diabetes impairs wnt3-induced neurogenesis in olfactory ... · 20/5/2016 · 2 abstract diabetes...

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Diabetes impairs Wnt3-induced neurogenesis in olfactory bulbs via glutamate transporter 1 inhibition

Tamami Wakabayashi1*, Ryo Hidaka1*, Shin Fujimaki1,2, Makoto Asashima1, and Tomoko

Kuwabara1

1Stem Cell Engineering Research Group, Biotechnology Research Institute for Drug Discovery,

Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science

and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan. 2Physical Education, Health and Sport Sciences, Graduate School of Comprehensive Human

Sciences, University of Tsukuba. 1-1-1 Tennodai, Tsukuba, Ibaraki, 303-8577, Japan.

Running Title: GAT1 suppression impairs neurogenesis in olfactory bulbs in diabetics

To whom correspondence should be addressed: Tomoko Kuwabara,

Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial

Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.

Tel: +81-29-861-2529; Fax: +81-29-861-2897; E-mail: [email protected]

Keywords: olfactory bulb; diabetes; neurogenesis; Wnt3, GAT1

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.672857The latest version is at JBC Papers in Press. Published on May 20, 2016 as Manuscript M115.672857

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT

Diabetes is associated with impaired

cognitive function. Streptozotocin (STZ)-

induced diabetic rats exhibit a loss of

neurogenesis and deficits in behavioral tasks

involving spatial learning and memory;

thus, impaired adult hippocampal

neurogenesis may contribute to diabetes-

associated cognitive deficits. Recent studies

have demonstrated that adult neurogenesis

generally occurs in the dentate gyrus of the

hippocampus, the subventricular zone, and

the olfactory bulbs (OB) and is defective in

patients with diabetes. We hypothesized that

OB neurogenesis and associated behaviors

would be affected in diabetes. In this study,

we show that inhibition of Wnt3-induced

neurogenesis in the OB causes several

behavioral deficits in STZ-induced diabetic

rats, including impaired odor

discrimination, cognitive dysfunction, and

increased anxiety. Notably, the sodium- and

chloride-dependent GABA transporters

(GATs) and excitatory amino acid

transporters (EAATs) that localize to

GABAergic and glutamatergic terminals decreased in the OB of diabetic rats.

Moreover, GAT1 inhibitor administration

also hindered Wnt3-induced neurogenesis in

vitro. Collectively, these data suggest that

STZ-induced diabetes adversely affects OB

neurogenesis via GABA and glutamate

transporter systems, leading to functional

impairments in olfactory performance.

INTRODUCTION

Mammalian neurogenesis occurs in the

subventricular zone (SVZ) of the lateral

ventricles and the sub-granular zone (SGZ) of

the dentate gyrus (DG) in the hippocampus (1-

3). In the SVZ, self-renewing multipotent

neural stem cells (NSCs) give rise to

neuroblasts, which migrate through the rostral

migratory stream into the olfactory bulb (OB)

where they differentiate into multiple types of

local interneurons. In addition to the SGZ and

SVZ, the OB core is another proposed source

of NSCs (4-8). NSC cultures have been derived

from the adult rodent and human OB (4,5);

thus, local NSCs in the OB may provide a

valuable source for autologous transplantation

in neurodegenerative disorders and other

diseases. NSCs possess self-renewal and

multipotency capacities and can differentiate

into neurons, astrocytes, or oligodendrocytes

(9,10). Insulin supports the function of basic

fibroblast growth factor (FGF2), which promotes maintenance of undifferentiated

NSCs (11), as well as triggers the

differentiation of multipotent NSCs into

oligodendrocytes (12). Newly formed neurons

are incorporated into the functional networks of

the OB and DG, demonstrating the substantial

impact of adult neurogenesis on brain functions

such as learning, memory processing, and odor

discrimination (13-17).

Diabetes—one of the most common

serious metabolic disorders in humans—is


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characterized by hyperglycemia that is caused

by defects in insulin secretion, activity, or both

(18). Diabetes is associated with cognitive

decline and increased risks of Alzheimer's

disease and dementia (19-23). Cognitive

deficits have also been reported in studies of

rodent models of diabetes. The cytotoxic agent

streptozotocin (STZ) selectively destroys

insulin-producing β-cells of the pancreas by

entering through the Glut2 glucose transporter.

(STZ)-induced type 1 diabetic rodents exhibit

deficits in behavioral tasks involving spatial

learning and memory, such as performance in

the Morris water maze and novel object

recognition tests (24-26). The blood–brain

barrier is devoid of Glut2 transporters (27),

suggesting that STZ has limited direct effects

on the brain. Impaired adult hippocampal

neurogenesis is also observed in diabetic

rodents, while abrogated long-term potentiation

(LTP), hippocampal neurogenesis, and

cognitive deficits were restored in STZ-

induced diabetic rats and db/db mice after

adrenalectomy and corticosterone replacement

at physiological concentrations (26). As such,

glucocorticoids were believed to be responsible

for the hindered neurogenesis and cognitive

dysfunction in diabetes.

We recently demonstrated that adult

hippocampal neurons and NSCs derived from

the hippocampus and OB express insulin at

detectable levels (28). In that study, NeuroD1

directly induced insulin (INS) gene expression

in NSCs isolated from the adult hippocampus

and OB. Wnt3 is an instructive factor secreted

by astrocytes and promotes adult neurogenesis

by stimulating NeuroD1 expression, a basic

helix-loop-helix transcription factor (29). Thus,

under Wnt3 activation, NeuroD1 promotes

neurogenesis and insulin production in the

adult OB and hippocampal NSCs. Indeed,

NSCs derived from the OB and hippocampus

of adult diabetic rats retained their intrinsic

capacity to produce insulin, making these cells

a useful source for autologous cell

transplantation in diabetes.

Dysfunctions in olfactory and OB

neurogenesis has been observed patients with

diabetes (30-32), and present clinically as

altered olfactory-related behaviors. The

characteristics of NSCs and their neurogenic

niche in diabetes are critical elements of insulin

function and neurogenesis; thus, it is important

to understand the mechanism of diabetes-

induced deficits in neuronal function. We

previously demonstrated great similarities in

the expression pattern of genes involved in

diabetes and Wnt signaling between NSCs of

the OB and hippocampus in diabetes. As such,

we hypothesized that adult OB NSC

neurogenesis and OB function are also affected

by diabetes (33). In this study, we evaluated the

morphology of the OB neurogenic niche. Most

importantly, Wnt3-induced neurogenesis was

inhibited in the OB of STZ-treated diabetic

rats, which also exhibited several behavioral

deficits, including loss of odor discrimination

ability, cognitive dysfunction, and increased


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anxiety. Sodium- and chloride-dependent

GABA transporter 1 (GAT1), which localizes

to GABAergic terminals, was inhibited in the

diabetic OB, and a GAT1 inhibitor suppressed

Wnt3-induced neurogenesis in vitro. From

these studies, we conclude that diabetes has an

adverse effect on OB neurogenesis and

function. Our findings provide evidence that

insulin deficiency leads to attenuation of

neurogenesis and GABA activity and glutamate

neurotransmission systems in the OB, thereby

causing functional impairment in olfactory

performance. In addition, insulin may promote

the neuronal differentiation of OB NSCs by

controlling GABA uptake by GAT1.

RESULTS

Strict regulation of NSC differentiation in the

OB

To analyze the cell fate specification of

NSCs in the OB, immunohistochemistry was

performed to identify stem cells, neuronal

progenitors, neurons, astrocytes, and

oligodendrocytes in 7-week-old rats. The

distribution of neural stem cells and mature

neurons was first confirmed by

immunostaining for Sox2 and NeuN, as

markers for stem cells and mature neurons,

respectively (Figure 1A). NeuN was

specifically expressed in the interneuron-rich

granule cell layer (GCL) and partially in the

glomerular layer (GL), but less in external

plexiform layer (EPL). In contrast, Sox2—a

multipotential NSC marker—was abundantly

expressed in the GL. No co-localization

between NeuN and Sox was observed in the

OB. Thus, the localization of multipotent

undifferentiated NSCs and mature neurons was

distinct in the OB.

To investigate neuronal differentiation

in OB, the distribution of Sox2-positive stem

cells and the early-stage neuronal

differentiation marker NeuroD1 was assessed

by IHC. Notably, NeuroD1-positive cells did

not co-localize with Sox2-positive cells in the

GL (Figure 1B left). To detect the neuronal

cells with further differentiation in the OB, the

presence of immature neurons and mature

neurons was further investigated with

antibodies directed toward the immature

neuronal markers TUJ1 and NeuN. TUJ1

(immature neuronal marker)-positive cells did

not co-localize with NeuN-positive cells

(Figure 1B, right), indicating that the OB

contained cells from each stage of neuronal

differentiation, but NSCs and early-stage

neuronal cells, immature, and mature neurons

did not co-localize with each other. The GL is

suggested to be another source of NSCs in OB.

In addition, neuronal differentiation from stem

cells to neurons is strictly controlled, similar to

that observed with hippocampal neuronal

differentiation.

Since NSCs can also generate

oligodendrocytes and astrocytes, we examined

neuronal morphology and glial lineages in OB.

Notably, positive cells for the immature


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oligodendrocyte marker Olig2 were observed

in GL where NSCs and neural progenitor cells

were found, but did not co-localize with NeuN-

positive cells (Figure 1C left). Alternatively,

cells positive for the mature oligodendrocyte

marker MBP were observed, but did not co-

localize with those expressing GFAP, a Wnt3-

producting astrocyte marker, in GCL (Figure

1C Right). GFAP-positive cells were also

found in the GL, but these cells did not co-

localize with NeuN-positive cells (Figure 1D

left). Mature astrocytes were also detected with

S100β. Most S100β-positive cells were co-

localized with GFAP-positive cells in the GCL

(Figure 1D right). Therefore, both mature and

immature oligodendrocytes and astrocytes exist

in OB. These results suggest that NSC

differentiation into neurons, astrocytes, or

oligodendrocyte is strictly regulated and that

once a differentiation path has been

determined, the cells no longer express markers

of other lineages.

Diabetes impairs neuronal and

oligodendrocyte differentiation

NSCs and other lineages were

observed in the normal OB. Then, the

expression of genes associated with each cell

lineage was investigated in STZ-induced

diabetic rats. Sox2 transcript levels in STZ-

induced diabetic rats were comparable to those

in the control rats (WT) 10 d after STZ

treatment, but expression increased by day 30

in the diabetic versus WT rats (Figure 2A left).

mRNA expression of another stem cell marker,

Nestin, markedly decreased on day 10, but was

comparable to that of the control at day 30

(Figure 2A right).

Expression of the immature neuronal

marker β-tubulin III (Tubb3) and mature neuronal marker synapsin 1 (Syn1) was

substantially lower in diabetic rats than WT

rats on days 10 and 30 (Figure 2B). Similar to

Tubb3 and Syn1, we observed a substantial

decrease in the expression of Mbp, a mature

oligodendrocyte marker, in diabetic rats on

days 10 and 30. The marker of immature

oligodendrocytes Olig2 was comparable in WT

and diabetic rats (Figure 2C). Expression of the

astrocyte markers GFAP and S100β markedly

decreased in diabetic versus WT rats on day 10,

and remained low on day 30 in both WT and

diabetic rats (Figure 2D). These data suggest

STZ-induced diabetes is associated with a

substantial reduction in the expression of

neuronal and mature oligodendrocyte lineage-

related transcripts in the OB over a relatively

long period of time.

Western blot analysis was performed to

examine the expression of lineage-specific

markers in the OB on day 30 after diabetes

induction. Sox2 protein expression was

comparable in WT and diabetic rats (Figure 3A

left). Nestin expression was also comparable in

WT and diabetic rats (Figure 3A right). In

contrast to the expression of stem cell markers,

that of neuron-specific markers TUJ1 and

SYN1 was substantially lower in diabetic than


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in WT rats (Figure 3B). The expression of the

oligodendrocyte markers Olig2 and MBP also

reduced in diabetic rats (Figure 3C).

Expression of the astrocyte marker protein

GFAP markedly decreased in diabetic versus

WT rats; however, expression levels of the

astrocyte marker S100β were comparable in

both groups (Figure 3D), consistent with the

previous study (34). These results suggest that

the differentiation of neuron and

oligodendrocyte lineages is impaired in

diabetic progression. However, diabetes may

not have a severe effect on the differentiation

and maturation of astrocytes, since S100β

characterizes a mature developmental stage in

the astrocytic lineage (35) and its protein

expression was similar in WT and diabetic rats

on day 30. There was no correlation between

gene and protein expression of several markers

of NSCs, astrocytes, and oligodendrocytes such

as Sox2, Olig2, and GFAP at 30 d after STZ

treatment. Transcript levels do not necessarily

correlate to the amount of expressed protein,

since the translation of mRNAs into proteins is

a highly regulated process. Although levels of

some transcripts did not correlate with protein

expression, it was clear that the expression of

neuronal and mature oligodendrocyte markers

was consistently reduced in diabetic rats at the

transcriptional and post-transcriptional levels.

These results suggest diabetes impairs neuronal

and oligodendrocyte differentiation.

Diabetes impairs NSC differentiation into

neurons

To detect the proliferation and lineage

specification rates of NSCs during diabetes

progression, proliferating cells were labeled

with BrdU on day 20 after STZ treatment. The

percentage of Sox2+ BrdU-labeled new cells

expressing the stem cell marker Sox2 was

similar in WT and diabetic rats (Figure 4A and

4C); however, the percentage of newly dividing

BrdU-labeled cells expressing the early

neuronal differentiation marker NeuroD1 was

remarkably lower in diabetic versus WT rats

(Figure 4B and 4C). Less than 10% of BrdU-

labeled cells expressed NeuroD1 in diabetic

rats, while more than 40% of BrdU-labeled

cells expressed this protein in WT rats (Figure

4C). The number of BrdU-labeled cells

expressing NeuN, a mature neuronal marker in

diabetes, was reduced slightly but not

significantly. No significant difference was

observed between the percentage of BrdU-

labeled cells expressing Olig2 and S100β in

WT and diabetic rats (Figure 4C). Thus, the

newly dividing BrdU+ cells that expressed

specific markers of early neuronal

differentiation and mature neurons were

partially suppressed in diabetic rats. Therefore,

diabetes impaired NSC differentiation into

neurons but had a minor influence on NSC

proliferation and the differentiation of

oligodendrocyte sand astrocytes.

Diabetes reduces Wnt3 expression in OB

Wnt3 is required to promote NeuroD1


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expression and neurogenesis in the adult

hippocampus (29,36,37). The impairment of

neuronal differentiation in diabetes (Figure 2–

4) prompted us to explore Wnt3 expression in

the OB at 10 and 30 d after STZ treatment. We

observed similar transcript and protein profiles

of specific markers for stem cell and neuronal

progenitors, neurons, astrocytes, and

oligodendrocytes on days 20 and 30 in diabetic

rats (data not shown); IHC was performed on

day 20 after STZ treatment. Although GFAP

expressing cells were present in both WT and

diabetic rats, Wnt3-expressing cells were

seems to be decreased in the GL and GCL

regions of diabetic versus WT rats on day 20

(Figure 5A). Indeed, the percentage of

Wnt3+GFAP+ astrocytes is dramatically

decreased in STZ-induced diabetes (Figure

5B). It showed that Wnt3-expressing astrocytes

in the OB neurogenic niche decrease severely

in diabetes. In addition, a substantial reduction

in Wnt3 transcript expression was observed in

diabetic versus WT rats on day 30 after STZ

treatment (Figure 5C). The reduction of Wnt3

transcript expression in diabetes occurred over

time after diabetes induction. Similarly, Wnt3

protein expression was remarkably inhibited on

days 10 and 30 in diabetic rats (Figure 5D).

These results indicated that Wnt3 production

was seriously inhibited in diabetic rats,

although expression of the astrocyte-specific

marker GFAP was observed in both WT and

diabetic rats, suggesting inhibition of Wnt3

production in astrocytes of the OB neurogenic

niche.

Diabetes inhibits Wnt canonical signaling

and its downstream targets in OB

We explored the activation of Wnt

signaling and Wnt target factors by real-time

qPCR and western blotting. In the canonical

Wnt signaling pathway, Wnt binds to its

receptor Frizzled (FZD) and a transmembrane

protein called low-density lipoprotein receptor-

related protein (LRP). Binding of Wnt to FZD

and LRP triggers a series of events that disrupts

the β-catenin destruction complex, resulting in

stabilization and accumulation of β-catenin in the cytoplasm. In the absence of a Wnt ligand,

cytoplasmic β-catenin is phosphorylated by

phosphorylated GSK3β (p-GSK3β) and is targeted for degradation by the proteasomal

machinery. The increased stability of β-catenin

following Wnt activation enables translocation

to the nucleus and binding to T cell-specific

transcription factor/lymphoid enhancer-binding

factor 1 (TCF/LEF) transcription factors and

mediates transcriptional induction of target

genes (38-40). GSK3β and p-GSK3β protein levels reduced on days 10 and 30 in diabetic

rats (Figure 6A). In addition, the expression of

β-catenin was reduced on day 30 after STZ treatment. These data suggest the reduction of

Wnt3 leads to downregulation of Wnt/β-catenin signaling in the OB.

NeuroD1 is a target of Wnt3 regulation

and induces terminal neuronal differentiation in

the OB (41). Transcript expression of NeuroD1


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was suppressed on days 10 and 30 after

diabetes induction and was accompanied by a

reduction in protein levels on day 30,

consistent with Wnt3 expression in diabetic

rats (Figure 6B). A neuronal precursor marker,

doublecortin (DCX) is also a Wnt3 target

(29,42). Expression of DCX transcripts was

reduced on day 10 and maintained at low levels

in WT and diabetic rats on day 30 (Figure 6C).

DCX protein expression was remarkably

reduced in diabetic rats on day 30 after STZ

treatment. These results suggest reduction of

Wnt3 expression leads to a reduction in the

transcript and protein expression of target

genes.

Behavioral alteration in STZ-induced

diabetes

Several lines of evidence suggest

inhibition of Wnt signaling causes impairment

of adult neurogenesis in the hippocampus and

results in cognitive decline (42-44). Since the

expression of Wnt3 and its target genes

reduced in the OB of diabetic rats (Figure 6),

behavioral alterations were evaluated using a

hole-board test for odor discrimination, a novel

object recognition test for recognition memory,

and an elevated plus maze test for anxiety. The

percentage of head-dips and time spent in the

food-scented hole was considerably reduced in

diabetic rats on days 10 and 30 after STZ

treatment (Figure 7A). The results indicated

poor odor discrimination and a severe loss of

olfactory function in diabetes. During

habituation in the novel object recognition test,

all groups of rats spent equivalent time

exploring the identical objects (left and right)

(data not shown). In the trial, the

discrimination index was significantly lower in

diabetic than WT rats on days 10 and 30

(Figure 7B). WT rats spent more time with the

novel object, while diabetic rats could not

discriminate between the novel and familiar

objects on days 10 and 30, consistent with prior

studies (26,45,46). In the elevated plus maze

test, the percentage of entries and time spent in

the open arms were significantly reduced in

diabetic rats on days 10 and 30 (Figure 7C).

These data suggest diabetes contributes to the

impaired olfactory-related exploratory

behaviors, reduces learning and memory

function, and increases anxious behaviors.

Thus, the decrease in neurogenesis in the OB

because of insulin deficiency may be involved

in the functional impairment of olfactory

performance.

GAT1 inhibition impairs Wnt3-stimulated

neuronal differentiation in STZ-induced

diabetes

Recent studies suggest GABA may

play a role in the regulation of adult

neurogenesis through GABAA receptors (47-

50). Alterations in GABAergic systems in the

OB of diabetic rats were examined by

analyzing the gene expression of GABAA

receptorα2 (GABAARα2) and of GABA

transporter (GAT)1 and 3, which remove


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extracellular GABA after its release from the

synaptic vesicle into the synaptic cleft. GAT1

transcript levels were substantially reduced on

days 10 and 30 after diabetic induction. GAT3

transcript expression was reduced on day 10 in

diabetic rats, but the difference was not

significant until day 30. Transcript levels of

GABAARα2 did not differ between WT and

diabetic rats (Figure 8A). Reductions in GAT1

and insulin protein expression were

demonstrated by immunohistochemistry in

diabetic rats (Figure 8B). The reduction of

GAT1 protein expression was validated by

western blotting (Figure 8C), suggesting that

insulin deficiency induces reduction in GAT1

expression and may impair GABA uptake.

GABA and glutamate are the principal

inhibitory and excitatory neurotransmitters,

respectively, in the mammalian central nervous

system (51), and are thereby involved in

cognition, memory, and learning. Excitatory

amino-acid transporters (EAATs) mediate

uptake of glutamate from the neural cleft, while

VGLUT transports glutamate into synaptic

vesicles. The glutamatergic system was

explored by assessing expression of (EAAT)1-

3, vesicular glutamate transporter (VGLUT)1,

VGLUT2, and N-methyl-D-aspartate receptor

R1 (NMDAR)1. EAATs mediated uptake of

glutamate from the neural cleft, while VGLUT

transported glutamate into synaptic vesicles. In

the diabetic rat group, transcript levels of

EAAT1, 2, and 3 were suppressed on day 10

and of EAAT 2 and 3 were suppressed on day

30 after STZ treatment (Figure 9A). In diabetic

rats, VGLUT1 and 2 mRNA expression levels

were markedly decreased on day 10, while

these expression levels were significantly

increased on day 30 in comparison to those for

the WT (Figure 9B left and middle). Transcript

expression of the glutamate receptor N-methyl-

D-aspartate receptor R1 (NMDAR1) did not

differ between WT and diabetic rats (Figure 9B

right). Thus, diabetes also modulates the

expression of glutamate transporters for the

first 10 days after induction, suggesting

diabetes-induced adverse effects may be seen

in not only GABAergic but also glutaminergic

transmission in the OB.

Since GAT1 expression was reduced

on days 10 and 30, we evaluated neuronal

differentiation after inhibiting GAT1 activity in

vitro. The GAT1-selective inhibitor

SKF89976A was added to NSCs at 0 to 100

µM in vitro. Expression of Wnt3 mRNA was

consistently down-regulated in the presence of

the GAT1 inhibitor (Figure 10). While,

astrocyte marker, GFAP mRNA expressions

were increased dose-dependently in the

presence of the GAT1 inhibitor. Consistent

with Wnt3 expression, the inhibition of GAT1

activity also resulted in decrease in NeuroD1

and TUBB3 transcripts in a dose-dependent

manner. These results demonstrate that GAT1

inhibition suppresses expression of Wnt3 and

NeuroD1, factors essential for early neuronal

differentiation, and of TUBB3, an immature

neuronal marker, but increase the expression of


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GFAP. The inhibition of Wnt3-induced

neuronal differentiation in diabetes is most

likely due to inactivation of GAT1.

To examine the importance of insulin

activity in neurogenesis, we evaluated neuronal

differentiation after insulin treatment at 0, 10,

100 ng/mL in vitro. On treatment with 100

ng/mL insulin, the mRNA expression of Wnt3,

GFAP, Tubb3, and Syn1 considerably increased;

moreover, the level of NeuroD1 expression was

significantly increased, albeit slightly, by

insulin treatment (Figure 11A). This finding

suggested that insulin acts as a supportive

factor to promote neurogenesis by the

activation of Wnt3-induced neurogenesis.

Then, to evaluate if insulin restored the

inhibition of neurogenesis induced by the

GAT1 inhibitor, insulin (0 or 100 ng/ml) was

added to NSCs after GAT1 inhibitor

administration (0 or 50 µM) in vitro. The GAT1

inhibitor considerably inhibited Wnt3 mRNA

expressions, while insulin recovered its

reduction by the GAT1 inhibitor (Figure 11B).

GFAP mRNA expressions were increased by

both GAT1 inhibitor and insulin. A profound

decrease in Neurod1 and Tubb3 expression was

also observed on the addition of the GAT1

inhibitor, the inhibition of Neurod1 and Tubb3

mRNA expression by the GAT1 inhibitor was

significantly moderated by insulin treatment.

This result suggests the potential function of

insulin in helping to recover the decrease in

neurogenesis. GFAP mRNA expressions were

increased by the presence of both the GAT1

inhibitor and insulin at considerable levels.

While, Wnt3 mRNA expressions were

increased by insulin, but decreased by GAT1

inhibitor. It is suggests that GAT1 inhibition

effects on Wnt3 production in astrocytes, but

insulin could help to promote the production.

DISCUSSION

The SVZ produces NSCs, which are

also produced in the OB core (5-8). In addition

to the OB core, this study demonstrated the

expression of multipotent stem cell marker

Sox2 in the GL of OB, suggesting GL is the

another source of NSCs in OB. In addition,

NSCs, neurons, astrocytes, and

oligodendrocytes in OB did not co-localize,

suggesting the lineage commitment is strictly

controlled.

Wnt3 is as an astrocyte-derived factor

that promotes neuronal differentiation of adult

hippocampal NSCs (29,36). In this study, Wnt3

expression was observed in the rat OB,

suggesting Wnt3 regulates the adult

neurogenesis there as it does in the

hippocampus. We also provide evidence of the

adverse effect of diabetes on neurogenesis in

the OB and on OB function, and identify the

molecular mechanism by which neurogenesis

is impaired in diabetes. STZ-induced diabetes

had a particular effect on OB neurogenesis due

to inhibition of Wnt3-mediated signaling and

expression of its target genes, NeuroD1 and

DCX. Downregulation of NeuroD1 inhibits


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neural differentiation and reduces the number

of mature neurons. In addition, STZ-induced

diabetic rats showed several behavioral

changes, including impaired olfactory function,

deficits in learning and memory, and an

increase in anxiety that can be attributed to the

inhibition of GABA and glutamate transporters.

Interestingly, inhibition of GAT1, which

modulates local levels of GABA, suppressed

Wnt3-regulated neuronal differentiation in

vitro. These data suggest the alterations in

GABAergic and glutamatergic neuronal

systems could contribute to the down-

regulation of Wnt3-induced neuronal

differentiation in the OB of diabetic rats, thus

leading to the impairment of olfactory function.

Furthermore, our results indicated that insulin

promotes the Wnt3-induced neurogenesis, even

in the presence of the GAT1 inhibitor in vitro,

supporting that insulin plays a supportive role

in neurogenesis.

Growing evidence suggests GABA has

a major role in survival, proliferation,

migration, synapse formation, and integration

of newly formed neurons into synaptic

networks (50,52-54). GABA is synthesized and

released by neuroblasts (47,49,50). GABAA

receptors are expressed in both neuroblasts and

stem cells, and are activated by local GABA

(47-50). In the SVZ, GABA serves as a

negative regulator for proliferation of NSCs

and neuroblasts (50,55) and migration (49).

Thus, GABA activation is an important

neurogenic niche signal to stop neuronal

production. In the SVZ, the GABA transporter

GAT3 is expressed in astrocyte-like NSCs and

GAT1 in neuroblasts (49,56).

Electrophysiological studies have indicated that

GAT1 and GAT3 in the SVZ reduce local

GABA levels and limits GABAA receptor

activation in astrocyte-like NSCs (50). These

resent studies suggest that GATs regulate

neurogenesis by maintaining ambient GABA

levels. We also demonstrated that impairment

of GAT1 in diabetic rats and a GAT1 inhibitor

disturbs Wnt3-induced neuronal differentiation

of NSCs in vitro. GAT1 and GAT3 are

localized in the OB (57). The expression of

these GATs and adult neurogenesis in the OB

were impaired in STZ-induced diabetes in the

present study. Therefore, we suggest GABA

uptake by GATs is critical for the maintenance

of adult neurogenesis in the OB, and insulin

regulates GAT1 activities. In addition, the

correlation of GAT1 reduction with lower

insulin expression was observed in OB,

suggesting that insulin plays a crucial role in

the maintenance of GAT1 function and GABA

uptake. The present study also showed that

insulin stimulates Wnt3-induced neurogenesis

and moderately inhibits the neurogenesis

induced by the GAT1 inhibitor in vitro. Thus, it

is suggested insulin may act as a supportive

factor to promote the adult neurogenesis. The

expression of GATs and the glutamate

transporters EAATs and vGLUTs changed in

diabetes in the present study. Glutamate

transporters may also be involved in the adult


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neurogenesis. Further studies are needed to

elucidate the possible roles of GABA and

glutamate transporters on the neurogenesis and

insulin regulation of GATs and EAATs.

Diabetes impairs neurogenesis and

synaptic neuronal plasticity, hampers

hippocampal learning and memory, and

increases anxiety-like behaviors in diabetic rats

and mice (24-26). STZ-induced animals

showed deficits in learning and memory in the

Morris water maze and novel object

recognition tests (26). Therefore, diabetes-

associated cognitive deficits may be attributed

to the reduction in hippocampal neurogenesis.

In the hippocampus of STZ-induced diabetic

rats, Wnt3 and insulin were reduced (28).

Reduction in Wnt3-regulated neurogenesis was

also observed in the OBs of STZ-induced

diabetic rats; thus, Wnt3-induced neurogenesis

in the OB and hippocampus of diabetic rats

may occur in a similar fashion.

Wnt3-producing astrocytes are present

in the only neurogenic niches. According to our

results, it is evident that the number of

Wnt3+GFAP+ astrocytes was dramatically

reduced in STZ-induced diabetes, and GAT1

inhibition is involved in the decrease of Wnt3

production in astrocytes during neuronal

differentiation. It suggests the involvement of

the GAT1 inhibition in the decrease of Wnt3-

producing GFAP+ astrocytes in STZ-induced

diabetes. In the study by Coleman et al, .insulin

treatment prevented decrease in GFAP

expression in the hippocampus of untreated

diabetic rats (58). The present study also

showed that insulin help to increase Wnt3 and

GFAP expressions and recovers from the

impaired neurogenesis by the GAT1 inhibition

in vitro. Insulin may increase the number of

Wnt3-secreting astrocytes in the neurogenic

niches and activates Wnt3-inducing

neurogenesis.

While, GFAP expression was shown to

be severely decreased in the OB (34) and

hippocampus in STZ-induced diabetes (59,60).

In the present study, GFAP mRNA expressions

were, however, increased by GAT1 inhibition

in vitro, suggesting that diabetic conditions

induces the decrease of GFAP expressions in

the STZ-induced diabetic rats. It was reported

that astrocytes are highly sensitive to acidic

conditions (58) and the junctional

communication among astrocytes is impaired

in STZ-induced diabetes and cultured

astrocytes (61). Accordingly, it is supposed that

acidic conditions in diabetes alter GFAP

expressions and astrocyte functions directly in

STZ-induced diabetes.

It is also evident that glucocorticoid-

mediated reduction of neurogenesis in DG and

SVZ in recent studies (62-64). Glucocorticoids

were shown to decrease astrocyte proliferation

(65,66). Interestingly, glucocorticoid treatment

in astrocyte culture showed decrease in Wnt7

expression (66). Wnt7 is a Wnt family member,

and promotes neurogenesis similar to Wnt3.

Wnt3 expression in STZ-induced diabetes may

also decrease in response to glucocorticoids. In


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addition to Wnt3, BDNF has also been

implicated in adult neurogenesis, survival, and

synaptic plasticity in the hippocampus (67,68).

Impairment of neurogenesis, spatial learning,

and memory are observed in a BNDF-deficient

animal model (69,70). BDNF expression also

decreased in the hippocampus of STZ-induced

diabetic rats (71). It is reported that elevated

levels of glucocorticoid reduce BDNF

expression (72,73). Thus, glucocorticoid-

mediated inhibition of Wnt7a and BDNF might

be involved in the suppression of neurogenesis

in the OB of STZ-treated rats.

The present study showed that the

neurogenic niche of OB NSCs was mostly

localized in the GL, while most mature neurons

were present in the GCL. NSCs, neurons,

astrocytes and oligodendrocytes in the OB

were morphologically distinct from each other,

which suggests the lineage commitment from

NSCs is strictly controlled. We also provide

evidence that diabetes inhibits Wnt3-induced

neuronal differentiation in the OB and alters

neurotransmitter systems such as GABA and

glutamate transporters. Diabetes led to several

behavioral deficits, including impaired odor-

mediated behavior, learning, and memory, and

increased anxiety. Thus, diabetes caused

decrease in neurogenesis and malfunction of

the olfactory and other brain areas, perhaps

contributing to these behavioral deficits. In

addition, the impairment GATs and EAATs was

found in the diabetic rats, and GAT1 inhibition

disturbed Wnt3-induced neuronal

differentiation of NSCs in vitro. Therefore, this

suggests that alterations in GABA transporters

lead to decrease in Wnt3-induced neurogenesis

and several behavioral deficits in diabetes. Not

only GABA but also glutamate transporter

systems were found to be altered in diabetes. It

is presumed that the regulation of local GABA

and glutamate neurotransmitter levels is

important for the maintenance of adult

neurogenesis in the OB and that insulin helps

to maintain GABA and glutamate transporter

activities. Thus, insulin is believed to be

essential for the control of GABA and

glutamate transporter activities, which in turn

affect Wnt3-induced neurogenesis and

olfactory functions. In addition, insulin was

found to help promote neurogenesis and

moderate the decrease in neurogenesis induced

by GAT1 inhibition in vitro. As such, insulin

may be an important factor for the maintenance

of neurogenesis.

EXPERIMENTAL PROCEDURES

Animals

Fischer 344 male rats (~4 months old)

weighing 70 to 100 g were used in this study.

All animals were maintained in a 12 h light/12

h dark cycle in a controlled temperature and

relative humidity environment with standard

rat chow and drinking water available ad

libitum. To induce diabetes, animals were

treated with a single intraperitoneal (i.p.)


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injection of 70 mg/kg STZ (Wako, Osaka,

Japan) dissolved in saline (74,75). Blood

glucose levels were measured for 3 d after STZ

administration. Rats with blood glucose levels

greater than 300 mg/dL were considered

diabetic. OBs were harvested from the rats at

10, 20, and 30 d after STZ injection. To label

proliferating progenitor cells, BrdU (200 mg/kg,

i.p.) was administered with the STZ injection

and analyzed in OBs isolated 20 d after

diabetes induction.

All animals were anesthetized with

pentobarbital sodium (100 mg/kg), quickly

perfused with saline. Collected brain and OB

samples were fixed in 4% PFA solution. All

animal protocols were approved by the

Institutional Animal Care and Use Committee

(IACUC) of the National Institute of Advanced

Industrial Science and Technology.

Immunohistochemistry

Samples fixed with 4% PFA overnight were

placed in 30% sucrose solution until the tissues

sank. Coronal sections (40 µm) were collected

and stored in tissue collection medium (25%

glycerin, 30% ethylene glycol, 0.05 M

phosphate) at -20°C. For the

immunohistochemical detection of BrdU,

sections were incubated in 50% formaldehyde

at 65°C for 90 min. After washing with Tris-

buffered saline containing 0.25% Tween 20

(TBS-T) for 15 min, the sections were placed

in 1 N HCl at 37°C for 15 min, and then

incubated in 0.1 M borate buffer (pH 8.5) for

10 min.

Sections were washed with 3% normal

donkey serum in 0.25% TBS-T for 15 min and

then incubated with primary antibodies at 4°C

for 3 d. After washing with 0.25% TBS-T for

15 min, sections were treated with secondary

antibodies overnight at 4°C. Finally, sections

were washed twice with 0.25% TBS-T and

mounted (Vectashield; Vector Laboratories).

Primary antibodies included the following:

rabbit anti-Sox2 (1:300, Millipore), mouse

anti-NeuN (1:100; clone A10), mouse anti-

NeuroD1 (1:100; Abcam), rabbit anti-TUJ1

(1:100; Promega, Madison, WI), mouse anti-

MBP (1:100; Abcam), guinea-pig anti-GFAP

(1:100; Advanced Immunochemical, Long

Beach, CA), rabbit anti-Olig2 (1:100;

Millipore), mouse anti-S100β (1:100; Abcam),

goat anti-Wnt3 (1:300; Everest Biotech Ltd.,

Oxfordshire, UK), rabbit anti-GAT1 (1:100;

Abcam), rabbit anti-insulin (1:100; Santa Cruz

Biotechnology) and DAPI (Wako, Osaka,

Japan), and rat anti-BrdU (1:400; Abcam). For

visualization, the following were used with

DAPI counterstaining: Cy3-conjugated donkey

anti-mouse, anti-rabbit, anti-rat, or anti-goat

IgG (1:500, Jackson ImmunoResearch

Laboratories); Cy5-conjugated donkey anti-rat

IgG (1:500, Jackson ImmunoResearch

Laboratories); and AlexaFluor 488-conjugated

donkey anti-mouse or anti-rabbit IgG (1:500;

Molecular Probes, Eugene, OR). Sections were

imaged with a Carl Zeiss LSM confocal

imaging system (LSM 510; Carl Zeiss, Tokyo,


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Japan) or an Olympus FV1000-D confocal

microscope (Olympus Corp., Tokyo, Japan).

For the quantification of Wnt3-secreting

astrocytes, the number of GFAP positive cells,

and Wnt3 and GFAP double positive cells were

counted, and then calculated the ratio of Wnt3-

GFAP-doouble positive cells /GFAP positive

cells in control and STZ-induced diabetes.

RNA extraction and qPCR

Isolated OB were immediately stored in

ISOGEN solution at -20°C. Total RNA was

isolated using ISOGEN (Wako) on ice

according to manufacturer instructions,

dissolved in DEPC-treated water, and stored at

−20°C. Extracted RNA was treated with DNase

I (Life Technologies) for 30 min at 37°C to

remove genomic DNA. cDNA synthesis was

performed with SuperScript III (Life

Technologies) or PrimeScript RT Master Mix

(TAKARA Bio., Otu, Japan) according to the

manufacturer’s protocols. Quantitative PCR

(qPCR) was performed with a Chromo4 system

(Bio-Rad) and Thunderbird SYBR qPCR Mix

(Toyobo, Osaka, Japan). All cDNAs were

diluted 50-fold with DEPC-treated water.

Primers were synthesized by Life Technologies

Japan (Table 1) and qPCR was performed as

follows: 40 cycles each of denaturation at 95°C for 15 s and annealing and extension at 60°C

for 40 s. Gapdh expression was used as the

internal control. The delta-delta Ct method was

used to calculate the relative quantity of each

target gene and normalized to Gapdh in each

sample.

Western blotting

Protein expression was analyzed as previously

described (76). OBs were homogenized in lysis

buffer (50 mM HEPES-KOH pH 7.4, 150 mM

NaCl, 10 mM EDTA, 10 mM NaF, 10 mM

Na4P2O7, 2 mM Na3VO4, 1% NP40, 1%

sodium deoxycholate, 0.2% SDS) containing

protease inhibitor mix (Nacalai Tesque, Kyoto,

Japan) on ice (77). Samples were then

centrifuged at 17,500 × g for 30 min at 4°C and the supernatants collected. Lysate protein

concentrations were quantified with a BCA

protein assay kit (Thermo Fisher Scientific,

Rockford, IL) and diluted with loading sample

buffer (62.5 mM Tris-HCl pH 6.8, 5% β-

mercaptoethanol, 2% SDS, 5% sucrose, and

0.005% bromophenol blue). Equal amounts of

protein were separated in SuperSep gels

(Wako) and transferred to PVDF membranes

(Millipore). After blocking in 10% Blocking

One (Nacalai Tesque) in 0.05% TBS-T for 1 h,

blots were incubated with primary antibodies at

4°C overnight. For immunodetection, the

following primary antibodies were used: rabbit

anti-GAPDH (1:5000; Santa Cruz

Biotechnology), rabbit anti-Sox2 (1:1000;

Millipore), mouse anti-Nestin (1:500; BD

Pharmingen), mouse anti-TUJ1 (1:10000;

Promega), mouse anti-Synapsin 1 (1:10000;

Transduction Lab), rabbit anti-Olig2 (1:1000;

Millipore), mouse anti-MBP (1:1000; Abcam),

rabbit anti-GFAP (1:10000; DAKO), mouse


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anti-S100β (1:1000; Abcam), rabbit anti-Wnt3

(1:5000; Santa Cruz Biotechnology), rabbit

anti-β-catenin (1:2000; Cell Signaling

Technology), rabbit anti-GSK3β (1:2000; Cell

Signaling Technology), rabbit anti-phospho-

GSK3β (1:2000; Cell Signaling Technology),

mouse anti-NeuroD1 (1:500; Abcam), rabbit

anti-Doublecortin (1:1000; Santa Cruz

Biotechnology), and rabbit anti-GAT1 (1:1000;

Abcam). After several washes with 0.05%

TBS-T, immunoreactive bands were detected

with HRP-conjugated secondary antibodies and

developed with SuperSignal West Femto

Maximum Sensitivity Substrate (Thermo

Fisher Scientific). The following HRP-

conjugated secondary antibodies were used:

donkey anti-rabbit and anti-mouse IgG

(1:10,000, GE Healthcare). Protein bands were

visualized with an LAS-3000 Imaging system

(Fuji Film Corp, Tokyo, Japan) and quantified

with ImageJ 1.32 software (National Institutes

of Health, Bethesda, MD).

Behavior analysis

Behavioral tests were performed in a gray

acrylic box (60 cm × 60 cm × 60 cm) or the

elevated plus maze and analyzed by

EthoVision XT (Noldus, Wageningen,

Netherlands). Cognitive function was assessed

in animals 10 or 30 d after diabetic induction.

Hole-board test

Olfactory-related exploratory behavior was

measured with a hole-board apparatus

consisting of a gray acrylic box with six round

holes in the floor (4 cm diameter). One hole

was food-scented and the others were empty;

thus, the rat is expected to spend more time at

and revisit the food-scented hole. In the trials,

animals were placed on the center of the

apparatus and allowed to freely explore for 10

min. Rodent behavior was recorded with a

digital camera placed above the apparatus. The

number of head-dipping events and time spent

at the scented hole were calculated. The hole-

board was carefully cleaned with 70% ethanol

after each observation and only one trial was

conducted for each rat.

Novel object analysis

The object recognition task evaluates a rodent’s

ability to recognize a novel object in the

environment as a way to assess recognition

memory. This experiment is based on the

observation that rats spend more time exploring

a novel object than a familiar one (78). Prior to

the test, rats were habituated to the test box for

10 min per day for two consecutive days. For

habituation, two identical objects were

positioned 15 cm away from the center. After a

retention interval of 24 h, rats were placed back

in the apparatus, wherein one of the objects

was replaced with a novel one. Rats freely

explored the objects and the open field for 10

min. Behavior and the time spent exploring

each object were recorded with a digital

camera placed above the apparatus. Each rat

was tested only once and objects were rinsed


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with 70% ethanol between trials and before the

first trial. A discrimination index [time spent

with object A – time spent with object B]/[total

time exploring both objects] was calculated

between the novel and familiar objects. The

index varies between +1 and −1, where

positive and negative scores indicate more or

less time spent with the novel object,

respectively. A discrimination index of 0

indicated equal exploration of both objects (79).

Elevated plus maze test

The elevated plus maze apparatus consists of

two open arms (50 cm × 10 cm) and two closed

arms (50 cm × 10 cm × 40 cm) extending from

a central platform elevated 50 cm above the

floor. The arms were connected with a central

quadrangular area (10 cm × 10 cm) to form a

cross. The rats were placed on the central

quadrangular area and allowed to move freely

on the arms for 10 min. The frequency of entry

into the open or closed arms and time spent on

each arm were recorded (80). The percentage

of time spent in the open arms was interpreted

as an index of fear and anxiety. Rodent

behavior was recorded by a digital camera

placed above the apparatus. The percentage of

time spent in the open arms (100 × open/total)

and the number of entries into the open and

closed arms were calculated. Each rat was

tested only once and the apparatus was

carefully cleaned with 70% ethanol between

each trial and before the first trial.

Cell cultures

The adult rat hippocampal neural (HCN) cell

line from the adult rat hippocampus was

cultured as described (81,82). Briefly, NSCs

were cultured in Dulbecco’s modified Eagle’s

medium/F12 medium (DMEM/F12, Life

Technologies) containing 1% antibiotic-

antimycotic (Life Technologies), 2 mM L-

glutamine (Life Technologies), 1% N2

supplement (Wako) and 20 ng/mL FGF-2

(Wako) in a 5% CO2 incubator at 37°C. For

neuronal differentiation, HCNs were cultured

in DEME/F12 containing 1 µM retinoic acid

(Sigma) and 1 µM forskolin (Sigma). Cells

were treated with the GAT1 selective inhibitor

SKF89976A (Abcam; 0, 1, 5, 10, 50, 100 µM)

(83-86) or insulin (0, 10, 100 ng/mL) during

the neuronal differentiation for the indicated

time periods, and then collected for RNA

isolation and qPCR analysis. To evaluate if

insulin rescued the neurogenesis hindered by

GAT1 inhibitor, HCNs were treated with

insulin (0 or 100 ng/mL) after GAT1 inhibitor

administration (0 or 50 µM) during neuronal

differentiation. The next day, cells were

collected for RNA isolation and qPCR analysis.

Statistical analysis

All animal experiments, the GAT1

inhibitor administration, and insulin treatment

experiments in culture were analyzed for

statistical significance using Student’s t tests.

All data are presented as mean ± standard

distribution (SD). P < 0.05 was considered to


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be significant. GAT1 inhibitor and insulin in

vitro co-treatment results were analyzed for

statistical significance by two-way ANOVA

and Bonferroni's post-hoc tests.

ACKNOWLEDGMENTS RH, SF, MA, and TK were supported by the

National Institute of Advanced Industrial

Science and Technology. TK was also

supported by a Grant-in-Aid for Scientific

Research on Innovative Areas, a Grant-in-Aid

for Scientific Research (B), the Naito

Foundation, the Mochida Memorial Foundation

for Medical and Pharmaceutical Research, the

Takeda Science Foundation, and the Mitsubishi

Foundation.

CONFLICT OF INTEREST

The authors declare no conflicts of interest

with the contents of this article.

AUTHORS' CONTRIBUTIONS

TK conceived and coordinated the study and

contributed to manuscript writing. RH and TW

designed, performed, and analyzed the

experiments and wrote the manuscript. SF

performed and analyzed the experiments. MA

supervised and supported the research. All

authors reviewed the results and approved the

final version of the manuscript.

Footnote

* These authors contributed equally to this

work.


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FIGURE LEGENDS

Figure 1. Morphological characteristics of stem cells, neurons, astrocytes, and

oligodendrocytes in the adult OB.

(A) The distribution of neuronal stem cells and mature neurons. Left: representative merged image

of Sox2 (red), NeuN (green), and DAPI-labeled nuclei (blue). Right: representative magnified

image of GL. (B) The distribution of neurons in different stages. Left: representative merged image

of NeuroD1 (red), Sox2 (green), and DAPI (blue). Right: the representative image of TUJ1 (red),

NeuN (green), and DAPI (blue). (C) The distribution of oligodendrocytes. Left: representative

image of Olig2 (red), NeuN (green), and DAPI (blue). Right: representative image of MBP (red),

GFAP (green), and DAPI (blue). (D) The distribution of astrocytes. Left: the representative image of

NeuN (red), GFAP (green), with DAPI (blue). Right: representative image of S100β (red), GFAP (green), and DAPI (blue).

Figure 2. Expression levels of specific marker genes for stem cells, neurons, astrocytes, and

oligodendrocytes in the adult OB of STZ-induced diabetes.

qRT-PCR analysis showing relative mRNA levels of specific marker genes for stem cells, neurons,

astrocytes, and oligodendrocytes in the OB of wild-type (WT) and diabetic (DB) rats at 10 d and 30

d after STZ treatment. (A) Gene expression of the stem cell markers Sox2 and Nestin. (B) Gene

expression of the neuronal markers β-tubulin3 (TUBB3) and synapsin1 (SYN1). (C) Gene expression of the oligodendrocyte markers Olig2 and MBP. (D) Gene expression of the astrocyte

markers GFAP and S100β. All data have been normalized to the levels of Gapdh mRNA as an internal control and indicated as the ratio relative to that in the WT at 10 d. White bars: WT, Black

bars: DB. Data are represented as mean ± SDM (n = 4). * P < 0.05 vs. corresponding WT, ** P <

0.01 vs. corresponding WT. Statistical significance was determined by Student’s t-test.

Figure 3. Expression of specific markers for stem cells, neurons, astrocytes, and

oligodendrocytes at the protein level in the adult OB in STZ-induced diabetes.

Western blot analysis of specific stem cell, neuron, astrocyte, and oligodendrocyte markers in the

OBs of wild-type (WT) and diabetic (DB) rats at 10 d and 30 d after STZ treatment. Quantification

of relative protein levels of Sox2 and Nestin (A), β-tubulin3 (Tubb3), and synapsin 1 (Syn1) (B),

Olig2 and Mbp (C), and Gfap and S100β (D). The protein expression levels were normalized to Gapdh expression and indicated as the ratio relative to that in WT at 10 d. Representative bands are

shown at the right in each graph. White bars: WT, Black bars: DB. Data are represented as mean ±


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SDM (n = 4). * P < 0.05, ** P < 0.01 vs. corresponding WT. Statistical significance was determined

by Student’s t-test.

Figure 4. Neuronal differentiation is impaired in the OB in STZ-induced diabetes.

STZ and BrdU (200 mg/kg) were administered to 4-week-old F344 rats. Differentiation potential of

the Sox2+ NSC cells into neural progenitors, mature neurons, astrocytes, and oligodendrocytes

were detected by the expression of specific markers and BrdU incorporation after 20 d in wild-type

(WT) and STZ-induced diabetic (DB) rats. (A) Representative images of Sox2 (red) and BrdU

(green) and DAPI (blue) in WT (left) and DB rats (right). (B) Representative images of NeuroD1

(red), BrdU (green), and DAPI (blue) in WT (left) and DB (right) rats. (C) The percentage of the

BrdU+ cells in each cell lineage population was quantified in the OB. White bars: WT, Black bars:

DB. Data are represented as mean ± SDM (n = 4). * P < 0.05 vs., ** P < 0.01 vs. corresponding

WT.

Figure 5. Diabetes causes severe reduction of Wnt3 expression in the OB.

Immunohistochemical analysis of Wnt3 in the OB of wild-type (WT) and diabetic (DB) rats at 20 d

after STZ treatment. Wnt3 (red) expression was abundant and co-localized with GFAP (green) in

the GL (left) and in GCL (right) of the WT (A, top) and DB rats (A, bottom). Nuclei were also

labeled by DAPI (blue). (B) The percentage of the number of Wnt3+GFAP+ astrocytes in OB. Wnt3

expression was abundantly reduced in the OB of DB rats. (C) Relative mRNA levels of Wnt3 in the

OB of WT (n = 4) and DB (n = 4) rats at 10 d and 30 d after STZ treatment, determined by qRT-

PCR analysis. All data were normalized to the levels of GAPDH mRNA as an internal control and

indicated as the ratio relative to that in the WT at 10 d. (D) Quantification of relative protein levels

of Wnt3 in the OB of WT (n = 3) and DB (n = 4) rats at 10 d and 30 d after STZ treatment. The

protein expression levels were normalized to GAPDH expression and indicated as the ratio relative

to that in the WT at 10 d. Representative bands are shown in the bottom of the graph. White bars:

WT, Black bars: DB. Data are represented as mean ± SDM (n = 4). * P < 0.05, ** P < 0.01 vs.

corresponding WT.

Figure 6. Downregulation of Wnt signaling and its targets in STZ-induced diabetes.

(A) Representative western blot bands of GSK3β, phosphorylated-GSK3β (p-GSK3β), β-catenin and GAPDH in the OB of wild-type (WT) and diabetic (DB) rats at 10 d and 30 d after STZ

treatment (left). Quantification of relative protein levels of GSK3β, p-GSK3β, and β-catenin in the OB of WT (n = 4) and DB (n = 4) rats at 10 d and 30 d after STZ treatment (right). The protein


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expression levels were normalized to Gapdh expression and indicated as the ratio relative to that in

the WT at 10 d. (B) Quantification of relative Neurod1 mRNA levels by qRT-PCR (left).

Quantification of relative NeuroD1 protein levels by western blotting analysis and its representative

western-blot bands (right). (D) Quantification of relative DCX mRNA levels by qRT-PCR (left).

Quantification of relative DCX protein levels by western blotting analysis and its representative

western-blot bands (right). Neurod1 and Dcx mRNA and protein expression was normalized to the

corresponding Gapdh mRNA and protein levels as an internal control and indicated as a ratio

relative to that in the WT at 10 d. White bars: WT, Black bars: DB. Data are represented as mean ±

SDM (n = 4). * P < 0.05, ** P < 0.01 vs. corresponding WT.

Figure 7. Behavioral analysis of rats with induced diabetes.

(A) Hole-board test. Percentage of the number of head dips and time in the odor hole performed by

the wild-type (WT, n = 6) and diabetic (DB, n = 6-8) rats at 10 d and 30 d after STZ treatment. (B)

Novel object recognition test. Discrimination index and percentage of time spent with the novel

object for the WT (n = 6) and DB (n = 6) at 10 d and 30 d after STZ treatment. (C) Elevated-plus

maze test. Percentage of the number of entries in open arms and time in open arms for WT (n = 4-5)

and DB (n = 4) rats at 10 d and 30 d after STZ treatment. White bars: WT, Black bars: DB. Data are

represented as mean ± SDM (n = 4-8). * P < 0.05 vs. corresponding WT, ** P < 0.01 vs.

corresponding WT. Statistical significance was determined by Student’s t-test.

Figure 8. Severe reduction in GATs in STZ-induced DB rats.

(A) Quantification of relative mRNA levels of GAT1, GAT3, and GABAARα2 in the OB of wild-

type (WT, n = 4) and diabetic (DB, n = 4) rats at 10 d and 30 d after STZ treatment, determined by

qRT-PCR analysis. All data were normalized to the levels of GAPDH mRNA as an internal control

and indicated as the ratio relative to that in the WT at 10 d. (B) Immunohistochemical analysis of

GAT1 (red) and insulin (green) in the OB of WT (left) and DB (right) rats at 20 d after STZ

treatment. Expression of GAT1 and insulin were abundant in the OB of the WT, but severely

inhibited in the DB. Nuclei were also labeled by DAPI (blue). (C) Quantification of relative GAT1

protein levels in the OB of WT and DB at 10 d and 30 d after STZ treatment by western blotting

analysis (left) and its representative western-blot bands (right). White bars: WT, Black bars: DB.

Data are represented as mean ± SDM (n = 3). ** P < 0.01 vs. corresponding WT.

Figure 9. Modification of glutamatergic neurotransmitter systems in the OB

Relative mRNA levels of Eaat1, Eaat2, and Eaat3 (A) and of Vglut1, Vglut2, and Nmdar1 (B) in


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the OB of wild-type (WT, n = 4) and diabetic (DB, n = 4) at 10 d and 30 d after STZ treatment, as

determined by qRT-PCR analysis. All data were normalized to the levels of Gapdh mRNA as an

internal control and indicated as the ratio relative to that in the WT at 10 d. White bars: WT, Black

bars: DB. Data are represented as mean ± SDM (n = 3-6). * P < 0.05, ** P < 0.01 vs. corresponding

WT.

Figure 10. GAT1 inhibition impairs Wnt3-induced neuronal differentiation in vitro.

Quantification of relative mRNA levels of Wnt3, NeuroD1 and TUBB3 in NSCs after 24-h

treatment with the GAT1 selective inhibitor SKF89976A (1, 5, 10, 50, and 100 µM). * P < 0.05 vs.

corresponding WT, ** P < 0.01 vs. corresponding values for medium alone (GAT1 inhibitor = 0).

Figure 11. Insulin promotes neurogenesis in vitro

(A) Quantification of relative mRNA levels of Wnt3, Neurod1, and Tubb3 in NSCs after 24-h

treatment with insulin (0, 10, 100 ng/mL). * P < 0.05, ** P < 0.01 vs. corresponding values for

medium alone (insulin = 0) Statistical significance was determined by Student’s t-test. (B)

Quantification of relative mRNA levels of Wnt3, GFAP, Neurod1 and Tubb3 after 24-h treatment

with GAT1 inhibitor (0 and 50 µM) and Insulin (0 and100 ng/mL). Statistical significance was

determined by two-way ANOVA with Bonferroni’s post hoc test. * P < 0.05, ** P < 0.01 vs.

corresponding values in the medium without insulin treatment in the same GAT1 inhibitor

treatment. # P < 0.05, ## P < 0.01 vs. corresponding values in the medium without GAT1 inhibitor

treatment in the same insulin treatment.


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Wakabayashi et al. Fig.1


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KuwabaraTamami Wakabayashi, Ryo Hidaka, Shin Fujimaki, Makoto Asashima and Tomoko

transporter 1 inhibitionDiabetes Impairs Wnt3-Induced Neurogenesis in Olfactory Bulbs via glutamate

published online May 20, 2016J. Biol. Chem.

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