National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGYProfessor

Associate Professor

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
NOMURA, Kengo
FUJIKAWA, Akihiro
SUZUKI, Ryoko
MATSUMOTO, Masahito
KUBOYAMA, Kazuya
LIN, Chia-Hao
MATSUDA, Takashi
YU, Yang
TANGA, Naomi
HIGASHI, Satoru
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
KODAMA, Akiko

NODA, Masaharu

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

SOKENDAI Graduate Student:

Technical Assistant:

Secretary:

SHINTANI, Takafumi

We have been studying the molecular and cellular mechanisms underlying the development of the vertebrate central
nervous system (CNS), mainly using mice. This researchcovers many developmental events including the patterningof the retina, neuronal terminal differentiation, axonal navigation, branching and targeting, synapse formation, refinement and plasticity. The scope of our interests also encompasses the mechanisms for various functions of the mature
brain, including body-fluid regulation, behavior control,
learning, and memory.

I. Mechanisms for neural circuit formation

Adenomatous polyposis coli 2 (APC2) is preferentiallyexpressed in the nervous system from early developmental
stages through to adulthood. The knockdown of Apc2 inchick retinas reduced the stability of microtubules in retinal
axons, and yielded abnormal growth cone behaviors including a reduced response to ephrin-A2 and misprojection in thetectum without making clear target zones. In Apc2-deficient
mice, robust defects in neuronal lamination were observedin the cortex, hippocampus, cerebellum, and olfactory bulb.
These laminary abnormalities are a result of dysregulated
neuronal migration by a cell-autonomous mechanism. APC2
is distributed along actin fibers as well as microtubules inneurons. Our investigation suggests that APC2 is involved in
the signaling pathway from membrane receptors for extracellular guidance factors to the intracellular migration machinery.
Very recently, two siblings with Sotos syndrome-likefeatures caused by homozygous frame shift mutation in theAPC2 gene were found by whole exome sequencing. Sotos
syndrome (OMIM #117550) is characterized by varying

degrees of mental retardation and a combination of typicalfacial features (prominent forehead with a receding hairline,
downslanting palpebral fissures, and a pointed chin) andlarge head circumference its estimated incidence is 1/14,000
live births. Sotos syndrome has been known to be causedby haploinsufficiency in the NSD1 gene. However, thesepatients had no mutations in NSD1 or other known susceptibility genes.
We performed an etiological study on these patients. Cellbased functional assays indicated that the mutant APC2 ofthese patients was a functionally null protein. Apc2-deficient
mice exhibited significantly impaired learning and memory
abilities, together with an abnormal brain structure and head
shape, indicating a high degree of concordance between
the phenotypes observed in human patients and knockout
mice. We demonstrated for the first time that the expression of APC2 was under the control of NSD1. The impaired
migration of cortical neurons was observed when Nsd1 wasknocked down in the embryonic mouse brain, as in the Apc2deficient brain, and this defect was rescued by the forced
expression of Apc2 (Figure 1). Our study explains molecularmechanisms of the intellectual disability associated withSotos syndrome.

Figure 1. Effects of Nsd1 knockdown on the migration of corticalneurons during development. The control miRNA, Apc2-miRNA, or
Nsd1-miRNA construct was electroporated into the ventricular regionof the forebrain at E15.5. For the rescue experiment, Nsd1-miRNA wasco-electroporated with an Apc2-expressing plasmid. At P7 the location oftransfected cells (GFP+) was assessed in coronal sections of the cortex.
Nuclei were stained with DAPI. The layers of the cortex are indicated on
the right side. WM, white matter. Scale bars, 100 mm.

II. Physiological roles of receptor-like protein
tyrosine phosphatases

Protein tyrosine phosphorylation plays crucial roles invarious biological events such as cellular proliferation, differentiation, survival, migration, and metabolism. Cellulartyrosine phosphorylation levels are governed by the opposing
activities of protein tyrosine kinases (PTKs) and protein
tyrosine phosphatases (PTPs). The physiological functions
and regulatory mechanisms of receptor-like PTPs (RPTPs)
are not fully elucidated. We have been making efforts toreveal the functional roles of RPTPs, especially of the R3and R5 subfamilies.

2-1 R3 RPTP subfamily

The R3 RPTP subfamily, which is comprised of PTPRB,PTPRH, PTPRJ, and PTPRO, reportedly plays pivotal rolesin the development of several tissues including the vascularand nervous systems. We performed a large scale examination of the enzyme-substrate interaction between theR3 RPTP members and representative RPTKs covering29

National Institute for Basic Biology

Neurobiology

all RPTK subfamilies. We revealed that multiple RPTKs
are recognized as substrates by the R3 RPTPs. Among theenzyme-substrate relationships identified, we examined theinteraction between the R3 RPTPs and insulin receptor (IR)in detail.
Co-expression of R3 RPTPs with the IR in HEK293T cells
suppressed the insulin-induced tyrosine phosphorylationof the IR. In vitro assays using synthetic phosphopeptides
revealed that R3 RPTPs preferentially dephosphorylatesparticular phosphorylation sites of the IR; Y960 in the juxtamembrane region and Y1146 in the activation loop. It has
been revealed that phosphorylation of Y1146 and Y960 arerequired for the full activation and the signal transductionof the IR, respectively. Therefore, R3 RPTPs are thought toeffectively dephosphorylate these sites of IR, and thereby
suppress insulin signaling.
RT-PCR and in situ hybridization showed that only PTPRJ
was expressed together with IR in major insulin target
tissues, such as skeletal muscle, liver, and adipose tissue.
Expectedly, Ptprj-deficient mice exhibited enhanced activation of IR and Akt by insulin, and improved glucoseand insulin tolerance (Figure 2). These results indicate thatPTPRJ is a physiological enzyme attenuating insulin signaling in vivo, and selective inhibitors of PTPRJ could beinsulin-sensitizing agents.

Figure 2. Improved glucose and insulin tolerance in Ptprj-KO mice. In
glucose (A) and insulin (B) tolerance tests, glucose (2 g/kg body weight)
and insulin (1 unit/kg body weight) were administered by intraperitoneal injection to fasted mice, respectively, and blood glucose levels wereassayed. Values are shown as the mean ± SEM. The asterisks indicate
significant differences from WT mice by Student’s t-test (*p < 0.05, **p< 0.01).

2-2 R5 RPTP subfamilyProtein-tyrosine phosphatase receptor type Z (PTPRZ)
is predominantly expressed in glial and neuronal cells
during development in the central nervous system (CNS).
Oligodendrocyte precursor cells (OPCs) are the principal
source of myelinating oligodendrocytes. Deficiencies inmyelination in diseases such as multiple sclerosis (MS) leadto serious neurological disorders. Two animal disease models
have been widely accepted for studying the clinical andpathological features of MS lesions. Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated inflammatory CNS demyelination model, and the cuprizone modelof demyelination is induced by a T-cell-independent mechanism through feeding of the copper chelator cuprizone. We
previously showed that adult Ptprz-deficient mice were lesssusceptible to the induction of EAE than wild-type controls.
This year, we revealed that cuprizone-fed Ptprz-deficient
mice exhibited severe demyelination and axonal damage

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in the corpus callosum, similar to wild-type mice, whereas
remyelination in Ptprz-deficient mice after cuprizoneinduced demyelination was significantly accelerated.
Accelerated remyelination was attributed to the higher differentiation potential of OPCs, because the number of oligodendrocyte-lineage cells recruited to the demyelinated area was
not altered. Importantly, pleiotrophin, one of the inhibitory
ligands for PTPRZ, was transiently expressed in the brain
upon demyelination, and gradually disappeared with remyelination. Pleiotrophin was detected in affected cortex neurons
and their axon fibers in the cuprizone model. Since pleiotrophin colocalized with a synaptic vesicle marker, the relevant
neurons are presumed to release pleiotrophin from theirdemyelinated axons (Figure 3). The treatment of a primaryculture of wild-type mouse brain cells with pleiotrophin itself
did not induce oligodendrocyte maturation, but enhanced
thyroid hormone-induced oligodendrocyte differentiation.
Of note, the differentiation of Ptprz-deficient cells was notfurther potentiated by pleiotrophin. Pleiotrophin released
from demyelinated fibers may stimulate the differentiation of
OPCs recruited in the demyelinated area in vivo.

Figure 3. Proposed mechanism for remyelination after demyelinating lesions. OPCs, but not mature oligodendrocytes (OLs), abundantly
express PTPRZ-A/B receptor proteins as chondroitin sulfate (CS) proteoglycans. The CS moiety of PTPRZ is important for achieving the highaffinity binding of PTN to the core protein. PTPRZ activity is requisite tomaintaining the immature state of OPCs. After cuprizone-induced oligodendrocyte death and demyelination, the expression of PTN is transiently
upregulated in damaged neurons. PTN may be released from demyelinated axons and bind to PTPRZ at the cell surface of OPCs that arerecruited to lesions, probably through PTPRZ-independent mechanisms.
The binding of PTN results in receptor dimerization or oligomerization,
thereby inhibiting its catalytic activity. PTPRZ inactivation releases theblock of differentiation in OPCs, so the remyelination of neighboringaxons is initiated.

We established immature oligodendrocytes (OL1 cells)
from p53-deficient mice. They were strongly positive forthe two receptor isoforms of PTPRZ-A and PTPRZ-B withchondroitin sulfate chains; however, their expression gradually decreased with differentiation, with only PTPRZ-B
being weakly detectable in mature oligodendrocytes. The
treatment of immature OL1 cells with pleiotrophin enhanced
the phosphorylation of p190 RhoGAP, which is a substratemolecule of PTPRZ. We found that pleiotrophin reduced
the expression of NG2 proteins in OL1 cells. Therefore, it isconceivable that the catalytic activity of PTPRZ functions tomaintain OPCs in an undifferentiated state, and the pleiotrophin-induced inactivation of PTPRZ releases this blockage(Figure 3).

III. Brain systems for body-fluid homeostasis

Sodium (Na) is a major electrolyte of extracellular fluids
and the main determinant of osmolality. Na homeostasis
is essential to life and Na+ concentrations in plasma andcerebrospinal fluid (CSF) are continuously monitored tomaintain a physiological level of Na+ in body fluids. We
have previously shown that Nax, which structurally resembles voltage-gated sodium channels (Nav1.1–1.9), is a Na+concentration ([Na+])-sensitive Na channel with a gatingthreshold of ~150 mM for extracellular [Na+] ([Na+]o) invitro. Nax is preferentially expressed in glial cells of sensory
circumventricular organs including the subfornical organ,
and is involved in [Na+] sensing for the control of salt-intake
behavior.
Although Nax is also expressed in neurons of some brain
regions including the amygdala and cerebral cortex, thechannel properties of Na x in neurons have not yet beenadequately characterized. To investigate the properties ofNax expressed in neurons, we established an inducible cellline of Nax using the mouse neuroblastoma cell line, Neuro2a, which is endogenously devoid of the expression of Nax(N2a-Mf1 cell). Functional analyses of this cell line revealed
that the [Na+]-sensitivity of Nax in neuronal cells was similar
to that expressed in glial cells.
Furthermore, we found that Nax bound to postsynaptic
density protein 95 (PSD95) through its PSD95/Disc-large/
ZO-1 (PDZ)-binding motif at the C-terminus. Nax co-localized with PSD95 clusters along dendrites of lateral amygdala

neurons. PSD95 is known as an anchoring and scaffoldingprotein for receptors and other postsynaptic proteins. Theinteraction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels by suppressing their endocytosis, because the depletion of endogenous
PSD95 resulted in a decrease in Nax at the plasma membrane(Figure 4). These results show for the first time that Nax
functions as a [Na+]-sensitive Na channel in neurons as wellas in glial cells.

Publication List:〔Original papers〕

M.*, Shintani, T.*, Fahiminiya, S.*, Fujikawa, A.,• Almuriekhi,
Kuboyama, K., Takeuchi, Y., Nawaz, Z., Nadaf, J., Kamel, H., Kitam,•••••A.K., Samiha, Z., Mahmoud, L., Ben-Omran, T., Majewski, J., andNoda, M. (2015). Loss-of-function mutation in APC2 causes Sotos
Syndrome features. Cell Repotrs 10, 1585-1598. *Co-first authors.
Fujikawa, A., Matsumoto, M., Kuboyama, K., Suzuki, R., and Noda,M. (2015). Specific dephosphorylation at Tyr-554 of Git1 by Ptprz
promotes its association with Paxillin and Hic-5. PLoS One 10,e0119361.
Kuboyama, K., Fujikawa, A., Suzuki, R., and Noda, M. (2015).
Inactivation of protein tyrosine phosphatase receptor type Z
by pleiotrophin promotes remyelination through activation ofdifferentiation of oligodendrocyte precursor cells. J. Neurosci. 35,
12162-12171.
Matsumoto, M., Hiyama, T.Y., Kuboyama, K., Suzuki, R., Fujikawa,A., and Noda, M. (2015). Channel properties of Nax expressed inneurons. PLoS One 10, e0126109.
Shintani, T., Higashi, S., Takeuchi, Y., Gaudio, E., Trapasso, F., Fusco,A., and Noda, M. (2015). The R3 receptor-like protein-tyrosine
phosphatase subfamily inhibits insulin signaling by dephosphorylating
the insulin receptor at specific sites. J. Biochem. 158, 235-243.
Sun, L.O., Brady, C.M., Cahill, H., Al-Khindi, T., Sakuta, H., Dhande,O.S., Noda, M., Huberman, A.D., Nathans, J., and Kolodkin, A.L.
(2015). Functional assembly of accessory optic system circuitry criticalfor compensatory eye movements. Neuron 86, 971-984.

〔Review articles〕

Noda, M., and Hiyama, T.Y. (2015). The Na channel: What it is and• whatit does. Neuroscientist 21, 399-412.
M., and Hiyama, T.Y. (2015). Sodium sensing in the brain.
• Noda,Pflügers Arch. – Eur. J. Physiol. 467, 465-474.
xFigure 4. Subcellular distribution of Nax in non-treated Neuro-2a (N2a)cells, or N2a-Mf1 cells transfected with control or PSD95 siRNA. Upper
panels: Immunostaining with anti-PSD95 and anti-Nax antibodies. In
order to inhibit endocytosis, cells expressing Nax were treated with 100
nM wortmannin or 200 μM dynasore for 6 h. Scale bars, 10 μm. Lowergraphs: Fluorescence intensity profiles along the white lines in the upperpanels. The profile was divided into three parts (P1 : C : P2 = 15 : 70 : 15in length). a.u., arbitrary unit.

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