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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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Sox2
+ Brd
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**
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GAT1
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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.
10.1074/jbc.M115.672857Access the most updated version of this article at doi:
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