Mol Neurobiol
DOI 10.1007/s12035-016-9777-6

Transduction Profile of the Marmoset Central Nervous
System Using Adeno-Associated Virus Serotype 9 Vectors
Yasunori Matsuzaki 1 & Ayumu Konno 1 & Ryo Mukai 2 & Fumiaki Honda 3 &Masafumi Hirato 3 & Yuhei Yoshimoto 3 & Hirokazu Hirai 1

Received: 2 July 2015 / Accepted: 3 February 2016
# Springer Science+Business Media New York 2016

Abstract The common marmoset is a small New World primate that has attracted remarkable attention as a potential experimental animal link between rodents and humans. Adenoassociated virus (AAV) vector-mediated expression of adisease-causing gene or a potential therapeutic gene in thebrain may allow the construction of a marmoset model of abrain disorder or an exploration of the possibility of genetherapy. To gain more insights into AAV vector-mediatedtransduction profiles in the marmoset central nervous system(CNS), we delivered AAV serotype 9 (AAV9) vectors expressing GFP to the cisterna magna or the cerebellar cortex.
Intracisternally injected AAV9 vectors expanded in the CNS
according to the cerebrospinal fluid (CSF) flow, by retrograde
transport through neuronal axons or via intermediarytranscytosis, resulting in diffuse and global transduction within the CNS. In contrast, cerebellar parenchymal injection intensely transduced a more limited area, including the cerebellar cortex and cerebellar afferents, such as neurons of the
pontine nuclei, vestibular nucleus and inferior olivary nucleus.
In the spinal cord, both administration routes resulted in labeling of the dorsal column and spinocerebellar tracts, presumably by retrograde transport from the medulla oblongata andcerebellum, respectively. Motor neurons and dorsal root

* Hirokazu Hirai
hirai@gunma-u.ac.jp

1

Department of Neurophysiology and Neural Repair, Gunma
University Graduate School of Medicine,
Maebashi, Gunma 371-8511, Japan2

Department of Ophthalmology, Gunma University Graduate Schoolof Medicine, Maebashi, Gunma 371-8511, Japan3

Department of Neurosurgery, Gunma University Graduate School of
Medicine, Maebashi, Gunma 371-8511, Japan

ganglia were also transduced, possibly by diffusion of thevector down the subarachnoid space along the cord. Thus,
these two administration routes led to distinct transduction
patterns in the marmoset CNS, which could be utilized togenerate different disease animal models and to deliver therapeutic genes for the treatment of diseases affecting distinct
brain areas.
Keywords Common marmoset . Adeno-associated virus
serotype 9 vector . Stereology . Cerebellum . Spinal cord .
Gene transfer

Introduction
The use of common marmosets (Callithrix jacchus) for neuroscience, including both basic and translational research, hasbeen expanding because marmosets have many advantages
over macaques, including (1) small size and easy handling,(2) high productive efficiency, and (3) a lack of fatal zoonotic
diseases such as herpes B virus [1]. Moreover, marmosets arepromising experimental animals that link mice to humans.
However, basic neuroanatomical and neurophysiological data
are still largely insufficient in marmosets relative to those in
macaques.
The abnormal accumulation of neurotoxic proteins in thecentral nervous system (CNS) causes a variety of disorders,
including lysosomal storage diseases [2, 3] and neurodegenerative diseases [4–6]. The efficient transfer of therapeutic
genes that ultimately reduce the level of accumulated proteinsin neurons of the CNS represents a promising approach for thetreatment of these diseases [3, 7–9]. Recent progress in thedevelopment of viral vectors has allowed the delivery oftransgenes into CNS cells with great efficiency. In particular,
adeno-associated virus (AAV) vectors, which are only 20 nm

Mol Neurobiol

in diameter [10], have the potential to transduce broad regionsin the CNS [11]. Previous studies used AAV serotype 9(AAV9) vectors to examine different administration routes in
non-human primates (NHPs) such as macaques and marmosets and showed local and retrograde gene transfer in the striatum and visual cortex (marmosets and macaques) [12] andwidespread transduction after intravascular and intracisternal
administration (macaques) [13–20]. Here, we used marmosets
to clarify the transduction profiles and pathways in the CNS
after intracisternal or cerebellar parenchymal injection of
AAV9 vectors. Our results will be useful for future applicationof AAV9 vector expression systems to the construction of a
marmoset model of a brain disorder as well as neuroanatomical and neurophysiological experiments of marmosets.

Results
Global Transduction of the CNS by Intracisternal
Injection of AAV9 Vectors
A previous study showed greater astrocytic tropism of
intracisternally administered AAV9 vectors [19]. Because
the application of AAV9 vectors for the production of neurodegenerative models and therapeutic gene delivery into neurons are future goals, we used a neuron-specific synapsin I(SynI) promoter. The sequence of the synapsin I promoter ishighly conserved among rodents and primates. Therefore, weused 1.0 kb of the rat SynI promoter augmented by the addition of the minimal cytomegalovirus (CMV) promoter(minCMV) sequence (rSynI-minCMV) [21]. The woodchuck
hepatitis virus posttranscriptional regulatory element (WPRE)
was placed in the main AAV plasmid downstream of the green
fluorescent protein (GFP) gene to stabilize the GFP mRNAand increase the expression level of the transgene [22]. An
AAV9 vector expressing GFP under the control of the rSynIminCMV promoter was injected into the cisterna magna of
two young adult marmosets approximately 1 year of age(H014 and H017) (Table 1). One marmoset (H014) weighed
250 g and received 150 μl of an AAV9 vector suspension witha titer of 1.5 × 1013 virus genomes (vg)/ml (9.2 × 1012 vg/kg
body weight). The second marmoset (H017) weighed 320 g
and received 200 μl of an AAV9 vector suspension with a titerof 6.9 × 1013 vg/ml (4.3 × 1013 vg/kg body weight). The behavior of the marmosets after the operation was almost indistinguishable from that of the naïve marmosets. The treatedmarmosets were euthanized 31 days after viral injection.
Observation of whole brains and spinal cords under a fluorescent stereoscopic microscope revealed remarkably diffused
GFP expression throughout the CNS (Fig. 1). Then, sagittal(H014) and coronal (H017) sections of the whole brains
were produced and double-immunostained for GFP and
the Nissl bodies.

Both the sagittal and coronal sections showed strong GFP
expression in the medulla oblongata that extended to the cerebellum through the cerebellar peduncle and moderate (orweak) disseminated GFP expression in various brain regions(Fig. 2a–d), such as the cerebral cortex (Fig. 2e), caudate
nucleus, hippocampus, the ventral posteromedial nucleus of
the thalamus, the gigantocellular reticular nucleus of the medulla oblongata (Fig. 2f), and the cerebellar cortex (Fig. 2g).
Interestingly, GFP expression in the cerebral cortex was characterized by a striped pattern that extended from the surface ofthe cerebral cortex vertically into the cerebral parenchyma.
Immunolabeling of blood vessels with an antibody against
factor VIII-related antigen showed that the GFP-expressing
cells surrounded penetrating arteries (Fig. 2h), suggesting that
the AAV9 vectors reached the cerebral parenchyma throughthe paravascular space (PVS) along the penetrating arteries aspreviously reported in cynomolgus macaques after AAV9vector injection into the cisterna magna [19].
Using stereology, we assessed the transduction efficiency
in various brain regions by determining the ratio of GFPexpressing neurons to total neurons present in the region ofinterest (Fig. 2i and Table 2). Consistent with the strong GFP
expression observed in the medulla oblongata (low magnification photos; Fig. 2a–d), approximately half of the neurons in
the medulla oblongata were transduced by intracisternal injection of the AAV9 vector. Approximately 10–20 % of the neurons in the frontal, parietal, occipital, and temporal lobes of thecerebral cortex, basal ganglia, and cerebellar cortex weretransduced, whereas only a small percentage of neurons in
the insular lobe, limbic lobe, thalamus, and hypothalamus
were transduced (Fig. 2i and Table 2). In the cerebellum, thelarge projection neurons in the deep cerebellar nuclei (DCN)
were efficiently transduced (approximately 50 %), followedby Purkinje cells (approximately 20 %). In contrast, the interneurons with small cell bodies in the DCN and the granulecells in the cerebellar cortex were rarely transduced.
Absence of Purkinje Cell Transduction
by Intraparenchymal Injection of an AAV9 Vector Withan rSynI-minCMV Promoter
To examine the transduction area and efficiency of the administration of the AAV9 vector directly into the interstitial spaceof the cerebellum, an AAV9 vector expressing GFP under thecontrol of the rSynI-minCMV promoter was injected into thebilateral cerebellar hemispheres of an adult marmoset (I4222;
Table 1). The marmoset received 100 μl (50 μl/hemisphere) ofan AAV9 vector suspension with a titer of 3.0 × 1013 (vg)/ml(8.8 × 1012 vg/kg body weight). The treated marmoset was
euthanized 555 days after the viral injection. Observation of
the whole brain and spinal cord with a fluorescent stereoscopic microscope showed remarkably strong GFP expression in
the cerebellum (Fig. 3a, b) and relatively strong GFPMol Neurobiol
Table 1

Summary of marmosets that received intracisternal and cerebellar parenchymal injections of AAV9 vectors

ID

Route

GenderAge (year)

Weight (g)Viral titer (vg/ml)

Volume (μl)

Dose(vg/kg BW)

Sacrifice(days after IJ)

Promoter

H014
H071
I4222
H016

IC
IC
CP
CP

M
F
F
M

0.8
1.5
5.0
1.2

250
320
341
324

1.5 × 1013
6.9 × 1013
3.0 × 1013
1.2 × 1014

150
200
100
100

9.0 × 1013
4.3 × 1013
8.8 × 1012
3.7 × 1013

31
31
555
32

rSynl-minCMV
rSynl-minCMV
rSynl-minCMV
MSCV

H018

CP

M

0.9

314

1.2 × 1014

341

13

100
100

3.8 × 1013
1.3 × 1013

28
30

MSCV
MSCV

H038

CP

F

2.2

4.5 × 10

IC intracisternal, CP cerebellar parenchymal, M male, F female, vg virus genomes, BW body weight, IJ injection.

expression along the spinal cord (Fig. 3c, d). Then, coronal
sections of the whole brain were produced and doubleimmunostained for GFP and the Nissl bodies (Fig. 4c–k).
These serial coronal sections showed highly efficient GFP
expression that occurred bilaterally in the hemispheres andthe vermal region of the cerebellum (Fig. 4a). Notably, highmagnification images showed an almost complete absence of
Purkinje cell transduction throughout the whole cerebellum(Fig. 4c–e, arrowhead), which was in sharp contrast to thestrong GFP expression in the granule cells beneath the
Purkinje cell layer. Additionally, we observed almost no transduced Purkinje cells in a marmoset treated with an AAV9vector expressing a different transgene under the control of
the rSynI-minCMV promoter (data not shown). These results
suggest that little or weak rSynI-minCMV promoter activity
occurred in marmoset Purkinje cells.
Fig. 1 Global transduction in the
marmoset CNS driven byintracisternal injection of an
AAV9 vector expressing GFP
under the control of the neuronspecific rSynI-minCMV
promoter. Whole brains andspinal cords from one noninjected control (Non-injected)
and two marmosets (H014 andH017) 1 month after the injectionof AAV9 vectors are presented.
a–f Bright field (a, c, e) and GFP
fluorescence (b, d, f) images ofwhole brains from non-injected(a, b) and AAV9 vector-treated(c–f) marmosets. g–l Bright field(g, i, k) and GFP fluorescence (h,j, l) images of spinal cords from
non-injected (g, h) and AAV9vector-treated (i, k, j, l)marmosets (scale bar, 1 cm)

In addition to the cerebellum, strong GFP expression
was observed in several regions of the brainstem andmidbrain that were closely associated with the cerebellar
cortex, including the red nucleus (Fig. 4b), inferiorolivary nuclei (Fig. 4f–h), and pontine nuclei (Fig. 4b,i–k).
Efficient Transduction of Purkinje Cells and Neuronsof the Brainstem Nuclei by Intraparenchymal Injectionof an AAV9 Vector With the Murine Stem Cell Virus
Promoter
Many types of spinocerebellar ataxia (SCA) primarily affect Purkinje cells in the cerebellum [23–25]. Therefore,
we were interested in expressing disease-causing and therapeutic genes in Purkinje cells. Because rSynI-minCMV

Mol Neurobiol
Fig. 2 Whole brain sections frommarmosets that received
intracisternal injections of an
AAV9 vector expressing GFP
under the control of the neuronspecific rSynI-minCMV
promoter and the transduction
efficiency in various brain
regions. Sections were
immunolabeled for GFP and
Nissl bodies (a–g) or factor VIIIrelated antigen (h). a, b Sagittal
sections +1.4 (a) and +4.0 mm (b)from the midline. c, d Coronal
sections −5.0 (c) and +5.0 mm (d)from the bregma. Note the striped
GFP expression pattern in the
cerebral cortex. e–h Enlarged
images of the cerebral cortex (e),reticular nucleus of the medulla
oblongata (f), and cerebellar
cortex (g). Arrows andarrowheads indicate pyramidal
neurons and Purkinje cells,
respectively. h An enlarged imageof a GFP stripe in the cerebral
cortex, showing many GFPexpressing cells around a bloodvessel immunolabeled for factor
VIII-related antigen. Scale bars,
1 mm (a–d) and 100 μm (e–h). i
Percent ratios of GFP (+) neurons
to total neurons in various brain
areas. The number of neurons ineach area was determined bystereology. DCN (large) andDCN (small); large projection
neurons and small interneurons,
respectively, in the deep
cerebellar nuclei

exhibited markedly low promoter activity in the Purkinjecells (Fig. 4c–e), we tested the constitutive murine stem
cell virus (MSCV) promoter, which had a preference for
Purkinje cells in the mouse cerebellum [26]. AAV9 vectors expressing GFP under the control of the MSCV promoter were injected into the bilateral cerebellar hemispheres of three young adult marmosets (H016, H018,
and H038; Table 1). These marmosets received 100 μl(50 μl/hemisphere) of an AAV9 vector suspension witha titer of 1.2 × 1014 vg/ml. The injected marmosets appeared to be slightly ataxic on the day of operation but
recovered and were almost indistinguishable from thenaïve marmosets by the next day.

The treated marmosets were euthanized approximately
1 month after the viral injection. Observation of the wholebrain and spinal cord with a fluorescent stereoscopic microscope showed remarkably strong GFP expression in the cerebellum (Fig. 3e, f, i, j) and relatively strong GFP expression
along the spinal cord (Fig. 3g, h, k, i). Sagittal (H018) andcoronal (H016) sections of the whole brains were produced
and double-immunostained for GFP and the Nissl bodies(Fig. 5a–h). These serial sagittal sections showed that thewide-ranging transduction of the cerebellar cortex and whitematter occurred primarily from the anterior to the central lobules (Fig. 5a, b), whereas the serial coronal sections showed
efficient GFP expression in the whole cerebellum (Fig. 5c).

159
156
315

121
1,473
1,594

218
91
309

29
702
731

Other neurons

Large neurons

Large neurons

Other neurons

4,567
26,564
Inferior olivary nucleus

1172
196,366
Vestibular nucleus

Neurons in WM

Interneurons
in ML
9,502
17,062167
64231

Large neurons

8,067
Pontine nuclei

892
7,175

4,693
390,612395,305

Granule cell

306
1,224
1,530

Cerebellum
Purkinje cell

29
330
359

Other neurons

122

Large neurons
in DCN
17
105

13,775
14,591

816

Interneurons

78,163
79,999

6,299

Small neurons
in DCN
612
5,687

102
102
204

DCN (Large)

55,40864,489

0
1,734
1,734

DCN (Small)

16,122
16,632510
57,65358,163

White matter

13,061
13,061

Hypothalamus
0

Results obtained from two marmosets following the intracisternal injection of an AAV9 vector expressing GFP under the control of the neuron-specific rSynI-minCMV promoter

Results obtained from two marmosets following cerebellar parenchymal injection of an AAV9 vector expressing GFP under the control of the MSCV promoterab

DCN deep cerebellar nuclei, GCL granule cell layer, ML molecular layer, WM white matter

Neurons were identified by NeuroTrace labeling, and the number (× 103 ) was determined by stereology. Each value is the average of two AAV9 vector-injected marmosets

GFP(+)GFP(−)
Total

Total

GFP(+)
408
306
2,959
GFP(−)
2857
3673
2,875
Total
3206
3979
5,816
Cerebellar parenchymal injection (MCSV promoter)b
Cerebellum
Purkenji cell
Granule cell
Interneurons
in GCLGFP(+)
962
5,722
542
GFP(−)
210
190,644
4,025

Medulla

5,510
5,612

Midbrain

58,16367,244

Brainstem
Pons

116,938
137,142

47,75556,836

Basal ganglia
9,081

70,40877,754

Limbic lobe
1,836

GFP(−)
Total

Insular lobe
102

Thlaramus
510

Temporal lobe
9,081

Frontal lobe
7,346

GFP(+)

Pariental lobe
9,081

Diencephalon

Telencephalon
Occipital lobe
20,204

Number of GFP-positive and total neurons in various regions of the marmoset brain

Intracisternal injection (rSynl-minCMV promoter)aTable 2

67,341
936,015
1,003,358

Total

Mol Neurobiol

Mol Neurobiol
Fig. 3 Robust transduction of thecerebellum, brainstem, and spinal
cord by cerebellar parenchymal
injection of an AAV9 vector.
AAV9 vectors expressing GFP
under the control of the rSynIminCMV or MSCV promoter
were injected bilaterally into the
cerebellar hemispheres of adultmarmosets (I4222, H018, andH016). The GFP expression
profiles in the brain and spinal
cord were examined 1.5 years(rSynI-minCMV) or 1 month(MSCV promoter) after viral
injection. The bright field and
GFP fluorescent images from
I4222 (a–d), H018 (e–h), andH016 (i–l) are presented (scalebar, 1 cm)

We produced coronal sections of the brain from another marmoset (H038) that showed a transduction pattern similar to
H016 (data not shown). A major difference in the transduction
patterns between the rSynI-minCMV and MSCV promoters is
the robust and efficient transduction of Purkinje cells by the
MSCV promoter (Fig. 5e), which is in contrast to the almost
complete lack of Purkinje cell transduction by the rSynIminCMV promoter (Fig. 4c–e). However, the brainstem andmidbrain, which are closely associated with the cerebellar cortex, expressed GFP at levels similar to the rSynI-minCMV
promoter in regions including the vestibular nuclei, inferiorolivary nuclei, pontine nuclei, and red nucleus (Fig. 5a, f–h).
Moreover, GFP expression was lower but clearly present inregions that received cerebellar efferents, such as the thalamic
nuclei (LP, VPL, VPPC, and VPM) [27] and dorsal lateralgeniculate nucleus (DLG) [28] (Fig. 5b, d). Interestingly, thevisual cortex (V1–V3) expressed GFP (especially V2 and V3)(Fig. 5b); these regions are also connected to the cerebellum
by a pontine nuclear relay [29].
Using stereology, we assessed the transduction efficiencyof neurons in various cerebellar regions by determining theratio of GFP-expressing neurons to total neurons present in theregion of interest (Fig. 5m and Table 2). Notably, more than
80 % of the Purkinje cells (Fig. 5e, m) and 50–70 % of thelarge projection neurons in the vestibular nucleus (Ve) (Fig. 5f,m), inferior olivary nucleus (Fig. 5g, m), and pontine nuclei(Fig. 5h, m) expressed GFP, whereas only limited percentagesof granule cells (Fig. 5m) and neurons in the DCN expressed
GFP, including large projection neurons and small interneurons (Fig. 5m).
Efficient Transduction of Motor Neurons and Sensory
Afferents in the Spinal Cord by Intracisternaland Cerebellar Parenchymal Injection of the AAV9 Vector
The transduction profiles of the spinal cord were examined at different spinal levels by double-immunolabelingof transverse sections for GFP and choline acetyltransferase. The fluorescent microscopic images revealed thatsimilar components were labeled by GFP following both
intracisternal injection and cerebellar parenchymal injection. The AAV9 vector transduced motor neurons in theanterior horns, dorsal root ganglia cell bodies, and sensory
projections, such as the dorsal column medial lemniscus,
spinocerebellar tracts, and spino-olivary fibers (Fig. 6).
These GFP-expressing sensory afferents projected into
the brainstem and cerebellum, both of which were close
to the viral injection sites. These results suggested theMol Neurobiol
Fig. 4 Coronal sections of thewhole brain and the magnified
images following cerebellar
parenchymal injection of an
AAV9 vector expressing GFP
under the control of the neuronspecific rSynI-minCMV
promoter. Sections were
immunostained for GFP and Nissl
bodies. a, b Coronal sections −5.5(a) and +3.5 mm (b) from thebregma. Pn, pontine nuclei; RN,
red nucleus. Note the high levelsof GFP expression in the
cerebellum and areas relevant tothe cerebellar cortex. c–k
Enlarged images of the cerebellar
cortex (c–e), inferior olivary
nucleus (f–h) and pontine nuclei(i–k). GFP, NeuroTrace, and themerged images were presented as
depicted. Note the complete
absence of GFP expression in the
Purkinje cells (arrowheads).
Scale bars, 1 mm (a, b) and100 μm (c–k)

occurrence of retrograde transport and the consequenttransduction of neurons whose axons constituted the dorsal column medial lemniscus, spinocerebellar tracts, andspino-olivary fibers (Fig. 6). In addition to the lower motor neurons and dorsal root ganglia, we examined the retinas of marmosets as an example of peripheral neuronal
structures after intracisternal or cerebellar parenchymal
injection but found no GFP-positive cells (data notshown).

Discussion
AAV is non-pathogenic for humans and measures only 20–
25 nm in diameter [10]. Thus, AAV9 vectors are diffusible inthe brain and allow the efficient delivery of transgenes to broad
regions of the CNS. These advantages allow the use of AAV9vectors for the generation of disease model animals for the
study of neurodegenerative diseases [30, 31] and gene therapy
vectors that may target brain disorders [8, 32, 33]. For theseMol Neurobiol

Fig. 5 Whole brain sections following cerebellar parenchymal injectionof an AAV9 vector expressing GFP under the control of the MSCV
promoter and the transduction efficiency in the cerebellum and the areas
associated with the cerebellum. Sections were immunostained for GFP
and Nissl bodies. a Sagittal sections +2.0 mm from the midline. IO,
inferior olive; Pn, pontine nuclei; RN, red nucleus; Ve, vestibular
nucleus. b Sagittal sections +4.0 mm from the midline. V1–V3, visualarea (1–3); LP, lateral posterior thalamic nucleus; VPL, ventral
posterolateral thalamic nucleus. c, d Coronal sections −6.0 mm (c) and+4.8 mm (d) from the bregma. DLG, dorsal lateral geniculate nucleus;
RN, red nucleus; VPM, ventral posteromedial thalamic nucleus; VPPC,

ventral posterior nucleus of the thalamus, parvicellular. Note the highlevels of GFP expression in the cerebellum and areas relevant to the
cerebellar cortex. e–h Enlarged images of the cerebellar cortex (e),vestibular nucleus (f), inferior olivary nucleus (g), and pontine nuclei(h). Scale bars, 1 mm (a–d) and 100 μm (e–h). i Percent ratios of GFP(+) neurons to total neurons in the cerebellum and brain stem areas
associated with the cerebellar cortex. The number of neurons wasdetermined by stereology. DCN, deep cerebellar nuclei; GCL, granulecell layer; IO, inferior olive; ML, molecular layer; Pn, pontine nuclei;
Ve, vestibular nucleus; WM, white matter

applications, it is important to clarify the relationship between
the administration route of the AAV9 vector and the consequenttransduction profiles in the CNS. Although such experiments
have been primarily conducted in rodents [30, 34–36], there are
significant merits to using NHP to replicate human disease

pathology and to assess the benefits and adverse reactions that
accompany therapeutic gene delivery.
Cerebrospinal fluid (CSF) is produced at the choroid plexus and flows through the cerebral ventricles and subarachnoid
space [37–39]. A recent study demonstrated that CSF flowedMol Neurobiol
Fig. 6 Immunohistochemistry of
the spinal cord at different levels.
Transverse sections of the cervical(a, d), thoracic (b, e), and lumbar(c, f) cords from marmosets
following intracisternal (H017),
and cerebellar parenchymal(H016) injection were doubleimmunolabeled for GFP (green)and choline acetyltransferase(red). The inset in each panel is anenlarged view of the ventral hornshowing efficient GFP expression
in motor neurons immunolabeled
for choline acetyltransferase(scale bars, 300 and 50 μm(insets))

into the PVS surrounding the arteries that penetrate the brain
tissue [40]. Intracisternally administered AAV9 vectors diffused via the CSF and flowed in the PVS that surrounded
the arteries entering the brain tissue. The inner and outer wallsof the PVS consist of vascular smooth muscle and astrocytic
endfeet, respectively. CSF is actively transferred from the PVS
to the interstitial space by the aquaporin 4 protein present on
the endfeet [40], whereas the AAV9 vectors must pass throughthe cleft between endfeet to reach the brain tissue. Because theastrocytic processes of primates are far more complex thanthose of rodents [41], the distance between the endfoot gap
and the permeability of the AAV9 vectors through it may
differ between primates and rodents. To answer these questions, we performed AAV9 experiments in marmosets.
We administered AAV9 vectors to the marmoset CNS via
two different injection routes (intracisternal and cerebellar parenchymal). To reach the brain parenchyma, intracisternallyinjected AAV9 vectors flowing in the CSF have to pass theendfoot gap, which is presumably 20 nm or less in NHPs. This
reduced gap suggests that only a portion or none of the AAV9vector may reach the brain tissue. The limited (striped) transduction along the penetrating arteries in the cerebral cortex(Fig. 2a–d) suggests that the gap between the astrocytic
endfeet is permeable for the AAV9 vectors but that the permeability is not very high. In the parenchymal injection, AAV9vectors are administered directly into the interstitial space,resulting in transduction of almost the entire cerebellar cortex(Fig. 7b, red arrow). Neurons in the inferior olivary nuclei,
pontine nuclei, and vestibular nuclei were highly efficiently
transduced (50–70 % of the projection neurons, Fig. 5m). This
result could be explained by the infection of AAV9 vectors
from the axon terminals projecting into the cerebellar cortex
and the subsequent retrograde transport (Fig. 7b, blue arrow).
The finding that intracisternal injection and cerebellar parenchymal injection resulted in essentially the same transduction patterns in the spinal cord was unexpected. Because bothadministration routes robustly transduced the cerebellar cortex
and the medulla oblongata, we postulated that the neurons
whose axons constituted the dorsal column medial lemniscus
and spinocerebellar tracts were retrogradely transduced byMol Neurobiol

Fig. 7 Schematic depicting a possible diffusion route of AAV9 vectors in
the marmoset CNS. a Intracisternally injected AAV9 vectors primarily
expand with the CSF flow (thick orange arrow) and enter into theparavascular space (PVS) along arteries penetrating into the cerebral tissue.
A portion of the AAV9 particles pass through the astrocytic endfoot gap toenter the interstitial space and transduce neurons near the penetrating arteries(thin orange arrow). Some AAV9 vectors enter the interstitial space of thebrainstem close to the injection site, probably due to the high viral density,and diffuse through the interstitial space to reach the cerebellum (red arrow).
Neurons in the spinal cord are transduced by two different routes: the first is
retrograde transport from axon terminals projecting into the cerebellum(spinocerebellar tract) and brainstem (cuneate fasciculus) (blue arrow) andthe second is infection by AAV9 vectors diffusing into the subarachnoid
space along the spinal cord (thick orange arrow), leading to the retrograde
transport and transduction of motor neurons and neurons of the dorsal root
ganglia. b Cerebellar parenchymal injection causes diffusion of AAV9vectors into the interstitial space throughout the cerebellar cortex (redarrow). Relevant brainstem nuclei are efficiently transduced in a retrograde
fashion (blue arrow). Neurons in the spinal cord are transduced similarly tothose intracisternally injected with AAV9 vectors (i.e., retrograde transport
via axons projecting into the cerebellum (spinocerebellar tract) andbrainstem (cuneate fasciculus) (blue arrow)). The vectors that diffuse
through the subarachnoid space (orange arrow) are re-retrogradelytransported through the axons of motor neurons and dorsal root ganglia.
Color figure online

infection with AAV9 vectors from the axon terminals in the
medulla oblongata and cerebellum (Fig. 7a, b, blue arrow). In
contrast, motor neurons in the ventral horn and dorsal root
ganglia were presumably transduced in a manner that differedfrom the dorsal column and spinocerebellar tracts; considering
the preference of AAV9 vectors for retrograde transport, thevectors descending in the subarachnoid space along the spinal
cord most likely infected the axons of motor neurons anddorsal root ganglia and were retrogradely transported to transduce those neurons.
It is likely that the similar administration of AAV9 vectors
results in different transgene expression patterns in rodents
and other species because the affinity of AAV9 vectors forneurons and glia may differ among species. Moreover, it ishighly probable that the activities of the cell type-specificpromoters used to drive gene expression in the AAV9 vectors
differ between rodents and other species, including primates.
Therefore, a number of studies have aimed to clarify the transduction profiles of AAV vectors in different species, such as
cynomolgus macaques [13, 16–19], rhesus macaques [14],pigs [13, 42], and cats [20]. Most of these studies employedintracisternal injection [13, 16–20]. The administration of the
AAV9 vector into the cisterna magna of marmosets in thisstudy was basically similar to studies that used cynomolgus
macaques in terms of global transduction and transduced cell
types, such as spinal motor neurons [16–18], dorsal root ganglia [18], and cerebral pyramidal neurons close to the bloodvessels [16, 18, 19]. It is difficult to compare retrograde transport between marmosets and other animals because previous
studies have not described the transduction pathway in detail.
One detectable difference between marmosets and cynomolgus macaques is marked GFP expression in the dorsal
column-medial lemniscus pathway (sensory afferents) in marmosets (Fig. 6) but not in cynomolgus macaques. In contrast,
high transduction was observed in the descending ventral
corticospinal tracts in cynomolgus macaques [18].
Transduction of the sensory afferents in marmosets is thought
to occur by the invasion of the medulla oblongata by highdensity AAV9 vectors, leading to retrograde axonal transport
through the dorsal column. Therefore, the absence of spinal
cord transduction in cynomolgus macaque may be attributable
to the lower density of AAV9 vectors in the medulla oblongata
due to rapid viral diffusion in the CSF rather than entry into
the medulla.
GFP fluorescence image from the marmoset (H014) treatedwith lower titer AAV9 vectors (Fig. 1d) was brighter than thatfrom the marmoset (H017) treated with higher titer vectors(Fig. 1f). We postulate that the posture after the viral injection
substantially influenced the viral diffusion in the CSF andconsequent transduction pattern. Indeed, the Trendelenburg
position after viral injection was shown to facilitate spreadingof the vector and significantly increase the transduction efficacy in cynomolgus macaques [17].

Mol Neurobiol

Many previous studies used constitutive promoters thatwere active in almost all cell types, such as the CMV promoter
and CMV early enhancer/chicken β actin (CAG) promoter. In
contrast, we used a neuron-specific rSynI-minCMV promoter
and the constitutive MSCV promoter and found that therSynI-minCMV promoter had no or very weak promoter activity in marmoset Purkinje cell (Fig. 4c–e), although thispromoter exhibited robust promoter activity in mouse
Purkinje cell [21, 30]. Thus, we have to be cautious in choosing promoters (especially cell type-specific promoters) because they can behave quite differently in monkeys and presumably in humans compared with rodents.
The present results suggest that intracisternal injection issuitable for diffuse and global transduction of the CNS,
whereas cerebellar parenchymal injection leads to more limited and robust transduction of the cerebellar cortex and cerebellar efferent neurons. Both administration routes transduced
motor neurons, dorsal root ganglia, the dorsal column medial
lemniscus and spinocerebellar tracts. The former route could
be utilized for gene therapy against diseases affecting broadb r a i n a r e a s , s u c h a s A l z h e i m e r ’s d i s e a s e a n d
mucopolysaccharidosis, whereas the latter route may be ofuse for gene therapy against disorders that primarily impair
the cerebellum, the associated brainstem nuclei, and the spinal
cord, such as spinocerebellar degeneration.

Materials and Methods
Marmosets
We used six common marmosets (C. jacchus; three males andthree females) in this study. Marmosets were purchased
through CLEA Japan (Tokyo, Japan) and were bred at the
Gunma University Bioresource Center. The animals weremaintained in rooms under controlled temperature (25 to
27 °C), humidity (40 to 50 %), and light cycle (12 h each oflight and dark) conditions. Water was provided ad libitum, and40 to 50 g of soaked monkey chow (CMS-1, CLEA Japan)
with vitamin supplementation and fruit, boiled chicken or
milk powder was provided daily. All efforts were made tominimize suffering and reduce the number of animals used.
Construction of Plasmids and AAV9 Production
We used two expression plasmids (pAAV-MSCV-GFP andpAAV-rSynI-minCMV-GFP-WPRE) [21] for AAV9 vector production. pAAV-MSCV-GFP was generated by subcloning the
MSCV-GFP fragment from pCL20c-MSCV-GFP [43] into
pAAV. The following two primer pairs were used to amplify
the inserts: XhoI-MSCV (forward) 5′-CGACTCGAGTAACGGGCCCAGCTTCGA-3′/GFP (reverse) 5′GTTTAAACTCATTACTTGTACAGCTCGTCCATG-3′ andX h o I - r S y n I ( f o r w a r d ) 5 ′ - AT G C T C TA G A C T C GAGGAAGAGGCTGAATACACATCAGAG-3′/KpnI-WPRE( r e v e r s e ) 5 ′ - C TA G A G G AT C C C C G G G TA C C A
TGCGGGGAGGCGGCCCAAA-3′.
The AAV9 vectors were produced as previously described[44]. Briefly, AAV9 vectors were generated by the cotransfectionof HEK293T cells (Thermo Fisher Scientific, Waltham, MA,
USA) with three plasmids: pHelper (Stratagene, La Jolla, CA,USA), pAAV2/9 (kindly provided by Dr. J. Wilson), and one of
the two expression plasmids. The viral particles were purified
using ammonium sulfate precipitation and iodixanol continuous
gradient ultracentrifugation as previously described [45]. The
genomic titer of the purified AAV9 vectors (Table 1) was determined by quantitative real-time PCR (Dice TP800, Takara Bio,
Shiga, Japan) using the THUNDERBIRD SYBR qPCR Mix(Toyobo, Osaka, Japan) with the following primers: 5′C T G T T G G G C A C T G A C A AT T C - 3 ′ a n d 5 ′ GAAGGGACGTAGCAGAAGGA-3′ targeting the WPRE sequence, or 5′-CACTCCCTTAAGTTTGACCTT-3′ and 5′GCCAAGGCTTCCCAGGTC-3′ targeting MSCV.
Viral Vector Injection
AAV9 vectors were injected into the cisterna magna or cerebellar cortex. Marmosets were anesthetized with ketamine (20–25 mg/kg) and xylazine (1.6–2.0 mg/kg), and anesthesia wasmaintained by inhalation of 2.0–2.5 % isoflurane (air flow,
0.5 l/min) in 80–100 % O2. The hair in the occipital region
was shaved prior to the skin incision. A 30-gauge needle attached to a 1-mm syringe was inserted into the cisterna magna
through the dura mater and arachnoid; the needle tip was monitored by radioscopy. We confirmed the entrance of the needle
tip into the subarachnoid space by gentle suction of CSF into
the syringe. Then, the syringe was replaced with a syringe containing the AAV9 vector, and 150 μl (H014) or 200 μl (H017)
of the viral suspension was injected into the cisterna magna.
For viral injection into the cerebellar parenchyma, the marmosets were fixed with a stereotaxic apparatus (SR-6C-HT;
Narishige, Tokyo, Japan). Muscles and ligaments over the occipital bone were carefully pushed aside without cutting to expose
the occipital bone. The bone was pierced using a 25-gauge needle(4.0 mm caudal to the torus occipitalis and 3.5–4.0 mm lateral tothe midline). The AAV9 vector was injected bilaterally into the
cerebellar hemispheres (5.0 mm deep from the occipital bone
surface). Fifty microliters of the viral solution was injected unilaterally into the hemisphere at a speed of 5 μl/min for 10 min using
a 33-gauge Hamilton syringe and a micropump (UltramicroPump
II; World Precision Instrument, Sarasota, FL, USA).
Histological Analysis
The marmosets were deeply anesthetized with a combinationof ketamine, xylazine, and isoflurane and transcardiallyMol Neurobiol

perfused with 1× phosphate-buffered saline (PBS(−)) and a
fixative containing 4 % paraformaldehyde in 0.1 M phosphatebuffer. Native GFP fluorescent images of whole brains wereobtained with a fluorescence stereoscopic microscope (VB7010; Keyence, Osaka, Japan). Then, the brains and spinal
cords were cut into 100-μm-thick sections using a Vibrating
Blade Microtome (VT1000; Leica, Wetzlar, Germany). Theeyes were cut into 8-μm-thick sections using the Cryostat(CM3050; Leica).
Immunohistochemistry
Sections were immunostained for GFP and Nissl bodies orcholine acetyltransferase. Blood vessels in the cerebral cortex
were visualized by immunolabeling for Factor VIII. Floating
brain and spinal cord sections were incubated in blocking
solution (2 % bovine serum albumin, 2 % normal donkey
serum (S30-100 ml; Merck Millipore, Billerica, MA, USA)and 0.4 % Triton X-100 in 0.1 M phosphate buffer) for 1 day
at 4 °C with the following primary antibodies: rat monoclonalanti-GFP (1:1000; 04404–84; Nacalai Tesque, Kyoto, Japan),rabbit polyclonal anti-Factor VIII-related antigen (vonWillebrand factor) (1:2; 220 M; Biomeda, Foster City, CA,USA), and goat polyclonal anticholine acetyltransferase(1:100; AB144P; Merck Millipore). The bound primary antibodies were visualized after incubation with Alexa Fluor 488conjugated donkey anti-rat IgG (1:1000; Thermo Fisher
Scientific), Alexa Fluor 594-conjugated donkey anti-rabbit
IgG (1:1000; Thermo Fisher Scientific), and Alexa Fluor
568-conjugated donkey anti-goat IgG (1:1000; Thermo
Fisher Scientific) in blocking solution for 2 h at room temperature. After the secondary antibody reaction, Nissl bodies
were stained with NeuroTrace 530/615 (1:200; Thermo
Fisher Scientific) or NeuroTrace 640/660 (1:200; Thermo
Fisher Scientific) in PBS for 1 h at room temperature. The
immunostained sections were mounted in the ProLong Goldor Diamond antifade reagent (Thermo Fisher Scientific). Low
magnification images of the whole brain and spinal cord sections were obtained using fluorescence microscopes (BZ9000 or BZ-X700, Keyence). High-magnification images
were obtained using a confocal laser scanning microscope(LSM 5 PASCAL; Carl Zeiss, Oberkochen, Germany).

the counting frame was 35 × 35 μm, and the dissector
height was 30 μm.
For the brains that received a cerebellar parenchymal injection, the cerebellum and brainstem were analyzed using different counting frames and grid sizes. For the examination of
the cerebellum, the interval was 1.2 mm (1-in-12 series, six
sections), the counting grid was 3500 × 2500 μm, the counting
frame was 100 × 100 μm, and the dissector height was 30 μm.
For the examination of the brainstem, the interval was 0.5 mm(1-in-5 series), the counting grid was 300 × 300 μm, thecounting frame was 35 × 35 μm, and the dissector heightwas 30 μm. The inferior olivary nucleus, pontine nuclei, andvestibular nucleus were examined using five, four, and sevensections, respectively.
To determine the ratio of GFP-expressing neurons to total
neurons present in the examined brain regions, neurons
double-labeled for GFP and Nissl bodies (NeuroTrace) andneurons labeled only with NeuroTrace were counted independently, and the ratios were calculated. In total, 9788 neurons
were counted for the intracisternal injection, and 13,895, 710,
258, and 830 neurons were counted in the cerebellum, inferiorolivary nucleus, pontine nuclei, and vestibular nucleus, respectively, for the cerebellar parenchymal injection.
Acknowledgments The authors are very grateful to the technicians
Motoko Uchiyama and Minako Noguchi for raising the marmosets.
This research is (partially) supported by the program for Brain Mappingby Integrated Neurotechnologies for Disease Studies (Brain/MINDS)
from the Ministry of Education, Culture, and Sports Science (MEXT)
and the Japan Agency for Medical Research and Development(AMED), MEXT KAKENHI grant number 26111701; grants from
Research on Measures for Intractable Diseases (Ataxic Diseases and
Neurodegenerative Diseases) from the Ministry of Health, Labor, andWelfare and the Gunma University Initiative for Advanced Research(GIAR) (to H. Hirai), MEXT KAKENHI grant number 26890005 (toY. Matsuzaki); and the Brain Sciences Project of the Center for Novel
Science Initiatives (CNSI), National Institutes of Natural Sciences (NINS;
grant numbers BS251006 and BS261011 to Y. Matsuzaki and A. Konno).
Compliance with Ethical Standards All procedures for the care andtreatment of animals were performed in accordance with the Japanese Act on
Welfare and Management of Animals, the Guidelines for the Proper Conductof Animal Experiments by the Science Council of Japan, and the Guide for
the Care and Use of Laboratory Animals of the National Research Councilof the United States. The experimental protocol was approved by theInstitutional Committee of Gunma University (No. 14-039).

Stereology

Conflict of Interest The authors have no potential conflicts of interest
to disclose.

The number and efficiency of GFP-expressing neurons invarious brain regions were analyzed statistically by stereology using Stereo Investigator ver. 11 (Micro Bright
Field Japan, Chiba, Japan). Following intracisternal injection, a 1-in-20 series (1 section per 2.0 mm) of 17 sections
immunostained for GFP and Nissl bodies (NeuroTrace)
were analyzed. The counting grid was 2500 × 2500 μm,

References1.

Kishi N, Sato K, Sasaki E, Okano H (2014) Common marmoset as
a new model animal for neuroscience research and genome editing

Mol Neurobiol

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

technology. Develop Growth Differ 56(1):53–62. doi:10.1111/dgd.
12109
Cox TM, Cachon-Gonzalez MB (2012) The cellular pathology oflysosomal diseases. J Pathol 226(2):241–254. doi:10.1002/path.
3021
Noh H, Lee JI (2014) Current and potential therapeutic strategies
for mucopolysaccharidoses. J Clin Pharm Ther 39(3):215–224. doi:
10.1111/jcpt.12136
Kesidou E, Lagoudaki R, Touloumi O, Poulatsidou KN,
Simeonidou C (2013) Autophagy and neurodegenerative disorders.
Neural RegenRes 8(24):2275–2283. doi:10.3969/j.issn.16735374.2013.24.007
Roussel BD, Kruppa AJ, Miranda E, Crowther DC, Lomas DA,
Marciniak SJ (2013) Endoplasmic reticulum dysfunction in neurological disease. Lancet Neurol 12(1):105–118. doi:10.1016/S14744422(12)70238-7
Ward SM, Himmelstein DS, Lancia JK, Binder LI (2012) Tau oligomers and tau toxicity in neurodegenerative disease. Biochem Soc
Trans 40(4):667–671. doi:10.1042/BST20120134
Nobrega C, Nascimento-Ferreira I, Onofre I, Albuquerque D, Hirai
H, Deglon N, de Almeida LP (2013) Silencing mutant ataxin-3
rescues motor deficits and neuropathology in Machado-Joseph disease transgenic mice. PLoS One 8(1):e52396. doi:10.1371/journal.
pone.0052396
Iwata N, Sekiguchi M, Hattori Y, Takahashi A, Asai M, Ji B,
Higuchi M, Staufenbiel M et al (2013) Global brain delivery of
neprilysin gene by intravascular administration of AAV vector inmice. Sci Rep 3:1472. doi:10.1038/srep01472
Torashima T, Koyama C, Iizuka A, Mitsumura K, Takayama K,
Yanagi S, Oue M, Yamaguchi H et al (2008) Lentivectormediated rescue from cerebellar ataxia in a mouse model ofspinocerebellar ataxia. EMBO Rep 9(4):393–399. doi:10.1038/
embor.2008.31
Flotte TR (2000) Size does matter: overcoming the adenoassociated virus packaging limit. Respir Res 1(1):16–18. doi:10.
1186/rr6
Murlidharan G, Samulski RJ, Asokan A (2014) Biology of adenoassociated viral vectors in the central nervous system. Front Mol
Neurosci 7:76. doi:10.3389/fnmol.2014.00076
Masamizu Y, Okada T, Kawasaki K, Ishibashi H, Yuasa S, Takeda
S, Hasegawa I, Nakahara K (2011) Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9. Neuroscience 193:249–258. doi:10.1016/j.
neuroscience.2011.06.080
Bevan AK, Duque S, Foust KD, Morales PR, Braun L, Schmelzer
L, Chan CM, McCrate M et al (2011) Systemic gene delivery inlarge species for targeting spinal cord, brain, and peripheral tissues
for pediatric disorders. Mol Ther 19(11):1971–1980. doi:10.1038/
mt.2011.157
Gray SJ, Matagne V, Bachaboina L, Yadav S, Ojeda SR, Samulski
RJ (2011) Preclinical differences of intravascular AAV9 delivery toneurons and glia: a comparative study of adult mice and nonhumanprimates. Mol Ther 19(6):1058–1069. doi:10.1038/mt.2011.72
Gray SJ, Nagabhushan Kalburgi S, McCown TJ, Jude Samulski R(2013) Global CNS gene delivery and evasion of anti-AAVneutralizing antibodies by intrathecal AAV administration in nonhuman primates. Gene Ther 20(4):450–459. doi:10.1038/gt.2012.
101
Hinderer C, Bell P, Vite CH, Louboutin JP, Grant R, Bote E, Yu H,
Pukenas B et al (2014) Widespread gene transfer in the central
nervous system of cynomolgus macaques following delivery of
AAV9 into the cisterna magna. Mol Ther Methods Clin Dev 1:
14051. doi:10.1038/mtm.2014.51
Meyer K, Ferraiuolo L, Schmelzer L, Braun L, McGovern V,
Likhite S, Michels O, Govoni A et al (2015) Improving single
injection CSF delivery of AAV9-mediated gene therapy for SMA:

18.

19.

20.

21.

22.

23.
24.

25.
26.

27.

28.
29.

30.

31.

32.

33.

a dose–response study in mice and nonhuman primates. Mol Ther
23(3):477–487. doi:10.1038/mt.2014.210
Samaranch L, Salegio EA, San Sebastian W, Kells AP, Bringas JR,
Forsayeth J, Bankiewicz KS (2013) Strong cortical and spinal cord
transduction after AAV7 and AAV9 delivery into the cerebrospinal
fluid of nonhuman primates. Hum Gene Ther 24(5):526–532. doi:
10.1089/hum.2013.005
Samaranch L, Salegio EA, San Sebastian W, Kells AP, Foust KD,
Bringas JR, Lamarre C, Forsayeth J et al (2012) Adeno-associatedvirus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther 23(4):382–389. doi:10.1089/
hum.2011.200
Yoon SY, Bagel JH, O’Donnell PA, Vite CH, Wolfe JH (2015)
Clinical improvement of alpha-mannosidosis cat following a single
cisterna magna infusion of AAV1. Mol Ther. doi:10.1038/mt.2015.
168
Matsuzaki Y, Oue M, Hirai H (2014) Generation of a neurodegenerative disease mouse model using lentiviral vectors carrying anenhanced synapsin I promoter. J Neurosci Methods 223:133–143.
doi:10.1016/j.jneumeth.2013.12.004
Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ (1999)
Enhanced expression of transgenes from adeno-associated virus
vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther
10(14):2295–2305. doi:10.1089/10430349950016942
Koeppen AH (2005) The pathogenesis of spinocerebellar ataxia.
Cerebellum 4(1):62–73. doi:10.1080/14734220510007950
Seki T, Adachi N, Abe-Seki N, Shimahara T, Takahashi H,
Yamamoto K, Saito N, Sakai N (2011) Elucidation of the molecularmechanism and exploration of novel therapeutics forspinocerebellar ataxia caused by mutant protein kinase C gamma.
J Pharmacol Sci 116(3):239–247
Zhang Y, Kaczmarek LK (2015) Kv3.3 potassium channels andspinocerebellar ataxia. J Physiol. doi:10.1113/JP271343
Takayama K, Torashima T, Horiuchi H, Hirai H (2008) Purkinjecell-preferential transduction by lentiviral vectors with the murine
stem cell virus promoter. Neurosci Lett 443(1):7–11. doi:10.1016/j.
neulet.2008.07.058
Gallay MN, Jeanmonod D, Liu J, Morel A (2008) Human
pallidothalamic and cerebellothalamic tracts: anatomical basis forfunctional stereotactic neurosurgery. Brain Struct Funct 212(6):443–463. doi:10.1007/s00429-007-0170-0
Vaudano E, Legg CR (1992) Cerebellar connections of the ventral
lateral geniculate nucleus in the rat. Anat Embryol 186(6):583–588
Glickstein M (2000) How are visual areas of the brain connected to
motor areas for the sensory guidance of movement? Trends
Neurosci 23(12):613–617
Huda F, Konno A, Matsuzaki Y, Goenawan H, Miyake K, Shimada
T, Hirai H (2014) Distinct transduction profiles in the CNS via three
injection routes of AAV9 and the application to generation of aneurodegenerative mouse model. Mol Ther Methods Clin Dev.
Article number: 14032. doi:10.1038/mtm.2014.32
Kirik D, Bjorklund A (2003) Modeling CNS neurodegeneration by
overexpression of disease-causing proteins using viral vectors.
Trends Neurosci 26(7):386–392. doi:10.1016/S0166-2236(03)00164-4
Wolf DA, Banerjee S, Hackett PB, Whitley CB, McIvor RS, Low
WC (2015) Gene therapy for neurologic manifestations of
mucopolysaccharidoses. Expert Opin Drug Deliv 12(2):283–296.
doi:10.1517/17425247.2015.966682
Heldermon CD, Ohlemiller KK, Herzog ED, Vogler C, Qin E,
Wozniak DF, Tan Y, Orrock JL et al (2010) Therapeutic efficacy
of bone marrow transplant, intracranial AAV-mediated gene therapy, or both in the mouse model of MPS IIIB. Mol Ther 18(5):873–
880. doi:10.1038/mt.2010.17

Mol Neurobiol
34.

Gholizadeh S, Tharmalingam S, Macaldaz ME, Hampson DR(2013) Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors inneonatal and juvenile mice. Human Gene Ther Methods 24(4):205–213. doi:10.1089/hgtb.2013.076
35. McLean JR, Smith GA, Rocha EM, Hayes MA, Beagan JA, Hallett
PJ, Isacson O (2014) Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven
AAV9 neonatal intracerebroventricular injection. Neurosci Lett
576:73–78. doi:10.1016/j.neulet.2014.05.044
36. Miyake N, Miyake K, Yamamoto M, Hirai Y, Shimada T (2011)
Global gene transfer into the CNS across the BBB after neonatal
systemic delivery of single-stranded AAV vectors. Brain Res 1389:
19–26. doi:10.1016/j.brainres.2011.03.014
37. Abbott NJ (2004) Evidence for bulk flow of brain interstitial fluid:
significance for physiology and pathology. Neurochem Int 45(4):545–552. doi:10.1016/j.neuint.2003.11.006
38. Koh L, Zakharov A, Johnston M (2005) Integration of the subarachnoid space and lymphatics: is it time to embrace a new conceptof cerebrospinal fluid absorption? Cerebrospinal Fluid Res 2:6. doi:
10.1186/1743-8454-2-6
39. Praetorius J (2007) Water and solute secretion by the choroid plexus. Pflugers Arch - Eur J Physiol 454(1):1–18. doi:10.1007/s00424006-0170-6
40. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA,
Benveniste H, Vates GE et al (2012) A paravascular pathway

facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med
4(147):147ra111. doi:10.1126/scitranslmed.3003748
41. Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, Xu Q,
Wyatt JD et al (2009) Uniquely hominid features of adult humanastrocytes. J Neurosci 29(10):3276–3287. doi:10.1523/
JNEUROSCI.4707-08.2009
42. Federici T, Taub JS, Baum GR, Gray SJ, Grieger JC, Matthews KA,
Handy CR, Passini MA et al (2012) Robust spinal motor neurontransduction following intrathecal delivery of AAV9 in pigs. Gene
Ther 19(8):852–859. doi:10.1038/gt.2011.130
43. Torashima T, Okoyama S, Nishizaki T, Hirai H (2006) In vivotransduction of murine cerebellar Purkinje cells by HIV-derived
lentiviral vectors. Brain Res 1082(1):11–22. doi:10.1016/j.
brainres.2006.01.104
44. Konno A, Shuvaev AN, Miyake N, Miyake K, Iizuka A, Matsuura
S, Huda F, Nakamura K et al (2014) Mutant ataxin-3 with an abnormally expanded polyglutamine chain disrupts dendritic development and metabotropic glutamate receptor signaling in mouse
cerebellar Purkinje cells. Cerebellum 13(1):29–41. doi:10.1007/
s12311-013-0516-5
45. Miyake K, Miyake N, Yamazaki Y, Shimada T, Hirai Y(2012) Serotype-independent method of recombinant adenoassociated virus (AAV) vector production and purification. J
Nippon Med School = Nippon Ika Daigaku zasshi 79(6):394–402