PGRN is not expressed in thethrombolysis without increasing the risk of hemorrhagic transformhealthy endothelium (Daniel et al., 2000). We speculated
ation in an embolic rat stroke model. Brain Res 2007; 1143: 221–7.
that T,theof PGRN
in endothelial
cells
may
Osada
Guexpression
YH, Kanazawa
M, Tsubota
Y, Hawkins BT,
Spatz
M,be
involvedin the vascular
protection
or repairagainstischaeetal. Interendothelial
claudin-5
expression
depends
on cerebral
endothelial
adhesion oedemaby b(1)-integrins.
J Cereb
mic injury.cell-matrix
Indeed, cerebral
volume inPgrn BloodknockFlow
Metab
2011;
31:
1972–85.
out mice was larger than that in wild-type mice after focalÖzdemir YG, Bolay H, Erdem E, Dalkara T. Occlusion of the MCA
cerebralischaemia.
Regardingmechanism
byhippowhichbyan intraluminalﬁlament
may causethe
disturbancesin the
PGRN
regulates
vascularpermeability,
weconsideredthatcampal blood ﬂow due to anomalies of circle of Willis and ﬁlament
cerebral oedema
not causedby recruitment of neutrophils
thickness.
Brain Resis1999;
822: 260–4.
Tangkeangsirisin
W,basedSerreroon
G. thePC-cellderivedfrom
growthfactor (PCDGF/
and microglia
ﬁndings
immunostainings
GP88)
stimulates
migration,
invasiveness
and
VEGF
in
using wild-type and Pgrn knockout mice. Aexpression
very recent
breast cancer cells. Carcinogenesis 2004; 25: 1587–92.
study demonstrated the involvement of the PDGF receptor
Toh H, Cao M, Daniels E, Bateman A. Expression of the growth
pathway
using Pgrn
knockout(Jackmanet andal., devel2013).
factorprogranulin
in endothelial
cellsmiceinﬂuences
growthWe proposed
PGRN
regulate
opmentof bloodanothervessels: apossibility
novel mousethat
model.
PLoSmight
One 2013;
8:e64989.
vascular permeability via VEGF pathway, because we
Shankaran SS, Capell A, Hruscha AT, Fellerer K, Neumann M,
observed more prominent expression of VEGF in Pgrn
Schmid B, et al. Missense mutations in the progranulin

M.M.
Kanazawa
Kanazawa
et etal. al.

Effects of progranulin on stroke

BRAIN 2015: 138; 1932–1948

Acknowledgements
We thank Dr Riuko Ohashi (Department of Cellular
Function, Division of Cellular and Molecular Pathology,
Niigata University) for her support in the confocal imaging
studies.

Funding
This work was supported by a Grant-in-Aid for ScientiﬁcResearch, a grant from Naito Foundation, Takeda Science
Foundation, Japan Cardiovascular Research Foundation,
Japan Brain Foundation, Yujin Memorial Grant, Japan
Medical Association (TS), and a Grant-in-Aid for
Scientiﬁc Research, a grant from SENSHIN Medical
Research Foundation, Uehara Memorial Foundation (MK).

Supplementary material
Supplementary material is available at Brain online.

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redistribution of nuclear TARDBP (Zhang et al., 2007,2009). Stated another way, the decreased level of PGRN
might cause loss of function of TARDBP in neurons, resulting in neuronal damage.
Finally, we demonstrated that intravenously administered
recombinant PGRN reduced volumes of cerebral infarct
and oedema, suppressed haemorrhagic transformation,and improved motor outcome with delayed tPA treatment
resulting in haemorrhagic transformation that is similar tohuman stroke, in a rat autologous embolic model(Kanazawa et al., 2011a; Kawamura et al., 2014). A previous study reported that intraventricular administration ofrecombinant PGRN reduced cerebral infarct and oedema ina mouse focal suture ischaemic model (Egashira et al.,2013), although this model and injection route poorly reﬂected the clinical practice. Our ﬁndings indicate the possibility that recombinant PGRN could be used as a novel
neurovascular protective drug with anti-inﬂammatory effectafter delayed tPA treatment.
Because of the importance of protein glycosylation inmediating a wide range of biological processes, characterization of glycan structures is necessary to understand thestructure–function relationship (Songsrirote et al., 2010).
Therefore, we chose the full-length glycosylated recombinant PGRN protein. We could not evaluate the impact ofglycosylated status of the PGRN on neuronal cells because
it is technically hard to use deglycosylated PGRN after
PNGase assay because of reagent toxicities for cells.
Further studies are needed to determine the impact of glycosylation status for recombinant PGRN on this effect.
In conclusion, we demonstrated dynamic changes of expression and localization of PGRN after acute focal cerebral ischaemia. PGRN may be a novel therapeutic target
that provides vascular protection, anti-neuroinﬂammation,and neuroprotection related in part to VEGF, IL10 and
TARDBP, respectively.

| 1947

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1932–1948

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A

F

B

－ 232 －RESEARCH ARTICLE

Clinicopathological Features in Anterior
Visual Pathway in Neuromyelitis Optica
Mariko Hokari, MD,1 Akiko Yokoseki, MD, PhD,1 Musashi Arakawa, MD, PhD,1
Etsuji Saji, MD, PhD,1 Kaori Yanagawa, MD, PhD,1 Fumihiro Yanagimura, MD,1
Yasuko Toyoshima, MD, PhD,2 Kouichirou Okamoto, MD, PhD,3Satoshi Ueki, MD, PhD,4 Tetsuhisa Hatase, MD, PhD,5 Riuko Ohashi, MD, PhD,6
Takeo Fukuchi, MD, PhD,5 Kohei Akazawa, PhD,7 Mitsunori Yamada, MD, PhD,8
Akiyoshi Kakita, MD, PhD,2 Hitoshi Takahashi, MD, PhD,2Masatoyo Nishizawa, MD, PhD,1 and Izumi Kawachi, MD, PhD1
Objective: Neuromyelitis optica spectrum disorder (NMOsd) is an autoimmune disorder of the central nervous system characterized by aquaporin-4 (AQP4) autoantibodies. The aim of this study was to elucidate the characteristics
of involvement of the anterior visual pathway (AVP) and neurodegeneration via glia–neuron interaction in NMOsd.
Methods: Thirty Japanese patients with serologically verified NMOsd were assessed with a neuro-ophthalmologicalstudy. Using 27 tissue blocks from 13 other cases of NMOsd, we performed neuropathological analysis of glial andneuroaxonal involvement in the AVP.
Results: The AVP involvement in NMOsd was characterized by the following, compared to multiple sclerosis: (1) longitudinally extensive optic neuritis (ON); (2) more severe visual impairment and worse prognosis for ON; (3) unique
AQP4 dynamics, including loss of AQP4 immunoreactivity on astrocytes with complement activation in ON lesions,loss of AQP4 immunoreactivity on M€
uller cells with no deposition of complement in the retinas, and densely packed
AQP4 immunoreactivity on astrocytes in gliosis of secondary anterograde/retrograde degeneration in the optic
nerves and retinal nerve fiber layer (RNFL); and (4) more severe neurodegeneration, including axonal accumulation ofdegenerative mitochondria and transient receptor potential melastatin 4 channel with complement-dependent astrocyte pathology in ON lesions, mild loss of horizontal cells, and RNFL thinning and loss of ganglion cells with abundance of AQP41 astrocytes, indicating secondary retrograde degeneration after ON.
Interpretation: Severe and widespread neuroaxonal damage and unique dynamics of astrocytes/M€
uller cells withalterations of AQP4 were prominent in the AVP and may be associated with poor visual function and prognosis in
NMOsd.
ANN NEUROL 2016;79:605–624

N

euromyelitis optica (NMO) spectrum disorder(NMOsd)1 and multiple sclerosis (MS) are the twomain autoimmune, inflammatory, and demyelinating
syndromes of the central nervous system (CNS). The
serum NMO immunoglobulin G autoantibody targets

the water channel aquaporin-4 (AQP4) and is a specificmarker of NMOsd.2,3 The anterior visual pathway(AVP), which includes the retinas and the pathway fromthe optic nerves to the lateral geniculate nuclei, is a frequent site of injury, as shown by the presence of optic

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24608
Received Aug 14, 2015, and in revised form Jan 28, 2016. Accepted for publication Jan 29, 2016.
Address correspondence to Dr Kawachi, Department of Neurology, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata
951-8585, Japan. E-mail: ikawachi@bri.niigata-u.ac.jp
From the 1Department of Neurology, Brain Research Institute, Niigata University, Niigata; 2Department of Pathology, Brain Research Institute, Niigata
University, Niigata; 3Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata; 4Center for Integrated Human Brain Science,
Brain Research Institute, Niigata University, Niigata; 5Department of Ophthalmology, Niigata University, Niigata; 6Department of Pathology, Niigata
University Medical and Dental Hospital, Niigata; 7Department of Medical Informatics, Niigata University Medical and Dental Hospital, Niigata; and8
Department of Brain Disease Research, Shinshu University School of Medicine, Matsumoto, Nagano, Japan.
Additional supporting information can be found in the online version of this article.

C 2016 American Neurological Association
V
605－ 233 －

ANNALS of Neurology

neuritis (ON) during the course of NMOsd. Comparedto MS, ON in NMOsd is characterized as follows: (1)visual loss is generally more severe in NMOsd4; (2) the
occurrence of bilateral simultaneous ON is more suggestive of NMOsd5; and (3) optic coherence tomography(OCT) assessment in NMOsd demonstrates a thinnerretinal nerve fiber layer (RNFL), suggesting more widespread axonal injury.6,7
In the optic nerves, chiasm, and tracts, expressionof AQP4 is generally enriched in astrocytic endfoot
membrane domains that face capillaries and the pia in
the CNS.8 In the retinas, a striking feature of AQP4
expression is its polarized distribution in 2 types of retinal glia: (1) M€
uller cells, which are a specialized form ofradial glia spanning the width of the retina from theinner limiting membrane to the outer limiting membrane, with the cell soma lying within the inner nuclear
layer (INL); and (2) retinal astrocytes, which are mostly
localized in the RNFL, which is composed predominantly of unmyelinated axons of retinal ganglion cells.
However, little is known about the details of the pathology in the AVP, including the dynamics of AQP4 expression in M€
uller cells and astrocytes in NMOsd. Here we
report that more severe and widespread neuroaxonal
damage and unique dynamics of AQP4 in astrocytes/
M€uller cells was prominent in the AVP and may be associated with visual function and prognosis in NMOsd byusing neuro-ophthalmological, radiological, and pathological assessments.

Patients and Methods
Neuro-ophthalmological and Neuroradiological
Assessments
We retrospectively reviewed the medical records of 79 Japanesepatients (67 women, 12 men; 30 patients with NMOsd, 49
patients with MS) between 2000 and 2013 at the MS/NMO
clinic in the Department of Neurology at Niigata University
Hospital. Patients were included in the study according to theseocular criteria: (1) intraocular pressure <20mmHg; (2) absenceof current or previous history of other ocular diseases, including
glaucoma, retinal detachment, early age-related macular degeneration, other macular degeneration, and retinal vascular diseases; and (3) adequate clinical data on follow-up(Supplementary Table 1). AQP4 autoantibodies were measured
in the sera of all patients using the method described in ourprevious reports.9,10 We stringently defined the definite form of
NMOsd as fulfilling all items of the 2006 NMO criteria,1 andlimited NMOsd to either: (1) ON and seropositivity for AQP4
autoantibodies but without spinal cord involvement or (2) myelitis with seropositivity for AQP4 autoantibodies but without
optic nerve involvement. As a disease control, MS was defined
as clinically definite MS according to the International Panelcriteria for MS,11 after excluding definite and limited NMOsd.

In NMOsd and MS patients, ON-affected eyes were defined aseyes with a previous clinical episode of ON or abnormalities in
the AVP on magnetic resonance imaging (MRI) findings. ONunaffected eyes were defined as eyes with no history of ON, noabnormalities in the AVP on MRI findings, and no abnormalities in the visual field (VF).
Visual function testing was performed using visual acuity(VA; Landolt broken ring chart), VF (automatic perimetry;
Humphrey; Carl Zeiss Meditec, Jena, Germany), and visualevoked potential (Neuropack X1; Nihon Kohden, Tokyo,
Japan). OCT was performed on both eyes using Fourier/spectral-domain (F/SD)-OCT with an RTVue-100 instrument(Optovue, Fremont, CA) with software version A4.0.
To detect ON lesions with MRI, coronal-oblique fat-saturated dual echo fast spin-echo images with 3mm-thick slices,
coronal-oblique 3-dimensional (3D) fast imaging employing
steady-state acquisition with 0.8- to 1.0mm-thick slices, andcoronal-oblique fat-saturated T1-weighted spin-echo images
with 3mm-thick slices before and after administration of
0.1mmol/kg intravenous gadolinium were acquired using a
1.5T scanner (GE Medical Systems, Milwaukee, WI; Philips
Achieve, Philips Medical Systems, Best, the Netherlands). The
3D double inversion recovery (DIR) images (repetition time 55,500 milliseconds, echo time 5 350 milliseconds, inversion
time 1 (TI1) 5 2,350 milliseconds; TI2 5 350 milliseconds,
number of excitations 5 2, echo train length 5 208, measured
voxel size 5 1.30 3 1.31 3 1.30mm) were acquired using a
1.5T scanner.
The present study was approved by the institutional
review board of the Niigata University School of Medicine, Niigata, Japan. Written informed consent was obtained from all
patients or their guardians.

T

C

Neuropathological Assessments
This study was conducted on retina and optic nerve tissues
from 13 patients with NMOsd (3 retinas, 13 optic nerves), 7
patients with MS (1 retina, 7 optic nerves), and 8 patients withother diseases (as controls; 4 retinas, 4 optic nerves; Supplementary Table 2). These individuals were different from those who
underwent neuro-ophthalmological and neuroradiologicalassessments. One to 4 blocks of retinas and optic nerves wereassessed per autopsy case. Four-micrometer-thick, paraffinembedded sections were obtained and stained with hematoxylin
and eosin, Kl€uver–Barrera staining, and Bodian silver impregnation. Selected sections were immunostained with polyclonal or
monoclonal antibodies (Supplementary Table 3).9,12,13 All
NMOsd and MS lesions were classified according to lesion
activity.14 The area of Wallerian degeneration (Wld) was
defined as an extensive loss of myelin and axons and profoundgliosis with densely packed glial fibrillary acidic protein (GFAP)
and AQP4 immunoreactivity (IR). For quantitative assessment,
the content of mitochondria or channel proteins was determined as the volume occupied in axons using X-Y and X-Z
confocal images, or as a percentage of the axonal volumes.
Serial optical sections at intervals of 0.49lm in the Z dimension were captured by using a LSM510 or LSM710 laser

606

Volume 79, No. 4－ 234 －

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Hokari et al: Lesions in NMO

TABLE 1. Clinical Characteristics of Neuro-ophthalmological Assessment

Characteristic

NMOsd Patients,
25 Eyes, 28 Attacks

MS Patients,
22 Eyes, 25 Attacks

Episodes of anterior visual pathway
involvement, No.a

1.1 6 0.4

1.2 6 0.4

Spherical equivalent, dioptersa

23.1 6 2.1

22.1 6 2.5

Optic nerve head involvement at ON attacks,No. (%)

10/28 (36)

4/25 (16)Visual acuity, logMAR, at ON attacks,
median [range]

1.52 [0.73–3.00]b

0.30 [0–1.35]b

Severe visual loss at ON attacks, No. (%)c20/28 (71)b

9/25 (36)b

Severe visual loss at 6 months after ON attacks,No. (%)c

9/28 (32)b0/25 (0)bChronic progressive visual disturbance,
No. (%)d7/28 (25)

2/25 (8)MD, dBa216.4 6 9.1b28.4 6 6.7b

PSD, dBa

11.0 6 3.8b6.0 6 3.8b

CPSD, dBa

10.6 6 4.6b

4.9 6 2.7b

I. Neurologic abnormalities, No. (%)

7/28 (25)

1/25 (4)I.1. Vertical step

0 (0)

1 (4)

I.2. Quadrant

0 (0)

0 (0)

I.3. Partial hemianopia

0 (0)

0 (0)

I.4. Hemianopia

3 (11)

Visual acuity analysis

Visual field defects at ON attacks

I.5. Three quadrant
II.1. Nerve fiber bundle abnormalities, No. (%)

4 (14)0 (0)b

0 (0)b5/28 (18)

3/25 (12)II.1.a. Temporal wedge

0 (0)

0 (0)

II.1.b. Enlarged blind spot

0 (0)

1 (4)

II.1.c. Nasal step

1 (4)

0 (0)

II.1.d. Paracentral

1 (4)

0 (0)

II.1.e. Partial arcuate

0 (0)

0 (0)

II.1.f. Arcuate

0 (0)

2 (8)

II.1.g. Altitudinal

3 (11)0 (0)0/28 (0)6/25 (24)II.2.a. Multiple foci

0 (0)

2 (8)

II.2.b. Widespread

0 (0)II.2. Diffuse abnormalities, No. (%)

II.3. Central abnormalities, No. (%)
II.3.a. Centrocecal
II.3.b. Central
II.4. Total loss of vision, No. (%)

4 (16)

4/28 (14)

b

2 (7)

13/25 (52)b
0 (0)

b

13 (52)b

12/28 (43)b

1/25 (4)b2 (7)April 2016

607

－ 235 －

ANNALS of Neurology

TABLE 1: Continued

T
In

Characteristic

NMOsd Patients,
25 Eyes, 28 Attacks

MS Patients,
22 Eyes, 25 Attacks

Artifactual abnormalities, No. (%)

0/28 (0)1/25 (4)Normal, No. (%)

0/28 (0)0/25 (0)12/13 (92)

15/16 (94)DI

VEP analysis at ON attacks
VEP abnormalities, No. (%)
aMean 6 standard deviation.
Statistically significant difference between the linked values (p < 0.05).
c
Severe visual loss was defined as visual acuity worse than 20/200 (1.0 logMAR).
d
Chronic visual disturbance was defined as progressive abnormalities of visual acuity (10.3 logMAR or more) and/or visual fieldfor >2 months.
CPSD 5 corrected pattern standard deviation; MD 5 mean deviation; MS 5 multiple sclerosis; NMOsd 5 neuromyelitis optica
spectrum disorder; ON 5 optic neuritis; PSD 5 pattern standard deviation; VEP 5 visual evoked potential.
bconfocal microscope (Carl Zeiss, Gottingen, Germany), andImaris software 7.6.4 (Bitplane, Zurich, Switzerland) was used
to generate the 3D reconstructed images.15 Sections with long(20lm) axonal segments were identified based on an established morphological study and after minimizing identificationof false-positive segments lacking mitochondrial elements
because of the naturally discontinuous distribution of mitochondrial elements and the variability in the intermitochondrial
distance within axons. To obtain these data, we quantified axonal morphology and the volume of mitochondria or channel
proteins in 262 images (3100 objective) acquired from 2 to 6
fields of each area in 20 tissue blocks of the optic nerves from
8 NMOsd cases, 4 MS cases, and 5 controls.

Statistical Analyses
Data analyses were performed using GraphPad Prism 5 software(GraphPad Software, San Diego, CA), IBM SPSS 19 (IBM, Chicago, IL), and Stata 11 software (StataCorp, College Station,
TX). Comparisons of medians and proportions between NMOsd
and MS groups were performed using the Mann–Whitney U
test or the Fisher exact test. Statistical tests among 3 subgroups
were performed using analysis of variance, the Kruskal–Wallis H
test, or the chi-square test. Multiple comparisons among subgroups were performed using the Bonferroni or Bonferroni–
Dunn tests. Generalized estimating equation (GEE) models,accounting for the history of ON, age, and the effects of withinpatient and intereye correlations, were used to determine the

TABLE 2. Clinical Characteristics of Neuroradiological Assessment: Contrast-Enhanced Magnetic ResonanceImaging Analysis of the Optic Nerves, Chiasma, and Tracts at the Time of ON Attack

Characteristic

NMOsd Patients,
21 Eyes, 28 Attacks

MS Patients,
18 Eyes, 21 Attacks

Optic perineuritis, No. (%)

13/28 (46)

6/21 (29)Simultaneous bilateral involvement, No. (%)

5/28 (18)

2/21 (10)Distribution of abnormal enhancement during
ON attacks, No. (%)
Nerve head segment involvement

0/28 (0)0/21 (0)Orbital segment involvementa24/28 (86)

10/21 (48)aCanalicular segment involvement

21/28 (75)

13/21 (62)Intracranial segment involvement

9/28 (32)

6/21 (29)

Optic chiasm involvement

1/28 (4)

2/21 (10)Optic tracts involvement

1/28 (4)

1/21 (5)aStatistically significant difference between the linked values (p < 0.05).
MS 5 multiple sclerosis; NMOsd 5 neuromyelitis optica spectrum disorder; ON 5 optic neuritis.

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Hokari et al: Lesions in NMO

TABLE 3. Clinical Characteristics of Neuroradiological Assessment: Magnetic Resonance Imaging Double
Inversion Recovery Analysis of the Optic Nerves, Chiasma, and Tracts in Remission

Distribution of Abnormal
Intensity Lesion

NMOsd Patients,
13 Eyes, 36 Attacks

MS Patients,
18 Eyes, 41 Attacks

Nerve head segment involvement, No. (%)

0/13 (0)0/18 (0)Orbital segment involvement, No. (%)

13/13 (100)17/18 (94)

Canalicular segment involvement, No. (%)

12/13 (92)

11/18 (61)a

5/18 (28)aIntracranial segment involvement, No. (%)

10/13 (77)

Optic chiasm involvement, No. (%)

9/13 (69)a3/18 (17)aOptic tracts involvement, No. (%)

6/13 (46)

2/18 (11)aStatistically significant difference between the linked values (p < 0.05). MS 5 multiple sclerosis; NMOsd 5 neuromyelitis optica
spectrum disorder.

relationship with clinical parameters, including neuroophthalmological and neuroradiological assessments (Tables 1–3;
Fig 1). For all GEE analyses, age was controlled in the models,
because this variable is an important factor in visual function
and RNFL thickness.16 Cumulative probabilities of the followup visual performance as assessed with time to recovery of VA
with 3 lines change from the nadir of ON attacks or to recovery
of VA to 20/25 (0.1 logMAR) were estimated by the Kaplan–
Meier method, and the significance test among curves of cumulative probabilities and survival rates was performed by the logrank test. All statistical analyses were considered significant at pvalues of <0.05.

Results
Neuro-ophthalmological Features of NMOsd at
the Time of ON Attacks
Of the 158 eyes that met the inclusion criteria in theseries, 31 of 60 eyes (20 of 30 patients, 67%, 56 attacks)
with NMOsd and 41 of 98 eyes (26 of 49 patients,
53%, 57 attacks) with MS were diagnosed as having
AVP involvement.
For the neuro-ophthalmological assessments at thetime of ON attacks, eyes were excluded17 if the affectedeyes had a prior ON attack and vision did not return to
20/20 (0 logMAR), mean deviation (MD) < 22.00dB at
a prior ON attack, or if optic disk pallor, a known
baseline-corrected VA < 20/40 (>0.3 logMAR), or VF
loss due to any other ophthalmic disorder was present, andthen we evaluated each attack of ON-affected eyes with
NMOsd (25 eyes, 28 attacks) and MS (22 eyes, 25 attacks;
see Table 1). Among ON-affected eyes with NMOsd andMS, the mean (standard deviation [SD]) number of episodes of AVP involvement in eyes with NMOsd and MS
was 1.1 (0.4) and 1.2 (0.4), respectively. In the VA analysis, the median VA (logMAR) at the time of ON attacks
was significantly impared. The frequency of eyes withsevere visual loss, which was defined as VA worse than 20/
200 (1.0 logMAR), at onset was significantly higher ineyes with NMOsd compared with those with MS. In the
VF analysis, the VF defects indicated by the average VFMD, pattern SD, and corrected pattern SD were significantly impaired in eyes with NMOsd compared with thosewith MS. VF morphological analysis using the Optic Neuritis Treatment Trial classification18 demonstrated that“neurologic abnormalities (I)” and “total loss of vision(II.4)” were prominent in ON-affected eyes with NMOsd,
whereas “central abnormalities (II.3)” were evident inthose with MS. “Altitudinal abnormalities (II.1.g)” were
present in 3 of 28 (11%) attacks with NMOsd, but 0 of
25 (0%) attacks with MS.
Intriguingly, 7 of 28 (25%) attacks in NMOsd and2 of 25 (8%) attacks in MS were associated with chronic
progressive visual deterioration, which was defined asprogressive abnormalities in VA (10.3 logMAR or more)
and/or VF for >2 months.
Neuroradiological Features on Enhanced MRI in
NMOsd at the Time of ON Attack
For the neuroradiological assessments at the time of ON
attacks, we evaluated the AVP using enhanced T1weighted images with fat suppression. Of 34 patients (17patients with NMOsd; 17 patients with MS) who underwent MRI examination at ON attack, abnormal enhancement was present in 16 of 17 (94%) patients (21 eyes,
28 attacks) with NMOsd, and in 16 of 17 (94%)
patients (18 eyes, 21 attacks) with MS (see Table 2; Fig1). MRI findings demonstrated that the length of abnormally enhanced lesions in NMOsd patients was significantly longer than those in MS patients, suggesting that
longitudinally extensive lesions were prominent in the
optic nerves in NMOsd. During attacks, radiologic

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T

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FIGURE 1: Radiological profiles of optic neuritis (ON) at the time of attacks in neuromyelitis optica (NMO) spectrum disorder(NMOsd) and multiple sclerosis (MS). Anterior visual pathways (AVP) during ON in NMOsd and MS were examined by using
axial and coronal views of enhanced T1-weighted magnetic resonance images with fat suppression. (A) They were divided into
6 segments: head, orbital, canalicular, and intracranial segments of the optic nerves, the optic chiasm, and the optic tracts.
Abnormal enhancement on fat-suppressed magnetic resonance imaging (MRI; arrows) was present in patients with NMOsd andMS. (B) The length of abnormally enhanced lesions in the AVP in NMOsd at the time of ON attacks (n 5 28 events) was significantly longer than in MS attacks (n 5 21 events). The pattern of abnormal enhancement in the AVP at the time of ON attacks
was generally homogeneous in most NMOsd and MS patients (A). However, the pattern of optic nerve sheath enhancement,
which is referred to as a “tram track sign” on axial views and a “doughnut sign” on coronal views, was also present in 46% ofattacks in NMOsd patients and 29% of attacks in MS patients (A). This was defined as optic perineuritis (OPN).19 The pathologic evidence (see Fig 4) indicated that OPN was associated with more widespread meningeal and perineural inflammation
around the optic nerves. (C, D) Three-dimensional (3D) double inversion recovery (DIR) MRI detected accumulation of archival
ON lesions in NMOsd and MS. DIR MRI simultaneously suppresses the signals from white matter and cerebrospinal fluid (CSF).
A substantial increase in cortical demyelinating lesions in patients with MS was found compared to more conventional sequences, including fluid-attenuated inversion recovery (FLAIR) imaging.49 Therefore, not only cortical demyelinating lesions but also
ON lesions, which usually contact the CSF, were detected with high sensitivity by using DIR imaging (C, arrows) compared to
FLAIR imaging in ON lesions in NMOsd and MS. The distribution of abnormal intensity on 3D-DIR images showed accumulationof archival lesions through the disease course, based on longitudinally evaluated MRI data. A representative case is shown in
D. A 38-year-old woman experienced 3 attacks of acute ON and 6 attacks of myelitis (M) during the 12 years after disease
onset. The distribution of abnormal intensity on 3D-DIR images 12 years after disease onset ($4, red, blue, and yellow arrowheads) equaled the addition of each abnormally enhanced lesion in 3 attacks of ON ($1, red arrowheads, 6 years after onset;$2, blue arrowheads, 7 years after onset; $3, yellow arrowheads, 11 years after onset; D). Gd T1WI 5 gadolinium-enhanced
T1-weighted image; Lt. 5 left eye; Rt. 5 right eye.

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TABLE 4. Clinical Characteristics of OCT Assessment: OCT Analysis of the Retinas in Remission

NMOsd Patients
Characteristic

MS Patients

Unaffected Eyes,
14 Eyes

Affected Eyes,
8 EyesUnaffected Eyes,
18 Eyes

Affected Eyes,
8 EyesMean RNFL, lma

95.9 6 9.4

63.9 6 10.2

96.5 6 9.7

78.9 6 9.5

Within normal limits, No. (%)

11/14 (79)

0/8 (0)11/18 (61)

1/8 (13)

Borderline, No. (%)

1/14 (7)0/8 (0)5/18 (28)

0/8 (0)Outside normal limits, No. (%)

2/14 (14)8/8 (100)

2/18 (11)

7/8 (88)

2.2 6 2.5

13.3 6 4.1

2.1 6 2.3

9.9 6 4.9

Mean GCC, lma

87.3 6 12.5

60.4 6 11.0

89.2 6 5.8

70.1 6 7.9Within normal limits, No. (%)

9/14 (64)0/8(0)

13/18 (72)

0/8 (0)Borderline, No. (%)

0/14 (0)0/8 (0)2/18 (11)

1/8 (13)

Outside normal limits, No. (%)

5/14 (36)

8/8 (100)

3/18 (17)7/8 (88)

Mean RNFL thickness

16-sector RNFL analysis
Average number of sectors withborderline or outside normal limitsa
Mean GCC thicknessaMean 6 standard deviation.
GCC 5 ganglion cell complex; MS 5 multiple sclerosis; NMOsd 5 neuromyelitis optica spectrum disorder; OCT 5 opticalcoherence tomography; RNFL 5 retinal nerve fiber layer.

characteristic features of optic perineuritis (OPN)19 weremarked in NMOsd patients (13 of 28 [46%] attacks)
compared to MS patients (6 of 21 [29%] attacks), but
the difference was not significant. Some patients with
NMOsd and MS retained abnormal enhancement with
the OPN pattern for >4 weeks.20 Five of 28 (18%)
attacks in NMOsd and 2 of 21 (10%) attacks in MS
simultaneously had bilateral optic nerve involvement.
Neuroradiological Features on 3D-DIR MRI in
NMOsd in Remission
Based on longitudinally evaluated MRI data, the distribution of abnormal intensity on 3D-DIR images showed
accumulation of archival lesions throughout the disease
course (see Fig 1C, D). In remission, 3D-DIR MRI
assessments were performed in 14 patients (28 eyes) with
NMOsd and 28 patients (56 eyes) with MS. Thirteen of
28 (46%) eyes with NMOsd and 18 of 56 (32%) eyeswith MS had abnormal intensities in the AVP. The numbers of segments with optic chiasma and intracranial
involvement were significantly higher in NMOsd patientscompared to MS patients (see Table 3).
OCT Analysis of Retinas with NMOsd inRemission
The cohort that underwent OCT assessment included 11
patients with NMOsd (4 with the definite form and 7with the limited form; 22 eyes; 14 ON-unaffected eyes
and 8 ON-affected eyes) and 13 patients with MS (26eyes; 18 ON-unaffected eyes and 8 ON-affected eyes;
Tables 4 and 5; Fig 2). In NMOsd and MS patients, the
RNFL and ganglion cell complex (GCC) were significantly thinner in ON-affected eyes than in ONunaffected eyes. For the ON-affected eyes, RNFL wassignificantly thinner in NMOsd patients than in MS
patients.
When damage was classified as “borderline” and“outside normal limits” for the RNFL or GCC thickness, the frequency of ON-unaffected eyes with RNFL
thinning was 21% in NMOsd patients and 39% in MS
patients, and the frequency of ON-unaffected eyes with
GCC thinning was 36% in NMOsd patients and 28%
in MS patients (see Table 4). In patients with the limited form of NMOsd with myelitis but no history ofON, the frequency of ON-unaffected eyes with RNFL
thinning was 30%, and the frequency of ONunaffected eyes with GCC thinning was 40% (see Table5). These data suggest that RNFL and GCC loss wasevident in ON-affected and ON-unaffected eyes, andnot only secondary retrograde changes after ON, butalso in vivo primary neuroaxonal pathology, regardlessof ON history, were present in retinas in NMOsd andMS.

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TABLE 5. Clinical Characteristics of OCT Assessment: OCT Analysis of the Retinas in Limited Formof NMOsd with Myelitis Patients in Remission

Characteristic

Limited Formof
NMOsd
with Myelitis
Patients,
Unaffected
Eyes, 10 Eyes

Mean RNFL thickness
Mean RNFL, lma

94.0 6 10.4

Within normal limits, No. (%)

7/10 (70)Borderline, No. (%)

1/10 (10)Outside normal limits, No. (%)

2/10 (20)16-sector RNFL analysis
Average number of sectors
with borderline or outside
normal limitsa

2.5 6 2.8

Mean GCC thickness
Mean GCC, lma

87.3 6 11.2

Within normal limits, No. (%)

6/10 (60)Borderline, No. (%)

0/10 (0)Outside normal limits, No. (%)

4/10 (40)aMean 6 standard deviation.
GCC 5 ganglion cell complex; NMOsd 5 neuromyelitis
optica spectrum disorder; OCT 5 optical coherence tomography; RNFL 5 retinal nerve fiber layer.

Follow-up Visual Performance in NMOsd
We evaluated the follow-up visual performance from thenadir of ON attacks in NMOsd and MS (Fig 3, Table1). The frequency of eyes with severe visual loss, which
was defined as VA worse than 20/200 (1.0 logMAR), at
6 months after ON onset was significantly higher in eyeswith NMOsd compared with those with MS (see Table1). Kaplan–Meier analysis of time to recovery of VA with
3 lines change from the nadir of ON attacks or to recovery of VA to 20/25 (0.1 logMAR) was performed.
Patients with NMOsd developed a significantly worse
outcome regarding visual performance over time thanthose with MS when we evaluated all cumulative attacks
of ON in patients with NMOsd and MS or all firstattacks of ON in each patient with NMOsd and MS(data not shown). Mean follow-up times between the
ON onset and assessment were 11.5 months in NMOsd
patients (25 attacks) and 9.2 months in MS patients (13attacks) or 11.5 months in NMOsd patients (25 attacks)

and 9.4 months in MS patients (14 attacks). All patientswith NMOsd and MS in the analysis were treated withmethylprednisolone pulse treatment (1g per day) for 3
days and/or high-dose oral prednisolone treatment(1.0mg/kg per day) followed by prednisolone tapered tomaintenance level. No patients in this study were treatedwith plasma exchange.
Astrocytopathy with Increased Meningeal
Inflammation in Plaques of the Optic Nerves,
Chiasm, and Tracts in NMOsd
Clinically, 85% (11 of 13) of NMOsd cases and 86% (6 of
7) of MS cases had a previous history of ON. Pathologically,
all cases in NMOsd and MS had several plaques in the AVP(Fig 4). The pathology in the AVP in NMOsd cases wasevaluated in 22 lesion areas, 13 areas of periplaque whitematter (PPWM), 11 areas of normal-appearing white matter (NAWM), and 10 areas of Wld. Similarly, in cases withMS, we evaluated 14 lesion areas, 11 areas of PPWM, and
11 areas of NAWM. All plaques in the optic nerves in
NMOsd cases showed hallmarks of NMO pathology,
including pattern-specific loss of AQP4 IR with thickened
hyalinized blood vessels.9,21 Moreover, thickening of theperioptic meninges with inflammatory infiltration including
CD45RO1 T cells and Iba-11 cells was prominent in themeninges adjacent to the plaques in NMOsd cases. The
meningeal infiltrates also extended into the pial septa andparenchyma of the optic nerves (see Fig 4). Therefore,
lesions in the AVP in NMOsd were pathologically definedas ON and/or OPN with meningeal inflammation, consistent with radiological findings (see Fig 1A).19

FIG
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Severe axonal damage with abnormal
mitochondrial dynamics and aberrant
expression of the TRPM4 cation channel in the
optic nerves, chiasm, and tracts in NMOsd
Axonal pathology was qualitatively and quantitatively
assessed with Bodian silver impregnation, and immunohistochemistry for phosphorylated neurofilaments (SMI31) and amyloid precursor protein. Axonal damage,including axonal loss, transections, swellings, and distorted axons, and axonal spheroids were evident in plaques and PPWM, and had accumulated toward
the plaque center of the AVP in NMOsd and MS (Figs
5–7). The axonal damage was the most prominent inplaques and areas of Wld in NMOsd. The density of
SMI-311 axons was significantly reduced, and the percentages of swollen axons and axonal spheroids were significantly increased in plaques, PPWM, and Wld in
NMOsd, and plaques, PPWM, and NAWM in MS compared to controls. Swollen axons and axonal spheroids
with no significant reduction in axonal density were alsopresent, even in the NAWM in NMOsd.

toati

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FIGURE 2: Optical coherence tomography (OCT) analysis in patients with neuromyelitis optica (NMO) spectrum disorder(NMOsd) and multiple sclerosis (MS). (A, B) The data shown are representative of optic neuritis (ON)-affected eyes with retinal
nerve fiber layer (RNFL) thinning (A) and ON-unaffected eyes with RNFL thinning (B) in NMOsd and MS patients. In the 16sector RNFL analysis, sectors with a classification of “within normal limits” (ie, sectors for which the probability of no damage 5%) are indicated in green, sectors with a “borderline” classification (probability < 5 but  1%) are indicated in yellow, andsectors with a classification of “outside normal limits” (probability < 1%) are indicated in red. The 16-sector RNFL analysis (A)revealed that sectors with a classification of “outside normal limits” (red) and “borderline” (yellow) were present in the ONaffected eyes of NMOsd and MS patients. (C–E) The average thicknesses of the RNFL (C) and ganglion cell complex (GCC; E)
were significantly decreased in the ON-affected eyes compared with the ON-unaffected eyes in patients with NMOsd and MS.
The 16-sector RNFL analysis (D) revealed the average number of normal sectors with significantly decreased thickness in the
ON-affected eyes compared with the ON-unaffected eyes in patients with NMOsd and MS. For the ON-affected eyes, RNFL
was significantly thinner in NMOsd patients than in MS patients (C). Intriguingly, the 16-sector RNFL analysis (B) revealed that
sectors with a classification of “outside normal limits” (red) and “borderline” (yellow) were present in the ON-unaffected eyesof NMOsd and MS patients. When damage was classified as “borderline” and “outside normal limits” for RNFL or GCC thickness, the frequency of ON-unaffected eyes with RNFL thinning was 21% in the NMOsd patients and 39% in the MS patients(see Table 4). The frequency of ON-unaffected eyes with GCC thinning was 36% in the NMOsd patients and 28% in the MS
patients (see Table 4). These data suggest that RNFL and GCC loss is present and is independent of ON in NMOsd and MS.
The peripapillary RNFL results were obtained based on Fourier/spectrum domain OCT using an RTVue-100 instrument inpatients with NMOsd and MS. GCC scanning using the OCT system encompasses the following 3 layers in the retina: (1) the
RNFL, which is composed of ganglion cell axons; (2) the ganglion cell layer, which is composed of ganglion cell bodies; and (3)the inner plexiform layer, which is composed of interlaced dendrites of ganglion cells and axons of bipolar cells with dendritesof amacrine cells (see Fig 8F). *p < 0.05, ***p < 0.001. IN 5 inferonasal; IT 5 inferotemporal; Lt 5 left eye; NL 5 nasal lower;
NU 5 nasal upper; Rt 5 right eye; SN 5 superonasal; ST 5 superotemporal; TL 5 temporal lower; TU 5 temporal upper.

Mitochondrial dynamics is an important contributor to damage and loss of axons in several neurodegenerative disorders.22 We thus assessed the dynamics ofvoltage-dependent anion channel (VDAC)/porin-labeled
mitochondria located in SMI-311 axons in NMOsd
compared to mitochondria in MS and controls (see Fig

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FIGURE 3: Follow-up visual performance in neuromyelitis optica (NMO) spectrum disorder (NMOsd) and multiple sclerosis (MS).
We performed Kaplan–Meier analysis of time to (A) recovery of visual acuity (VA) with 3 lines change and (B) recovery of VA to
20/25 (0.1 logMAR) from the nadir of optic neuritis (ON) attacks in NMOsd and MS patients. Eyes were excluded if deterioration of VA with <3 lines change was present at presentation (A) or VA was >20/25 (<0.1 logMAR) at presentation (B). Patientswith NMOsd developed significantly worse visual performance outcomes over time than those with MS, when we evaluated
not only all accumulative attacks of ON in patients with NMOsd and MS, but also all first attacks of ON in each patient with
NMOsd and MS (data not shown).

6). First, morphological analysis of VDAC-11 mitochondria showed that shortened, fragmented, or swollen mitochondria, indicating extensive fission of mitochondria
and mitochondrial degeneration, accumulated withinaxons in all areas including plaques, PPWM, NAWM,
and Wld in NMOsd and MS compared to controls.
Abnormal morphological changes were more evident inaxons in NMOsd compared to those in MS, and weremore prominent in swollen axons compared to nonswollen axons in NMOsd and MS. Second, compared tocontrols, quantitative analysis of the mitochondrial volume in axons demonstrated that the VDAC-11 mitochondrial content in SMI-311 axons was significantly
increased in swollen axons of PPWM and NAWM andin areas of Wld in NMOsd, but not in these areas inMS. The VDAC-11 mitochondrial content in the axonsof active plaques (17.1%, 6.6; mean, SD) was increased
compared to inactive plaques (6.1%, 3.4) in NMOsd.
These qualitative and quantitative data suggest that theaccumulation of mitochondria with abnormal morphology in axons was more extensive in NMOsd comparedto MS and controls and was dependent on morphological changes in axons (swollen axons) and on the diseasestage (active stage) from the lesion center to even
NAWM in NMOsd (see Figs 5–7).
Consistent with the dynamics of degenerative mitochondria in axons, the axon-specific mitochondria docking protein, syntaphilin,23 was also significantly increased
in axons in plaques, PPWM, NAWM, and Wld in
NMOsd and MS, suggesting immobile mitochondria
or decreased mitochondrial movement within axons (seeFig 6C).

Next, to investigate the pathophysiological mechanisms of axonal damage in NMOsd, we assessed thetransient receptor potential melastatin 4 (TRPM4). This
protein crucially contributes to axonal damage, and pharmacological inhibition of TRPM4 using the antidiabetic
drug glibenclamide reduces axonal degeneration in an
animal model of MS, experimental autoimmune encephalomyelitis (EAE).24 Quantification of axonal TRPM4
expression revealed significantly more TRPM4 IR inareas of plaques and Wld and in the swollen axons of
PPWM and NAWM in NMOsd and MS compared tocontrols, in which we detected only a few TRPM41
axons (see Figs 6B and 7D, E). TRPM41 axons weremore evident in swollen axons than in nonswollen axons
in NMOsd and MS. Therefore, aberrant axonal accumulation of mitochondria with abnormal morphology anddecreased movement, and abnormal axonal expression of
TRPM4 extended over wider areas in NMOsd comparedto MS.
€ ller Cell
Neuroaxonal Damage with Mu
Pathology in the Retina in NMOsd
The retina is anatomically isolated and represents a
unique unmyelinated model in which to study neurodegeneration and inflammation, as well as the distal effects
of demyelination of optic nerves in inflammatory anddemyelinating syndromes of the CNS. All retina samples
from NMOsd autopsy cases showed severe RNFL thinning with an abundance of GFAP1 and AQP41 astrocytes and neuronal loss of ganglion cells in the retina,
suggesting the presence of secondary retinal retrograde
degeneration after ON (Fig 8). The calcium-binding

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FIGURE 4: Characterization of typical pathological findings of neuromyelitis optica (NMO) spectrum disorder (NMOsd) in optic
nerves including pattern-specific loss of aquaporin-4 (AQP4) immunoreactivity (IR) in inflammatory lesions. (A, B) All patientswith NMOsd in this study had typical pathological findings of NMOsd in optic nerves (arrowheads), including pattern-specificloss of AQP4 and glial fibrillary acidic protein (GFAP) IR and immunoglobulin deposits colocalizing with a product of complement activation, C9neo, in a vasculocentric pattern around thickened hyalinized vessels in inflammatory demyelinating lesions.
Moreover, Iba-11 microglia, CD45RO1 T cells, and CD201 B cells were abundant in inflammatory demyelinating lesions. In particular, thickening of the perioptic meninges with inflammatory infiltration including CD45RO1 T cells and Iba-11 cells wasprominent in the meninges adjacent to the plaques in NMOsd patients. (C) The meningeal infiltrates also extended into thepial septa and parenchyma of the optic nerves. HE 5 hematoxylin and eosin; KB 5 Kl€uver–Barrera; MBP 5 myelin basic
protein.

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FIGURE 5: Severe axonal damage in the optic nerves in neuromyelitis optica (NMO) spectrum disorder (NMOsd). Axonal
pathology of the optic nerves in NMOsd and multiple sclerosis (MS) was qualitatively assessed with Bodian silver impregnation,and immunohistochemistry for phosphorylated neurofilaments (SMI-31) and amyloid precursor protein (APP). Axonal damage,including axonal loss, transections, swellings, and distorted axons, and axonal spheroids were evident in plaques and periplaque white matter (PPWM) and had accumulated toward each plaque center in the optic nerves in NMOsd and MS compared tocontrol. Areas of Wallerian degeneration (Wld), which were defined by an extensive loss of myelin and axons, and profoundgliosis with densely packed glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) immunoreactivity (IR), were present in
NMOsd. Axonal damage, including axonal loss, transections, swellings, and distorted axons, and axonal spheroids were mostprominent in plaques and areas of Wld in NMOsd and MS. All NMO and MS lesions were classified according to lesion activity,
as previously described.14 In brief, active plaques were characterized by macrophages with minor myelin products (cyclic nucleotide phosphodiesterase) or myelin basic protein (MBP) and profound inflammation, and inactive plaques were characterizedby macrophages without minor or major myelin products within their cytoplasm. PPWM was defined as normal myelin, microglial activation, and few inflammatory infiltrates adjacent to the plaque. Additionally, PPWM in NMOsd included loss of AQP4
IR adjacent to the plaque.14 Normal-appearing white matter (NAWM) was defined as myelin integrity and at least 1cm awayfrom a plaque’s edge.50 Additionally, NAWM in NMOsd included preservation of AQP4 IR. *Plaque. KB 5 Kl€uver–Barrera; neg
5 negative.

protein calbindin is a known marker of cone photoreceptors, horizontal cells, and subpopulations of bipolar cells
and amacrine cells in the human retina. In the retina in

FIG
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NMOsd cases, we observed mild loss of calbindin1 horizontal cells in the INL and unique dynamics of M€
ullercells including scattered loss of AQP4 IR in M€uller cells

an
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Hokari et al: Lesions in NMO

FIGURE 6: Abnormal axonal mitochondrial dynamics and aberrant axonal expression of transient receptor potential melastatin
4 (TRPM4) cation channel in the optic nerves in neuromyelitis optica (NMO) spectrum disorder (NMOsd). (A) We assessed the
dynamics of mitochondria labeled with voltage-dependent anion channel (VDAC)/porin (green) located in SMI-31 (blue)1 axons
in NMOsd compared to controls. Myelin basic protein (MBP; red)1 myelinated and MBP2 demyelinated axons were identified
based on the lesion classification. Morphological analysis of VDAC-11 mitochondria showed shortened, fragmented, or swollen
mitochondria, indicating extensive fission of mitochondria and mitochondrial degeneration. These mitochondria accumulated
within axons in all areas including plaques, periplaque white matter (PPWM), normal-appearing white matter (NAWM), andWallerian degeneration (Wld) in NMOsd and multiple sclerosis (MS) compared to controls (arrowheads in A). (B) We thenassessed the dynamics of the TRPM4 cation channel (green) in SMI-31 (red)1 axons in NMOsd and MS compared to controls.
TRPM41 axons were more evident in areas of plaques and Wld and in the swollen axons of PPWM and NAWM in NMOsd andMS compared to controls, in which we detected only a few TRPM41 axons. (C) Consistent with the dynamics of degenerative
mitochondria in axons (A), the axon-specific mitochondrial docking protein, syntaphilin (red), was also increased within SMI-311
axons (blue) in plaques, PPWM, NAWM, and Wld in NMOsd and MS, suggesting immobile mitochondria or decreased mitochondrial movement within axons. Therefore, aberrant accumulation of mitochondria with abnormal morphology and withdecreased movement in axons was evident in the optic nerves in NMOsd and MS. Scale bars 5 50lm.

riler
lls

and mild to moderate loss of glutamine synthetase(GS)1 M€uller cell soma in the INL. Iba-11 microglia
were present in the inner retina in NMOsd cases; how-.4

April 2016

ever, CD45RO1 T cells, CD201 B cells, and perivascular deposition of complement, C9neo, were not observedin all retina samples from NMOsd cases (data not617－ 245 －

ANNALS of Neurology

shown). The retinas of MS cases showed severe RNFL
thinning with an abundance of GFAP1 and AQP41
astrocytes and neuronal loss of ganglion cells in the ganglion cell layer and calbindin1 horizontal cells in the
INL. However, retinas from MS cases showed no changes
in AQP4 or GS IR in M€
uller cells. Reported evidence
supports the involvement of AQP4 in retinal inflammatory responses,25 retinal swelling,25 and light-induced retinal neurodegeneration.26 Moreover, optic nerve

transection in animal models results in RNFL thinningand ganglion cell loss but not in atrophy or degenerationof deep retinal layers including the INL.27 If this is thecase in the eyes of NMO patients, the M€
uller cells withunique dynamics observed in NMO patients may berelated to primary retinal degeneration. These data suggest that the characteristics of the retinal pathology
observed in NMOsd were strikingly distinct not only
from the characteristics of the retinas in MS but also

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Hokari et al: Lesions in NMO

from myelitis or ON in NMOsd (see Fig 4), and the retina of NMOsd cases was characterized by primary as
well as secondary neuronal and axonal loss with uniquedynamics of M€
uller cells.

Discussion
The optic nerves are the major site of involvement in
NMOsd and have several distinguishing structures. First,
the long, thin cylindrical dimension of the optic nerveswith the cul-de-sac anatomy of the subarachnoid space
may cause unique cerebrospinal fluid (CSF) dynamics,
including restricted diffusion of proinflammatory elements (eg, AQP4 antibodies), retained inflammation,and limited clearance of myelin and axonal debris, followed by enhanced NMOsd lesion formation.28,29 We
revealed prominent pathological meningeal thickening
with inflammation (see Fig 4) and radiologically abnormal enhancement of the OPN pattern in NMOsd (seeFig 1). Inflammatory processes may contribute to theclosing of the arachnoid apertures that drain CSF into
the meningeal lymphatics, thus adding to CSF compartmentalization.29 Second, the absence of classical blood–
brain barrier (BBB) properties with nonspecific permeability of microvessels in the prelaminar region in the
optic nerve head30 may cause transmigration of AQP4
antibodies. Third, the unique structure of pial septa thatpartition each optic nerve fiber bundle from the adjoining glial limitans (a network of AQP41 astrocytic footprocesses) may form an immunological barrier that prevents immune cells from entering the CNS parenchyma.
Penetration of this barrier by encephalitogenic T lymphocyte requires the activity of matrix metalloproteinases,

which are produced by myeloid cells in an EAE model.31
Thus, the presence of CD45RO1 T cells and Iba-11
macrophages in the pial septa in NMOsd (see Fig 4)
may increase the chance of breaking the barrier and promote effector responses for AQP4 on astrocytes. Finally,
abundant large orthogonal arrays of particles (OAPs) inastrocytic endfeet of the optic nerves enhance binding of
AQP4 antibodies and complement-dependent astrocyte
damage.28 These unique structures of the optic nerves
may promote typical NMOsd lesions that show apattern-specific loss of AQP4 with complement
activation.
Importantly, we demonstrated that severe and widespread neurodegeneration with aberrant accumulation ofdegenerative mitochondria and TRPM4 channels wasevident in the optic nerves in NMOsd. In the pathogenesis of MS, key elements that drive neurodegenerationand that can result in permanent clinical deficits32
include accumulation of axonal mitochondrial damage,ion channel dysfunction including TRPM4, and involvement of Wld in axons of the CNS as follows.24,33,34
First, axons are extremely susceptible to mitochondrial
dysfunction, because the geometry of the neuron–axon
unit presents a substantial challenge for efficient distribution of mitochondria and adenosine triphosphate (ATP)production within axons, which can reach 1m or morein length in humans.33 “Fusion and fission” and an“increased number” of mitochondria play critical roles inmaintaining functional mitochondria in the presence ofenvironmental stress.22 These dynamics of mitochondria
fine-tune fundamental cellular processes such as calcium
homeostasis and the generation of ATP and reactive

FIGURE 7: Quantitative analysis of axonal damage in the optic nerves in neuromyelitis optica (NMO) spectrum disorder(NMOsd). (A–D) Axonal pathology in the optic nerves in NMOsd was quantitatively assessed by immunohistochemistry for SMI31 (A, B), voltage-dependent anion channel -1 (VDAC-1; C), and transient receptor potential melastatin 4 (TRPM4; D). Axonal
damage was classified as normal axons (score 0; green), swollen axons with or without spheroids (score 1; yellow), and fragmented axons (score 2; red), as modified from a previous report35 (B). The density of SMI-311 axons was significantly reduced(A), and percentages of swollen axons (score 1; yellow) and fragmented axons (score 2; red; B) were significantly increased inplaques and periplaque white matter (PPWM) in NMOsd and multiple sclerosis (MS) compared to controls. Swollen or fragmented axons with no significant reduction in axonal density were present in normal-appearing white matter (NAWM) in
NMOsd, whereas both types of morphological changes and reduced axonal density were evident in NAWM in MS (A, B). Quantitative analysis of the mitochondrial volume in axons demonstrated that the VDAC-11 mitochondrial content in SMI-311 axons
was significantly increased in swollen axons of PPWM and NAWM, and in areas of Wallerian degeneration (Wld) in NMOsd
compared to controls (C). In contrast, quantitative analysis of the mitochondrial volume in axons demonstrated that the VDAC11 mitochondrial content in SMI-311 axons was not increased in plaques, PPWM, or NAWM in MS (C). Quantification of axonal
TRPM4 expression revealed significantly more TRPM4 immunoreactivity in areas of plaques and in swollen axons of PPWM and
NAWM in NMOsd and MS compared to controls, in which we detected only a small proportion of TRPM41 axons (D). TRPM41
axons were more evident in swollen axons than in nonswollen axons in NMOsd and MS (D). (E) When the severity of axonal
damage was compared between NMOsd and MS, more severe axonal damage, including swollen axons, increased axonal mitochondrial content, and aberrant TRPM4 cation channel expression, was prominent in plaques, PPWM, and NAWM in NMOsd
compared to MS, except that reduced axonal density and swollen axons were more evident in NAWM in MS compared to
NMOsd. These qualitative (see Figs 5 and 6) and quantitative (current figure) data suggest that the accumulation of mitochondria with abnormal morphology in the axons was more extensive in NMOsd compared to controls and depended on morphological changes in axons (swollen axons) and the disease stage (active stage) from the lesion center to NAWM in NMOsd. *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; vs control in A–D, MS vs NMO in E.

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oxygen species and consequently play important roles in
apoptosis, mitophagy, and oxygen sensing.22 Intriguingly,
in the EAE model, focal axonal degeneration/swelling(FAD) with focal intra-axonal accumulation of dysmorphic and swollen mitochondria, but with intact myelin
sheaths, is the earliest ultrastructural sign of damage,

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Volume 79, No. 4－ 248 －

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Hokari et al: Lesions in NMO

which progresses to axonal fragmentation and demyelination.35 These axonal changes that are consistent with
FAD are present in acute MS lesions.35 In addition, lackof mitochondrial respiratory chain complex IV activity ina proportion of Na1/K1 ATPase a-11 demyelinatedaxons supports axonal dysfunction as a contributor toneurological impairment in MS.36 Second, TRPM4 contributes crucially to inflammation-induced neurodegeneration including glutamate excitotoxicity, which is
downstream of glutamatergic Ca21 influx and depends
on calcium-activated TRPM4-mediated inward currents.
This excitotoxicity mediates axonal swelling and neuronalcell death in the EAE model and MS.24 Third, Wld,
which was traditionally thought to result from passive
degeneration of axons due to a lack of cell body-derived
nutrients,37 is a prominent early feature of most neurodegenerative disorders, including amyotrophic lateral sclerosis. However, analysis of the slow Wallerian degeneration(WldS) mutant mouse has recently elucidated that Wld ismediated by an active autodestruction program via axon
death signaling pathways, including the Toll-like receptor
adaptor SARM1.38 Early during Wld, mitochondria
swell, fragments accumulate at paranodes, and mitochondria lose their membrane potential.38 In the EAE model,
WldS mutant mice show a modest attenuation of axonal
loss, suggesting that EAE-associated axonal damage may

occur by a mechanism that is partially similar to Wld.39
In early MS, Wld is a major component of axonal
pathology in plaques and PPWM.34 Collectively, degenerative mitochondria, accumulation of TRPM4, and Wld
may be mutually interrelated and mediate processes ofsevere neurodegeneration in MS and EAE. In this study,
we showed that more severe and widespread neurodegeneration, including Wld with accumulation of degenerative mitochondria and TRPM4 channels, was evident in
NMOsd compared to MS. These factors may contributeto clinical features such as the profound and persistentvisual loss and chronic progressive deterioration seen in
ON in NMOsd.
We here propose that the pathogenesis of the AVP
in NMOsd may be caused by several neurodegenerative
processes, including not only secondary anterograde(Wld) or retrograde changes after ON, but also in vivo
primary neuroaxonal pathology via AQP4 antibodymediated pathology of astrocytes/M€
uller cells, regardlessof ON history. The presence of neurodegeneration in
AQP4-deficient regions including (1) myelin-preserved
PPWM adjacent the optic nerve plaques and (2) the retina, which is physiologically devoid of myelin, in
NMOsd may indicate that demyelination is not necessarily essential for processes of neurodegeneration in
NMOsd. A marked reduction in the astrocytic Na1-

FIGURE 8: Retinal nerve fiber layer (RNFL) thinning and mild loss of calbindin1 horizontal cells with unique dynamics of M€
ullercells in the retina of neuromyelitis optica (NMO) spectrum disorder (NMOsd). (F) The retina consists of 2 major parts: the retinal pigment epithelium and the neural retina. The neural retina can be divided into 9 layers: (1) photoreceptors (rods [r] andcones [c]); (2) external limiting membrane (attachment sites of adjacent photoreceptors and M€
uller cells [m]); (3) outer nuclear
layer (ONL; nuclei of photoreceptors); (4) outer plexiform layer (OPL; axonal extensions of photoreceptors), which contains the
middle limiting membrane (desmosomelike attachments of photoreceptor synapse expansions); (5) inner nuclear layer (INL;
nuclei of bipolar [b], horizontal [h], amacrine [a], and M€
uller cells); (6) inner plexiform layer (IPL; mostly synapses of bipolarcells, ganglion cells [g], and amacrine cells); (7) ganglion cell layer (GCL); (8) RNFL (axons of ganglion cells); and (9) internal limiting membrane (basement membrane of M€
uller cells). In addition to M€
uller cells, astroglial (as) and microglial cells (mi) arefound in the inner retina. In normal conditions, aquaporin-4 (AQP4) water channels are expressed by astrocytes within the
RNFL and by M€
uller cell membranes especially within the RNFL and OPL, and the cell soma of M€
uller cells lies within the INL.
The calcium-binding protein calbindin is a known marker of cone photoreceptors, horizontal cells, and subpopulations of bipolar cells and amacrine cells in the human retina. The human retina is devoid of myelin. Ganglion cell complex (GCC) scanning
using a Fourier/spectrum domain optical coherence tomography system encompasses the 3 layers, including the RNFL, GCL,
and IPL in the retina. We uncovered 3 important findings in NMOsd retinas. (A) First, all NMOsd and multiple sclerosis (MS)autopsy cases showed severe thinning of the RNFL with an abundance of glial fibrillary acidic protein (GFAP)1 and AQP41
astrocytes, and neuronal loss in the GCL in the retina, suggesting the presence of secondary retinal degeneration after optic
neuritis (ON). (B, D) Second, we observed scattered loss of AQP4 immunoreactivity (IR) in the INL, OPL, and ONL of M€
ullercells and mild to severe reduction in M€
uller cell nuclei with glutamine synthetase (GS)-IR-rich cytoplasm in the INL of NMOsd
cases. Conversely, the GS1 cell soma of M€
uller cells lay within the INL in control and MS retinas (arrowheads). (C) Third, all retinas in the NMOsd and MS patients showed mild loss of calbindin1 horizontal cells (arrows) in the INL. (E) Schematic lesion
maps of the retina in each case with NMOsd are shown. Lesions with loss of AQP4 IR in M€
uller cells in the INL, OPL, and ONL
are indicated in red, lesions with loss of soma of GS1 M€
uller cells in the INL are indicated in blue, and lesions with loss of
calbindin1 horizontal cells in the INL are indicated in green. All NMOsd patients showed mild neuronal loss in the INL withunique dynamics in M€
uller cells. Iba-11 microglia were present in the inner retina of NMOsd cases, but CD45RO1 T cells,
CD201 B cells, and perivascular deposition of complement, C9neo, were not observed (data not shown). These data suggestthat the characteristics of the retinal pathology observed in NMOsd cases were strikingly distinct not only from those of theretina in MS cases but also from those of myelitis or ON in NMOsd cases (see Fig 4). Therefore, the retina in NMOsd cases
was characterized by primary as well as secondary neuronal and axonal loss with unique dynamics of M€
uller cells. The data
shown are representative of NMOsd, MS, and control retinas (A–D). HE 5 hematoxylin and eosin; I 5 INL; O 5 ONL; R 5
RNFL.

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ANNALS of Neurology

dependent excitatory amino acid transporter 2,40,41 anddownregulation of the NMDA-receptor subunit NR1,41
are present in AQP4-deficient regions in NMOsd
patients and a rat passive transfer model using AQP4
antibodies, even when myelin IR is preserved.41 These
data suggest that disruption of glutamate homeostasis40,41
may play a role in primary neurodegeneration via glia–
neuron interactions in NMOsd pathogenesis. Further
research is required to investigate all aspects of NMOsd
pathogenesis, including modulation of synaptic function/
network activity and/or myelin maintenance caused byastrocytopathy in NMOsd.
The basic effector mechanisms of autoantibody
binding to its cellular target may be: (1) functional blockof the target, (2) target internalization, 3) complementdependent cytotoxicity (CDC), and (4) antibodydependent cell-mediated cytotoxicity (ADCC) in autoimmune disorders. Previous studies in rodent models andin vitro assays have shown indirect evidence that CDC,42
ADCC,43 and internalization of AQP4 channels44 may
play important roles in NMOsd pathogenesis. CDC is
the most important immune mechanism of AQP4 antibodies in NMOsd, because a hallmark of NMOsd
pathology is pattern-specific loss of AQP4 IR on astrocytes with complement activation. This hallmark wasprominent at 2 major sites, the optic nerves (see Fig 4)
and spinal cord.9,21 Conversely, the pathology in the retina (see Fig 8) and cortical gray matter12,45 in NMOsd isdistinct from that in the optic nerves and spinal cord.
The retinal pathology in NMOsd was characterized bysecondary retrograde degeneration of RNFL axons withgliosis (an abundance of AQP41 astrocytes) following
ON, loss of horizontal cells in the INL, and scatteredloss of AQP4 IR on M€
uller cells, but no deposition ofcomplement in situ. The cortical gray matter pathology in
NMOsd is characterized by substantial cortical neuronalloss in layers II, III, and IV with nonlytic reaction of
AQP4-negative astrocytes in layer I, but no cortical demyelination nor deposition of complement.12 Thus, the pathological processes in the retina and cortical gray matter in
NMOsd may consist of an entirely different mechanism
than CDC or ADCC in the spinal cord and optic nerves.
These region-specific pathologies in NMOsd may dependon the following factors: (1) anatomical structures, including the glial framework and BBB integrity; (2) the complement regulators CD46, CD55, and CD59, which
protect the brain from complement-mediated damage46;
and (3) the cluster size of OAPs containing AQP4 tetramers, which is influenced by maturational differences in
the ratio of the AQP4 M1 isoform to M23. OAPs contribute to effective complement activation on astrocytic
membranes,44 because OAP assemblies are required for

AQP4 antibodies to recognize AQP4.47 The binding of
AQP4 antibodies may cause incomplete internalization of
AQP4 into the endolysosomal compartment in astrocytes,
followed by disruption of water and/or glutamate homeostasis with excitotoxic consequences for neurons in the retina and cortical gray matter.
This study has some limitations. The subjects who
were evaluated for neuro-ophthalmological assessments
were different from those who were pathologically examined, and the cohort of individuals who were pathologically assessed was small. Our study has intrinsic
limitations owing to the limited availability of autopsied
samples and the partial postmortem detachment that
occurs in the retina in NMOsd, similar to the situationin previous reports describing MS eyes.48 In the future,prospective, long-term follow-up and large cohort studies
are needed to further characterize NMOsd pathogenesis.
In summary, our findings are consistent with a newconcept in which severe neurodegeneration via abnormal
AQP4 dynamics in astrocytes/M€
uller cells is prominent
in NMOsd pathogenesis. Based on this study, future
therapeutic strategies designed to limit the deleterious
effects of not only AQP4 antibodies and damage toastrocytes but also neurodegeneration via glia-neuron
interactions (eg, mitochondrial protection and replenishment strategies for degenerative axonal mitochondria33;
TRPM4 inhibition strategies for accumulation of
TRPM424; and WldS/nicotinamide mononucleotide
adenylyltransferase-mediated axonal protection strategies
for Wld38) are worthy of further investigation.

Po

Acknowledgment

12This work was supported in part by JSPS KAKENHI(grant number 26461289, I.K.), and a Health and Labor
Sciences Research Grant on Rare and Intractable Diseases(Evidence-Based Early Diagnosis and Treatment Strategies for Neuroimmunological Diseases) from the Ministry of Health, Labor, and Welfare of Japan (M.N.).
We thank S. Kawaguchi and M. Kaneko for technicalassistance with assays for AQP4 autoantibodies, Dr. K.
Tanaka for advice on neurological and immunologicalassessments, Dr. M. Tsujita and Dr. K. Sakimura for providing an AQP4 expression vector, and Dr. B. P. Morgan
for providing the complement antibody.

No

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2.

3.

4.

5.

6.

7.

8.

9.

10

11

13

14

15

16

17

Author Contributions
M.H. and A.Y. contributed equally to this work. Conception and design of the study: M.N., I.K. Acquisition
and analysis of data: all authors. Drafting the text or preparing the figures: M.H., A.Y., M.A., E.S., K.Y., M.N.,
I.K.

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Hokari et al: Lesions in NMO

Potential Conflicts of Interest

Hickman SJ, Toosy AT, Jones SJ, et al. A serial MRI study following optic nerve mean area in acute optic neuritis. Brain 2004;
127(pt 11):2498–2505.

21.

Roemer SF, Parisi JE, Lennon VA, et al. Pattern-specific loss ofaquaporin-4 immunoreactivity distinguishes neuromyelitis optica
from multiple sclerosis. Brain 2007;130(pt 5):1194–1205.

Nothing to report.

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Volume 79, No. 4－ 252 －RESEARCH ARTICLE

Effects of Alda-1, an Aldehyde
Dehydrogenase-2 Agonist, on Hypoglycemic
Neuronal Death
Tetsuhiko Ikeda1, Tetsuya Takahashi1, Mika Tsujita2, Masato Kanazawa1,
Masafumi Toriyabe1, Misaki Koyama1, Kosuke Itoh2, Tsutomu Nakada2,
Masatoyo Nishizawa1, Takayoshi Shimohata1*
1 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan, 2 Department ofCenter for Integrated Human Brain Science, Brain Research Institute, Niigata University, Niigata, Japan* t-shimo@bri.niigata-u.ac.jp

Abstract

OPEN ACCESS
Citation: Ikeda T, Takahashi T, Tsujita M, Kanazawa
M, Toriyabe M, Koyama M, et al. (2015) Effects of
Alda-1, an Aldehyde Dehydrogenase-2 Agonist, on
Hypoglycemic Neuronal Death. PLoS ONE 10(6):e0128844. doi:10.1371/journal.pone.0128844
Academic Editor: M.Faadiel Essop, Stellenbosch
University, SOUTH AFRICA
Received: January 3, 2015
Accepted: April 30, 2015
Published: June 17, 2015
Copyright: © 2015 Ikeda et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.

Hypoglycemic encephalopathy (HE) is caused by a lack of glucose availability to neuronalcells, and no neuroprotective drugs have been developed as yet. Studies on the pathogenesis of HE and the development of new neuroprotective drugs have been conducted using
animal models such as the hypoglycemic coma model and non-coma hypoglycemia model.
However, both models have inherent problems, and establishment of animal models thatmimic clinical situations is desirable. In this study, we first developed a short-term hypoglycemic coma model in which rats could be maintained in an isoelectric electroencephalogram (EEG) state for 2 min and subsequent hyperglycemia without requiring anti-seizure
drugs and an artificial ventilation. This condition caused the production of 4-hydroxy-2nonenal (4-HNE), a cytotoxic aldehyde, in neurons of the hippocampus and cerebral cortex,
and a marked increase in neuronal death as evaluated by Fluoro-Jade B (FJB) staining. We
also investigated whether N-(1,3-benzodioxole-5-ylmethyl)-2,6-dichlorobenzamide (Alda1), a small-molecule agonist of aldehyde dehydrogenase-2, could attenuate 4-HNE levels
and reduce hypoglycemic neuronal death. After confirming that EEG recordings remained
isoelectric for 2 min, Alda-1 (8.5 mg/kg) or vehicle (dimethyl sulfoxide; DMSO) was administered intravenously with glucose to maintain a blood glucose level of 250 to 270 mg/dL.
Fewer 4-HNE and FJB-positive cells were observed in the cerebral cortex of Alda-1-treatedrats than in DMSO-treated rats 24 h after glucose administration (P = 0.002 and P = 0.020).
Thus, activation of the ALDH2 pathway could be a molecular target for HE treatment, andAlda-1 is a potentially neuroprotective agent that exerts a beneficial effect on neurons when
intravenously administered simultaneously with glucose.

Data Availability Statement: All relevant data arewithin the paper.
Funding: This work was supported by a grant fromGrant for Promotion of Niigata University Research
Projects.

Introduction

Competing Interests: The authors have declaredthat no competing interests exist.

Transient hypoglycemic episodes occur during treatment of diabetes mellitus with insulin ororal hypoglycemic drugs and in cases involving insulinoma, alcoholism, anorexia nervosa, or

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Effects of Alda-1 on Hypoglycemic Neuronal Deathothers. Although many patients recover with appropriate treatment, transient hypoglycemic
episodes can result in coma, seizures, and myriad other global and focal neurological deficits[1–3]. In an analogy to hypoxic encephalopathy, this syndrome has been termed hypoglycemic
encephalopathy (HE). The only treatment for HE is blood glucose (BG) correction by glucose
administration, and no neuroprotective drugs have been developed as yet.
Studies on the pathogenesis of HE and the development of neuroprotective drugs have been
conducted using animal models. There are two conventional animal models of HE: one inwhich an isoelectric electroencephalogram (EEG) is maintained for a long period (hypoglycemic coma model) [4, 5] and the other involving euthanization or glucose administration before
the isoelectric EEG (hypoglycemic non-coma model) [6–8]. In the hypoglycemic coma model,
the isoelectric EEG manifesting after the BG decrease is maintained for at least 30 min to ensure that the brains are in a state of severe hypoglycemia. However, once changes in the brain
due to hypoglycemia become apparent, seizures and respiratory arrest that cause additional
neuronal damage are unavoidable. Model animals must therefore be treated with an anti-seizure drug and an artificial ventilator beforehand. Regarding a prolonged, markedly hypoglycemic state with an anesthetic agent and an artificial ventilator use, the pathogenesis of HE in
animal models may differ from that of human HE experienced in clinical practice. However,
the hypoglycemic non-coma model can also be problematic in that the various indices used byresearchers as alternatives to isoelectric EEG, to indicate degrees of severity and courses, varyconsiderably, reflecting differences in individual responses to hypoglycemic loads. Thus, inorder to elucidate the pathogenesis of HE and develop neuroprotective drugs, there is a need toestablish animal models using isoelectric EEG as a quantitative index of hypoglycemic loads,
without the use of anti-seizure drugs and an artificial ventilation.
Regarding the pathological conditions of HE, neuronal death in animal models is reportedly
induced by oxidative stress generated after glucose administration [5, 9, 10]. This phenomenon
is termed “glucose reperfusion injury”, and its severity increases with higher BG levels after BG
correction [10]. In this context, we recently confirmed higher BG levels after correction in 47
consecutive patients who experienced hypoglycemic episodes (271.1 ± 128.5 mg/dL) [3].
Therefore, an animal model that experiences hypoglycemia followed by similar higher BG levels is preferred.
Studies on the development of neuroprotective drugs for HE have aimed to improve hypoenergetic conditions associated with hypoglycemia or to inhibit oxidative stress caused by glucose reperfusion injury. Among factors causing oxidative stress, 4-hydroxy-2-nonenal(4-HNE), a cytotoxic aldehyde that is a lipid oxidation product, has been intensively investigated in myocardial ischemia [11, 12]. It has been shown that 4-HNE is produced from ω-6 polyunsaturated fatty acids, such as arachidonic acid, and can cause mitochondrial disorders topotentially induce neuronal death [13]. Interestingly, N-(1,3-benzodioxole-5-ylmethyl)2,6-dichlorobenzamide (Alda-1), an aldehyde dehydrogenase 2 (ALDH2) agonist that increases the activity of mitochondrial ALDH2, inhibits myocardial damage caused by 4-HNE
after ischemia [12, 14]. Therefore, this raises the possibility that Alda-1 may inhibit oxidative
stress caused by glucose reperfusion injury via suppression of 4-HNE levels.
Based on the aforementioned observations, this study had the following three objectives: (1)to establish a novel animal model of HE that can be maintained in an isoelectric EEG state for ashort period without the use of anti-seizure drugs and an artificial ventilation, enabling the isoelectric EEG to be used as an index of HE (we termed this model “a short-term hypoglycemic
coma model”), (2) to confirm whether oxidative stress and neuronal death occur in this model,and (3) to reveal whether Alda-1 exerts protective effects against neuronal death associatedwith glucose reperfusion injury thereby demonstrating its potential as a neuroprotective drug.

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Effects of Alda-1 on Hypoglycemic Neuronal Death

Materials and Methods
The study protocol was approved by the Niigata University Administrative Panel on Laboratory Animal Care. All operations concerning animals were performed according to ARRIVE(Animal Research: Reporting of In Vivo Experiments) guidelines [15]. All efforts were made tominimize the number of animals used and their suffering. Alda-1 and vehicle (dimethyl sulfoxide, DMSO) were coded and administered by a blinded observer.

EEG recordings
Male Sprague-Dawley rats (260–310 g, 8–10 weeks old, Charles River Japan, Inc.) were used.
After sebum had been removed from both the shaved head and anterior neck regions by scrubbing with cotton balls soaked in alcohol, both areas were abraded with EEG abrasive gel (Skin
Pure, Nihon Kohden Corporation). EEG disc electrodes (LEAD110A, BIOPAC Systems, Inc.)
to which EEG disc electrode paste had been applied (Elefix, Nihon Kohden Corporation) werethen attached to the forehead (near the nose) and the occiput (between the porus acusticus) of
the rat, for use as EEG recording electrodes; the former served as the reference. Another discelectrode was also attached to the anterior neck to be used as the earth electrode. After the electrodes were attached, we confirmed with an alternating current (AC) resistance measuring instrument (Model 1089ES Checktrode, UFI Inc.) that the contact resistance between eachelectrode was 10 kO or less and that the variation in contact resistance at each electrode was
3 kO or less. An EEG amplifier (EEG100C, BIOPAC Systems, Inc.) was used for EEG amplification, and an analog-digital (A/D) converter (MP100System, BIOPAC Systems, Inc.) andwaveform analysis software (AcqKnowledge, BIOPAC Systems, Inc., version 3.7.2) were used
to collect and analyze the data. A bandpass of 0.1 to 100 Hz was used to filter the amplified
EEG, and the sampling frequency of A/D conversion was 200 Hz.

Short-term hypoglycemic coma model
The aforementioned rats were used. Each rat was fed a restricted diet of 20 g/day [16] for atleast three consecutive days and then fasted, being allowed only water, for one day before theoperation. To prevent changes in food consumption due to stress caused by an increase or decrease in the number of rats in each cage [17], the number per cage was fixed at three. The remaining amount of food was measured once a day before feeding to confirm that there were nochanges in food consumption.
Hypoglycemic loads were applied under anesthesia maintained with a mixed gas of 70% nitrous oxide and 30% oxygen with 1.0% to 1.5% halothane added while rectal temperature wasmonitored. The rectal temperature was maintained at 37.0 ± 0.5°C with a temperature control
mat, a fan, and a heating lamp. Under direct visualization using a surgical microscope, the left
inguinal region was incised to expose the left femoral vein. A polyethylene catheter (Clay
Adams Intramedic polyethylene tube PE50, Becton Dickinson & Co.) filled with 1% heparinized saline was then placed in the vein and used to collect blood samples for BG measurement.
A BG monitoring instrument (Ascensia BREEZE 2, Bayer Health Care) was used for BG measurement. Next, the left cervical region was incised to expose the left anterior facial vein. Another tube, as described above, was placed in the vein and used to administer glucose andexperimental drugs. After a resting EEG had been recorded, rapid-acting insulin (Novolin R
100 IU/mL Injection, Novo Nordisk Pharma Co., Ltd.) was intraperitoneally administered at adose of 15 IU/kg [5, 18], and blood samples were then collected every 15 min. If the EEG did
not become isoelectric by more than 180 min after insulin administration, the experiment was
discontinued. After 2 or 10 min of isoelectric EEG state, 50% glucose was intravenously injected at a dose of 0.2 mL (Fig 1A). Then, 50% glucose was intravenously infused at a rate of

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Effects of Alda-1 on Hypoglycemic Neuronal Death

Fig 1. Short-term hypoglycemic coma model. (A) Protocol for hypoglycemic loading and induction of the hyperglycemic state. Rapid-acting insulin was
intraperitoneally administered at a dose of 15 IU/kg. After 2 or 10 min of the presence of an isoelectric electroencephalogram (EEG), 50% glucose wasintravenously injected at a dose of 0.2 mL and intravenously infused at a rate of 2.2 mL/h. Rats were sacrificed after 24 h of glucose reperfusion. (B) Alda-1
treatment protocol. After 2 min of an isoelectric EEG state, Alda-1 was administered intravenously at a dose of 8.5 mg/kg with 50% glucose (Alda-1 group) or
the same dose of dimethyl sulfoxide (DMSO) was administered (control group).
doi:10.1371/journal.pone.0128844.g001

2.2 mL/h and again intravenously injected at a dose of 0.2 mL three times every 5 min. Furthermore, while blood samples were collected every 15 min, an additional 50% glucose solution
was administered at doses of 0.2 mL if necessary, to maintain BG levels at the target of 250 to
270 mg/dL for 3 h to create a hyperglycemic state that would cause glucose reperfusion injury
in HE. BG levels after correction by glucose administration were determined by reference to
our clinical trials [3]. Disc electrodes and catheters were removed 3 h after the start ofglucose administration.

Intravenous administration of an aldehyde dehydrogenase agonist
An ALDH2 agonist (Alda-1, Merck KGaA), an activator of aldehyde dehydrogenase, was dissolved in DMSO at a concentration of 15 mg/mL. At the end of the isoelectric EEG period,
Alda-1 was administered to the Alda-1 group animals at a dose of 8.5 mg/kg with glucose into
the left anterior facial vein, and the same dose of DMSO was administered to the control group(Fig 1B). The dose of Alda-1 was determined according to the optimal dosage described in previous reports of myocardial ischemia in rats [12, 14]. Alda-1 and DMSO were administered torandomly selected rats.

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Immunostaining for 4-HNE, a lipid oxidation productRats were euthanized with halothane 24 h after glucose administration. The left ventricle was
perfused with 200 mL of cold saline and 100 mL of 4% paraformaldehyde (PFA) [19]. Removed
whole brains were fixed in 4% PFA at 4°C for 12 h [20]. After rinsing with 0.1 M phosphatebuffered saline (PBS) at 4°C overnight, brains were immersed in 15% sucrose/0.1 M PBS at 4°C
overnight and then 30% sucrose/0.1 M PBS at 4°C for 24 h. Four coronal sections were collected from each animal starting 4.0 mm posterior to Bregma. They were frozen in isopentane, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek Inc.),
and stored at -80°C. Brain samples were cut with a cryostat (Cryotome E Electronic Cryostat,
Thermo Electron Co.) into sections of 20 μm thickness, placed on slides, and dried for 2 h.
Samples were immersed in 0.3% hydrogen peroxide dissolved in absolute methanol for 30 min
to inhibit peroxidase activity. After rinsing with PBS, sections were incubated with mousemonoclonal anti-4-HNE antibody (HNEJ-2, JalCA, 1:50) at 4°C overnight. The secondary antibody (#BA-2000, Vector Laboratories, Inc.) was applied at room temperature for 2.5 h. Afterincubation with an ABC Elite kit (Vectastain ABC, Vector Laboratories, Inc.), sections were developed in 3,3'-diaminobenzidine. Cerebral cortices (hippocampus level) from each rat wereviewed using a light microscope (Eclipse E600W, Nikon Co.). The number of 4-HNE-positive
cells was counted in five non-overlapping areas of 0.25 mm2 in both parietal and temporal cortical sections, and findings were confirmed in triplicate. Cells were counted in both hemispheres by a blinded observer.

Assessment of neuronal death by Fluoro-Jade B
The 25-μm sections were placed on slides, dried, then immersed in 0.1 M PBS, dried at 50°C or
higher for 30 min, and immersed in 80% ethanol containing 1% sodium hydroxide. Next, samples were immersed in 70% ethanol solution for 2 min and in purified water for 2 min. After
immersion in 0.06% potassium permanganate solution for another 10 min, samples wererinsed with purified water for 2 min. After immersion in degenerating neuron binding fluorescent derivative staining solution (Fluoro-Jade B [FJB], Histo-Chem Inc., 0.0004%) for 20 min,a 1-min rinse with purified water was repeated three times; samples were then dried at 50°C for
15 min. Next, samples were immersed in ethanol and xylene, dehydrated, and mounted withDPX (Fluka; Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Cerebral cortices (hippocampus level) from each rat were observed with a fluorescence microscope (excitation wavelength, 480 nm; fluorescence wavelength, 525 nm). The number of FJB-positive cells wascounted in five non-overlapping areas of 0.25 mm2 in both parietal and temporal cortical sections, and findings were confirmed in triplicate. Cells were counted in both hemispheres by ablinded observer.

Statistical analysis
Results are expressed as means ± standard deviation (SD). A t-test was used for statistical analyses. A P value < 0.05 was considered statistically significant.

Results
Changes in BG levels before and after insulin administration
We first measured BG levels of rats before and after insulin administration. The mean BG level
before insulin administration was 112.4 ± 26.1 mg/dL (Fig 2). After insulin administration, BG
levels gradually decreased and stabilized around 20 mg/dL 60 min after insulin administration.
Since their EEGs did not become isoelectric more than 180 min after insulin administration,

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Effects of Alda-1 on Hypoglycemic Neuronal DeathFig 2. Temporal changes in blood glucose levels after administration of insulin and glucose. Blood glucose levels were measured every 15 min before
and after injection of rapid-acting insulin (from -15 min to 120 min; n = 48) as well as after injection of 50% glucose (from 15 min to 180 min and 24 h; n = 9).

Fig 3.
mean
precisdoi:10.

doi:10.1371/journal.pone.0128844.g002

the experiment was discontinued in 3 of 30 rats to which glucose was to be administered 2 minafter an isoelectric EEG state was observed (2-min isoelectric EEG group) and 4 of 48 rats towhich glucose was to be administered after 10 min of an isoelectric EEG state (10-min isoelectric EEG group). After glucose administration, BG levels immediately increased and remainedin the 250 to 270 mg/dL range 30 min after the start of glucose administration and beyond.

Changes in EEG and neurological findings
We next investigated EEG findings and neurological signs associated with hypoglycemia andglucose reperfusion (Fig 3). Resting EEGs before insulin administration exhibited amplitudesof 10 to 50 μV and a frequency range of 4 to 8 Hz. There were no pupil abnormalities, and thelight reflex was rapid. High-amplitude slow waves (amplitude, 100–200 μV; frequency, 0.1–4Hz) occurred 69 ± 29 min after insulin administration. At this time, BG levels were 21.4 ±4.9 mg/dL, pupil diameter was increased, and the light reflex was slow. Mean EEG amplitudeand frequency then both gradually decreased, and the EEG became isoelectric 92 ± 33 minafter insulin administration. At this time, BG levels were 18.3 ± 7.8 mg/dL, pupils were dilated,
and the light reflex disappeared. When glucose was administered after isoelectric EEG, the appearance of the EEG was similar to that at rest (recovering EEG). Following glucose administration, BG levels were 245.8 ± 92.7 mg/dL, dilatation of the pupils disappeared, and the lightreflex was rapid. Since pupil diameter and light reflexes were not maintained at a constant level
during each phase, these findings were not suitable for predicting precise BG levels. Frequencies of seizure and subsequent death in the isoelectric EEG 2-min group were 6.7% (2/30) and16.7% (5/30), respectively. By contrast, these were 100% (48/48) and 87.5% (42/48), respectively, in the 10-min isoelectric EEG group.

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Effects of Alda-1 on Hypoglycemic Neuronal Death

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Fig 3. Temporal changes in electroencephalogram findings and neurological signs. EEG; Electroencephalogram, N/A; not applicable. Data aremeans ± SD. Since pupil diameter and light reflexes were not maintained at a constant level during each phase, these findings were not suitable for predicting
precise BG levels.
doi:10.1371/journal.pone.0128844.g003

Assessment of 4-HNE-positive cells in the short-term hypoglycemic
coma model
The number of 4-HNE-positive cells was compared among the sham group (to which insulin
was not administered), the 2-min isoelectric EEG group, and the 10-min isoelectric EEG group(Fig 4). There were no significant differences in body weight, food consumption, or BG levels
before or after the experiment among the three groups. In the sham group, no 4-HNE-positive
cells were detected in any of the brain regions examined. In the 2-min isoelectric EEG group,
4-HNE-positive cells were detected in the frontal to parietal-temporal cortex and in the hippocampus. The number of 4-HNE-positive cells in the hippocampus was less than that in the cerebral cortex. The frequency of 4-HNE-positive cells in the parietal-temporal cortex at the levelof the hippocampus was significantly higher in the 10-min isoelectric EEG group than in the
2-min isoelectric EEG group (217.9 ± 27.1 cells/0.25 mm2 vs. 104.8 ± 8.9 cells/0.25 mm2;
P < 0.001).

Assessment of FJB-positive cells in the short-term hypoglycemic comamodel
The frequency of hypoglycemia-induced neuronal death, evaluated by FJB staining of the parietal-temporal cortex at the level of the hippocampus, was significantly higher in the 10-min isoelectric EEG without glucose reperfusion group than in the 2-min isoelectric EEG without
glucose reperfusion group (109.9 ± 45.4 cells/0.25 mm2 vs. 53.3 ± 15.9 cells/0.25 mm2;
P = 0.009, S1 Fig).
Next, the number of FJB-positive cells was compared among the sham, 2-min isoelectric
EEG, and 10-min isoelectric EEG groups (Fig 5). In the sham group, there were no FJB-positive
cells in any of the brain regions examined. In the 2-min isoelectric EEG group, FJB-positive

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Effects of Alda-1 on Hypoglycemic Neuronal DeathFig 4. Distribution of 4-HNE-positive cells in the short-term hypoglycemic coma model. (A) Representative images of 4-HNE-positive cells in the shamgroup, the 2-min isoelectric EEG (iso-EEG) group, and the 10-min isoelectric EEG group. Higher magnification views of 4-HNE-positive cells are shown in thesmall frames. Scale bar: 100 μm. (B) The number of 4-HNE-positive cells in the sham group, the 2-min isoelectric EEG group, and the 10-min isoelectric EEG
group. Data are means ± SD (n = 15 fields). *P < 0.001.
doi:10.1371/journal.pone.0128844.g004

Fig 5.
groupin thedoi:10.

cells were observed in the frontal to parietal-temporal cortex and hippocampus. The number
of FJB-positive cells in the hippocampus was less than that in the cerebral cortex. The frequency of FJB-positive cells in the parietal-temporal cortex at the level of the hippocampus was significantly higher in the 10-min isoelectric EEG group than in the 2-min isoelectric EEG group(150.7 ± 19.8 cells/0.25 mm2 vs. 89.9 ± 6.8 cells/0.25 mm2; P < 0.001).

Effects of Alda-1 on 4-HNE-positive cells
To examine the effects of Alda-1 on glucose reperfusion-associated 4-HNE expression, thenumber of 4-HNE-positive cells was examined in rats to which Alda-1 had been administeredafter 2 min of isoelectric EEG (Alda-1 group) and in rats to which DMSO, a solvent, had been
administered (control group). There were no significant differences in body weight, food consumption, or BG levels between the two groups. The number of 4-HNE-positive cells was significantly lower in the Alda-1 group than in the control group (101.5 ± 5.6 vs. 92.9 ± 8.1 cells/
0.25 mm2; P = 0.002) (Fig 6). We did not examine the effect of Alda-1 in the 10-min isoelectric
EEG group because a large number of rats developed seizures before administration of glucose
and Alda-1.

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DeathEffects of Alda-1 on Hypoglycemic Neuronal Deathshamin theEEG

Fig 5. Fluoro-Jade B-positive cells in the short-term hypoglycemic coma model. (A) Representative images of Fluoro-Jade B-positive cells in the shamgroup, the 2-min isoelectric EEG (iso-EEG) group, and the 10-min isoelectric EEG group. Scale bar: 100 μm. (B) The number of Fluoro-Jade B-positive cells
in the sham group, the 2-min isoelectric EEG group, and the 10-min isoelectric EEG group. Data are means ± SD (n = 15 fields). *P < 0.001.

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Effects of Alda-1 on FJB-positive cells
Finally, to ascertain whether Alda-1 exerts protective effects against glucose reperfusion-associated neuronal death, the number of FJB-positive cells was compared in the Alda-1 and control
groups. Notably, the number of FJB-positive cells was significantly lower in the Alda-1 groupthan in the control group (50.9 ± 11.1 vs. 41.1 ± 6.3 cells/0.25 mm2; P = 0.020) (Fig 7).

Discussion
In this study, we first attempted to develop a short-term hypoglycemic coma model that could
be maintained in an isoelectric EEG state for a short period. The isoelectric EEG period was reduced from 30 min, which is the isoelectric period used in a conventional hypoglycemic comamodel, to 2 min. We confirmed that seizures did not occur in this condition. The advantages ofthis model are as follows. (i) The degree of severity induced by the hypoglycemic load is relatively uniform, while measurements of the light reflex and pupil diameter lack objectivity andquantitativity. (ii) BG levels during the hyperglycemic state are determined on the basis of actual human clinical data [3]. (iii) This model does not require the use of anti-seizure drugs andan artificial ventilator. (iv) The severity of oxidative stress and neuronal death are quantifiable
by measuring 4-HNE- and FJB-positive cells, respectively.

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Effects of Alda-1 on Hypoglycemic Neuronal DeathFig 6. Effects of Alda-1 on 4-HNE-positive cells. (A) Representative images of 4-HNE-positive cells
treated with vehicle (dimethyl sulfoxide; DMSO) or Alda-1. A higher magnification view of a 4-HNE-positive
cell is shown in the small frame. Scale bar: 100 μm. (B) Number of 4-HNE-positive cells in groups treated withvehicle (DMSO) or Alda-1. Data are means ± SD (n = 15 fields). *P = 0.002.
doi:10.1371/journal.pone.0128844.g006

Next, we investigated the characteristics of glucose reperfusion injury using this model, andnoted the following observations. (i) We found that the appearance of 4-HNE-positive cells inthe brain even in the 2-min isoelectric EEG group. This suggests that the conditions present in
the 2-min isoelectric EEG group are enough to cause glucose reperfusion injury. Since oxidative stress is not caused by hypoglycemic loads alone [7, 10], this raises the possibility that hyperglycemia associated with glucose administration causes glucose reperfusion injury. (ii) We
observed fewer 4-HNE-positive cells in the 2-min isoelectric EEG group than in the 10-min
isoelectric EEG group, although BG levels during the hyperglycemic state were similar. Thisfinding suggests that the severity of glucose reperfusion injury may be influenced by the degreeof severity of the hypoglycemic load-induced neuronal damage. However, to our knowledge,
no reports have determined the effect of the severity of the hypoglycemic load on glucose reperfusion injury. Future studies need to address this issue. (iii) We observed fewer 4-HNE-positive
cells in the hippocampus than in the cerebral cortex. Previous studies demonstrated similarfindings in the non-coma model [7, 8], although the mechanism underlying the difference in
4-HNE production in different brain regions remains unknown.
Finally, we showed that administration of Alda-1 inhibits both the production of 4-HNE
and neuronal death associated with glucose reperfusion injury, similar to previous reports in

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Fig 7. Effects of Alda-1 on Fluoro-Jade B-positive cells. (A) Representative images of Fluoro-Jade Bpositive cells treated with vehicle (DMSO) or Alda-1. Scale bar: 100 μm. (B) Number of Fluoro-Jade Bpositive cells in groups treated with vehicle (DMSO) or Alda-1. Data are means ± SD (n = 15 fields).
*P = 0.020.
doi:10.1371/journal.pone.0128844.g007

which Alda-1 inhibits myocardial damage caused by 4-HNE after myocardial ischemia [12,14]. Previous studies show that treatments devised to attenuate oxidative stress associated withglucose reperfusion injury may be therapeutic candidates, including therapeutic hypothermia[19], administration of nitric oxide synthase inhibitors [21], and nicotine adenine dinucleotide
phosphate oxidase inhibitors [10]. In addition, Alda-1 may be a therapeutic candidate. Activation of ALDH2 by Alda-1 increases detoxification of reactive aldehydes, such as 4-HNE, acytotoxic aldehyde that accumulates during glucose reperfusion injury as a by-product of
ROS-induced lipid peroxidation [14]. Enhanced ALDH2 activity reduces 4-HNE protein adduct formation and increases cell survival. We suggest that activation of the ALDH2 pathway
could be a molecular target for HE treatment, and that Alda-1 is a potentially neuroprotective
agent that exerts a beneficial effect on neurons when intravenously administered simultaneously with glucose.
This study has a number of limitations. First, with regard to the FJB-positive cells in the
10-min isoelectric EEG group, the seizures associated with a prolonged hypoglycemic state
may affect the results. Second, we did not investigate the effect of other neuroprotective drugs
that inhibit oxidative stress on neuronal death. Future studies are required to determine themost suitable drug for treatment of glucose reperfusion injury.

PLOS ONE | DOI:10.1371/journal.pone.0128844 June 17, 201511 / 13－ 263 －

Effects of Alda-1 on Hypoglycemic Neuronal Death

In conclusion, we established a short-term hypoglycemic coma model. In this model, we
confirmed both the appearance of 4-HNE-positive cells and neuronal death associated withglucose reperfusion injury. Furthermore, with this model we demonstrated that Alda-1 inhibits
4-HNE production and exerts a protective effect against neuronal death. Thus, Alda-1 may bea candidate neuroprotective agent for cases involving HE.

Supporting Information
S1 Fig. Hypoglycemia-induced neuronal death evaluated by Fluoro-Jade B staining. (A)Representative images of Fluoro-Jade B-positive cells in the sham group, the 2-min isoelectric
EEG (iso-EEG) without glucose reperfusion group, and the 10-min isoelectric EEG without
glucose reperfusion group. Scale bar: 100 μm. (B) The number of Fluoro-Jade B-positive cells
in the sham group, the 2-min isoelectric EEG without glucose reperfusion group, and the
10-min isoelectric EEG without glucose reperfusion group. Data are means ± SD (n = 15
fields).  P = 0.009.
(TIF)

Acknowledgments
The authors gratefully thank Prof. Hironaka Igarashi (Department of Center for Integrated
Human Brain Science, Brain Research Institute, Niigata University) for his support.

Author Contributions
Conceived and designed the experiments: TI TT TS. Performed the experiments: TI TT M. Kanazawa M. Toriyabe M. Koyama. Analyzed the data: TI TT M. Kanazawa TS. Contributed reagents/materials/analysis tools: M. Tsujita KI TN. Wrote the paper: TI MN TS.

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Effects of Alda-1 on Hypoglycemic Neuronal Death

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d. Kare-

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PLOS ONE | DOI:10.1371/journal.pone.0128844 June 17, 201513 / 13－ 265 －RESEARCH ARTICLE

Reduced CSF Water Influx in Alzheimer’s
Disease Supporting the β-Amyloid Clearance
Hypothesis
Yuji Suzuki1, Yukihiro Nakamura1, Kenichi Yamada1, Hironaka Igarashi1,
Kensaku Kasuga2, Yuichi Yokoyama3, Takeshi Ikeuchi2, Masatoyo Nishizawa4, Ingrid
L. Kwee5, Tsutomu Nakada1,5*
1 Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata, Niigata, Japan,
2 Department of Molecular Genetics, Brain Research Institute, University of Niigata, Niigata, Japan,
3 Department of Psychiatry, Faculty of Medicine, University of Niigata, Niigata, Japan, 4 Department of
Neurology, Brain Research Institute, University of Niigata, Niigata, Japan, 5 Department of Neurology,
University of California Davis, Davis, California, United States of America* tnakada@bri.niigata-u.ac.jp

Abstract
Objective
OPEN ACCESS
Citation: Suzuki Y, Nakamura Y, Yamada K, Igarashi
H, Kasuga K, Yokoyama Y, et al. (2015) Reduced
CSF Water Influx in Alzheimer’s Disease Supportingthe β-Amyloid Clearance Hypothesis. PLoS ONE 10(5): e0123708. doi:10.1371/journal.pone.0123708
Academic Editor: Stephen D Ginsberg, Nathan
Kline Institute and New York University School of
Medicine, UNITED STATES
Received: October 12, 2014
Accepted: March 5, 2015
Published: May 6, 2015
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise usedby anyone for any lawful purpose. The work is madeavailable under the Creative Commons CC0 publicdomain dedication.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: The study was supported by grants from
the Ministry of Education, Culture, Sports, Science,
and Technology (Japan) (TN).
Competing Interests: The authors have declaredthat no competing interests exist.

To investigate whether water influx into cerebrospinal fluid (CSF) space is reduced in Alzheimer’s patients as previously shown in the transgenic mouse model for Alzheimer’s
disease.

Methods
Ten normal young volunteers (young control, 21-30 years old), ten normal senior volunteers(senior control, 60-78 years old, MMSE  29), and ten Alzheimer’s disease (AD) patients(study group, 59-84 years old, MMSE: 13-19) participated in this study. All AD patients werediagnosed by neurologists specializing in dementia based on DSM-IV criteria. CSF dynamics were analyzed using positron emission tomography (PET) following an intravenous injection of 1,000 MBq [15O]H2O synthesized on-line.

Results
Water influx into CSF space in AD patients, expressed as influx ratio, (0.755 ± 0.089) was
significantly reduced compared to young controls (1.357 ± 0.185; p < 0.001) and also compared to normal senior controls (0.981 ± 0.253, p < 0.05). Influx ratio in normal senior controls was significantly reduced compared to young controls (p < 0.01).

Conclusion
Water influx into the CSF is significantly reduced in AD patients. β-amyloid clearance has
been shown to be dependent on interstitial flow and CSF production. The current study indicates that reduction in water influx into the CSF may disturb the clearance rate of β-amyloid,
and therefore be linked to the pathogenesis of AD.

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Trial Registration
UMIN Clinical Trials Registry UMIN000011939

eIntroduction

apan,

lz-

eers
ts
were
amin-

as
omon-

as
indiyloid,

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The brain lacks conventional lymphatics for clearing interstitial fluid of solutes not absorbed
across capillaries. The cerebrospinal flow (CSF) system has long been suggested to play a roleequivalent to the systemic lymphatic system. Nevertheless, the role of CSF as the brain’s lymphatic system has not received much attention. Recently, however, the classic CSF circulation
theory where CSF is almost exclusively produced by choroid plexus is found to be incomplete.
It is now understood that influx from the peri-capillary (Virchow-Robin) space into the CSF
system, classically referred to as interstitial flow, plays a major role in CSF production [1–3].
Furthermore, a number of studies have now shown that this interstitial flow plays a critical rolein the clearance of β-amyloid [4–8]. Prealbumin (transthyretin) in the CSF has been identified
to play the role of chaperon to β-amyloid, and prevents β-amyloid's natural tendency to aggregate and form plaques [9]. It is highly conceivable, therefore, that disturbance of interstitial
flow may play a significant role in the pathogenesis of senile plaque (SP) formation.
Water homeostasis of the Virchow-Robin space and, therefore, interstitial flow is regulated
by aquaporin-4 (AQP-4) [2,3,10], an isoform of water channels abundant in the brain. Using JJ
vicinal coupling proton exchange (JJVCPE) imaging [11,12], a novel non-invasive magnetic
resonance imaging (MRI) method capable of tracing exogenously applied substrates in a manner similar to positron emission tomography (PET), we had previously demonstrated thatwater influx into the CSF system is significantly reduced in senile plaque bearing transgenic
AD model mice to the extent similar to AQP-4 knockout mice [12,13]. Using PET, we confirmed in this study that water influx into CSF is also reduced in AD patients.

Materials and Methods
Subjects
Ten normal young volunteers (10 males, ages 21–30 years), ten normal senior volunteers (7males and 3 females, ages 60–78 years), and ten Alzheimer’s disease (AD) patients (6 males
and 4 females, ages 59–84 years) participated in this study. Participant recruitment was completed between October 1, 2013, and April 4, 2014 (Fig 1). All volunteers were free of any significant medical conditions such as hypertension, diabetes, chronic pulmonary diseases, andwere not taking any prescribed, over the counter, or herbal medications. AD patients were recruited from the Memory Clinic, Niigata University Hospital. The diagnosis of AD was determined based on DSM-IV criteria by neurologists specializing in dementia. The neurologists
further determined whether the potential subject had decision making capacity to consent forthe study. Age-matched senior volunteers were assessed to have no functional and no cognitive
impairment (Mini-Mental State Examination (MMSE) score 29), and had no neurologicaldisease. In compliance with the Institutional Review Board of University of Niigata, the study
protocol was explained in detail to all potential subjects, or their proxy where appropriate.
Written informed consent was obtained from all subjects or, where the Alzheimer’s subject wasdeemed to have no decision making capacity to consent for the study, by his/her proxy. All imaging studies were performed between October 2013 and April 2014. The study was approvedby the Ethics Review Board of the Internal Review Board of University of Niigata. The project

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Fig 1. Consort Flow Diagram providing details of participant enrolment.
doi:10.1371/journal.pone.0123708.g001

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was registered at the UMIN Clinical Trials Registry as UMIN000011939 (http://www.umin.ac.
jp/ctr/index.htm). Participants were provided with contact names and telephone numbers in
the event of any adverse event related to the study. All participants were followed up within
one month of the study to further confirm the absence or occurrence of adverse events related
to the study, not otherwise self-reported by the study participant or proxy to the study coordinator. Alzheimer’s patients are additionally followed up annually at the Memory Clinic.

PET imaging
PET imaging was performed using a combined PET/CT scanner (Discovery ST Elite, GE
Healthcare, Schenectady NY, USA) with a 15 cm field of view (FOV) positioned in the regionof the cerebrum. To correct for photon attenuation, low-dose CT imaging was acquired in helical mode. The subject’s head rested on a foam cushioned headrest. A head strap was applied tominimize head movement.
1000 MBq [15O]H2O, synthesized on-line ([15O]CO2 + 4H2 ! 2[15O]H2O + CH4) was injected intravenously into an antecubital vein via an automatic water injection system (AMWR01, JFE Technos, Yokohama, Japan). The system delivered a 10 ml bolus over 10 seconds at
1 ml/sec with both pre- and post- flush of an inert saline solution. Immediately after starting
the administration, emission data were acquired over 20 minutes in three-dimensional list
mode with a 25.6 cm axial FOV and sorted into 40 time frames (40 × 30 seconds).
All emission scans were normalized for detector inhomogeneity and corrected for random
coincidences, dead time, scattered radiation, and photon attenuation. For optimization ofimage quality, the 40 frames of the dynamic emission scans were reconstructed using
3D-OSEM (Ordered-Subset Expectation Maximization) with 2 iterations and 28 subsets. The
resultant image quality allowed manual identification of regions of interest (ROIs). For the reconstruction algorithms, the data were collected in a 128 × 128 × 47 matrix with a voxel size of
2.0 × 2.0 × 3.27mm.

Data Analysis
The CT and PET image data were transferred to a Xeleris 1.1 workstation (GE Healthcare) for
PET data analysis. Manually defined ROIs on the attenuation corrected axial images and CT
images (lateral and third ventricle, cortex of occipital lobe) were used to obtain the time-activity data of the scans of each subject. The tissue activity concentration in each ROI was expressedas the standardized uptake value (SUV, g/ml), corrected for the subject’s body weight and administrated dose of radioactivity. Each tissue time activity concentration was determined by fitting the data using the following equation:yðtÞ ¼ y0 þ ae�bt
where y0 is the baseline SUV. Subsequently, the ratio between SUVs of ventricle and cortex was
deﬁned as the inﬂux ratio. The numerical data were subjected to the Mann-Whitney-Wilcoxon
rank sum test for group analysis using IBM SPSS version 19.5 (IBM Corporation, Armonk,
NY, USA).

Results
Results are shown in Table 1 and summarized in Fig 2. Water influx into CSF space in AD patients, expressed as influx ratio (0.755 ± 0.089), was significantly reduced compared to young
controls (1.357 ± 0.185, p < 0.001) and senior controls (0.981 ± 0.253, p < 0.05). Furthermore,
the influx ratio of senior controls compared to that of young controls was significantly reduced

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Table 1.

Young

SeniorAD

Age

Inﬂux Ratio

21

1.1062021

1.3560821

1.4500921

1.1426022

1.16180221.6207922

1.2499024

1.4121324

1.4895930

1.5787360

0.6829963

0.9272863

1.0398763

1.0572065

1.4419568

0.94050681.2752669

0.8139674

1.0305078

0.5989259

0.69261

61

0.7932163

0.8610563

0.62796710.7008871

0.73065730.7451079

0.6623280

0.8438984

0.88935

doi:10.1371/journal.pone.0123708.t001(p < 0.01) as well. The observed large range of influx ratio (0.599–1.442) in the senior controls
suggested that the reduction in water influx into CSF represented an aging process.

Discussion
Fluid-filled canals surrounding perforating arteries and veins in the brain parenchyma wererecognized in the early era of modern medicine. They became known as the Virchow Robinspace, so named after the first two scientists who described the structure in detail, namely,
Rudolph Virchow and Charles Philippe Robin [14,15]. It was soon recognized that fluid in the
Virchow Robin space may play a role similar to systemic lymphatics [4–8,16]. Studies based onmodern technologies disclosed some of the physiological roles of the Virchow Robin space andinterstitial fluid flow, ranging from the regulation of regional blood flow to β-amyloid clearance
during sleep [17–19].
The unique anatomical features of the brain’s vascular system ensure a properly functioning
blood brain barrier (BBB) and are a result of modification of the systemic circulation system. A
critical property of the BBB is prevention of free water permeability across capillary walls (tight

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Fig 2.
(IR): threducdata pas we

doi:10.

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Ratio

20

08

09

60

80

79

90

13

59

73

99

28

87

20

95

50

26

96

50

92

61

21

05

96

88

65

10

32

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Fig 2. Schematic presentation of the results with mean (circle) and standard deviation (bar). Water influx into CSF space is expressed as influx ratio(IR): the ratio between the standardized uptake value (SUV, g/ml) of the ventricle to that of cortex. IR in Alzheimer’s disease patients (AD) is significantly
reduced compared to both young controls (p < 0.001) and senior controls (p < 0.05), Mann-Whitney-Wilcoxon rank sum test. Note that there is no overlap indata points between AD and young controls. Reduction of influx ratio in senior controls compared to that in young control is found to be significant (p < 0.01)
as well. A large range of influx ratio in senior controls suggests that the observed reduction likely represents one of the aging processes.
doi:10.1371/journal.pone.0123708.g002

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endothelium). This is in strong contrast to systemic capillaries which possess a leaky endothelium. The molecular basis of the tight endothelium is the formation of tight junctions. Various
tight junction proteins such as claudin and occludin play the role of “gating control” of paracellular transport. Passive water movement across brain capillaries is strongly restricted by thesetight junctions. In systemic capillaries, active water transport across endothelial cells is accomplished by the water channel aquaporin 1 (AQP-1). In brain capillaries, such water movement
is totally abolished through the active suppression of AQP-1 [20]. Accordingly, water motion
between capillary lumen into the peri-capillary Virchow Robin space is highly limited to restricted passive movement.
In contrast to the above situations, the peri-capillary Virchow Robin space receives constantwater influx through AQP-4, another isoform of the aquaporin family, abundantly expressedin the perivascular endfeet of astrocytes [21]. Virchow Robin space water homeostasis and,hence, interstitial flow is primarily dependent on water influx through AQP-4 [2,3,10]. Accordingly, brain interstitial flow, which plays a role equivalent to the systemic lymphatic system, isnow considered to be an AQP-4 dependent system [13]. The basic function of lymphatics isdrainage of cellular debris that has been subjected to molecular scrutiny before being returned
to venous circulation. β-amyloid has been shown to be essential for synaptic formation [22].
Disturbance in the proper clearance of β-amyloid, and negative balance between its clearance
and production has been implicated to play a significant role in senile plaque formation and ultimately the pathogenesis of Alzheimer’s disease. The various components of amyloid homeostasis have become a target of therapeutic strategies [23–25]. Since under physiologicalconditions interstitial flow plays a critical role in the clearance of β-amyloid [4–8], disturbancein AQP-4 functionality and resultant reduction in interstitial flow could cause significant βamyloid accumulation. Transgenic mice lacking endothelial cell expression of Agrin are shownto have reduced AQP4 while BBB remained intact. These mice have significant accumulationof β-amyloid[26].
Although it is difficult to determine whether the observed reduction in water influx into the
CSF system in AD patients is the primary abnormality or merely related to β-amyloid deposits
in the brain, the clear cut differences between young normal volunteers and AD patients unequivocally indicated that AD patients have significant reduction in interstitial flow. Furthermore, the theoretical possibility of contribution by choroid plexus or the leptomeningeal
vasculature to the water detected in the CSF compartment cannot be totally excluded. Nevertheless, the observed large range of influx ratios in senior controls without cognitive dysfunction, strongly suggests that reduction of water influx into the CSF itself is not a sufficient factor
for the pathogenesis of AD, a situation similar to senile plaque formation. There must be additional factors that eventually lead to neural death and cortical dysfunction.
Drainage of β-amyloid by interstitial flow through the Virchow Robin space into CSF is essential for maintaining proper homeostasis between β-amyloid production and clearance. Thebalance between β-amyloid production and its clearance appears to be vital for maintaining
proper neural function. Disruption in β-amyloid homeostasis may play a critical role in thepathogenesis of senile plaque formation, and, hence, the development of Alzheimer’s disease.
Assessment of the dynamic indices of production and clearance of β-amyloid in various cohort
groups, including mild cognitive impairment (MCI), is warranted.

Supporting Information
S1 TREND Checklist.
(PDF)

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Reduction of Water Influx into CSF in Alzheimer's Disease

Acknowledgments
The study in part was presented at the 66th AAN Annual Meeting of the American Academyof Neurology, April 2014, Philadelphia, USA and Alzheimer’s Imaging Consortium, July 2014,
Copenhagen, Denmark.

Author Contributions
Conceived and designed the experiments: TN. Performed the experiments: YS YN KY KK YY
TI. Analyzed the data: YS HI MN ILK TN. Wrote the paper: YS ILK TN.

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4.

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19002474

7.

Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, et al. (2012) A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid
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8.

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9786239

9.

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22112803

10.

Kitaura H, Tsujita M, Huber VJ, Kakita A, Shibuki K, Sakimura K, et al. (2009) Activity-dependent glial
swelling is impaired in aquaporin-4 knockout mice. Neurosci Res 64: 208–212. doi: 10.1016/j.neures.
2009.03.002 PMID: 19428702

11.

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JJVCPE amyloid imaging in transgenic mice. J. Neuroimag 2014; 24:595–598.

12.

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14.

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427–462

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Physiol Homme Animaux 2: 537–548

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PLOS ONE | DOI:10.1371/journal.pone.0123708 May 6, 2015

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seasear0

The Molecular Mechanisms of Neural Flow Coupling:
A New Concept
Tsutomu Nakada
From the Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata, Niigata, Japan.

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ABSTRACTThe phenomenon known as neural ﬂow coupling (NFC) occurs at the capillary level where there are no known pressure controlling structures. Recent developments in advanced magnetic resonance imaging technologies have made possible in vivo direct
investigations of water physiology that have shed new insight on the water dynamics of the cortical pericapillary space and theircomplex functionality in relation to NFC. Neural activities initiate a chain of events that ultimately affect NFC. First, neuralactivities generate extracellular acidiﬁcation. Extracellular acidosis in turn produces inhibition of aquaporin-4 (AQP-4) locatedat the end feet of pericapillary astrocytes, the water channel which regulates water inﬂux into the pericapillary space and, hence,
interstitial ﬂow. Reduction of pericapillary water pressure results in a negative balance between pericapillary and intraluminal
capillary pressure, allowing for capillary caliber expansion. Proton permeability through the tight junctions of the blood brain
barrier is signiﬁcantly high owing to the Grotthuss proton “tunneling” mechanism and, therefore, carbonic anhydrase (CA) typeIV (CA-IV) anchored to the luminal surface of brain capillaries functions as scavenger of extracellular protons. CA-IV inhibitionby acetazolamide or carbon dioxide results in the accumulation of extracellular protons, causing AQP-4 inhibition and a secondary
increase in rCBF.
Keywords: neuro flow coupling, aquaporin 4, interstitial flow, protons, neural activation.
Acceptance: Received December 10, 2014. Accepted for publication December 10, 2014.
Correspondence: Address correspondence to Tsutomu Nakada, MD, PhD, FAAN, FANA, Center for Integrated Human Brain Science, Brain ResearchInstitute, University of Niigata, 1-757 Asahimachi, Niigata, 951-8585, Japan. E-mail: tnakada@bri.niigata-u.ac.jp.
J Neuroimaging 2015;25:861-865.
DOI: 10.1111/jon.12219

Introduction
Recent studies have shown that the classic circulation modelof cerebrospinal fluid (CSF) is incomplete.1 Production of CSF
is not only dependent on choroid plexus but also on water
flux in the pericapillary (Virchow Robin) space.2,3 Historically,
CSF flow through the pericapillary space is known as interstitialflow, and is considered to play a role equivalent to the systemic
lymphatic system.4–10 Advancements in modern magnetic resonance imaging (MRI) technologies have allowed for thenoninvasive investigation of water flow in vivo. These studies
revealed that water dynamics of the pericapillary space, ie, interstitial flow, is controlled by aquaporin-4 (AQP-4), the main
subset of the aquaporin water channel family in the brain.11–13
It has also been demonstrated that inhibition of AQP-4 isstrongly coupled with an increase in regional cerebral bloodflow (rCBF).14 These observations have led to a better understanding of the architectural significance and functionality of
the cerebrovascular system.15,16 This article is a concise review of the modern concept of neural flow coupling (NFC)
and its relationship to water dynamics in the pericapillary
space.5–10,16

Cerebral Autoregulation: Upstream Control
Cerebral autoregulation signifies an intrinsic ability of thecerebral vasculature to maintain cerebral blood flow at a
relatively constant rate of approximately 50 ml per 100 g brain
tissue per minute in the face of blood pressure changes.17–22

Autoregulation generally functions between mean bloodpressures of 60 to 150 mmHg. It is maintained in parasympathetically and/or sympathetically denervated animals,21 and thesystem is independent from extrinsic neural control. Instead, intrinsic neural nitric oxide (NO) control22 and release of vasoactive substrates by the brain are believed to play essential roles inmaintaining constant cerebral perfusion.19,20 Perfusion is held
constant by means of the cerebral vasculature smooth muscle
that constricts and dilates in response to elevated and decreased
systemic pressure, respectively.17–22 Although this “upstream”
control of inflow pressure appears to be rather straightforward,
the physiologic mechanisms underlying NFC, neural activityassociated rCBF increase, were until now poorly understood.

Virchow Robin Space and Interstitial Flow:
Cerebral Lymphatic Equivalent
Fluid-filled canals surrounding perforating arteries and veins in
the parenchyma of the brain were recognized in early modern
medicine and described in detail by Rudolph Virchow andCharles Philippe Robin.23,24 The space is commonly referred
to as the Virchow Robin space. It is now clearly understoodthat the fluid in the Virchow Robin space constitutes interstitial
flow that drains into the CSF system (Fig 1). Virchow Robininterstitial flow is believed to play a role similar to systemic
lymphatics.2–10
The basic function of systemic lymphatics is drainage ofcellular debris subjected to molecular scrutiny before returningThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
9/9◦ 2015 The Authors. Journal of Neuroimaging published by Wiley Periodicals, Inc. on behalf ofCAmerican Society of Neuroimaging－ 275 －

861

Active suppression of AQP-1 expression in brain capillaries is essential for proper maintenance of the blood brain barrier, preventing excessive movement of water across capillary
walls.27,28 Active water influx into the CSF system from theblood stream has been shown to be regulated by AQP-4, not
AQP-1, indicating that interstitial flow plays an important rolein CSF dynamics.2
As cerebral equivalent of the systemic lymphatic system,
interstitial flow dysfunction can be expected to result in reduction of β-amyloid clearance. Indeed, senile plaque bearing
transgenic mice showed significant decline of water influx into
the CSF system, to the extent similar to that found in AQP-4
knockout mice.29 Positron emission tomography studies in AD
patients have shown virtually identical results.14

NFC and rCBF
Fig 1. Virchow Robin space and interstitial flow. The ventricles andsubarachnoid space represent the cerebrospinal fluid (CSF) spacein the brain. The Virchow Robin space is a continuous canal surrounding penetrating vessels. Interstitial flow runs within the Virchow
Robin space and drains into the subarachnoid space. Contrary to
the classical concept of CSF flow, water CSF within the subarachnoid space is now believed to be dependent on interstitial flow in the
Virchow Robin space. Although not as yet mainstream, the Virchow
Robin space likely surrounds the medullary veins and subependymal
veins as well. As shown in Figure 3, water influx from the systemic
circulation into CSF is strongly dependent on interstitial flow in the
Virchow Robin space through aquaporin-4 (AQP-4).

to the venous circulation. Although there is no conventional
lymphatic system in the brain, interstitial flow of the Virchow
Robin space constitutes its equivalent, and plays an essential
role in clearing toxic proteins from brain parenchyma. Interstitial flow, and, hence, CSF circulation, eventually drains into thevenous system through the cerebral dural sinuses (Pacchionianbodies), the latter playing a role similar to the thoracic duct of
the systemic lymphatic system.
An important and intriguing example of interstitial flow protein clearance is β-amyloid clearance.3,5,7,8,14–16 β-amyloid is essential for synaptic formation.25 Nonetheless, excess β-amyloid
can result in aggregation of the protein and senile plaque formation. Drainage of β-amyloid by interstitial flow through the
Virchow Robin space into CSF is likely critical for maintaining proper homeostasis of β-amyloid production and clearance. The role of prealbumin, abundant in CSF, as β-amyloid
chaperon and in preventing β-amyloid’s natural tendency toplaque formation, further supports the β-amyloid clearance
hypothesis.26

Pericapillary Water Dynamics and AQP-4
The aquaporin family is a large collection of integral membraneproteins that enable the movement of water across biological membranes. Three isoforms, namely AQP-1, AQP-4, and
AQP-9, have been identified in brain in vivo. Expression of
AQP-1 within CNS capillaries is actively suppressed, and AQP1 in the brain is uniquely found in the choroid plexus epithelium. AQP-9 is only scarcely expressed in the CNS and is considered to have no significant role.12,27 By contrast, AQP-4 isexpressed abundantly in the brain and has a specific distribution: the subpial and perivascular endfeet of astrocytes.2,10–14
862

Increased rCBF associated with brain activation is a wellrecognized phenomenon that is known as NFC. Since thisis a micro-, rather than macroenvironmental event occurring
within an area limited to 250 μm around the site of neuralactivity, the regulatory mechanism for NFC should be within
the capillaries.30
Considering blood flow to be steady, laminar flow withina long cylindrical pipe (Fig 2), the Hagen-Poiseuille equation
gives volumetric blood flow, �, as
�=

Fig

of m
lum
equ

π �P 4R
8η L

where �P is pressure loss (differences in inflow and outflow
pressure), L is the length of the vessel tube, η is blood viscosity,
and R is the radius of the vessel.31
Given that steady inflow pressure is rigorously controlled
by upstream arterial autoregulation17,18 and constant venous
pressure, under physiological conditions, cerebral blood flow isvirtually determined by the radius of the vessel and increases
parallel to its fourth power� ∼ R4
The relationship implies that even small changes in capillary
caliber have significant effects on rCBF.
Capillaries are devoid of muscle and, hence, are not underneural control. The perforating vessels of the cerebral cortex
are surrounded by a fluid-filled perivascular (Virchow Robin)
space. At the capillary level, fluid pressure within the vessel lumen is directly opposed by pericapillary fluid pressure. Therefore, the parameter that determines capillary caliber is thepressure balance between luminal (intracapillary) and outer(pericapillary) fluid pressures. Since intracapillary pressure reflects the inner pressure of arterioles, and is therefore a functionof upstream arterial autoregulation,17,18 it is the fluid pressureof the pericapillary space that inversely determines cerebral
capillary caliber changes and, hence, rCBF, as follows:� ∼ R4 ∼ 1/Pperi−capillary
AQP-4 controls the water dynamics of the pericapillary
space in the brain.2,10,14 Therefore, AQP-4 activities play a rolein controlling rCBF. Simply put, under physiological conditions, rCBF correlates inversely to AQP-4 activities.
rCBF ∼ 1/AQP 4activities

Journal of Neuroimaging Vol 25 No 6 November/December 2015

－ 276 －

Fig

cap
junc
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and
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and

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Fig 2. Vessel diameter is determined by tension of smooth muscle in artery, arteriole, venule, and vein (Brain Vessels). Capillaries are devoidof muscle and in capillaries with tight endothelium such as brain capillaries, capillary caliber is determined by the pressure balance between
luminal and outer fluid pressures (Brain Capillary). For capillaries with leaky endothelium (Common Capillary), pressure balance is quickly
equalized without capillary caliber changes.

Neural Activities and Extracellular Acidosis

n
n

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,

d
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-

Fig 3. Pericapillary water dynamics. Water permeability of brain
capillaries is restricted due to the tight endothelium, presence of tightjunctions and active suppression of AQP-1. By contrast, significant
water flow is present in the Virchow Robin space (interstitial flow)
and is supported by active water inflow through AQP-4. Although it
has not been clearly confirmed (?), interstitial flow may similarly bepresent along the medullary and subependymal veins.

Indeed, reduced AQP-4 activities by its inhibitor TGN-020
effectively increased rCBF in mice.13 Under physiological conditions, AQP-4 is believed to be inhibited by extracellular
protons similar to other AQP isoforms.32 Therefore, rCBF is
predicted to correlate with extracellular proton density (Figs 3
and 4).
rCBF ∼ [H + ]extra
The steps outlined above provide a fresh understanding of
the underpinnings of NFC.

Since the original description by Urbanics et al33 extracellular(interstitial) acidification associated with neural activities has
been extensively studied by various investigators.34–36 Modern
MRI technologies demonstrated unequivocally that regional
neural activities in humans are accompanied by extracellularacidosis found in a virtually identical distribution as the neural activity-induced increase in rCBF detected by blood oxygenation level-dependent contrast.37 Therefore, at least froma phenomenological stand point, neural activity-induced extracellular acidification plays a role in NFC. Although the precise underlying mechanisms remain to be elucidated, it appears
clear that neural activity-induced interstitial acidification, andthe resultant inhibition of AQP-4, is indeed a main mediatorof neural activity-associated rCBF increase. Further support
for this concept comes from the “Diamox effect.” Acetazolamide (Diamox) is a carbonic anhydrase (CA) inhibitor anda powerful agent for increasing rCBF. This “Diamox effect”
is well known to be accompanied by interstitial acidosis in
the brain.38
Within the large CA family, CA type IV (CA-IV) represents the dominant CA in the cerebral cortex and is anchoredto the luminal surface of cerebral capillaries.39 It has beenshown that interstitial CA activity in the brain is attributable to
CA-IV.40 The human NBC1 sodium bicarbonate cotransporter
directly interacts with CA-IV. The tethering of intracellular CA
type II (CA-II) and extracellular CA-IV in proximity to theNBC1 HCO3 − transport site maximizes the transmembrane
HCO3 − gradient local to NBC1 and thereby activates the transport rate.41
Since proton permeability through the tight junctions is significantly higher than for other small molecules, owing to the
Grotthuss proton tunneling mechanism,42 capillary CA-IV with
NBC1 and CA-II effectively function as scavenger of extracellular protons generated by neural activation (Fig 5). CA inhibitionby acetazolamide or excess of carbon dioxide (CO2 ) in capillary
blood results in accumulation of extracellular protons which inturn inhibit water flux through AQP-4. The resultant negative
pressure relation with respect to intraluminal capillary pressure
affects capillary dilatation and an increase in rCBF.

Nakada: The Molecular Mechanisms of Neural Flow Coupling－ 277 －

863

intr
trus
exp
link
ral
ilar
to b

The
Cul

Re
1.

2.

Fig 4. Neural activation. Neural activation produces extracellular acidification accompanied by increase in rCBF and astrocyte swelling.
Proton inhibition of AQP-4 results in a reduction of water flow from astrocytes into the pericapillary Virchow Robin space, astrocyte swellingand capillary expansion due to reduction of pericapillary fluid pressure.

3.

4.
5.

6.
7.

8.

9.

10.

11.

12.
13.

Fig 5. CA-IV system. Complex of CA-IV anchored to luminal surface of cerebral capillary, human NBC1 sodium bicarbonate cotransporterand intracellular CA-II. Their proximity maximizes the transmembrane HCO3 − gradient local to NBC1 and thereby activates the transport rate.
Because of the high proton permeability through tight junctions, capillary CA-IV with NBC1 and CA-II effectively function as scavenger ofextracellular protons generated by neural activation. CA inhibition by acetazolamide or excess of CO2 in capillary blood results in accumulationof extracellular protons.

Summary: A New Concept
Technological advancements, especially those in the fieldof MRI, have led to a new level of understanding of thephysiologic underpinnings of neural activation-induced rCBF
increase, a phenomenon known as NFC. The main player mediating NFC is the proton. Neural activities produce interstitial acidification. Excess protons inhibit AQP-4 activities in the
pericapillary Virchow Robin space, resulting in a reductionin the pericapillary pressure. The negative balance between

864

pericapillary and intraluminal capillary pressure induces dilatation of capillaries and an increase in rCBF. Acetazolamide orexcess CO2 blocks active clearance of interstitial protons which
are ordinarily highly permeable through the tight junctions and,similar to neural activities, causes interstitial acidification andan increase in rCBF.
The precise molecular mechanism of extracellular acidification associated with neural activities remains to be elucidated. Such acidification has been shown to be associated with

Journal of Neuroimaging Vol 25 No 6 November/December 2015

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14.
15.
16.

17.
18.

19.
20.

.
g

r
.
f
n

r
h
,
d

h

intracellular alkalinization of astrocytes.36 Active proton extrusion by astrocytes appears to be the most attractive
explanation.14,36 The functional significance of NFC has been
linked to elimination of heat production brought about by neural activities. A heat-sensitive voltage-gated proton channel similar to neutrophil Hv1 may play a role, although much remains
to be investigated.14,43
The work was supported by grants from the Ministry of Education,
Culture, Sports, Science, and Technology (Japan).

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type IV. J Neurosci 2000;20:8247-53.
41. Alvarez BV, Loiselle FB, Supuran CT, et al. Direct extracellularinteraction between carbonic anhydrase IV and the human NBC1
sodium/bicarbonate co-transporter. Biochemist 2003;42:12321-9.
42. Angelow S, Kim KJ, Yu ASL. Claudin-8 modulates paracellularpermeability to acid and basic ions in MDCK II cells. J Physiol
2006;571.1:15-26.
43. Fujiwara Y, Kurokawa T, Takeshita K, et al. The cytoplasmic
coiled-coil mediates cooperative gating temperature sensitivity inthe voltage-gated H+ channel Hv1. Nat Commun 2012;3:816.

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Magn Reson Med Sci, Vol. 14, No. 1, pp. 13–24, 2015

doi:10.2463/mrms.2014-0039
doi:10.2463/mrms.2014-0039

Magn
MedSociety
Sci, Vol.
No. 1, pp.
13–24, 2015
2014Reson
Japanese
for14,
Magnetic
Resonance
in Medicine©
© 2014 Japanese Society for Magnetic Resonance in Medicine

14

REVIEWREVIEWN-acetylaspartate Decrease in Acute Stage of Ischemic Stroke:A Perspective fromN-acetylaspartate Decrease in Acute Stage of Ischemic Stroke:A Perspective from
Experimental and Clinical Studies
Experimental and Clinical Studies
Hironaka IGARASHI11*, Yuji SUZUKI11, Vincent J. HUBER11, Masahiro IDA22,
Hironaka IGARASHI *, Yuji SUZUKI , Vincent J.1HUBER , Masahiro IDA ,and Tsutomu NAKADA1
and Tsutomu NAKADA
1Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata
1Center for Integrated Human Brain Science, Brain Research Institute, University of Niigata
1–757 Asahimachi-dori, Chuo-ku, Niigata City 951–8585, Japan
1–757
Asahimachi-dori,Chuo-ku,
City 951–8585,
Japan
2
Departmentof Radiology,
Tokyo Niigata
Metropolitan
Ebara Hospital2
Department of Radiology, Tokyo Metropolitan Ebara Hospital(Received March 25, 2014; Accepted July 16, 2014; published online December 15, 2014)(Received March 25, 2014; Accepted July 16, 2014; published online December 15, 2014)

N-acetylaspartate (NAA) appears in a prominent peak in proton magnetic resonanceN-acetylaspartate(NAA)
appears
a prominent
peak inmagneticattenuation
resonance
spectroscopy( 11 H-MRS)of thebrain.inExhibition
by NAAof proton
time-dependent
spectroscopy(
H-MRS)
ofthebrain.
Exhibition
by
NAA
of
time-dependent
attenuationthat reﬂects energy metabolism during the acute stage of cerebral ischemia makes thisthatreﬂectsa energy
duringthe acutestage stroke.
of cerebral
ischemia
makesresothis
metabolite
unique metabolism
biomarker forassessingischemic
Althoughmagnetic
metabolite
unique biomarker
for techniqueassessing forischemic
stroke.
Although magnetic
nance (MR)a imaging
is a powerfulinspecting
the pathologicalchangesresothat
nance
(MR)
imagingisapowerfultechniqueforinspecting
thepathologicalchangesthat
occur during ischemic stroke, biomarkers that directly reﬂect the drastic metabolicchangesoccurduringischemic
stroke,biomarkers
thatdirectly
reﬂectthedrastic
metabolicchanges
associated with acute-stage ischemia are strongly warranted for appropriate therapeutic
associatedwith acute-stage
ischemia
are strongly
warranted
for appropriate
therapeutic
decision-making
in daily clinicalsettings.
In this review,
we provide
a brief overview
ofdecision-making
indailyclinicalsettings.
In
thisreview,
we
provideabrief
overviewof
NAA metabolism and focus on the use of attenuation in NAA as a means for assessing the
NAA
metabolism
and
focus
on
the
use
of
attenuation
in
NAA
as
a
means
for
assessingthepathophysiological changes that occur during the acute stage of ischemic stroke.
pathophysiological changes that occur during the acute stage of ischemic stroke.
Keywords: brain, ischemia, magnetic resonance spectroscopy, N-acetylaspartate
Keywords: brain, ischemia, magnetic resonance spectroscopy, N-acetylaspartate
to a combination of brain tissue necrosis and neuroto
combinationof braintissueand neuronala loss
within several
days
to a necrosis
week following
thenalloss within
to a week
followingischemic
eventseveral
unlessdays
energymetabolism
is thereischemic
eventtheunlessenergyhours.
metabolism is resumed withinﬁrst several
sumed
withinthe ﬁrst
severalIschemic
strokehas been
the hours.
most widely studied
Ischemic
strokehas
been
the most
widelyof pathological conditions thatexhibitacutestudied
NAAof
pathologicalconditions
thatexhibit
acuteattenuation in both animal models and humans,NAA
priattenuationin bothmodels andincidence.
humans, pri1
marily becauseof itsanimalhigh worldwide
H1marilybecauseofits
highworldwide
incidence.
MRS is useful for visualizing the progression Hof
MRS
useful for visualizing
the progressionofthose ispathologicalconditions following
severely
thosepathologicalconditions
following
severelycompromised
cerebral
blood ﬂow
resultingfromcompromised
cerebralblood
ﬂow
resultingfromvascular occlusion. Lactate appears within minutes
vascularocclusion.
Lactateappears
withinminutesof the occlusion due to an acceleration of anaerobicof
the occlusion
to an accelerationof anaerobic
glycolysis,
whichdue
compensatesfor hampered
mitoglycolysis,
which
compensatesfor
hampered
mitochondrial oxidation. Because NAA synthesisischondrial
oxidation.
Because
NAA
synthesisisadenosine triphosphate (ATP)-dependent (energyadenosine
triphosphate(ATP)-dependent(energy6
dependent), 6 impaired energy availability initiates
dependent),
a continuous impaired
decline inenergy
levels availabilityof NAA, theinitiates
rate ofawhichcontinuous
declineinlevelsofNAA,
therate ofdepends in part on severity, within hoursof
which
dependsevent.
in parton severity,
withinresonancehours ofthe ischemic
Althoughmagnetic
theischemic
event.
Althoughmagnetic resonance(MR)
imaging,
especiallydiffusion-weighted
im(MR)
imaging,
especiallydiffusion-weighted
images (DWI) and perfusion images (PI), is a powerful
ages
(DWI)
and
perfusion
images(PI),
isapowerfultechnique for visualizing pathological changes intechniqueforbevisualizingchangesinvivo that canapplied to pathologicalischemic stroke,
we bevivo
that
can
be
appliedtoischemic
stroke,webelieve observation of biomarkers that directly reﬂect
lieve observation of biomarkers that directly reﬂect

Introduction
Introduction

N-acetylaspartate (NAA) is the acetylated derivN-acetylaspartate
the acetylated
derivativeof L-aspartate 11 (NAA)
and theissecond
most abundant
ativeof
L-aspartateandthesecondmostabundantamino acid-related metabolite in the mammalianaminoacid-related
in thethereason
mammalian
2,3
brain after
glutamate.metabolite
Althoughfor its
2,3
brain
after
glutamate.
Althoughthereasonfor its
abundance in the brain remains to be elucidated,
abundance
in
the
brain
remainstobeelucidated,
NAA exists mainly in neurons at sufﬁcient concenNAA
existssufﬁcientconcentrations
to bemainly
visibleininneurons
proton atmagnetic
resonance
trations
to
1 be visible in proton magnetic resonancespectra ( 1 H-MRS) of normal brain, where the methspectraof normal
brain, wheretheatmethyl group( H-MRS)of NAA shows
a prominent
peak
2.01
yl
group
of
NAA
shows
a
prominentpeak
at 2.01ppm (Fig. 1). Furthermore, NAA levels attenuateinppm1).pathological
Furthermore,
NAArelatinglevels attenuateinmany(Fig.
of thestatesto impaired
many
of
the
pathologicalstatesrelating
toimpaired
cellular metabolism and neuronal loss. Pathologicalcellularmetabolism
and neuronal
Pathologicalconditions
characterized
by acuteloss.
failureof energy
conditions
characterizedbyacutefailureenergymetabolism, i.e., ischemic stroke, hypoxia,ofandtraumetabolism,i.e.,ischemic
stroke,hypoxia,
andtraumatic brain injury, exhibit drastic NAA attenuation
matic
brain
injury,exhibitdrastic
NAA
attenuationfrom within minutes to hours following the speciﬁcfromwithinminutes
to hours
following
thethespeciﬁc
4,5
initiating
event.
This
attenuation
duringacute
4,5
initiating
attenuation
during thestage
mayevent.
initiallyThis
involvea deteriorationof acute
NAA
stage
may acceleration
initially involvea deterioration
synthesis,of NAA
catabolism,ofor NAAtheir
synthesis,
accelerationof NAAconditions
catabolism,or theircombination.
These pathologic
often
lead
combination. These pathologic conditions often lead*Corresponding author, Phone: +81-25-227-0676, Fax: +81*Corresponding
author,higara@bri.niigata-u.ac.jp
Phone: +81-25-227-0676, Fax: +8125-227-0822, E-mail:25-227-0822, E-mail: higara@bri.niigata-u.ac.jp
13
13－ 280 －

met
high
ther
brie
que
sess
stag

Dis

Dis
H
mog
brai
the
sph
imm
Add
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ther
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cell
dete

0039
0039

14

H. Igarashi et al.

omomurourothe
the
rere-

died
died
AA
AA
pripri1
H1
n Hof
nrely
of
rely
rom
rom
utes
utes
obic
obic
itositoiss isrgyrgyates
eates
of
es of
of
snce
of
nce
imimrful
rful
s in
sbein
beﬂectﬂect

Fig. 1. Chemical structure of N-acetylaspartate (NAA) and representative proton magnetic resonance spectroscopy (1H-MRS). The methyl group of NAA (a)shows the most prominent singlet peak at 2.01 ppm in 1H-MRS of normal brain(b). NAA reduces and lactate appears (c) in the acute ischemic lesion delineatedby diffusion-weighted imaging (DWI) (d). tCHO, total choline compounds; tCr,
sum of creatine and phosphorcreatine.

metabolic changes during the acute stage will behighly valuable for making the most appropriate
therapeutic decisions. In this review, we present a
brief overview of NAA metabolism with subsequent focus on the use of NAA visualization to assess pathophysiological changes during the acutestage of ischemic stroke.

Distribution, Biochemistry, and Functions
Distribution of NAA
Human studies using MRS show a relatively homogenous distribution of NAA throughout thebrain, with slightly higher concentrations found in
the occipital cortex, thalamus, and cerebellar hemisphere. 7,8 These results agree with those of previous
immunohistochemical studies using rodent brain. 9
Additional immunohistochemical studies have demonstrated the presence of NAA predominately inneuronal cell bodies and their processes. 4,9,10 Further conﬁrmation of this distribution was obtainedwith high performance liquid chromatography(HPLC) and high resolution nuclear magnetic resonance (NMR) studies. 11–13 Oligodendrocyte type2 astrocyte progenitor cells (O-2A progenitors, NG2cells) 14 were also found to express NAA at easily
detectable levels. 12,13 NG2 cells in postnatal brain

have the potential to differentiate into oligodendroglia in gray and white matter 15 and have been recently conﬁrmed to make up about 5% of all cells in
the adult central nervous system (CNS). 16 Although
the expression of NAA by NG2 cells in the adultbrain has not been conﬁrmed, some caution is warranted in interpreting changes in NAA concentration as a pure neuronal marker.
Tricellular compartment metabolism of NAA
NAA and its glutamate dipeptide derivative, Nacetylaspartylglutamate (NAAG), which supposedly works as a cotransmitter with glutamate and £aminobutyric acid (GABA), 17 form the unique tricellular compartment metabolism involving neurons, oligodendrocytes, and astrocytes (Fig. 2). 18
Both NAA and NAAG are synthesized in the neuron, but neither are catabolized in the neuron itself.
NAA is either released from neurons or is transported to oligodendrocytes, where it is metabolized into
acetate and aspartate by aspartoacylase (ASPA). 19
NAA-derived acetate in the oligodendrocytes isconverted to acetyl coenzyme A (acetyl CoA) withacetyl CoA synthetase, which serves primarily as aprecursor in myelin lipid synthesis. 20,21 NAAG isreleased from the axon terminal in response to neuronal activity, and most is subsequently hydrolyzed－ 281 －

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NAA Attenuation in Acute Ischemic Stroke

15

Fig. 2. Tricompartment metabolism of N-acetylaspartate (NAA) and N-acetylaspartylglutamate synthetase (NAAG). Both NAA and NAAG are synthesized inneurons. NAA is mainly released from neurons or transported to oligodendrocytes, where it is metabolized into acetate and aspartate by aspartoacylase.
NAA-derived acetate in oligodendrocytes is mainly used as a precursor of myelin
lipid synthesis. NAAG is released from axonal terminals in concordance withneuronal activity, and most of them are hydrolyzed into NAA and glutamate withglutamate carboxypeptidase II (GCPII) at the surface of astrocytes. AAT, aspartate
aminotransferase; acetyl CoA, acetyl coenzyme A; ¡-KG, ¡-keto glutarate; APSA,
aspartoacylase; Asp-NAT, L-aspartate N-acetyltransferase; GS, glutamine synthetase; OAA, oxaloacetate.

into NAA and glutamate at the surface of the astrocyte membrane by glutamate carboxypeptidase II(GCPII). 22
Synthesis, catabolism, and functions
The majority of NAA is thought to be synthesizedin neuronal mitochondria23 from aspartate and acetyl-CoA.24,25 Aspartate is converted in the mitochondria from malate, which enters the mitochondria
through the mitochondrial transporter, aralar1.26 The
conversion of malate to aspartate occurs via aspartate aminotransferase (AAT) and part of the tricarboxylic acid (TCA) cycle (Fig. 2). 27 Finally, NAA
is synthesized by membrane-bound L-aspartate Nacetyltransferase (Asp-NAT) 28 in an energy-dependent manner. 6,12,29 Partly because of that energy dependence and the involvement of the TCA cycle inthe conversion of malate to aspartate in mitochondria, NAA biosynthesis is very susceptible to energy failure. 6 Once synthesized, NAA is transported
to the cytosol by the dicarboxylic acid transporter. 30
A recent report identiﬁed the synthesis of a smallVol. 14 No. 1, 2015

but distinct amount of NAA in cytosol. 31,32 In thissecondary pathway, Asp-NAT in the cytosol isthought to utilize aspartate sequestered from the mitochondria through aralar1. 33 However, the signiﬁcance of the 2 distinct NAA synthetic pathways remains unknown.
NAA synthesized in the neuron is transported toeither the extracellular space (ECS) or oligodendroglia. Because it exists as a dianion at physiologicalpH, the nonspeciﬁc diffusion of NAA through thecell membrane is unlikely. No transporter of NAA
has been identiﬁed, but Fujita and Ganapathy suggest that Na + /carboxylate cotransporter 3 (NaC3)
is a likely candidate. 34 It has been estimated thatapproximately 5% of the total NAA in the CNS is
excreted from the ECS into the blood presumably
via putative NAA transporters in the astrocytes.35
NAA is hydrolyzed by aspartoacylase, the distribution of which is restricted to oligodendrocytes. 36
Aspartoacylase activity is reported to increase inparallel with white matter myelination during development, 37 and NAA is hydrolyzed into aspartate－ 282 －

16

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15

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H. Igarashi et al.

and acetate, the latter being an important component of myelin lipid. Therefore, it is believed thatpart of the NAA synthesized in the neuron serves asa source of building blocks for myelin synthesis inoligodendrocytes. 20,21,38
Other roles for NAA in the brain are still debated.
However, there is evidence that NAA is importantin maintaining the microenvironment of the brain
parenchyma. 4 Two tissues outside the CNS, peritoneal mast cells 39 and ocular lenses, 40 have beenfound to contain NAA, and in both examples, NAA
appeared to play an important role in osmoregulation and the control of water distribution. Likewise,
NAA is also thought to participate in controlling
osmolality and water distribution in the CNS. 41 In
this context, Baslow claimed that NAA may be released from neurons in some controlled fashion,
which can differ from that of neurotransmitters, andserve in intercellular signaling. 19 A high concentration of NAA has also been considered to compensate for the anion deﬁcit in the CNS. 2,4 Nakada suggested that the negatively charged NAA in neuronalcytosol can facilitate passive diffusion of high energy phosphates from the mitochondria to the cell
membrane to fulﬁll the high energy demands ofneuronal cell membranes. 42,43

NAA Attenuation in Acute-stage Ischemic
StrokeConcept of experimental ischemic stroke
Experimental models of cerebral ischemia havebeen developed to assess pathophysiologicalchanges resulting from ischemia. The universal feature of the various models is the interruption of cerebral blood ﬂow that feeds a part of the brain,
which, in turn, elicits a depletion of oxygen andglucose and disrupts tissue metabolism. 44 At present, rodent models are widely used because their
preparation is convenient, and multiple means ofanalysis are available. 45
Rodent models of ischemia can be divided into 2classes, global and focal. The global ischemia model is characterized by temporary pan-forebrain ischemia of relatively short duration, typically 5 to 30
min, that is induced by occlusion of the bilateralinternal carotid arteries 46,47 alone or with the simultaneous occlusion of the bilateral vertebral arteries. 48 This model elicits a profound ischemia ofshort duration over the forebrain, so it is considered
to be a model of short-duration cardiac arrest or
severe hypotension. Infarction is not the primary
result of this insult, which instead leads within aweek of the ischemic injury to patchy neuronal loss
with several degrees of reactive gliosis in vulnera-

ble regions, such as the hippocampus, striatum andcortex.
Focal ischemia models are used to mimic clinicalischemic stroke. In these models, permanent (no reperfusion) or transient (reperfusion typically withinseveral hours) ischemia are induced in a certain vascular territory, commonly that of the middle cerebral artery (MCA), by arterial occlusion using a silicon-coated suture, blood clot, or clipping. In thismodel, energy failure from severe blood cessationin the core of the circumscribed lesion (ischemiccore) produces irreversible biological changes within a short time. Reduction of blood ﬂow is relatively
mild in the rim of the lesion, and that tissue can besalvaged if blood ﬂow can be restored (reperfusion)
within a few hours. 49 In this salvageable area, called
the “penumbra” after the half-shaded area of an
eclipse, blood ﬂow is too low to maintain electricactivity but remains sufﬁcient to preserve the function of energy-dependent ion channels. 50 The term
“penumbra” is now generally used to deﬁne ischemic but salvageable cerebral tissue that requires immediate reperfusion.51
MRS ﬁndings and gradual attenuation of NAA in theacute stage of ischemic stroke
Typical chronological changes visible by MRS
during acute ischemic stroke have been identiﬁed
in several animal studies 52–55 and subsequently conﬁrmed in human clinical studies. 56 –59 Typically,
chronological changes of four MRS visible metabolites can be seen following cerebral ischemia,
namely rise of lactate, and attenuations of NAA,
creatine and phosphocreatine (Cr), and choline
compounds (Cho). Lactate appears immediately after cerebral blood ﬂow is compromised and usuallyreaches a concentration plateau within 24 hours depending on several factors, such as the severity ofhypoperfusion and plasma glucose concentrations.
Attenuation of NAA levels becomes evident within
an hour and continues almost linearly during theﬁrst 24 hours (Fig. 3). 54,56 –59 After 24 hours, lactate
tends to cover the entire topographical area of hypoperfusion, including the penumbra. Part of the“tissue at risk” can be identiﬁed as areas in which
lactate is present but there is no attenuation of NAA(Fig. 3b). 60 Respective peaks caused by Cr and Cho
begin to gradually fall after cell death begins, andtissue necrosis becomes obvious, typically several
hours following ischemia.
The changes in relaxation affect the attenuationsof metabolite peaks mainly in subacute to chronic
stage of ischemia. In experimental study, van der
Toorn and associates measured T 1 and T 2 of themetabolites and concluded that relaxations of NAA－ 283 －

Magnetic Resonance in Medical Sciences

NAA Attenuation in Acute Ischemic Stroke

17

18

Fig. 3. Chronological changes in N-acetylaspartate (NAA) of rat brain withpermanent focal ischemia. In a diffusion-weighted imaging (DWI)-delineated areaof ischemia (a), NAA visually decreased on proton magnetic resonance spectroscopy ( 1H-MRSI), and lactate appeared in the area at risk (b). NAA declined more
steeply in the ischemic core (lateral caudo-putamen [LCP] and somatosensory
cortex [SSC]) than the ischemic rim (MCS). MCP, medial caudo-putamen; R,
reference area.

did not change within 24 hours after the inductionof MCA occlusion in the ischemic core of the rat
brain. 61 These results agree with the clinical study
which concluded that the T 2 of NAA decreased during the period between 5 and 20 days after the insult
but did not change earlier.62
Our laboratory conﬁrmed a linear decrease in
NAA levels within the ischemic core and rim instudies utilizing both rodent models and clinicalcases. 55,63 In both studies, no reperfusion was attempted at the point where MRS imaging (MRSI)
was carried out. The observation of a steeper slope
in the decline in NAA in the core than the penumbra(Figs. 3, 4) indicates that the rate of NAA attenuation in acute-stage ischemia depends in part on theseverity of hypoperfusion. Some of the data in thehuman study deviated lower (open circle, Fig. 4),
and most of those cases demonstrated hyperglycemia (>120 mg/dL), which adversely affects metabolic deterioration 64 and stroke outcome. 65 Therefore, we undertook a partial correlation of the normalized NAA concentration, time from stroke onset, and initial plasma glucose concentration. That
correlation revealed that both the amount of time
elapsed from the ischemic event and the plasmaVol. 14 No. 1, 2015

concentration of glucose in the ischemic core contributed to the rate of NAA decline (Fig. 4b). The
reason NAA declines faster in response to hyperglycemia remains unknown. However, hyperglycemia is thought to facilitate the release of cytoplasmic amino acids and their derivatives into the ECS
during ischemia, 66 thereby raising the intracellularconcentration of calcium ions, which can, in turn,activate aspartoscylase. 67 These mechanisms may
affect the acceleration of NAA decline.
In our human study, the rate of NAA attenuation
was calculated as 0.67 μg/kg/h; this included compensation from the concentration of plasma glucose
in the brain tissue, which was calculated with an
NAA concentration of 9.2 mmol/g (Fig. 2b) revealed by 13 C-MRS study in human. 68 The result
was somewhat larger than the rate of NAA synthesis
in normal adult human brain (0.55 μg/kg/h) calculated from a human 13 C NMR study that measured
the turnover of NAA in brain tissue. 42,68 These results imply the occurrence of either NAA catabolism or its excretion and cleavage products, namely
aspartate and acetate, during ischemic stroke, even
if NAA production has failed completely. The
13
C NMR study in rats demonstrated that NAA syn-－ 284 －

thes
min
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17

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18

H. Igarashi et al.

Fig. 4. Chronological changes in N-acetylaspartate (NAA) in clinical ischemic
stroke. As observed in rat brain with permanent focal ischemia, NAA declines
more steeply in the ischemic core than the ischemic rim (a). A closed circle shows
the result from euglycemic cases (¯120 mg/dL), and an open circle denotes theresults from hyperglycemic (>120 mg/dL) cases in the ischemic core. NAA decline was partly determined with time and plasma glucose concentration in theischemic core (b).

thesis continued within the ischemic core at 120
min after induction of the MCA occlusion, albeit
at a signiﬁcantly impaired level. 69 A massive NAA
release into the ECS was observed in rat models of
permanent focal ischemia, with the concentration of
NAA in the ECS increasing to 10 times that of anormal rat. However, NAA was undetectable in the
blood with ischemic stroke, 70 so the direct excretionof NAA into the residual blood ﬂow within the ischemic core can be expected to play only a minor
role on NAA attenuation in acute sgate of ischemia.
Furthermore, no rise in NAA levels were found in
the CSF of rat models of focal ischemia 71,72 despite
its massive release into the ECS. These results suggest a previously unknown NAA catabolic pathway
in brain tissue that is activated during ischemia. Indeed, complete global ischemia achieved by decapitation, in which there is a lack of residual blood
ﬂow, leads to a reciprocal accumulation of aspartate
and acetate over NAA loss. 72 Aspartacylase activated by Ca 2+ accumulated in cytoplasm as a result ofischemia may play the dominant role in NAA catabolism in complete global ischemia. 68 Paradoxically, the levels of both aspartate and acetate decreased in the ischemic lesion during focal ischemia. 72,73 One important difference between these 2models of ischemia is the complete absence ofblood ﬂow in the global model and the maintenanceof residual blood ﬂow to the ischemic core of the
focal model. 74 A massive amount of aspartate isalso released into the ECS during ischemia. 75 Excretion of the aspartate from the ECS into the blood
can occur through alanine-serine-cysteine transporter 2 (ASCT2) channels located on the basal
membranes of the vascular epithelium, which areactivated by ischemic acidosis. 76 Indeed, serum
concentrations of aspartate were reported to riseduring focal ischemia. 77 From these reports, we
can ascertain that part of aspartate is released into
residual blood ﬂow from the CNS. Acetate may also
be released into blood or further metabolized bysurviving cells, possibly astrocytes that exclusively
metabolize external acetate under normal physiological conditions. 78 Though the fate of acetate isunclear compared to that of aspartate, we conﬁrmedthat during focal ischemia, the NMR signal fromacetate was detectable in the center of the ischemic
core (Fig. 5a-c). In summary, attenuation of NAA
during ischemic stroke can result from suppressionof NAA synthesis, acceleration of NAA catabolism,excretion of NAA-related derivatives into blood,and further metabolism (Fig. 5d, e).

－ 285 －

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NAA Attenuation in Acute Ischemic Stroke

19

20

Fig. 5. Magnetic resonance spectroscopy (MRS) at 10 hours after induction of
focal ischemia (a-c) and suspected metabolic state of N-acetylaspartate (NAA)
and its derivatives (d,e). Proton MRS (1H-MRS) (a, dashed line), ﬁtted curve (a,solid line, and c) and ﬁtted residual (d). An acetate peak was seen at 1.9 ppm(arrow), but a suspected aspartate peak (2.8 ppm) was absent (arrowhead). 1, totalcholine compounds (tCho); 2, creatine and phosphocreatine (Cr); 3, glutamate; 4,
NAA; 5, acetate; 6, lactate. Reduction of NAA in ischemic stroke can result in the
suppression of NAA synthesis, acceleration of NAA catabolism, release intoblood of the derivatives, and further metabolism (d,e).

to 1
rebr

Association and Dissociation between NAA
Attenuation and DWI Findings
Cross-sectional studies: topographical NAA concentration as a complement of DWI
Diffusion-weighted imaging is a powerful modality for visualizing focal ischemic lesions in acuteischemic stroke. It can be used to delineate the regional reduction of the apparent diffusion coefﬁcient (ADC) of water, which can be calculated fromseveral DWI images. Though energy failure fromischemia reduces both ADC and NAA, ADC is reduced primarily in the areas in which cellular structure is altered by Na + /K + ATP pump failure, which
may only reﬂect an indirect disruption of energy
metabolism. On the other hand, a reduction of NAA
directly reﬂects metabolic changes, speciﬁcally energy failure, in brain tissue during acute-stage ischemia. Based on that difference, we can expect
NAA levels to reﬂect the condition of the ischemic
area more sensibly than ADC. However, because
NAA metabolism is comparatively slow, the delineation of the ischemic lesion based on NAA attenuation relating to metabolic energy failure lags behind the reduction of ADC. In clinical cases ofVol. 14 No. 1, 2015

proximal MCA occlusion, Nicoli reported that within 7 hours from onset, MRSI could indicate a large
heterogeneity in NAA concentration in ischemic
areas showing even the lowest ADC values.79 Moreover, some reports suggest a wide range of NAA
concentrations, from 40 to more than 100% of thenormal range, in acute-stage ischemic lesions having an unperturbed DWI. 59,80 Although it is difﬁcult
at present to ascribe a threshold NAA concentration
for making an accurate tissue prognosis, i.e., whether it will infarct or be salvageable, during the acutestages of ischemia, it has been suggested that salvageable areas might be characterized by the presence of lactate with no or only subtle NAA attenuation. 80 In cases with hemispheric transient ischemic attack (TIA), NAA was observed to attenuate
about 10 to 15% in the acute stage, which recoveredin 12 to 18 months after the incidents. 81 Only thereport of a case with occlusion of the brachiocephalic artery described the reversibility of NAA
in the acute stage of ischemia, with the ratio of
NAA to Cr attenuated 10% at the initial measurement normalized after recanalization. 82 Though alarger study is warranted for conﬁrmation, these reports suggest that NAA attenuation by less than 10－ 286 －

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H. Igarashi et al.

Fig. 6. Chronological changes of proton magnetic resonance spectroscopy (1HMRS), apparent diffusion coefﬁcient (ADC) map, and regional cerebral blood
ﬂow (rCBF) map before, during, and after short-term focal ischemia.92 ADC restored at one hour after recirculation, but N-acetylaspartate (NAA) demonstrated
continuous attenuation after the induction of ischemia. Open square delineates theregion of interest for magnetic resonance spectroscopy (MRS).

to 15% may be reversible in the acute stage of cerebral ischemia.
Longitudinal studies: perpetual NAA attenuation
against temporary restoration of ADC
NAA levels decline at an almost linear rate overtime from the onset of ischemia. This time-dependent decline distinguishes NAA as a useful biomarker for the imaging of acute-stage ischemic stroke.
Cvoro described a moderate correlation between the
degree of NAA reduction and the time from onset. 59
Because the rate at which NAA levels decline islargely governed by the time from onset, residual
blood ﬂow and initial plasma glucose concentration, which are thought to be the most importantfactors determining the fate of ischemic tissue, 83
NAA levels can be used to predict the fate of tissue
with an acute ischemic lesion independently. Combined DWI and PI studies may not be sufﬁcient topredict tissue outcome successfully 83,84 except insome limited situations. 85 Thus, a study combining
DWI, PI, and MRSI can be expected to improve theprediction of tissue outcome, particularly given that
NAA ﬁndings complement those of DWI and PI. 86
A representative situation in which dissociation
between NAA and ADC is often observed is thecase of transient ADC restoration following earlyreperfusion in both experimental models87,88 andclinical 89,90 settings. Following early reperfusion
within a few hours of onset, DWI suggested resolution of the ischemia within several hours of reperfusion; however, the area re-emerged several hours
later on DWI and became infarcted within 24 hours.
We tested the chronological changes in NAA levels
in a rat model of focal ischemia using 90-min MCA
occlusion. 91 Though ADC was restored to the normal range within one hour of reperfusion in the partof ischemic temporal cortex and lateral basal ganglia in which ischemic insult should be mild, NAA
levels continued to show a decline in the same area
(Fig. 6). The mechanism of NAA attenuation despite ADC resolution has not been solved in detail.
However, one clue may be found in reports suggesting that energy restoration after recirculation could
re-establish membrane potential (which is maintained by Na + /K + ATP pump activity) to a marginally survivable level following ischemic insult andthereby normalize DWI. Nevertheless, energy production following reperfusion may not be sufﬁcient
to maintain other energy-dependent mechanisms,
such as NAA synthesis, protein synthesis, 92 andDNA repair via poly(ADP-ribose) polymerase(PARP), 93 at a level sufﬁcient to ensure long-term
cell survival.

－ 287 －

Magnetic Resonance in Medical Sciences

NAA Attenuation in Acute Ischemic Stroke

Conclusion
Direct reﬂection of energy metabolism and nearlinear time-dependency during acute-stage cerebral
ischemia make NAA a unique biomarker for theclinical assessment of ischemic stroke. Despite thechallenges remaining for the application of MRS(I)in the daily clinical setting, the levels of NAA withthose of lactate measured in the CNS can provide
one of the most promising biomarkers available forthe assessment of pathophysiological changes that
occur during acute-stage ischemic stroke.

11.

12.

13.

Acknowledgements
This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.
We would like to thank Professor Akiko Nishiyama, Department of Physiology and Neurobiology, University of Connecticut, for suggestive advice
about NG2 cells.
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Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

Reduced plasma desmosterol-to-cholesterol ratio and longitudinalcognitive decline in Alzheimer’s disease
Yoshiaki Satoa, Francois Berniera, Yasukazu Yamanakab, Ken Aoshimaa, Yoshiya Odac,
Martin Ingelssond, Lars Lannfeltd, Akinori Miyashitae, Ryozo Kuwanoe, Takeshi Ikeuchie,*aEisai Co., Ltd., Tsukuba, Ibaraki, JapanEIDIA Co., Ltd., Inashiki-gun, Ibaraki, Japanc
Eisai Inc., Andover, MA, USAdGeriatrics, Department of Public Health and Caring Sciences, Uppsala University, Uppsala, SwedeneDepartment of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japanb

Abstract

Background: We here examined whether plasma desmosterol-to-cholesterol ratio (DES/CHO) is
decreased in patients with Alzheimer’s disease (AD) and investigated the association between plasma
DES/CHO and longitudinal cognitive decline.
Methods: Plasma DES/CHO of AD patients and age-matched controls in a Japanese cross-sectional
cohort was determined. Plasma DES/CHO at baseline and follow-up visits was assessed in relation tocognitive decline in Japanese and Swedish longitudinal cohorts.
Results: Plasma DES/CHO was signiﬁcantly reduced in Japanese AD patients and signiﬁcantly
correlated with Mini-Mental State Examination (MMSE) score. The longitudinal analysis revealed
that plasma DES/CHO in AD patients shows a signiﬁcant decrease at follow-up intervals. The decline
in plasma DES/CHO is larger in the AD group with rapid progression than in that with slow progression. The changes in plasma DES/CHO signiﬁcantly correlated with changes in the MMSE score.
Conclusion: Plasma DES/CHO is decreased in AD patients and may serve as a longitudinal surrogate marker associated with cognitive decline.
 2015 The Alzheimer’s Association. Published by Elsevier Inc. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:

Alzheimer’s disease; Mild cognitive impairment; Blood-based biomarker; Desmosterol; Longitudinal biomarker

1. Introduction
Alzheimer’s disease (AD) is one of the most common anddebilitating neurodegenerative disorders of the aging population. AD manifests itself as a progressive decline in memory
accompanied by other cognitive and functional disabilities[1]. From the viewpoint of clinical practice and therapeuticclinical trials in AD, biomarkers are becoming increasingly
important particularly when disease-modifying drugs will
become available. Numerous studies have shown that tau,phosphorylated tau, and amyloid-b (Ab) 42 in cerebrospinal*Corresponding author. Tel.: 181-25-227-2343; Fax: 181-25-2270793.
E-mail address: ikeuchi@bri.niigata-u.ac.jp

ﬂuid (CSF) are reliable biomarkers for AD diagnosis [2–4].
However, the CSF examination of AD patients has not beenbroadly applied in general clinical practice because lumbar
puncture to obtain CSF is relatively invasive and time
consuming. Moreover, these CSF markers do not seem tobe associated with longitudinal cognitive decline inpatients with AD [5]. Thus, there is a compelling need toestablish a noninvasive biomarker for AD that follows thedisease progression. Efforts to ﬁnd reliable blood-based biomarkers for AD have met with little success [6]. Several reports have been published describing altered levels ofproteins, peptides, or metabolites in patients with AD, but
those blood-based biomarkers have proven difﬁcult to replicate in independent studies [6], highlighting the importanceof multiple validations.

http://dx.doi.org/10.1016/j.dadm.2014.11.009
2352-8729/ 2015 The Alzheimer’s Association. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

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Y. Sato et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

Table 1
Demographic characteristics of AD patients and age-matched cognitively
normal controls in the Japanese cross-sectional cohort
Variable
Female (%)
Age, mean (SD)Age at examination (y)Age at onset (y)MMSE, mean (SD)APOE genotype
2*3
2*4
3*3
3*4
4*4
DES/CHO (1026), mean (SD)Control (n 5 201)

AD (n 5 200)7275

77.6 (4.7)
n/a28.7 (1.5)

77.6 (5.4)
73.5 (5.0)
17.0 (5.2)**

15
2
151
32
1
456 (119)

6
1
71
99
23
357 (134)**

performed longitudinal studies to determine the association
between plasma DES/CHO and cognitive decline in patientswith AD over time.
2. Materials and methods

Abbreviations: AD, Alzheimer’s disease; n/a, not available; MMSE,
Mini-Mental State Examination; DES/CHO, desmosterol-to-cholesterol
ratio.
**P , .01.

In our previous report, we found that the plasma
desmosterol-to-cholesterol ratio (DES/CHO) is signiﬁcantly
decreased in Caucasian patients with AD and subjects withmild cognitive impairment (MCI) [7]. Desmosterol is themost abundant precursor but rarely exceeds 1% of total brain
sterols because the conversion from desmosterol to cholesterol is tightly regulated by the enzyme 3-hydroxysterol
24-reductase (DHCR24) [8]. A substantially higher desmosterol concentration in the hippocampus could be attributed toneurogenesis and synaptic plasticity that take place in theadult dentate gyrus [9]. Conversely, a decrease in desmosterol level in the hippocampus could at least in part correlatewith the reduced number of progenitor cells differentiating
into neurons [10]. These reports suggest an important roleof desmosterol in the brain.
With this background, we here measured plasma DES/
CHO of samples from a large Japanese cohort to extend
our previous result that plasma DES/CHO is decreased in patients with AD in a different ethnic group. Furthermore, we

2.1. Subjects
For cross-sectional analysis, plasma samples of 200 patients with AD and 201 age-matched cognitively normal
elderly individuals (older than 65 years) were collectedfrom seven clinical institutions in Japan (Table 1). The diagnosis of AD was made on the basis of the criteria of the National Institute of Neurological and Communicative
Diseases and the Stroke–Alzheimer’s Disease and Related
Disorders Association [11]. Each participant was asked tocomplete the Mini-Mental State Examination (MMSE)[12]. APOE genotyping was performed as previously reported [13].
For longitudinal analysis, we used 17 subjects with AD(Japanese longitudinal cohort collected at Niigata University
Hospital) and 28 subjects (Swedish longitudinal cohort consisting of 6 control, 12 MCI, and 10 AD subjects collected at
Uppsala University Hospital), whose blood was drawn at
two different time points (Table 2). Additional longitudinal
plasma samples of 30 subjects at least at 3 different time
points were obtained from Uppsala University Hospital(AD, n 5 6; MCI, n 5 6; control, n 5 2) or purchasedfrom PrecisionMed, Inc. (AD, n 5 12; control, n 5 4)(San Diego, CA, USA). The criteria of Petersen et al.
[14,15] were used for the diagnosis of MCI. To beconsidered as having MCI, the patients had to be free ofsigniﬁcant underlying medical, neurologic, or psychiatricillness and meet the following criteria: (1) subjective
memory complaint, (2) objective signs of decline in any
cognitive domain, (3) intact activities of daily living, and(4) clinical features not fulﬁlling the DSM-IV/ICD-10
criteria for dementia [16]. The two AD/MCI groups withslow and rapid progression were classiﬁed on the basis of

Table 2
Demographic characteristics of subjects in the longitudinal study

Variable
Female (%)
Age, mean (SD)
Follow-up time, y, mean (SD)
MMSE, mean (SD)
Baseline
Follow-up
DMMSE
DES/CHO (1026), mean (SD)
Baseline
Follow-up% Change

Swedish cohort

Japanese cohort,
AD (n 5 17)

Control (n 5 6)

MCI (n 5 12)

AD (n 5 10)

Combined cohort,
MCI/AD (n 5 39)

71
68 (8)
2.0 (1.0)

75
67 (9)
2.7 (0.8)

33
62 (8)
1.8 (0.9)30
66 (10)2.2 (0.9)

56
66 (9)
2.1 (1.0)

20 (5)
16 (7)
24 (4)**

30 (1)
30 (0)0 (1)

28 (2)
28 (2)0 (2)

25 (3)
22 (3)
23 (3)*

24 (6)
21 (7)
22 (4)**

329 (103)
290 (79)
210 (14)*654 (146)
661 (130)
1.7 (5.1)

660 (200)
607 (237)
28.3 (21)556 (232)
487 (276)
218 (27)489 (225)
438 (237)
211 (20)**

Abbreviations: AD, Alzheimer’s disease; MCI, mild cognitive impairment; MMSE, Mini-Mental State Examination; DES/CHO, desmosterol-to-cholesterol
ratio.
*P , .05, **P , .01.

－ 293 －

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69

Fig. 1. Decreased plasma DES/CHO in patients with AD. (A) Comparison of plasma desmosterol-to-cholesterol ratio (DES/CHO) between 201 control and 200
AD subjects from a large Japanese cohort. (B) Comparison of plasma DES/CHO among male control (M_Cont), female control (F_Cont), male AD (M_AD),and female AD (F_AD) subjects. (C) Comparison of plasma DES/CHO between control subjects without APOE ε4 (nonE4_Cont) and with APOE ε4 (E4_Cont),
and AD patients without APOE ε4 (nonE4_AD) and with APOE ε4 (E4_AD). (D) Comparison of plasma DES/CHO among groups classiﬁed as MMSE groups.
*P , .05; **P , .01.

the mean change in MMSE score (change from the baseline)
as threshold. Written informed consent was obtained fromeach of the participants (or the respective legal guardian);
the study was approved by the appropriate university, hospital, and company institutional ethics committees.
2.2. Blood sampling and laboratory measurement
Peripheral blood samples were obtained from each participant using the commercially available blood collection
tubes (Terumo Venoject for Japanese cohort and BD Vacutainer for Swedish cohort) containing EDTA as the anticoagulant. Plasma was separated by centrifugation at 1500 g for
15 minutes at room temperature before being aliquoted andstored at 280 C until analysis.
The concentrations of desmosterol and cholesterol were
measured with a liquid chromatography mass spectrometer(LC/MS), as described previously [7]. Brieﬂy, 25 mL ofplasma was spiked with cholesterol-25,26,26,26,27,27,27D7 and desmosterol-26,26,26,27,27,27-D6 as internal standards. Fifty percent potassium hydroxide was then added
to the solution, which was then mixed thoroughly and incu-

bated at 70 C for 60 minutes. After the incubation, 2 mL ofhexane and 0.5 mL of phosphate-buffered saline (pH 6.8)
were added and mixed well. The solution was centrifuged
for 10 minutes at 2000 g, and the upper organic phase wastransferred to a new tube. The lower layer was extractedwith an additional 1 mL of hexane, which was also added
to the organic-phase extract. The solvents were evaporated
to dryness under a nitrogen gas stream at 40 C, and the obtained pellet was reconstituted in ethanol and the resulting
solution was subjected to liquid chromatography/atomospheric pressure chemical ionization-mass spectrometer
analysis as described previously [7].
2.3. Statistical analysis
Values are shown as mean 6 standard deviation. Correlations between different variables were assessed using thePearson correlation coefﬁcient on log-transformed data.
The t test or analysis of variance was carried out to determine
differences between two or more groups. Nonparametrictests (Mann-Whitney U test) were carried out when the variables were not normally distributed. The paired t test was－ 294 －

70

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Y. Sato et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

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Fig. 2. Change in plasma DES/CHO between baseline and follow-up visits. AD/MCI subjects were classiﬁed into two groups, namely, those with slow progression (A, D, G) and those with rapid progression (B, E, H) on the basis of the mean DMMSE between baseline and follow-up visits in a Japanese cohort(A–C), a Swedish cohort (D–F), and a combined cohort (G–I). Plasma DES/CHO remains stable in the group with slow progression (A, D, G), whereas in
the group with rapid progression, a signiﬁcant decline over time was found (B, E, H). The change in plasma DES/CHO was signiﬁcantly larger in the groupwith rapid progression than in the group with slow progression (C, F, I). *P , .05; **P , .01.

carried out to see the difference in the individual values between baseline and follow-up visits. The statistical signiﬁcance was set at P , .05.
3. Results
3.1. Cross-sectional study of plasma desmosterol
The characteristics of the AD and age-matched control
subjects included in this cross-sectional study are listed inTable 1. A signiﬁcant decline in plasma DES/CHO wasobserved in patients with AD compared with control subjects (P , .01; Fig. 1A). The decline in plasma DES/CHO
in AD patients was signiﬁcant regardless of gender or
APOE ε4 status (Fig. 1B and C). There was a signiﬁcantdecline in plasma DES/CHO in AD patients with both the
APOE ε3/3 and ε3/4 genotypes (data not shown; see
Supplementary Fig. 1A and B for review). Plasma DES/

－ 295 －CHO of female subjects in both the AD and control groups
was signiﬁcantly lower than that of male subjects (Fig. 1B,
P , .01). No signiﬁcant correlation of plasma DES/CHO
with age was observed (data not shown). A signiﬁcant correlation between plasma DES/CHO and MMSE score in bothmales and females was observed (data not shown; see
Supplementary Fig. 1C and D). We divided subjects of thiscohort into four MMSE score groups: high MMSE score(30 S MMSE S 26), middle MMSE score (25 S MMSE
S 20), low MMSE score (19 S MMSE S 10), and very
low MMSE score (9 S MMSE S 0). We then compared
DES/CHO among these groups. A signiﬁcant difference between the high MMSE score group and the other MMSE
score groups was observed (Fig. 1D). The linear trend analysis revealed that there was also a signiﬁcant change
showing that groups with lower MMSE scores had lower
DES/CHO (trend t test: P , .01).

Y. Sato et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

71

72

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4.
Fig. 3. Comparison of longitudinal change in plasma DES/CHO between control and AD subjects. (A) Longitudinal changes in plasma DES/CHO at two
different points were compared between control subjects and AD patients from the combined cohort. *P , .05. (B) Longitudinal changes in plasma DES/
CHO at multiple points were compared between control subjects and AD patients from the Swedish cohort and a commercially available resource. **P , .01.

3.2. Longitudinal analysis
Longitudinal plasma samples were collected from two
clinical institutes, namely, Niigata University Hospital (Japanese cohort) and Uppsala University Hospital Memory
Clinic (Swedish cohort). Forty-seven participants composedof 17 subjects in the Japanese cohort and 30 subjects in the
Swedish cohort were included, and the demographic characteristics of these subjects are listed in Table 2. At baseline,
DES/CHO in Japanese AD patients was signiﬁcantly lower
than that of Swedish AD patients (P , .01).
In the Japanese longitudinal cohort, the average change in
the MMSE score (DMMSE) between the baseline andfollow-up visits was 24 6 4 with a change in DES/CHO

Fig. 4. Correlation between longitudinal changes in MMSE and plasma
DES/CHO (%) in AD patients, MCI patients, and normal subjects. There
were 122 points for blood collection, consisting of 30 baselines and 92
follow-ups (AD, 52 follow-ups; MCI, 13 follow-ups; and control, 27
follow-ups). There was a signiﬁcant correlation between changes in
MMSE and plasma DES/CHO (r 5 0.37, P , .01).

of 210 6 14% (Table 2). Both MMSE score and plasma
DES/CHO signiﬁcantly decreased between the two visits
in the Japanese cohort (paired t test, P , .05). In the Swedishcohort, the MMSE score (23 6 3) decreased signiﬁcantly in
AD patients between the baseline and follow-up visits(P , .05). However, in this cohort, there was no signiﬁcantchange in DES/CHO in both the AD patients and MCI subjects (Table 2). In the combined AD/MCI cohort, MMSE
score (22 6 4) and plasma DES/CHO (211 6 20%)decreased signiﬁcantly between the two visits (Table 2).
Next, we divided the AD/MCI subjects into two groups,
namely, those with slow or rapid progression, on the basisof their mean DMMSE in the Japanese (cutoff score, 24)
and Swedish cohorts (cutoff score, 22) and compared thelongitudinal change in plasma DES/CHO between groups(Fig. 2). Although the AD/MCI group with slow progression
did not show any signiﬁcant change in plasma DES/CHO(Fig. 2A, D, and G), groups with rapid progression showeda signiﬁcant decrease in plasma DES/CHO between thebaseline and follow-up visits (Fig. 2B, E, and H). In addition,
the change in plasma DES/CHO was signiﬁcantly larger inthe AD/MCI group with rapid progression than in the groupwith slow progression in the Japanese, Swedish, and combined cohorts (Fig. 2C, F, and I).
We further compared the longitudinal change in plasma
DES/CHO in control subjects and AD patients (Fig. 3).
The change in DES/CHO between the two visits was
212.8 6 19.7 in the AD patients, which was signiﬁcantly
larger than that in the control subjects (1.7 6 5.3;
Fig. 3A). These results suggest that although plasma DES/
CHO in normal subjects remained stable, plasma DES/
CHO in AD patients tended to decline over time.
Finally, we performed another longitudinal study todetermine the association between plasma DES/CHO andDMMSE in 30 participants, including 18 AD, 6 MCI, and6 control subjects from either the Swedish cohort or from
PrecisionMed. Blood samples were collected every year or－ 296 －

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71

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72

Y. Sato et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

every 6 months from the participants. There were 122 points
for blood collection, consisting of 30 baselines and 92
follow-ups. The correlation between the DMMSE and thechange in plasma DES/CHO (compared with baseline)
determined using all 92 follow-up points is shown in
Fig. 4. There was a signiﬁcant correlation between the longitudinal DMMSE and the change in DES/CHO from thebaseline (P 5.01, r 5 0.37). All the changes in MMSE scoreand plasma DES/CHO in each of the participants are shown(see Supplementary Fig. 2). In addition, AD patients showeda signiﬁcant decrease in plasma DES/CHO at follow-up intervals (27.7 6 36.8%) compared with the control subjects(6.4 6 12.3%) in this cohort (Fig. 3B).
4. Discussion
The present cross-sectional study using a Japanese cohort
was undertaken to replicate our previous ﬁnding that plasma
DES/CHO is decreased in Caucasian patients with AD. The
following points were conﬁrmed in the present Japanese andpreviously reported Caucasian cross-sectional cohorts: (1)plasma DES/CHO was decreased in patients with AD incomparison with control subjects, (2) the decrease in plasma
DES/CHO in AD patients was independent of gender and
APOE genotype, (3) female subjects tended to have a lower
plasma DES/CHO than male subjects, and (4) plasma DES/
CHO correlated signiﬁcantly with the MMSE score. Taken
together, the results suggest that plasma DES/CHO may be
a potential diagnostic biomarker reﬂecting cognitive
dysfunction in AD patients.
Recently, Popp et al. [17] have reported that the plasma
desmosterol level does not change in AD patients. The
discrepancy in ﬁnding between that and the present study
may be explained by differences in the analytical methods
used to determine the concentration of desmosterol. We previously showed that the LC/MS method that we used in thepresent study enables the puriﬁcation of desmosterol inplasma more efﬁciently than the gas chromatographymethod used in the study by Popp et al. [7,17]. Thus, the
LC/MS method is likely to be more suitable for themeasurement of plasma desmosterol concentration.
Here, we determined for the ﬁrst time the longitudinalchange in plasma DES/CHO and examined a possible association with concurrent cognitive decline in AD/MCI patients. Our results revealed (1) that plasma DES/CHO wasrelatively stable over time in cognitively normal controls,
whereas it signiﬁcantly decreased in AD patients; (2) amore pronounced decline in plasma DES/CHO in the AD/
MCI group with rapid progression than in the group withslow progression; and (3) that the longitudinal change inplasma DES/CHO positively correlated with the change in
the MMSE score. These results suggest that the plasma
DES/CHO change is associated with the cognitive decline
in AD and might be used to monitor the progression of
cognitive decline in patients with AD. It will be interesting
to clarify the usefulness of monitoring plasma DES/CHO－ 297 －

as a surrogate marker for evaluating the effects of clinicaldrug trials in patients with AD.
Our longitudinal study suggests that the plasma DES/CHO
changes before the appearance of clinical symptoms, as determined by MMSE in some cases (see Supplementary Fig. 2 forreview; subjects A, C, and H). The result obtained from thesubject with MCI that converted to AD (see Supplementary
Fig. 2 for review; subject S) may suggest that plasma DES/
CHO is useful as a progression marker to monitor the conversion from MCI to AD. In this connection, recent lipidomic
analysis showed that the quantiﬁcation of several lipid metabolites in plasma, such as phosphatidylcholine and acylcarnitine, is useful for predicting phenoconversion to amnestic
MCI or AD in cognitively normal subjects [18].
There is now accumulating evidence that cholesterol
metabolism may be relevant to the production and clearanceof Ab and thus to the Ab-related toxicity in the pathogenesisof AD [19]. The strongest genetic risk factor for sporadic AD
is the ε4 allele of APOE, which encodes apolipoprotein E(apoE), with a crucial role in cholesterol metabolism [20].
The presence of APOE ε4 may contribute to the pathologic
accumulation and deposition of cerebral Ab at early preclinical disease stages [21]. A recent study has shown that CSF
apoE levels are decreased in patients with AD and that MCI
in subjects with a low CSF apoE level will more likely
convert to AD [22]. An interaction between APOE genotype
and plasma desmosterol level may be postulated because
desmosterol is the immediate precursor of cholesterol. However, the plasma desmosterol level was not clearly associatedwith APOE genotype in this study.
It is of particular interest that the level of desmosterol in
the AD brain was found to be lower than that of control brain[7,23]. It has been demonstrated that the levels of steroid
hormones (e.g., progesterone, pregnenolone, and 17aOHprogesterone) that exhibit inhibitory activity against
DHCR24 are decreased in the AD brain, particularly in thevicinity of plaques and neuroﬁbrillary tangles [24]. Notably,
the concentration of desmosterol is 100-fold higher in the rat
brain than in the rat liver [25], which implies that most of thedesmosterol in the blood might originate from the brain.
Taken together, it could be speculated that brain desmosterollevel may decrease with an increase in DHCR24 activity in
the AD brain; this may subsequently result in a change inplasma DES/CHO. The question of why plasma DES/CHO
decreases in patients with AD and is associated with longitudinal cognitive decline in the course of the disease warrants further investigation.
Although our ﬁndings, using samples from cross-sectional
and longitudinal cohorts, are interesting, our study has some
limitations. We did not analyze the samples from other typesof dementia, including dementia with Lewy bodies, frontotemporal dementia, and vascular dementia. An additionalcross-sectional study that includes samples from other typesof dementia will be necessary. The number of samples fromlongitudinal cohorts in this study is relatively small. Longitudinal studies with a prospective design using a larger number

Y. Sato et al. / Alzheimer’s & Dementia: Diagnosis, Assessment & Disease Monitoring 1 (2015) 67–74

of samples should be performed to conﬁrm the utility ofplasma DES/CHO as a longitudinal biomarker. Moreover, it
is important to understand how early plasma DES/CHO starts
to decline using longitudinal samples from asymptomatic AD
subjects with amyloid deposition conﬁrmed by amyloidpositron emission tomography imaging. Although our ﬁndings need to be validated in independent cohorts, our datasuggest that the use of plasma desmosterol as a bloodbiomarker can be useful in the diagnosis of AD and also inmonitoring disease progression.
Acknowledgments
The authors would like to thank the subjects who participated in this study. We would like to thank Dr. Makoto
Ogo and Mr. Mamoru Yanagimachi who provided valuablecomments. Special thanks also go to Mr. Kentaro Matsuura
and Mr. Masayuki Ikawa whose support and information
have helped us throughout this study. This study was fundedby grants from Eisai Co. Ltd., Japanese Society of Promotion of Science, Ministry of Health, Labour and Welfare,Japan, and Uppsala University.
Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.dadm.2014.11.009.

RESEARCH IN CONTEXT

1. Systemic review: There is a compelling need toestablish blood-based biomarker to diagnose Alzheimer’s disease (AD) and monitor the disease progression. A previous study reported that plasma
desmosterol-to-cholesterol ratio (DES/CHO) issigniﬁcantly decreased in Caucasian patientswith AD.
2. Interpretation: We found that plasma DES/CHO wassigniﬁcantly reduced in Japanese AD patients. The
longitudinal study revealed (1) that plasma DES/
CHO was relatively stable in normal controls,
whereas it signiﬁcantly decreased in AD patients;(2) a more pronounced decline in plasma DES/
CHO in the AD/MCI group with rapid progression
than in that with slow progression; and (3) that thelongitudinal change in plasma DES/CHO positively
correlated with the change in MMSE score.
3. Future directions: A future cross-sectional study that
includes samples from other types of dementia andlongitudinal studies with a prospective design using alarger number of samples need to be performed to
conﬁrm the utility of plasma DES/CHO.

73

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[23] Wisniewski T, Newman K, Javitt NB. Alzheimer’s disease: brain desmosterol levels. J Alzheimers Dis 2013;33:881–8.

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iﬁab-

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ular
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EG,
and

BT,
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etama-

TR,
em-

es-

ath44.
H,mer－ 299 －

[24] Lindenthal B, Holleran AL, Aldaghlas TA, Ruan B, Schroepfer GJ Jr,
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[25] Smiljanic K, Vanmierlo T, Djordjevic AM, Perovic M, LoncarevicVasiljkovic N, Tesic V, et al. Aging induces tissue-speciﬁc changes
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－ 300 －Plasma DES/CHO (10-6)

250

500

750

1000

0

10

P<0.0120(n=71)MMSE score(n=151)

**

30

D

B

250

500

750

0

10

P<0.0120(n=99)MMSE score(n=32)**

30

Figure legend Supplementary Fig. 1:(A) Comparison of plasma DES/CHO between control subjects and AD patients with APOE H3/3. **, p<0.01.
(B) Comparison of plasma DES/CHO between control subjects and AD patients with APOE H3/4. **, p<0.01.
(C) Correlation between plasma DES/CHO and MMSE score in males (n=105).
(D) Correlation between plasma DES/CHO and MMSE score in females (n=281).

Plasma DES/CHO (10-6)

C

A
Plasma DES/CHO (10-6)

Supplementary Fig. 1. Sato et al. (for review)

Plasma DES/CHO (10-6)

(B) Comparison of plasma DES/CHO between control subjects and AD patients with APOE H3/4. **, p<0.01.
(C) Correlation between plasma DES/CHO and MMSE score in males (n=105).
(D) Correlation between plasma DES/CHO and MMSE score in females (n=281).

Supplementary Fig. 2. Sato et al. (for review)

Figure legend for Supplementary Fig. 2:
Changes in plasma DES/CHO and MMSE scores in AD patients (A-R), MCI subjects (SX), and normal subjects (Y-IV). The longitudinal change in plasma DES/CHO (red line)
was compared with the baseline value. Longitudinal MMSE scores are shown in blue.

－ 301 －

Supplementary Figure 2. Sato et al. (continued)－ 302 －

Journal of Human Genetics (2015) 60, 281–283& 2015 The Japan Society of Human Genetics All rights reserved 1434-5161/15

OPEN

www.nature.com/jhg

SHORT COMMUNICATION

Systematic review and meta-analysis of Japanesefamilial Alzheimer’s disease and FTDP-17
Kensaku Kasuga1,2,6, Masataka Kikuchi1,3,6, Takayoshi Tokutake4, Akihiro Nakaya1,5, Toshiyuki Tezuka4,
Tamao Tsukie1,3, Norikazu Hara1, Akinori Miyashita1, Ryozo Kuwano1 and Takeshi Ikeuchi1
Mutations in APP, PSEN1 and PSEN2 as the genetic causes of familial Alzheimer’s disease (FAD) have been found in various
ethnic populations. A substantial number of FAD pedigrees with mutations have been reported in the Japanese population;
however, it remains unclear whether the genetic and clinical features of FAD in the Japanese population differ from those inother populations. To address this issue, we conducted a systematic review and meta-analysis of Japanese FAD and
frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) by literature search. Using this analysis, we
identiﬁed 39 different PSEN1 mutations in 140 patients, 5 APP mutations in 35 patients and 16 MAPT mutations in 84
patients. There was no PSEN2 mutation among Japanese patients. The age at onset in Japanese FAD patients with PSEN1
mutations was signiﬁcantly younger than that in patients with APP mutations. Kaplan–Meier analysis revealed that patients with
MAPT mutations showed a shorter survival than patients with PSEN1 or APP mutations. Patients with mutations in different
genes exhibit characteristic clinical presentations, suggesting that mutations in causative genes may modify the clinicalpresentations. By collecting and cataloging genetic and clinical information on Japanese FAD and FTDP-17, we developed
an original database designated as Japanese Familial Alzheimer's Disease Database, which is accessible at http://alzdb.bri.
niigata-u.ac.jp/.
Journal of Human Genetics (2015) 60, 281–283; doi:10.1038/jhg.2015.15; published online 19 February 2015

Mutations in APP, PSEN1 and PSEN2 as the genetic causes of familial
Alzheimer’s disease (FAD) have been found in various ethnic
populations.1,2 In addition, patients with mutations in MAPT associated with frontotemporal dementia with parkinsonism linked tochromosome 17 (FTDP-17) have been shown to exhibit Alzheimer’s
disease (AD)-like phenotypes.3,4 Although a substantial number of
FAD pedigrees have been reported in Japan, it is not yet clear whether
the genetic and clinical features of FAD in the Japanese populationdiffer from those in other ethnic populations. To characterize thegenetic and clinical features of Japanese FAD and FTDP-17, we here
performed a systematic review and meta-analysis, and developed an
original database of Japanese FAD and FTDP-17.
To comprehensively review the previously reported Japanese FAD
and FTDP-17 cases, we performed a systematic search for publicationsin PubMed and Ichushi, a bibliographic database of medical literature
in Japanese. The terms ‘familial Alzheimer’, ‘familial AD’, ‘FTDP-17’,‘presenilin’, ‘PSEN1’, ‘PSEN2’, ‘APP’ and ‘MAPT’ were used to searchin PubMed, and the equivalent terms in Japanese were used to searchin Ichushi. From the literature searches we found 60 English and 29
Japanese articles and/or abstracts that reported on Japanese FAD and
FTDP-17 pedigrees bearing the causative mutations (Supplementary

Table 1). Using the information obtained by the systematic literature
review, we developed an original database for Japanese FAD and
FTDP-17 designated as Japanese Familial Alzheimer's Disease database(JFADdb). In the database, each of the mutations in APP, PSEN1/2,
MAPT and GRN was described in accordance with the reference
sequences.5 Information on age at onset, clinical manifestations,age at death and APOE genotype were included in the database(Supplementary Figure 1).
We identiﬁed 39 different PSEN1 mutations in 140 patients, 5 APP
mutations in 35 patients and 16 MAPT mutations in 84 patients(Table 1). Among them, 10 PSEN1 mutations, 5 APP mutations and
11 MAPT mutations were not included in the well-known Alzheimer
Disease and Frontotemporal Dementia Mutation database (http://
www.molgen.ua.ac.be/ADMutations/).6 No PSEN2 mutation has beenfound in Japanese FAD. The frequency of mutated genes in FAD
patients in the Japanese population was not signiﬁcantly different fromthose in other populations (χ2, P = 0.99).6 Most FAD pedigrees show
autosomal dominant inheritance; however, an APP ΔE693 mutation
was responsible for a recessively inherited FAD.7 Sporadic occurrencesof mutations were observed: ﬁve patients with PSEN1 mutations, onepatient with APP mutation and two patients with MAPT mutations.

1

Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan; 2Center for Transdisciplinary Research, Niigata University, Niigata, Japan;
Research Association for Biotechnology, Tokyo, Japan; 4Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan and 5Department of Genome
Informatics, Graduate School of Medicine, Osaka University, Osaka, Japan
6These authors contributed equally to this work.
Correspondence: Dr T Ikeuchi, Department of Molecular Genetics, Brain Research Institute, Niigata University, 1-757 Asahimachi, Niigata 951-8585, Japan.
E-mail: ikeuchi@bri.niigata-u.ac.jp
Received 7 November 2014; revised 7 January 2015; accepted 13 January 2015; published online 19 February 20153

－ 303 －

Systematic review of Japanese FAD and FTDP-17
K Kasuga et al
282

Table 1 Summary of genetic features of Japanese FAD and FTDP-17

DiseaseGenesmutationspedigrees

patients

FAD

PSEN1

39

40

140

PSEN2
APP

0
5a

0
13

0
35

MAPT
GRN

16
2

29
2

84
2

FTDP-17

Age of symptom onset (yrs)

Number of

Abbreviations: FAD, familial Alzheimer’s disease; FTDP-17, frontotemporal dementia withparkinsonism linked to chromosome 17.
aAPP duplication was included.

In our analysis, the majority of mutations (73%) was observed in asingle small pedigree. Considering that novel mutations in FAD tend
to be reported rapidly, note that there may be publication bias in thefrequency of mutations in the database. Although rare, there were two
GRN mutations in patients with primary progressive aphasia andfrontotemporal lobar degeneration (FTLD).8 Because the number of
GRN mutations was too small, the patients with GRN mutations were
excluded from further meta-analysis.
The ages at onset were 44 ± 8 years (mean ± s.d.) in patients with
PSEN1 mutations (n = 87), 54 ± 9 years in APP mutations (n = 23)
and 45 ± 10 years in MAPT mutations (n = 51). These ages at onset of
FAD in our analysis are consistent with those reported in other ethnic
populations.6,9 The age at onset in patients with APP mutations was
signiﬁcantly older than those in patients with PSEN1 or MAPT
mutations (Figure 1a). The clinical phenotypes of patients with MAPT
mutations were classiﬁed into three subgroups: FTLD,10 AD-like3,4
and progressive supranuclear palsy (PSP) phenotypes.8 The age at
onset in patients with the FTLD or PSP phenotype was signiﬁcantly
younger than that with the AD-like phenotype (Supplementary Figure2). APOE genotypes did not signiﬁcantly modify the age at onset inpatients with causative mutations (Supplementary Figure 3). There
was no signiﬁcant difference in age at death among the patients with
mutations in the three genes (Figure 1b). The disease duration fromage at onset to death in patients with MAPT mutations was
signiﬁcantly shorter than that with PSEN1 mutations (Figure 1c).
The survival of patients after the onset was analyzed by Kaplan–Meierestimation, which revealed that patients with MAPT mutations
showed a shorter survival than patients with PSEN1 or APP mutations(Supplementary Figure 4).
The clinical diagnosis of AD before the genetic testing was
performed in 96% of patients with PSEN1 mutations and 97% of
patients with APP mutations (Supplementary Table 2). Notably, only
57% of patients with MAPT mutations were clinically diagnosed ashaving FTLD; 19% and 12% of patients with MAPT mutations
were clinically diagnosed as having AD and PSP, respectively(Supplementary Table 2). This ﬁnding suggests that mutational
screening of clinically diagnosed FAD patients should not only include
APP and PSEN1/2 mutations but also include MAPT mutations.
We next analyzed the frequency of each of the clinical manifestations including psychiatric symptoms, mood disorders, spastic
paraparesis, parkinsonism and epilepsy/seizure (Figure 2). As expected,
the frequencies of psychiatric symptoms and parkinsonism weresigniﬁcantly higher in patients with MAPT mutations. Spastic
paraparesis, which is a characteristic symptom of ‘variant AD withcotton-wool plaque pathology’11,12 was observed in 15% of patientswith PSEN1 mutations, whereas none of the patients with APP
mutations exhibited spastic paraplegia. Epilepsy/seizure was described
in 8% of patients with PSEN1 and 6% of patients with APP mutations,

**

60

40

20
PSEN1
(n=87)

Age at death (yrs)

Number of

APP

MAPT

APP

MAPT

(n=23)(n=51)

80

60

40

PSEN1
(n=35)(n=10)(n=35)*

Disease duration (yrs)

Number of

**

80

20

10

0
PSEN1
(n=23)

APP

(n=8)

MAPT
(n=26)

Figure 1 Age at onset and death, and disease duration in Japanese FAD and
FTDP-17 patients. (a) Age at onset for patients grouped on the basis of
PSEN1, APP and MAPT mutations. The horizontal line in the box indicatesthe median, the lower and upper boundaries of the box represent the lower
and upper quartile boundaries, respectively, and whiskers are 1.5 times theinterquartile range. Patients with PSEN1 and MAPT mutation showedsigniﬁcantly younger age at onset than patients with APP mutations(**Po0.01, ANOVA with post hoc Tukey’s test). (b) Age at death of threegroups with gene mutations. There was no signiﬁcant difference in age atdeath among the groups. (c) Disease duration was deﬁned as the period fromage at onset to death. The disease courses of patients with MAPT mutations(7 ± 4 years, mean ± s.d.) were signiﬁcantly shorter than those with PSEN1
mutations (11 ± 5) (*Po0.05, ANOVA with post hoc Tukey’s test). ANOVA,
analysis of variance; FAD, familial Alzheimer’s disease; FTDP-17,
frontotemporal dementia with parkinsonism linked to chromosome 17.

Journal of Human Genetics－ 304 －

Systematic review of Japanese FAD and FTDP-17
K Kasuga et al
283

**

**

40%
30%

*
*

**

*

10%

MAPT

APP

MAPT

APP

PSEN1

*
MAPT

APP

PSEN1

MAPT

APP

PSEN1

MAPT

*
APP

0%

PSEN1

20%

PSEN1

Frequency of symptoms

50%

Psychiatric symptomMood disorderSpastic paraparesis
Parkinsonism/Rigidity
Epilepsy/Seizure
Psychiatric
Mood
Spastic
Parkinsonism/
Epilepsy/
symptom
disorderparaparesis
Rigidity
SeizureFigure 2 Frequency of each clinical manifestation in patients with Japanese FAD and FTDP-17. We investigated the presence or absence of psychiatric
symptoms, mood disorders, spastic paraplegia, parkinsonism and epilepsy/seizure by careful reading of the original papers. We determined the frequency ofeach of the clinical manifestations by counting the number of patients for whom the presence of the manifestation was described in literature. In case there
was no description of the manifestation, the patient was not counted as manifesting the manifestation. The observed frequencies of causative gene mutation
were signiﬁcantly different from the expected frequencies determined by residual analyses for χ2 statistical analysis (*Po0.05, **Po0.01). FAD, familial
Alzheimer’s disease; FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome 17.

whereas none of the patients with MAPT mutations exhibited
epilepsy/seizure. Previous studies showed that the frequency of seizure
was relatively high in patients with early onset of AD,13,14 and low inpatients with MAPT mutations.15 These ﬁndings suggest that epilepsy/
seizure is closely associated with amyloid pathology, and that tauopathy alone may not be sufﬁcient to cause epilepsy. Taken together,mutations in causative genes may modify the clinical presentations inpatients with familial dementia.
In summary, we have comprehensively collected, cataloged andsystematically meta-analyzed the data from currently available data on
Japanese FAD and FTDP-17. We made all the results publicly available
on the online database ‘JFADdb’. The database may provide information useful for estimating the age at onset and the natural course of
disease in future preventive or therapeutic trials of Japanese FAD.

This work is supported in part by Grants-in-Aid for scientiﬁc research fromJapan Society of Promotion of Science, Japan (26870209 to KK, 23591234 and20372469 to TI) and Grant-in-Aid from the Ministry of Health, Labour andWelfare of Japan. We are grateful to Dr Hasegawa and Ms Fukaumi for theirtechnical assistance.

The role of PSEN1 and MAPT R406W mutations. Dement. Geriatr. Cogn. Disord. 26,
43–49 (2008).
5 Pruitt, K. D., Brown, G. R., Hiatt, S. M., Thibaud-Nissen, F., Astashyn, A. &Ermolaeva, O. et al. RefSeq: an update on mammalian reference sequences. Nucleic
Acids Res. 42, D756–D763 (2014).
6 Cruts, M., Theuns, J. & Van Broeckhoven, C. Locus-speciﬁc mutation databases forneurodegenerative brain diseases. Hum. Mutat. 33, 1340–1344 (2012).
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Ann. Neurol. 63, 377–387 (2008).
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13 Noebels, J. A perfect storm: converging paths of epilepsy and Alzheimer's dementia
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Seizures and epileptiform activity in the early stages of Alzheimer disease. JAMA
Neurol. 70, 1158–1166 (2013).
15 Sperfeld, A. D., Collatz, M. B., Baier, H., Palmbach, M., Storch, A. & Schwarz, J. et al.
FTDP-17: an early-onset phenotype with parkinsonism and epileptic seizures caused bya novel mutation. Ann. Neurol. 46, 708–715 (1999).

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disease: beyond APP, PSENs and APOE. Neurobiol. Aging 33, 437–456 (2012).
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Mutational analysis in early-onset familial dementia in the Japanese population.

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CONFLICT OF INTEREST
The authors declare no conﬂict of interest.
ACKNOWLEDGEMENTS

Supplementary Information accompanies the paper on Journal of Human Genetics website (http://www.nature.com/jhg)

Journal of Human Genetics－ 305 －

Biochemical and Biophysical Research Communications 456 (2015) 482–488

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

ApoE-isoform-dependent cellular uptake of amyloid-b is mediatedby lipoprotein receptor LR11/SorLA
Ryuji Yajima a, Takayoshi Tokutake a, Akihide Koyama b, Kensaku Kasuga a,b,c, Toshiyuki Tezuka a,c,
Masatoyo Nishizawa a, Takeshi Ikeuchi c,⇑a

Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan
Center for Transdisciplinary Research, Niigata University, Niigata, Japanc
Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japanba r t i c l e

i n f oArticle history:
Received 7 November 2014
Available online 5 December 2014
Keywords:
Alzheimer’s diseaseLR11/SorLAb-Amyloid
Apolipoprotein E

a b s t r a c t
The formation of senile plaques composed of b-amyloid (Ab) in the brain is likely the initial event in
Alzheimer’s disease (AD). Possession of the APOE e4 allele, the strong genetic factor for AD, facilitates
the Ab deposition from the presymptomatic stage of AD in a gene-dosage-dependent manner. However,the precise mechanism by which apoE isoforms differentially induce the AD pathology is largely
unknown. LR11/SorLA is a type I membrane protein that functions as the neuronal lipoprotein endocytic
receptor of apoE and the sorting receptor of the amyloid precursor protein (APP) to regulate amyloidogenesis. Recently, LR11/SorLA has been reported to be involved in the lysosomal targeting of extracellularamyloid-b (Ab) through the binding of Ab to the vacuolar protein sorting 10 (VPS10) protein domain of
LR11/SorLA. Here, we attempted to examine the human-apoE-isoform-dependent effect on the cellular
uptake of Ab through the formation of a complex between an apoE isoform and LR11/SorLA. Cell culture
experiments using Neuro2a cells revealed that the cellular uptake of secreted apoE3 and apoE4 wasenhanced by the overexpression of LR11/SorLA. In contrast, the cellular uptake of apoE2 was not affectedby the expression of LR11/SorLA. Co-immunoprecipitation assay revealed that apoE-isoform-dependent
differences were observed in the formation of an apoE-LR11 complex (apoE4 > apoE3 > apoE2). ApoEisoform-dependent differences in cellular uptake of FAM-labeled Ab were further investigated by coculture assay in which donor cells secrete one of the apoE isoforms and recipient cells express FL-LR11. The
cellular uptake of extracellular Ab into the recipient cells was most prominently accentuated when cocultured with the donor cells secreting apoE4 in the medium, followed by apoE3 and apoE2. Taken together,
our results provide evidence for the mechanism whereby human-apoE-isoform-dependent differences
modulate the cellular uptake of Ab mediated by LR11/SorLA.
 2014 Elsevier Inc. All rights reserved.

1. Introduction
Alzheimer’s disease (AD) has emerged as the most prevalent
form of dementia in adults. The cardinal pathological features ofAD brain are the loss of synapses and neurons as well as abnormal
accumulation of misfolded proteins such as b-amyloid (Ab) and
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid-b; APP, amyloid precursor
protein; VPS10, vacuolar protein sorting 10; TACE, tumor necrosis factora-converting enzyme; FL, full-length; CSF, cerebrospinal ﬂuid; PCR, polymerase
chain reaction; GFP, green ﬂuorescent protein; HEK, human embryonic kidney;
SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; co-IP,
coimmunoprecipitation.
⇑ Corresponding author at: Department of Molecular Genetics, Brain ResearchInstitute, Niigata University, 1-757 Asahimachi, Niigata 951-8585, Japan. Fax: +81
25 227 0793.
E-mail address: ikeuchi@bri.niigata-u.ac.jp (T. Ikeuchi).

phosphorylated tau. The deposition of Ab in the brain is considered
to be the earliest event in AD [1] and is substantially affected by the
APOE genotype, which has been shown to be a strong genetic risk
factor for AD in various ethnic populations [2,3]. The presence of
the APOE e4 allele markedly increases the risk of developing AD
and decreases the age at onset by 10 to 15 years; in contrast, thee2 allele confers protection against AD development [4,5]. Although
there have been numerous studies attempting to elucidate themechanism underlying the increase or decrease in the risk of AD
posed by different apoE isoforms [6], the exact mechanisms are still
not completely understood. Thus, to address the differential effects
of apoE isoforms on the amyloid pathology and Ab metabolism isimportant in the elucidation of the AD pathogenic pathway.
LR11, also known as SorLA, is a type I membrane protein thathas homology to members of the LDL receptor family and vacuolar

http://dx.doi.org/10.1016/j.bbrc.2014.11.1110006-291X/ 2014 Elsevier Inc. All rights reserved.

prote
nantl
pus [
patie
repor
LR11
ﬁndin
patho
phism
assoc
[3]. F
tor-a
We psigniﬁ
AD [1levels
gests
AD in
LR
recep
curso
Golgi
sized
into
quen
amyl
LR11/
LR11/
ated
prote
in lys
recep
attem
the c
betw

2. Ma

2.1. c

Co
ampl
LR11
tor (Lclone
allelic
genes
(Clon
PCR p

2.2. C

M
kidne
[16].
out b
stableapoE2
Pepti
FAMdisso

2.3. CCo
ously－ 306 －

ent inlitates
wever,
argelyocyticloidoellularmain ofellularulture4 was
fectedndent
ApoEcocul1. The
coculgether,renceserved.

deredby thec risknce ofng ADt, the
hough
e the
of AD
e stillffectssm isn thatcuolarR. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

protein sorting 10 (VPS10) protein family. LR11/SorLA is predominantly expressed on neurons in the cerebral cortex and hippocampus [7]. Reduced expression of LR11/SorLA in the brains of AD
patients and subjects with mild cognitive impairment has beenreported [8]. Furthermore, Ab deposition is more prominent in
LR11 knockout mice crossed with a mouse AD model [9]. Theseﬁndings suggest the protective role of LR11/SorLA against AD
pathology. In addition, some of the single-nucleotide polymorphisms (SNPs) in SORL1 encoding LR11/SorLA were found to be
associated with a risk of sporadic AD in various ethnic backgrounds[3]. Full-length (FL) LR11/SorLA is cleaved by tumor necrosis factor-a-converting enzyme (TACE) to generate soluble LR11 [10].
We previously reported that the level of soluble LR11/SorLA wassigniﬁcantly high in the cerebrospinal ﬂuid (CSF) of patients withAD [11]. The APOE e4 carriers among AD patients showed higher
levels of soluble LR11/SorLA than the e4 noncarriers [11]. This suggests that the level of soluble LR11/SorLA in CSF is associated withAD in an apoE-isoform-dependent manner.
LR11/SorLA functions as the neuronal lipoprotein endocytic
receptor of ApoE [12] and the sorting receptor of the amyloid precursor protein (APP) to regulate amyloidogenesis in endosomal and
Golgi compartments [13]. LR11/SorLA interacts with newly synthesized APP in the Golgi apparatus and prevents the trafﬁcking of APP
into the cellular compartment where secretases reside. Consequently, the overexpression of LR11/SorLA in cultured cells reducesamyloidogenic processing [14]. Very recently, a novel function of
LR11/SorLA has been reported. Caglayan et al. reported that
LR11/SorLA plays a role in the lysosomal targeting of newly generated Ab through the binding of Ab to the amino-terminal VPS10
protein domain of LR11 [15]. This binding enhances Ab clearance
in lysosomes by internalization of extracellular Ab through thereceptor LR11/SorLA. With this as a background, we here
attempted to determine the apoE-isoform-dependent effects on
the cellular uptake of Ab through the formation of a complex
between an spoE isoform and LR11/SorLA.
2. Materials and methods
2.1. cDNA cloning and construction of expression plasmids
Complementary DNA (cDNA) of human full-length LR11 wasampliﬁed by PCR using pCMV6 plasmid cDNA containing human
LR11 (Origene, Rockville, USA) and cloned into the pcDNA3.1 vector (Life Technologies, Carlsbad, USA). Human APOE e3 cDNA wascloned and inserted into the pcDNA3.1 vector. We generated twoallelic variants of human APOE, namely, APOE e2 and e4, by mutagenesis. Each isoform of APOE was cloned into the pEGFP vector(Clontech, Mountain View, USA). All constructs generated from
PCR products were veriﬁed by DNA sequencing.

483

with cDNA encoding human apoE2, apoE3, apoE4, apoE2-GFP,
apoE3-GFP, or apoE4-GFP were cultured on a dish with a 1.0 lm
ﬁlter insert (BD Bioscience, San Diego, USA) for 24 h. The donor
HEK293T cells on the dish were placed in a six-well culture dishcontaining the recipient N2a cells mock-transfected or transiently
transfected with the FL-LR11 cDNA construct. After 24 h ofcoculture, the lysate of the recipient cells and the medium were
subjected to Western blot analysis.
2.4. Western blot analysis
The cells were solubilized using a lysis buffer (150 mM NaCl,
50 mM Tris-HCl, pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate,and 5 mM EDTA). The protein concentration of the detergentextracted lysates was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, USA). Equal amounts of protein
were boiled in Laemmli sample buffer and then subjected to
Tris–glycine or Tris-tricine SDS–PAGE system. The separated proteins were transferred to a polyvinylidene diﬂuoride membrane(Millipore, Billerica, USA) and then incubated with appropriate primary antibodies. FL-LR11 and sLR11 were detected using themonoclonal antibody 48/LR11 (BD Biosciences), which recognizes
the extracellular domain of LR11. ApoE was visualized by the
monoclonal antibody E6D7 (Sigma, St. Louis, USA). Ab polypeptides
were detected by the monoclonal antibody 6E10 (Covance,
Berkeley, USA). GFP-fusion proteins were detected by the monoclonal antibody1E4 (MBL, Japan). Actin was detected using the goatanti-actin antibody I-19 (Santa Cruz Biotechnology, Dallas, USA).
Immunoreactive bands were detected using an Immobilon
Western Chemiluminescent HRP substrate (Millipore). The band
intensities were quantiﬁed using a LAS system (GE Healthcare,
Pittsburgh, USA).
2.5. Co-immunoprecipitation assays
HEK293T cells were cotransfected with the cDNA encoding
apoE2, apoE3, or apoE4 and with FL-LR11 cDNA. The cells were solubilized using co-IP buffer (1% CHAPS, 150 mM NaCl, 50 mM
HEPES, pH 7.5). The supernatant was incubated with either ananti-LR11 antibody (48/LR11) or an anti-apoE antibody (AB947,
Millipore) at 4 C for 16 h. To immunoprecipitate the protein complex, 25 lL of protein G Mag Sepharose beads (GE Healthcare) was
added to the mixture, and the mixture was incubated on a rotator
at 4 C. The beads were collected on a magnetic stand and washedthree times with the solubilization buffer. The immunoprecipitatedproteins were released from the beads by incubation in Laemmli
sample buffer. The obtained samples were analyzed by SDS–PAGE
followed by Western blot analysis.
2.6. Fluorescence confocal laser scanning microscopy and image
analysis2.2. Cell culture and transfection
Mouse neuroblastoma Neuro2a (N2a) and human embryonickidney (HEK)293T cells were cultured as previously described[16]. Transient transfection of plasmid DNA into cells was carriedout by using Lipofectamine 2000 (Life Technologies). To generate
stable cell lines, N2a cells transfected with cDNA encoding human
apoE2, apoE3, apoE4 or FL-LR11 were selected using G418.
Peptides of Ab40 were purchased from Wako (Tokyo, Japan) andFAM-labeled Ab40 from AnaSpec (Fremont, USA). They weredissolved in 1% ammonium hydroxide.
2.3. Coculture system
Coculture experiments were performed essentially as previously described [16]. Donor HEK293T cells transiently transfected

N2a cells treated with GFP-labeled apoE3 or FAM-labeled Ab40
were cultured. All images were obtained using an inverted
microscope (TE-300NT, Nikon, Japan) and a confocal microscope(CSU-10, Yokogawa Electric Corp, Japan). The obtained images
were further analyzed with a quantitative analysis system(AquaCosmos, Hamamatsu Photonics, Japan).
2.7. Statistical analyses
Data are shown as the mean ± standard error of the mean
(SEM). Statistical comparison between two groups was carriedout by the Student t-test. For statistical comparison among several
groups, we used one-way analysis of variance (ANOVA) followed－ 307 －

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R. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

by the Tukey post hoc test. The statistical analyses were performed
using SPSS ver. 12.0 J (SPSS Japan).
3. Results
3.1. Isoform-dependent change in level of secreted apoE by expressionof LR11/SorLAWe ﬁrst determined whether the expression of FL-LR11/SorLA
affects the levels of apoE in the lysate and medium. N2a cells stably
expressing human apoE3 were mock-transfected or transiently
transfected with human FL-LR11 cDNA. The levels of apoE3 in
the medium and lysate were determined using the anti-apoE antibody at different time points from 2 to 24 h after the medium was
replaced. The level of apoE3 migrating at 34 kDa and secreted
into the medium increased after the medium replacement in atime-dependent manner (Fig. 1A). The level of apoE3 in the medium was signiﬁcantly lower in N2a cells transfected with FL-LR1124 h after the medium change compared with mock-transfected
cells, whereas the level of apoE in the lysate was comparable
between the mock- and FL-LR11-transfected cells (Fig. 1B).
Next, we investigated whether apoE-isoform-dependent differences are observed in this study. N2a cells stably expressing
human apoE2, apoE3, or apoE4, were mock-transfected ortransiently transfected with FL-LR11, and the levels of apoE in
the medium and lysate were determined by immunoblot analysis.
The apoE ratio (medium/lysate) signiﬁcantly decreased in apoE3and E4-expressing cells that were transiently transfected with
FL-LR11 (Fig. 1C and D), whereas the apoE ratio (medium/lysate)
was unchanged in apoE2-expressing cells transfected with
FL-LR11. These ﬁndings suggest that the effect of LR11/SorLA on
apoE ratio (medium/lysate) is dependent on the apoE isoform.

N2a apoE3 stable cell

A

mock

(kDa)

2

4

8

FL-LR11

12

2

24

4

8

12

24 (hrs)260

FL-LR1140

apoE (lysate)

40

apoE (medium)
actin

40

*

apoE ratio(medium/lysate)

B

1
mock

0.8

FL-LR11

0.6
0.4
0.2
0
2

C
(kDa)

4

8

apoE2
mock

24 (hrs)12
apoE3

FL-LR11

mock

apoE4

FL-LR11mock260

FL-LR1140

apoE (lysate)

40

apoE (medium)
actin

40

apoE2

D

apoE ratio(medium/lysate)

FL-LR11

apoE3

apoE4

*

1.2
1.2

1.2
1.2

0.8
0.8

0.8
0.8

0.8
0.8

0.4
0.4

0.4
0.4

0.4
0.4

00

00

00

mock LR11

*

1.2
1.2

mock LR11

mock LR11

Fig. 1. Human-apoE-isoform-dependent differences in apoE ratio mediated by LR11/SorLA. (A) N2a cells stably expressing human apoE3 were mock-transfected ortransiently transfected with human FL-LR11. The levels of apoE3 in the medium and cell lysate were determined by immunoblot analysis at different time points ranging
from 2 to 24 h after the medium change. Note that the level of apoE in the medium of cells expressing FL-LR11 was decreased compared with mock-transfected cells. The
equivalence of protein loading is shown in the b-actin blot. (B) Results of semiquantitative analysis by densitometry of apoE level. The apoE ratio (medium/lysate) was deﬁned
as the ratio of the level of apoE in the medium to that in the cell lysate. The apoE ratio (medium/lysate) signiﬁcantly decreased 24 h after the medium change in cells
transfected with FL-LR11 compared with mock-transfected cells. Results of three independent experiments are shown as mean ± SEM. ⁄p < 0.05 by Student t-test. (C) N2a cells
stably expressing human apoE2, apoE3, or apoE4 were mock-transfected or transiently transfected with human FL-LR11. The levels of apoE in the medium and the lysate ofcells expressing each of the apoE isoforms were determined 24 h after the medium change. (D) Results of semiquantitative analysis of the apoE ratio (medium/lysate).
The apoE ratio (medium/lysate) was signiﬁcantly decreased in apoE3- or apoE4-expressing cells transfected with FL-LR11, whereas the apoE ratio did not change in
apoE2-expressing cells. ⁄p < 0.05.

－ 308 －

Fig. 2.
FL-LR1mediu(mediucocultuwith F
expresdonor
examinThe lev

cted oranginglls. Thedeﬁnedin cells
2a cells
ysate oflysate).
ange in

apoE2

LR11

mock

LR11
FL-LR1130

apoE (lysate)30

apoE (medium)

40

actin

apoE2

apoE/actin

B

mock

apoE3

apoE4
1.5
1.5

1.5
1.5

1.5
1.5

11

11

11

0.5
0.5

0.5
0.5

0.5
0.5

00
2
1.5
1.5
1
0.5
0.5
0

C donor :00

mock LR11

mock LR11

(kDa)

LR11

00

mock LR11

*

2
1.5
1.5
1
0.5
0.5
0

mock LR11

apoE2-GFP

recipient : mock***

mock LR11

*

2
1.5
1.5
1
0.5
0.5
0

mock LR11

apoE3-GFP

apoE4-GFP

mock

mock

LR11

LR11

260
70

FL-LR1170

apoE-GFP (medium)apoE-GFP (lysate)

actin

40

D

485

apoE4

260

apoE ratio(medium/lysate)

A

apoE3

LR11

(kDa)

apoE2-GFP
apoE-GFP ratio(medium/lysate)differessing
ed or
oE in
alysis.
poE3with
ysate)
with
LA on
m.

donor :

recipient : mockapoE-GFP/actin

med-LR11
ectedarableR. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

apoE3-GFP

apoE4-GFP

1.5
1.5

1.5
1.5

11

11

11

0.5
0.5

0.5
0.5

0.5
0.5

00

2
1.5
1.5
1
0.5
0.5
0

mock LR11

mock LR11

00

2
1.5
1
0.5
0

**

1.5
1.5

00

mock LR11**

mock LR11

2
1.5
1.5
1
0.5
0.5
0

*

mock LR11**

mock LR11

Fig. 2. ApoE isoform-dependent cellular uptake of apoE by LR11/SorLA in coculture system. (A) The recipient N2a cells mock-transfected or transiently transfected with
FL-LR11 were cocultured with the donor HEK293T cells that were transiently transfected with apoE2, apoE3, or apoE4 for 24 h. Cellular uptake of secreted apoE from themedium into the recipient N2a cells was examined by Western blot analysis. (B) Results of semiquantitative analysis of cellular uptake of apoE are shown. The apoE ratio(medium/lysate) signiﬁcantly decreased in the recipient cells transfected with FL-LR11 compared with the mock-transfected recipient cells when the recipient cells were
cocultured with the donor HEK293T cells expressing apoE3 or apoE4 (upper panels). The apoE level normalized by b-actin level in the lysate of the recipient cells transfectedwith FL-LR11 showed a signiﬁcant increase compared with that of mock-transfected recipient cells when the recipient cells were cocultured with the donor HEK293T cells
expressing apoE3 or apoE4 (lower panels). ⁄p < 0.05; ⁄⁄p < 0.01. (C) The recipient N2a cells mock-transfected or transiently transfected with FL-LR11 were cocultured with thedonor HEK293T cells that secreted apoE2-GFP, apoE3-GFP, or apoE4-GFP in the medium for 24 h. Cellular uptake of apoE from the medium into the recipient N2a cells was
examined. (D) Results of semiquantitative analysis of cellular uptake of apoE-GFP are shown. The apoE ratios (medium/lysate) for the apoE isoforms are shown (upper panels).
The levels of apoE-GFP normalized by b-actin level for the apoE isoforms are shown (lower panels). ⁄p < 0.05; ⁄⁄p < 0.01.

－ 309 －

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R. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

3.3. ApoE-isoform-dependent cellular uptake of apoE in coculture
systemCo-IP

A

medium(kDa)

IP : apoE (AB947)
IB : LR11
IP : LR11
IB : apoE (E6D7)

260
40

IP : apoE (AB947)
IB : LR11
IP : LR11
IB : apoE (E6D7)

40

1

3

4

5

6

Sample : medium
IP : anti-apoE Ab

3
0

2
2

4
2
0

3
4
3 4
apoE

Sample : lysate
IP : anti-apoE Ab

*

0

2
2

3
4
3 4
apoE

3.4. ApoE-isoform-dependent binding afﬁnity to LR11/SorLA

3
4
3 4
apoE

We speculated that the differential cellular apoE uptake in therecipient cells may be explained by a difference in binding afﬁnity
between LR11/SorLA and each of the apoE isoforms. To test thishypothesis, we examined the ability of LR11/SorLA to form a complex with each of the apoE isoforms by co-immunoprecipitation(co-IP) assay. We collected the detergent-extracted lysate of
HEK293T cells that were cotransfected with FL-LR11 and each of
the apoE isoforms. Co-IP assay using the anti-apoE or anti-LR11
antibody was performed using the medium and cell lysate. This
assay revealed that LR11/SorLA bound to each isoform of apoE withdifferent efﬁciencies (Fig. 3A). ApoE4-expressing cells showed thehighest level of the LR11-apoE complex, followed by apoE3- andapoE2-expressing cells (Fig. 3B).

*

9

*

2

2
2

Sample : lysate
IP : anti-LR11 Ab

Co-IP/input(apoE)

Co-IP/input(LR11)

4

*

6

*

6

7

Sample : medium
IP : anti-LR11 Ab**

9
Co-IP/input(LR11)

2

Co-IP/input(apoE)

lysate260

B

We next examined whether the enhanced uptake of apoE by
FL-LR11 is modiﬁed by apoE isoforms using the coculture system.
First, we attempted to conﬁrm whether apoE3 and GFP-labeled
apoE3 secreted from the donor HEK293T cells are taken up into
the recipient cells. This experiment revealed that apoE3 and GFPlabeled apoE3 were detected in the lysate of the recipient cells(Supplementary Fig. 2B), whereas GFP alone without apoE was
not detected in the recipient cells.
Having established that apoE secreted from the donor cells istaken up into the recipient cells, we next asked if isoforms of apoE
differently affect the cellular uptake of apoE in the recipient N2acells mock-transfected or transiently transfected with FL-LR11.
The level of apoE taken up into the recipient N2a cells was determined by immunoblot analysis using the anti-apoE antibody(Fig. 2A). The cellular uptake levels of apoE3 and apoE4 were signiﬁcantly higher in the recipient cells that express FL-LR11 compared with the mock-transfected cells (Fig. 2B). In a similar
experiment using donor cells that express apoE2-GFP, apoE3-GFP,
or apoE4-GFP, the cellular uptake levels of apoE3-GFP and apoE4GFP were increased by the expression of FL-LR11 (Fig. 2C and D).
These results suggest that FL-LR11 enhances the cellular uptakeof apoE3 and apoE4 but not that of apoE2.

6
3
0

2
2

3
4
3 4
apoE

Fig. 3. Human-apoE-isoform-dependent differences in formation of complex
between apoE and LR11/SorLA examined by co-IP assay. (A) HEK293T cells were
cotransfected with each of the human apoE isoforms and human FL-LR11. The
formed complex between apoE and LR11/SorLA was immunoprecipitated using theanti-apoE antibody (AB947) or anti-LR11 antibody (48/LR11), followed by immunoblot analysis. The lysates (lanes 1–3) and immunoprecipitated samples (lanes 4–7) were run on the same gel. (B) Results of semiquantitative analysis of
immunoprecipitated apoE and LR11/SorLA. ⁄p < 0.05; ⁄⁄p < 0.01 by Tukey test
following ANOVA.

3.2. Cellular uptake of apoE examined by ﬂuorescence confocal laser
microscopy
The ﬁnding that the level of apoE in the medium of cells transfected with FL-LR11 decreased raises the possibility that apoE in
the medium was taken up into cells by the lipoprotein receptor
LR11/SorLA. To explore this possibility, N2a cells were incubatedwith the medium prepared from cells that transiently expressed
the apoE3-GFP fusion protein. Fluorescence confocal microscopy
revealed apoE-GFP signals inside the cells transfected with
FL-LR11 and incubated with the medium of cells expressing
apoE3-GFP (Supplementary Fig. 1B). This ﬁnding suggests thatthe cellular uptake of apoE from the medium was enhanced by
the expression of LR11/SorLA.

3.5. ApoE-isoform-dependent cellular uptake of Ab
Given the physical interaction between LR11/SorLA and apoE,
we next considered the possibility that FL-LR11 may be involvedin the cellular uptake of the apoE-Ab complex, because apoE isknown to form a complex with Ab. To address this issue, N2a cells
were incubated with FAM-labeled Ab40 at various concentrationsof ranging from 0.5 to 3.0 lM. By ﬂuorescence confocal laser
microscopy, we detected the cellular uptake of FAM-labeled Ab
at concentrations above 1.0 lM (Supplementary Fig. 3A). We
observed the cellular uptake of FAM-labeled Ab at a low concentration of 0.25 lM when the recipient cells were transfected with
FL-LR11 and cocultured with the donor cells expressing apoE4(Supplementary Figs. 3B, 4A and 4B). The ﬁnding suggests thatthe cellular uptake of Ab was enhanced in the presence of both
FL-LR11 and apoE.
We ﬁnally performed coculture experiments in which the recipient N2a cells mock-transfected or transiently transfected with
FL-LR11 were cocultured with the donor cells expressing apoE2,
E3, or E4 in the presence of the Ab peptides at 0.25 lM. The cellular
uptake level of apoE and Ab were highest when the recipient cells
were cocultured with the donor cells expressing apoE4, followed
by apoE3 and apoE2 (Fig. 4A and B).

－ 310 －

Fig. 4.
with drecipiethe ap

4. DisDelogica
accum
wealt
genet
unde
ious
dence
aggre
may
how
logicaregar
differ
focus
LR11/
Fir
enhan
gests
was s
surfa
we h
differ
was
suppo
LR11/
differR. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

reDonor apoE: mock E2 E3 E4
Aβ peptide (0.25 μM)

A

oE by
stem.
beled
p into
GFPcells
E was

apoE,
olved
poE isa cells
ations
laser
ed Ab
. We
entrawith
apoE4s thatbothe reciwith
poE2,
ellulart cells
owed

Recipient: mock

FL-LR11(kDa)

apoE (medium)

30

260

3.5

Aβ dimerAβ monomer

40

actin

*

4.5
4.5
Aβ / actin

apoE ratio(lysate / medium)

apoE

30

*

B
10
10
7.5
7.5
5
5.0
2.5
2.5
00

FL-LR11

**
** **

3
3.0

1.5
1.5

12

23

apoE

34

00

2

3

apoE

4

A dimer / monomer

recipient cells

ells is
apoE
t N2a
LR11.
deteribody
e sigcommilar
3-GFP,
poE4nd D).
ptake

n the
ﬁnity
t thiscomtation
te of
ach of-LR11. ThisE withed the
- and

487

1.5
1.5
1
1.0
0.5
0.5
00

12

23 34
apoE

Fig. 4. ApoE-isoform-dependent differences in cellular uptake of Ab in cells expressing LR11/SorLA. (A) Recipient N2a cells stably expressing human FL-LR11 were cocultured
with donor N2a cells stably expressing human apoE2, apoE3, or apoE4 in the presence of Ab40 peptides at 0.25 lM for 24 h. The cellular uptake of Ab and apoE into therecipient cells was examined by Western blot analysis. (B) Results of semiquantitative analysis of cellular uptake of Ab and apoE are shown as -fold increase compared withthe apoE2 isoform. ⁄p < 0.05; ⁄⁄p < 0.01 by Tukey test following ANOVA.

4. Discussion
Despite signiﬁcant advances in our understanding of the pathological events occurring in the AD brain, the factors leading to Ab
accumulation in sporadic AD patients are not well understood. A
wealth of evidence has conﬁrmed that APOE e4 is the strongest
genetic risk factor for sporadic AD [2,3]. Although it is not fully
understood how human apoE isoforms differentially affect the various pathogenic processes implicated in AD, several lines of evidence suggest that the effects of apoE on Ab accumulation,
aggregation, clearance, neurotoxicity, and neuroinﬂammation
may play a role in AD pathogenesis [6]. Hence, understanding
how each of the apoE isoforms differentially plays a pathophysiological role in AD is an important question to address. In thisregard, this study provided new insights into how apoE isoforms
differentially modify the AD pathogenic pathway with particularfocus on the cellular uptake of Ab via the lipoprotein receptor
LR11/SorLA.
First, we demonstrated that the overexpression of LR11/SorLA
enhanced the cellular uptake of apoE3 and apoE4. This ﬁnding suggests that LR11/SorLA functions as the apoE receptor. This notion
was suggested by previous studies showing that FL-LR11 at the cellsurface was capable of cellular uptake of apoE [12]. Importantly,
we here showed that the cellular uptake of apoE is modiﬁed bydifferent apoE isoforms. The cellular uptake of apoE4 and apoE3
was signiﬁcantly enhanced by the expression of LR11/SorLA. In
support of this notion, the co-IP assay revealed that the apoELR11/SorLA complex formation showed apoE-isoform-dependent
differences in efﬁciencies, that is, apoE4 > apoE3 > apoE2. Major

apoE receptors belong to the LDL receptor family including
the LDL receptor and LDLR-related protein (LRP1). Different efﬁciencies in the formation of complexes between the LDL receptor
and the apoE isoforms were observed. Similar to our ﬁnding, thecellular uptake levels of apoE3 and apoE4 were previously foundto be higher than that of apoE2 in cells expressing the LDL receptor[17].
Secondly, our cell culture experiments revealed that extracellular Ab was taken up into cells overexpressing LR11/SorLA in an
apoE-isoform-dependent manner. A recent study has shown that
LR11/SorLA binds to Ab directly through the VPS10 protein domainof LR11/SorLA [15]. Importantly, we demonstrated that the cellular
uptake of extracellular Ab was apoE-isoform-dependent, that is,
apoE4 > apoE3 > apoE2. Although this ﬁnding is potentiallyinteresting, how this ﬁnding is relevant to AD pathogenesis is notpresently understood. Enhancement of cellular uptake of Ab may
have two different consequences. A harmful consequence would
be that an increased intracellular Ab concentration through theuptake of extracellular Ab may provide seeds of Ab oligomers, a
toxic forms of Ab [18]. Some studies suggested that intracellular
Ab oligomers may be a major cause of synaptic dysfunction [19].
On the other hand, a beneﬁcial consequence would be that the cellular uptake of extracellular Ab may enhance the clearance of Ab inlysosomes [20]. Our ﬁnding favors the former possibility, that is,cellular uptake of Ab may exert a detrimental effect on neurons
because this phenomenon is most closely associated with the presence of apoE4. Further studies delineating the precise consequences of increased intracellular Ab level caused by enhanced
cellular uptake of Ab will be required.

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R. Yajima et al. / Biochemical and Biophysical Research Communications 456 (2015) 482–488

ApoE is predominantly generated by astrocytes and microglia inthe brain and is subsequently lipidated by ABCA1 to form lipoprotein particles [21]. It has been demonstrated that lipoprotein
receptors including LDLR and LRP on the neuronal surface are able
to take apoE-Ab complexes into neurons after apoE is lipidated[22]. The lipidation of apoE has been shown to inﬂuence its isoform-speciﬁc afﬁnity to Ab. The efﬁciency of complex formation
between lipidated apoE and Ab is in the order of apoE2 > apoE3 >apoE4 [23]. In addition, the variable levels of apoE oxidation may
affect the properties of apoE binding to Ab. In this study, we did
not fully address the lipidation or oxidation status of apoE secreted
from HEK293T cells, which should be taken into account wheninterpreting our results. To minimize confounding factors, it isdesirable to use apoE prepared under conditions that preserve
apoE lipidation and oxidation status found in the brain. Collectively, our study indicated that LR11/SorLA is capable of cellular
uptake of Ab in an apoE-isoform-dependent manner; however, its
pathophysiological role requires further investigation.
Acknowledgments
This work is supported in part by JSPS KAKENHI (23591234 and20372469 to T.I.) and a Grant-in-Aid from the Ministry of Health,Labour and Welfare of Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bbrc.2014.11.111.
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Cerebral Cortex Advance Access published October 6, 2015
Cerebral Cortex, 2015, 1–13
doi: 10.1093/cercor/bhv209
Original Article

ORIGINAL ARTICLE

Dopamine D1 Receptor-Mediated Transmission

Satomi Chiken1,2,†, Asako Sato3,4,†, Chikara Ohta1,5, Makoto Kurokawa5,
Satoshi Arai3, Jun Maeshima3, Tomoko Sunayama-Morita4,6,
Toshikuni Sasaoka2,3,4,7, and Atsushi Nambu1,2
1

Division of System Neurophysiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan,
School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki 444-8585, Japan,
3
Department of Laboratory Animal Science, Kitasato University School of Medicine, Sagamihara 252-0374, Japan,
4
National Institute for Basic Biology, Okazaki 444-8585, Japan, 5Department of Biological Sciences, Tokyo
Metropolitan University, Tokyo 192-0397, Japan, 6Department of Life Sciences, Graduate School of Arts andScience, The University of Tokyo, Tokyo 153-8902, Japan and 7Department of Comparative and Experimental
Medicine, Brain Research Institute, Niigata University, Niigata 951-8585, Japan
2

Address correspondence to Atsushi Nambu, Division of System Neurophysiology, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji,
Okazaki 444-8585, Japan. Email: nambu@nips.ac.jp; Toshikuni Sasaoka, Department of Comparative and Experimental Medicine, Brain Research Institute,
Niigata University, Niigata 951-8585, Japan. Email: sasaoka@bri.niigata-u.ac.jp†

These authors contributed equally to this work.

Abstract
In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deﬁciency results in severe motor
dysfunctions as seen in Parkinson’s disease. According to the well-accepted model of the BG, dopamine activates striatal direct
pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits
striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role ofdopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally andreversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired
motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, wasrecorded in awake conditions to examine the mechanism of motor deﬁcits. Cortically evoked inhibition in the EPN mediated by thecortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous ﬁring rates andpatterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediateddopaminergic transmission maintains the information ﬂow through the direct pathway to appropriately release motor actions.
Key words: basal ganglia, conditional knockdown, dopamine receptor, extracellular recording, mouse© The Author 2015. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
For commercial re-use, please contact journals.permissions@oup.com

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Maintains Information Flow Through the CorticoStriato-Entopeduncular Direct Pathway to Release
Movements

2| 2Cerebral| Cerebral
Cortex
CortexIntroduction
Introduction

entopeduncularentopeduncular
nucleusnucleus(EPN) (homologous(EPN) (homologous
to the internal
to the internalsegment
segmentof the globusof the globus
pallidus,
pallidus,GPi) to GPi)
cortical
to corticalstimulation
stimulation(Cx Stim.)(Cx with
Stim.)early
withexcitation,
early excitation,
inhibition,
inhibition,
and andlate excitation,late excitation,
which which
are mediatedare mediatedby the byi) cortico-subthalamo
the i) cortico-subthalamo(STN)-EPN(STN)-EPN
hyperdirect,
hyperdirect,ii) cortico-striato-EPNii) cortico-striato-EPN
direct, direct,
and iii)andcortico-striato-externaliii) cortico-striato-external
pallidopallido(GPe)- (GPe)STN-EPN
STN-EPN
indirectindirectpathways,pathways,respectively.
respectively.
Red, blue,
Red,andblue,
green
and triangles
green triangles
represent
represent
glutamatergic
glutamatergic
excitatory,
excitatory,
GABAergic
GABAergic
inhibitory,
inhibitory,
and dopaminergicand dopaminergic
projections,projections,
respectively.
respectively.
D2Rs, dopamine
D2Rs, dopamine
D2 receptors;
D2 receptors;
SNc, substantia
SNc, substantia
nigra parsnigra
compacta.
pars compacta.
(B) Schematic(B) Schematic
diagramdiagramof Dox-regulatedof Dox-regulated
D1R expression
D1R expression
in D1RKD
in D1RKD
mice. Before
mice. Before
Dox Doxtreatment
treatment(left), tetracycline(left), tetracyclinetransactivatortransactivator(tTA) binds(tTA)tobinds
the tetracycline
to the tetracycline
responsive
responsive
element
element(TRE), and(TRE),
D1Rs
andand
D1Rs
lacZ
and
are
lacZ
transcribed.
are transcribed.
Dox treatment
Dox treatment
interferesinterfereswith withtTA bindingtTA binding
to TREto(right),TRE (right),
and suppresses
and suppresses
D1R and
D1R
lacZ
andexpression
lacZ expression(Tet-off(Tet-off
system).
system).
(C) D1R(C)immunoreactivity
D1R immunoreactivity
in the instriatum
the striatum(Str) of(Str)wild-typeof wild-type(WT, top),(WT,D1Rtop), D1R
knockout
knockout(D1RKO,(D1RKO,
middle),middle),and D1RKD
and D1RKD(before(before
Dox treatment,
Dox treatment,bottom)bottom)mice shown
mice shownin frontal
in frontal
sections.
sections.
The pattern
The patternof D1R of
expression
D1R expression
in D1RKD
in D1RKD
mice wasmice
similarwas similartotothat in that
WT in
mice,
WTwhereas
mice, whereas
D1R immunoreactivity
D1R immunoreactivity
was notwasobserved
not observedin D1RKO
in D1RKO
mice. Themice.
dorsolateral
The dorsolateralmotor areas
motorofareas
the Strof theare Strdeﬁnedare deﬁned
by rectangles
by rectangles(left) and(left)
shown
and shownat higher
at higher
magniﬁcation
magniﬁcation(right).(right).
Scale bars,
Scale1bars,
mm on
1 mm
the on
left,
the
100
left,
μm100
on μm
the onright.
the (D)
right.
Western(D) Western
blot analysis
blot analysisof D1Rofprotein
D1R protein
expression
expression
in the instriatum
the striatumof a D1RKO
of a D1RKO
mousemouse(n = 1, number(n = 1, numberof miceofused),mice D1RKD
used), D1RKD
mice before,
mice before,
during,during,
and afterandDoxafter
treatment
Dox treatment(Days 0,(Days7, 14,0,21,
7, 14,
28, 21,
different
28, differentdays after
daysstarting
after startingDox treatment;
Dox treatment;
After, 7After,days 7 days
after cessation
after cessationof Dox oftreatment
Dox treatment
for 14 days;for 14ndays;= 3–4 nper
= 3–4
condition),
per condition),and WT
andmice
WT(nmice= 3). (n = 3).

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Figure 1.
FigureDopamine
1. Dopamine
D1 receptor
D1 receptor(D1R) suppression(D1R) suppression
in D1R in
knockdown
D1R knockdown(D1RKD)(D1RKD)mice withmice
doxycyclinewith doxycycline(Dox) treatment.
(Dox) treatment.
(A) Schematic(A) Schematic
diagramdiagramshowing
showing
the cortico-basal
the cortico-basal
gangliaganglia
pathway
pathway
and stimulating
and stimulating(Stim.) (Stim.)
and recording
and recording(Rec.) sites(Rec.)insites
the electrophysiologicalin the electrophysiologicalexperiments
experiments(left), along(left), withalonga withtypicala typical
response
response
patternpattern(right) (right)
in the in the

stri
cre

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segment
segmentof theof
globusthe globus
pallidus
pallidus(GPe),(GPe),whichwhichis alsoiscalled
also called
the glothe globus pallidus
bus pallidus
in rodents.
in rodents.
Such Such
differential
differential
effects
effectsof dopamineof dopamine
In theInbasal
the basal
gangliaganglia(BG), dopaminergic(BG), dopaminergictransmission
transmission
plays playsa piv-a pivthroughthrough
D1Rs D1Rs
and D2Rs
and D2Rs
were wereoriginallyoriginallyproposed
proposed
basedbasedon onotal role
otalinrole
theincontrol
the controlof voluntaryof voluntary
movements
movementsand motor
and motor
learn-learnchanges
changesin genein geneexpression,
expression,
glucoseglucoseutilization,utilization,and receptor
and receptor
ing (Albin
ing (Albinet al. et1989;
al. 1989;
DeLong
DeLong1990; 1990;
GerfenGerfenet al. et1990;
al. 1990;
Graybiel
Graybielbindingbindingin thesein thesepathwayspathways
underunderconditions
conditionsof dopamineof dopamine
deple-deple2005; 2005;
JoshuaJoshuaet al. et2009;
al. 2009;
EnomotoEnomotoet al. et2011).
al. 2011).
Dopamine
Dopaminedeﬁ- deﬁtion (Gerfen
tion (Gerfen
et al. et1990;
al. 1990;
HirschHirschet al. et2000)
al. 2000)
and haveand havebeen beenrein- reinciency,
ciency,as occurs
as occursin Parkinson’sin Parkinson’s
diseasedisease(PD), results(PD), results
in severein severe
forcedforced
by recentby recentin vitro
in electrophysiologicalvitro electrophysiologicalstudiesstudies(Surmeier(Surmeiermotormotor
and nonmotor
and nonmotor
dysfunctions
dysfunctions
includingincludingbradykinesia,
bradykinesia,
rigid-rigidet al. et2007;
al. 2007;Day etDay
al. et2008;
al. 2008;
Flores-Barrera
Flores-Barrera
et al. et2011;
al. 2011;
GerfenGerfenand andity, tremor,
ity, tremor,autonomic
autonomic
abnormalities,
abnormalities,
cognitive
cognitive
dysfunction,
dysfunction,
and andSurmeier
Surmeier
2011; 2011;
PlanertPlanertet al. et2013).
al. 2013).
The striato-EPN/SNr
The striato-EPN/SNr
directdirect
depression
depression(Fahn(Fahnet al. 2011;
et al. 2011;
SeppiSeppi
et al. 2011).
et al. 2011).
Dopaminergic
Dopaminergic
in- inand striato-GPe-subthalamo(STN)-EPN/SNr(STN)-EPN/SNr
indirectindirectpathwayspathwaysputs fromputs fromthe substantia
the substantia
nigra nigra
pars compacta
pars compacta
terminateterminatein thein theand striato-GPe-subthalamo
opposite
oppositeroles roles
in controllingin controlling
movements.
movements.
The signals
The signals
striatumstriatumand areandthought
are thought
to differentiallyto differentiallymodulate
modulatethe excitabilthe excitabil-play playthroughthe direct
the direct
pathway
pathway
reducereduceactivity
activityof theof
EPN/SNrthe EPN/SNr
and inand inity of ity
2 typesof 2 typesof striatalof striatal
projection
projection
neurons
neurons
throughthroughdifferent
different
re- re-throughthalamocorticalthalamocorticalactivity
activity
via disinhibition,
via disinhibition,
resultingresultingin re-in receptors
ceptors(Albin(Albinet al.et1989;
al. 1989;
DeLong
DeLong1990; 1990;
MalletMallet
et al.et2006)
al. 2006)creasecreaseof movements.
of movements.
In contrast,
In contrast,
signals
signals
throughthroughthe indirect
the indirect(Fig. 1A,
(Fig.left).
1A, Oneleft).isOne
excitatory
is excitatory
effects
effectsthroughthroughdopamine
dopamine
D1 re-D1 re-lease lease
pathway
pathway
increaseincrease
activity
activityof
theof
EPN/SNr,the
EPN/SNr,resultingresultinginsuppresin suppresceptors
ceptors(D1Rs)(D1Rs)
on direct
on direct
pathway
pathway
neurons
neurons
that directlythat directly
project
projectsion
movements
of movements(Albin(Albinet al. et1989;
al. 1989;
DeLong
DeLong1990; 1990;
GerfenGerfenet al. et al.
to thetooutput
the output
nucleinucleiof theofBG,
thetheBG,entopeduncular
the entopeduncular
nucleusnucleussion of
Mink Mink
1996; 1996;
Nambu
Nambu
2007; 2007;
Kravitz
Kravitz
et al. et2010;
al. 2010;
Sano Sanoet al. et al.
(EPN),(EPN),
whichwhich
is homologous
is homologous
to thetointernal
the internalsegment
segmentof theof
globusthe globus1990; 1990;
Thus,Thus,
the lossthe ofloss
dopaminergicof dopaminergic
inputsinputsto bothto pathway
both pathway
pallidus
pallidus(GPi) in(GPi)
primates,in primates,and substantia
and substantia
nigra nigra
pars reticulata
pars reticulata2013).2013).
neurons
is consideredis considered
to increase
to increase
ﬁring ﬁring
rates ofrates
EPN/SNrof EPN/SNrneurons
neurons(SNr).(SNr).
The other
The otheris inhibitory
is inhibitory
effects
effectsthroughthrough
D2 receptors
D2 receptorsneurons(D2Rs)(D2Rs)
on indirect
on indirect
pathway
pathway
neurons
neurons
that projectthat projectto thetoexternal
the externalthroughthroughthe inhibitory
the inhibitory
striato-EPN/SNrstriato-EPN/SNr
directdirect
and netandexcitatory
net excitatory

mis
bee
sho
pha
sio
et a
dow
rev
D1R
al a
sen
spo
D1R
log
neu
ona
ma
D1R
the
wa
wh
ma
mo
EPN

Ma

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and
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acc
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on

Ge

We
and
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tran
mo
(BA
Rec
tion
cod
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of t
frag
and
tec
me
mo
pol
of D
era
D1T
olo

use
al B

Role ofRole
Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al. et| al.3 | 3

Dox Treatment
Dox Treatment
Generation
Generationof Conditionalof Conditional
and Reversible
and Reversible
D1RKD
D1RKD
Mice Mice

Dox (2.0
Doxmg/mL)(2.0 mg/mL)
was mixed
was mixed
in drinking
in drinking
waterwatercontaining
containing
5% su5% suand deliveredand delivered
to thetoD1RKD
the D1RKD
and WTandmice
WT micethroughthrougha watera water
We developed
We developed
D1RKD
D1RKD
mice micein which
in which
D1Rs D1Rs
can becanconditionallybe conditionallycrose croseand reversibly
and reversibly
regulated
regulatedby DoxbyusingDox using
the Tet-Off
the Tet-Offsystemsystemafter deafter de-bottle.bottle.
letionletionof theofendogenous
the endogenous
D1Rs D1Rs(Fig. 1B).
(Fig.To
1B).
generate
To generate
tetracycline
tetracyclinetransactivator-VP16
transactivator-VP16(tTA)-expressing(tTA)-expressing
mice miceusingusing
the D1R
the proD1R proImmunohistochemistry
Immunohistochemistry
motermoter(transactivator(transactivatorline), line),
bacterial
bacterial
artiﬁcialartiﬁcialchromosome
chromosome(BAC) (BAC)
clonesclones
containing
containing
mouse
mouse
D1R wereD1R modiﬁed
were modiﬁedwith awith
2-stepa 2-stepWe used
We used
4 D1RKD,
4 D1RKD,
3 WT,3andWT,1and
D1RKO
1 D1RKOmice micefor immunohistofor immunohistoRec ARecstrategy
A strategy
for BACformodiﬁcation
BAC modiﬁcation(Yang(Yanget al. 1997).
et al. 1997).
A modiﬁcaA modiﬁca-chemicalchemicalanalysis.
analysis.
Mice were
Mice weredeeplydeeplyanesthetizedanesthetizedby injection
by injectionof tribromoethanol(400 mg/kg(400 mg/kg
body bodyweight,weight,i.p.) andi.p.)perfused
and perfusedtion cassettetion cassettethat introduces
that introduces
the tTAtheintotTAtheintoendogenous
the endogenous
D1R D1Rof tribromoethanol
codingcodingsequence
sequence
was constructed
was constructed(see Supplementary(see Supplementary
Fig. 1A).
Fig. 1A).transcardiallytranscardiallywith 4%withparaformaldehyde
4% paraformaldehydein phosphatein phosphatebufferbuffer
A DNA
A fragment
DNA fragment(0.92 kb,
(0.92
A-arm)
kb, A-arm)
containing
containing
the upstream
the upstream
regionregion(PB, pH
(PB,
7.4).
pHThe
7.4).brains
The brains
were were
postﬁxed
postﬁxed
overnight
overnight
at 4°C,
attrans4°C, transof theoftranslation
the translation
initiation
initiationsite ofsite
D1R
ofwas
D1Rligated
was ligatedto thetoDNA
the DNAferredferred
to a 30%
to asucrose
30% sucrose
solution,solution,immersed
immersedin OCTin compound
OCT compoundfragment
fragment(1.5 kb)(1.5containingkb) containing
the coding
the coding
sequence
sequenceof theoftTA
the tTA(Sakura(SakuraFinetek),
Finetek),frozen,frozen,and stored
and storedat −80°C
at −80°C
until until
use. Frontal
use. Frontal
sections
were were
cut atcut
25-μm
at 25-μm
thicknessthicknessand stored
and stored
in PBSinatPBS
4°C.
at 4°C.
and theandSV40
the SV40
poly Apoly
signalA signal( poly (A)polyof theA) ofpTet-off
the pTet-off
vectorvector(Clon-(Clon-sectionstech),tech),and theandresulting
the resulting
fragment
fragment
was ligated
was ligatedto thetoDNA
the fragDNA frag-Free-ﬂoating
Free-ﬂoating
sections
sections
were wereincubatedincubatedwith 1%
withbovine1% bovine
serumserum
al- almentment(0.96 kb,
(0.96
B-arm)
kb, B-arm)of exonof exon2 of D1R
2 oftoD1R
generate
to generate
D1Txx,
D1Txx,
the thebumin
bumin
containing
containing
antibody
antibody
for D1R
for(1:1000;
D1R (1:1000;
Frontier
Frontier
Institute)
Institute)
modiﬁcation
modiﬁcationcassette
cassette(3.38 kb).
(3.38Becausekb). Because
the tTAtheandtTAtheandSV40
the SV40overnight
overnight
at 4°C.atD1R
4°C.antibody
D1R antibody
bindingbinding
was visualized
was visualizedusingusing
the thepoly Apoly
signalA signal
sequences
sequenceswere introduced
were introduced
at theatinitiation
the initiation
codoncodonVectastain
Vectastain
Elite Elite
ABC System
ABC System(Vector(VectorLaboratories)
Laboratories)and 3,
and3′- 3, 3′diaminobenzidine.
of D1R,
ofthe
D1R,
expression
the expressionof endogenousof endogenous
D1R wasD1Rdisrupted.
was disrupted.
To genTo gen-diaminobenzidine.
erate erate
the pSV1-RecA-D1T
the pSV1-RecA-D1T
shuttleshuttlevector,vector,the DNA
the DNA
fragment
fragmentofof
D1TxxD1Txx
was inserted
was inserted
into theintopSV1-RecA
the pSV1-RecA
vectorvector
to facilitate
to facilitate
hom-homWestern
Western
Blot Analysis
Blot Analysis−
ologousologousrecombination
recombination
in RecAin −RecA
Escherichia
Escherichia
coli. coli.
The BAC
Theclone,
BAC clone,
BAC4-D1R
BAC4-D1R(80 230(80bp)
230(Researchbp) (Research
Genetics),
Genetics),
was wasWe used
We 19
used
D1RKD
19 D1RKD(3–4 in(3–4
each
in condition,
each condition,
0, 7, 14,0, 21,
7, 14,
and
21,28
and
days
28 days−used (seeusedSupplementary(see Supplementary
Fig. 1B).
Fig.
RecA
1B).−RecA
starting
starting
Dox treatment
Dox treatment
and 7anddays
7 afterdays cessation
after cessationof Doxof DoxE. coliE.
carryingcoli carrying
the originthe origin-after after
treatment
for 14fordays),
14 days),
3 WT,3 and
WT, 1and
D1RKO
1 D1RKOmice micefor western
for western
al BAC
alclone
BAC clone
were transformed
were transformed
with thewithpSV1-RecA-D1T
the pSV1-RecA-D1T
shuttleshuttletreatment－ 315 －

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on at on8:00atAM)
8:00andAM)given
and givenfood andfooddrinking
and drinking
waterwater
ad libitum.
ad libitum.

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vector.
The resulting
The resulting
clonesclones
were subjected
were subjected
to Southern
to Southern
analysisanalysis
striato-GPe-STN-EPN/SNrstriato-GPe-STN-EPN/SNr
indirectindirectpathways,pathways,resultingresultingin theindethe de-vector.
eithereithera 5′ probea 5′ probe
or tTAorprobetTA probe
to conﬁrm
to conﬁrm
properproperrecombinrecombincreased
creasedmotormotor
activity
activity
seen inseen
PD in(AlbinPD (Albin
et al. 1989;
et al. 1989;
DeLong
DeLong1990).1990).usingusing(see Supplementary(see Supplementary
Fig. 1A).
Fig.The1A).BAC
Thetransgenic
BAC transgenic(Tg) con(Tg) conHowever,
However,the exact
the exactrole ofrole
D1R-mediatedof D1R-mediateddopaminergic
dopaminergictrans-trans-ation ation
BAC4-tTA,
BAC4-tTA,
was obtained
was obtained(see Supplementary(see Supplementary
Fig. 1B).
Fig. 1B).
missionmissionin vivo
inisvivo
notis
well
notunderstood.
well understood.
Contradicting
Contradicting
results
results
have havestruct,struct,been been
observed
observed
following
following
D1R blockade.
D1R blockade.
D1R knockout
D1R knockout(KO) mice(KO) mice The Tg
The
construct,
Tg construct,
D1R-tetracycline
D1R-tetracycline
responsive
responsive
element
element(D1R-(D1Rwas generated
was generated
for tetracycline
for tetracycline
operator
operator(tetO)-target(tetO)-target
Tg Tg
showshowincreases
increasesin spontaneousin spontaneous
locomotor
locomotor
activity,
activity,
althoughalthoughTRE), TRE),
mouselines using
lines using
the following
the following
DNA fragments
DNA fragments(see Supplemen(see Supplemenpharmacologicalpharmacologicalblockadeblockadeof D1Rsof D1Rs
induces
induces
locomotor
locomotor
suppressuppres-mouse
tary
1C):
Fig.a 1C):
9.7-kb
a 9.7-kb
fragment
fragment
includingincludingthe entire
the entire
D1R coding
D1R coding
se- sesion insion
agreement
in agreementwith withthe above
the above
explanation
explanation(Waddington(Waddingtontary Fig.
quence,a 0.64-kb
a 0.64-kb
fragment
fragment
encoding
encoding
the TRE
theandTREbidirectionaland bidirectional
et al. 2005).
et al. 2005).
In theInpresent
the present
study,study,
we developed
we developed
novelnovel
D1R knockD1R knock-quence,
promoterfrom the
from
pBI
the
Tet
pBI
vector
Tet vector(Clontech),(Clontech),a 3466-bp
a 3466-bp
fragment
fragment
downdown(D1RKD)(D1RKD)mice micein which
in whichthe D1Rs
the D1Rs
can becanconditionallybe conditionallyand andpromoter
encodinglacZ fromlacZ fromthe pBI-GL
the pBI-GL
Tet vector
Tet vector(Clontech),(Clontech),
a 0.98-kb
a 0.98-kb
reversibly
reversibly
regulated
regulatedby doxycycline
by doxycycline(Dox) (Dox)treatment
treatment(Fig. 1B).
(Fig.The
1B). Theencoding
fragment
containing
containing
rabbitrabbit
beta-globinbeta-globin
poly A
poly
andASV40
and SV40
poly A
poly A
D1RKD
D1RKD
mice enabled
mice enabled
us to examine
us to examine
motormotor
behaviors
behaviorsand neuronand neuron-fragment
signalsfrom thefrompBstN
the pBstN
plasmid,
plasmid,and aand0.3-kb
a 0.3-kb
fragment
fragment
encoding
encoding
al activities
al activities
in theincortico-BG
the cortico-BG
pathwayspathwaysin theinpresence
the presence
and aband ab-signals
the chicken
beta-globinbeta-globin
insulator
insulator
sequences
sequencesfrom fromthe plasmid
the plasmid
sencesenceof D1Rsof D1Rsin theinsame
the samemice.mice.
The mice
The mice
exhibitedexhibiteddecreased
decreasedthe chicken
pUC19
INS240-SNNS.
INS240-SNNS.
spontaneous
spontaneous
motormotor
activity
activity
and impaired
and impaired
motormotor
abilityability
whenwhenpUC19
The BAC4-tTA
and D1R-TRE
and D1R-TRE
DNAsDNAs(10 ng/μL(10 ng/μLeach) each)were wereused used
D1R expression
D1R expression
was suppressed,
was suppressed,
consistentconsistent
with thewithpharmacothe pharmaco- The BAC4-tTA
mouse
Tg mouse
generation
generation
usingusing
standard
standard
techniques
techniques(Nagy(Nagyet al. et al.
logicallogicalblockadeblockadestudystudy(Waddington(Waddington
et al. et2005).
al. 2005).
To examine
To examine
the thefor Tgfor
The BAC4-tTA
The BAC4-tTA
and D1R-TRE
and D1R-TRE
DNAsDNAs
were independently
were independently
mi- mineuralneuralmechanismmechanismof theofmotor
the motor
deﬁcits,
deﬁcits,
we next
we recorded
next recorded
neur-neur-2002).2002).
croinjected
into fertilized
into fertilized
mouse
mouse
oocytes
oocytes
isolatedisolatedfrom crosses
from crossesofofonal activity
onal activity
in theinEPN
thein
EPN
awake
in awake
mice,mice,
becausebecausethe EPN
theisEPN
theis thecroinjected
D1R homozygous
knockout
knockout(D1RKO)(D1RKO)
femalefemaleand maleand malemice (Tranmice (Tranmain main
outputoutputnucleusnucleusof theof
BG
the
to BG
theto
thalamusthe thalamus
and theandtargetthe target
ofofD1R homozygous
2008).
al. 2008).
ThreeThreeTg lines
Tg forlines
BAC4-tTA
for BAC4-tTA
and 15
and
Tg 15
lines
Tg forlines
D1Rfor D1RD1R-expressing
D1R-expressing
striatal
striatal
neurons.
neurons.
CorticallyCorticallyevokedevokedinhibition
inhibitionininet al. et
TRE were
independently
independently
generated.
generated.
Each BAC4-tTA
Each BAC4-tTA
Tg lineTgwasline wasthe EPN,
thewhich
EPN, whichis mediatedis mediatedby theby
cortico-striato-EPN
the cortico-striato-EPN
directdirect
path- path-TRE werecrossedwith eachwith each
D1R-TRE
D1R-TRETg line.
Tg Theline.progeny
The progeny
from fromcrossescrossesway, way,
was mostly
was mostly
lost during
lost during
suppression
suppressionof D1Rof expression,
D1R expression,crossedof BAC4-tTA
and D1R-TRE
and D1R-TRE
Tg mice
Tg micewas subjected
was subjected
to analyses
to analysesofofwhereaswhereasspontaneous
spontaneousﬁring ﬁring
rates andratespatterns
and patternsof EPNofneurons
EPN neurons
re- re-of BAC4-tTA
Dox-controllable
expression
expressionof lacZof usinglacZ using
X-gal X-gal
staining
stainingofofmainedmainedunchanged.
unchanged.
TheseThese
results
results
indicate
indicatethat D1R
thatsuppression
D1R suppressionDox-controllable
sections
sections
usingusing
a standard
a standard
methodmethodand expression
and expressionof D1Rof D1R
mostlymostlyblocksblocks
the information
the information
ﬂow throughﬂow throughthe cortico-striatothe cortico-striato-brain brain
in theinstriatum
the striatum
usingusing
westernwesternblottingblottingwith anti-D1R
with anti-D1R
antibody
antibody
EPN direct
EPN direct
pathway
pathway
and reduces
and reduces
spontaneous
spontaneous
motormotor
activity.
activity.
(Sigma).
(Sigma).
Two TgTwolines
Tg lines
harboringharboring
BAC4-tTA
BAC4-tTAand D1R-TRE
and D1R-TRE(desig(designatednated442–43
442–43and 442–112)
and 442–112)
were wereconsistently
consistently
foundfound
to exhibit
to exhibit
Materials
Materials
and and
Methods
Methods
distinct,
distinct,comparable
comparable
expression
expressionof lacZ
ofin
lacZ
theinregion
the region
wherewhereen- endogenous
D1R wasD1Rexpressed
was expressedand Dox-controllable
and Dox-controllable
expression
expression
AnimalAnimalprotocols
protocols
were approved
were approvedby theby
Institutional
the Institutional
AnimalAnimalCare Caredogenous
lacZD1R.
and Therefore,
D1R. Therefore,
the 442–112
the 442–112
line waslineused
was forusedfurther
for furtherand UseandCommittee
Use Committeeof Nationalof National
Institutes
Institutesof Naturalof Natural
Sciences,
Sciences,of lacZofand
analyses
as D1RKD
as D1RKD
mice.mice.
Kitasato
Kitasato
University
University
and Niigata
and Niigata
University,
University,and were
and were
conducted
conductedanalyses
Wild-type(WT) (WT)C57BL/6J
C57BL/6J
and D1RKO
and D1RKO(Tran(Tran
et al. et2008)
al. 2008)
mice miceaccordingaccordingto thetoguidelines
the guidelinesof theofNational
the National
Institutes
Institutesof Healthof Health Wild-type
also usedalso forused
comparison
for comparison
in immunohistochemical,
in immunohistochemical,
west-westGuideGuide
for theforCare
the and
CareUseandofUseLaboratoryof Laboratory
Animals.
Animals.
One toOne
5 miceto 5 micewere wereern andblot,behavioral
and behavioral
analyses.
analyses.
were werehousedhousedin eachin cageeach undercage undera 12-ha light–dark
12-h light–darkcycle cycle(lights(lightsern blot,

2| 4Cerebral| Cerebral
Cortex
Cortexofchronicallythe globus pallidus(GPe),
whichis also forelimb
called theandglo-orofawere
implanted
into
the caudal
blot analysis as described previously (Tran et al. 2008) with minorsegment
Introduction

Data Analysis
Spontaneous discharge rates were calculated from continuous
digitized recordings for 50 s. The following parameters characterizing ﬁring patterns were calculated from the ﬁrst 30 s of thesame recordings: the coefﬁcient of variation (CV) of interspike inElectrophysiology
tervals (ISIs), the burst index (Hutchison et al. 1998; Sano et al.
2013), and the percentage of spikes in bursts detected by the PoisSurgery
Figure 1. Dopamine D1 receptor (D1R) suppression in D1R knockdown (D1RKD) mice with doxycycline (Dox) treatment. (A) Schematic diagram showing the cortico-basal
son surprise method (Legéndy and Salcman 1985; Chiken et al.
We used 4 D1RKD (mouse W, K, T, and O, 20–50 weeks old, males)
ganglia pathway and stimulating (Stim.) and recording (Rec.) sites in the electrophysiological experiments (left), along with a typical response pattern (right) in the2008; Sano et al. 2013) (Poisson surprise value (−log10andand 3 age-matched WT mice for the electrophysiological experiP) ≥2.0;
entopeduncular nucleus (EPN) (homologous to the internal segment of the globus pallidus, GPi) to cortical stimulation (Cx Stim.) with early excitation, inhibition,
ments. Under
anesthesia
withhydrochloride(100hyperdirect,
mg/kg,
minimum numberof spikes
during bursts was3). Autocorrelate excitation,
which aremediated by
the ketaminei) cortico-subthalamo(STN)-EPNii) thecortico-striato-EPN
direct, andiii) cortico-striato-external
pallido(GPe)i.p.)
and xylazine(4–5
mg/kg,
i.p.),
a smallrepresent
U-frameSTN-EPN
indirectpathways,hydrochloride
respectively. Red,blue,
and green
triangles
glutamatergic
excitatory,
GABAergic
dopaminergic
projections,lograms(bin widthof 0.5inhibitory,ms) wereandconstructedfromcontinuous
respectively.
D2Rs, dopamine
D2 receptors;
SNc, substantia
nigra pars compacta.
Schematic
diagramrecordingsof Dox-regulated
D1Rs.expression in D1RKD mice. Before Dox
polyacetal
head holder
was ﬁxed
to the exposedskull of (B)thedigitized
for 50treatment(left), withtetracyclinetransactivator(tTA) resinbinds to(forthe tetracycline
responsive
element (TRE), and D1Rs and lacZ are transcribed. Dox treatment interferes withmouse
transparent
acrylic
details, see
ChikenResponses to cortical stimulation were examined by contTA binding to TRE (right), and suppresses D1R and lacZ expression (Tet-off system). (C) D1R immunoreactivity in the striatum (Str) of wild-type (WT, top), D1R
et al. 2008; Sano et al. 2013). After recovery from the ﬁrst surgery,structing peristimulus time histograms (PSTHs; bin width ofknockout (D1RKO, middle), and D1RKD (before Dox treatment, bottom) mice shown in frontal sections. The pattern of D1R expression in D1RKD mice was similar tolightanesthesia
with ketaminehydrochloride(30–50mg/
1 ms) for100areas
stimulus
trials.
The meanvalue (left)
andandSDshownof the disthat in under
WT mice,whereasD1R immunoreactivity
was not
observed in D1RKO
mice.
The dorsolateral
motorof the Str
are deﬁned
by rectangles
kg,magniﬁcationi.p.), a portionof Scale
the skull
theright.
motor
chargerate during
100-msperiodat higher(right).
bars, 1was
mm removed
on the left,to100accessμm on the(D) Western
blot analysisof D1Rthe
proteinexpression
in preceding
the striatumtheof astimulation
D1RKO
EPN, of
and
GPe.
Two
pairs
of before,
bipolarstimulatingmousecortex,(n = 1, number
miceused),
D1RKD
mice
during,and after electrodes
Dox treatment (Days0, 7, 14,21, 28,calculateddifferent daysfor
after
starting
Dox treatment;
After, 7 days
onset
were
each
PSTH
and consideredas theafter cessation of Dox treatment for 14 days; n = 3–4 per condition), and WT mice (n = 3).

－ 316 －

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tated at 4 rpm, and then was accelerated at a constant rate from4 to 40 rpm over 4 min, with the ﬁnal speed maintained for 1 min.
The time spent on the rotarod was measured in 3 trials every day
between 1:00 PM and 7:00 PM and averaged.

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in rodents.
Such differential
dopaminecial regionsof the motor
cortex (for effects
details,ofsee
Chiken et al.
modiﬁcations. Brieﬂy, after euthanasia by cervical dislocation,bus pallidus
In the basal ganglia (BG), dopaminergic transmission plays a piv2008).
the striatum was dissected and homogenized in lysis buffer.throughD1Rs and D2Rs were originally proposed based on
otal role in the control of voluntary movements and motor learnTotal lysates were resolved with 10% SDS-PAGE and transferredchanges in gene expression, glucose utilization, and receptor
ing (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Graybiel
Recordingofpathways
Neuronal Activity
in the Awake
State
to a polyvinylidene diﬂuoride membrane (Millipore). The mem-binding
in these
under conditionsof dopamine
deple2005; Joshua et al. 2009; Enomoto et al. 2011). Dopamine deﬁAfter fullthesurgery,
neuronal
recordingbrane was blocked with 5% skim milk and incubated with anti-tion (Gerfen
et recovery
al. 1990; from
Hirschetsecond
al. 2000)
and havebeen reinciency, as occurs in Parkinson’s disease (PD), results in severe(fordetails,see Chiken et al.studies
2008; Sanoet al. 2013).
D1R (1:5000; Sigma) or anti-actin (as a protein loading control;forcedwas
by started
recent in
vitro
electrophysiological(Surmeiermotor and nonmotor dysfunctions including bradykinesia, rigid1:500; Sigma) antibodies, followed by incubation with secondaryet al. The
kept quiet etin al.
a stereotaxic
2007;awake
Day et mouseal. 2008;was
Flores-Barrera
2011; Gerfenapparatus
andity, tremor, autonomic abnormalities, cognitive dysfunction, andwith2011;
its head
restrained
painlessly
using the U-frame
antibodies conjugated to horseradish peroxidase (Sigma). SignalsSurmeier
Planertet al. 2013).
The striato-EPN/SNr
direct head
depression (Fahn et al. 2011; Seppi et al. 2011). Dopaminergic inholder. A glass-coated Elgiloy-alloy
microelectrodewas inserted
were developed with an ECL detection kit (GE Healthcare). Theand striato-GPe-subthalamo(STN)-EPN/SNr
indirect pathways
puts from the substantia nigra pars compacta terminate in the
intothe
EPN/GPethroughthe dura mater.
Unit activity
density of the bands was determined with CS Analyzer softwareplay vertically
oppositeroles
incontrolling
movements.
The
signals
striatum and are thought to differentially modulate the excitabilwas
isolated
andconvertedtodigitalpulsesusingawindow(Atto).
through the direct pathway reduce activity of the EPN/SNr and in- disity of 2 types of striatal projection neurons through different recriminator. Spontaneous discharges and responses to the corcrease thalamocortical activity via disinhibition, resulting in receptors (Albin et al. 1989; DeLong 1990; Mallet et al. 2006)
tical stimulation (200-μs duration, single pulse, 50-μA strength)
leaseof movements. In contrast, signals through the indirect
Behavioral
Analyses(Fig. 1A, left). One is excitatory effects through dopamine D1 rethrough the electrodes implanted in the motor cortex were repathway
increase activity of the EPN/SNr, resulting in suppresceptors(D1Rs)
onage-matcheddirect pathway
that(7–28
directly
project
D1RKD
and
WTneuronsmale miceweeksold) werecorded in the EPN/GPe from the same mouse in 3 conditions: besion of movements (Albin et al. 1989; DeLong 1990; Gerfen et al.
to theused
outputnuclei
of
the
BG,
the
entopeduncular
nucleusfor behavioral analyses. D1RKD and WT mice were randomfore, during, and after Dox treatment. We ﬁrst recorded neuronal
Mink 1996; Nambu 2007; Kravitz et al. 2010; Sano et al.
(EPN),lywhich
is homologous
to theandinternal
segmentof thedividedinto Dox-treateduntreatedgroups.
Allglobus
behavioral1990; activity
before Dox treatment (“before” condition) and then2013). Thus, the loss of dopaminergic inputs to both pathway
pallidusin primates,
substantia
data(GPi)
are presented
as andthe mean± SD. nigra pars reticulata
started Dox treatment. Recording of neuronal activity was reneurons is considered to increase ﬁring rates of EPN/SNr neurons(SNr). The other is inhibitory effects through D2 receptors
sumed 5 days after starting Dox treatment and continued till
(D2Rs) on indirect pathway neurons that project to the external
through the inhibitory striato-EPN/SNr direct and net excitatory
22 days after starting Dox treatment (“during Dox” condition)
Spontaneous Motor Activity
when D1R expression was greatly suppressed and spontaneous
We assessed spontaneous motor activity of 9 Dox-treated D1RKD,
motor activity was distinctly decreased (Figs 1D, 2A). Finally, we
4 untreated D1RKD, and 5 Dox-treated WT mice. The mice were
stopped Dox treatment and resumed recording 15 days after cesindividually housed in an 11.8 cm (L) × 20.8 cm (W) × 14.5 cm (H)sation of Dox treatment (“after” condition) when D1R expression
home cage, and movements of each mouse were detected before,
and spontaneous motor activity had fully returned to the level
during, and after Dox treatment with a pyroelectric infrared senprior to Dox treatment (Figs 1D and 2C). During the recording sessor installed above the cage (O’hara) as reported previously
sion, we carefully monitored vigilance state of the mouse by vis(Nakamura et al. 2014). Movements were continuously counted
ual inspection.
in 10-min bins, and spontaneous motor activity per day was calculated as the cumulative number of movements in 24 h beginHistology
ning at 8:00 AM. The bedding was replaced every 7 days, andIn the ﬁnal experiment, several sites of neuronal recording weremotor activity was increased after bedding replacement because
marked by passing cathodal DC current (20 μA for 30 s) throughof exploratory behavior. Thus, data on these days were excluded
the recording electrodes. The mice were anesthetized deeply
when calculating the mean weekly spontaneous motor activity.
Spontaneous motor activity was classiﬁed into 3 levels basedwith sodium pentobarbital (120 mg/kg, i.p.) and perfused transcardially with 0.1 M PB ( pH 7.3) followed by 10% formalin in
on counts per 10 min: inactive (≤9), low (10–199), and high
0.1 M PB, and then 0.1 M PB containing 10% sucrose. The brains(≥200). We also assessed spontaneous motor activity of anotherwere removed immediately and saturated with the same buffer
4 Dox-treated D1RKD and 4 Dox-treated WT mice until 10 days
containing 30% sucrose. They were cut into frontal 50-μm-thickafter cessation of Dox treatment for 14 days.
sections on a freezing microtome. The sections were mounted
onto gelatin-coated glass slides, stained with 0.7% neutral red,
Rotarod Testdehydrated, and coverslipped. The sections were observed
We used another 13 Dox-treated D1RKD, 5 untreated D1RKD, 8
under a light microscope, and the recording sites were reconDox-treated WT, and 15 untreated WT mice for the rotarod test.
structed according to the lesions made by current injection andDox treatment was started 35 days before the test and continued
traces of electrode tracks. The sites of stimulation in the motor
during the test periods for Dox-treated groups. Mice were placedcortex were also examined histologically.
on a rotating rod (32 mm diameter, O’hara), which initially ro-

Figu

D1R
(gra

mot

repr

trea
(Bon

27) D

inac

Spo
repl

trea

num

Role ofRole
Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al. et| al.3 | 5

Animal protocols were approved by the Institutional Animal Careand Use Committee of National Institutes of Natural Sciences,
Kitasato University and Niigata University, and were conductedaccording to the guidelines of the National Institutes of Health
Guide for the Care and Use of Laboratory Animals. One to 5 mice
were housed in each cage under a 12-h light–dark cycle (lights
on at 8:00 AM) and given food and drinking water ad libitum.

Dox Treatment
Generation of Conditional and Reversible D1RKD Mice

Dox (2.0 mg/mL) was mixed in drinking water containing 5% sucrose and delivered to the D1RKD and WT mice through a water
We developed D1RKD mice in which D1Rs can be conditionallybottle.
and reversibly regulated by Dox using the Tet-Off system after deletion of the endogenous D1Rs (Fig. 1B). To generate tetracyclinetransactivator-VP16 (tTA)-expressing mice using the D1R proImmunohistochemistry
moter (transactivator line), bacterial artiﬁcial chromosome(BAC) clones containing mouse D1R were modiﬁed with a 2-step
We used 4 D1RKD, 3 WT, and 1 D1RKO mice for immunohistoRec A strategy for BAC modiﬁcation (Yang et al. 1997). A modiﬁcachemical analysis. Mice were deeply anesthetized by injectionof tribromoethanol (400 mg/kg body weight, i.p.) and perfusedtion cassette that introduces the tTA into the endogenous D1R
coding sequence was constructed (see Supplementary Fig. 1A).
transcardially with 4% paraformaldehyde in phosphate buffer
A DNA fragment (0.92 kb, A-arm) containing the upstream region(PB, pH 7.4). The brains were postﬁxed overnight at 4°C, transof the translation initiation site of D1R was ligated to the DNA
ferred to a 30% sucrose solution, immersed in OCT compoundfragment (1.5 kb) containing the coding sequence of the tTA(Sakura Finetek), frozen, and stored at −80°C until use. Frontal
sections were cut at 25-μm thickness and stored in PBS at 4°C.
and the SV40 poly A signal ( poly A) of the pTet-off vector (ClonFigure 2. Spontaneous motor activity and rotarod performance during D1R suppression. (A) Spontaneous motor activity of Dox-treated D1RKD (D1RKD Dox (+)), untreatedtech), and the resulting fragment was ligated to the DNA fragFree-ﬂoating sections were incubated with 1% bovine serum alD1RKD (D1RKD Dox (−)), and Dox-treated WT (WT Dox (+)) mice in their home cages. Dox treatment was started on Day 0 and continued for 4 weeks for Dox-treated groups
ment(gray(0.96
kb,
B-arm)
of
exon
2
of
D1R
to
generate
D1Txx,
thebumin
containing
antibody
for D1R
bar). Daily changes in spontaneous motor activity during Dox treatment were observed.
Bedding was
replaced every
7 days(1:1000;(Days 0, 7,Frontier
14, and 21),Institute)and spontaneous
modiﬁcationcassette(3.38 kb).
Becausethe tTAbehavior.
and theSV40
overnight
at 4°C.
D1Rfrom
antibody
visualizedusing the areas
motor activity
was increased
becauseof exploratory
Thus,
data on thesedays were
excludedfurther binding
analyses. wasColoredlines and light-coloredand ± SD,and ﬁlled
circles andrepresentmean weekly
spontaneous Elite
motor ABC
activityand ±SD.(Vector*P < 0.05; signiﬁcantly
different
frompoly Arepresent
signal mean
sequenceswereintroduced
atwhiskers
the initiation
codon
Vectastain
SystemLaboratories)
and
3, before
3′- Doxtreatment(Bonferroni∫ P < 0.05, signiﬁcantly
different fromcontrol (WT
Dox (+) and D1RKD Dox (−) mice), which is indicated by the corresponding color
diaminobenzidine.
of D1R,the expressionof test).
endogenous
D1R was disrupted.
To gentest). (B) Classiﬁcations of spontaneous motor activity of WT Dox (+), D1RKD Dox (−), and D1RKD Dox (+) mice before (Days –6 to –1) and during (Days 22–
erate (Bonferroni
the pSV1-RecA-D1T
shuttle vector, the DNA fragment of27) Dox treatment. Spontaneous motor activity was classiﬁed into 3 levels based on counts per 10 min: inactive (≤9), low (10–199), and high (≥200). *, the percentage ofD1Txx was inserted into the pSV1-RecA vector
to facilitate hominactive (≤9) time signiﬁcantly −increased (χ 2 test with Bonferroni correction, Western
P = 0.008), andthatAnalysis
of high-active (≥200) time signiﬁcantly decreased (P = 0.0004). (C)Blotologousrecombination
in RecA
Escherichia
coli.WT Dox (+) mice after cessation of Dox treatment. The 2-week Dox treatment was stopped on Day 0. Bedding was
Spontaneous
motor activityof D1RKD
Dox (+) andThe
BAC clone,(80 230
Genetics),
was of D1RKD
We used
19D1RKD
D1RKD
(3–4
each
0, 7, 14,anddays
replacedevery 7BAC4-D1R
days (Days –14,
–7, 0,bp)
and(Research7). (D) Rotarod
performanceDox (+),
Dox(−),inWT
Dox condition,(+), and untreated
WT21,
(WT
Dox28
(−))
mice. Dox−†treatment
was started 35Fig.
days1B).
beforethe rotarod
P < 0.01,
control,which
is indicatedandby thecolor (Bonferronitest). The
used (seeSupplementary
RecA
afterfromstartingDox
treatment
7 correspondingdays after cessationof DoxE. coli test.
carrying
thesigniﬁcantly
origin- different
numbermice transformed
used for each experimentindicated by n.
treatment for 14 days), 3 WT, and 1 D1RKO mice for western
al BAC
cloneofwerewith theispSV1-RecA-D1T
shuttle－ 317 －

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Materials and Methods

vector. The resulting clones were subjected to Southern analysis
using either a 5′ probe or tTA probe to conﬁrm proper recombination (see Supplementary Fig. 1A). The BAC transgenic (Tg) construct, BAC4-tTA, was obtained (see Supplementary Fig. 1B).
The Tg construct, D1R-tetracycline responsive element (D1RTRE), was generated for tetracycline operator (tetO)-target Tg
mouse lines using the following DNA fragments (see Supplementary Fig. 1C): a 9.7-kb fragment including the entire D1R coding sequence, a 0.64-kb fragment encoding the TRE and bidirectional
promoter from the pBI Tet vector (Clontech), a 3466-bp fragment
encoding lacZ from the pBI-GL Tet vector (Clontech), a 0.98-kb
fragment containing rabbit beta-globin poly A and SV40 poly A
signals from the pBstN plasmid, and a 0.3-kb fragment encoding
the chicken beta-globin insulator sequences from the plasmid
pUC19 INS240-SNNS.
The BAC4-tTA and D1R-TRE DNAs (10 ng/μL each) were used
for Tg mouse generation using standard techniques (Nagy et al.
2002). The BAC4-tTA and D1R-TRE DNAs were independently microinjected into fertilized mouse oocytes isolated from crosses of
D1R homozygous knockout (D1RKO) female and male mice (Tran
et al. 2008). Three Tg lines for BAC4-tTA and 15 Tg lines for D1RTRE were independently generated. Each BAC4-tTA Tg line wascrossed with each D1R-TRE Tg line. The progeny from crosses
of BAC4-tTA and D1R-TRE Tg mice was subjected to analyses of
Dox-controllable expression of lacZ using X-gal staining of
brain sections using a standard method and expression of D1R
in the striatum using western blotting with anti-D1R antibody(Sigma). Two Tg lines harboring BAC4-tTA and D1R-TRE (designated 442–43 and 442–112) were consistently found to exhibit
distinct, comparable expression of lacZ in the region where endogenous D1R was expressed and Dox-controllable expressionof lacZ and D1R. Therefore, the 442–112 line was used for further
analyses as D1RKD mice.
Wild-type (WT) C57BL/6J and D1RKO (Tran et al. 2008) mice
were also used for comparison in immunohistochemical, western blot, and behavioral analyses.

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striato-GPe-STN-EPN/SNr indirect pathways, resulting in the decreased motor activity seen in PD (Albin et al. 1989; DeLong 1990).
However, the exact role of D1R-mediated dopaminergic transmission in vivo is not well understood. Contradicting results havebeen observed following D1R blockade. D1R knockout (KO) miceshow increases in spontaneous locomotor activity, althoughpharmacological blockade of D1Rs induces locomotor suppression in agreement with the above explanation (Waddington
et al. 2005). In the present study, we developed novel D1R knockdown (D1RKD) mice in which the D1Rs can be conditionally andreversibly regulated by doxycycline (Dox) treatment (Fig. 1B). The
D1RKD mice enabled us to examine motor behaviors and neuronal activities in the cortico-BG pathways in the presence and absence of D1Rs in the same mice. The mice exhibited decreased
spontaneous motor activity and impaired motor ability when
D1R expression was suppressed, consistent with the pharmacological blockade study (Waddington et al. 2005). To examine theneural mechanism of the motor deﬁcits, we next recorded neuronal activity in the EPN in awake mice, because the EPN is themain output nucleus of the BG to the thalamus and the target of
D1R-expressing striatal neurons. Cortically evoked inhibition in
the EPN, which is mediated by the cortico-striato-EPN direct pathway, was mostly lost during suppression of D1R expression,
whereas spontaneous ﬁring rates and patterns of EPN neurons remained unchanged. These results indicate that D1R suppression
mostly blocks the information ﬂow through the cortico-striatoEPN direct pathway and reduces spontaneous motor activity.

2| 6Cerebral| Cerebral
Cortex
Cortexthe globus
palliduswhich
also called
gloandofinactive
times
were(GPe),observed
inisneither
WTthe
Dox
(+) norbaseline discharge rate. Changes in neuronal activity in responsesegment
Introduction

We developed D1RKD mice in which D1Rs were conditionally andreversibly regulated by Dox using the Tet-Off system (Fig. 1B, seealso Materials and Methods). We ﬁrst examined the distributionof D1Rs in D1RKD mice immunohistochemically (Fig. 1C) using
D1R antibodies. The speciﬁcity of the antibodies was examined
in WT C57BL/6J mice and D1RKO mice (Fig. 1C, WT and D1RKO).
D1Rs were expressed at a high level in the striatum including
the ventral striatum and at moderate levels in the cortex of
D1RKD mice (Fig. 1C, D1RKD). The distribution of D1Rs was similar to that of WT mice (Fig. 1C, WT) and that reported previously(Fremeau et al. 1991; Weiner et al. 1991; Gaspar et al. 1995). We
next quantitatively evaluated D1R expression in the striatum of
D1RKD mice before, during, and after Dox treatment using western blot analysis (Fig. 1D). Before Dox treatment, D1R expression
in the striatum was 27-fold higher than that in WT mice (Fig. 1D,
Day 0 of D1RKD and WT). Dox treatment rapidly and completely
suppressed D1R expression (Day 7, 27.1 ± 4.2%; Day 14, 3.1 ± 1.4%;
Days 21 and 28, undetectable). After cessation of Dox treatment,
D1R expression recovered to the level prior to Dox treatment over
7 days (Fig. 1D, After).

127 and 89 EPN neurons before and during Dox treatment, respectively, in 4 awake D1RKD mice. EPN neurons continuously
and irregularly ﬁred at a high discharge rate (53.9 ± 14.8 Hz) before
Dox treatment (Table 1, Fig. 3A1) as observed in WT mice (Chiken
et al. 2008). Dox treatment did not change either the ﬁring rate
(54.2 ± 13.5 Hz; Table 1; 1-way ANOVA, P = 0.93) or pattern (Table 1,
Fig. 3A2). The CV of ISIs, the burst index, and percentage of spikes
in bursts, which characterize ﬁring patterns, also did not change.
These results indicate that D1R suppression had little inﬂuenceon the spontaneous activity of EPN neurons. More than 15 days
after the cessation of Dox treatment, we recorded 36 EPN neurons
and found that the ﬁring rates and percentage of spikes in bursts
remained unchanged, whereas the CV of ISIs and burst index
were increased (Table 1; 1-way ANOVA with Tukey’s post hoc test,
P < 0.001 for the CV of ISIs and burst index).
For comparison, we also examined the activity of GPe neurons
in the same 4 D1RKD mice, because striato-GPe neurons are assumed to express D2Rs, not D1Rs (Fig. 1A). We found no signiﬁcant differences in the ﬁring rate or pattern among the 3
conditions (Table 1; 1-way ANOVA, P = 0.84 for the ﬁring rate,

Table 1 Spontaneous ﬁring rates and patterns of EPN and GPe neurons
before, during, and after Dox treatment in D1RKD mice

Motor Behaviors During D1R Suppression

BeforeDuring Dox

After

Before Dox treatment, D1RKD mice showed normal behaviors(5–22 days afterand similar spontaneous motor activity to WT mice (Days −6 tostarting Dox−1 in Fig. 2A), despite the higher D1R expression in D1RKD
treatment)
mice. Dox treatment signiﬁcantly decreased spontaneous
EPN
motor activity in D1RKD mice from the ﬁrst week (Days 1–6 of
No. of neurons
127
89
36
D1RKD Dox (+) in Fig. 2A) and monotonically decreased their acFiring
rate
(Hz)
53.9
±
14.8
54.2
±
13.5
53.8 ± 19.0
tivity during the second, third, and fourth weeks. Dox had little
CV of ISIs
0.53 ± 0.15* 0.49 ± 0.15#
0.65 ± 0.19*,#
effect on WT mice (WT Dox (+)), and spontaneous motor activity
Figure 1. Dopamine D1 receptor (D1R) suppression in D1R knockdown (D1RKD) mice with doxycycline
treatment. (A) Schematic
diagramshowing
Burst(Dox)index
1.33 ± 0.36*
1.23± 0.30#the cortico-basal
1.91 ± 1.19*,#in untreated D1RKD mice (D1RKD Dox (−)) was also unchanged
ganglia pathway and stimulating (Stim.) and recording (Rec.) sites in the electrophysiological experiments(left), (%)
along withpattern (right)1.16
in theSpikes in bursts
0.82 a± typical2.02 response
0.69 ± 1.27
± 1.70(repeated-measures 2-way ANOVA, P = 0.0024 for genotype–day
entopeduncular nucleus (EPN) (homologous to the internal segment of the globus pallidus, GPi)
GPeto cortical stimulation (Cx Stim.) with early excitation, inhibition, andinteraction;
test,
0.013 for Days (STN)-EPN
1–6, P < 0.01
forlate excitation,
which Bonferroni
are mediated by
the P
i) =
cortico-subthalamo
hyperdirect,ii) cortico-striato-EPN direct, and iii) cortico-striato-external pallido (GPe)No. of neurons
120
100
38
Days
8–13,
15–20 and
22–27). Finally,in thesponSTN-EPN
indirectpathways,respectively.
Red, blue,theand difference
green triangles
represent
glutamatergic excitatory, GABAergic inhibitory, and dopaminergic projections,Firing rate (Hz)51.0 ± 15.8
51.1 ± 15.5
52.9 ± 16.4
respectively.
D2Rs,motor
dopamine
D2 receptors;
SNc, substantia
nigra pars
Schematic diagram of Dox-regulated D1R expression in D1RKD mice. Before Doxtaneousactivityof D1RKD
Dox (+) miceandcompacta.
that of (B)WT
CV of ISIs
0.63 ± 0.21
0.68 ± 0.24
0.69 ± 0.15
treatmenttetracyclinetransactivator(tTA)binds to signiﬁcantthe tetracycline
element (TRE), and D1Rs and lacZ are transcribed. Dox treatment interferes with
Dox(left),(+) and
D1RKD
Dox (−) mice
becamein responsive
the fourth
Burst
index
1.61
±
1.08
1.54
±
0.47
1.69
± 0.43tTA binding to TRE (right), and suppresses D1R and lacZ expression (Tet-off system). (C) D1R immunoreactivity in the striatum (Str) of wild-type (WT, top),
D1R
week (Days 22–27) (Bonferroni test, P < 0.01). Decreased spontanSpikes inThebursts(%)of D1R
1.43
± 3.17 in1.12± 1.66
1.33 ±
knockout (D1RKO, middle), and D1RKD (before Dox treatment, bottom) mice shown in frontal sections.
pattern
expression
D1RKD
mice was similar
to1.80motor
activity
D1RKD Dox (+)
mice
was dueinto
an increasethat ineous
WT mice,whereasD1R inimmunoreactivity
was
not observed
D1RKO
mice. The dorsolateral motor areas of the Str are deﬁned by rectangles (left) and shownin magniﬁcation
inactive timeand Scalea decreasein highly
activetime
2B; (D)χ 2 Western*,#P <blot
0.01,analysis
signiﬁcantly
differenteach other
ANOVAofwith
Tukey’s postat higher(right).
bars, 1 mmon the left,
100 μmon (Fig.
the right.
of D1R
proteinfrom
expressionin (1-waythe striatum
a D1RKO
hoc
mousetest(n = 1,withnumberof mice used),
D1RKD mice
during,after Dox0,test).
7, 14, 21, 28, different days after starting Dox treatment; After, 7 days
Bonferroni
correction,
P =before,
0.008);
suchandchanges
intreatment
active (Daysafter cessation of Dox treatment for 14 days; n = 3–4 per condition), and WT mice (n = 3).

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Biochemical Characteristics of D1RKD Mice

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in rodents.
of dopamine
D1RKD Dox(−) mice.Such
Afterdifferential
cessation ofeffects
Dox treatment,
spontanto cortical stimulation were judged signiﬁcant if the dischargebus pallidus
In the basal ganglia (BG), dopaminergic transmission plays a piveousD1Rs
motor
activity
increased
and thenreturnedtorate during at least 2 consecutive bins (2 ms) reached a signiﬁ-throughand
D2Rstemporarily
were originallyproposedbasedonotal role in the control of voluntary movements and motor learnthe innormalover 7 days
(Fig. utilization,
2C).
cance level of P < 0.05 (Nambu et al. 2000; Chiken et al. 2008; Ta-changes
gene level
expression,
glucoseand receptor
ing (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Graybiel
chibana et al. 2008; Sano et al. 2013). The latency of eachbinding in
Motor
was also
impaired
duringDox treatment
as evitheseability
pathwaysunderconditionsof dopamine
deple2005; Joshua et al. 2009; Enomoto et al. 2011). Dopamine deﬁresponse was deﬁned as the time at which the ﬁrst bin exceededtion (Gerfen
denced etbyal.
therotarod
test.
timeandspent
onbeenthe rotarod
1990;
Hirschet Theal. 2000)haverein- byciency, as occurs in Parkinson’s disease (PD), results in severe
this level. The responses were judged to end when 2 consecutiveforcedD1RKD
Doxin(+)vitro
miceelectrophysiological
was signiﬁcantly shorter
thanthat for WT
by recentstudies(Surmeiermotor and nonmotor dysfunctions including bradykinesia, rigidbins fell below the signiﬁcance level. The end point was deter-et al. Dox
WT(+), and
D1RKD Doxet(−)
2D; repeated2007;(−),
Day
et Doxal. 2008;
Flores-Barrera
al.mice2011;(Fig.
Gerfenandity, tremor, autonomic abnormalities, cognitive dysfunction, andmined as the time at which the last bin exceeded this level. TheSurmeier
measures
ANOVA,
P < 0.0001
genotype–day interaction;
2011; 2-way
Planertet al. 2013).
Theforstriato-EPN/SNr
directdepression (Fahn et al. 2011; Seppi et al. 2011). Dopaminergic inamplitude of each component of cortically evoked responses wasand striato-GPe-subthalamo
Bonferroni test, P < 0.01).(STN)-EPN/SNr indirect pathways
puts from the substantia nigra pars compacta terminate in thedeﬁned as the number of spikes during the signiﬁcant responseplay opposite roles in controlling movements. The signals
striatum and are thought to differentially modulate the excitabilminus that of the baseline discharge in the PSTH (i.e., the areathrough the direct pathway reduce activity of the EPN/SNr and inity of 2 types of striatal projection neurons through different reof the response; positive and negative values indicate excitation
creaseSpontaneousthalamocorticalactivityofvia
disinhibition,
resulting During
in reActivity
EPN
and GPe Neurons
ceptors (Albin et al. 1989; DeLong 1990; Mallet et al. 2006)
and inhibition, respectively). If no signiﬁcant changes werelease D1R
of movements.
In contrast, signals through the indirect
Suppression(Fig. 1A, left). One is excitatory effects through dopamine D1 refound, the amplitude was set to zero. For population PSTHs, thepathway increase activity of the EPN/SNr, resulting in suppresceptors (D1Rs) on direct pathway neurons that directly project
To assess the mechanism of the decreased motor activity in
PSTH of each neuron with a signiﬁcant response was averagedsion of movements (Albin et al. 1989; DeLong 1990; Gerfen et al.
to the output nuclei of the BG, the entopeduncular nucleus
D1RKD mice following D1R suppression, we examined neuronaland ﬁltered with a Gaussian ﬁlter (σ = 1.6 ms). All electrophysio1990; activity
Mink 1996;
Nambu
2007; Kravitz
et motor-relatedal. 2010; Sano area
et al.of the(EPN), which is homologous to the internal segment of the globus
in awake
conditionsin thelogical data are presented as the mean ± SD.
2013).EPN
Thus,
the lossof 2008),
dopaminergic
to bothpathway
pallidus (GPi) in primates, and substantia nigra pars reticulata(Chikenet al.
which is inputs
the targetof D1R-expressing
neurons
is considered
increase
ﬁring
rates of
EPN/SNr(SNr). The other is inhibitory effects through D2 receptors
striatal
neurons toandthe mainoutputnucleusof neurons
the BG to the(D2Rs)Results
on indirect pathway neurons that project to the external
throughthe inhibitory
striato-EPN/SNr
directspontaneousand net excitatory
thalamus(Fig. 1A).
We ﬁrst recorded
activity of

Figu

and

P=
the

Cor
Du

Aft
am
of t
et a
al a
vol
San
(75
tex
com
cita
ple
mic
Dox
Tab
are
cha
mo
of e
biti
ex,
24%
cha
hib

Role ofRole
Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al. et| al.3 | 7

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－ 319 －

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vector. The resulting clones were subjected to Southern analysis
using either a 5′ probe or tTA probe to conﬁrm proper recombination (see Supplementary Fig. 1A). The BAC transgenic (Tg) construct, BAC4-tTA, was obtained (see Supplementary Fig. 1B).
The Tg construct, D1R-tetracycline responsive element (D1RTRE), was generated for tetracycline operator (tetO)-target Tg
mouse lines using the following DNA fragments (see Supplementary Fig. 1C): a 9.7-kb fragment including the entire D1R coding sequence, a 0.64-kb fragment encoding the TRE and bidirectional
promoter from the pBI Tet vector (Clontech), a 3466-bp fragment
encoding lacZ from the pBI-GL Tet vector (Clontech), a 0.98-kb
fragment containing rabbit beta-globin poly A and SV40 poly A
signals from the pBstN plasmid, and a 0.3-kb fragment encoding
the chicken beta-globin insulator sequences from the plasmid
pUC19 INS240-SNNS.
The BAC4-tTA and D1R-TRE DNAs (10 ng/μL each) were used
for Tg mouse generation using standard techniques (Nagy et al.
2002). The BAC4-tTA and D1R-TRE DNAs were independently microinjected into fertilized mouse oocytes isolated from crosses of
D1R homozygous knockout (D1RKO) female and male mice (Tran
et al. 2008). Three Tg lines for BAC4-tTA and 15 Tg lines for D1RTRE were independently generated. Each BAC4-tTA Tg line wascrossed with each D1R-TRE Tg line. The progeny from crosses
of BAC4-tTA and D1R-TRE Tg mice was subjected to analyses of
Dox-controllable expression of lacZ using X-gal staining of
brain sections using a standard method and expression of D1R
in the striatum using western blotting with anti-D1R antibody(Sigma). Two Tg lines harboring BAC4-tTA and D1R-TRE (designated 442–43 and 442–112) were consistently found to exhibit
Materials and Methods
distinct, comparable expression of lacZ in the region where enFigure
3. Spontaneous
activity of EPN
and Institutional
GPe neurons during
D1R suppression.
Digitized spikes(top)
andexpressed
autocorrelograms(bottom) of spontaneous
activity of EPN (A)dogenous
D1R
wasand Dox-controllable
expression
Animalprotocols
were approvedby theAnimalCareand GPe (B) neurons before (1) and during (2) Dox treatment in D1RKD mice are shown.
of lacZ and D1R. Therefore, the 442–112 line was used for furtherand UseCommittee of National Institutes of Natural Sciences,
analyses as D1RKD mice.
Kitasato University and Niigata University, and were conducted
Wild-type (WT) C57BL/6J and D1RKO (Tran et al. 2008) miceaccording to the guidelines of the National Institutes of Healthalso used
for comparison
immunohistochemical,
westdisappearanceof theininhibition
was already observedin= 0.26CVUseof ISIs,P = 0.69 forthe burst
index,= 0.73 forwere The
GuidePforthe forCaretheandof Laboratory
Animals.
One
to 5 Pmiceern blot,
the andﬁrstbehavioral
half (5–14 analyses.
days) of Dox treatment (see Supplementary
percentage
spikesin bursts;
3B).
were thehousedin eachof
cage
under
a 12-hFig.
light–darkcycle (lights
Fig. 3A). Population PSTHs constructed for each mouse (Fig. 4B)
on at 8:00 AM) and given food and drinking water ad libitum.
evidenced that the disappearance of the inhibition was commonCortically Evoked Responses of EPN and GPe Neurons Dox Treatmently observed in all 4 mice. Quantitative analysis showed that theGeneration
Conditional
and Reversible D1RKD Mice
Duringof
D1R
Suppression
durationandamplitude
the inhibition
were markedly
diminDox (2.0mg/mL)
wasmixed inof
drinking
water containing
5% suduring Dox
treatment
2; 1-way
ANOVA awith
Tukey’sand delivered
to the
D1RKD (Table
and WT
mice throughwater
We developed
After recording
D1RKD
spontaneous
mice in which
activity
D1Rsofcan
EPNbe
neurons,
conditionally
we next ex-crose ishedbottle.post hoc test, P < 0.001 for the duration and amplitude). On theand reversibly
amined theregulated
responses
by Doxof theseusingneurons
the Tet-Off
to electricalsystem after
stimulation
deother hand, the latency, duration, and amplitude of the early exletionofofthe
theforelimbendogenous
and orofacial
D1Rs (Fig.
regions
1B). Toofgenerate
the motor
tetracycline
cortex (Chiken
citation remained unchanged. The latency of the late excitationtransactivator-VP16
et al. 2008) (Fig. 4A),(tTA)-expressing
because corticalmicestimulation
using theinduces
D1R proneuronImmunohistochemistry
was decreased, and its amplitude was increased during Dox
moteral (transactivator
activity in the BGline),and mimics
bacterialinformation
artiﬁcial chromosome
processing during
treatment(P <30.001
duration,P < for
0.01immunohistofor the amplitude),(BAC) voluntary
clones containing
movements
mouse(Chiken
D1R were
et al.modiﬁed
2008; Tachibana
with a 2-step
et al. 2008;We used
4 D1RKD,
WT, forandthe1 D1RKOmice
probably
becausediminutionof inhibition bymay
unmask the
Rec ASanostrategy
et foral. BAC2013).
modiﬁcation
Before Dox(Yangtreatment,
et al. 1997).
60% AofmodiﬁcaEPN neuronschemical
analysis.
Micethewere
deeply anesthetizedinjectionlate excitation. (400
Moremg/kg
than 15
daysweight,
after thei.p.)
cessationof Dox treatbody
and perfusedtion cassette(75/125 cells,
that introduces
Table 2) responded
the tTA tointostimulation
the endogenousof the motor
D1R cor-of tribromoethanol
ment, a similar
percentageof neuronsin(73%,
Table 2)buffer
respondedcodingtex.
sequence
The most
wascommonconstructed(64%) (seeresponse
Supplementary
was a triphasic
Fig. 1A).
responsetranscardiallywith 4%paraformaldehyde
phosphate
to 7.4).
the motor
corticalstimulation,and the mostcommonA DNA
composedfragmentof
(0.92
early
kb,excitation,
A-arm) containing
followed theby inhibition
upstream region
and late ex-(PB, pH
The brains
were
postﬁxed overnightat 4°C,trans- (58%)
wasagain triphasic,
was similar
to thatobserved beof thecitationtranslation(ex-inh-ex)
initiation
as observedsite of D1R
in PSTHs
was ligated(Fig. 4A1,
to theleft,
DNA
see Sup-ferredpattern
to a 30%
sucrosesolution,and
immersedin OCT
compoundfore
Dox treatment(Fig.stored
4A3, left,
see Supplementary
Fig. 2A,
fragment
plementary(1.5 kb)Fig.
containing
2A, Before),
thewhichcodingis thesequence
typicalof
response
the tTAin WT(Sakura
Finetek),frozen, andat −80°C
until use. Frontal
After).
Such
recovery
was also andevident
in in
population
PSTHs
were
cut at25-μm thickness
storedPBS at 4°C.
and themice
SV40(Chikenpoly A
etsignalal. 2008,( polyseeA)
also
of Supplementary
the pTet-off vector
Fig.(Clon3B). Duringsections(Fig. 4A3,
right) andquantitative(Table 2).
tech),Dox
andtreatment,
the resulting
a similarfragment
percentage
was ligatedof neurons
to the(72%,
DNA 63/88
frag- cells,Free-ﬂoating
sections
were
incubated analyses
with 1% bovine
serum alWe also examined
corticallyevokedresponses
of GPementTable(0.96 2)kb,responded
B-arm) oftoexon
the motor
2 of D1R
corticalto generate
stimulation
D1Txx,
in thethesamebumin containing
antibody thefor D1R(1:1000;
Frontier
Institute)neuronsin D1R
the same
4 D1RKDmice
comparison.
modiﬁcation
area of thecassette
EPN (Fig.
(3.38
5A);
kb).
however,
Becauseresponse
the tTA patterns
and the SV40
drasticallyovernightat 4°C.
antibody
binding
wasforvisualizedusingThethe most
commoncorticallyevoked(Vector
response
was triphasic
withpoly Achanged.
signal sequences
The inhibition
were introduced
was mostly
at thelostinitiation(Fig. 4A2,
codonleft). TheVectastain
Elite
ABC System
Laboratories)
and3, early
3′- excitation, followed by inhibition and late excitation throughout
diaminobenzidine.
of D1R,mostthe common
expression(65%)
of endogenous
response was
D1Rbiphasic
was disrupted.
excitation
To consisting
genthe 3 conditions (Fig. 4C, left; 67% before, 67% during, and 61%
erate oftheearly
pSV1-RecA-D1T
and late excitation,
shuttleandvector,the percentage
the DNA fragmentof neuronsof exhiafter Dox treatment, see Supplementary Fig. 2B); this is the typD1Txxbiting
was inserted
responses
intowiththeinhibition,
pSV1-RecAsuch
vectoras toex-inh-ex,
facilitateex-inh,hom- inhWestern Blot Analysis
ical response in WT mice (Chiken et al. 2008). In addition, popuologous
ex, recombinationand inh, was signiﬁcantly
in RecA− Escherichia
decreased(before, 84%; during Dox,coli.
2lationPSTHs (3–4
(Fig. in4C,
right)and the0,amplitudeand28duration
of
24%;
The
BACχclone,(80see230 bp)(Research Genetics),
wasTheseWe used
test,BAC4-D1R
P < 0.0001,
Supplementary
Fig. 2A).
19 D1RKD
each
condition,
7, 14, 21, anddays
component(Table 2;
1-way
ANOVA;
excitation,
P = 0.51
used (seeSupplementary
Fig. 1B).
RecA− E.inchanges
were also clearlyobserved
population
PSTHs.
The in-after eachstarting
Dox treatment
and7 daysafterearlycessationof Doxcoli
carrying theoriginfor thefor
duration,
P =30.42
forandthe 1amplitude;
inhibition,
P = 0.67 forhibition
disappeared
duringDox
treatment(Fig. 4A1,
A2, right).treatment
14 days),WT,
D1RKO micefor western
al BAC
clone weretransformed
withthepSV1-RecA-D1T
shuttlestriato-GPe-STN-EPN/SNr indirect pathways, resulting in the decreased motor activity seen in PD (Albin et al. 1989; DeLong 1990).
However, the exact role of D1R-mediated dopaminergic transmission in vivo is not well understood. Contradicting results havebeen observed following D1R blockade. D1R knockout (KO) miceshow increases in spontaneous locomotor activity, althoughpharmacological blockade of D1Rs induces locomotor suppression in agreement with the above explanation (Waddington
et al. 2005). In the present study, we developed novel D1R knockdown (D1RKD) mice in which the D1Rs can be conditionally andreversibly regulated by doxycycline (Dox) treatment (Fig. 1B). The
D1RKD mice enabled us to examine motor behaviors and neuronal activities in the cortico-BG pathways in the presence and absence of D1Rs in the same mice. The mice exhibited decreased
spontaneous motor activity and impaired motor ability when
D1R expression was suppressed, consistent with the pharmacological blockade study (Waddington et al. 2005). To examine theneural mechanism of the motor deﬁcits, we next recorded neuronal activity in the EPN in awake mice, because the EPN is themain output nucleus of the BG to the thalamus and the target of
D1R-expressing striatal neurons. Cortically evoked inhibition in
the EPN, which is mediated by the cortico-striato-EPN direct pathway, was mostly lost during suppression of D1R expression,
whereas spontaneous ﬁring rates and patterns of EPN neurons remained unchanged. These results indicate that D1R suppression
mostly blocks the information ﬂow through the cortico-striatoEPN direct pathway and reduces spontaneous motor activity.

2| 8Cerebral| Cerebral
Cortex
CortexIntroduction

after (3) Dox treatment in D1RKD mice. Raster and peristimulus time histograms (PSTHs; 100 trials; bin width, 1 ms) for typical examples (left) and population PSTHs (right,
with Gaussian ﬁlter) are shown. Stimulation was delivered at time 0 (arrows). The mean ﬁring rate and statistical levels of P < 0.05 (1-tailed t-test) are indicated in PSTHs bya black and white dashed line (mean) and black (upper limit) and white (lower limit) solid lines, respectively. The number of neurons used for population PSTHs isindicated by n, and the shaded areas represent ±SD. (B) Population PSTHs (with Gaussian ﬁlter) of cortically evoked EPN responses constructed for each mouse (mouse
W, K, T, and O). Blue and red lines represent before and during Dox treatment, respectively.

the duration, P = 0.91 for the amplitude; late excitation, P = 0.72 forobserved in WT mice (Chiken et al. 2008). The most common rethe duration, P = 0.83 for the amplitude) were similar throughout
sponse before Dox treatment was a triphasic response composed
the 3 conditions.
of early excitation, followed by inhibition and late excitation
Finally, we examined whether the effects of Dox to EPN(Fig. 5A1). During Dox treatment, cortically evoked responses inneurons described above were speciﬁcally observed in D1RKD
the same area mostly changed to biphasic excitation consisting
mice, but not in WT mice. We recorded neuronal activity in theof early and late excitation (Fig. 5A2). GPe neurons that responded
motor-related area of the EPN in 3 age-matched WT mice before
to motor cortical stimulation were distributed in the middle andlateral parts of the GPe as observed in WT mice (Chiken et al. 2008;
and during Dox treatment. In WT mice, Dox treatment did not
Sano et(Dox)al. 2013).
The
commonresponsewastriphasic bothchangetheD1corticallyevokedresponse
the EPN
as observed
Figure 1.
Dopamine
receptor (D1R)
suppression
in D1R in
knockdown(D1RKD)mice with doxycycline
treatment.
(A)mostSchematic
diagramshowing thecortico-basal
gangliain
pathwaystimulating(Stim.)
and recording(Rec.) sites in theFig.
electrophysiologicalexperiments(left),and
along
with a (Fig.
typicalresponse
pattern (right) in the
before(Fig. 5B1)
during
5B2)
Dox treatment.
PSTHsand
and
population
PSTHs(see Supplementary
3B).
entopeduncular nucleus (EPN) (homologous to the internal segment of the globus pallidus, GPi) to cortical stimulation (Cx Stim.) with early excitation, inhibition, andlate excitation, which are mediated by the i) cortico-subthalamo (STN)-EPN hyperdirect, ii) cortico-striato-EPN direct, and iii) cortico-striato-external pallido (GPe)STN-EPN indirect pathways, respectively. Red, blue, and green triangles represent glutamatergic excitatory, GABAergic inhibitory, and dopaminergic projections,
Locationof Recorded
Neurons
respectively.
D2Rs, dopamine
D2 receptors;
SNc, substantia nigra pars compacta. (B) Schematic diagram of Dox-regulated D1R expression in D1RKD mice. Before Dox

Discussion

treatment (left), tetracycline transactivator (tTA) binds to the tetracycline responsive element (TRE), and D1Rs and lacZ are transcribed. Dox treatment interferes with

Recording sites in the EPN (mouse W) and GPe (mouse K) of
The present study examined motor behaviors and neuronal acD1RKD mice are shown in frontal sections using different symtivity of the BG in the presence and absence of D1Rs using
knockout (D1RKO, middle), and D1RKD (before Dox treatment, bottom) mice shown in frontal sections. The pattern of D1R expression in D1RKD mice was similar to
based
on corticallyevoked response
patterns(Fig. 5).
EPN
novel D1RKD
mice
that
in whichthat in bols
WT mice,whereasD1R immunoreactivity
was not observedin D1RKO
mice.
The dorsolateralmotor areasof theStrweare developed
deﬁned by rectangles(left)D1R
and expression
shown
neurons
that responded
stimulation
ofleft,the100motor
were
canblot
be analysis
conditionallyand reversibly
by Doxtreatment.
at higher
magniﬁcation(right). Scaletobars,
1 mm on theμm oncortex
the right.
(D) Westernof D1R protein
expression regulated
in the striatumof a D1RKO
mousedistributed(n = 1, numberthroughoutof mice used),the
D1RKD
mice before,
during,parts
and afterDox EPN
treatment
0, 7, 14, 21, 28, different
after startingby
DoxDoxtreatment;
After, 7 days
middleand lateralof theas (Days
Suppressionof D1Rdays
expression
treatment
severelytTA binding to TRE (right), and suppresses D1R and lacZ expression (Tet-off system). (C) D1R immunoreactivity in the striatum (Str) of wild-type (WT, top), D1R

after cessation of Dox treatment for 14 days; n = 3–4 per condition), and WT mice (n = 3).

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Figure 4. Cortically evoked responses of EPN and GPe neurons during D1R suppression. Cortically evoked responses of EPN (A) and GPe (C) neurons before (1), during (2), and

Tab
dur

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In the basal ganglia (BG), dopaminergic transmission plays a pivotal role in the control of voluntary movements and motor learning (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Graybiel
2005; Joshua et al. 2009; Enomoto et al. 2011). Dopamine deﬁciency, as occurs in Parkinson’s disease (PD), results in severe
motor and nonmotor dysfunctions including bradykinesia, rigidity, tremor, autonomic abnormalities, cognitive dysfunction, anddepression (Fahn et al. 2011; Seppi et al. 2011). Dopaminergic inputs from the substantia nigra pars compacta terminate in the
striatum and are thought to differentially modulate the excitability of 2 types of striatal projection neurons through different receptors (Albin et al. 1989; DeLong 1990; Mallet et al. 2006)(Fig. 1A, left). One is excitatory effects through dopamine D1 receptors (D1Rs) on direct pathway neurons that directly projectto the output nuclei of the BG, the entopeduncular nucleus(EPN), which is homologous to the internal segment of the globus
pallidus (GPi) in primates, and substantia nigra pars reticulata(SNr). The other is inhibitory effects through D2 receptors(D2Rs) on indirect pathway neurons that project to the externalsegment of the globus pallidus (GPe), which is also called the globus pallidus in rodents. Such differential effects of dopamine
through D1Rs and D2Rs were originally proposed based onchanges in gene expression, glucose utilization, and receptor
binding in these pathways under conditions of dopamine depletion (Gerfen et al. 1990; Hirsch et al. 2000) and have been reinforced by recent in vitro electrophysiological studies (Surmeier
et al. 2007; Day et al. 2008; Flores-Barrera et al. 2011; Gerfen and
Surmeier 2011; Planert et al. 2013). The striato-EPN/SNr direct
and striato-GPe-subthalamo (STN)-EPN/SNr indirect pathways
play opposite roles in controlling movements. The signals
through the direct pathway reduce activity of the EPN/SNr and increase thalamocortical activity via disinhibition, resulting in release of movements. In contrast, signals through the indirect
pathway increase activity of the EPN/SNr, resulting in suppression of movements (Albin et al. 1989; DeLong 1990; Gerfen et al.
1990; Mink 1996; Nambu 2007; Kravitz et al. 2010; Sano et al.
2013). Thus, the loss of dopaminergic inputs to both pathway
neurons is considered to increase ﬁring rates of EPN/SNr neurons
through the inhibitory striato-EPN/SNr direct and net excitatory

EPN
N
re
n
te
Ear
L
D
A
(s
Inh
L
D
A
(s
Late
L
D
A
(s

GPe
N
re
n
te
Ear
L
D
A
(s
Inh
L
D
A
(s
Late
L
D
A
(s

*,#P <

hoc

imp
itio
dire
exp
EPN
D1R
for
to a

Mo
EPN

Bas
exe
thr

Role ofRole
Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al. et| al.3 | 9

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Table 2 Cortically evoked
responses
of EPNresulting
and GPe neurons
before, vector.
1990).
Thus, theloss wereof dopaminergic
inputs throughThe resulting
clonessubjected to Southern
analysis D1Rs
striato-GPe-STN-EPN/SNr
indirectpathways,in the deduring,andafter Doxtreatment
in D1RKD
mice DeLong 1990).
expected
increaseﬁringproperof EPN recombinneurons and reusingwould
either be
a 5′
probe ortotTAprobe mean
to conﬁrm
creasedmotor
activityseen
in PD (Albinet al. 1989;
decreased motor
Increasedmean ﬁring
ation sult(seeinSupplementary
Fig.activity.
1A). The
BAC transgenic(Tg) rates
con- were
However, the exact role of D1R-mediated dopaminergic transBefore
During Dox (5–22 Afterreportedin the GPi
in rodents) ofprimatestruct,originally
BAC4-tTA,
was obtained(see(EPN
SupplementaryFig.
1B). models
mission in vivo is not well understood. Contradicting results havedays after startingof Tg
PD construct,(Miller andD1R-tetracycline
DeLong 1987; Filion
and Tremblay
1991;
Boraudbeen observed following D1R blockade. D1R knockout (KO) miceThe
responsive
element(D1RDox treatment)al. 1998;
Heimerettetracycline
al. 2002; Wichmannet (tetO)-target
al. 2002). However,TRE), etwas
generatedfor
operator
Tg reshow increases in spontaneous locomotor activity, although
centstudiesfailed to detect
the expected
rate increase
mouse
lines
usinghavethe following
DNA fragments(seeﬁring
Supplemenpharmacologicalblockade of D1Rs induces locomotor suppresEPN
in the(Wichmann
et includingal. 1999; Raz
al. 2000;1C):GPia 9.7-kb
fragment
theet
entireD1R Tachibana
coding se- et al.
sion inNo.
agreementwiththe (60%)
above explanation
75/12563/88 (72%) (Waddington
24/33 (73%) tary Fig.
of2011).
Instead,abnormalﬁring patterns,
bursts and oscilquence,
a 0.64-kb
fragment
encoding
the TREsuch
andas
bidirectional
et al. 2005).
In the present study, we developed novel D1R knockresponded
lations,
werein the(Clontech),
BG of PD animals
andpatients (Razpromoterfromthe recorded
pBI Tet vector
a 3466-bp
fragment
down (D1RKD)
miceofin which the D1Rs can be conditionally andneurons/No.
testedneurons
et al.lacZ
2000;
Brown
et al. 2001;
Tachibanaet al. 2011),
and synencoding
fromthe pBI-GL
Tet vector(Clontech),
a 0.98-kb
reversibly
regulatedby doxycycline (Dox) treatment (Fig. 1B). The
Early
excitation
chronous
activationmay
disable thepoly
abilityof individual
fragment
containing
rabbitbeta-globin
A and
SV40 polyneurons
A
D1RKD
mice
enabled us to examine motor behaviors and neuronLatency(ms)cortico-BG
3.7 ±pathways
1.0
3.5± 0.9presence and3.4
to from
process
relay
motor-related
information,in failtheand
pBstN
plasmid,and a 0.3-kb
fragmentresulting
encoding
al activities
in thein theab-± 0.8 signals
Duration± 2.5 The mice
4.5 ±exhibited
2.7
3.8 ± 2.6 the chicken
ure of appropriate(Bergman etal. 1998;
Brown 2007).
beta-globinmovements
insulator sequencesfromthe plasmid
sence of
D1Rs in(ms)the same3.8
mice.
decreased
Amplitude
43.1 ±and30.2impaired
58.1 ±motor
54.0 ability when
64.7 ± 56.4 pUC19
The
present study revealed that spontaneous motor activity in
INS240-SNNS.
spontaneous
motor activity(spikes)theBAC4-tTA
mice wasanddecreasedof D1RThe
D1R-TREduring
DNAs suppression(10 ng/μL each)wereexpression
used
D1R expression was suppressed, consistent with the pharmacoInhibition2) without
anyusing
prominent
effects
on spontaneous
for Tg(Fig.
mousegeneration
standard
techniques(Nagy et al.ﬁring
logicalblockade study (Waddington et al. 2005). To examine theLatency (ms)± 2.5
10.5 ± 2.5
10.3 ± 3.2
in either the
EPN
or GPeDNAs(Tablewere
1, Fig.
3). The resultsmiindicate2002).rates
The BAC4-tTA
and
D1R-TRE
independently
neural mechanismof the11.0
motor
deﬁcits,
we next recorded neurDuration (ms)6.1 ± 5.9*
0.8 ± 1.5*,#
9.1 ± 6.2#
that thedeﬁcits
duringtheisolated
absenceof crosses
D1R-mediatedcroinjected
intomotor
fertilizedmouse
oocytesfromofonal activity in the EPN in awake mice, because,# the EPN is the#
Amplitude−27.4 ± 24.4*−3.2 ± 7.7*
−39.3 ± 33.9
dopaminergictransmission
be explained
simply by
D1R homozygous
knockout(D1RKO)cannot
female andmale mice (Tranmain output nucleus of the BG to the thalamus and the target of(spikes)in spontaneousﬁring ratesandin theEPN.
et al. changes2008). ThreeTg lines for BAC4-tTA
15 Tg
lines for D1RD1R-expressing striatal neurons. Cortically evoked inhibition in
Late excitation
TRE were independently generated. Each BAC4-tTA Tg line was
the EPN, which is mediated by the cortico-striato-EPN
direct pathLatency (ms)19.2 ± 3.8*
14.1 ± 3.2*,#
21.0 ± 5.4#
crossed with each D1R-TRE Tg line. The progeny from crosses
way, was mostly lost during suppression of D1R expression,
Duration (ms)7.7 ± 5.9
9.8 ± 6.1
7.1 ± 7.4
Cortically Evoked Responses of EPN and GPe Neurons
whereasAmplitude
spontaneous ﬁring
rates
and patternsof EPN
neurons
re-± 71.6#of BAC4-tTA and D1R-TRE Tg mice was subjected to analyses of,#54.3± 54.2*92.4 ± 79.3*
51.2
Dox-controllable
expressionof lacZusing
mained(spikes)
unchanged. These results indicate that D1R suppression
The BG receive
inputs froma wideareaX-galof thestaining
cerebralofcortex
brain (Minksectionsusing
a standard
methodandexpressionis
ofprocessed
D1R
mostly blocks the information ﬂow through the cortico-striato1996;
Nambu
et al. 2002).
The
information
GPe pathway and reduces spontaneous motor activity.
in thethroughstriatumthe
usingwestern blottingwith anti-D1R
antibody
EPN direct
cortico-STN-EPN
hyperdirect,
cortico-striato-EPN
No. of
67/114 (59%) 45/82 (55%)
18/30 (60%) (Sigma).
TwoandTg lines
harboring BAC4-tTA andindirect
D1R-TRE(desig- anddirect,cortico-striato-GPe-STN-EPN
pathwaysresponded
natedreaches
442–43theandEPN,
442–112)
werestationconsistently
foundto exhibit
the outputof the BG(Fig. 1A).
During volMaterials
and of
Methods
neurons/No.
distinct,comparable
expression
ofsignals
lacZ in originating
the region inwhere
en- areuntarymovements,
neuronal
the cortex
neurons
dogenous
D1R wasexpressed
and throughDox-controllable
expression
considered
to betransmitted
these pathways
and reach
Animaltested
protocols
were approved by the Institutional Animal Care
Early excitationof lacZthe
and
D1R.
Therefore,
the how
442–112line wasusedoriginating
for furtherin the
EPN.
Thus,
evaluatingneuronal
signals
and UseCommittee of National Institutes of Natural Sciences,
Latency (ms)
3.9 ± 1.3
4.1 ± 1.0
4.0 ± 1.4 analyses
as D1RKD
mice.
motor
cortex are
transmitted through the BG is essential for asKitasato University and Niigata University, and were conducted
Duration (ms)5.4 ± 2.7
6.0 ± 3.5
5.9 ± 1.9
Wild-type(WT)
C57BL/6J and
D1RKO (Tran
et al.
2008) micesessing themechanismof abnormal
motor
behaviors.
Corticalaccording to the guidelines of the National Institutes of Health
Amplitude
80.5 ± 48.7
83.5 ± 40.7
96.3 ± 42.8
were stimulation
also used forinducescomparison
in immunohistochemical,
west-inforneuronal
activity in the BG mimicking
Guide for the Care and Use of Laboratory Animals. One to 5 mice(spikes)ern blot,and behavioral
mationprocessing analyses.
during voluntary movements and providing
were housed in each cage under a 12-h light–dark cycle (lights
Inhibition
important clues for understanding the changes in information
on at 8:00
AM)
and
given
food
and
drinking
water
adlibitum.
Latency (ms)10.8 ± 2.4
11.2 ± 2.6
11.8 ± 1.9
processing through the BG (Chiken et al. 2008; Tachibana et al.
Duration (ms)6.1 ± 5.4
7.3 ± 6.7
6.6 ± 10.6 Dox Treatment
2008; Sano et al. 2013). Cortically evoked responses in the BG
Amplitude−31.8 ±and30.6 Reversible−32.3 ± 24.9
−36.0 ± 74.7
Generationof Conditional
D1RKD Mice
Dox (2.0
was mixed
in indrinking
containing 5%
suaremg/mL)
dramaticallyalteredhyper-waterand hypokinetic
movement(spikes)(Chikenet D1RKD
al. 2008;
Kita
and
Kita
2011; Nishibayashi
crose disorders
and delivered
to theandWT
mice
througha water
We developed
D1RKD mice in which D1Rs can be conditionally
Late excitation
and reversiblybyDoxusing theTet-Off
de-± 3.8 bottle.et al. 2011).
Latencyregulated
(ms)
20.2
± 4.2
19.9
± 4.0 system after
19.8
Before Dox treatment, cortical stimulation induced a triphasic
letion ofDuration
the endogenous
D1Rs(Fig. 1B).17.2
To generate
tetracycline
(ms)
21.6
± 36.0± 25.117.5 ± 16.5
response composed of early excitation, followed by inhibitiontransactivator-VP16(tTA)-expressing
mice± using
pro-± 177.6
196.1 ± 392.0 159.5
292.7 the D1R162.5
Amplitude
Immunohistochemistry
and late excitation in the EPN of D1RKD mice (Fig. 4A1); this ismoter (spikes)(transactivator line), bacterial artiﬁcial chromosome
the typicalresponse
in WT
mice (Chiken
et immunohistoal. 2008, see Supple(BAC) clones containing mouse D1R were modiﬁed with a 2-step
We used
4 D1RKD,
3 WT, and1 D1RKOmice for,#P < 0.01, signiﬁcantly
different from (Yangeach other(1-wayANOVAwith Tukey’s postchemical
mentary
Fig. 3B).
D1RKD
also
showed normal
behaviors and
Rec A*strategy
for BAC modiﬁcation
et al.
1997).
A modiﬁcaanalysis.
Miceweremice
deeplyanesthetizedby injection
hoc test). that introduces the tTA into the endogenous D1Rof tribromoethanol(400 mg/kg
body
weight,i.p.)miceand despite
perfuseda highsimilar spontaneous
motor
activity
to WTtion cassettelevel of D1R
expression,suggesting compensatory
mechanisms,
coding sequence was constructed (see Supplementary Fig. 1A).
transcardiallywith
4% paraformaldehydein phosphatebuffersuch
asThedesensitizationof D1Rs (Staunton
1982)
and deA DNA fragment (0.92 kb, A-arm) containing the upstream region(PB, pH
7.4).
brains were postﬁxed
overnightet
atal.
4°C,
transimpaired motor behaviors in the mice. Cortically evoked inhibof dopamine
release. immersed
Transient increaseof spontaneousof the translation initiation site of D1R was ligated to the DNA
ferredcrease
to a 30%
sucrose solution,
in OCT compound
ition in the EPN, which is mediated by the cortico-striato-EPN
motor
activity
after cessation
of at
Dox
treatment(Fig. Frontal
2C) also sugfragment (1.5 kb) containing the coding sequence of the tTA(SakuraFinetek),frozen,and stored−80°C
until use.
direct pathway, was mostly lost during suppression of D1R
sections
were
cut at 25-μm
thickness andstored in PBS
at 4°C.
gests
involvementof compensatory
mechanisms,
such
as sensiand the SV40 poly A signal ( poly A) of the pTet-off vector (Clonexpression, whereas spontaneous ﬁring rates and patterns oftizationsectionsof D1Rswere
andincubatedincrease withof dopamine
Duringtech), and the resulting fragment was ligated to the DNA fragFree-ﬂoating
1% bovinerelease.
serum alEPN neurons remained unchanged. These results suggest thatsuppression
D1R expression,
corticallyevokedinhibition inment (0.96 kb, B-arm) of exon 2 of D1R to generate D1Txx, thebumincontaining ofantibody
for D1R (1:1000;
Frontier
Institute)
D1R-mediated dopaminergic transmission maintains the inthe EPN
wasD1R
mostlylost (Fig.
4A2,wasTable
2, see Supplementary
modiﬁcation cassette (3.38 kb). Because the tTA and the SV40
overnight
at 4°C.
antibody
bindingvisualizedusing theformation ﬂow through the cortico-striato-EPN direct pathway
Fig. 2A).
On thehand,
corticalLaboratories)
stimulation induced
triphapoly A signal sequences were introduced at the initiation codon
Vectastain
Elite
ABCother
System(Vectorand 3, a3′to appropriately release motor actions.
diaminobenzidine.
sic response composed of early excitation, followed by inhibitionof D1R, the expression of endogenous D1R was disrupted. To genand late excitation in the GPe that was not changed during D1Rerate the pSV1-RecA-D1T shuttle vector, the DNA fragment ofMotor Behaviors and Spontaneous Activity ofsuppression (Fig. 4C, Table 2, see Supplementary Fig. 2B).
D1Txx was inserted into the pSV1-RecA vector to facilitate homEPN and GPe Neurons
Western Blot Analysis
Many studies have revealed that cortically evoked early exciologous recombination in RecA− Escherichia coli.
tation,inhibition,
latecondition,
excitation 0,
in7,
themediatedThe
BAC clone,
230 bp)(Research
Genetics), wasBasedon theBAC4-D1R
classical (80modelof the
BG, dopaminergic
inputsWe used
19 D1RKD(3–4 andin each
14,EPN/GPi
21, and are
28 days−
the hyperdirect,
direct,indirectused (seeSupplementary
Fig. 1B).
exert
excitatory effects
onRecAstriatal
pathway
neuronsafter bystarting
Dox treatment
andand7 daysafterpathways,
cessation respectively
of DoxE. colidirectcarrying
the origin(Maurice
et al.
1999;
Nambu
2000, mice
2002;for
Tachibanathrough
D1Rs(Albin et al.
1989;
DeLong 1990; Gerfen
for 14days),
3 WT,
andet1 al.
D1RKO
western et al.
al BAC
clone weretransformed
withthe pSV1-RecA-D1T
shuttleet al.treatment

2

| 10
Cerebral| Cerebral
CortexCortex

Introduction

cortically evoked response patterns. ex, excitation; ic, internal capsule; inh, inhibition; ot, optic tract; Rt, reticular thalamic nucleus; Str, striatum.

cortico-striatal synapses through long-term potentiation (Calab2008) (Fig. 1A). Thus, the loss of inhibition in the EPN during D1R
resi et al. 2007; Gerfen and Surmeier 2011). A signiﬁcant loss of
suppression indicates that information ﬂow through the corticodendritic spines on striatal projection neurons in PD has alsostriato-EPN direct pathway was strongly suppressed. Several posbeen reported (Villalba and Smith 2010). Second, other extrastriasible mechanisms for this suppression can be considered. First,
tal mechanisms can also be considered. A previous study (Kliem
the excitability of striatal direct pathway neurons may be decreased during D1R suppression. The heads of dendritic spines
et al. 2007) revealed that D1Rs are also expressed in striatal axon
Figure 1. Dopamine D1 receptor (D1R) suppression in D1R knockdown (D1RKD) mice with doxycycline (Dox) treatment. (A) Schematic diagram showing the cortico-basal
terminals in the GPi, and their activation increases GABA releaseof striatal projection neurons receive excitatory inputs from corganglia pathway and stimulating (Stim.) and recording (Rec.) sites in the electrophysiological experiments (left), along with a typical response pattern (right) in thefrom the striato-GPi axon terminals. Decreased GABA releasetical neurons, with the neck of the spine receiving dopaminergic
entopeduncular nucleus (EPN) (homologous to the internal segment of the globus pallidus, GPi) to cortical stimulation (Cx Stim.) with early excitation, inhibition, andthe striato-EPN
to GPi) terminals
maycontribinputs which
throughsynapses
volume transmission
late excitation,
are mediatedby and/or
the i) cortico-subthalamo(STN)-EPN(Herschhyperdirect, ii) fromcortico-striato-EPN
direct,(homologous
and iii) cortico-striato-external
pallido(GPe)ute to excitatory,
loss of corticallyevokedinhibitionin the EPN.
The EPN also
et indirect
al. 1995;
Smithrespectively.
and Kieval
Arbuthnott
andrepresent
Wickens
STN-EPN
pathways,
Red,2000;
blue, andgreen triangles
glutamatergic
GABAergic
inhibitory,
and dopaminergic
projections,
respectively.
D2 receptors; SNc,
substantia
nigra pars
compacta. (B)Schematic
diagramGABAergicof Dox-regulated
D1R expression
in D1RKD
BeforeHowever,Doxreceives
inhibitory
inputsfrom mice.
the GPe.
2007).D2Rs,
Thisdopamine
spatial arrangement
allowsdopamine
to modulate
intreatment(left), tetracycline(tTA)drive.
binds tothe tetracycline
responsive(TRE), and D1Rs
andlacZthat
are transcribed.
Dox treatment
interfereswithcorticonsideringthefactboth spontaneousﬁring
and thecomingexcitatorytransactivator
glutamatergic
D1Rs
are coupled
to Gs/olfelement,tTA binding to TRE (right), and suppresses D1R and lacZ expression (Tet-off system). (C) D1R immunoreactivity in the striatum (Str) of wild-type (WT, top), D1R
cally evoked response in the GPe remained unchanged during
which activates adenylyl cyclase and facilitates intrinsic excitknockout (D1RKO, middle), and D1RKD (before Dox treatment, bottom) mice shown in frontal sections. The pattern of D1R expression in D1RKD mice was similar to
D1R suppression (Fig. 3B, Fig. 4C, Tables 1 and 2, see Supplemenandglutamate
receptor-mediatedresponses
in striatal
that in ability
WT mice,whereasD1R immunoreactivity
was not observedin D1RKO
mice. The dorsolateral motor areas of the Str are deﬁned by rectangles (left) and shown
taryblot
Fig.analysis2B), thebe responsible
for the attenuatedneurons(Hervé
et al.
1995;
al.1002007;
Gerfenandat higher
magniﬁcation(right).
Scalebars, Surmeier
1 mm on theetleft,
μm on
the right.
(D) WesternofGPe
D1R cannot
protein expression
in the striatumof a D1RKO corticallyevokedinhibition
theEPN. Dox treatment; After, 7 days
2011).
D1R
activation
synapticin (Days
mouseSurmeier(n = 1, numberof miceused),
D1RKD miceincreases
before, during,
and afterefﬁcacy
Dox treatment
0, 7, 14,21, 28, different
daysinafter
starting
after cessation of Dox treatment for 14 days; n = 3–4 per condition), and WT mice (n = 3).

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Figure 5. Recording sites in the EPN (A) (Mouse W) and GPe (B) (Mouse K) of D1RKD mice before (1) and during (2) Dox treatment. Frontal sections are arranged
rostrocaudally from left to right, and the distance from bregma to each section is indicated. Locations of recorded neurons are indicated by different symbols based on

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In the basal ganglia (BG), dopaminergic transmission plays a pivotal role in the control of voluntary movements and motor learning (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Graybiel
2005; Joshua et al. 2009; Enomoto et al. 2011). Dopamine deﬁciency, as occurs in Parkinson’s disease (PD), results in severe
motor and nonmotor dysfunctions including bradykinesia, rigidity, tremor, autonomic abnormalities, cognitive dysfunction, anddepression (Fahn et al. 2011; Seppi et al. 2011). Dopaminergic inputs from the substantia nigra pars compacta terminate in the
striatum and are thought to differentially modulate the excitability of 2 types of striatal projection neurons through different receptors (Albin et al. 1989; DeLong 1990; Mallet et al. 2006)(Fig. 1A, left). One is excitatory effects through dopamine D1 receptors (D1Rs) on direct pathway neurons that directly projectto the output nuclei of the BG, the entopeduncular nucleus(EPN), which is homologous to the internal segment of the globus
pallidus (GPi) in primates, and substantia nigra pars reticulata(SNr). The other is inhibitory effects through D2 receptors(D2Rs) on indirect pathway neurons that project to the externalsegment of the globus pallidus (GPe), which is also called the globus pallidus in rodents. Such differential effects of dopamine
through D1Rs and D2Rs were originally proposed based onchanges in gene expression, glucose utilization, and receptor
binding in these pathways under conditions of dopamine depletion (Gerfen et al. 1990; Hirsch et al. 2000) and have been reinforced by recent in vitro electrophysiological studies (Surmeier
et al. 2007; Day et al. 2008; Flores-Barrera et al. 2011; Gerfen and
Surmeier 2011; Planert et al. 2013). The striato-EPN/SNr direct
and striato-GPe-subthalamo (STN)-EPN/SNr indirect pathways
play opposite roles in controlling movements. The signals
through the direct pathway reduce activity of the EPN/SNr and increase thalamocortical activity via disinhibition, resulting in release of movements. In contrast, signals through the indirect
pathway increase activity of the EPN/SNr, resulting in suppression of movements (Albin et al. 1989; DeLong 1990; Gerfen et al.
1990; Mink 1996; Nambu 2007; Kravitz et al. 2010; Sano et al.
2013). Thus, the loss of dopaminergic inputs to both pathway
neurons is considered to increase ﬁring rates of EPN/SNr neurons
through the inhibitory striato-EPN/SNr direct and net excitatory

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RoleRoleof Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al.et al.
| 3|

11

Dox Treatment
Notes

During D1R suppression, cortically evoked inhibition in the EPN
mixed
drinking
water containing
5% suwas mostly lost (Fig. 4A,B), and motor behaviors were severelyDox (2.0
Wemg/mL)
thank M.was
Katsuki,A.in
Aiba,
K. Nakamura,and K. Nakao
for techimpaired D1RKD(Fig. 2).mice
UponinD1R
re-expression,
the inhibitioncrose nicaland delivered
to criticalthe D1RKD
and WTonmicethroughofa water
We developed
which
D1Rs can bebothconditionallyadvice anddiscussion
generation
the D1RKD
and motor
behaviors
restored.
This observation
candeexplainbottle.mice, K. Miyamoto, S. Sato, K. Awamura, H. Isogai, M. Goto, andand reversibly
regulatedbywereDox using
the Tet-Offsystem after
mechanismof reduced
motor
activity
during
D1R suppresletiontheof theendogenous
D1Rs (Fig.
1B). To
generate
tetracycline
members of the Department of Comparative and Experimentalsion (see Supplementary
Fig. 4). Under
normaltransactivator-VP16(tTA)-expressing
mice using
theD1R
D1Rexpression
proMedicine, Brain Research Institute, Niigata University for technical
Immunohistochemistry
Supplementary
Fig. 4,bacterial
left), signals
throughthe cortico-striamoter(see(transactivatorline),
artiﬁcialchromosome
assistance. We also thank M. Kimura, Y. Kubo, and Y. Yamagata forpathway
inducein theEPN.
The phasicWe used
(BAC) to-EPN
clones directcontainingmouse
D1R inhibition
were modiﬁedwith
a 2-step
4 D1RKD,
WT,
and 1 D1RKO
mice
for immunohistocommentson 3
themanuscript.
Conﬂictof Interest:
None declared.
inBAC
the modiﬁcation
EPN increases(Yangthalamocorticalactivityby disin-chemical analysis. Mice were deeply anesthetized by injection
Rec Ainhibition
strategy foret al. 1997). A
modiﬁcahibitory thatmechanismand thereleases
motor
actions(Albin etal. 1989;of tribromoethanol (400 mg/kg body weight, i.p.) and perfusedtion cassette
introducestTA into
theendogenous
D1R
References
DeLong1990;wasGerfenet al. 1990;
Mink
1996; Hikosaka
al. 2000;transcardially with 4% paraformaldehyde in phosphate buffer
codingsequence
constructed(see
SupplementaryFig.et1A).
Nambu
et al.
2000,
Nambu
2007; Kravitz
et al. 2010).
During(PB, pH
A DNA
fragment(0.92
kb,2002;
A-arm)
containing
the upstream
region7.4).RL,
The
brainspostﬁxed
overnight
at 4°C, transAlbin
Young
AB,werePenneyJB. 1989.
The functional
anatomy of
suppression(see site
Supplementary
Fig. 4,to right),
signalsferred tobasalof theD1Rtranslation
initiationof D1R was ligatedthe DNA
a 30%ganglia
sucrosesolution,Trends
immersedin OCT
compounddisorders.
Neurosci.
12:366–375.
throughdirectsequencepathway arestrongly
fragment(1.5thekb) cortico-striato-EPN
containing the codingof thetTA sup-(Sakura
Finetek), GW,
frozen,and storedat −80°C
Arbuthnott
Wickens
J. 2007.
Space,until
timeuse.
andFrontal
dopamine.
were cutat 25-μm
thickness and stored in PBS at 4°C.
pressedto induce
the EPN,
resultingand theSV40 andpolyfail
A signal( polyinhibition
A) of the in
pTet-off
vector(Clon-in thesections Trends
Neurosci.
30:62–69.
activity.
Moreover,this toobservationsuggestsFree-ﬂoatingtech),reduced
and the motor
resultingfragment
was ligatedthe DNA fragsections
were incubatedwith
al- M,
Bergman
H, Feingold
A, Nini A,
Raz1%
A,bovine
Slovinserum
H, Abeles
theof theofphasic
in generate
the EPN throughmentthat(0.96
kb,loss
B-arm)
exon inhibition
2 of D1R to
D1Txx, thethedirectbumin containing
antibody
for D1R (1:1000;
Institute)
Vaadia E. 1998.
Physiologicalaspects Frontierof information
processpathway,cassette
but not (3.38
the ﬁringrate or patterna funda-overnighting
modiﬁcationkb). Because
the tTAchanges,
and the is
SV40
at in
4°C.
antibody
wasvisualizedusingprimates.
thetheD1R
basal
gangliabindingof normal
andparkinsonian
in reduced
motor
activity
during
D1R sup-Vectastain
poly Amental
signal phenomenon
sequences wereintroduced
at theinitiationcodon
EliteNeurosci.
ABC System(Vector Laboratories) and 3, 3′Trends
21:32–38.
pression.
The reductionof spontaneous
motor To
activityof D1R,the expressionof endogenous
D1R was disrupted.
gen- wasdiaminobenzidine.
Boraud T, Bezard E, Guehl D, Bioulac B, Gross C. 1998. Effects of Leven in the shuttle
ﬁrst weekof Doxtreatment(Fig. 2A)
erate observed
the pSV1-RecA-D1T
vector,the
DNA fragmentofwhenDOPA on neuronal activity of the globus pallidus externalis
levelof D1R
expression
was stillhigherthan thatin WT
D1Txxthewasinserted
into
the pSV1-RecA
vectorto facilitate
hom(GPe) and globus pallidus internalis (GPi) in the MPTP-treatedWestern Blot Analysis−
mice(Fig. 1D). The inloss
of corticallyevokedologousrecombination
RecA
monkey. Brain Res. 787:157–160.
Escherichia
coli. inhibition in EPN neuBrown
P. 2007.
oscillatory
in therons
alreadyobserved
in bp)
the(Researchﬁrst half Genetics),(5–14 days)of DoxWe used
The
BACwasclone,
BAC4-D1R(80 230
was
19 D1RKD(3–4Abnormal
in each condition,
0, 7,synchronisation
14, 21, and 28 days−
treatment(see Supplementary
3A).
These theresultssuggestafter starting
motorDoxsystemleadsandto 7impaired
used (seeSupplementary
Fig. 1B). RecAFig.
treatmentdays aftermovement.
cessation ofCurr
Dox OpinE. colicarrying
originthat
relativedecrease of D1R
would beshuttle
responsibletreatment
Neurobiol.
17:656–664.
for 14 days),
3 WT, and 1 D1RKO mice for western
al BACclone
were transformed
withexpression
the pSV1-RecA-D1T

Generation of Conditional and Reversible D1RKD Mice

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Release Motor Actions

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forThetheresulting
changes clones
in motorbehavior
and toneuronal
activity.
Considvector.
were
subjected
Southernanalysis
striato-GPe-STN-EPN/SNr
The cortically evokedindirect
triphasic
pathways,response
resultingin the GPein theis mediateddefactthatorexpression
not onlyin thestriatum buteitherthe
a 5′
probe
tTA probeoftoD1Rs
conﬁrmproperrecombincreased
sequentiallymotor activity
by theseen
cortico-STN-GPe,in PD (Albin etcortico-striato-GPe,al. 1989; DeLong 1990).
and corti-usingering
the striatum
wasThesuppressed
during Dox(see outside
Supplementary
Fig. 1A).
BAC transgenic(Tg) treatment
conHowever,co-striato-GPe-STN-GPe
the exact role of D1R-mediatedpathways (Maurice
dopaminergic
et al. 1999;
transNambuation also
D1RKD mice1C), wecannotrule out thepossibilitythatwas(Fig.
obtained(see
Supplementary
Fig.
1B).
missionet al.
in 2000,
vivo is2002;
not well
Kita etunderstood.
al. 2004) (Fig.
Contradicting
1A). The maintained
results have
triphasicstruct,inBAC4-tTA,
loss
extrastriatal
D1Rs were also
involvedelementin their (D1Rbehavioral
been response
observedin
following
the GPe in
D1R
theblockade.
absence ofD1R
D1Rs
knockout(Fig. 4C, Table(KO) mice
2, see Sup- The
Tgof
construct,
D1R-tetracycline
responsive
observed
the current study.
Fig.spontaneous
2B) suggests thatthe excitability
these compo-TRE), changes
was generated
forintetracycline
operator (tetO)-target Tg
showplementary
increases inlocomotor
activity,ofalthough
Theusing
present
study revealeda crucial (seerole Supplemenfor dopamine in
nents, including
the cortex,
striatal
indirectpathwaysuppresneurons, GPe,mouse lines
the following
DNA fragments
pharmacologicalblockadeof D1Rs
induces
locomotor
maintaining
dynamicsof thethe
BGentire
circuitry:D1R-mediatedandagreement
STN, remains
unchanged
despitethe fact that
D1Rs are also ex-tary Fig.
1C): a 9.7-kbthe
fragment
including
D1R coding
sesion inwiththe above
explanation(Waddington
dopaminergictransmission
maintains
the bidirectional
information ﬂow
pressedmoderate
inwe
thedeveloped
cortex (Fig.novel
1C; Fremeau
et al. 1991;quence,
a 0.64-kb fragment
encoding
the TRE andet al. 2005).
Inatthepresentlevels
study,
D1R knockthrough(GPi) direct
pathway
to approprietmiceal. 1991;
Gasparet D1Rsal. 1995).
hybridization
fromthethecortico-striato-EPN
pBI Tet vector (Clontech),a 3466-bp
fragment
downWeiner(D1RKD)in whichthecan Inbesituconditionally
andstudypromoter
atelylacZ
releaseactions
throughusingregulateda D2R-speciﬁc
riboprobe followed
by immunohistochemistry
encoding
frommotor
the pBI-GL
Tet
vector disinhibitory(Clontech), a mechanisms
0.98-kb
reversibly
by doxycycline(Dox) treatment(Fig. 1B). The(see containing
Supplementary4). The present
results
with
Cre-speciﬁc
indicatedthat D1Rs andmayneuronalso be aber-fragment
rabbitFig.
beta-globin
poly A and
SV40also
polysuggest
A
D1RKD
mice
enabled usantibody
to examine
motor behaviorsthatphasicactivitychangesthe EPN
throughthe corticofrom
the pBstN
plasmid,andin
a 0.3-kb
fragment
encoding
rantly expressedin striato-GPe
indirectpathway
neuronsin trans-signals
al activities
in the cortico-BG
pathwaysin thepresence
and abstriato-EPN
direct pathway
fundamental
to plasmid
both normal
beta-globin
insulator are
sequencesfrom themice
Cre recombinase
under thecontrol of thethe chicken
sencegenicof D1Rs
inexpressing
the same amice.
The mice exhibiteddecreased
functionsof the BG and the pathophysiology of movement
INS240-SNNS.
D1R regulatory(Lemberger
et al.
2007).
However,neitherpUC19
spontaneous
motor elements
activity andimpairedmotor
abilitywhen
disorders.
Dopaminedeﬁciency
phasic
BAC4-tTAand D1R-TRE
DNAsimpairs(10 ng/μLeach)activation
were usedof the
spontaneousdischarges
nor corticallyevokedof GPe neu- The
D1R expression
was
suppressed,
consistent
with responses
the pharmacorelease of
motor
actions,
for Tgcortico-striato-EPN
mouse generation directusing pathway
standardandtechniques(Nagy
et al.
rons
changedduring
D1R suppressionin our current
electrophysiologicalblockade
study(Waddington
et al. 2005).
To examine
thebe involvedin theDNAs
bradykinesia
seen in PD. mi2002).and
Thewould
BAC4-tTAand D1R-TRE
were independently
study (Figs
3Bmotorand 4C,
Tableswe
1 and
see Supplementaryneurallogicalmechanismof thedeﬁcits,
next2,recorded
neurcroinjected into fertilized mouse oocytes isolated from crosses ofFig. 2B), suggesting
they areless because
functional.
onal activity
in the EPNthat
in awakemice,
the EPN is theknockout Material(D1RKO) female and male mice (Tran
Recentstudieshave
results
regardingmain output
nucleusof theBGshown
to the conﬂicting
thalamus andthe targetof theD1R homozygous
Supplementary
et al. 2008). Three Tg lines for BAC4-tTA and 15 Tg lines for D1Rclassical direct
andindirectCorticallypathwaysevokedmodel.inhibition
Striatal neurons
D1R-expressing
striatal
neurons.
inSupplementarymaterial
can befound
at: http://www.cercor.
independently
generated.
Each
BAC4-tTATg line wasprojecting
the EPN also
haveaxon collateralsdirect
to thepathGPe, indi-TRE were
the EPN,
which istomediatedby thecortico-striato-EPN
oxfordjournals.org/.
no clearanatomical
separation ofand indirectcrossed with each D1R-TRE Tg line. The progeny from crosses
way, cating
was mostly
lostduring suppressionof theD1Rdirect
expression,of BAC4-tTA and D1R-TRE Tg mice was subjected to analyses ofpathways(Graybiel
2005;
andof
Parent2005; Fujiyama
whereasspontaneous
ﬁring
ratesLévesqueand patterns
EPN neurons
reFunding expression of lacZ using X-gal staining of
et al.
2011). Somestriatalprojection
both D1RsDox-controllable
mainedunchanged.
These
results
indicateneurons
that D1Rexpress
suppression
andD2Rsthe(Surmeieret al. 1996).
However,thethecortico-striatopresent results sup-brain sections using a standard method and expression of D1R
mostlyblocksinformation
ﬂow throughThis study was supported by JSPS KAKENHI (#23115721,
port theoriginal
that
striatal projection
neurons can bein the striatum using western blotting with anti-D1R antibody
EPN direct
pathway
andnotion
reducesspontaneous
motor activity.
#21500378 and #25430021 to S.C.), JSPS KAKENHI (#22500343
functionally separated into 2 groups, that is, one with D1Rs pro-(Sigma). Two Tg lines harboring BAC4-tTA and D1R-TRE (desigand #26290029), the Collaborative Research Project (#2011-2221)
jecting to the EPN (GPi) and the other with D2Rs projecting to thenated 442–43 and 442–112) were consistently found to exhibitof Brain Research Institute, Niigata University and the MEXTMaterials
andMethods
GPe (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Hersch et al.distinct, comparable expression of lacZ in the region where enSupported Program for the Strategic Research Foundation
D1R was expressed and Dox-controllable expression
1995;
Sano etwere
al. 2013).
The axon
terminals
of striatal
neurons
Animalprotocols
approved
by theInstitutional
AnimalCare thatdogenous
at Private Universities, 2011-2015 (to T.S.), and JSPS KAKENHI
and D1R. Therefore, the 442–112 line was used for further
express
D1Rs inoftheGPe maybe minorless functionaland UseCommittee
National
Institutes
ofor
NaturalSciences, com-of lacZ(#21240039,#26250009 and #15H05873), CREST, Strategic
analyses as D1RKD mice.
paredwith those
D2Rs.
Kitasato
University
andwithNiigata
University, and were conductedJapanese-German Cooperative Programme and the Takeda SciWild-type (WT) C57BL/6J and D1RKO (Tran et al. 2008) miceaccording to the guidelines of the National Institutes of Healthence Foundation (to A.N.). Funding to pay the Open Access publiwere also used for comparison in immunohistochemical, westGuide for the Care and Use of Laboratory Animals. One to 5 micecation charges for this article was provided by the JSPS KAKENHI
D1R-Mediated Dopaminergic Transmission Maintains
ern blot, and behavioral analyses.
were housed in each cage under a 12-h light–dark cycle (lights(#15H05873).
the Information Flow Through the Direct Pathway toon at 8:00 AM) and given food and drinking water ad libitum.

2

| 12
Cerebral| Cerebral
CortexCortex

Hikosaka
Takikawa
Y, Kawagoe
R. 2000.
Roleof theof the O,
globuspallidus(GPe), whichis alsocalledthebasal
glo- ganBrown P, Oliviero A, Mazzone P, Insola A, Tonali P, Di Lazzaro V.segment
Introduction

at higher magniﬁcation (right). Scale bars, 1 mm on the left, 100 μm on the right. (D) Western blot analysis of D1R protein expression in the striatum of a D1RKO
mouse (n = 1, number of mice used), D1RKD mice before, during, and after Dox treatment (Days 0, 7, 14, 21, 28, different days after starting Dox treatment; After, 7 days
after cessation of Dox treatment for 14 days; n = 3–4 per condition), and WT mice (n = 3).

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Nis

Pla

Raz

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knockout (D1RKO, middle), and D1RKD (before Dox treatment, bottom) mice shown in frontal sections. The pattern of D1R expression in D1RKD mice was similar tothat in WT mice, whereas D1R immunoreactivity was not observed in D1RKO mice. The dorsolateral motor areas of the Str are deﬁned by rectangles (left) and shown

Nam

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glia in rodents.
the control
of differential
purposive saccadic
movements.
Sucheffects ofeye
dopamine
2001. Dopamine dependency of oscillations between subtha-bus pallidus
In the basal ganglia (BG), dopaminergic transmission plays a pivRev.D2Rs
80:953–978.
lamic nucleus and pallidum in Parkinson’s disease.through Physiol
D1Rs andwere originally proposed based on
otal role in the control of voluntary movements and motor learnHirsch
EC, Périer
C, Orieux
G, François
C, FégerandJ, Yelnik
J, Vila M,
J Neurosci. 21:1033–1038.
changesin geneexpression,
glucoseutilization,
receptoring (Albin et al. 1989; DeLong 1990; Gerfen et al. 1990; Graybiel
Levy
R, TolosaES, Marin
et al. 2000.
Calabresi P, Picconi B, Tozzi A, Di Filippo M. 2007. Dopamine-binding inthesepathways
under C,
conditionsof Metabolic
dopamineeffects
deple- of ni2005; Joshua et al. 2009; Enomoto et al. 2011). Dopamine deﬁgrostriatal
denervationin al.
basal
ganglia.
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Figure 1. Dopamine D1 receptor (D1R) suppression in D1R knockdown (D1RKD) mice with doxycycline (Dox) treatment. (A) Schematic diagram showing the cortico-basal
ed. New York: Cold Spring Harbor Press.
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motor impairmentsof dopamine
the whichdorsalandsynaptic relationships
withlate excitation,
arestriatum
mediated by
the theiri) cortico-subthalamo(STN)-EPN hyperdirect,ii) cortico-striato-EPN
direct,Distinct
and iii) cortico-striato-external
pallido(GPe)D1excitatory,and D2 receptor
mice
revealed projections,by three types
motorpathways,
corticostriatal
afferents.
J Neurosci.
STN-EPN indirect
respectively.
Red, blue,and green15:5222–5237.
triangles represent glutamatergic
GABAergic knockout
inhibitory, anddopaminergic
respectively.
dopamine
receptors; SNc,M.
substantia
nigra pars compacta.
(B)of
Schematic diagramof Dox-regulated
D1R expression
in D1RKD 8:56.
mice. Before Doxof motorbehavior. Front
Integr Neurosci.
HervéD2Rs,
D, Rogard
M,D2Lévi-Strauss
1995. Molecularananysistreatment (left),tetracycline Golf
transactivator(tTA) binds mRNAs
to the tetracycline
elementNambu(TRE), andandlacZ aretranscribed.
Dox treatment
interfereswith Res.
A.D1Rs2007.
Globuspallidusinternal
segment.
Prog Brain
the multiplealpha subunitin the responsive
rat brain.
tTA binding to TRE (right), and suppresses D1R and lacZ expression (Tet-off system). (C) D1R immunoreactivity in the striatum (Str) of wild-type (WT, top), D1R
160:135–150.
Brain Res Mol Brain Res. 32:125–134.

San

Sep

Sm

Sta

Sur

RoleRoleof Dopamineof Dopamine
D1 Receptors
D1 Receptors
in Motorin Motor
Control
Control
ChikenChikenet al.et al.
| 3|

13

We developed D1RKD mice in which D1Rs can be conditionallyand reversibly regulated by Dox using the Tet-Off system after deletion of the endogenous D1Rs (Fig. 1B). To generate tetracyclinetransactivator-VP16 (tTA)-expressing mice using the D1R promoter (transactivator line), bacterial artiﬁcial chromosome(BAC) clones containing mouse D1R were modiﬁed with a 2-step
Rec A strategy for BAC modiﬁcation (Yang et al. 1997). A modiﬁcation cassette that introduces the tTA into the endogenous D1R
coding sequence was constructed (see Supplementary Fig. 1A).
A DNA fragment (0.92 kb, A-arm) containing the upstream regionof the translation initiation site of D1R was ligated to the DNA
fragment (1.5 kb) containing the coding sequence of the tTA
and the SV40 poly A signal ( poly A) of the pTet-off vector (Clontech), and the resulting fragment was ligated to the DNA fragment (0.96 kb, B-arm) of exon 2 of D1R to generate D1Txx, themodiﬁcation cassette (3.38 kb). Because the tTA and the SV40
poly A signal sequences were introduced at the initiation codonof D1R, the expression of endogenous D1R was disrupted. To generate the pSV1-RecA-D1T shuttle vector, the DNA fragment of
D1Txx was inserted into the pSV1-RecA vector to facilitate homologous recombination in RecA− Escherichia coli.
The BAC clone, BAC4-D1R (80 230 bp) (Research Genetics), wasused (see Supplementary Fig. 1B). RecA− E. coli carrying the original BAC clone were transformed with the pSV1-RecA-D1T shuttle

Dox (2.0 mg/mL) was mixed in drinking water containing 5% sucrose and delivered to the D1RKD and WT mice through a waterbottle.

Immunohistochemistry
We used 4 D1RKD, 3 WT, and 1 D1RKO mice for immunohistochemical analysis. Mice were deeply anesthetized by injectionof tribromoethanol (400 mg/kg body weight, i.p.) and perfusedtranscardially with 4% paraformaldehyde in phosphate buffer(PB, pH 7.4). The brains were postﬁxed overnight at 4°C, transferred to a 30% sucrose solution, immersed in OCT compound(Sakura Finetek), frozen, and stored at −80°C until use. Frontal
sections were cut at 25-μm thickness and stored in PBS at 4°C.
Free-ﬂoating sections were incubated with 1% bovine serum albumin containing antibody for D1R (1:1000; Frontier Institute)overnight at 4°C. D1R antibody binding was visualized using the
Vectastain Elite ABC System (Vector Laboratories) and 3, 3′diaminobenzidine.

Western Blot Analysis
We used 19 D1RKD (3–4 in each condition, 0, 7, 14, 21, and 28 days
after starting Dox treatment and 7 days after cessation of Dox
treatment for 14 days), 3 WT, and 1 D1RKO mice for western－ 325 －

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Dox Treatment

Generation of Conditional and Reversible D1RKD Mice

Downloaded from http://cercor.oxfordjournals.org/ at Niigata Univ Asahimachi on October 6, 2015

Downloaded from http://cercor.oxfordjournals.org/ at Niigata Univ Asahimachi on October 6, 2015

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WJ,wereYan subjected
Z. 1996. Coordinatedof
The resulting
clonesto Southern expression
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striato-GPe-STN-EPN/SNr
Nambu A, Tokuno H,indirect
Hamadapathways,I, Kita H, resulting
Imanishiin
M,theAkazawadeT,vector.
dopamine
receptors
in neostriatal
mediumspiny neurons.
a 5′ probe
or tTA probe
to conﬁrm
proper recombincreased motor
Ikeuchiactivity
Y, Hasegawa
seen inN.
PD2000.
(AlbinExcitatory
et al. 1989;
corticalDeLonginputs1990).to pal-using either
16:6579–6591.
Supplementary
Fig. 1A). The BAC transgenic (Tg) conHowever,lidaltheneurons
exact role
via ofthe
D1R-mediatedsubthalamicdopaminergic
nucleus in thetransmonkey.ation (seeJ Neurosci.
IwamuroH, KitaTakada M, Nambu
A. 2011. Substruct,Tachibana
BAC4-tTA,Y,wasobtained(seeH,
Supplementary
Fig. 1B).
mission in
J Neurophysiol.
vivo is not well84:289–300.
understood. Contradicting results havethalamo-pallidal
interactions responsive
underlying element
parkinsonian
been Nambu
observedA,following
Tokuno H,
D1R
Takadablockade.
M. 2002.
D1RFunctional
knockout (KO)signiﬁcancemiceof The Tg
construct, D1R-tetracycline(D1R-neuronaloscillationsin the primate
basal ganglia.
Eur J Neurosci.
the cortico-subthalamo-pallidal
pathway.TRE), wasgenerated
for tetracycline
operator(tetO)-target
Tg
show increases
in spontaneous locomotor ‘hyperdirect’
activity, although
34:1470–1484.
Neurosciblockade
Res. 43:111–117.
mouse lines
using the following DNA fragments (see Supplemenpharmacologicalof D1Rs induces locomotor suppresTachibana
Y, Kita
H, Chiken
S, Takada
M, Nambu
A. 2008.
Nishibayashi
Ogura
Kakishita
K, Tanaka
S, Tachibana Y,tary Fig.
1C): a 9.7-kb
fragment
includingthe entire
D1R coding
se-Motorsion inagreementH,
with
theM,above
explanation(Waddington
corticalof internal
pallidalactivitythrough glutamaNambu
Kita H, study,
ItakuraweT. developed
2011. Cortically
responsesquence, a0.64-kbcontrol
fragment
encoding
the TREand bidirectional
et al. 2005).
In theA,present
novelevoked
D1R knockandpBIGABAergic
inputs in aawake
monkeys.
of humanpallidalduring stereotactic
promotertergic
from theTet vector (Clontech),
3466-bp
fragmentEur J
down (D1RKD)
mice inwhich neurons
the D1Rs recorded
can be conditionallyand
Mov Disord. (Dox)26:469–476.
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the pBI-GL Tet vector (Clontech), a 0.98-kb
reversiblyneurosurgery.
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Trancontaining
AH, Uwanorabbit
T, KimuraT, Hori E,
Katsuki
M,SV40
Nishijo
H,AOno T.
Planert
H, Berger
Silberberg
G. 2013.
Membrane
properties offragment
beta-globin
poly
A and
poly
D1RKD
mice enabled
usTK,
to examine
motor
behaviorsand neuron2008.theDopamine
D1 receptor
modulates
hippocampal
pBstN plasmid,
and a 0.3-kb
fragment
encoding restriatal
directand indirect
pathway
neuronsin mouse
and ratsignals fromal activities
in thecortico-BG
pathwaysin thepresence
and abpresentation
plasticity
spatial novelty.
J plasmid
Neurosci. 28:
beta-globin
insulatortosequencesfrom thetheirmodulation
bymicedopamine.
PLoS
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et al. 2005). To examine the
O’TuathaighC, O’Sullivan
G, Tomiyama
K,
The BAC4-tTAJL,
and
D1R-TRE DNAs
were independently
mihydropyridine
parkinsonism.
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neural mechanismof thevervet
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N, CrokeDT.oocytes
2005. Phenotypicstudies
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into fertilizedmouse
isolated fromcrosses
20:8559–8571.
onal activity
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knockout(D1RKO)and malemice(TranH,nucleus
Chiken S,
K, Nambu
A.target2013. of
SignalsD1R homozygous
main Sano
outputof Hikida
the BGT,
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the thalamus
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insights
andBAC4-tTA
challenges
frompsychoet al. 2008).
Three Tglines forand
15 10
Tgyears
lines atfortheD1Rthroughstriatal
the striatopallidal
indirect evoked
pathwayinhibition
stop movements
D1R-expressing
neurons. Corticallyininterface.Tg
Psychopharmaindependently generated.biology
Each BAC4-tTAline wasby phasic
excitationin cortico-striato-EPN
the substantia nigra.
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Weinerand
DM,D1R-TRE
Levey AI,RK, Niznik
HB, O’Dowd
Tg Sunahara
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to analyses
of BF,Seppi
K, Weintraub
D, Coelho
M, Perez-Lloret
S, Foxwhereasspontaneousﬁring rates
and patternsof EPN neurons
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30:228–235.

A Modification cassette construct ATG
Mouse
D1R gene

Nc

S E

EB

Xh

Nc

E Xb

E

B

E

BB

coding regionS

non-coding region
1kb

5’ probe
Xh

B A

Xb

D1Txx

Xh#

tTA-pAA-armtTAprobe B-arm

tTA-pA, tetracycline transactivator and SV40 polyAA, Asc I; B, Bam HI; E, Eco RI; Nc, Nco I; S, Sau 3AI; Xb, Xba I; Xh, Xho I; Xh#, Xho I (artificial)

B

BAC4-tTA construct
D1R gene

Mouse
chr. 1364.1kb

16.1kb

20kb

BAC4-D1R
BAC4-tTA

tTA-pA:tetracycline transactivator and SV40 polyAtTA-pA
1.5 kbC

D1R-TRE construct

Mouse
D1R gene

Nc

S E

E

E

TRE

Nc

E Xb

E

E

S

coding region
non-coding region

Xb

Nc

E Xb

E

E

N#

lacZ

D1R-TRE

Xh

1kb

TRE, tetracycline responsive element and bidirectional promoter
E, Eco RI; Nc, Nco I; N#, Not I (artificial); S, Sau 3AI; Xh, Xho I; Xb, Xba I－ 326 －

rabbit β-globin polyA
and SV40 polyA
chicken β-globin
insulator

A EPN% of neurons
0

20

40

60

80

100

BeforeDuring
Dox
After

B GPe

ex-inh-ex

ex-inhinh-ex

inh

ex-ex

ex

% of neurons
0

20

40

60

BeforeDuringDox
After－ 327 －

80

100

A D1RKD EPN
1 During Dox (5–14 days)

2 During Dox (15–22 days)
250

200

Firing rate (Hz)Firing rate (Hz)250

n = 36

150
100
50
0
-25

0

Stim.

25

50

200

n = 27

150
100
50
0
-25

75
ms

0

Stim.

25

50

75
ms

B WT EPN
1 Before
Firing rate (Hz)Firing rate (Hz)

250

400
300
200
100
0
-25

0

Stim.

25

50

200

100
50
0
-25

75
ms

n = 30

150

0

Stim.

25

50

75
ms

2 During Dox
Firing rate (Hz)Firing rate (Hz)

250

400
300
200
100
0
-25

0

Stim.

25

50

75
ms

－ 328 －

200

n = 41

150
100
50
0
-25

0

Stim.

25

50

75
ms

Normal D1R

D1R supression

Cortex

Cortex

D1R

D1R

EPN (GPi) activity

Time

Directpathway

Striatum

Directpathway

EPN (GPi) activity

Striatum

Time

Reducemotor activity

Releasemotor actions－ 329 －

Supplementary Figure 1. Construction of BAC4-tTA and D1R-TRE. (A) Schematic
representation of mouse D1R and the modification cassette D1Txx for bacterial artificial
chromosome (BAC) modification using a two-step Rec A strategy. The 5’ probe, tTA probe,
A-arm and B-arm were used for Southern analysis. (B) Schematic representation of the BAC
clone containing mouse D1R and the construct for generation of tTA-expressing transgenic (Tg)mice. BAC4-D1R was modified using homologous recombination with the pSV1-RecA-D1T
shuttle vector (see Materials and Methods) to generate BAC4-tTA. (C) Schematic representationof the D1R-TRE construct for the generation of tetracycline operator-target Tg mice. The
bidirectional tetracycline-responsive promoter leads to simultaneous expression of both D1R
and lacZ depending on the action of tTA.

Supplementary Figure 2.

Proportions of neurons in the entopeduncular nucleus (EPN, A)

and external pallidum (GPe, B) classified according to the cortically evoked response patterns
before, during, and after doxycycline (Dox) treatment in Dopamine D1 receptor knockdown(D1RKD) mice.

ex, excitation; inh, inhibition.

Response patterns can be grouped into 2

groups: responses with inhibition, such as ex-inh-ex, ex-inh, inh-ex and inh, and responses
without inhibition, such as ex-ex and ex.

Supplementary Figure 3.

(A) Cortically evoked responses of EPN neurons in D1RKD micein the first half (5–14 days after starting Dox treatment, 1) and in the latter half (15–22 days
after starting Dox treatment, 2) of Dox treatment. Population peristimulus time histograms－ 330 －

(population PSTHs, with Gaussian filter) are shown. Stimulation was delivered at time 0(arrows). The number of neurons is indicated by n, and the shaded areas represent ± SD. (B)Cortically evoked responses of EPN neurons before (1) and during (2) Dox treatment inwild-type (WT) mice. Raster and PSTHs (100 trials; bin width, 1 ms) for typical examples (left)
and population PSTHs are shown. The mean firing rate and statistical levels of p < 0.05
(one-tailed t-test) are indicated in PSTHs by a black and white dashed line (mean) and black(upper limit) and white (lower limit) solid lines, respectively.

Supplementary Figure 4.

D1R-mediated dopaminergic transmission maintains theinformation flow through the cortico-striato-EPN (homologous to GPi) direct pathway toappropriately release motor actions. Under normal D1R expression (left), signals through thecortico-striato-EPN (GPi) direct pathway induce inhibition in the EPN (GPi) and release motor
actions by disinhibiting the thalamus. During D1R suppression (right), the information flow
through the cortico-striato-EPN (GPi) direct pathway is strongly suppressed and fails to induce
inhibition in the EPN (GPi), resulting in the reduced motor activity. Open and shaded circles
represent excitatory and inhibitory neurons, respectively.

－ 331 －

ORIGINAL RESEARCH
published: 17 June 2015
doi: 10.3389/fcell.2015.00038

Expression pattern of immediate
early genes in the cerebellum of D1R
KO, D2R KO, and wild type mice
under vestibular-controlled activity
Toru Nakamura 1, 2, 3 , Asako Sato 4, 5 , Takashi Kitsukawa 1, 3 , Toshikuni Sasaoka 4, 5, 6 andTetsuo Yamamori 1, 2, 7*
1
Division of Brain Biology, National Institute for Basic Biology, Okazaki, Japan, 2 Department of Basic Biology, Graduate
University for Advanced Studies (SOKENDAI), Okazaki, Japan, 3 KOKORO-Biology Group, Laboratories for Integrated Biology,
Graduate School of Frontier Biosciences, Osaka University, Suita, Japan, 4 Laboratory of Neurochemistry, National Institutefor Basic Biology, Okazaki, Japan, 5 Department of Laboratory Animal Science, Kitasato University School of Medicine,
Sagamihara, Japan, 6 Department of Comparative and Experimental Medicine, Brain Research Institute, Niigata University,
Niigata, Japan, 7 Laboratory of Molecular Analysis for Higher Brain Function, RIKEN Brain Science Institute, Wako, Japan

Edited by:
Kathleen Dave,St. Luke’s University Health Network,
USA
Reviewed by:William Wisden,
Imperial College London,
United Kingdom
Cathryn Kubera,
Monmouth University, USA*Correspondence:
Tetsuo Yamamori,RIKEN Brain Science Institute, Wako,
Saitama 351-0198, Japan
yamamori@nibb.ac.jp;
tetsuo.yamamori@riken.jp
Specialty section:
This article was submitted to
Systems Biology,
a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 24 March 2015
Accepted: 01 June 2015
Published: 17 June 2015
Citation:
Nakamura T, Sato A, Kitsukawa T,
Sasaoka T and Yamamori T (2015)
Expression pattern of immediate earlygenes in the cerebellum of D1R KO,
D2R KO, and wild type mice undervestibular-controlled activity.
Front. Cell Dev. Biol. 3:38.
doi: 10.3389/fcell.2015.00038

We previously reported the different motor abilities of D1R knockout (KO), D2R KO andwild-type (WT) mice. To understand the interaction between the cerebellum and thestriatal direct and indirect pathways, we examined the expression patterns of immediate
early genes (IEG) in the cerebellum of these three genotypes of mice. In the WT naive
mice, there was little IEG expression. However, we observed a robust expression of c-fos
mRNA in the vermis and hemisphere after running rota-rod tasks. In the vermis, c-fos was
expressed throughout the lobules except lobule 7, and also in crus 1 of the ansiform
lobule (Crus1), copula of the pyramis (Cop) and most significantly in the flocculus in
the hemisphere. jun-B was much less expressed but more preferentially expressed in
Purkinje cells. In addition, we observed significant levels of c-fos and jun-B expressions
after handling mice, and after the stationary rota-rod task in naive mice. Surprisingly, weobserved significant expression of c-fos and jun-B even 30 min after single weighing.
Nonetheless, certain additional c-fos and jun-B expressions were observed in threegenotypes of the mice that experienced several sessions of motor tasks 24 h after
stationary rota-rod task and on days 1 and 5 after rota-rod tasks, but no significant
differences in expressions after the running rota-rod tasks were observed among thethree genotypes. In addition, there may be some differences 24 h after the stationaryrota-rod task between the naive mice and the mice that experienced several sessions ofmotor tasks.
Keywords: IEG, c-fos, jun-B, rota-rod, striatum

Introduction
We have recently reported the distinct motor impairments of congenic dopamine D1 and D2
receptor KO mice in their performance of three different types of motor behavioral tasks, that is,
spontaneous motor activity tasks in the home cage, rota-rod tasks, and our own invented StepWheel task (Kitsukawa et al., 2011). In the Step-Wheel task, mice run as they adjust their steps on

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Nakam

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Nakamura et al.

IEG expression in the cerebellum

foothold pegs to drink water (Nakamura et al., 2014). Durieux
et al. (2012) showed that the ablation of D1R medium spiny
neurons (MSNs) in the dorsolateral striatum (DLS) of micecorresponding to the primate putamen decreases the learning
curve in rota-rod tasks. They also showed that the ablationof D2R MSN in the dorsomedial striatum (DMS) of micecorresponding to the primate caudate decreases the initial
learning curve of the task. These results of local ablation are
generally consistent with those of the entire striatal ablation of
D1R and D2R neurons (Durieux et al., 2012) and the resultsof our behavioral experiments using general D1R and D2R KO
mice, suggesting that DLS and DMS play major roles in rota-rod
tasks. However, there are also differences in the results between
local ablation and general D1R KO and D2R KO mice (Nakamura
et al., 2014). The ablation of D1R MSNs in either DMS or theentire striatum reduced locomotion in the open field, which
behavior is the reverse of general D1R KO mice. The ablation of
D2R MSNs in either DMS or the entire striatum does not affectthe late-phase performance (Durieux et al., 2012), whereas thegeneral D2R KO mice showed poorer leaning than WT mice in
the Step-Wheel tasks (Nakamura et al., 2014). These differences
between local ablation of DRs and general KO suggest that the
involvement and interaction between the striatum and otherbrain regions.
To explore this possibility further, in this study we employed
the immediate early gene (IEG) mapping technique, with which
the neuronal activity in the cerebellum is monitored on thebasis of IEG expressions. Among eight IEGs we examined, cfos and jun-B showed marked expression and specific induction,
respectively, in the cerebellum immediately after the runningrota-rod tasks. Interestingly, by examining c-fos and jun-B
expressions in the vermis, Crus1, Cop and the flocculus in the
cerebellar hemisphere, we found significant expressions of c-fos
and jun-B 24 h after the stationary rota-rod tasks, during whichtime the mice did not perform any motor tasks but were allowed
to move freely. We consider that this may be consistent with theidea that motor sequence learning and adaptation progress withtime, through “consolidation” and “automatization” (Doyonet al., 2009).
We therefore examined in detail the expression patterns ofc-fos and jun-B in the congenic D1R KO, D2R KO, and WT
mice after the stationary and running rota-rod tasks in orderto determine how cerebellar activities related to the D1R- andD2R-dependent pathways. Although we have not confirmed thealteration of c-fos expression patterns in the cerebellar flocculus
on days 1 and 5 after the rotating rota-rod tasks among thethree genotypes of mice, D1R KO, D2R KO, and WT mice,
we found significant expressions of c-fos and jun-B not only
immediately (30 min) after the mice ran on the rota-rod but also
after handling them, and 30 min and unexpectedly even 24 h after
the stationary rota-rod task. This observation led us to examine aseries of examination for c-fos and jun-B expression patterns. We
found that vestibular-controlled activities such as handling mice
and weighing caused significant c-fos and jun-B expressions in
cerebellar flocculus. However, there may be still some differencesin expressions 24 h after the stationary rota-rod task between thenaive and trained mice. Our results indicate that the analysis of

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c-fos and jun-B expression patterns is a useful tool for studying
cerebellar activity, which may play a role complementary to
imaging studies such as functional MRI, because of the fineresolution at the cellular level, in understanding the sequential
events of motor learning in the cerebellum.

Materials and Methods
The expression of eight IEGs was first examined in the cerebellumof naive WT mice, that is, mice that did not experience any
motor tasks but were allowed to move freely. The IEG expression
patterns were examined in the mice that performed a rotarod task (Experiment 1). Next, we compared c-fos and jun-B
mRNA expression levels, in the cerebellar hemisphere, especiallythe flocculus among the three genotypes of mice: D1R KO,
D2R KO, and WT mice (Experiment 2). Additionally, we
examined the expressions patterns of c-fos and jun-B in the mice
30 min and 24 h after the stationary rota-rod tasks and 1-weekhandling.
For Experiment 1, adult male mice (8–9 week old, n = 5,C57BL/6J, Charles river laboratories Japan Inc.) were purchased.
For Experiment 2, mice lacking either D1R (n = 7) or D2R(n = 9) were generated in accordance with the protocol
previously published (Yamaguchi et al., 1996; Tran et al., 2008)and backcrossed for up to 10 generations with C57BL/6J (CLEA
Japan Inc.) mice. Their genotypes were determined by PCR
analysis of genomic DNA extracted from the tail of each mouse.
As a control, C57BL/6J WT mice were purchased. All mice
were housed individually in a plastic cage under 12 h light/darkcycle (9:00–21:00) and given food (Rodent Diet CA-1, CE-2,
CLEA Japan Inc.) and water ad libitum. To encourage foodintake to maintain their health (Drago et al., 1994; McNamara
et al., 2003), D1R KO mice were additionally given palatable
food (Rodent Diet B-F, CLEA Japan Inc.) on the floor of theircages. All the experiments were performed in accordance withthe guidelines of the National Institutes of Health and theMinistry of Education, Culture, Sports, Science and Technology(MEXT) of Japan, and were approved by the Institutional Animal
Care and Use Committees of the National Institutes of Natural
Sciences, Kitasato University School of Medicine, and Frontier
Biosciences of Osaka University. We made all efforts to minimize
the number of animals used and the incidence or severity of
distress experienced by the animals.

Motor Behavior Tasks
In Experiment 1, WT naive mice were initially group housed for
1 week until they were segregated and individually housed during
motor task training and testing as described in the Results. In
Experiment 2, the same mice that experienced series of two typesof motor tasks (rota-rod and Step-Wheel tasks) over a period of
4 months as described previously were used (Nakamura et al.,2014, detailed history is shown in Figure 3 and Supplementary
chart 1). In these series of experiments, the mice were divided
into two groups for the rota-rod tasks: fast-slow (15–5 rpm)
and slow-fast (5–15 rpm) groups (Nakamura et al., 2014). The
mice were sacrificed at 10 months of age. We examined theexpression patterns of c-fos and jun-B in the WT mice that did

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not previously experience any motor task at 30 min (n = 3)
and 24 h (n = 3) after the stationary (0 rpm) rota-rod task. The
rota-rod apparatus (MK-660A, Muromachi Kikai Co., Ltd.) wasused. The rod diameter was 3 cm and the rotation speed was
10 rpm (Experiment 1) or 5 rpm (Experiment 2). In the runningrota-rod tasks, a trial was considered when any one of thefollowing three events occurred: The mouse (1) fell, (2) remainedon the rod up to 60 s (Experiment 1) or 120 s (Experiment 2),or (3) clung to the rod for two complete turns, in which themouse was considered to have fallen. The duration (retention
times) that the mouse remained on the rod was recorded as
the score. Five trials (Experiment 1) or three trials (Experiment2) per day were performed and the interval between the trials
was set at 30–60 s, during which time, the mice were placedin their home cages. Presessions consisted of trials for 1 day(Experiment 1) or 3 days (Experiment 2), during which the mice
were placed on a stationary rod for habituation to the apparatus.
Following the presessions, the running experiments were started.
In Experiment 1, the mice were sacrificed 30 min after the last
trial on day 1 (n = 2). In Experiment 2, the mice were divided
into 3 groups: Pre (WT, n = 2; D1R KO, n = 2; D2R KO, n = 3),
day 1 (WT, n = 3; D1R KO, n = 3; D2R KO, n = 3), and day
5 (WT, n = 3; D1R KO, n = 2; D2R KO, n = 3) groups. The
mice of the Pre group were sacrificed on day 1, that is, the next
day after the presessions (24 h after riding on the stationary rod).
For the days 1 and 5 groups, the mice were sacrificed 30 min after
the last trial. The rota-rod tasks were performed during the lightphase (13:00-19:00). For this series of experiments, we weighed
30 min before the decapitation except for naive mice in which noweighing was done at least for a week before sacrifice.

and 500 µg/ml salmon sperm DNA] for 30 min. DIG-labeledcRNA probes were denatured for 2 min at 82◦ C and chilled on ice.
The sections were covered with hybridization buffer containing
1 µg/ml of the cRNA probes, added dropwise, and coverslipped.
The information of each probes used in this study is shown in
Supplementary Table 1. The slides were incubated overnight (14–18 h) at 72◦ C in a humidified chamber (50% formaldehyde, 5 ×SSC), washed three times with 0.2 × SSC at 72◦ C, and then rinsed
with TBS, pH 7.5 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl). To
detect the hybridized probes, the sections were blocked with 1×blocking solution (Roche) in TBS, pH 7.5, for 30 min, and thenincubated in the blocking solution with an alkaline phosphatase(AP)-conjugated anti-DIG antibody (1:1000 dilution, Roche)
overnight at 4◦ C. The sections were rinsed three times with
TBS, pH 7.5, and equilibrated in TBS, pH 9.5 (100 mM TrisHCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2 ). Enzymatic activity
was visualized by staining with 0.2 mM 5-bromo-4-chloro-3indolyl-phosphate, 0.2 mM nitroblue tetrazolium (NBT/BCIP) in
TBS, pH 9.5, in the dark, until the signal reached a satisfactoryintensity. After a brief wash in PBS and distilled water, thesections were dehydrated in a series of gradually increasing
concentrations of ethanol solutions. After immersion in xylene,
the sections were mounted in Entellan, a new rapid mounting
medium (Merck).

Results
IEG Expression Patterns in the Cerebellum

Handling
To investigate the effects of handling, WT mice (n = 2) that
did not experience any motor tasks were handled for 1 week.
The mice were placed on the palm of an experimenter for about
3 min per day. On the last day, the mice were decapitated 30 min
after they were handled. On the other hand, the naive mice(n = 2) were fed ad libitum, and exposed to external factors as
minimally as possible including experimenters and immediately
decapitated.

In Situ Hybridization (ISH)
In situ hybridization (ISH) was performed as described
previously (Schaeren-Wiemers and Gerfin-Moser, 1993; Lianget al., 2000; Komatsu et al., 2005) with some modifications.
The mice were quickly decapitated. The brain was removed,
embedded in O.C.T. compound (Sakura) and quickly frozen in
isopentane cooled with liquid nitrogen. Sections (10 µm thick)were cut on a cryostat (CM3050, Leica), thaw-mounted onslides (MAS coated glass slides, Matsunami), and air-dried. The
sections were fixed in 4% paraformaldehyde (PFA) in 0.1 M
phosphate buffer (PB, pH 7.3) for 15 min, washed three times
in PBS, pH 7.4, acetylated for 10 min in 0.25% acetic anhydridein 0.1 M triethanolamine-HCl, pH 8.0, and washed three times
with PBS. Prehybridization was performed using hybridizationbuffer [50% formamide, 5 × SSC (20 × SSC in 3 M NaCl, 0.3 M
sodium citrate, pH 7.0), 5× Denhardt’s, 250 µg/ml yeast tRNA,

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Nakam

We examined the IEG expression patterns in the cerebellum.
In our previous study, we examined the expression patternsof IEGs including the fos and jun families in cerebellar slices
without and with treatment with α-amino-3-hydroxy-5-methyl4-isoxazole-propionate (AMPA) and/or 8-bromo-cGMP (8-BrcGMP) (Nakazawa et al., 1993). That study showed that untreatedslices express low levels of IEGs but slices treated with AMPA
and 8-Br-cGMP express high levels of c-fos, jun-B and zif268 (NGFI-A). Following that study, we first examined IEG
expression patterns in the cerebellum. Naive mice that weremoving freely but doing no specific tasks were sacrificed, and the
IEG expression patterns of the fos family (c-fos, fos-B), jun family(jun-B, c-jun, and jun-D), and other IEGs of zif-268 (egr 1, NGFIA), krox 20 (egr 2, NGFI-B), and arc were examined immediately
after the sacrifice (see Materials and Methods). Consistent withthe previous results, we observed little expression of these eight
IEGs in the cerebellum of naive mice (Figures 1, 2). We next
examined the expression patterns of these IEGs immediately
after the mice performed five trials of running rota-rod tasks(Figure 3A). The mice were sacrificed 30 min after the last
trial and the IEG expression patterns in the cerebellum were
examined (Figures 4, 5). Among the members of the fos andjun families, c-fos was expressed in almost all the lobules of thevermis (except lobule 7), and Crus1, Cop and most significantlyin the flocculus in the hemisphere. It is strongly expressed in
the granular and Purkinje layers, and some intense signals wereobserved in the molecular layer. jun-B was also expressed in
the same regions as c-fos was expressed but more preferentiallyin Purkinje cells and some sparsely scattered intense signalsof junsome
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IEG expression in the cerebellum

abeledon ice.
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cerebellar slices, although we previously observed significant zif268 expression (Nakazawa et al., 1993), here we observed only

ticle 38

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FIGURE 1 | Expression patterns of eight IEGs in the cerebellumof naive mice. Vermis (left panel), hemisphere (middle panel),and the magnified image within the box in the middle panel

showing flocculus (right panel). Each row shows expression
patterns of eight IEGs (from top to bottom: c-fos, fos-B, jun-B,
c-jun, jun-D).

a generally moderate expression of zif-268 under the present
condition. krox 20 was also expressed to some extent in theflocculus. Note that the adjacent external layer of the inferior
colliculus expressed arc and zif-268 at certain levels, which may

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Nakam

FIGURE 2 | Expression patterns of eight IEGs in the cerebellum of naive mice. Vermis (left panel), hemisphere (middle panel), and the magnified image
within the box in the middle panel showing flocculus (right panel). Each row shows expression patterns of eight IEGs (from top to bottom: zif-268, krox 20, arc).

be used as internal controls for evaluating the IEG expression
levels in the cerebellum. Overall, c-fos and jun-B among the IEGs
examined in the flocculus showed most prominent expressions
immediately (30 min) after the rota-rod running tasks. Therefore,
hereinafter, we focused on the expression patterns of c-fos andjun-B in the flocculus.

c-fos and jun-B expression Patterns in Stationary
Rota-Rod Task in Naive Mice
As we mentioned in Introduction, we were interested in thefunctional relationship between the cerebellum and the striatum
and explored a model experimental system. Dopamine receptors
are distributed in certain brain regions (Gingrich and Caron,
1993). However, our previous studies demonstrated that D1R
KO and D2R KO mice showed distinguished motor impairments(Nakamura et al., 2014). In the running rota-rod tasks, theimpairments were largely similar to those resulting from local
ablation of D1R neurons and D2R neurons in DLS and DMS(Durieux et al., 2012), respectively. These results suggest thatwhen using D1R KO and D2R KO mice in the rota-rod
tasks, the main target is the striatum. We therefore used D1R
KO and D2R KO mice to examine the cerebellar flocculus

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IEG (c-fos and jun-B) expression patterns and the relationshipbetween the striatum and the cerebellum in relation to motor
learning.
We first examined the expression patterns of c-fos and junB in the stationary rota-rod task. In this task, a mouse wasplaced on a non-rotating bar with a thickness about half thelength of the mouse’s body. This task was not too difficult formice to perform, and not only WT mice but also D1R KO
and D2R KO mice were able to stay for the time examined(Supplementary Figure 2). Nonetheless, this task requires mice tobalance on the bar and therefore activates related brain regions.
In parallel, we first examined the c-fos and jun-B expression
patterns in the cerebellum of WT mice after they were handled
for 1 week (Figure 6). Herein, from Figures 6–10 for all the
ISH data, we examined 6–13 sections of the cerebellums ofindividual mice under each motor condition as described foreach figure. We examined two to five sections of the vermis
and four to nine sections of the hemisphere in each mouse, andhere used the sections that showed the highest signal intensityof the IEG expression in the flocculus among the sections of thecerebellum under each condition. We were surprised to find highexpression levels of c-fos and jun-B in the cerebellum immediately

(30 m
mode
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jun-B
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WT n
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IEG expression in the cerebellum

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d june waslf theult forR KO
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FIGURE 3 | Experimental schedules. Schematic experimental proceduresfor Experiments 1 and 2. (A) Experiment 1. (B) Experiment 2, WT, D1R KO,
and D2R KO mice, training schedule. (C) Experiment 2, sampling schedule.
(D) Experiment 2, 30 min and 24 h after stationary rota-rod task.

Abbreviations: nor Br, normal breeding; Se., session group; RR, rota-rod;
SW, Step-Wheel; w, week; d, day; h, hour; m, minute; s, second. The widthand height of the black bar represent task time and speed of rota-rod,
respectively.

(30 min) after handling (Figure 6). In the hemisphere, we foundmoderately high expression levels in Crus1, and Cop and highexpression levels in the flocculus. The expressions of c-fos andjun-B were probably induced by the attempts of mice to maintaintheir balance on the palm of the hand during the handling. The
expression patterns of c-fos and jun-B appeared to be overallsimilar between the conditions (handling, stationary and runningrota-rod), although there may have been some differences. We
also found the expression of these genes not only immediately(30 min) after but also 24 h after the stationary rota-rod task for
WT naive mice (Figures 3D, 7A,B) and for the same duration in
the D1R KO, D2R KO, WT trained mice (Figures 3B,C, 8, three
sessions as the trained mice). The expressions of c-fos and jun-Bin the flocculus even 24 h after the stationary rota-rod task was
unexpected because IEG expressions would not last for such a

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long time. We weighed the mice 30 min prior to increase theirsacrifice 24 h after the stationary rota-rod task. The weighing
involved placing the mice on the scales of a balance, which may
have activated the vestibular systems. It was indeed the casebecause we observed significant expression of c-fos and jun-B
30 min after only single weighing (Supplementary Figure 3A).
We also examined the expression patterns of these two IEGs 24 hafter the stationary rota-rod task in the mice that experienced
several sessions of the wheel running and rota-rod tasks (rotatingrota-rod and Step-Wheel tasks, see Materials and Methods fordetails, Figure 8). In the naive mice, which only experienced thestationary rota-rod task, the expression levels of c-fos and jun-B
were reduced to some extent but were still significant. In the micethat experienced several sessions of motor tasks, the expressions,in particular that of jun-B, appeared to be more sustained

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Nakam

FIGU
mice
that p

Doyon et al. (2009) proposed that the basal ganglia arefunctionally related to motor learning. They propose that motor
sequence learning and adaptation progress with time through

the “
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FIGURE 4 | Expression patterns of eight IEGs in the cerebellum of mice that performed rota-rod tasks. IEG expression patterns in the mice that performed
three trials of the rota-rod tasks (see Materials and Methods). The figures are lined up in the same order as in Figure 1.

c-fos and jun-B Expression Patterns in Stationary
and Rotating Rota-Rod-Tasks In Trained Micethan those in the naive mice (Figures 7, 8). We also noticed
that the expression pattern of c-fos and jun-B may be different
between those of 30 min after only weighing in naive mice and24 h after stationary rota-rod in trained mice (Supplementary
Figures 4, 5).

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IEG expression in the cerebellum

FIGURE 5 | Expression patterns of eight IEGs in the cerebellum of
mice that performed rota-rod tasks. IEG expression patterns in the micethat performed three trials of the rota-rod tasks (see Materials and Methods).

a are
motor
rough

the “consolidation” and “automatization” processes. During
the course of these processes, the basal ganglia, cerebellum,
hippocampus and cerebral cortex interact with each other, and,with time, the sites of plasticity are shifted to the cerebral
cortex and striatum for motor sequence learning and the cerebral
cortex and cerebellum for motor adaptation. We thus examinedthe cerebellar IEG expression patterns 24 h after the stationaryrota-rod task in the mice that experienced several motor tasks(rotating rota-rod and Step-Wheel tasks, see Materials andMethods for details) in order to determine whether there areany differences in cerebellar activity among the three different
genotypes of mice immediately (30 min) after they performed
running rota-rod tasks on days 1 and 5 (Figures 9, 10).

ticle 38

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med

nary
e

The figures are lined up in the same order as in Figure 2. The last (4th) row
shows the signals hybridized with a sense probe of c-fos, indicating thebackground level.

Figures 9, 10 show the expression patterns of c-fos 24 h after
the stationary rota-rod task in the three genotypes of mice after
the rota-rod tasks on days 1 and 5, respectively. We observed
significant expressions of c-fos in the hemisphere even 24 hafter the stationary rota-rod task. Among the regions in thehemisphere, the expression in the flocculus was most significant.
The c-fos expression is generally similar between days of 1 and5. Figures 9, 10 show cerebellar jun-B expression patterns in
WT mice. jun-B was expressed in the cerebellar vermis, andCrus1 and Cop of cerebellar hemisphere. Although jun-B was
expressed to some extent in the molecular layer and at a lower
level in the granular layers, the expression was most prominent
in Purkinje cells. jun-B expression in the Purkinje cells in the8－ 339 －

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IEG expression in the cerebellum

Nakam

FIGURE 6 | Expression levels of c-fos and jun-B mRNAs in the
cerebellum of mice handled for a week. (A) Mice handled for a week (seeMaterials and Methods). (B) Naive mice that were allowed to move freely but
were not handled by humans. Sections of the hemisphere that showed thehighest signal intensity for each gene are shown (left panel), Right panels show
magnified images of floccular signals enclosed in a box in the top image of theleft panel.

flocculus seemed to be similarly enhanced in the trained mice
24 h after the stationary rota-rod task in contrast to the naive
mice, and immediately after the running rota-rod tasks on days 1
and 5.
D1R KO, D2R KO, and WT mice expressed c-fos in the
cerebellar hemisphere, particularly in flocculus 24 h after the

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FIGURE 7 | Expression levels of c-fos and jun-B mRNAs in the
cerebellum of mice after stationary rota-rod task or single weighing.
Mice that did not experience any motor tasks (untrained) were sacrificed (A)30 min, (B) 24 h after the stationary rod task (see Materials and Methods), or(C) 30 min after single weighing. Right panels show magnified images offloccular signals.

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FIGU
cereb

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ng.
A)
, or

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Nakamura et al.

IEG expression in the cerebellum

FIGURE 8 | Continued(B) D1R KO, and (C) D2R KO mice were trained to perform a series ofrota-rod and Step-Wheel tasks (see Materials and Methods). These mice weresacrificed 24 h after the stationary rota-rod task and designated as the Pregroup for the running rota-rod tasks (see Materials and Methods). The left
panels show the sections from the hemisphere that showed the highest signal
intensity among the sections examined in each row. The right panels show themagnified images of the flocculus enclosed in a box in the first row of the left
panel.

stationary rota-rod tasks and on days of 1 and 5 after the rotatingrota-rod tasks. The expression levels of c-fos and jun-B 24 h after
the stationary rota-rod task in D1R KO mice appeared lower thanthose in D2R KO and WT mice, but since we failed in the stainingof ISH in one of D1R KO mice and we were unable to make aconclusion on the significance of this finding. The expression ofc-fos in the granular layer of the flocculus of D2R KO mice might
be more enhanced on day 1 after the running rota-rod tasks
than under the other conditions (Figures 8–10). jun-B expressionin D2R KO mice was also enhanced 24 h after stationaryrota-rod and on days 1 and 5, and may be more enhanced
on day 1. These observation, however, were not statisticallyconfirmed any further owing to the lack of a sufficient numberof mice of these three genotypes available for series of motor
tasks.

Discussion
We examined cerebellar expression patterns of eight different
IEGs in D1R KO, D2R KO, and WT mice following stationary
and rotating rota-rod tasks. c-fos expression was enhanced in all
the lobules of the vermis except lobule 7 and in Crus1, Cop andthe flocculus in the hemisphere, whereas jun-B expression was
only enhanced in some of the Purkinje cells in these regions.
Furthermore, in this study we surprisingly found that c-fos andjun-B in the cerebellar vermis and flocculus were significantly
expressed in mice after handling and 24 h after the stationaryrota-rod task. We also found robust expressions of c-fos and junB after the running rota-rod tasks on days 1 and 5 in D1R KO,
D2R KO, and WT mice.

IEG Expression Patterns in the Cerebellum
We examined the IEG expression patterns in the cerebellum.
Among the eight IEGs we examined, c-fos was significantly
expressed in all lobules of the cerebellar vermis except lobule 7
and in Crus1, Cop and the flocculus of the cerebellar hemisphere.
jun-B was preferentially expressed in the Purkinje cells inthese regions. arc is known to be induced by various typesof neural activities including visual stimulation (e.g., Nakagami
et al., 2013); there is virtually no induction in the cerebellum(Figure 5). zif-268 is also known to be induced upon various
neural activation, but was little induced in the cerebellum(Figure 5). One plausible reason why arc and zif-268 are notinduced in the cerebellum is that cerebellar Purkinje cells do
not express functional NMDA receptors (Perkel et al., 1990;
Llano et al., 1991). For example, arc transcription is regulated

FIGURE 8 | Expression levels of c-fos and jun-B mRNAs in the
cerebellum of trained mice after riding on stationary rota-rod. (A) WT,(Continued)

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IEG expression in the cerebellum

FIGURE 9 | Expression patterns of c-fos and jun-B mRNAs in the
cerebellum of trained mice after rota-rod task on day 1. (A) WT, (B)D1R KO, and (C) D2R KO mice were trained to perform a series of rota-rod
and Step-Wheel tasks. After the presessions (0 rpm, 3 days), the mice
performed rota-rod tasks (5 rpm) and were sacrificed 30 min after the session
on the day 1. The left panels show the sections from the hemisphere, theright panels show magnified images of the part enclosed in a box in the firstrow of the left pane that shows the flocculus. (B) D1R KO mice, the top row
shows c-fos expression of the mice under the fast-slow (15–5 rpm) conditionat the training sessions, and the second row shows that under the slow-fast(5–15 rpm) conditions (see Materials and Methods). Bottom two rows show
jun-B expressions in this order. (C) D2R KO mice, on the day 1 after arotating rota-rod task (5–15 rpm). The 15-5 rpm task was not done for thisgroup of mice (D2R KO, day 1).

by NMDA- or voltage-gated calcium channel (VGCC)- mediatedmembrane depolarization, cAMP-dependent pathways andbrain-derived neurotrophic factor (BDNF)-mediated pathways(Zheng et al., 2009). However, the exact mechanisms underlying
non-expression of arc in the cerebellum need to be elucidated infuture studies.
c-fos mapping showed the regions activated immediately after
the running rota-rod tasks. In the vermis, c-fos was moderately
induced in all the lobules except lobule 7 throughout the
cerebellar vermis. In the hemisphere, significant expressions of cfos were observed in Crus1, Cop, and flocculus. This observation
indicates that the cerebellar vermis plays a central role invisually guided movements (Edge et al., 2003) and receives limb

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Nakam

and visual inputs (see Edge et al., 2003 for reference). Crus1
also receives visuomotor inputs (Edge et al., 2003; Cerminara
et al., 2005). Hindlimb stimulation primary activates the medialaspect of Cop (Santori et al., 1986). Our c-fos mapping resultsin this report well match those of the previous studies thatexamined the cerebellar areas activated by visuomotor inputs.
One new observation in c-fos mapping may be the neuronal
activation in the flocculus in the cerebellar hemisphere after thestationary and running rota-rod tasks. This is probably because
the rota-rod tasks required mice to maintain their balance, which
likely activated eye movements and the vestibular systems. The
vestibular-ocular reflex presumably activates the flocculus (Ito,1982).

FIGU
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and S
rota-r
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IEG expression in the cerebellum

w
on
st

Crus1
minaramedialesultss thatnputs.
uronaler thecause
which
s. Thes (Ito,ticle 38

FIGURE 10 | Expression patterns of c-fos and jun-B mRNAs in the
cerebellum of trained mice after rota-rod task on day 5. (A) WT, (B)D1R KO, and (C) D2R KO mice were trained to perform a series of rota-rod
and Step-Wheel tasks. After Pre sessions (0 rpm, 3 days), mice performedrota-rod tasks (5 rpm) and were sacrificed 30 min after the session on day 5.
The left panels show the sections from the hemisphere, the right panels

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show magnified images of the flocculus enclosed in a box in the top image ofthe left panel, which shows the flocculus. (B) D1R KO and (C) D2R KO mice.
Top row shows the sections from mice under fast-slow (15–5 rpm) conditions
during the training sessions, and the second row shows the sections frommice under slow-fast (5–15 rpm) conditions (see Materials and Methods fordetails).

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c-fos and jun-B Expression Patterns after
Stationary Rota-Rod Task
We first examined the c-fos and jun-B expression patterns in the
cerebellum immediately and 24 h after the mice performed thestationary rota-rod task in which mice were required to stay on
the non-rotating (stationary) rota-rod. This is not a very difficult
task for mice to perform, and both D1R KO and D2R KO mice
were able to stay as long as WT mice did (Supplementary Figure2). However, to our surprise, even 24 h after performing thestationary rota-rod task, we observed significant c-fos and jun-B
expressions in the cerebellum. It was puzzling why the cerebellum
was activated 24 h after the task, during which time the mice did
not perform any specific tasks. One possible explanation for thiswould be as follows: there were other brain regions that wereactivated, which in turn activated the cerebellum. Interaction
among the cerebellum, basal ganglia and cerebral cortex has been
proposed (Doya, 2000; Hikosaka et al., 2002; Doyon et al., 2009;
Bostan et al., 2013). It is therefore possible that the cerebellum
was activated by other brain areas such as the basal ganglia. We
therefore examined the c-fos and jun-B expression patterns in
D1R KO and D2R KO mice after the running rota-rod tasks.
We weighed the mice 30 min before sacrifice after 24 h after
stationary rota-rod tasks. The weighing involved placing the miceon the scales of a balance, which may have activated the vestibular
systems. It was indeed the case as shown in Supplementary Figure
3. However, the enhancement of c-fos and jun-B 24 h after thestationary rota-rod task in the mice that experienced several
sessions of the motor tasks seems to be higher than that of 24 hafter the stationary rota-rod for naive mice (Figures 7, 8). We
also noticed the different IEG expression pattern between after
only single weighing in naive mice and 24 h after stationary rotarod in trained mice although both groups of mice were sacrificed
30 min after the last weighing (Supplementary Figures 4, 5). Thisdifference may reflect the shift of cerebellar activation by forminginternal model within the cerebellum (Bursztyn et al., 2006).
The expression of c-fos did not appear to be altered between
days 1 and 5 after the running rota-rod tasks in D1R KO, D2R
KO, and WT mice. However, there may be some difference in

IEG expression in the cerebellum

Nakam

jun-B expression under our conditions. Durieux et al. (2012)
and Nakamura et al. (2014) demonstrated that D1R KO mice
and, mice with complete ablation of D1R neurons in the
striatum showed decreased learning curve and performanceafter learning of the rota-rod tasks, whereas mice with ablated
D2R neurons in the DMS and the entire striatum showeddecreased initial performance of the rota-rod tasks. It has beenreported that cerebellar activity reflects an acquired internal
model (Imamizu et al., 2000). At the time when the impairmentof striatal functions is severe, the cerebellar region works tocompensate for the impairment and will presumably be mostactivated.
The expression of IEG and the downstream regulated genes
in the striatum of D1R KO, D2R KO mice has been reported
upon pharmacological stimulation (Drago et al., 1996; Aoyama
et al., 2000; Welter et al., 2007). In D1R KO mice cocaine did
not induce c-fos and zif-268 expressions in the striatum (Dragoet al., 1996). In D2R KO mice cocaine did not induce c-fos but
induce zif-268 in the striatum (Welter et al., 2007). In the striatum
D1R is coexpressed with substance P (SP) and D2R is coexpressedwith encephalin (ENK). In D1R KO mice SP expression was
reduced and ENK expression was unaltered. Cocaine induced SP
but not ENK in the striatum of D1R KO mice. In D2 KO mice
ENK expression was increased and SP expression was decreased(Aoyama et al., 2000). In D1R KO mice cocaine induced SP
and but did not affect ENK expression (Drago et al., 1996). In
D2 KO mice cocaine induced SP expression. Therefore, Theseprevious studies indicated that by in large the direct (D1R) andindirect pathways (D2R) are affected as expected as we write in
the Introduction in D1R KO and D2R KO mice in terms of the
IEG expression. Compared to these previously reported resultsin the striatum where the D1R and D2R play direct roles in theregulation of IEGs, in the cerebellum there is little or only indirect
effect in the IEG expression, if any, in D1R and D2R KO mice as
we showed in this paper.
We have been using chemical methods for detecting ISH
because of their high spatial resolution. However, for quantitativeevaluation, chemical detection methods are not as quantitative

as radradiatVario
exact
regard
on da
and D
us to
the sa
contr
It
Purki
is qu
highlyWe p
conju
cerebe1995)
rota-rmotor
differenumb

TABLE 1 | c-fos and jun-B expressions in cerebellar flocculus.
WT
c-fos

D1R KO

D2R KO

jun-B

c-fos

jun-B

c-fos

jun-B

Naïve

−

−

NT

NT

NT

NT

Handling (for 1 week)

++

++

NT

NT

NT

NT

Stationary RR 30 min

+++

++

NT

NT

NT

NT

Stationary RR 24 h after (for naive mice)

++

+

NT

NT

NT

NT

Stationary RR 24 h (for trained mice)

+++

++

++*

+*

+++

++

Running RR Day 1

+++

++

+++

+ + ∼ + + + **

+++

+++

+++

++

+++

++

+++

++

+∼ + +***

+***

NT

NT

NT

NT

Running RR Day 5
Weighing

Summary of the expression levels of c-fos and jun-B in the cerebellar flocculus are represented; −, background level; +, weak positive; ++, positive; + + +, highly positive signal.
* Only one mouse and somewhat weaker staining than other samples. ** Some variations between two mice. *** Uneven expression within floccullus. NT: not tested. Note that all animals

were sacrificed 30 min after final task except for the naive mice that were sacrificed without weighing. The evaluation was done subjectively with agreement of by two of authors (ToruNakamura and Tetsuo Yamamori).

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Impl
Expr
and

In th
cerebethe stexpre
cerebepoint
severacerebe

Refe

Aoyam
in d
anta
Bostanthe
10.1
BursztyNeu
10.1
Cerminlate
10.1
Doya, K
and
438
Doyon
Con
to m
11.0
Drago,
P. E

Frontieebellum

Nakamura et al.

2012)
mice
n the
mance
blatedhowedbeen
ternalrment
rks tomost

as radioisotope detection methods, in which the amount ofradiation is strictly proportional to that of radioactive decay.
Various conditions could affect chemical reactions; therefore,
exact quantitation is difficult without an internal control. In thisregard, our visual evaluation of c-fos expression in the flocculus
on days 1 and 5 after the running rota-rod tasks in D1R KO
and D2R KO mice should be more reliable because it enabled
us to compare the expression with that in other lobules ofthe same section of the cerebellar hemisphere as an internal
control.
It should be noted that jun-B was preferentially expressed in
Purkinje cells after the rota-rod tasks. This expression patternis quite in contrast to that of c-fos, which was particularly
highly expressed in the granular layer and other layers as well.
We previously reported that jun-B is selectively induced by
conjunctive stimulation of climbing fibers and AMPA in the
cerebellum, which mimics cerebellar LTD (Yamamori et al.,1995). jun-B expression level in the mice 24 h after the stationaryrota-rod task may be higher in the mice that experienced several
motor tasks than in the naive mice. However, these apparent
differences should be examined by statistical analysis with a larger
number of mice in future studies.

genes
portedyama
ne didDrago
os butiatum
ressed
n was
ed SP
mice
eased
ed SP
6). InThese) andrite inof theesultsin thedirectice asg ISHtativetative

IEG expression in the cerebellum

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NT
NT
++
+++
++

NT

Supplementary Material
The Supplementary Material for this article can be foundonline at: http://journal.frontiersin.org/article/10.3389/fcell.
2015.00038/abstract

References

ticle 38

NT

We thank Dr. Motoya Katsuki for scientific advice and critical
discussion on the generation of D1R KO and D2R KO mice.
This research is part of a project supported by a Grant-inAid for Scientific Research on Innovative Areas (Neocortical
Organization) (#22123009 to T.Y.), a Grant-in-Aid for Scientific
Research (B) (#26290029 to T.S.), and a Grant-in-Aid for
Scientific Research (C) (#T25430009 to T.K.).

In this study, we applied techniques of IEG mapping in the
cerebellum to detect the altered cerebellar neuronal activity after
the stationary and running rota-rod tasks. We summarized theexpression patterns of c-fos and jun-B in the flocculus in the
cerebellum in Table 1. We consider that there are three main
points in our studies reported in this paper. First, we foundseveral different levels of expressions of c-fos and jun-B in the
cerebellum in various conditions of weighing and handling,e signal.
animalsrs (ToruNT

Acknowledgments

Implications of Studies of c-fos and jun-B
Expression Patterns in the Cerebellum in D1R KO
and D2R KO Mice

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in the trained mice even considering the weighing effect for
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D1R KO, D2R KO, and WT mice after the stationary and runningrota-rod tasks. We did not observe significant differences among
the three genotypes although this does not still exclude thepossibility that there are some differences among them. To
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10.1038/sj.npp.1300008
Nakagami, Y., Watakabe, A., and Yamamori, T. (2013). Monocular inhibition
reveals temporal and spatial changes in gene expression in the primary visualcortex of marmoset. Front. Neural Circuits 7:43. doi: 10.3389/fncir.2013.00043
Nakamura, T., Sato, A., Kitsukawa, T., Momiyama, T., Yamamori, T., and Sasaoka,
T. (2014). Distinct motor impairments of dopamine D1 and D2 receptor
knockout mice revealed by three types of motor behavior. Front. Integr.
Neurosci. 8:56. doi: 10.3389/fnint.2014.00056
Nakazawa, K., Karachot, L., Nakabeppu, Y., and Yamamori, T. (1993). The
conjunctive stimuli that cause long-term desensitization also predominantly
induce c-Fos and Jun-B in cerebellar Purkinje cells. Neuroreport 4, 1275–1278.
doi: 10.1097/00001756-199309000-00017
Perkel, D. J., Hestrin, S., Sah, P., and Nicoll, R. A. (1990). Excitatory
synaptic currents in Purkinje cells. Proc. Biol. Sci. 241, 116–121. doi:
10.1098/rspb.1990.0074

Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2015 Nakamura, Sato, Kitsukawa, Sasaoka and Yamamori. Thisis an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) or licensor are credited and that theoriginal publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.

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ARTICLE
Received 25 Nov 2014 | Accepted 24 Feb 2015 | Published 13 Apr 2015

DOI: 10.1038/ncomms7745

G-CSF supports long-term muscle regenerationin mouse models of muscular dystrophy
Nozomi Hayashiji1, Shinsuke Yuasa1, Yuko Miyagoe-Suzuki2, Mie Hara1, Naoki Ito2, Hisayuki Hashimoto1,
Dai Kusumoto1, Tomohisa Seki1, Shugo Tohyama1, Masaki Kodaira1, Akira Kunitomi1, Shin Kashimura1,
Makoto Takei1, Yuki Saito1, Shinichiro Okata1, Toru Egashira1, Jin Endo1, Toshikuni Sasaoka3,
Shin’ichi Takeda2 & Keiichi Fukuda1

Duchenne muscular dystrophy (DMD) is a chronic and life-threatening disease that is initially
supported by muscle regeneration but eventually shows satellite cell exhaustion andmuscular dysfunction. The life-long maintenance of skeletal muscle homoeostasis requires
the satellite stem cell pool to be preserved. Asymmetric cell division plays a pivotal role in themaintenance of the satellite cell pool. Here we show that granulocyte colony-stimulating
factor receptor (G-CSFR) is asymmetrically expressed in activated satellite cells. G-CSF
positively affects the satellite cell population during multiple stages of differentiation in ex vivo
cultured ﬁbres. G-CSF could be important in developing an effective therapy for DMD basedon its potential to modulate the supply of multiple stages of regenerated myocytes. This study
shows that the G-CSF–G-CSFR axis is fundamentally important for long-term muscle
regeneration, functional maintenance and lifespan extension in mouse models of DMD withvarying severities.

1 Department of Cardiology, Keio University School of Medicine, 35-Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan. 2 Department of Molecular Therapy,
National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan. 3 Department of Comparative and Experimental
Medicine, Brain Research Institute, Niigata University, Niigata 951-8585, Japan. Correspondence and requests for materials should be addressed to
S.Y. (email: yuasa@a8.keio.jp).

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dult skeletal muscle has its own stem cell population
known as satellite cells (SCs). After muscle injury,
quiescent SCs are activated and then proliferate anddifferentiate into mature skeletal muscle to ensure that muscle
function is recovered. SCs are at the top of the hierarchy ofdifferentiating stages in adult myocytes, followed by myoblasts,
immature myocytes and matured myocytes. Thus, maintaining
the homoeostasis of skeletal muscle function over the long term
requires a well-preserved population of SCs. However, SCs arestill heterogenic, and among the distinct differentiation stages, thesatellite stem cell population must be maintained to preserve theirstemness, that is, long-term self-renewal and differentiation
abilities. Accumulating data indicate that SCs maintain their
population by asymmetric cell division1–4. The different
differentiation stages of SCs are distinguishable by theexpression of speciﬁc markers such as PAX7 and MYOD;
PAX7 þ /MYOD � cells are quiescent, PAX7 þ /MYOD þ cells
are activated and PAX7 � /MYOD þ cells are differentiating.
Interestingly, some activated SCs exhibit asymmetric MYOD
distribution within the cells, and each daughter cell is either
PAX7 þ /MYOD þ or PAX7 þ /MYOD � , the latter of which
retains the quiescent state5. Such evidence indicated thatasymmetric protein distribution produces asymmetric cell
division, which has a critical role in maintaining the SC
population. In our previous screen for myocyte differentiationpromoting factors6,7, we noted markedly elevated expression of
granulocyte colony-stimulating factor receptor (G-CSFR, encodedby csf3r) in the developing somite8. Furthermore, G-CSFR wastransiently expressed in regenerating myocytes of adult injured
skeletal muscle, and extrinsic G-CSF supported short-termmuscle regeneration in cardiotoxin-induced skeletal muscle
injury9 and crush injury10. However, although we found high
G-CSFR expression in regenerating immature myocytes, whether
G-CSFR would be expressed in SCs and whether G-CSF
signalling would affect the SC population in skeletal muscle
remain unclear.
Duchenne muscular dystrophy (DMD) is one of the mostcommon inheritable muscle diseases in humans11,12. It is an
X-linked progressive disease that affects approximately one in
3,500 male live births13. DMD is caused by a mutation in the geneencoding dystrophin, which links the internal muscle cytoskeleton
to the extracellular matrix, enabling the lateral transmission offorce from within muscle cells to the surrounding matrix.
Mutations in the dystrophin gene lead to dystrophin deﬁciency
at the myoﬁbre membrane and progressive muscle ﬁbre
degeneration14,15. The absence of dystrophin increases thefragility of the sarcolemma, which is susceptible to even mild
stress16. Subsequent injury results in myoﬁbre necrosis,
followed by repetitive degeneration and regeneration supported
by innate SCs. In DMD, muscle regeneration initially supports
the mutation-driven functional impairment; however, suchregeneration is eventually attenuated due to stem cell
exhaustion17. Therapy that induces effective long-termskeletal muscle regeneration is, therefore, crucially needed
for DMD.
Long-term muscle regenerative therapy for conditions such as
DMD relies on increasing the pool of functional myocytes andmaintaining the pool of SCs. We showed that G-CSFR isasymmetrically distributed in activated SCs, and that G-CSF
increases the population of SCs via the G-CSF–G-CSFR axis in
ex vivo cultured myoﬁbres. On the basis of this, we hypothesizedthat G-CSF could be important in developing an effective therapy
for DMD based on its potential to modulate the supply of
multiple stages of regenerated myocytes. It is surprising that
haploinsufﬁciency of G-CSFR leads to early lethality in DMD
model mice, indicating that dose reduction of the G-CSF/G-CSFR
2

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system could not support the balance between degeneration andregeneration. We also showed that G-CSF administration in micewith severe DMD markedly improves the phenotype, including
muscle pathology, functioning and lifespan. Together, our datasuggested that the G-CSF–G-CSFR axis is fundamentally
important for long-term muscle regeneration, functional maintenance and lifespan extension in DMD mouse models withvarying severities.
Results
G-CSFR is expressed in activated SCs. To explore the precise
expression pattern of G-CSFR in skeletal muscle, we examined
the SCs on isolated myoﬁbres from the extensor digitorum longus(EDL) muscles of wild-type mice. This model preserves thephysiological behaviour of SCs through activation, proliferationand differentiation on myoﬁbres. In the isolated myoﬁbres,
PAX7 þ quiescent SCs showed no G-CSFR expression byimmunostaining at the time of myoﬁbre isolation (Fig. 1a).
G-CSFR expression was observed in the PAX7 þ cells after 6 h(Fig. 1a,c), when MYOD expression also appeared (Fig. 1b;
Supplementary Fig. 1a). G-CSFR expression was still apparent at
48 h in the MYOGENIN þ SCs, which are committed to differentiation (Supplementary Fig. 1b,c)18. By 24 and 48 h, the numberof G-CSFR-expressing cells increased to B94% of PAX7 þ cells(Fig. 1c).
Asymmetrically expressed G-CSFR in activated SCs.
Unexpectedly, we found that G-CSFR is asymmetrically distributed within migrating SCs obtained from isolated myoﬁbres at
day 5 (Fig. 2a). This asymmetric distribution of G-CSFR was
apparent in B20% of PAX7 þ SCs at 72 h (Fig. 2b). Syndecan-4(SDC4) was expressed in the activated SCs until mature myocyte
fusion occurred; thus, paired SDC4 þ cells were characterized as
the daughter cells of SC division19,20. The majority of paired
SDC4 þ cells showed equivalent expression of G-CSFR (94.4%;
Fig. 2c), but in the remaining 5.6% of paired SDC4 þ cells,
G-CSFR was detectable in only one daughter cell (Fig. 2d).
Similarly, single SDC4 þ SCs either express G-CSFR or not(Fig. 2e,f). In B5% of paired PAX7 þ /SDC4 þ cells, one
daughter cell highly expressed G-CSFR; in the other daughter
cell, G-CSFR expression was not observed (Fig. 2g). In addition,
in B5% of paired PAX7 þ cells, one G-CSFR þ daughter cellhighly expressed MYOD, whereas no MYOD expression observedin the other (Fig. 2h). These data suggested that G-CSFR isexpressed from the activation of SCs through asymmetrical
distribution during the ﬁrst divisions but is not expressed in the
self-renewing PAX7 þ /MYOD � SCs (Supplementary Fig. 2).
G-CSF increases SCs via the G-CSF–G-CSFR axis. To elucidate
whether G-CSFR signalling affects the populations of SCs, we
examined the PAX7 þ cell population in cultured myoﬁbres fromwild-type and G-CSFR-mutated (csf3r � / � ) mice. At 0 and 24 h,there was no signiﬁcant difference in the number of PAX7 þ cells
between wild-type and csf3r � / � mice; however, at 48 h, thecsf3r � / � mice showed a signiﬁcantly smaller number of
PAX7 þ cells than that of wild-type mice (Fig. 3a,b). At 24 h,most PAX7 þ cells express MYOD, are activated and thereafter
proliferate and commit to differentiation21–23. In the csf3r � / �
mice, the number of MYOD þ /PAX7 þ cells was signiﬁcantly
decreased in PAX7 þ cells activated for 24 h (Fig. 3c,d),suggesting that the genetic ablation of G-CSFR signalling
negatively affects the population of activated SCs. We thenexamined the direct role of G-CSF on SCs from wild-type mice.
At 72 h, the subpopulation of activated SCs had lost MYOD
expression in vivo and on cultured myoﬁbres21–23. Surprisingly,

G-C
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(hours)Figure 1 | G-CSFR is expressed in activated satellite cells. (a) Immunostaining of satellite cells on myoﬁbres isolated from EDL muscles for PAX7(red) and G-CSFR (green), including nuclei stained with DAPI (blue) and bright-ﬁeld images, at 0 h (T ¼ 0), 6 h (T ¼ 6) and 12 h (T ¼ 12). Scale bar, 10 mm.
(b) Immunostaining of satellite cells on myoﬁbres for MYOD (red) and G-CSFR (green) at 6 h (T ¼ 6), 24 h (T ¼ 24) and 48 h (T ¼ 48). Scale bar, 10 mm.
(c) The G-CSFR þ /PAX7 þ cell ratio at 0, 6, 12, 24 and 48 h (T ¼ 0: n ¼ 53; T ¼ 6: n ¼ 73; T ¼ 12: n ¼ 45; T ¼ 24: n ¼ 41; T ¼ 48: n ¼ 297; each myoﬁbre:n ¼ 20–30). All myoﬁbres were obtained from B10-week-old wild-type mice. Data are shown as mean±s.d. DAPI, 4’,6-diamidino-2-phenylindole
dihydrochloride.

G-CSF signiﬁcantly increased the population of SCs throughout
the differentiation stages, including self-renewal SCs, as shown by
the PAX7 þ /MYOD � cells at 72 h in wild-type mice; in
addition, csf3r � / � mice showed signiﬁcantly decreased
numbers of SCs throughout the differentiation stages (Fig. 3e,f;Supplementary Table 1). To elucidate the effect of G-CSF on the

population of PAX7 þ cells in vivo, we examined SC behaviour ina cardiotoxin injury model (Fig. 3g). In the csf3r � / � mice, thepopulation of PAX7 þ -expressing cells was signiﬁcantly
decreased at 14 days after cardiotoxin injection (Fig. 3h,i),suggesting that genetic G-CSFR signal ablation negatively affects
the population of PAX7 þ cells in vivo.

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SDC4

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G-CSFR

PAX7

SDC4

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PAX7

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5 µm
Figure 2 | G-CSFR is asymmetrically expressed in activated satellite cells. (a) Immunostaining of satellite cells for MYOD (red), G-CSFR (green)
and DAPI (blue) at day 5. Scale bar, 20 mm. (b) Immunostaining of satellite cells for G-CSFR (purple), PAX7 (green) and DAPI (blue) at day 3. Scale bar,
5 mm. (c) Immunostaining of dividing satellite cells on myoﬁbres for G-CSFR (green), SDC4 (red) and DAPI (blue), as well as bright-ﬁeld (BF) images,
at 48 h (satellite cells with symmetric G-CSFR expression: n ¼ 51; satellite cells with asymmetric expression: n ¼ 3). Scale bar, 10 mm. (d) Immunostaining ofdividing satellite cells on myoﬁbres for G-CSFR (green), SDC4 (red) and DAPI (blue), as well as BF images, at 48 h. Scale bar, 10 mm. (e,f) Immunostainingof satellite cells on myoﬁbres for SDC4 (red), G-CSFR (green) and DAPI (blue), as well as BF images, at 48 h. Scale bar, 10 mm. (g) Immunostainingof cultured dividing satellite cells for G-CSFR (purple), PAX7 (red), SDC4 (green) and DAPI (blue). Scale bar, 5 mm. (h) Immunostaining of cultured dividing
satellite cells for G-CSFR (purple), PAX7 (red), MYOD (green) and DAPI (blue) at day 3 (among 273 dividing satellite cells, dividing satellite cellswith symmetric G-CSFR expression: n ¼ 5; dividing satellite cells with asymmetric expression: n ¼ 268). Scale bar, 5 mm. All myoﬁbres were obtained
from B10-week-old wild-type mice. DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.
4

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Figure 3 | G-CSF increases satellite cells in multiple differentiation stages. (a) Immunostaining of satellite cells on myoﬁbres of B10-week-old WT andcsf3r � / � mice for PAX7 (red) and DAPI (blue) at 48 h. Scale bar, 50 mm. (b) Time course of the number of satellite cells (PAX7 þ ) at 0, 24 and 48 h per
myoﬁbre of WT and csf3r �/ � mice. Data are shown as mean±s.d. **Po0.01; equal variance Student’s t-test. Each ﬁbre number: n ¼ 20–29.
(c) Immunostaining of satellite cells on myoﬁbres of WT and csf3r � / � mice for PAX7 (red), MYOD (green) and DAPI (blue) at 24 h. Scale bar, 50 mm.
(d) The percentage of MYOD þ / PAX7 þ satellite cells at 24 h. Data are shown as mean±s.d. **Po0.01; unequal variance Student’s t-test. Eachﬁbre number: n ¼ 29). (e) Immunostaining of satellite cells on myoﬁbres of WT mice administered control and G-CSF and csf3r �/ � mice after being
cultured for 72 h for PAX7 (red), MYOD (green) and DAPI (blue), as well as bright-ﬁeld images. Scale bar, 50 mm. (f) Quantiﬁcation of the number of
PAX7 � /MYOD þ (green), PAX7 þ /MYOD þ (red) or PAX7 þ /MYOD � (blue) satellite cells per myoﬁbre. Data are shown as mean±s.d. One-way
analysis of variance with Bonferroni’s post hoc test. Quantiﬁcation information in Supplementary Table 1. (g) Time course of cardiotoxin (CTX) injectionand examination for 8-week-old WT and csf3r � / � mice. (h) Immunostaining of cross-sections of TA muscle of WT and csf3r �/ � mice at 14 days after
CTX injection for laminin, PAX7 and DAPI. Upper panel: merged with laminin (green), PAX7 (red) and DAPI (blue). Lower panel: merged with laminin(green) and DAPI (white). Arrows indicate PAX7 þ /DAPI þ cells. Scale bar, 20 mm. (i) Number of PAX7 þ /DAPI þ cells per 100 myocytes of WT andcsf3r � / � mice. Data are shown as mean±s.d. **Po0.01; unequal variance Student’s t-test. n ¼ 5–6 per group. DAPI, 4’,6-diamidino-2-phenylindole
dihydrochloride; WT, wild type.

d

tions

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Figure 4 | G-CSF increases satellite cells through the G-CSF–G-CSFR–JNK axis. (a) Immunostaining of satellite cells for G-CSFR (purple), PAX7 (red),pJNK (green) and DAPI (blue) at day 3. Scale bar, 5 mm. (b) Immunostaining of dividing satellite cells for G-CSFR (purple), PAX7 (red), pJNK (green)
and DAPI (blue) at day 3. Scale bar, 5 mm. (c) Immunostaining of satellite cells on myoﬁbres of WT mice for PAX7 (red), MYOD (green) and DAPI (blue),as well as bright-ﬁeld images, following JNK inhibitor administration with or without G-CSF at 72 h. Scale bar, 50 mm. (d) Quantiﬁcation of the number of
PAX7 � /MYOD þ (green), PAX7 þ /MYOD þ (red) or PAX7 þ /MYOD � (blue) satellite cells per myoﬁbre. Data are shown as mean±s.d. One-way
analysis of variance with Bonferroni’s post hoc test. Quantiﬁcation information in Supplementary Table 1. DAPI, 4’,6-diamidino-2-phenylindole
dihydrochloride.

We previously showed that G-CSF apparently activates JNK
and p38 signalling in cultured myoblasts9. Therefore, we
examined whether these pathways are active in activated SCs.
Immunostaining for pJNK and pp38 revealed that both JNK and
p38 are activated in G-CSFR þ SCs (Fig. 4a; Supplementary
Fig. 3a). The p38 pathway is required for SC activation and
MYOD induction following proliferation24; herein, p38 inhibitionin single myoﬁbres decreased the population of PAX7 � /MYOD þ SCs with or without G-CSF (Supplementary
Fig. 3b,c; Supplementary Table 1). Although JNK signalling is
involved in myoblast differentiation25, its role in SC behaviour
remains unclear. In paired PAX7 þ cells, the G-CSFR-expressing
daughter cells highly expressed activated JNK, whereas the otherdaughter cells without G-CSFR expression did not (Fig. 4b). JNK
inhibition also strongly reduced the number of PAX7 þ /MYOD � and PAX7 þ /MYOD þ cells in single myoﬁbres(Fig. 4c,d; Supplementary Table 1). These data indicated that
G-CSF increases both activated and self-renewal SCs through the
G-CSF–G-CSFR–JNK axis, suggesting that G-CSF could increaselong-term regeneration in chronic skeletal muscle injury throughmaintenance of the SC pool.
G-CSF increases muscle regeneration in mdx mice. Patientswith DMD show progressive physical impairment leading to thedeath by 20–30 years of age, whereas a mouse model of DMD(mdx mice) harbouring a dystrophin mutation has minimal
physical impairment and a normal lifespan26. Indeed, mdx miceappear grossly normal (Supplementary Fig. 4a). However,pathological analysis revealed apparent muscle degeneration
and regeneration in these mdx mice from 3 to 5 weeks of age
6(Supplementary Fig. 4b). Myocyte numbers in the tibialis anterior(TA) muscle were decreased at 3 weeks of age, but were thereafter
recovered (Supplementary Fig. 4c). Generally, healthy myocytes
have peripherally located nuclei, and regenerating andregenerated myocytes have central nuclei. The mdx mice
myocytes showed central nuclei from 4 weeks of age andincreased in number up to 12 weeks of age (Supplementary
Fig. 4d). These data suggested initial injury to the muscle ﬁbreswith subsequent regeneration to an almost normal state in
the mdx mice. The histological observation of TA muscle
showed strong G-CSFR expression in regenerating myocytes(Supplementary Fig. 4e), implicating the possible role of
G-CSF in the muscle regeneration process. We then examined
whether G-CSF administration would improve skeletal muscle
regeneration in mdx mice. We examined the gross appearance
and functional differences following daily intraperitoneal (i.p.)
G-CSF injections from 3 to 5 weeks of age (Fig. 5a).
Immunostaining for laminin showed the typically ﬁne cellulararchitecture of TA muscle (Fig. 5b). The number of myocyteswith central nuclei signiﬁcantly increased with G-CSF
administration at 5 weeks of age (Fig. 5c). The BF-45 antibody
recognizes myosin heavy chain present during embryonic andneonatal skeletal muscle development and in newly formed adultregenerating myocytes27. Immunostaining for BF-45 in 4-weekold mice treated with G-CSF revealed signiﬁcantly increased
numbers of regenerating myocytes (Fig. 5d,e). Measuring theindividual cross-sectional area of the myocytes further revealed asigniﬁcantly increased proportion of small myocytes, indicating asubstantial amount of newly formed regenerated myocytes at 5
weeks of age (Fig. 5f). Given that muscle size is a predictive value
for muscle function, the demonstrated increase in the TA muscle

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Figu
(b) I
(c) T
varia
of ag
**Po
of T
Data
at 5
sign
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dihy

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NATU

tionslaminin

k
ee
w
5

k
ee
w
3

k
ee
w
0

(%)

*

40
30

200 µm20

G-CSF administration

100

100 µmExamination
Control
Control

Control

G-CSF

G-CSF

G-CSF

DAPI

laminin

DAPI

laminin

BF-45

MERGE

BF-45

MERGE

BF-45+ myocytes
(%)
**
30
20
10

100 µm0

100 µmMuscle perimeter(mm)

Myocyte size
1000

(mN)

*

600
400

Tetanic force
NS

60

8

800

40

4

20

0

0

4000 <

(n)
6

120

4

*

80

2
31 – 35

26 – 30

40

0

21 – 25(µm2)

Specific force

Exercise tolerance test

15 – 203501 – 4000

3001 – 3500

2501 – 3000

1001 – 2500

1501 – 2000

0

501 – 1000

200
1001 – 1500

rior
after
ytes
and
mice
and
tary
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ned
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ance
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5a).
ular
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and
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eekased
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ed a
ng a
at 5
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scle

Centralnuclear myocytes

mdx

100 – 500

red),
en)
lue),
er of
way

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745

Myocyte number

7745

0
(min)

Figure 5 | G-CSF increases skeletal muscle regeneration in mdx mice. (a) Time course of G-CSF administration and examination in mdx mice.
(b) Immunostaining of cross-sections of TA muscle for laminin at 5 weeks of age mdx mice. Scale bar, 200 mm (upper panel); 100 mm (lower panel).
(c) The percentages of myocytes with central nuclei in cross-sections of TA muscle at 5 weeks of age. Data are shown as mean±s.d. *Po0.05; equalvariance Student’s t-test. n ¼ 8 per group. (d) Immunostaining of cross-sections of TA muscle for BF-45 (red), laminin (green) and DAPI (blue) at 4 weeks
of age. Scale bar, 100 mm. (e) The percentages of BF-45-positive myocytes in cross-sections of TA muscle at 4 weeks of age. Data are shown as mean±s.d.
**Po0.01; unequal variance Student’s t-test. n ¼ 5 per group. (f) The individual ﬁbre cross-sectional area of individual myocytes in cross-sectionsof TA muscles at 5 weeks of age. Data are shown as mean. n ¼ 6 per group. (g) Muscle perimeters of cross-sections of TA muscles at 5 weeks of age.
Data are shown as box plot. Top, maximum; bottom, minimum. *Po0.05; equal variance Student’s t-test. n ¼ 8 per group. (h) Exercise tolerance test results
at 5 weeks of age. n ¼ 9 per group. (i) Tetanic force examination of ex vivo EDL muscle tension at 5 weeks of age. Data are shown as mean±s.d. No
signiﬁcant difference; unequal variance Student’s t-test; n ¼ 6 per group. (j) Speciﬁc forces, which are the ratios of tetanic force to muscle size, measured at
5 weeks of age. Data are shown as mean±s.d. *Po0.05; unequal variance Student’s t-test. n ¼ 5 per group. DAPI, 4’,6-diamidino-2-phenylindole
dihydrochloride; NS, not signiﬁcant.

perimeter in G-CSF-treated mice at 5 weeks of age suggested
enhanced muscle function recovery (Fig. 5g). G-CSF alsosigniﬁcantly improved exercise capacity in the mice by 5 weeks
of age based on in vivo exercise tolerance testing (Fig. 5h). In apure muscle function test, ex vivo tetanic force measurements andspeciﬁc force examinations showed signiﬁcantly improved muscle
tension in the treated mice at 5 weeks of age (Fig. 5i,j).
Next, we examined whether G-CSF treatment would accelerate
the regeneration process following cardiotoxin injury in mdx
mice (Fig. 6a). The number of myocytes with central nuclei
signiﬁcantly increased by G-CSF administration in cardiotoxininjured mdx mice (Fig. 6b,c). The number of BF-45 þ myocytes

was also signiﬁcantly increased by G-CSF treatment (Fig. 6d,e).
The TA muscle perimeter was also increased by G-CSF treatment
in cardiotoxin-injured mdx mice (Fig. 6f). These data suggested
that G-CSF signiﬁcantly improves muscle function throughmyocyte regeneration in mdx mice.
Haploinsufﬁciency of G-CSFR induces lethality in mdx mice.
To clarify the roles of G-CSF in DMD, we next used csf3r � / �
mice, which show a slight haematological disorder and anormal lifespan28,29. We hypothesized that breeding G-CSFR
homozygote-knockout mdx mice (mdx/csf3r � / � ) would produce

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ARTICLE
mice with severely impaired muscle regeneration and function.
Surprisingly, G-CSFR heterozygote mdx (mdx/csf3r þ / � ) mice
showed early partial lethality after birth and late partial lethality

3
ay

NAT

at 3–5 weeks of age (Fig. 7a). These striking data indicatedthe importance of proper G-CSF signalling in DMD. The
mdx, csf3r þ / � and mdx/csf3r þ / � mice were of normal gross

app
mdx
com
of e
of
stain
and

Control

2
ay

D

1
D

0

ay
D

D

ay

mdx

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745(%) 80Centralnuclear myocytes*

60

100 µm40

CTX injection

Examination

20

G-CSF

G-CSF administration

0
100 µmControl

Control G-CSF

G-CSF

BF-45

lamininBF-45

laminin

DAPI

MERGE

DAPI

MERGE

100 µm(%)
60

BF-45+ myocytes

100 µm

(mm)
12

*

40

10

20

8

0

Muscle perimeterNS*

6
Control G-CSF

CTX

Control G-CSF

Control

Figure 6 | G-CSF increases muscle regeneration in mdx mice after CTX injury. (a) Time course of CTX and G-CSF administration, and examination in
8-week-old mdx mice. (b) Haematoxylin and eosin staining of cross-sections of TA muscle at day 3 after CTX injection. Scale bar, 100 mm. (c) The
percentages of central nuclear myocytes in cross-sections of TA muscle at day 3 after CTX injection. Data are shown as mean±s.d. *Po0.05; unequalvariance Student’s t-test. n ¼ 4 per group. (d) Immunostaining of cross-sections of TA muscle for BF-45 (red), laminin (green) and DAPI (blue) at day 3
after CTX injection. Scale bar, 100 mm. (e) The percentages of BF-45-positive myocytes in cross-sections of TA muscle at day 3 after CTX injection.
Data are shown as mean±s.d. *Po0.05; unequal variance Student’s t-test. n ¼ 5 per group. (f) Muscle perimeters of cross-sections of TA muscle at day 3
after CTX injection. Data are shown as box plot. Top, maximum; bottom, minimum. *Po0.05; Student’s t-test. n ¼ 4–6 per group. CTX, cardiotoxin;
DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; NS, not signiﬁcant.

Figure 7 | G-CSF signalling is essential for the survival in mdx mice. (a) Kaplan–Meier survival curve of csf3r þ / � (red, n ¼ 50), mdx (yellow, n ¼ 50)
and mdx/csf3r þ / � (blue, n ¼ 53) mice. (b) Gross appearance of mdx, csf3r þ / � , mdx/csf3r þ / � and WT mice at post-natal day 1. (c) Body weights of
mdx (n ¼ 7), csf3r þ / � (n ¼ 9), mdx/csf3r þ / � (n ¼ 11) and WT (n ¼ 15) mice at post-natal day 1. Data are shown as box plot. Top, maximum; bottom,
minimum. **Po0.01; unequal variance Student’s t-test. (d) Evans blue staining of the diaphragm of mdx, csf3r þ / � , mdx/csf3r þ / � and WT mice at postnatal day 1. (e) Haematoxylin and eosin staining of the diaphragm of mdx, csf3r þ / � , mdx/csf3r þ / � and WT mice at post-natal day 1. Scale bar, 50 mm.
(f) Diaphragm thickness of mdx, csf3r þ / � , mdx/csf3r þ / � and WT mice at post-natal day 1 (n ¼ 5 per group). Data are shown as mean±s.d. **Po0.01;
Student’s t-test; n ¼ 5 per group. (g) Immunostaining of cross-sections of the diaphragm for PAX7 (red), laminin (green) and DAPI (blue) at post-natal day
1 of mdx, csf3r þ / � , mdx/csf3r þ / � and WT mice. Scale bar, 50 mm. (h) Number of PAX7 þ /DAPI þ cells per 100 myocytes at post-natal day 1 of mdx,
csf3r þ / � , mdx/csf3r þ / � and WT mice. Data are shown as mean±s.d. **Po0.01; Student’s t-test; n ¼ 5 per group. (i) Whole-body plethysmography to
examine the respiratory function of individual mice. (j) Breath per min (BPM) in 8-week-old mdx and mdx/csf3r þ / � mice. Data are shown as mean±s.d.
No signiﬁcant difference; unequal variance Student’s t-test; n ¼ 4 per group. (k) Tidal volume of 8-week-old mdx and mdx/csf3r þ / � mice. Data areshown as mean±s.d. No signiﬁcant difference; unequal variance Student’s t-test; n ¼ 4 per group. (l) Body weight growth curves of csf3r þ / � andmdx/csf3r þ / � mice from 2 to 8 weeks of age. Data are shown as mean±s.d. *Po0.05, **Po0.01; unequal variance Student’s t-test; n ¼ 7 per group.
DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; NS, not signiﬁcant; WT, wild type.
8

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7745

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745

ated
The
ross

appearance at post-natal day (P) P1 (Fig. 7b); however,
mdx/csf3r þ / � mice had signiﬁcantly reduced body weights
compared with mdx littermates (Fig. 7c). To elucidate the causeof early lethality, we examined the mice for gross appearanceof the diaphragm and degenerative lesions by Evans blue
staining. Lesions were largely absent from the mdx, csf3r þ / �
and wild-type mice, whereas the mdx/csf3r þ / � mice showed

mdx/ csf3r +/–WTmdx/ csf3r +/–

WT

0.6
0.4
0.2
20

40

csf3r +/–

(g)
1.6

mdx

60 (Day)
mdx/ csf3r +/–Body weight**csf3r +/–

mdx

1.2
0.8

2 mm

0.4
csf3r +/–

mdx/ csf3r +/–

WT

W

T

+/
–
dx
/c
sf
3r
+/
–

mdx

m

cs
f3
r

m

dx

0

50 µm

Diaphragm thickness
**

(µm)

NS

mdx

csf3r +/–

mdx/ csf3r +/–WT

PAX7 laminin DAPIPAX7 laminin DAPIPAX7 laminin DAPIPAX7 laminin DAPI

50 mm

50 mm

50 mm

50 mm

300
200
100
/–
3r
+

W
T

/–

BPM

(ml/g)

Tidal volume

200

0.8

100

0.4

0

0
3r
sf
/c

cs
W
T

m

m

dx

dx
/
m

dx

/c

sf

3r
+

/–

/–
f3
r+

cs

m

dx

0

+/

dx

f3

m

10

r+

dx

20

1.2

m

**

30

300

/–

PAX7 positive cell

–

m
dx

/c

cs

sf

f3

r+

dx

0

Body weight

(g)
25
20

*

15
10
5
0

oup.

tions

csf3r +/–

0.8

qual
ay 3
n.
ay 3

50)
s of
om,
postmm.
0.01;
day
mdx,
hy to
±s.d.
e

mdx

Survival curve

1.0

m

on in

obvious degenerative lesions (Fig. 7d,e). Diaphragm thickness
was also signiﬁcantly decreased in mdx/csf3r þ / � micecompared with mdx littermates (Fig. 7f). The population of
PAX7 þ cells in diaphragm was also signiﬁcantly decreased in
mdx/csf3r þ / � mice (Fig. 7g,h). Whole-body plethysmography
indicated that mdx/csf3r þ / � mice showed mildly impaired
respiratory function (Fig. 7i–k). In addition, the surviving

**

*

2

4

csf3r +/–

6

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－ 355 －

8 (Weeks)

mdx/ csf3r +/–9

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745

P7

P13
leth
with
age
num
decr
Ex
decr
com
cells
(Fig
for

P13
mdx

mdx/ csf3r +/–mdx/ csf3r +/–mdx/ csf3r –/–mdx/ csf3r –/–NAT

mdx/ csf3r +/–100 µm50 µm

5 weeks

Tetanic force(mN)
60

Specific force

*

80

1000

4

20

40

0

0

0

0

mdx/ csf3r +/–mdx

PAX7 laminin DAPI PAX7 laminin DAPI
m

m
dx
dx
/c
sf
3r
+/
–

Pax7+ cells (5 weeks) Pax7+ cells (8 weeks)12128laminin

DAPI

laminin

DAPI

laminin

DAPI

4

4

0

0

–

DAPI

**

*

20 µm

20 µm

/c
dx
m

m

dx

/c

sf

sf

3r

+/

m
dx

laminin8 weeks

5 weeks

8
20 µm

20 µm

–

mdx/ csf3r +/–PAX7 laminin DAPI PAX7 laminin DAPI

*

m

m

m
dx
dx
/c
sf
3r
+/
–
m

m

mdx

m
dx
dx
/c
sf
3r
+/
–

40

m
dx
dx
/c
sf
3r
+/
–

8

+/

**

8 weeks3r

Muscle perimetert
(mm)
12
*

Myocytes number
3000
2000

4 weeksdx

3 weeksm

2 weeks

Figure 8 | Haploinsufﬁciency of G-CSFR reduces muscle regeneration in mdx mice. (a) Gross appearance analyses showing body size differencesamong mdx, mdx/csf3r þ / � and mdx/csf3r � / � mice at post-natal day 7 (left panel) and between mdx/csf3r þ / � and mdx/csf3r � / � mice at post-natal
day 13 (right panel). (b) Haematoxylin and eosin staining of cross-sections of TA muscle from mdx/csf3r þ / � mice from 2 to 8 weeks of age are shown in
the lower (upper panel) and higher (lower panel) magniﬁed images. Scale bar, 100 mm (upper panel); 50 mm (lower panel). (c) The total numbers of
myocytes in cross-sections of TA muscle at 8 weeks. Data are shown as mean±s.d. **Po0.01; unequal variance Student’s t-test; n ¼ 5 per group.
(d) Perimeters of cross-sections of TA muscle at 8 weeks of age. Data are shown as mean±s.d. *Po0.05; unequal variance Student’s t-test; n ¼ 5
per group. (e) Tetanic force examination of ex vivo EDL muscle tension at 8 weeks of age. Data are shown as mean±s.d. *Po0.05; unequal varianceStudent’s t-test; n ¼ 5 per group. (f) Speciﬁc forces, represented by tetanic force per muscle size ratios, at 8 weeks of age. Data are shown as mean±s.d.
*Po0.05; unequal variance Student’s t-test; n ¼ 6 per group. (g) Immunostaining of cross-sections of TA muscle from mdx and mdx/csf3r þ / � miceat 5 and 8 weeks of age for laminin, PAX7 and DAPI. Upper panel: merged with laminin (green), PAX7 (red) and DAPI (blue). Lower panel: mergedwith laminin (green) and DAPI (white). Arrows indicate PAX7 þ /DAPI þ cells. Scale bar, 20 mm. (h) Number of PAX7 þ /DAPI þ cells per 100 myocytesof mdx and mdx/csf3r þ / � mice at 5 and 8 weeks of age. Data are shown as mean±s.d. *Po0.05, **Po0.01; unequal variance Student’s t-test; n ¼ 5 pergroup. DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; NS, not signiﬁcant; WT, wild type.

mdx/csf3r þ / � mice showed consecutive body weight loss
compared with csf3r þ / � mice (Fig. 7i). Because most mdx/
csf3r þ / � mice with a strong phenotype died before 5 weeks ofage and relative healthy mdx/csf3r þ / � mice survive, thedifferences in body weight and respiratory function would bedecreased thereafter.
10

In mdx mice, skeletal muscle degeneration and regeneration isprominent at 3–5 weeks of age. We therefore proposed that
mdx/csf3r þ / � mice might show deteriorating muscle regeneration and function around that age. Gross examination revealed asmaller body size in mdx/csf3r þ / � and mdx/csf3r � / � mice at
P7, and apparently smaller body size in mdx/csf3r � / � mice at

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－ 356 －

Figu
stain
pane
mini
show
lami
4 we
the m
5 we
repr
(j) K
(n ¼

NATU

natal
wn in
of

ce
±s.d.
e
d
ytes
5 per

n is
that
eraed a
e at
e at

tions

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745

P13 (Fig. 8a). Mdx/csf3r þ / � mice that survived the initial early
lethality prevalence showed signiﬁcantly increased degeneration
with decreased numbers of regenerating myocytes at B4 weeks ofage that remained at B8 weeks of age (Fig. 8b). The myocyte
number and TA muscle perimeter were also signiﬁcantly
decreased in mdx/csf3r þ / � mice at 8 weeks of age (Fig. 8c,d).
Ex vivo muscle tension examination also indicated signiﬁcantly
decreased tetanic and speciﬁc forces in the mdx/csf3r þ / � micecompared with mdx mice (Fig. 8e,f). The population of PAX7 þcells was also signiﬁcantly decreased in mdx/csf3r þ / � mice
(Fig. 8g,h). These results conﬁrmed that G-CSF signal is crucial
for skeletal muscle regeneration in mdx mice.

G-CSF supports the survival of severe DMD model mice. The
physically normal appearance of mdx mice could reﬂect a
redundant function of the utrophin gene, a dystrophin paralogue.
The mdx/utrn � / � mice that express neither dystrophin
nor utrophin manifest severe dystrophy resembling human
DMD30,31. These mice showed an apparently small body size
compared with the mdx mice (Supplementary Fig. 5a), withprominent skeletal muscle degeneration that remained to a latestage of post-natal growth (Supplementary Fig. 5b). Total
myocyte number in TA muscle was also decreased from 2 to
5 weeks of age in these mdx/utrn � / � mice, and recovered
thereafter (Supplementary Fig. 5c), and central nuclear myocytes

k

th
ea

k

ee
w

D

10

k
w
8

ee

k
w
5

ee

k

ee
w
3

ee
w
0

Muscle perimeter
G-CSF

Daily

G-CSF
G-CSF

200 µm

Daily
Daily

Twice a week

G-CSF

0

0.4
0.2
0

TA

Gas

Control
G-CSF

G-CSF

SalineMoist muscle weight ratio
*
*

0.6

4

50 µm

Twice a week
Control

0.8

**

8

G-CSF

BF-45

lamininBF-45

laminin

DAPI

Merge

DAPI

Merge
50 µm

50 µm

Quad

Myocyte size

4001 <

3501 – 4000

3001 – 3500

2501 – 3000

2001 – 2500

0201501 – 2000

10(mN) Tetanic force
40
**
30

10001 – 1500

20

600
500
400
300
200
100
0

100 – 500(%) BF-45+ myocytes
30*

501 – 1000

Myocyte number

7745

10
0

(µm2)

Survival curve

1.0

Specific force

0.8

120

0.6

80

0.4

40

0.2
0

*

0
50
Saline

100
G-CSF

150
(Day)

200

250

300

G-CSF from day 100

Figure 9 | G-CSF improves the survival of mice with severe muscular dystrophy. (a) Time course of G-CSF administration. (b) Haematoxylin and eosin
staining of cross-sections of TA muscle from mdx/utrn � / � mice at 5 weeks of age (left: control; right: G-CSF administration). Scale bar, 200 mm (upper
panel); 50 mm (lower panel). (c) Perimeters of cross-sections of TA muscle at 5 weeks of age. Data are shown as box plot. Top, maximum; bottom,
minimum. **Po0.01; unequal variance Student’s t-test; n ¼ 6 per group. (d) Ratios of moist muscle weight to body weight at 5 weeks of age. Data areshown as mean±s.d. *Po0.05; equal variance Student’s t-test; n ¼ 9 per group. (e) Immunostaining of cross-sections of TA muscle for BF-45 (red),laminin (green) and DAPI (blue) at 4 weeks of age. Scale bar, 50 mm. (f) The percentages of BF-45-positive myocytes in cross-sections of TA muscle at
4 weeks of age. Data are shown as mean±s.d. **Po0.01; equal variance Student’s t-test; n ¼ 5 per group. (g) The individual ﬁbre cross-sectional areas ofthe myocytes in TA muscle at 5 weeks of age. Data are shown as mean; n ¼ 6 per group. (h) Tetanic force examination of ex vivo EDL muscle tension at
5 weeks of age is presented. Data are shown as mean±s.d. **Po0.01; unequal variance Student’s t-test; n ¼ 5 per group. (i) Speciﬁc force results,
represented by tetanic force per muscle size ratios. Data are shown as mean±s.d. *Po0.05; unequal variance Student’s t-test; n ¼ 5 per group.
(j) Kaplan–Meier survival curve of mdx/utrn � / � mice without G-CSF (n ¼ 13), with G-CSF (n ¼ 10) or with G-CSF administration starting at day 100(n ¼ 9). DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.
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ARTICLE
were apparent after 3 weeks (Supplementary Fig. 5d). G-CSFR
was also expressed in regenerating myocytes in the mdx/utrn � / �
mice (Supplementary Fig. 5e).
To elucidate whether G-CSF improves skeletal muscle
regeneration in these mice, we i.p. injected G-CSF using several
schedules (Fig. 9a). After daily G-CSF injection from 3 to 5 weeks
of age, muscle size was signiﬁcantly increased in the G-CSFtreated animals at 5 weeks of age (Fig. 9b,c). G-CSF administration also signiﬁcantly increased the moist muscle weight ratio of
the gastrocnemius and quadriceps muscles in mdx/utrn � / � mice
(Fig. 9d), as well as the numbers of regenerated myocytes andtotal myocytes in TA muscle (Supplementary Fig. 6a,b; Fig. 9g).
The number of BF-45 þ myocytes was signiﬁcantly increased by
G-CSF treatment (Fig. 9e,f). The population of PAX7 þ cell was
also signiﬁcantly increased by G-CSF treatment (Supplementary
Fig. 6c,d). Ex vivo muscle functional analysis showed that tetanicand speciﬁc forces were signiﬁcantly increased by G-CSF
administration (Fig. 9h,i). These data suggested that G-CSF
improves skeletal muscle regeneration and muscle function in
mdx/utrn � / � mice.
The mdx/utrn � / � mice died at B3 to 5 weeks of age and after
100 days of age, resulting in 100% lethality by 180 days (Fig. 9j,
blue line). Finally, we showed that long-term G-CSF treatment
could notably improve the survival of mdx/utrn � / � mice, with
G-CSF injected daily from 3 to 5 weeks of age and twice a week
after 10 weeks of age (Fig. 9j, red line). It is clinically important totreat developed DMD; hence, the G-CSF was also injected from
10 weeks of age. G-CSF administration prolonged the survival ofdeveloped DMD model mice (Fig. 9j, yellow line). Whole-bodyplethysmography then indicated that G-CSF administration
signiﬁcantly increased respiratory function, measured by tidal
volume (TV), in the mdx/utrn � / � mice injected daily from 3
to 8 weeks of age (Supplementary Fig. 6e). The G-CSF-treatedmdx/utrn � / � mice also showed improved physical activity at
150 days of age (Supplementary Movie). Although cardiomyopathy is one of the fatal phenotypes of DMD, heart morphology is
not affected by G-CSF treatment (Supplementary Fig. 6f,g). These
data strongly suggested that G-CSF improves skeletal muscle
regeneration and function, and supports the survival of DMD
model mice.
Discussion
Research over many years has sought to understand themechanism of skeletal muscle stem cell behaviour and elucidate
the precise pathogenesis underlying DMD and, in turn, todevelop effective therapies. Although many promising therapeuticstrategies have been developed in animal experiments32, mosthuman clinical trials failed to show signiﬁcant efﬁcacy in patientswith DMD12,14,33–35. In addition, with the exception of
glucocorticoids, beneﬁcial results with immunosuppressantswere inconclusive36–39. Indeed, the precise mechanism bywhich glucocorticoids increase muscle strength in DMD is notknown and its clinical effects are generally not sufﬁcient.
Improved or additional therapies for DMD are therefore
urgently needed. G-CSF is a well-known cytokine that recruits
hematopoietic cells40. The safety and side effects in humans were
widely known from previously established clinical applications,which enable us to use G-CSF early in clinics. Our studies now
highlight apparently encouraging the effects of G-CSF on DMD.
In this work, we showed that G-CSFR is asymmetrically
distributed in some activated SCs, and G-CSF successfullyincreases the population of SCs at the multi-differentiatingstages. We successfully showed that G-CSF administration
markedly improves the phenotype of DMD model mice.
The quiescent state in SC is required for the long-term
maintenance of skeletal muscle41. We showed that G-CSF
12

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NAT

increased the PAX7 þ /MYOD � self-renewal SCs. There arequiescent SCs among PAX7 þ /MYOD � SCs, but it remains
unclear whether G-CSF would affect the quiescent SC population.
Because G-CSFR is not expressed in PAX7 þ /MYOD � SCs,
G-CSF signalling would not be directly involved in the transition
from activated SCs to PAX7 þ /MYOD � SCs. Furthermore, itremains unclear how G-CSF signalling increases the populationof PAX7 þ /MYOD � SCs. And intrinsic differences among
heterogenic satellite stem cells are still unclear and a practicalmarker to distinguish the subpopulations of satellite stem cells
is still missing. Further precise elucidation of molecularmechanisms may directly facilitate control of the quiescent
SC pool.
As a study limitation, we could not directly show that bonemarrow cells would not be involved in the phenotype of DMD
model. To determine whether bone marrow cells have a role inearly lethality of haploinsufﬁciency for G-CSFR in the dystrophic
background, we performed bone marrow transplantation using
mdx/csf3r þ / � mice of B3–4 weeks of age. There is noestablished method of bone marrow transplantation for pups
earlier than those ages. We selected active eight mdx/csf3r þ / �
mice of B3–4 weeks of age and successfully performed bonemarrow transplantation. One of eight mdx/csf3r þ / � mice diedat 3 weeks after wild-type bone marrow transplantation by 8
weeks of age, which is similar to the survival rate of active mdx/
csf3r þ / � mice without bone marrow transplantation. Althoughwild-type bone marrow cells could not rescue the lethality of
mdx/csf3r þ / � mice, bone marrow transplantation would
preferably be performed in mdx/csf3r þ / � mice 0 days of age;alternatively, skeletal muscle-speciﬁc G-CSFR-deﬁcient micewould ideally be generated.
We conﬁrmed that the G-CSF/G-CSFR system is criticallyinvolved in muscle regeneration during the course of DMD inseveral mouse models. Given our ﬁndings, it is surprisingthat haploinsufﬁciency of G-CSFR leads to early lethality in
mdx mice, indicating that dose reduction of the G-CSF/G-CSFR
system cannot support the balance between degeneration
and regeneration in these mouse models. We also showed that
G-CSF administration markedly improves the phenotype of mdx/
utrn � / � mice. Together, our data suggested that G-CSF could
improve the phenotype of human DMD and could consequently
form the basis of novel and effective therapies in the near future.

utrop
mou
comp
the P
GAG
35 cy
deter
csf3r
to wi
allele
both
30 se
for t
react
Japan

Methods

Mice. C57BL/6-background mdx mice were provided by Dr T. Sasaoka (Niigata
University Niigata, Japan). The utrophin-knockout mice were a kind gift fromDr Kay E Davies (University of Oxford, Oxford, UK)31. The dystrophin–utrophin
double-knockout male homozygous (dystrophin � /Y (mdx), utrophin � / � ) mice
were obtained by crossing those heterozygous (mdx, utrophin þ / � ) mice. The
csf3r � / � mice were kindly gifted by Dr Daniel C. Link (Washington University
School of Medicine, St Louis)28. Mice deﬁcient in both dystrophin and csf3r were
derived by the following breeding program: male csf3r-knockout (csf3r � / � ) mice
were mated with female C57B/6 mdx (dystrophin � /X) mice, resulting in all
F1-generation mice being heterozygous for csf3r (csf3r þ / � ) and either
heterozygous for the mdx mutation (dystrophin � /X, females) or hemizygous for the
mdx mutation (dystrophin � /Y, males). All data were obtained from P1 to 20 weeks
of age male mice. All experimental procedures and protocols were approved by the
Animal Care and Use Committee of Keio University and conformed to the NIH
Guidelines for the Care and Use of Laboratory Animals.
Genotyping. DNA was isolated from mouse tails using the Easy-DNA Kit(Invitrogen, CA, USA). PCR analysis to identify mutant dystrophin alleles used aforward primer complementary to mouse dystrophin (50 -CATAGTTATTAATGCATAGATATTCAG-30 ) and reverse primers complementary to the wild-typeallele (50 -GTCACTCAGATAGTTGAAGCCATTTAG-30 ) or mouse mutant dystrophin allele (50 -GTCACTCAGATAGTTGAAGCCATTTAA-30 ). PCR cycling
conditions for both the reactions were as follows: 95 C for 4 min; 34 cycles of 95 C
for 1 min, 55 C for 1 min and 72 C for 1 min, and 72 C for 10 min. Each reaction
yielded a 275-bp product. All PCR reactions were performed using the GoTaq
DNA Polymerase kit (Promega, Madison, WI, USA). PCR analysis to assess the

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－ 358 －

G-CS
in 1
mice
27-g
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treat
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NATU

ARTICLE

7745

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7745

are
ains
ion.
SCs,
tion
e, it
tion
ong
tical
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utrophin-knockout status used a forward primer complementary to exon 7 ofmouse utrophin (50 -GTGAAGGATGTCATGAAAG-30 ) and reverse primers
complementary either to intron 7 (50 -TGAAGTCCGAAAGAGATACC-30 ) or to
the PGK promoter located within the Neo-knockout cassette (50 -ACGAGACTAGTGAGACGTGC-30 ). PCR reactions were carried out under the following conditions:
35 cycles of 94 C for 30 s, 57 C for 30 s and 72 C for 25 s. PCR analysis todetermine mutant csf3r alleles used a forward primer complementary to mouse
csf3r (50 -AGTTCACCAGGCAGGTGAGT-30 ) and reverse primers complementary
to wild-type alleles (50 -GTAGGCCTAGTTCATACCTG-30 ) or mouse mutant csf3r
alleles (50 -TCCAGACTGCCTTGGGAAAA-30 ). The PCR cycling conditions forboth reactions were as follows: 95 C for 4 min, followed by 30 cycles of 95 C for
30 sec, 60 C for 30 sec and 72 C for 45 sec. Each reaction yielded a 279-bp productfor the wild-type allele and a 366-bp product for the mutant csf3r allele. All PCR
reactions were conducted using the Taq DNA Polymerase kit (TaKaRa, Shiga,
Japan).

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G-CSF treatment. G-CSF (Neutrogin, 100 mg; Chugai, Tokyo, Japan) was diluted
in 1 ml of saline (concentration of 100 mg ml � 1). Short-term treatment for mdx
mice involved daily i.p. injections of 5 mg G-CSF from 3 to 5 weeks of age using a
27-gauge needle. Middle-term treatment for mdx/utrn � / � mice involved daily i.p.
injections from 3 to 8 weeks of age at 5 mg using a 27-gauge needle. Long-term
treatment for mdx/utrn � / � mice involved daily i.p. injections from 3 to 5 weeks ofage and twice weekly from 10 weeks of age at 5 mg using a 27-gauge needle. Latetreatment for mdx/utrn � / � mice involved twice weekly i.p. injections from 10
weeks of age at 5 mg using a 27-gauge needle.
Histological analysis. Frozen section of TA muscle was stained by haematoxylin
and eosin to measure the ratio of muscle regeneration. The ﬁbre cross-sectional
area was measured using software (BZ-H1C: Keyence, Osaka, Japan) from lamininstained cross-sections of TA muscle. Micrographs were taken from each section at,
� 10 and � 20 magniﬁcation with a digital camera (BIOREVO: Keyence). Evansblue (Sigma-Aldrich, MO, USA) was dissolved in PBS (0.15 M NaCl, 10 mM
phosphate buffer, pH 7.0). Mice were i.p. injected with 0.5 ml 0.1% Evans blue at P1
using a 27-gauge needle and were killed 3 h later.
Immunoﬂuorescence. All immunoﬂuorescence was carried out on 6-mm-thickfrozen sections, myoﬁbres and SCs. The frozen sections were ﬁxed with acetone(WAKO, Osaka, Japan) for 20 min at � 30 C. Myoﬁbres and SCs were ﬁxed with
4% paraformaldehyde for 20 min at room temperature. All samples were incubatedwith 0.1% Triton X-100 for 5 min at room temperature, washed and then incubatedwith the following primary antibodies: anti-G-CSF receptor (sc-9173, 1:50: Santa
Cruz Biotechnology, CA, USA; sc-323898, 1:100, Santa Cruz Biotechnology), antilaminin 2 (L0663, 1:1000, Sigma-Aldrich), anti-laminin (L9393, 1:1000, SigmaAldrich), anti-BF-45 (1:30, Developmental Studies Hybridoma Bank, IA, USA),anti-MyoD (sc-32758, 1:200, mouse; sc-304, 1:200, Santa Cruz Biotechnology),
anti-Pax7 (sc-81648, 1:100, 1:40, Santa Cruz Biotechnology), anti-myogenin(ab1835, 1:100, Abcam, Camb, UK; sc-576 1:100, Santa Cruz Biotechnology),
anti-syndecan-4 (ab24511, 1:100, Abcam), anti-phospho-SAPK/JNK (#9251 1:100,
rabbit, Cell Signaling Technology, MA, USA) and anti-phospho-p38 (sc-7973,
1:100, mouse, Santa Cruz Biotechnology). After overnight incubation, boundantibodies were visualized with a secondary antibody conjugated with Alexa 488,
Alexa 546 or Alexa 633 (Life Technologies, CA, USA). Nuclei were stained with
4’,6-diamidino-2-phenylindole dihydrochloride (Life technologies). For Pax7
staining, an M.O.M. kit (Vector Laboratories) was used to block the endogenous
mouse IgG. The images were recorded using a confocal laser microscope system(Carl Zeiss, Jena, Germany) and BIOREVO (Keyence).
Muscle force measurements. EDL muscles were isolated and removed from
5-week-old mice. The muscles were carefully mounted in a chamber ﬁlled with PBS(95% O2 and 5% CO2) and maintained at 30 C. One tendon of the muscle was
attached to a steel hook in the chamber, and the other was tied to the lever arm of adual-mode servomotor system (Electronic Stimulator; NIHON KOHDEN, Tokyo,
Japan) via 5-0 surgical silk. The muscles were stretched to the length at which asingle twitch and tetanic force showed the highest amplitude (optimal length; Lo).
The corresponding tetanic force was then measured at 150 Hz over 5-s intervals fora total of 20 min, with a rest period of 120 s to change the buffer. The muscles were
adjusted to the optimum length (Lo) before all force measurements. For comparative purposes, all force measurements were expressed per the total muscle unit
cross-sectional area, which was calculated by dividing the muscle mass by the
product of the length and the mammalian skeletal muscle density (1.056 mg mm � 3).
The speciﬁc force (N cm � 2) was calculated with the muscle density assumed as
1.056 g cm � 3.
Exercise tolerance tests. Mice were subjected to an exhaustion treadmill test byplacing them on the belt of a one-lane motorized treadmill (MK-680S Treadmill for
Rats & Mice; Muromachi Kikai, Tokyo, Japan). The test was started at an incline of
0 at 5 m min � 1 for 5 min; thereafter, the speed was increased by 1 m min � 1 every

minute. The end point of the test was when the mouse remained on the shocker
plate for 420 s.
Whole-body plethysmography. Animals were placed in a free-moving chamberthat was connected to a high-gain differential pressure transducer (Valydine MP45,
Validyne, North Ridge, CA, USA). As the animal breathed, changes in the pressure
were converted to signals representing the TV; these signals were ampliﬁed (BMA
830; CWE, Ardmore, PA, USA), recorded on a strip-chart recorder (Dash 10;
Astro-Med, West Warwick, RI, USA) and stored in a computer with respiratory
acquisition software for analysis. O2 consumption and CO2 production were
measured by the open-circuit method using Beckman OM-14 and LB-2 analysers.
The parameter TV was analysed in real time, then average values were calculated
ﬁve times every 2 min for each serial 3-h period. The TV was normalized by
dividing by the body weight.
Myoﬁbre isolation and culture. Single myoﬁbres were isolated from EDL muscles
and digested in DMEM GltaMax (Gibco, CA, USA) with 0.2% type 1 collagenase at
37 C for 90 min. Fibres were liberated by trituration in DMEM medium with
Pasteur pipettes. Isolated myoﬁbres were cultured in suspension in serum-coateddishes. The ﬁbre medium contained 20% foetal bovine serum (Sigma-Aldrich),
1% chick embryo extract (US Biological, MA, USA) 1% penicillin–streptomycin(Gibco), and DMEM GltaMax (Gibco). Single myoﬁbres were incubated at 37 Cwith 5% CO2 for the indicated times. For the treatments, G-CSF (Neutrogin;
Chugai) was added to the ﬁbre medium to a concentration of 0.375 ng ml � 1, p38
inhibitor (SB203580, CST) was added to the ﬁbre medium at a concentration of
10 mM and JNK inhibitor (SP600125, Cell Signaling Technology) was added to theﬁbre medium at a concentration of 5 mM.
Isolation of SCs. We obtained SCs using single myoﬁbre culture, during which theSCs began to migrate on Matrigel (BD Biosciences, CA, USA)-coated dishes. Single
SCs were then placed in Matrigel-coated glass-base dishes (Asahi Glass, Tokyo,
Japan) and were maintained in ﬁbre medium at 37 C with 5% CO2 for 3 days toallow SCs to migrate off the ﬁbre and onto the Matrigel-coated dishes.
Statistical analysis. Values are reported as the means±s.d. The data wereanalysed using StatView J-4.5 software. Comparisons between the two groups were
performed with Student’s t-test. Comparisons among the groups were performedby one-way analysis of variance with Bonferroni’s post hoc test. Scheffe’s F-test wasused to determine the level of signiﬁcance. The probability level accepted forsigniﬁcance was *Po0.05, **Po0.01.

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Acknowledgements
This study was supported in part by research grants from The Ministry of Education,
Science and Culture, Japan; The Program for Promotion of Fundamental Studies in
Health Science of the National Institute of Biomedical Innovation; The Ministry of
Health Labour and Welfare; Health Labour Sciences Research Grant; Nakatomi
Foundation; Japan Heart Foundation/Novartis Grant for Research Award on Molecular
and Cellular Cardiology; SENSHIN Medical Research Foundation; Kimura Memorial
Heart Foundation Research Grant; Japan Intractable Diseases Research Foundation,
Japan; The Cell Science Research Foundation; The Tokyo Biochemical Research
Foundation; Suzuken Memorial Foundation; and The Japan Foundation for Applied
Enzymology.

Author contributions

Er
re
dy

Noz
Dai
Yuk

Natu

The
prod
publ

S.Y. designed the research, N.H., S.Y., Y.S., M.H., N.I., H.H., D.K., T.S., S.T., M.K., A.K.,
S.K., M.T., Y.S., S.O., T.E., J.E. and T.S. performed the research, N.H., S.Y., Y.S., S.T. and
K.F. analysed the data and S.Y. wrote the paper.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Hayashiji, N. et al. G-CSF supports long-term muscle
regeneration in mouse models of muscular dystrophy. Nat. Commun. 6:6745
doi: 10.1038/ncomms7745 (2015).

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J. 4,

DOI: 10.1038/ncomms8295

gical

ancet

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Erratum: G-CSF supports long-term muscle
regeneration in mouse models of muscular
dystrophy
Nozomi Hayashiji, Shinsuke Yuasa, Yuko Miyagoe-Suzuki, Mie Hara, Naoki Ito, Hisayuki Hashimoto,
Dai Kusumoto, Tomohisa Seki, Shugo Tohyama, Masaki Kodaira, Akira Kunitomi, Shin Kashimura, Makoto Takei,
Yuki Saito, Shinichiro Okata, Toru Egashira, Jin Endo, Toshikuni Sasaoka, Shin’ichi Takeda & Keiichi Fukuda
Nature Communications 6:6745 doi: 10.1038/ncomms7745 (2015); Published 13 April 2015; Updated 18 Jun 2015.

ed

The immunoﬂuorescence images in Fig. 1b of the PDF version of this Article were inadvertently duplicated from Fig. 1a during the
production process. The correct version of Fig. 1 appears below; the HTML version of the paper was correct from the time of
publication.

A.K.,
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NATURE COMMUNICATIONS | 6:7295 | DOI: 10.1038/ncomms8295 | www.nature.com/naturecommunications& 2015 Macmillan Publishers Limited. All rights reserved.

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Brain Pathology ISSN 1015–6305

RESEARCH ARTICLE

Targeting Notch Signaling and Autophagy Increases
Cytotoxicity in Glioblastoma Neurospheres
Manabu Natsumeda1,2; Kosuke Maitani1; Yang Liu1; Hiroaki Miyahara1; Harpreet Kaur1; Qian Chu1,3;
Hongyan Zhang1; Ulf D. Kahlert1,4; Charles G. Eberhart1
1 Department of Pathology, Division of Neuropathology, Johns Hopkins Hospital, Baltimore,MD.

2 Department of Neurosurgery, Brain Research Institute, Niigata University, Niigata, Japan.
3

Department of Oncology, Tongji Hospital, Wuhan, China.

4 Department of Neurosurgery, University Medical Center Du€sseldorf, Du€sseldorf, Germany.

Keywords
autophagy, chloroquine, combination treatment, gamma-secretase inhibitor,
glioblastoma.
Corresponding author:
Manabu Natsumeda, MD, PhD, Departmentof Pathology, Division of Neuropathology,
Johns Hopkins University School of Medicine,
Ross Building 558, 720 Rutland Ave.,
Baltimore, MD 21205 (E - mail:mnatsumeda@bri.niigata-u.ac.jp)
Received 07 August 2015
Accepted 25 November 2015
Published Online Article Accepted
27 November 2015

Abstract
Glioblastomas are highly aggressive tumors that contain treatment resistant stem-like cells.
Therapies targeting developmental pathways such as Notch eliminate many neoplastic
glioma cells, including those with stem cell features, but their efﬁcacy can be limited byvarious mechanisms. One potential avenue for chemotherapeutic resistance is the inductionof autophagy, but little is known how it might modulate the response to Notch inhibitors. We
used the g-secretase inhibitor MRK003 to block Notch pathway activity in glioblastoma
neurospheres and assessed its effects on autophagy. A dramatic, several fold increase of
LC3B-II/LC3B-I autophagy marker was noted on western blots, along with the emergence ofpunctate LC3B immunostaining in cultured cells. By combining the late stage autophagy
inhibitor chloroquine (CQ) with MRK003, a signiﬁcant induction in apoptosis and reduction
in growth was noted as compared to Notch inhibition alone. A similar beneﬁcial effect on
inhibition of cloogenicity in soft agar was seen using the combination treatment. These
results demonstrated that pharmacological Notch blockade can induce protective autophagy
in glioma neurospheres, resulting in chemoresistance, which can be abrogated by
combination treatment with autophagy inhibitors.

doi:10.1111/bpa.12343

INTRODUCTION
Glioblastomas are the most common primary adult brain malignancy, comprising more than 50% of all gliomas (23). Despitesome advances in therapeutic options, outcomes remain dismal
with a median overall survival of 14.6 months and a 2-year survival
rate of 26.5% (33). Treatment resistant stem-like glioma cells havebeen proposed as one reason for treatment failure (4, 8, 10, 25, 36,38). We (3, 9) and others (30, 34) have demonstrated the efﬁcacyof g-secretase inhibitors (GSI), which block Notch pathway activity, using in vitro and in vivo glioma models. However, success has
been limited in clinical trials using GSIs as a single agent in gliomas (17), and in other solid tumors alone (17, 35) or in combination with other drugs (5, 26, 29). We, therefore, examined if
induction of autophagy might be limiting the therapeutic efﬁcacyof Notch inhibitors.
Autophagy is a dynamic process by which cellular organelles
and proteins are sequestered, delivered to lysosomes and digested(37). Induction of autophagy has been identiﬁed as a potential
mechanism for resistance to chemotherapy (16) including temozolomide (14, 18, 19, 22, 42) as well as radiation treatment (13, 41),
which represent the standard therapies for glioblastoma. Manipulation of autophagy has increasingly been explored as a treatment

strategy in many cancers (21). However, there is minimal data on
the effects of the Notch pathway on autophagy in cancer (12). We
examined if Notch pathway blockade using the GSI MRK003
would induce autophagy in glioma neurosphere lines, and foundthat it does. We next explored a therapeutic regimen using the latestage autophagy inhibitor, chloroquine (CQ), in conjunction with
MRK003, and showed that the combination induced apoptosis
and more effectively decreased cell growth, proliferation andclonogenicity.

MATERIALS AND METHODS
Cell culture conditions and drug preparation
HSR-GBM1 and JHH520 glioblastoma neurosphere lines wereused, and their identity was authenticated at the Johns Hopkins
Core Laboratory through short tandem repeat (STR) analysis.
HSR-GBM1 was a kind gift from Dr. Angelo Vescovi, JHH520
from Dr. Gregory Riggins. The HSR-GBM1 is completely methylated at the MGMT locus but resistant to temozolomide in vitro,lacks mutation at the IDH1 and alterations in p53. For treatment
studies, neurospheres were disassociated into single cell
1

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Targeting Notch and Autophagy in GBM

Natsumeda et alsuspensions using Accutase (Sigma Aldrich, St Louis, MO, USA),counted, plated and allowed to grow overnight in Neural Stem Cell
medium supplemented with 20 ng/mL of human epidermal growth
factor and 10 ng/mL of human ﬁbroblast growth factor (Peprotech,
Rocky Hill, NJ, USA). All cells were grown in a humidiﬁed atmosphere containing 5% CO2 at 378C. The next morning, GSI
MRK003, generously provided by Merck Research Laboratories(Boston, MA, USA), dissolved in dimethyl sulfoxide (DMSO)(Sigma Aldrich, St Louis, MO, USA) at ﬁnal concentrations of 0.5
to 5 lM, chloroquine diphosphate (CQ) (Sigma Aldrich, St Louis,
MO, USA), dissolved in phosphate buffered saline (PBS) at a ﬁnal
concentration of 10 lM, or DMSO (1:1000 dilution) was added tothe media. Baﬁlomycin A1 (BafA1) (Sigma-Aldrich, St Louis,
MO, USA) was dissolved in DMSO at ﬁnal concentrations of 0.5–
5 nM. 3-methyladenine (3-MA) (Selleckchem, Houston, TX, USA)was diluted in DMEM at a ﬁnal concentration of 10 mM.

Quantitative real-time PCR analyses
Cells were collected after treatment with 0–5 mL of MRK003 for
48 h. RNA extracted was performed using the RNeasy Mini Kit(Quiagen) with on-column DNA digestion to prevent ampliﬁcationof genomic DNA. Reverse trancription into complementary DNA(cDNA) was performed. cDNA levels were analyzed by quantitative real-time PCR analysis with SsoAdvanced Universal SYBRGreen Supermix (Bio-Rad, Hercules, CA, USA) on an iQ5 Multicolor real-time PCR detection system. Standard curves were used
to determine expression levels and all values were normalized tob-actin. Statistical comparisons were between at least three biological replicates, each with triplicate technical replicates. Primer
sequences were as follows: HES1 forward F: 50 -GTG-AAG-CACCTC-CGG-AAC-30 , reverse R: 50 -CGT-TCA-TGC-ACT-CGCTGA-30 ; HES5 forward F: 50 -GTG-CCT-CCA-CTA-TGA-TCCTTA-AA-30 , reverse R: 50 -AGT-ACA-AAG-TCG-TGC-CCA-CA30 ; HEY1 forward F: 50 -TCT-GAG-CTG-AGA-AGG-CTG-GT-30 ,reverse R: 50 -CGA-AAT-CCC-AAA-CTC-CGA-TA-30 ; b-actin
forward F: 50 -CCC-AGC-ACA-ATG-AAG-ATC-AA-30 , reverse
R: 50 -CGA-TCC-ACA-CGG-AGT-ACT-TG- 30 .

Western blot analysis
Proteins were extracted from HSR-GBM1 and JHH520 after treatment with MRK003 and/or CQ for 48–72 h, unless otherwise indicated. The cells were lysed in RIPA buffer containing proteaseinhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) and phosphatase inhibitors on ice for 10 minutes then centrifuged at 48C and14,000 rpm for 5 minutes to remove cellular debris. Protein quantiﬁcation was done using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Subsequently, protein separation was performedon 12% (for LC3B protein analysis) or 4%–12% (for analysis ofother proteins) SDS-polyacrylamide gel, and transferred onto nitrocellulose membranes (Invitrogen, Carlsbad, CA, USA). Membranes
were then probed with primary antibodies at 48C overnight. The
following primary antibodies were used: LC3B, P62/SQSTM1,
phospho-AKT (Ser 473), AKT (C6E7), cleaved PARP, cleaved
NOTCH1 (Val1744)(D3B8) (1:1000 dilution, Cell Signaling, Danvers, MA, USA), HES1 (1:800 dilution, Aviva Systems Biology,
San Diego, CA, USA), b-actin (1:5000 dilution, Sigma-Aldrich, St
Louis, MO, USA). Secondary antibodies conjugated to horseradishNatperoxidase (KPL, Gaitersberg, MD, USA) were incubated for 1 h
at room temperature and detected with a Western Lightning Plus
ECL chemiluminescent kit (PerkinElmer, Waltham, MA). All blots
were repeated at least three times and representative blots areshown. Densitometry was performed using Image J Ver. 1.440 software (http://rsb.info.nih.gov/ij/) (31).

Immunofluorescence and immunohistochemical
analyses
HSR-GBM1 cells were treated with DMSO or 5 mM MRK003 for
48 h, or starved for 4 h in Earle’s Basic Salt Solution (EBSS). The
cells were subsequently pelleted, ﬁxed in 10% formalin and embedded in parafﬁn. Antigen retrieval was performed by heating in apressure cooker for 30 minutes in Target Retrieval Solution (Dako,
Glostrup, Denmark). Sections were stained using anti-LC3B rabbit
polyclonal antibodies (1:10,000 dilution, ab51520, Abcam, Cambridge, UK and 1:500 dilution, Cell Signaling, Danvers, MA,
USA) at 48C overnight. The sections were stained by avidin-biotinperoxidase complex method (Vector, Burlingame, CA, USA) withsecondary antibody incubation for 30 minutes at a dilution of
1:5000, diaminobenzidine as the chromogen and counterstainedwith hematoxylin.
For immunoﬂuorescence staining, HSR-GBM1 and JHH520
cells were grown in 10% FBS DMEM in 12 well plates on coverslips. From the next day, the cells were treated with DMSO or
5 mM MRK003 for 48 h. The cells were subsequently ﬁxed in 4%
paraformaldehyde, permeabilized in ice cold (2208C) methanol for
15 minutes, blocked for 30 minutes with 5% normal goat serum(NGS) with 0.3% Triton X solution. Sections were stained using
anti-LC3B rabbit monoclonal antibody (1:2000 dilution, ab51520,
Abcam, Cambridge, UK), which was added in 5% NGS with 0.3%
Triton X solution at 48C overnight. After washing with PBS, cells
were incubated for 90 minutes at a dilution of 1:400 in Cy-3 (Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA)
conjugated secondary antibody. After washing with PBS, nuclei
were counterstained with DAPI 40 , 6-diamindino-2-phenylindoleand coverslips were mounted on slides with antifade (Vectastain
Elite, Vector Laboratories, Burlingame, CA, USA). Confocal imaging was done using the Fluoview 1000 (Olympus America, Melville, NY, USA).

Determination of cell growth
3-(4,5-dimethylthiazol-2-yl)25-(3-carboxymethoxyphenyl)22-(4sulfophenyl)-2H-tetrazolium (MTS) assays were performed todetermine growth in viable cell mass. Cells were dissociatedand seeded into 96-well plates at a density of 5000 per well in
200 mL of medium and treated with DMSO, 5 mM MRK003
and/or 10 mM CQ and incubated in 5% CO2 at 378C. For theplate readings, 20 mL of MTS solution was added to each wellafter 0–96 h postplating and incubated for 1 h, protected from light.
The optical density at 490 nm was subsequently measured by spectrophotometer. The experiments were repeated at least three times
for each cell line.

Cell proliferation assay
Cells were treated with DMSO, 5 mM MRK003 and/or 10 mM CQ
for 72 h. Cells were subsequently ﬁxed and stained for Ki67 per

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perFigure 1. MRK003 inhibits Notch pathway. A. Treatment with
MRK003 inhibited downstream Notch pathway transcription factors
HES1, HES5, HEY1 in JHH520 and HSR-GBM1 lines at the mRNAlevel. A statistically significant up to 50% reduction of HES1 wasseen after treatment with 5 lM MRK003. Likewise, an approximately
75% decrease of HES5 and approximately 80% decrease of HEY1

compared to DMSO control was seen after 2 lM MRK003 treatment
in both lines. Stars denote P values vs. DMSO. B. Treatment with 0.5
lM MRK003 almost completely inhibited cleaved (active) Notch1
expression in JHH520 and HSR-GBM1 lines. Inhibition of HES1 wasobserved after treatment with 5 lM MRK003 in both cell lines.

manufacturer’s instructions using the Muse Ki67 Proliferation
Assay (Millipore, Billerica, MA, USA). Cells incubated with IgG1
control in parallel were used to set the gates. The percentage ofKi67 positive cells was determined using the Muse Cell Analyzer.
Analyses were repeated at least three separate times for each cellline.

Apoptosis assay

Cell cycle analyses
Cells were treated with DMSO, 5 mM MRK003 and/or 10 mM CQ
for 72 h. Cells were then ﬁxed with 70% ethanol for at least 3 hand stained with Muse Cell Cycle Reagent. Cell cycle analyses
were performed using the Muse Cell Analyzer and percentages of
sub-G1, G0/G1, S and G2/M populations were determined using
FlowJo software. Analyses were repeated at least three separate
times for each cell line.

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Apoptosis was measured using the Muse Annexin V and Dead Cell
Assay per the manufacturer’s protocol. Neurospheres were treatedwith DMSO, 5 mM MRK003 and/or 10 mM CQ for 72 h. Cells were
resuspended in DMEM/F12 without phenol red with 1% Bovine
Serum Albumin (BSA) before analysis. Percentage of apoptotic anddead cells were analyzed using the Muse Cell Analyzer. Analyses
were repeated at least three separate times for each cell line.

Clonogenic assays
Neurosphere media was mixed 3:1 with 4% agarose (Invitrogen,
Carlsbad, CA, USA) to make the bottom layer, which was used tocoat each well of a six-well plate. Neurospheres treated with
DMSO, 2 mM MRK003 or a combination of 2 mM MRK003 and10 mM CQ for 72 h were dissociated and added to 7:1 media and3－ 365 －

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agarose mixture at a concentration of 5000 cells per 2 mL to makethe top layer. After the agarose gelled, 2 mL of fresh media andDMSO or drug were added to the top of each well. Media and drug

were changed every 1–4 days during the growth phase of the clonogenic assay. The cells were allowed to form colonies for 19–23
days. Colonies were visualized by staining with nitroblue

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Figure 2. Autophagy is induced after MRK003 treatment in vitro. A.
Western blot showed induction of autophagy as seen by the increaseof the LC3B-II band after GSI treatment in JHH520 and HSR-GBM1
lines in a dose-dependent manner. B. Quantification of LC3B-II/LC3B-I
from three separate blots. C. HSR-GBM1 cells were treated with
DMSO, 5 lM MRK003, or EBSS, then pelleted and stained for LC3B.
Note the punctate staining of the cytoplasm of glioma cells after treatment with MRK003 and EBSS, but not DMSO (original magnification
all 3400). D. HSR-GBM1 and JHH520 cells were treated with DMSO

or 5 lM MRK003 and stained for LC3B. Note the markedly increased
punctate staining in the cytoplasm of glioma cells after treatment with
MRK003 compared to DMSO by immunofluorescence analysis of
LC3B (original magnification all 3640). E. Accumulation (loss of clearance) of LC3B-II after treatment with chloroquine (CQ). A decrease in
p62 after MRK003 treatment alone and increase of p62 was observed
after combined treatment with MRK003 and CQ, suggesting inhibitionof autophagy by CQ.

tetrazolium (NBT) in a tissue culture incubator overnight at 378C
after which they were imaged. Colonies greater than 50 mm indiameter were scanned and counted using MCID Elite software(Cambridge, England, UK). For each well, at least three images
were scanned and counted. Triplicates of each condition wereplated and the experiment was repeated three times.

nature resulting in further migration on electrophoresis. A signiﬁcant (P < 0.05) increase in LC3B-II/LC3B-I ratio averaging overthreefold was seen after 5 mM treatment with MRK003 in JHH520;
likewise, over fourfold (P < 0.05) and sixfold (P 5 0.0008)
increases were seen after 2 and 5 mM treatment in HSR-GBM1(Figure 2B).
Having observed an induction of autophagy after treatment with
MRK003 by western blotting, we next sought to determine if
induction of autophagy could be conﬁrmed by immunohistochemical analysis in HSR-GBM1 cells and by immunoﬂourescence analysis in both cell lines. Increased punctate staining of LC3B in thecytoplasm of HSR-GBM1 cells, suggesting mature autophagosomes, was observed after 48 h treatment with 5 mM MRK003 and4 h starvation in EBSS media, but not in DMSO control (Figure2C). Immunoﬂourescence analysis showed markedly increased
punctate staining for LC3B in the cytoplasm of tumor cells compared to DMSO control in both cell lines (Figure 2D).
Given the induction of potentially protective autophagy after
treatment with MRK003, a combinatorial regimen using the latestage autophagy inhibitor CQ in conjunction with MRK003 was
explored. CQ blocks LC3B clearance via lysosomal inhibition,
causing its accumulation in tumor cells (1). Consistent with thismechanism, we saw a very strong induction of the LC3B-II band
after CQ treatment in both lines, indicating in this context a blockage of late autophagy (Figure 2E). We chose a dosage of 5 mM
MRK003 for combination experiments, because maximal suppression of HES1 protein and induction of autophagy was seen at thislevel. An increase in LC3B-II/LC3B-I ratio was also seen after
treatment with the two drugs in combination (Figure 2E). To help
determine if this reﬂected an ongoing increase in autophagy or alate blockade of the process, we assessed expression of p62, which
recognizes autophagic cargo and mediates formation of selective
autophagy (27). p62 is known to be upregulated when autophagy isinhibited, resulting in intracellular waste accumulation. A 40%
decrease in p62 was seen after induction of autophagy by MRK003
and was subsequently increased 2.4 fold after treatment with both 5
mM MRK003 and 10 mM CQ in HSR-GBM1, suggesting that thedominant process when both agents are used is inhibition of
autophagy (Figure 2E). Interestingly, p62 was decreased by only
20% in JHH520 after treatment with 5 mM MRK003, and theincrease in p62 after combination MRK003 and CQ treatment wasa modest 1.8-fold compared to DMSO control (Figure 2E). Likewise, a similar lack of p62 decrease after MRK003 treatment andonly modest (1.6-fold compared to 3.9-fold for HSR-GBM1)
increase of p62 was seen after combination MRK003 and BafA1
treatment in JHH520 (Supporting Information Figure 1B), suggesting a cell line dependent variability in effects.

Statistical analyses
Differences between three or more groups were assessed using one
way ANOVA test with post hoc Tukey’s multiple comparison test.
Error bars represent standard error of means. All statistical tests
were performed using the GraphPad Prism 6 software (GraphPad
Software, La Jolla, CA, USA). P values less than 0.05 were considered statistically signiﬁcant.

RESULTS
MRK003 inhibits Notch pathway targets
Notch pathway activity was assessed using quantitative RT PCR tomeasure levels of the canonical Notch pathway transcriptional targets
HES1, HES5 and HEY1 after 48-h treatment of glioblastoma neurospheres with MRK003. A statistically signiﬁcant, dose dependent,
up to 50% reduction of HES1 was seen after treatment with 5 mM
MRK003. Likewise, an approximately 75% decrease of HES5 andapproximately 80% decrease of HEY1 was seen after 2 mM
MRK003 treatment in both cell lines (Figure 1A). Interestingly, thetreatment with MRK003 at a high dose (5 mM) resulted in a parodoxical increase in the mRNA levels of HES5 and HEY1. We have previously observed this paradoxical increase when blocking Notch
activity in brain tumors with relatively high concentrations of GSI or
arsenic trioxide, and speculated a feedback or resistance mechanismof cells surviving maximal therapy [(6) and unpublished data]. We
next assessed the effects of MRK003 on Notch pathway activity at
the protein level. Treatment with 0.5 mM MRK003 almost completely inhibited cleaved (active) Notch1 expression in JHH520 and
HSR-GBM1; inhibition of HES1 was also observed after treatment
with 5 mM MRK003 in both neurosphere lines (Figure 1B).

Notch inhibition induces autophagy inglioblastoma
Induction of autophagy, evidenced by a dose-dependent increase in
LC3B-II/LC3B-I ratio, was seen after 48-h treatment with 0.5–5
mM of MRK003 in both glioblastoma neurosphere lines (Figure
2A,B). In mature autophagosomes, LC3B-I undergoes a posttranslational modiﬁcation to LC3B-II increasing its hydrophobic•–••

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Figure 3. Combination treatment with MRK003 and chloroquine more
effectively inhibits glioma neurosphere growth and proliferation. A.
5,000 JHH520 and HSR-GBM1 cells per well were plated in a 96-wellplate and cell viability was assessed by MTS assay after treatment
with DMSO, 5 lM MRK003, 10 lM CQ, and a combination of 5 lM
MRK003 and 10 lM CQ at days 0–5 of treatment. Combination treatment markedly reduced growth compared to DMSO (JHH520;

P 5 0.0004, HSR-GBM1; P 5 0.0002) and MRK003 alone (JHH520;
P < 0.01, HSR-GBM1; P < 0.05) in both neurosphere lines. Stars
denote P values vs. DMSO unless otherwise specified. B. Ki67
MUSE assays were performed to assess effects on proliferation. A
modest but significant decrease in the percentage of Ki67-positive
cells was observed after combination treatment compared to DMSO
control in the HSR-GBM1 line.

Combination treatment with MRK003 andchloroquine inhibits growth and proliferation

5 days of treatment. However, while the addition of BafA1 to 2
mM MRK003 resulted in fewer viable cells, this increase over the
GSI alone was not statistically signiﬁcant, perhaps because the antigrowth effects of MRK003 were particularly prominent in thesestudies. Similar effects were seen with combination of 2 mM
MRK003 and 5 mm of BafA1 (data not shown). However, no
reduction in p62 was noted in JHH520 cells after MRK003 treatment. Increases in LC3B-II/LCB-I ratio and p62 were seen after
combination treatment with MRK003 and BafA1, suggesting inhibitory effects of BafA1 at the late stage of autophagy similar to CQ(Supporting Information Figure 1B).
A combination of 2 mM MRK003 and 10 mM of the early stage
autophagy inhibitor, 3-MA, showed signiﬁcant reduction of cell
growth compared to DMSO control (JHH520; P 5 0.002, HSRGBM1; P 5 0.002) and MRK003 alone (JHH520; P 5 0.03, HSRGBM1; P 5 0.0009) after 5 days of treatment (data not shown).
Western blots showed a 50% decrease in p62 expression compared
to DMSO after MRK003 treatment and a 3.2-fold increase in p62
after combination MRK003 and 3-MA treatment in HSR-GBM1

The effects of combination treatment with MRK003 and CQ on
growth of the two glioblastoma neurosphere lines were examinedusing the MTS assay. In both lines, 10 mM CQ did not have a signiﬁcant effect on the viable cell mass, while Notch inhibition
caused a moderate slowing (P < 0.05) in the glioma growth (Figure
3A). In both cell lines, culture growth was signiﬁcantly reduced
compared to DMSO controls (JHH520; P 5 0.0004, HSR-GBM1;
P 5 0.0002) and MRK003 alone (JHH520; P < 0.01, HSR-GBM1;
P < 0.05) when MRK003 treatment was paired with inhibition of
autophagy (Figure 3A).
We sought to determine whether combination treatment of
MRK003 with another late stage autophagy inhibitor, BafA1,
would exhibit increased cytotoxicity. Combination treatment with
2 mM of MRK003 and 0.5 mM of BafA1 showed a drastic reduction of cell growth compared to DMSO (JHH520; P < 0.0001,
HSR-GBM1; P < 0.001) (Supporting Information Figure 1A) after
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Figure 4. Induction of apoptosis is observed after combination
treatment with MRK003 and chloroquine. A. Western blot showedapproximately fourfold increase of cleaved PARP after 5 lM MRK003
treatment compared to DMSO control in both cell lines and 12.7-foldto 21.1-fold increase after combination treatment compared to DMSO

control. B. Cell cycle analysis and C. Annexin V MUSE assays were
performed to assess effects on apoptosis. An approximately 10-foldincrease in percentage of sub-G1 population and modest but significant increase in percentage of Annexin V-positive cells were seen
after combination treatment compared to DMSO control.

(Supporting Information Figure 2). A 40% reduction in LC3B-II/I
ratio was seen after 10 mM 3-MA treatment compared to DMSO
control and 30% decrease in LC3BII/I ratio was seen after combination MRK003 and 3-MA compared to MRK003 alone, suggesting the inhibition of early stage autophagy.

To determine if combination treatment altered the proliferationof glioblastoma neurospheres, the percentage of Ki67-positive cells
was assessed using the MUSE Ki67 assay. A modest but signiﬁcantreduction in the percentage of Ki67-positive cells was observed
after combination treatment with 5 mM MRK003 and 10 mM CQ•–••

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Figure 5. Combination treatment with MRK003 and chloroquine
reduces clonogenicity in glioblastoma neurosphere lines. A.
Representative images of nitroblue tetrazolium-stained colonies
formed by JHH520 and HSR-GBM1 cells after treatment with DMSO,

2 lM MRK003, 10 lM CQ and combination MRK003 and CQ are
shown. B. Quantification of number of colonies depicted in A, showing markedly reduced colony formation after combination therapy.

compared to DMSO control in the HSR-GBM1 line (P < 0.05)(Figure 3B). However, this reduction in proliferation following
combination therapy was not signiﬁcantly greater than noted after
Notch inhibition alone, suggesting that it was not the only mechanism by which overall growth was affected.

age of G0/G1 population was noted after 72-h combination treatment of HSR-GBM1 (Supporting Information Figure 2C).
Although cell cycle arrest was not noted after 72-h combination
treatment, G2/M arrest was seen after 48-h combination treatment
in line (data not shown), suggesting that cells in G2/M arrest eventually undergo apoptosis. Finally, a signiﬁcant but less pronounced
increase in the percentage of total apoptotic cells was observed after
combination treatment compared to DMSO control in both lines
using the Annexin V assay (JHH520; P < 0.05, HSR-GBM1;
P < 0.01) (Figure 4C). These data support the concept that induction of autophagy prevents apoptosis following Notch blockade inglioma cells, and that CQ can suppress this and increase the cytotoxicity of GSI.

Combination treatment with MRK003 andchloroquine induces apoptosis
Given the limited effect of combination treatment on proliferation,
we examined changes in apoptosis. An approximately fourfold
induction in the apoptotic mediator cleaved PARP was noted after
5 mM MRK003 treatment in both lines, with a lesser increase seen
after phamacological inhibition of autophagy (Figure 4A). In contrast, a dramatic 12-fold to 21-fold induction was seen after combination treatment. Only a minor, nonsigniﬁcant increase wasobserved in the percentage of sub-G1 population by cell cycle analysis after treatment with 5 mM of MRK003 compared to DMSO. In
contrast, an 8.9-fold to 10.6-fold increase in percentage of sub-G1
population was observed after combination treatment (Figure 4B,
Supporting Information Figure 2A,B).
With respect to other cell cycle alterations, no signiﬁcant change
in percentage of G0/G1, S, or G2/M population was noted after 72h combination treatment of JHH520; a modest reduction of percent-Clonogenic capacity is reduced after
combination treatment with MRK003
and chloroquine
Having conﬁrmed the increase of apoptosis caused by inhibition of
autophagy by CQ, we next looked at clonogenic capacity after
monotherapy and combination treatment using the soft agar assay.
These were performed using 2 mM rather than 5 mM MRK003, as
the higher GSI dose resulted in too few colonies for an additiveeffect of the autophagy inhibitor to be examined. A representative

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Figure 6. Phospho-AKT is inhibited by MRK003 and MRK003 induces
autophagy in vivo. A, B. Phospho-AKT (Ser 473) was significantly
inhibited by MRK003 after treatment with 0.5–5 lM MRK003 in HSRGBM1 for 48 h (P  0.0001). However, inhibition of phospho-AKT was

not seen in JHH520 after 5 lM MRK003 treatment for 48 h. C.
Phospho-AKT was most inhibited by 5 lM MRK003 treatment for
24 h, but subsequently upregulated after 48–72 h treatment in
JHH520 line.

experiment shows an approximately 40%–50% decrease in thenumber of colonies after treatment with 2 mM MRK003 alone compared to DMSO in both cell lines (Figure 5A,B). However, withcombination therapy a 75%–85% reduction in clonogenicity wasobserved, and this was signiﬁcantly more pronounced than theeffect with 2 mM MRK003 alone (both P < 0.0001). The clonogenic assays were repeated three times for each cell line, and all ofthese experiments showed a signiﬁcantly lower number of colonies
for combination treatment than the GSI alone.

MRK003, we found that phospho-AKT levels decreased dramatically 48 h after Notch inhibition at concentrations as low as 0.5 uM
in HSR-GBM1 neurospheres (Figure 6A,B). JHH520 cells showedmodest inhibition of AKT activity with greatest inhibition after
24 h treatment with 5 mM MRK003 (Figure 6C). However, further
investigation is needed to elucidate whether autophagy after
MRK003 treatment is mediated by mTORC2 inhibition.

DISCUSSION
Inhibition of AKT following Notch inhibition
AKT activity is known to suppress autophagy (39), and is also
modulated by Notch in cancers other than glioma (7, 24). We havepreviously shown that GSIs other than MRK003 can inhibit phosphorylation and activation of AKT (9) in glioblastoma. Using•–••

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Despite some recent advances in treatment, the prognosis for glioblastoma patients remains dismal. Induction of autophagy has been
implicated as a potential resistance mechanism to general treatments in gliomas, and its manipulation has actively been investigated as a therapeutic strategy (15). However, little is known about
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the role of Notch in regulating autophagy in gliomas, or the potential for autophagy to mediate resistance to pathway inhibitors suchas GSIs. Increasing evidence suggests that the Notch pathway
affects autophagosomal trafﬁcking (2), and that inhibition of the
Notch pathway induces autophagy in some types of cancer (12)and during differentiation of stem cells (32).
Our data provide initial evidence that pharmacological Notch
pathway blockade can induce autophagy in gliomas. When neurosphere lines were treated with GSI, a dramatic several fold induction of the LC3B-II protein compared to LC3B-I was noted, as wellas the emergence of punctate immunohistochemical LC3B staining
in ﬁxed cells, consistent with an increase in autophagic ﬂux. However, a robust decrease in p62 following Notch blockade was only
seen in one line, thus, clear induction of autophagy may be
restricted to a subset of cases. Autophagy may also be present in amore complex in vivo treatment milieu, as analysis of orthotopic
glioblastoma xenografts treated with the GSI from a prior study (3)has revealed focal punctate LC3B staining, but too little material
was present for deﬁnitive conclusions to be drawn. Our ﬁndings
are consistent with a very recent report demonstrating induction of
autophagy after NOTCH1 knockdown by shRNA in a single glioma cell line (40).
The induction of autophagy is of potential clinical relevance,
as combining the late stage autophagy inhibitor CQ with
MRK003 resulted in a signiﬁcant induction in apoptosis andreduction in growth and clonogenicity as compared to Notch
inhibition alone. Similar growth changes were noted when combining Notch inhibition with the early stage autophagy inhibitor
3-MA. A few autophagy inhibitors have been clinically tested
in gliomas. CQ is an antimalarial drug with lysosomotropic
properties that inhibit lysosomal function. It is known to havecentral nervous system penetration and has been shown to effectively inhibit autophagy in vitro (28). A previous trial using
radiation and carmustine with or without CQ in glioblastoma
patients showed a trend toward increased median overall survival that was not signiﬁcant (18). Hydroxychloroquine (HCQ),
a derivative of CQ, shows equal potency to inhibit autophagy
in vitro and has demonstrated less retinal toxicity. A trial of
HCQ in conjunction with radiation therapy and temozolomide
showed biological response caused by HCQ, but failed to
improve overall survival in glioblastoma (28). New and morepotent inhibitors of autophagy such as Lys05 (20) and VATG027 (11) have been tested preclinically and await clinicalvalidation.
We have demonstrated that pharmacological Notch blockade
can induce protective autophagy in glioma neurospheres, which
can be abrogated by combination treatment with CQ. Together, thetwo drugs efﬁciently induce apoptosis and decrease cell growth,proliferation and clonogenicity. A range of options for pharmacological inhibition of both Notch and autophagy exist, and combinatorial blockade of these two pathways represents a promising newtreatment strategy for glioblastoma.

ACKNOWLEDGMENTS
We would like to acknowledge Dr. Jeff Mumm for assistance withthe confocal microscope. The authors declare no conﬂict of interest.
This study was funded by grant R01NS055089 to CGE.

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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Figure 1. A Combination treatment with 2 mM MRK003 andBaﬁlomycin A1 (BafA1) signiﬁcantly decreased cell growthcompared to DMSO in both cell lines. B Increase in LC3B-II/
LC3B-I ratio expression and marked elevation of p62 is noted
after treatment with BafA1, suggesting late stage inhibition of
autophagy. Conversely, LC3B-II/LC3B-I ratio decreases andp62 increases after treatment with 3-MA, suggesting early stage
autophagy inhibition.
Figure 2. A, B Representative cell cycle analysis data depicting
marked increases in percentages of sub-G1 population in
JHH520 (A) and HSR-GBM1 (B) lines after combination treatment with MRK003 and CQ. C Cell cycle arrest was notobserved after 72-hour treatment with MRK003 and CQ.

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