Mood Stabilizing Drugs Expand the Neural Stem Cell Pool in the
Adult Brain Through Activation of Notch Signaling
MIKITO HIGASHI,a,b
NORIKO MARUTA,c
ALAN BERNSTEIN,d
KAZUHIRO IKENAKA,a,b
SEIJI HITOSHI
a,b
a
Department of Physiological Sciences, School of Life Sciences, Graduate University for Advanced Studies,
Kanagawa, Japan; b
Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences,
Okazaki, Japan; c
Department of Psychiatry, Health Service Center, University of Tokyo, Tokyo, Japan; d
Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
Key Words. Lithium • Valproic acid • Carbamazepine • Neural stem cell • Self-renewal • Notch
ABSTRACT
Neural stem cells (NSCs) have attracted considerable attention
as a potential source of cells for therapeutic treatment
of impaired areas of the central nervous system. However,
efficient and clinically feasible strategies for expansion of the
endogenous NSC pool are currently unavailable. In this
study, we demonstrate that mood stabilizing drugs, which
are used to treat patients with bipolar disorder, enhance the
self-renewal capability of mouse NSCs in vitro and that this
enhancement is achieved at therapeutically relevant concentrations
in the cerebrospinal fluid. The pharmacological
effects are mediated by the activation of Notch signaling in
the NSC. Treatment with mood stabilizers increased an
active form of Notch receptor and upregulated its target
genes in neural stem/progenitor cells, whereas coculture
with ␥-secretase inhibitor or the presence of mutation in the
presenilin1 gene blocked the effects of mood stabilizers. In
addition, chronic administration of mood stabilizers expanded
the NSC pool in the adult brain, which subsequently
increased the cell supply to the olfactory bulb. We suggest
that treatment with mood stabilizing drugs could be used to
facilitate regeneration following insult to the central nervous
system. STEM CELLS 2008;26:1758–1767
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION
Neural stem cells (NSCs) reside in the adult mammalian brain
and provide new neurons in neurogenic regions, such as the
olfactory bulb and dentate gyrus of the hippocampus [1, 2].
Although the physiological roles of adult neurogenesis are not
yet fully understood, they may contribute to olfaction [3, 4] and
a certain type of memory [5]. In addition, NSCs in the adult
brain may play significant roles in pathological conditions. In
the rodent brain, ischemic insult induces the proliferation of
neural progenitor cells, progeny of NSC, in the subventricular
zone (SVZ) and subgranular layer of the hippocampal dentate
gyrus [6–8]. Neural progenitor cells in the SVZ were recruited
to the lesion, differentiated to neurons, and contributed to functional
recovery [9, 10]. If endogenous NSCs could be efficiently
activated to proliferate and differentiate to neurons through the
use of currently available pharmacologic agents, it would open
novel treatment modalities for the reversal of neurologic dysfunction.
However, no drugs are currently known that directly
act on the NSC and induce proliferation of neural stem/progenitor
cells in the adult brain.
Studies of NSC proliferation have been hampered by the
cells’ characteristics. That is, adult NSCs in the SVZ divide
slowly (estimated cell cycle time Ն15 days) and asymmetrically
to self-renew and produce transit-amplifying neural progenitor
cells [11]. Therefore, even with extensive labeling with 5-bromo-2Ј-deoxyuridine
(BrdU), only a small portion of NSCs can
be labeled, which makes it difficult to determine the absolute
number of NSCs in the adult brain. Nevertheless, BrdUϩ
cells
that remain in the SVZ 4 weeks after BrdU injections are
considered to be relatively quiescent NSCs because rapidly
proliferating neural progenitor cells dilute the label below the
threshold of immunofluorescent detection, leave the SVZ, or die
[12, 13]. An alternative way to assess NSCs is an in vitro
colony-forming neurosphere assay, in which NSCs from the
adult SVZ proliferate in serum-free media in the presence of
growth factors and form floating colonies called neurospheres
[14]. Neurospheres may be derived from subpopulations of
transit amplifying neural progenitor cells in addition to bona
fide NSCs [15]. Although the nature of epidermal growth factor
(EGF)-responsive neurosphere-forming cells in vivo remains to
be investigated, the neurosphere assay is useful for determining
the effects of drugs on NSC self-renewal.
Lithium, valproic acid (VPA), and carbamazepine (CBZ),
collectively called mood stabilizers, are used to treat patients
with bipolar disorder. However, the molecular mechanisms underlying
their pharmacological actions and the disease itself
remain largely unknown. Based largely on in vitro evidence,
several molecular models for the therapeutic actions of mood
Author contributions: M.H.: collection and assembly of data, data analysis and interpretation, manuscript writing; N.M.: collection and
assembly of data, data analysis and interpretation; A.B.: provision of study material or patients; K.I.: conception and design, administrative
support; S.H.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
Correspondence: Seiji Hitoshi, M.D., Ph.D., Division of Neurobiology & Bioinformatics, National Institute for Physiological Sciences, 5-1
Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Telephone: ϩ81-564-59-5246; Fax: ϩ81-564-59-5247; e-mail: shitoshi-tky@
umin.ac.jp Received December 7, 2007; accepted for publication April 28, 2008; first published online in STEM CELLS EXPRESS May 8,
2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-1032
TISSUE-SPECIFIC STEM CELLS
STEM CELLS 2008;26:1758–1767 www.StemCells.com
stabilizers have been proposed. These include depletion of inositol
[16–19], inhibition of glycogen synthase kinase-3␤ (GSK-
3␤) [20–23], and modification of MAPK/ERK pathways [24,
25]. In addition, at least one of mood stabilizers (VPA) exhibits
activities as a histone deacetylase inhibitor, which may mediate
some of mood stabilizing effects [26]. Interestingly, most, if not
all, of those studies made their observations at much higher
doses than are commonly used to treat bipolar disorder. When
an in vitro system is used to identify clinically relevant cellular
and molecular targets of mood stabilizers, several caveats are
necessary [27]. First, because mood stabilizers are assumed to
act in the central nervous system, the effects of the drugs should
be observed at doses relevant to therapeutic concentrations
found in the cerebrospinal fluid (CSF) or brain. Second, the
effects of mood stabilizers are observed only after chronic
exposure. Recently, it has been suggested that mood stabilizers
enhance neurogenesis, both in vivo [28, 29] and in vitro [29–
31]. This model is attractive because it takes several weeks
before new neurons mature and are integrated into existing
neural networks and this time frame corresponds to that of
observed clinical benefits [32]. The precise manner in which
mood stabilizing drugs modulate neurogenesis remains to be
documented.
MATERIALS AND METHODS
Animals and Drug Administration
CD1 (ICR) mice (Japan SLC, Japan) were kept in the institutional
Center for Experimental Animals with free access to food and
water. Presenilin1 mutant mice (C57BL/6 background) were genotyped
as described [33]. Because the number of neurosphere-initiating
NSCs is relatively stable since 6 weeks of age [34], 8–15
weeks of age mice were used in this study. Eight-week-old adult
male mice were given 3 g/L LiCl (Wako Chemicals, Osaka, Japan,
http://www.wako-chem.co.jp), supplemented with 0.6% NaCl, or
0.5 g/L VPA (Sigma, St. Louis, http://www.sigmaaldrich.com) by
drinking water for 3 weeks. CBZ (Sigma, 1 mg/kg i.p. dissolved in
DMSO at 0.5 mg/mL) was given to mice twice daily for 3 weeks.
Serum drug concentrations were measured by atomic-absorption
spectrometry for lithium or by enzyme immunoassay for VPA and
CBZ (Mitsubishi BCL, Japan). All experiments were carried out
under the permission of the institutional Animal Research Commit-
tee.
Isolation of Forebrain NSCs and the Neurosphere
Assay
The protocol used to generate neurospheres in vitro from adult and
embryonic brains has been described [13]. Cells from adult SVZ or
embryonic medial ganglionic eminence (MGE) were triturated in
serum-free media (SFM) [35] and cultured in the presence of 20
ng/mL EGF and 10 ng/mL fibroblast growth factor-2 (FGF-2)
together with 2 ␮g/mL heparin (Sigma) [14]. After 6 days in vitro,
the numbers of floating sphere colonies (neurospheres) possessing a
diameter Ͼ0.08 mm were counted. The total number of neurosphere-forming
NSCs in the adult brain was calculated from the
following: number of neurospheres, volume applied in the culture,
and volume in which cells were suspended (usually 700 ␮L)
[34, 36].
BrdU Labeling and Immunohistochemistry
Mice were injected with BrdU (Sigma) (65 mg/kg, i.p.) every 3
hours for a total of five injections, and sacrificed 1 hour or 4 weeks
after the last injection [13]. Coronal cryosections at 14-␮m thickness
were immunostained with mouse anti-BrdU antibody 1:1,000
(BD Biosciences, San Diego, http://www.bdbiosciences.com) at
4°C overnight, followed by secondary Alexa594-conjugated goat
anti-mouse IgG antibody (Molecular Probes Inc., Eugene, OR,
http://www.probes.invitrogen.com). Sections were counter-labeled
with the nuclear stain Hoechst 33,342 (1 ␮g/mL; Sigma), and the
total number of BrdU-labeled cells in the SVZ was estimated using
the optical dissector method.
Reverse Transcription-PCR
Total RNA isolation, cDNA synthesis, and RT-PCR analysis were
carried out as described [13]. cDNA was amplified in a thermal
cycler with denaturation at 95°C for 30-second, annealing at 56°C
for 40-second and extension at 72°C for 40-second for 32 (Hes1,
Hes5, and ␤-actin) or 35 cycles (the others). Quantitative RT-PCR
analyses for Hes5 and ␤-actin were performed using the LightCycler
system (Roche, Tokyo, http://www.roche-diagnostics.jp) as
described [34, 37].
Luciferase Reporter Assay
Firefly luciferase reporter plasmids [38] were generously provided
by Dr. Kageyama (Kyoto University). Primary neurospheres were
triturated into single cells and then transfected with the luciferase
reporter plasmid together with synthetic Renilla luciferase reporter
plasmid (Promega) as an internal control. Transfection was performed
using Lipofectamine 2000 (Invitrogen) for 3 hours at 37°C
and the cells were cultured to generate secondary neurospheres with
or without drugs. Four days after the transfection, neurospheres
were collected and cell extracts were assayed for luciferase activity
(Luciferase Assay System, Promega, Madison, WI, http://www.
promega.com). The firefly luciferase activities were normalized by
Renilla luciferase activities in each sample.
SDS-PAGE and Immunoblotting
Primary neurospheres or tissues from the adult SVZ were homogenized
in lysis buffer and subjected to SDS-PAGE and Western
blotting. Primary antibodies used are as follows: anit-Hes1 (1:1,000;
gift by Dr. Sudo, Toray Corporation) [39], anti-cleaved Notch1
(1:1,000; Cell Signaling Technology, Beverly, MA, http://www.
cellsignal.com), anti-Notch1 (1:1,000; Santa Cruz Biotechnology
Inc., Santa Cruz, CA, http://www.scbt.com), anti-actin (AC-15,
1:20,000; Sigma), anti-phospho-GSK3␤ (Ser9) (1:1,000; Cell Signaling
Technology), and anti-GSK3␤ (1:5,000; Cell Signaling
Technology).
In Vitro ␥-Secretase Assay
␥-Secretase was prepared from secondary neurospheres derived
from the adult SVZ using a method described in [40]. The membrane
fraction that contains ␥-secretase was solubilized in 1%
CHAPSO. Full-length Notch1 tagged with myc-epitope [41, 42]
was expressed in Neuro2a cells and the membrane fraction of the
cells were prepared as ␥-secretase. The ␥-secretase solution and
myc-tagged Notch substrate were mixed, and the reaction was
stopped by adding Laemmli sample buffer at zero time points or
after the incubation at 4°C for 16 hours. The reaction samples were
separated by SDS-polyacrylamide gel and blotted with anti-c-Myc
antibody (9E10, Biomol, Plymouth Meeting, PA, http://www.
biomol.com).
In Situ Hybridization
Digoxigenin (DIG)-labeled single strand riboprobes for Hes1 or
Hes5 genes were synthesized using DIG RNA labeling mix
(Roche). In situ hybridization was performed as described previously
[43]. DIG-labeled RNA probes were visualized with alkaline
phosphatase-conjugated anti-DIG antibody (1:2000; Roche) and
NBT/BCIP reaction, followed by anti-BrdU immunostaining and
visualization using the ABC Elite kit (Vector Laboratories, Burlingame,
CA, http://www.vectorlabs.com) and diaminobenzidine as a
chromogen.
Statistics
Statistical analysis was performed using one-way ANOVA followed
by Scheffe’s or Dunnett’s post hoc comparison or using an
unpaired two-tailed Student’s t-test if applicable. Level of significance
was set at p Ͻ .05.
1759Higashi, Maruta, Bernstein et al.
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RESULTS
In Vitro Effects of Mood Stabilizers
To examine the effects of mood stabilizing drugs on primary
neurosphere formation, cells from the SVZ of 8-week-old male
mice were cultured at low cell density (five cells/␮L) in the
presence of the drugs. LiCl at 0.5–1.0 mM (within the therapeutically
relevant concentrations in the CSF) (Table 1), increased
the number of primary neurospheres, whereas LiCl at
concentrations greater than 3.0 mM showed toxic effects on
primary neurospheres (F(5, 61) ϭ 24.69, p Ͻ .001) (Fig. 1A).
Similarly, at doses equivalent to therapeutically relevant CSF
concentrations, both VPA (F(5, 57) ϭ 35.85, p Ͻ .001) and CBZ
(F(4, 56) ϭ 11.96, p Ͻ .001) increased the number of primary
neurospheres from the adult SVZ (Fig. 1B, 1C).
These results could be interpreted in two ways: mood stabilizers
could enhance the survival of cultured NSCs, [1] or
mood stabilizers could increase the self-renewal capability of
neural stem/progenitor cells [2] and thereby increase the number
of neurosphere-initiating cells, although these possibilities are
not mutually exclusive. To distinguish between those possibilities,
we first investigated whether or not mood stabilizers
prevent cell death by means of counting dead cells by trypan
blue exclusion. The number of dead cells 24 hours after the
plating was comparable between drug-treated and non-treated
cells (14.71 Ϯ 4.07% for control, 15.29 Ϯ 4.18% for LiCl,
13.47 Ϯ 3.81% for VPA, 13.88 Ϯ 3.60% for CBZ, mean Ϯ
SEM, F(3, 64) ϭ 0.75, p ϭ .53). We then subcloned primary
Figure 1. Effects of mood stabilizers on neurosphere formation in vitro. (A–C): Cells from the adult SVZ were cultured in the presence of
LiCl (A), VPA (B), or CBZ (HBC complex, Sigma) (C). All drugs increased the numbers of primary neurospheres when added at therapeutic
concentrations and decreased the numbers at high concentrations as compared to control (n Ն 6 for each concentration from at least three
independent experiments). (D–F): The resultant primary neurospheres were subcloned to generate secondary and tertiary neurospheres in the
absence of the drugs. (D): Single primary neurospheres were triturated and one tenth of the cells were cultured to form secondary neurospheres.
The numbers of resultant secondary neurospheres are expressed as per single primary neurospheres. (n ϭ 5 animals, 4–8 neurospheres from
each animal). (E): Neurospheres were serially passaged in bulk and cultured in the absence of the drugs at a clonal density (two cells/␮L). Cells
from the primary neurospheres treated with 0.5 mM LiCl, 60 ␮M VPA or 5 ␮M CBZ, produced more secondary and tertiary neurospheres than
their control counterpart (n ϭ 5). (F): Cells from the primary neurospheres grown in the presence of 0.5 mM LiCl, 60 ␮M VPA or 5 ␮M CBZ
were plated at 0.5 cells/␮L and cultured in the absence of the drugs. Total cell numbers after 4 or 7 days in vitro were counted (n ϭ 4). (G):
Effects of mood stabilizers at the therapeutic concentrations (0.5 mM LiCl, 60 ␮M VPA and 5 ␮M CBZ) were examined using cells from the
E14.5 medial ganglionic eminence. Mood stabilizer treatment increased primary neurosphere formation, whereas 10 ␮M PHT or 1.0 ␮M FLX
showed no effect (n Ն 4). Data represent means Ϯ SEM. *, p Ͻ .05. Abbreviations: CBZ, carbamazepine; FLX, fluoxetine; PHT, phenytoin;
VPA, valproic acid.
Table 1. Therapeutic concentrations of mood stabilizers
Lithium VPA CBZ
Serum levels 0.4–1.0 mEq/L 50–125 ␮g/␮L (0.35–0.87 mM) 4–12 ␮g/␮L (17–51 ␮M)
CSF/Serum ratio 0.5859
0.1560
0.2161
CSF levels 0.23–0.58 mEq/L 52–130 ␮M 3.6–10.7 ␮M
1760 Mood Stabilizers Activate Adult Neural Stem Cells
neurospheres that had been grown in the presence of 0.5 mM
LiCl, 60 ␮M VPA, or 5 ␮M CBZ and cultured the cells in the
absence of the drugs to generate secondary neurospheres. Because
primary neurospheres are clonally derived from single
NSCs when the adult SVZ cells are cultured at low cell density
[44] (see Discussion), the number of secondary neurospheres
derived from each primary neurosphere represents the number
of symmetric expansive divisions of the original NSCs during
the culture. We cultured one-tenth of the cells from each primary
neurosphere to keep the cell densities below two cells/␮L
and found that a greater number of secondary neurospheres were
generated from single primary neurospheres treated with mood
stabilizers than those without treatment (F(3, 16) ϭ 7.024, p ϭ
.003) (Fig. 1D). It is unlikely that these effects are mediated by
histone deacetylase inhibitor activities because trichostatin A,
another histone deacetylase inhibitor, neither increased the number
of primary neurosphere nor enhanced the self-renewal but,
inversely, alleviated the primary neurosphere formation at 10
nM or higher concentrations (F(4, 11) ϭ 35.46, p Ͻ .001)
(supplemental online Fig. 1).
The self-renewal capacity of NSCs was also analyzed
through serial bulk passaging of neurospheres, in which drugtreated
or control neurospheres were dissociated in bulk and
cultured at a constant low cell density (two cells/␮L) in the
absence of the drugs (Fig. 1E). Bulk passaging demonstrated
that NSCs treated with mood stabilizers in the primary culture
exhibited enhanced self-renewal, with increased numbers of
both secondary (F(3, 16) ϭ 10.36, p Ͻ .001) and tertiary neurospheres
(F(3, 16) ϭ 5.46, p ϭ .009) as compared to controls (Fig.
1E). The proliferation of cells from drug-treated primary neurospheres
was examined by plating cells at a very low density
(0.5 cells/␮L) and counting cell numbers 4 or 7 days later (Fig.
1F). Cells obtained from primary neurospheres grown in the
presence of mood stabilizers proliferated more rapidly than
those from control neurospheres (F(3, 12) ϭ 10.45, p ϭ .001)
(Fig. 1F). These results suggest that in vitro treatment with
therapeutically relevant doses of mood stabilizers enhances the
self-renewal of NSCs from the adult brain.
The effect of mood stabilizers on NSCs derived from the MGE
ventricular zone of E14.5 embryos was also evident (F(4, 64) ϭ
10.41, p Ͻ .001), whereas another anti-convulsant, phenytoin, or
anti-depressant, fluoxetine, had no such effect (Fig. 1G), suggesting
that the effect was specific to mood stabilizing drugs.
Individual neurospheres treated with mood stabilizers produced
␤III tubulinϩ
neurons, GFAPϩ
astrocytes, and O4-antigenϩ
oligodendrocytes under differentiation conditions (online supplemental
Fig. 2), suggesting that the multipotentiality of NSCs
was not altered by the drug treatment.
In Vivo Effects of Mood Stabilizers
Next, we used the neurosphere assay to determine whether lithium
treatment increased the number of neurosphere-forming NSCs in
the SVZ of the adult brain. Because each primary neurosphere is
clonally derived from a single NSC when cultured at low cell
densities, the total number of NSCs in the SVZ can be estimated by
counting primary neurospheres [34, 36, 45]. When LiCl was administered
to mice for 3 weeks (3 g/L in drinking water), serum
lithium concentrations reached 0.86 Ϯ 0.23 mEq/L (mean Ϯ SD),
a value close to that observed during therapeutic management of
bipolar patients [32]. The total number of neurosphere-forming
NSCs in the lateral portion of the SVZ was increased in lithiumtreated
animals compared to controls, and this increase was statistically
significant only after 3-week treatment but not after 1- or
2-week treatment (F(3, 42) ϭ 8.36, p Ͻ .001) (Fig. 2A). In contrast,
treatment with higher doses of LiCl, which subsequently resulted in
higher serum concentrations of 4.36–6.59 mEq/L, reduced the
number of neurosphere-forming NSCs in the adult brain (F(1, 6) ϭ
8.12, p ϭ .036) (Fig. 2B).
We asked if a similar stimulatory effect occurred following treatment
with VPA or CBZ. Mice given VPA (0.5 g/L in drinking water)
or CBZ (1 mg/kg body weight by intraperitoneal injection, twice daily)
achieved much lower drug concentrations in their sera (ϳ6.0 ␮g/mL
for VPA and 0.5 ␮g/mL or lower for CBZ) than observed during
treatment of bipolar patients [32]. Nevertheless, administration of VPA
and CBZ produced similar increases in the numbers of neurosphereforming
NSCs. Compared to controls, the total number of NSCs in the
SVZ was increased in both VPA-treated mice and in CBZ-treated
mice (F(1, 12) ϭ 11.10, p ϭ .006 and F(1, 16) ϭ 9.38, p ϭ .007,
respectively) (Fig. 2C, 2D). In contrast, the number of NSCs in the
SVZ from mice treated with phenytoin (200 mg/L drinking water, 3
weeks), an anticonvulsant lacking mood stabilizing action, was comparable
to control (95.1 Ϯ 7.6% of control, n ϭ 3).
Figure 2. Number of neural stem cells in the adult brain subventricular
zone was determined by the neurosphere assay. (A): The total number
of neural stem cells in the lateral portion of the SVZ was increased in
mice treated with LiCl (0.86 Ϯ 0.23 mEq/l, mean Ϯ SD) for 3 weeks
(n ϭ 10) but not for 1 week (n ϭ 9) or 2 weeks (n ϭ 6) as compared
to controls (n ϭ 21). (B): The total numbers of neural stem cells in the
mice, which were treated with higher doses of LiCl and subsequently
showed toxic levels of lithium in their sera (4.36–6.59 mEq/l) (n ϭ 3),
were reduced relative to control (n ϭ 4). (C, D): Mice were given VPA
or CBZ for 3 weeks and were subjected to the neurosphere assay. The
total numbers of neural stem cells in the lateral portions of the SVZ were
increased in mice treated with VPA (n ϭ 6) as compared to controls
(n ϭ 8) (C), and in mice treated with CBZ (n ϭ 10) as compared to
DMSO-injected controls (n ϭ 8) (D). (E): Serial bulk passages of
neurospheres were performed at a clonal density (two cells/␮L) every 6
days. The primary neurospheres derived from mice given LiCl (n ϭ 9,
serum Liϩ
concentrations at 0.22–1.19 mEq/L) or VPA (n ϭ 9) or CBZ
(n ϭ 3) produced more secondary and tertiary neurospheres than their
control counterpart. Data represent means Ϯ SEM. *, p Ͻ .05. Abbreviations:
CBZ, carbamazepine; DMSO, dimethyl sulfoxide; VPA, valproic
acid.
1761Higashi, Maruta, Bernstein et al.
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To determine the effect of mood stabilizer administration
on the self-renewal capacity of NSCs, primary neurospheres
were serially passaged in bulk and cells were cultured at low
cell density (two cells/␮L) in the absence of the drugs. NSCs
derived from the SVZ of mice which had been given mood
stabilizers and showed serum drug concentrations at or below
the therapeutic doses, exhibited enhanced self-renewal. That
is, we observed increased numbers of both secondary (F(2, 21) ϭ
10.36, p Ͻ .001 for LiCl and VPA and t(4) ϭ 5.85, p ϭ .004 for
CBZ) and tertiary neurospheres (F(2, 20) ϭ 9.74, p ϭ .001 for
LiCl and VPA and t(4) ϭ 9.10, p Ͻ .001 for CBZ) as compared
to appropriate controls (Fig. 2E). Thus, these results suggest that
administration of mood stabilizers enhances the self-renewal of
NSCs in the adult brain and expands the NSC pool in the SVZ.
At this point, we focused on lithium treatment for further in
vivo analyses, because administration of LiCl by drinking water
for 3 weeks or longer consistently resulted in serum Liϩ
concentrations
in the range 0.4–1.2 mEq/L, whereas 3-week treatments
with VPA or CBZ produced low and variable drug
concentrations. In these trials, the number of NSCs in the SVZ
of treated mice was assessed by a different method. After 3
weeks of treatment, mice received 5 BrdU injections every 3
hours and were sacrificed 4 weeks after the last injection.
During the 4-week period following the last injection, transitamplifying
neural progenitor cells, having incorporated BrdU,
diluted the label by further divisions, differentiated and migrated
away from the SVZ, or died [46]. BrdUϩ
cells that remained in
the SVZ for 4 weeks are considered relatively quiescent NSCs.
BrdUϩ
cells in the SVZ were increased in number in the mice
given LiCl as compared to control (t(10) ϭ –3.97, p ϭ .003) (Fig.
3A).
Next, we asked if the lithium-induced increase of NSCs in
the SVZ also increased the number of neural progenitors and
subsequent neurogenesis. After 3 weeks of lithium treatment,
mice received 5 BrdU injections every 3 hours and were sacrificed
1 hour (total 13 hours, Fig. 3B) or 7 days (Fig. 3C) after
the last injection. BrdU labeling over 13 hours that encompasses
estimated cell cycle times of neural progenitor cells [12] demonstrated
more dividing cells in the SVZ of lithium-treated mice
than control (F(1, 15) ϭ 5.13, p ϭ .039 for SVZ) (Fig. 3B),
suggesting that neural progenitor cells were also increased after
chronic lithium treatment. As a result of this increase in proliferating
cells in the SVZ, more BrdUϩ
cells survived and migrated
to the olfactory bulb (t(8) ϭ –2.82, p ϭ .022) of LiCltreated
mice than that of control (Fig. 3C). These findings
demonstrate that lithium administration expanded the size of the
NSC pool and increased neural progenitor proliferation in the
SVZ, resulting in increased cell supply to the olfactory bulb in
the adult brain.
Notch Signaling Mediates the Effects of Mood
Stabilizers.
Recent studies suggest that Notch signaling plays a critical role
in the maintenance of NSCs [13, 47, 48]. Therefore, we investigated
whether mood stabilizers activate Notch signaling in
neurospheres. RT-PCR analyses revealed that two downstream
genes of the Notch pathway, Hes1 and Hes5, were upregulated
in all three-drug-treated neurosphere cultures relative to control
(Fig. 4A), which was confirmed by quantitative RT-PCR (F(3, 20) ϭ
4.94, p ϭ .01) (supplemental Fig. 3A). We next performed a
reporter assay using a luciferase gene plasmid, in which the
firefly luciferase gene was placed downstream of either the
Hes1 or Hes5 gene promoter [38]. Treatment with 0.5 mM LiCl,
or 60 ␮M VPA, or 5 ␮M CBZ enhanced both Hes1 (F(3, 44) ϭ
68.34, p Ͻ .001) and Hes5 (F(3, 44) ϭ 23.48, p Ͻ .001) gene
promoter activities (Fig. 4B and online supplemental Fig. 3B).
Western blotting revealed that all three mood stabilizers increased
Hes1 protein in neurosphere cultures (F(3, 8) ϭ 10.78,
p ϭ .004) (Fig. 4C and online supplemental Fig. 3C). Thus,
mood stabilizing drugs at therapeutic concentrations upregulate
the expression of Hes1/5 genes, suggesting activation of the
Notch signaling pathway.
To obtain direct evidence of Notch signaling activation, we
quantified amounts of an active form of Notch1 receptor in
neurospheres by Western blotting. Anti-cleaved Notch1 antibody
recognizes the Notch intracellular domain, which is a
direct measure of the amount of Notch signaling activation.
Primary neurospheres exposed to LiCl, VPA, or CBZ contained
significantly more cleaved Notch1 than the control neurospheres
(F(3, 12) ϭ 11.63, p Ͻ .001) (Fig. 4D and online supplemental
Fig. 3D).
If mood stabilizers enhance NSC expansion through Notch
signaling, then inhibition of Notch signaling should block this
enhancement. Indeed, addition of 1 ␮M ␥-secretase inhibitor,
Figure 3. Lithium increases BrdUϩ
cells in the adult brain. Mice were
administered LiCl for 3 weeks, received 5 BrdU injections (65 mg/kg)
every 3 hours, and then sacrificed 4 weeks (A), 1 hour (B), or 7 days (C)
after the last injection. Cryosections were immunostained for BrdU
incorporation, which overlapped the nuclear staining with Hoechst
33,342. (A): More BrdUϩ
cells which remained in the SVZ for 4 weeks
after the labeling were detected in the mice given LiCl (n ϭ 6) than in
control (n ϭ 6). (B): BrdU labeling demonstrated more dividing cells in
the SVZ of lithium-treated mice (n ϭ 10) than control (n ϭ 7). (C):
BrdUϩ
cells that migrated to the GCL of the olfactory bulb 7 days after
the labeling were counted. Representative higher magnification images
for BrdU positivity (red, right panels) show a similar region as indicated
by a box in a lower magnification image for Hoechst 33,342 nuclear
staining (blue, left panel). More BrdUϩ
cells were observed in the GCL
of lithium-treated mice (n ϭ 4) than in that of control mice (n ϭ 6).
Scale bars ϭ 100 ␮m. Data represent means Ϯ SEM. *, p Ͻ .05.
Abbreviations: RMS, rostral migratory stream; BrdU, 5-bromo-2Ј-deoxyuridine;
GCL, granular cell layer.
1762 Mood Stabilizers Activate Adult Neural Stem Cells
L-685,458, to cultures abolished the effects of LiCl, VPA, and
CBZ on primary neurosphere formation (Fig. 4E). Presenilin1 is
a major component of the ␥-secretase complex or ␥-secretase
itself [49] and homozygous mutation for the presenilin1 gene
results in the depletion of NSCs due to the impaired activation
of Notch signaling [13]. We examined the effects of mood
stabilizers on neurosphere formation from MGE cells of E14.5
presenilin1Ϫ/Ϫ
embryos because those embryos die around the
time of birth [33]. In contrast to wild-type controls, where mood
stabilizers increased the number of primary neurospheres, those
drugs exhibited few effects, if any, on primary neurosphere
numbers from presenilin1Ϫ/Ϫ
NSCs (Fig. 4F). Finally, we examined
whether or not mood stabilizers modify ␥-secretase
activities using ␥-secretase source from neurospheres and myctagged
full-length Notch1 as a substrate. Indeed, ␥-secretase
activities were enhanced in the presence of therapeutic doses of
LiCl and VPA by ϳ60%, but not in the presence of CBZ (Fig.
4G, 4H). These results suggest that mood stabilizers enhance the
self-renewal capability of NSCs by activating Notch signaling
and that these effects are mediated by, at least in part, the
augmentation of ␥-secretase activities.
Both lithium and VPA have been shown either directly or
indirectly to inhibit GSK-3␤ [20–23], a key mediator of Wnt
and other signal pathways, and inhibit inositol monophosphatase
(IMPase), leading to inositol depletion [16–19]. Therefore,
we determined whether or not effects of mood stabilizers
on neurospheres were mediated by the inhibition of GSK-3␤
signaling or inositol depletion. We examined levels of phosphorylated
GSK-3␤ at Ser9, an inactive form of GSK-3␤, in the
neurospheres by Western blotting following drug treatment.
Treatment with therapeutic doses of mood stabilizers did not
alter the ratio of phosphorylated to total GSK-3␤ (Fig. 5A, 5B),
whereas high doses of mood stabilizers increased the phosphorylation
of GSK-3␤ (F(3, 16) ϭ 4.86, p ϭ .014) (Fig. 5C, 5D).
Similarly, therapeutic concentrations of these drugs did not
change the expression of IMPase 1/2 genes (Fig. 5E). By
Figure 4. Mood stabilizers activate Notch signaling in neurospheres. (A, C, D): Primary neurospheres were grown in the presence of 0.5 mM LiCl,
60 ␮M VPA, or 5 ␮M CBZ. (A): RNA from the neurospheres was subjected to RT-PCR analysis. RT Ϯ indicates the presence/absence of reverse
transcriptase in the assay. (B): Firefly luciferase reporter assay was performed using secondary neurospheres. Treatment with 0.5 mM LiCl, 60 ␮M
VPA, or 5 ␮M CBZ enhanced Hes1 gene promoter activities. (C, D): Protein extracts from the primary neurospheres were separated on SDS-PAGE,
and then immunoblotted with anti-Hes1 (C) and anti-cleaved Notch1 (D) antibody. (E): Primary neurospheres were formed in the presence of
therapeutic doses of mood stabilizers together with 1.0 ␮M ␥-secretase inhibitor, L-685,458, which inhibits the activation of Notch signaling. (F):
The medial ganglionic eminence cells from E14.5 presenilin1Ϫ/Ϫ
(n ϭ 8) and their wild-type littermates (n ϭ 8) were cultured. Mood stabilizers
enhanced the formation of primary neurospheres from wild-type cells but showed few effects on those from presenilin1Ϫ/Ϫ
cells. (G, H): ␥-Secretase
assay was performed in the presence of 0.5 mM LiCl, 60 ␮M VPA, 5 ␮M CBZ, or 10 ␮M L-685,458. Myc-tagged cleaved Notch1 intracellular
domain was immunoblotted with anti-c-myc antibody (G). (H): The bands were quantified and relative levels of myc-tagged cleaved Notch1 to control
as 100% are shown. All experiments were carried out four or more times. Data represent means Ϯ SEM. *, p Ͻ .05. Abbreviations: CBZ,
carbamazepine; RT, reverse transcription; VPA, valproic acid.
1763Higashi, Maruta, Bernstein et al.
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contrast, coculture with higher concentrations of mood stabilizers
lowered the expression levels of those genes (Fig. 5F).
Addition of myo-inositol to cultured cells rescue the depletion
of inositol [18]. Adult SVZ cells were cultured to generate
primary neurospheres in the presence of 1.0 mM myo-inositol
together with lithium, VPA and CBZ. Myo-inositol itself
showed virtually no ability to increase the number of neurospheres
and did not reverse the effects of mood stabilizers at
therapeutic concentrations (Fig. 5G). By contrast, coculture with
1.0 mM myo-inositol reversed the toxic effects of mood stabilizers,
observed in the presence of 5.0 mM LiCl, 300 ␮M of
VPA, and 50 ␮M of CBZ (Fig. 5H). Together, these results
suggest that Notch signaling activation in the neurospheres by
therapeutic doses of mood stabilizers is not mediated by the
alteration of GSK-3␤ or inositol signaling.
Notch Signaling Activation In Vivo
Activation of Notch signaling in NSCs by mood stabilizers was
evident not only in vitro but also in vivo. In situ hybridization
assessment of Hes1 and Hes5 gene expression showed that
Hes1/5-expressing cells were increased in the SVZ of mice
treated with LiCl for 3 weeks (Fig. 6A, 6B). The lateral portion
of the SVZ was excised and then homogenized in lysis buffer.
Comparable amounts of protein were obtained from each mouse
(control, 1.79 Ϯ 0.42 mg (n ϭ 12) vs. LiCl, 1.77 Ϯ 0.30 mg
(n ϭ 12), mean Ϯ SD), verifying equivalent tissue isolation.
Samples of 20 ␮g protein were separated on SDS-PAGE and
immunoblotted with anti-cleaved and anti-total Notch1 antibody
(Fig. 6C, 6D). Higher amounts of cleaved Notch1 were detected
in the SVZ of the mice treated with lithium for 3 weeks relative
to control mice (F(1, 18) ϭ 75.38, p Ͻ .001) (Fig. 6E) and an
insignificant increase of total Notch1 protein was also observed
(Fig. 6F). Increase of cleaved Notch1 protein level was only
detected after 3-week, but not 1-week, treatment by lithium
(online supplemental Fig. 4A, 4B). These results indicate that
lithium treatment results in either increased amounts of activated
Notch in individual neural stem/progenitor cells or increased
numbers of neural stem/progenitor cells with equivalent
levels of activated Notch. In contrast, GSK-3␤ signaling was not
altered by lithium treatment in vivo (online supplemental Fig.
4C, 4D).
DISCUSSION
In the present study, we demonstrated that three different mood
stabilizing drugs directly enhanced NSCs’ self-renewal capability
through activation of Notch signaling. Furthermore, lithium
administration expanded the size of the NSC pool in the SVZ of
the adult brain, resulting in an increased cell supply to the
olfactory bulb.
Several intrinsic and extrinsic factors are known to alter
neurogenesis in the adult brain, especially in the dentate gyrus of
the hippocampus [11]. However, the growth kinetics of NSCs in
the adult brain are largely unknown, at least in part because
there are no specific markers currently available that discriminate
NSC from transit amplifying neural progenitor cells. In this
study, we utilized an in vitro colony-forming neurosphere assay
in which NSCs respond to growth factors in serum-free media,
proliferate and form clonal neurospheres. The clonality of the
neurosphere has recently been challenged as demonstrated by an
observation that cells separately prepared from neurospheres
constitutively expressing EGFP and neurospheres constitutively
expressing ␤-galactosidase generated a substantial percentage of
chimeric neurospheres even when cultured at low cell densities
(0.5 cells/␮L) [50]. However, in mixing experiments using
wild-type and GFP-expressing cells from the adult SVZ, we
have never observed primary neurospheres that consisted of
both wild-type and GFPϩ
cells (online supplemental Fig. 5A,
5B). Those results suggest that adhesion and mixing events
between two or more neurosphere-initiating NSCs are rare or, at
least, negligible when estimating the number of NSCs in the
Figure 5. Effects of mood stabilizers on GSK-3␤ signaling and the
inositol pathway. (A): Primary neurospheres from the adult SVZ were
generated in the presence of 0.5 mM LiCl, 60 ␮M VPA, or 5 ␮M CBZ.
Protein extracts of the neurospheres were separated on SDS-PAGE and
then immunoblotted with anti-GSK-3␤ or anti-phospho-GSK-3␤ (Ser9)
antibodies. (B): The bands were quantified and the ratio of phosphorylated
to total GSK-3␤ was calculated. (C): Primary neurospheres were
grown and toxic doses of mood stabilizers were added to the culture at
5 days in vitro. After 15-minute treatment, protein extracts of the
neurospheres were subject to Western blotting. (D): The bands were
quantified and the ratio of phosphorylated to total GSK-3␤ was calculated.
(E, F): Total RNA was extracted from primary adult neurospheres
cultured with mood stabilizers at therapeutic (E) or higher concentrations
(F) and subject to RT-PCR analysis. Mood stabilizers at the
therapeutic concentrations showed few effects on the expression levels
of IMPA1/2 in the neurospheres (E), whereas the drugs at the higher
concentrations downregulated gene expression (F). RT Ϯ indicates the
presence/absence of reverse transcriptase in the assay. (G, H): Effects of
inositol rescue were determined by adding 1 mM myo-inositol in cultures
of the neurosphere assay. Myo-inositol did not alter the effects of
0.5 mM LiCl, 60 ␮M VPA, or 5 ␮M CBZ, which increased the number
of primary neurospheres (G), but resumed the toxic effects of 5 mM
LiCl, 300 ␮M VPA or 50 ␮M CBZ (H). Data represent means Ϯ SEM.
from 3–5 independent experiments. *, p Ͻ .05. Abbreviations: CBZ,
carbamazepine; VPA, valproic acid.
1764 Mood Stabilizers Activate Adult Neural Stem Cells
SVZ. The in vivo BrdU labeling analyses support our conclusion
that chronic lithium treatment expands the size of the NSC
pool in the SVZ. The discrepancy between the data from Singec
et al. and our data may be due to the fact that cells derived from
neurospheres that keep proliferating in vitro [50] are highly
motile and adhere to each other more easily than primary cells
from the SVZ. Alternatively, non-adhesive culture plates used in
the study by Singec et al. [50] may substantially enhance the cell
movement and fusion events during the neurosphere formation
(online supplemental Fig. 5C, 5D).
Notch signaling plays critical roles in the development of
the central nervous system, as revealed by studies of mice
deficient for Notch pathway molecules [51]. Attenuation of
Notch signaling leads to the reduction of neural stem/progenitor
cells and precocious neuronal differentiation in developing
brains, suggesting that activation of Notch signaling keeps the
NSC undifferentiated and expands the NSC pool by promoting
symmetrical and self-renewing divisions. In addition, NSCs are
decreased by 40% in the brains of adult presenilin1 heterozygous
mutant mice, and conversely, introduction of an active
form of Notch1 receptor by retrovirus infection increases the
number of NSCs in the adult brain [13]. Thus, the modification
of Notch signaling activities alters the size of the NSC pool
postnatally. Our present results suggest that treatment with
mood stabilizers at therapeutic concentrations activates Notch
signaling in neural stem/progenitor cells in the adult brain.
Activation of Notch signaling results in the expansion of the
NSC pool and subsequently increased production of neural
progenitor cells in the SVZ and the olfactory bulb. Interestingly,
Notch activation is reported in human neuroblastoma cells after
treatment with VPA in vitro [52], although this effect was only
observed at very high doses (1–2 mM). It remains to be investigated
whether sensitivity to the drug differs between NSCs in
the adult brain and cancerous cells or whether neuroblastoma
cells lose their sensitivity after transformation and extended
culture in vitro.
Consistent with other findings [28, 29], our data indicate
that all three mood stabilizers increase the number of NSCs and
that they therefore enhance neurogenesis in vivo. Increased
neurogenesis in the olfactory bulb after chronic treatment
with mood stabilizers may simply reflect the increase of
NSCs, or alternatively, mood stabilizers may have direct
effects on neurons to facilitate their survival or maturation
[53]. A multitude of studies suggest that mood stabilizing
drugs inhibit GSK-3␤ activities in neurons [20–23] and that
this event may alter several different signaling cascades. One
of the main effects of GSK-3␤ inhibition is accumulation of
␤-catenin, which has antiapoptotic effects and may enhance
the survival of neurons [54, 55]. Mood stabilizers also inhibit
IMPase and cause inositol depletion [16–19], which subsequently
inhibits the collapse of sensory neuron growth cones
and may facilitate axonogenesis in vitro [18]. Although we
could detect few effects on GSK-3␤ or inositol signaling
activities in neural stem/progenitor cells, it remains formally
possible that the drugs have less apparent but biologically
significant effects on these signaling in postmitotic neurons.
Our data do not exclude a possibility that mood stabilizers
modify other signal pathways than Notch signaling in NSCs
as well as non-stem cells in the adult brain.
It is currently unknown whether the effects of mood stabilizers
on the NSC and neurogenesis in the adult brain are related
to the pharmacological actions of the drugs to ease symptoms of
bipolar disorder. It is well documented that neurogenesis is
suppressed in animal models of stress or depression [56, 57].
Importantly, hippocampal neurogenesis is necessary for antidepressant
drugs to improve behavioral deficits in stressed mice
[58]. Therefore, it would be reasonable to hypothesize that
impaired neurogenesis is causally related to the depressive state
and that mood stabilizers attenuate the symptoms of at least the
depressive phase of bipolar disorder by enhancing neurogenesis
in the hippocampus. It is currently difficult to explain how mood
stabilizers prevent bipolar disorder patients from proceeding to
the manic phase, because the molecular and cellular mechanisms
underlying the pathogenesis of mania are largely unknown
and animal models that fully reproduce the symptoms of
mania are not available. Nevertheless, our working hypothesis
that the pharmacological action of mood stabilizers is mediated
by the time-consuming expansion of NSC pool in the adult
brain, which is achieved by Notch signaling activation, is attractive
because it may explain why clinical benefits of the
drugs are observed only after chronic treatment [32]. It is
tempting to investigate the modification of neuronal network
following the expansion of NSC pool and enhanced neurogenesis
by mood stabilizer treatment.
Figure 6. Lithium administration activates
Notch signaling in the SVZ of the adult
brain. (A, B): Mice were given LiCl for 3
weeks and received 5 BrdU injections. Cryosections
were analyzed by in situ hybridization
for Hes1 (A) and Hes5 (B) gene expression
(purple) and double labeled for BrdU
incorporation (brown). Lithium treatment
for 3 weeks increased the number of Hes1/
5-expressing cells in and close to the SVZ,
where BrdUϩ
neural stem/progenitor cells
resided. Scale bar ϭ 50 ␮m (lower magnification)
or 10 ␮m (higher magnification).
(C–F): The SVZ tissue was subjected to
Western blotting. Treatment with lithium for
3 weeks resulted in a significant increase of
cleaved Notch1 (n ϭ 8) (C, D) and an insignificant
increase of total Notch1 protein
(n ϭ 4) (E, F). Data represent means Ϯ
SEM. *, p Ͻ .05. Abbreviations: CC, corpus
callosum; STR, striatum.
1765Higashi, Maruta, Bernstein et al.
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ACKNOWLEDGMENTS
We thank R. Kageyama for luciferase reporter plasmids, T.
Tomita, H. Okano, and J. Nye for full-length Notch1 plasmid,
and T. Sudo for anti-Hes1 antibody. This work was supported
by Grants-in-Aid for Scientific Research (B) (#19300136) and
for Scientific Research on Priority Areas—Research on Pathomechanisms
of Brain Disorders (#18023035)—from the Ministry
of Education, Culture, Sports, Science and Technology of
Japan.
DISCLOSURE OF POTENTIAL CONFLICTS
OF INTEREST
The authors indicate no potential conflicts of interest.
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1767Higashi, Maruta, Bernstein et al.
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