tion to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and
provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete
isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3? small interfering RNA (siRNA) and/or GSK-3? siRNA
mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate,
HDAC inhibitors potentiated ?-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine
synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder.
Lithium and valproic acid (VPA) are two first-line treatment
drugs for bipolar disorder. The mechanisms underlying their
clinical efficacy, however, remain essentially unknown. One of
the common effects of lithium and VPA is their ability to protect
against apoptotic insults in vitro and in vivo (for review, see
protects cultured brain neurons from glutamate-induced apo-
ptosis (Nonaka et al., 1998; Hashimoto et al., 2002; Leng and
beneficial effects in cellular and animal models of neurodegen-
erative diseases such as stroke, Alzheimer’s disease, Parkinson’s
atrophy, retinal degeneration, and human immunodeficiency
virus-1 infection (for review, see Tariot et al., 2002; Chuang and
Lithium is known to directly inhibit glycogen synthase
kinase-3 (GSK-3) activity (Klein and Melton, 1996; Stambolic et
al., 1996). GSK-3 is generally considered to have a proapoptotic
role, and its inhibition results in cytoprotection (for review, see
Bijur and Jope, 2003; Doble and Woodgett, 2003). Lithium also
indirectly inhibits GSK-3 by triggering phosphorylation of GSK-
3?Ser21/?Ser9(Chalecka-Franaszek and Chuang, 1999; De Sarno
et al., 2002; Zhang et al., 2003). VPA, also an anticonvulsant, has
been reported to inhibit GSK-3? enzymatic activity and induce
GSK-3?Ser9phosphorylation in some, but not all, neurally re-
al., 2001; Phiel et al., 2001). HDAC inhibitors, including phenyl-
butyrate (PB), sodium butyrate (SB), and trichostatin A (TSA),
regulate expression of neuroprotective/neurotrophic proteins
and proapoptotic/proinflammatory proteins (for review, see
Langley et al., 2005).
Several lines of evidence suggest that neuroprotective/neuro-
content (Moore et al., 2000a) and enhances levels of N-acetyl-
Institutes of Health (NIH). We sincerely thank Dr. Weihan Wang of Uniformed Services University of the Health
Sciences (Bethesda, MD) for his valuable assistance in the course of this study. We also thank the NIH Fellows
2576 • TheJournalofNeuroscience,March5,2008 • 28(10):2576–2588
aspartate, a marker of neuronal viability, in
the brain of bipolar patients (Moore et al.,
2000b). Moreover, bipolar subjects with
past lithium or VPA exposure tend to have
greater amygdalar gray volume than con-
trol patients without such an exposure
(Chang et al., 2005). Interestingly, the loss
of the subgenual prefrontal cortex volume
found in bipolar patients was essentially
suppressed in patients receiving protracted
lithium or VPA (Drevets, 2001).
Despite the prominent roles of lithium
and VPA in treating bipolar disorder, a sig-
adequate response to either drug. Com-
bined treatment with mood stabilizers has
been a frequently used strategy to control
bipolar syndromes resistant to mono-
therapy. One of the most efficacious and
safe mood stabilizer combinations appears
to be a mixture of lithium and anticonvul-
sants, notably VPA (for review, see Free-
man and Stoll, 1998; Lin et al., 2006). The
present study was undertaken to search for
an experimental paradigm in which the
neuroprotective actions of lithium and
VPA or other HDAC inhibitors can be dra-
matically potentiated and to determine
synergistic neuroprotection elicited by
combined drug treatment.
Primary cultures of cerebellar granule cells and drug treatment. Cerebellar
granule cells (CGCs) were prepared from 8-d-old Sprague Dawley rats
and cultured as described previously (Nonaka et al., 1998), with some
modification. Specifically, the dissociated cells were resuspended in
serum-free B27/neurobasal medium and plated at a density of 1.2 ? 106
cells/ml on 0.01% poly-L-lysine precoated plates. Cytosine arabino-
furanoside (10 ?M) was added to the cultures 24 h after plating to arrest
the growth of non-neuronal cells. Cultures were routinely pretreated
with the indicated concentrations of LiCl, sodium VPA, PB, SB, TSA, or
a combination of lithium with any of the HDAC inhibitors for indicated
times, starting from 1 or 6 d in vitro (DIV), and then exposed to 50 ?M
glutamate for 24 h to induce neurotoxicity. At the time of experimenta-
tion, ?92% of cells were CGC neurons.
Measurement of cell viability. To determine cell survival in a quantita-
tive colorimetric assay, the mitochondrial dehydrogenase activity that
reduces 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bro-
mide (MTT) was assayed (Nonaka et al., 1998). CGCs cultured on 96-
well plates were incubated with MTT (125 ?g/ml) added directly to the
formazan product was dissolved in dimethylsulfoxide and quantified
age of viability of the control culture.
Lactate dehydrogenase assay. Cell viability was also quantified with a
cytotoxicity detection kit that measures lactate dehydrogenase (LDH)
Science, Indianapolis, IN). Briefly, an aliquot of 100 ?l of culture me-
dium was taken from the CGC culture grown on a 96-well plate and
control cells and compared with LDH levels in treated cell lysates. LDH
release into the medium was expressed as a percentage of total LDH.
Analysis of chromatin condensation. Chromatin condensation was de-
six-well plates were washed with ice-cold PBS and fixed with 4% form-
aldehyde in PBS. Cells were then stained with Hoechst 33258 (5 ?g/ml)
for 5 min at 4°C. Nuclei were visualized under an inverted fluorescence
microscope at a wavelength of 360 nm.
Western blotting. CGC neurons cultured in six-well plates were de-
previously (Leng and Chuang, 2006). Protein concentration was deter-
mined with a BCA protein assay kit (Pierce, Rockford, IL). Aliquots
containing equal amounts of protein (10 ?g) from each sample were
mixed with an equal volume of SDS sample buffer, loaded into a 4–12%
Nupage Bis-Tris gel, and then subjected to electrophoresis. After separa-
which was incubated for 1 h with a primary antibody against p53 (1:
1000), GSK-3?/? antibody (1:2000), ?-catenin (1:3000) (all from Santa
Cruz Biotechnology, Santa Cruz, CA), phospho-GSK-3?Ser21/?Ser9(1:
1000, Cell Signaling, Beverly, MA), acetylated histone-H3 against both
Lys9 and Lys14 acetylation (1:3000), acetylated histone-H3 against Lys9
acetylation (1:2000), acetylated histone-H3 against Lys14 acetylation (1:
2000), HDAC1 antibody (1:1000) (all from Millipore, Temecula, CA),
phospho-TauSer400, phospho-TauThr205, and total Tau (1:1000; Invitro-
gen, Carlsbad,CA), glyceraldehyde-3-phosphate
(GAPDH) (1:5000; Advanced Immunochemical, Long Beach, CA), or
with an HRP-labeled secondary antibody (1:2000; GE Healthcare, Little
minescence on the membrane.
prepared from the cerebral cortex of 17-d-old Sprague Dawley rat em-
were dissected from embryonic brain, and the meninges were removed.
The cells were dissociated by trypsinization and trituration, followed by
neurobasal medium and plated at a density of 3 ? 105cells/cm2on
dishes precoated with 0.01% poly-L-lysine. The cells were maintained at
37°C in the presence of 5% CO2and 95% air in a humidified incubator.
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors J.Neurosci.,March5,2008 • 28(10):2576–2588 • 2577
for the determination of levels of phospho-GSK-3?Ser21/?Ser9and
GSK-3? kinase activity assay. GSK-3? activity was measured in a cell-
free system, using an immune complex kinase assay. Lysates from CGCs
at 7 DIV were prepared in lysis buffer as described previously (Leng and
Chuang, 2006). An aliquot of 100 ?g of protein extract was incubated at
4°C overnight with 2 ?g of the anti-GSK-3? antibody (1:200; BD Bio-
science, Palo Alto, CA). The immunocomplex was bound to protein G
three times with kinase assay buffer (Cell Signaling Technology). Phos-
the kinase was performed by incubation for 30 min at 37°C in 40 ?l of
reaction mixture, containing 2 ?g of substrate, 30 mM Tris-HCl, pH 7.5,
1 ?Ci [?-32P]ATP, 4 mM MgCl2, 2.5 mM ?-glycerophosphate, 1 mM
DTT, 0.05 mM Na3VO4, 40 ?g/ml bovine serum albumin, 100 ?M ATP,
and GSK-3? immunocomplex in the absence or presence of 3 mM LiCl,
0.8 mM VPA (sodium salt), or a combination of lithium and VPA. The
32P-labeled peptides were recovered on a p81 phosphocellulose paper,
Transfection of small interference RNA specific for GSK-3?, GSK-3?, or
or scrambled small interference RNA (siRNA) immediately before plat-
ing using electroporation with the Nucleofector apparatus in conjunc-
tion with a Rat Neuron Nucleofector kit (Amaxa, Gaithersburg, MD)
according to the instructions of the manufacturer. The sequence for rat
GSK-3? siRNA (?P1269) is CTTCAGTCCTGGTGAACTT, whereas
that for rat GSK-3? (?P555) is CCTCTTGCTGGATCCTGAT, as de-
scribed previously (Liang and Chuang, 2006). Rat HDAC1-targeted
siRNA was purchased from Dharmacon (Lafayette, CO). A scrambled
siRNA (Dharmacon) was used as an RNA inter-
ference control for all siRNA transfection
Transfection of GSK-3? and GSK-3? plasmid
pMT2) and a dominant-negative mutant of
GSK-3? (hGSK-3?/pMT2-KR) were obtained
from Dr. Peter S. Klein (University of Pennsyl-
vania School of Medicine, Philadelphia, PA)
with the permission of Dr. James R. Woodgett
(Ontario Cancer Institute, Toronto, Ontario,
traZeneca R & D, So ¨derta ¨lje, Sweden). Two
dominant-negative mutants (pAdTrack–CMV–
GSK-3?–K85R and pAdTrack–CMV–GSK-3?–
R96A) for GSK-3? were generated using the
QuikChange II site-directed mutagenesis kit
(Stratagene, La Jolla, CA). Transfection of
dominant-negative mutants) into CGC neurons
was conducted at the time of plating using the
Nucleofector apparatus (Amaxa), according to
the instructions of the manufacturer. The trans-
fluorescence protein (eGFP) was cotransfected
to ensure that the transfection efficiencies were
similar between drug-treated and untreated
in six-well plates for 7 d were washed once with
PBS and then buffer A (20 mM HEPES, pH 7.5,
mM EGTA, 0.5 mM DTT, 2 mM Na3VO4, 50 mM
sodium fluoride, 100 ?M phenylmethylsulfonyl
fluoride, 10 ?g/ml leupeptin, 10 ?g/ml aproti-
Cells were then lysed in buffer A with 20 strokes
using a Dounce homogenizer. Cell lysates were
collected by centrifugation (1000 ? g for 10 min) at 4°C in a microcen-
trifuge tube. The nuclear pellet was washed two times by gently resus-
phenylmethylsulfonyl fluoride, 10 ?g/ml leupeptin, 10 ?g/ml aprotinin,
wild-type Lef binding sites (Lef-OT) was a kind gift from Dr. Bert Vo-
gelstein (Johns Hopkins University, Baltimore, MD). CGCs were trans-
fected with the Lef-OT reporter construct immediately before plating
ing to the instructions of the manufacturer. The transfection efficiencies
were ?30%. eGFP and secreted alkaline phosphatase were cotransfected
to ensure that the transfection efficiencies were similar between drug-
treated and untreated cultures. Six days after transfection, cultures were
treated with the indicated concentrations of drugs for 24 h. Cells were
then harvested, lysed, and assayed for luciferase activity using the lucif-
with a luminometer (Packard Lumicount; Global Medical Instrumenta-
tion, Ramsey, MN). As a control for the luciferase basal expression, an
empty vector (promoterless plasmid) was also cotransfected, and the
relative activity of luciferase was calculated.
Lef-OT cell culture. Lef-OT cell line was a kind gift from Dr. Peter S.
Klein. The OT cell line was derived by stable transfection of human
embryonic kidney 293 (HEK 293) cells with a reporter (OT-luciferase)
containing three wild-type Lef binding sites regulating luciferase expres-
independent cultures. Note that maximal synergism was observed with 0.8 mM VPA and 3 mM LiCl. C, CGCs at 6 DIV were
with LiCl (3 mM), VPA (0.8 mM), or both for 6 d before glutamate (50 ?M) treatment for 24 h. Cells were then harvested for
Western blotting of p53. Shown are results from a typical experiment that was repeated three times. Arrows, Note that
2578 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors
sion. Cells were selected and maintained in
DMEM supplemented with 10% fetal bovine se-
rum and 0.04% G418, an antibiotic used for the
selection of eukaryotic cells stably transfected
with neomycin resistance genes. After reaching
60–70% confluency, cells were treated with the
indicated concentration of drugs and then lysed
Animals and animal treatments. All proce-
dures were performed in accordance with the
National Institutes of Health Guidelines for the
Care and Use of Laboratory Animals. Two-
month-old male CD-1 mice (20–25 g; Charles
water and food under a 12 h light/dark cycle.
After a 7 d acclimation period, mice were fed
with a chow containing bacon flavor alone, ba-
con lithium carbonate (3 g/kg), bacon sodium
VPA (25 g/kg), or a combination of bacon lith-
ium carbonate and sodium VPA. The control
and drug-containing chows were purchased
from Bio-Serv (Frenchtown, NJ). These doses of
lithium and VPA were chosen because they pro-
duced serum drug levels within therapeutic con-
centrations (Einat et al., 2003). Mice were killed
after dietary treatment for 20 d. The brains were
removed and dissected, followed by homogeni-
zation and sonication for 40 s in lysis buffer as
described previously (Leng and Chuang, 2006).
An aliquot of 15 ?g of total protein extract was
used for Western blot detection.
Statistical analyses. Data are expressed as
means ? SEM from at least three independent
experiments. Statistical significance was ana-
lyzed by one-way ANOVA and the Bonferroni’s
post hoc test. A p value of ?0.05 was considered
tures. We thus compared the vulnerability
of young versus aging CGC cultures to glu-
tamate and their responsiveness to lithium
and VPA pretreatment. Young CGCs were
pretreated with various concentrations
and then exposed to 50 ?M glutamate for
24 h (7 to 8 DIV) to induce excitotoxic
death. Glutamate exposure induced ?50%
neuronal death as measured by MTT assay,
and this excitotoxicity, which was NMDA
receptor-mediated, was prevented by lith-
ium pretreatment in a concentration-
dependent manner (Fig. 1A). However,
pretreatment of aging CGCs with lithium
(from 6 to 12 DIV) failed to effectively pro-
tect against 50 ?M glutamate (24 h, 12–13
(?80%) than that observed in young cul-
and examined under an inverted fluorescence microscope. Scale bar, 30 ?m. B, MTT staining. Experimental conditions are as
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors J.Neurosci.,March5,2008 • 28(10):2576–2588 • 2579
roprotection was found with VPA in the
concentration range examined (0.1–0.8
mM) (Fig. 1B). Because the maturing cul-
tures (7–8 DIV) were more resistant than
the aging cultures
glutamate-induced insult, we examined
whether the neuroprotection elicited by
atively weak excitotoxicity (Fig. 1C). The
pressing excitotoxicity elicited by gluta-
mate in a wide concentration range (50–
800 ?M) in the young cultures.
ing preincubation of CGCs from 6 to 12
DIV caused a concentration-dependent
the cell viability at 1, 2, and 3 mM lithium,
respectively (Fig. 2A). Similarly, the pres-
incubation resulted in a dose-dependent
synergistic neuroprotection that reached a
complete rescue at 0.8 mM (Fig. 2B). Mea-
surement of cell death by LDH release con-
ited by cotreatment with lithium (3 mM)
showed that glutamate-induced excitotoxicity in CGCs involves
p53 upregulation (Chen and Chuang, 1999; Chen et al., 2003).
The glutamate-induced increase of p53 protein level was unaf-
fected by treatment with either lithium or VPA alone but was
blocked by their cotreatment (Fig. 2D).
CGCs were pretreated with lithium (3 mM) or VPA (0.8 mM)
either alone or in combination from 6 to 12 DIV, followed by
glutamate (50 ?M) exposure for 24 h. When viable cells were
detected by incubation with calcein-AM, little or no neuropro-
tection was elicited by either lithium or VPA; however, we ob-
nal cell bodies and processes (Fig. 3B). When Hoechst 33258 dye
was used to identify neurons undergoing chromatin condensa-
glutamate-induced apoptosis was also evident (Fig. 3C).
GSK-3 is an evolutionary conserved kinase consisting of ? and ?
teins, notably transcription factors (for review, see Grimes and
Jope, 2001). Lithium inhibits GSK-3 activity by binding to the
magnesium-sensitive active site of the enzyme (Klein and Mel-
ton, 1996; Stambolic et al., 1996) and also indirectly through
inducing phosphorylation of GSK-3?Ser21and GSK-3?Ser9
(Chalecka-Franaszek and Chuang, 1999; De Sarno et al., 2002;
Zhang et al., 2003). Emerging evidence supports the idea that
GSK-3 inhibition is involved in the neuroprotective effects of
lithium (Bhat et al., 2000; De Sarno et al., 2002; Li et al., 2002;
Phiel et al., 2003; Liang and Chuang, 2007). Therefore, we first
examined the effects of VPA on levels of lithium-induced GSK-
3?/? serine phosphorylation (Fig. 4). Levels of phospho-GSK-
3?Ser9, the predominant phosphorylated GSK-3 isoform present
in CGCs, were markedly enhanced by lithium treatment for 30
GSK-3?Ser9phosphorylation levels but did potentiate lithium-
points. Relatively weak levels of phospho-GSK-3?Ser21were also
detected in the untreated conditions, which were enhanced by
lithium treatment. Similar to the effects on GSK-3?Ser9phos-
phorylation, the copresence of VPA potentiated the effects of
Because GSK-3 is a major kinase that phosphorylates the cy-
toskeletal protein Tau (Hong et al., 1997), the effects of lithium
and/or VPA on Ser/Thr phosphorylation levels of Tau were also
examined. Treatment with lithium or VPA decreased levels of
both drugs decreased the phosphorylation levels by almost 90%
(Fig. 5A,B). Cotreatment with lithium and VPA also reduced
phospho-TauThr205levels more than either drug alone. In rat
ing from 6 DIV also increased phospo-GSK-3?Ser21/?Ser9and
decreased phospho-TauSer400more than either drug alone (Fig.
2580 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors
immunoprecipitated from CGC lysates using an anti-GSK-3?
antibody, and the immunocomplex was assayed for the ability of
GSK-3? to phosphorylate its protein substrate. The GSK-3? en-
zymatic activity was inhibited ?40% by lithium and ?20% by
VPA, whereas the copresence of lithium and VPA inhibited the
activity by ?70% (Fig. 5D).
tamate excitotoxicity in aging CGCs, we first examined whether
this inability is related to the ineffectiveness of lithium to inhibit
GSK-3. CGCs at 3, 6, or 13 DIV were treated with 3 mM LiCl for
30 min, and then levels of phospho-GSK-3?Ser9were measured.
culture-time-dependent manner (Fig. 6A,B). These results are
reminiscent of age-induced dephosphorylation of phospho-
suggest an increase in GSK-3? activity. Moreover, lithium-
induced elevation of phospho-GSK-3?Ser9also diminished with
the age of the culture, notably from 6 to 13 DIV. The dose re-
neuroprotection in aging CGCs were also studied. The optimal
dose of lithium to increase phospho-GSK-3?Ser9and to rescue
neurons from glutamate excitotoxicity remained at 3 mM (Fig.
6C–E), thus indicating that the decreased response of GSK-3 to
lithium cannot be overcome by increasing
the concentration of this drug.
Although GSK-3 is likely the major tar-
get of lithium to elicit neuroprotection,
other targets of this drug have also been
suggested for other actions (for review, see
Gould et al., 2004; Chuang and Priller,
2006; Rowe et al., 2007). To provide addi-
tional evidence that synergistic neuropro-
tection provided by lithium and VPA is re-
lated to GSK-3 inhibition, CGCs were
transfected with GSK-3? siRNA and/or
GSK-3? siRNA before plating, followed by
at 6 DIV. Our previous study has shown
that these siRNAs for GSK-3? and GSK-3?
are isoform specific in silencing their ex-
pression in rat cortical neurons (Liang and
Chuang, 2007). Consistent with these re-
sults, transfection of CGCs with a mixture
of GSK-3? siRNA and GSK-3? siRNA
of both GSK-3 isoforms measured 6 d later
(Fig. 7A). Treatment with either GSK-3?
siRNA or GSK-3? siRNA alone failed to
show neuroprotection against glutamate
to GSK-3? siRNA or GSK-3? siRNA fol-
lowed by VPA treatment caused a signifi-
cant increase in cell viability from ?20 to
scrambled siRNA, used as a control, was
ineffective. In CGC cultures transfected
with a mixture of GSK-3? siRNA and
GSK-3? siRNA in conjunction with VPA
treatment, an additional increase in cell vi-
the synergistic effects of lithium and VPA in neuroprotection.
Similar results were observed when a dominant-negative mutant
of GSK-3?, pMT2-KR, was used to replace GSK-3? siRNA and a
dominant-negative mutant of GSK-3?, K85R, was used to re-
place GSK-3? siRNA (data not shown). In another experiment,
the synergistic neuroprotective effects of lithium and VPA were
found to be reduced by overexpressing wild-type GSK-3? or
GSK-3? but, as expected, unaffected by overexpression of a
dominant-negative mutant of GSK-3?, pMT2-KR, or two
dominant-negative mutants of GSK-3?, K85R and R96A (Fig.
7C). Neither wild-type nor dominant-negative mutants of
GSK-3? and GSK-3? alone significantly affected glutamate-
induced neuronal death. Together, these results lend additional
support to the view that GSK-3 is a molecular target for lithium
and VPA to induce synergy in their neuroprotective actions.
Because VPA is an inhibitor of HDAC, we attempted to investi-
gate whether the synergy in neuroprotection is related to this
ability. Our recent study showed that silencing the expression of
HDAC1 isoform with its specific siRNA mimics the ability of
VPA to activate BDNF promoter IV in rat cortical neurons (Ya-
suda et al., 2008). Therefore, we first tested the effects of HDAC1
siRNA on neuroprotection against glutamate excitotoxicity.
three independent experiments) are in B. *p ? 0.05, **p ? 0.01 between the indicated groups. Note that p-TauSer400,
7 DIV were immunoprecipitated with anti-GSK-3? antibody. The immunocomplex was then assayed for GSK-3? enzymatic
activity in the absence or presence of LiCl (3 mM), VPA (0.8 mM), or a combination of both. The data are means ? SEM of
percentage of control from three independent experiments. *p ? 0.05, **p ? 0.01, ***p ? 0.001 between the indicated
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitorsJ.Neurosci.,March5,2008 • 28(10):2576–2588 • 2581
Transfection of CGCs with HDAC1 siRNA be-
fore plating resulted in a marked decrease in
HDAC1 protein level measured 6 d later (Fig.
conjunction with subsequent lithium treatment
caused robust neuroprotection. In contrast,
no effect. Next, we examined the effects of PB
and SB, which are fatty acid derivatives that are
structurally similar to VPA. Reminiscent to the
effects of VPA, preincubation with PB or SB
CGCs against glutamate excitotoxicity (Fig.
8C,D). More importantly, preincubation with
protected CGCs from excitotoxicity. Like VPA,
PB and SB did not change GSK-3?Ser21/?Ser9
phosphorylation levels but potentiated lithium-
induced serine phosphorylation levels of GSK-
3?/?, after a 1 d treatment (Fig. 8E–H). Similar
synergism in neuroprotection and potentiation
in lithium-induced GSK-3?Ser21/?Ser9phos-
phorylation was observed when TSA, a hydrox-
amate, was used as an HDAC inhibitor during
pretreatment (Fig. 9A–C).
and hence stabilization of this transcription fac-
tor (Hedgepeth et al., 1997). ?-Catenin then ac-
cumulates in the cytoplasm and translocates to
the nucleus with another transcription factor
Lef/Tcf to activate transcription (Barker et al.,
2000). Therefore, we investigated the effects of
scription. CGCs were transfected by electropo-
ration with a firefly luciferase reporter contain-
ing three wild-type Lef binding sites (Lef-OT)
before plating and then treated with lithium (3 mM) and/or VPA
VPA alone resulted in a fivefold to sevenfold increase in Lef-
elicited a more than additive increase in the activation of Lef-
ment. As expected, incubation with lithium (3 mM) for 1 d en-
hanced nuclear ?-catenin levels, and this effect was enhanced by
cotreatment with VPA (Fig. 10B).
inhibitors. Treatment of HEK 293 cells with lithium for 24 h
caused a concentration-dependent increase in Lef-luciferase ac-
tivity with a ?30-fold increase at 20 mM (Fig. 11A). Incubation
with VPA also induced a similar dose-dependent increase in Lef-
luciferase activity, and the presence of both VPA and lithium
together at equal concentrations triggered a synergy (up to 170-
fold) in Lef-dependent transcription (Fig. 11B), reminiscent of
the previously reported results (Phiel et al., 2001). We further
expanded the above results to show that similar synergism with
lithium occurred when other HDAC inhibitors (PB and TSA)
were used in combination with lithium during incubation (Fig.
11C,D). Together, these results further suggest that the GSK-3-
mediated ?-catenin/Lef signaling cascade contributes to the syn-
ergism in neuroprotection elicited by cotreatment with lithium
and an HDAC inhibitor.
Given that HDAC is a target of VPA, we also examined whether
VPA or other HDAC inhibitors. Treatment of CGCs with VPA
for 24 h (from 6 to 7 DIV) induced a marked increase in levels of
histone-H3 Lys9 and Lys14 acetylation, suggesting HDAC inhi-
bition (Fig. 12A). Although lithium by itself failed to change
histone Lys9 and Lys14 acetylation levels, it slightly enhanced
VPA-induced histone-H3 acetylation, when used in combina-
tion. Qualitatively similar results were obtained when levels of
CGCs at 3, 6, or 13 DIV were treated with vehicle or 3 mM LiCl for 30 min and then harvested for Western blotting of
SEM from 4 independent cultures) are shown in B. **p ? 0.01 between the indicated groups. Note that basal and
2582 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors
histone acetylation at either Lys9 or Lys14 were separately mea-
tion at either residue. Similar slight potentiation of histone acet-
ylation was observed when other HDAC inhibitors (SB, PB, and
TSA) were used in conjunction with lithium (Fig. 12B–D).
To assess the effects of mood stabilizers in vivo, mice were sub-
jected to dietary treatment with lithium carbonate (3 g/kg)
that lithium markedly increased the levels of phospho-GSK-
3?Ser9in the lysate of the frontal cortex (Fig. 13A,C). VPA treat-
ment also significantly increased cortical phospho-GSK-3?Ser9
VPA elicited a more than additive effect. Similar potentiation of
GSK-3?Ser9phosphorylation was observed in the cerebellum of
these mice treated with lithium and/or VPA (Fig. 13B,D).
In this study, we showed for the first time that cotreatment of
effects in a time-dependent manner. Thus, pretreatment with
either drug alone provided little or no neuroprotection against
glutamate-induced excitotoxicity in aging CGCs, whereas their
copresence produced a complete blockade of glutamate-induced
neuronal death, as assessed biochemically and morphologically.
Our results demonstrated that potentiation of GSK-3 inhibition
is closely associated with the synergy of neuroprotection. For
example, the presence of VPA enhanced lithium-induced in-
crease in GSK-3?Ser21/?Ser9phosphorylation in both CGCs and
cortical neurons. Lithium and VPA cotreatment caused an addi-
tive decrease in GSK-3 activity in CGCs, as assayed by TauSer400
and TauThr205phosphorylation levels in intact cells and GSK-3?
enzymatic activity in a cell-free system. Furthermore, chronic
resulted in an additive increase in phospho-GSK-3?Ser21/?Ser9
that GSK-3 inhibition has a neuroprotective role and is most
likely a major mechanism responsible for the neuroprotective
2004; Liang and Chuang, 2007). Thus, potentiation of GSK-3
inhibition by the combination of lithium and VPA treatment
appears to be an important target involved in the synergism of
neuroprotection. This notion is further supported by our obser-
vation that transfection with siRNAs or dominant-negative mu-
tants for GSK-3? and/or GSK-3? mimicked the synergistic neu-
roprotection of lithium with VPA and that overexpression of
wild-type GSK-3? suppressed the synergistic effects of these
Research on the effects of VPA on GSK-3 activity has gener-
ated inconsistent results. VPA inhibits GSK-3 through an in-
neuroblastoma cells overexpressing GSK-3?, as assessed by Tau
phosphorylation (Grimes and Jope, 2001). In contrast, other
studies suggested that this drug fails to affect GSK-3 activity
(Phiel et al., 2001; Hall et al., 2002; Eickholt et al., 2005; Jin et al.,
2005). In CGCs, VPA alone did inhibit GSK-3 activity, although
serine phosphorylation, in contrast to an increase in the phos-
phorylation found in the mouse brain after VPA dietary treat-
ment. Together, these results suggest that the effects of VPA on
the GSK-3 activity and the regulatory mechanisms, such as its
serine phosphorylation, depend on the experimental paradigms
and conditions used.
induced excitotoxicity. Cells were transfected with 100 nM siRNA specific to either GSK-3?
Western blotting revealed that protein levels of both GSK-3? and GSK-3? were markedly
decreased 6 d after transfection with a mixture of siRNA for GSK-3? and siRNA for GSK-3?
independent experiments. *p ? 0.05, **p ? 0.01, ***p ? 0.001 between the indicated
with a plasmid of wild-type GSK-3? (wt-GSK-3?), GSK-3? dominant-negative mutant (dn-
GSK3?-pMT2-KR), wild-type GSK-3? (wt-GSK-3?), or GSK-3? dominant-negative mutant
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors J.Neurosci.,March5,2008 • 28(10):2576–2588 • 2583
Transfection of CGCs with HDAC1
siRNA mimicked the ability of HDAC in-
hibitors to synergistically protect neurons
in the presence of lithium, suggesting that
potentiation of lithium-induced GSK-3
serine phosphorylation and neuroprotec-
tion by VPA is also mimicked by lithium
HDAC inhibitors PB and SB and structur-
ally dissimilar TSA, a hydroxamate. These
results suggest that the potentiation is a
common feature for all HDAC inhibitors
of the initial actions of VPA and other
HDAC inhibitors. Cotreatment of HDAC
inhibitors with lithium appeared to have
only slight effects on HDAC inhibitor-
induced histone hyperacetylation. How-
ever, given that Western blotting measures
genome-wide levels of histone acetylation
in bulk chromatin and that HDAC inhibi-
tors are likely to affect a small fraction of
nucleosomes to regulate gene transcrip-
tion, additional experiments are necessary
to address the issue regarding the roles of
HDAC-regulated genes in mediating the
neuroprotective synergy. Both the nuclear
levels of ?-catenin protein and ?-catenin-
Lef/Tcf-mediated transcriptional activation
were enhanced by the combined treatment
with lithium and VPA. Moreover, cotreat-
ment with lithium and VPA or other HDAC
inhibitors, both at relatively high concentra-
tions, caused a robust synergism in Lef/Tcf-
dependent transcription in HEK 293 cells. It
requires additional investigation as to
ity, and Lef/Tcf transcriptional activity
requires the intrinsic activity of VPA to
that GSK-3 inhibition is related to the
antidepressant-like and antimania-like ef-
fects in rodent models. For example, treat-
ment with a GSK-3 peptide inhibitor or
mice shows antidepressant-like effects in a
forced swim test (Kaidanovich-Beilin et al., 2004; O’Brien et al.,
produces antidepressant-like behavior in rats (Gould et al.,
2004). In addition, the same GSK-3 inhibitor reduces
amphetamine-induced hyperactivity in rats, a mania-like behav-
ior (Gould et al., 2004). Reducing GSK-3 activity by pharmaco-
logical inhibition or genetic deletion has also been reported to
suppress dopamine-dependent locomotor hyperactivity (Beau-
lieu et al., 2004).
decrease in the density of glial cells and neurons, as well as neu-
ronal atrophy in the prefrontal, orbital, and cingulate cortices,
(for review, see Manji et al., 2003). Structural imaging studies
showed a reduction in gray matter volume in orbital and medial
prefrontal cortices, ventral striatum, and hippocampus as well as
suppress the loss of gray matter volume in the prefrontal cortex
and amygdala compared with patients receiving no such treat-
suggest that neuroprotective and neurotrophic effects of mood
stabilizers, most likely through GSK-3 inhibition, enhance cellu-
lar resilience and plasticity and, in turn, contribute to their clin-
ical efficacy (for review, see Manji et al., 2001; Bachmann et al.,
transfected with 100 nM siRNA specific to rat HDAC1 isoform (siHDAC1) before plating by electroporation. Scrambled siRNA
ylation in CGCs treated with lithium and/or PB. H, Quantified results of GSK-3? and GSK-3? serine phosphorylation in CGCs
2584 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors
2005), although additional longitudinal clinical studies are re-
quired to firmly establish this linkage.
Combined treatment with lithium and VPA has been used
frequently to treat bipolar disorder resistant to monotherapy
with either drug (for review, see Lin et al., 2006). It is recognized
that lithium and VPA combination is the first-line treatment for
some bipolar subtypes such as those characterized by rapid cy-
cling or mixed episodes and substance abuse comorbidity (for
review, see Lin et al., 2006). Bipolar patients receiving a lithium-
VPA cotherapy also seem to be less likely to develop a relapse
compared with patients receiving lithium monotherapy (for re-
and its receptor hyperactivity have been suggested to be a critical
factor involved in the pathogenesis of stress-related depression
(for review, see Zarate et al., 2003). Thus, synergistic neuropro-
tective effects against glutamate excitotoxicity elicited by lithium
that the combination of lithium and VPA reportedly causes an
C kinase substrate, a potential molecular target of mood stabiliz-
ers (Lenox et al., 1996). Several atypical antipsychotic drugs have
also been used in conjunction with lithium or VPA to maximize
the clinical efficacy for bipolar disorder (for review, see Freeman
and Stoll, 1998; Zarate and Quiroz, 2003; Bachmann et al., 2005;
the synergy in neuroprotection can be elicited by antipsychotic
drug cotreatment with lithium/VPA.
Glutamate-induced excitotoxicity has been implicated in
ischemic stroke and many neurodegenerative diseases, including
Alzheimer’s disease, Huntington’s disease, amyotrophic lateral
kinson’s disease, and cerebellar degeneration (for review, see
Chuang, 2004b; Chuang and Priller, 2006). Pre- or post-insult
Kim et al., 2007). These two drugs also exhibit neuroprotective
effects in animal models of a variety of neurodegenerative dis-
eases from several independent studies (for review, see Tariot et
al., 2002; Chuang and Priller, 2006). Notably, lithium, by acting
on GSK-3, decreases the production of ?-amyloid peptide from
amyloid precursor protein (APP) (Phiel et al., 2003), lowers the
phosphorylation and aggregation of Tau protein (Noble et al.,
2005), and provides neuroprotective effects in APP transgenic
mice (Rockenstein et al., 2007). HDAC inhibitors have also been
reporter construct before plating, using an Amaxa Nucleofector. At 6 DIV, CGCs were treated
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitorsJ.Neurosci.,March5,2008 • 28(10):2576–2588 • 2585
shown to enhance learning, memory, and synaptic plasticity
through chromatin remodeling and cAMP response element-
scriptional activation (Fischer et al., 2007; Vecsey et al., 2007). In
light of the novel findings in the present study, it may be sug-
gested that combined use of lithium and VPA or other HDAC
inhibitors may be a rational strategy in clinical trials for neuro-
degenerative diseases, particularly those linked to glutamate
robust synergistic neuroprotective effects
against glutamate excitotoxicity when the
mood stabilizers lithium and VPA were
used in combination. We also provided ev-
idence that GSK-3 inhibition was corre-
lated with the synergy of neuroprotection
elicited by these two drugs. Our findings
have implications in the combined use of
both drugs in treating bipolar disorder
and further suggest potential utility of a
combination of lithium and HDAC in-
hibitors in intervening glutamate-related
(2005) Mood stabilizers target cellular plastic-
ity and resilience cascades: implications for the
Barker N, Morin PJ, Clevers H (2000) The yin-
yang of TCF/?-catenin signaling. Adv Cancer
Beaulieu JM, Sotnikova TD, Yao WD, Kockeritz L,
Woodgett JR, Gainetdinov RR, Caron MG
dependent behaviors mediated by an AKT/gly-
cogen synthase kinase 3 signaling cascade. Proc
Natl Acad Sci USA 101:5099–5104.
Bhat RV, Shanley J, Correll MP, Fieles WE, Keith
RA,ScottCW,LeeCM (2000) Regulationand
localization of tyrosine216 phosphorylation of
glycogen synthase kinase-3? in cellular and an-
imal models of neuronal degeneration. Proc Natl Acad Sci USA
BijurGN,JopeRS (2003) Glycogensynthasekinase-3?ishighlyactivatedin
nuclei and mitochondria. NeuroReport 14:2415–2419.
Chalecka-Franaszek E, Chuang DM (1999) Lithium activates the serine/
threonine kinase Akt-1 and suppresses glutamate-induced inhibition of
Akt-1 activity in neurons. Proc Natl Acad Sci USA 96:8745–8750.
Chang K, Barnea-Goraly N, Karchemskiy A, Simeonova DI, Barnes P, Ketter
T, Reiss AL (2005) Cortical magnetic resonance imaging findings in fa-
milial pediatric bipolar disorder. Biol Psychiatry 58:197–203.
Chen G, Huang LD, Jiang YM, Manji HK (1999) The mood-stabilizing
agent valproate inhibits the activity of glycogen synthase kinase-3. J Neu-
Chen G, Bower KA, Ma CL, Fang SY, Thiele CJ, Luo J (2004) Glycogen
ronal death. FASEB J 18:1162–1164.
ChenRW,ChuangDM (1999) Longtermlithiumtreatmentsuppressesp53
and Bax expression but increases Bcl-2 expression: a prominent role in
neuroprotection against excitotoxicity. J Biol Chem 274:6039–6042.
DM (2003) Regulationofc-JunN-terminalkinase,p38kinaseandAP-1
and lithium neuroprotection. J Neurochem 84:566–575.
Chuang DM (2004a) Neuroprotective and neurotrophic actions of the
mood stabilizer lithium: can it be used to treat neurodegenerative dis-
eases? Crit Rev Neurobiol 16:83–90.
ChuangDM (2004b) Lithiumneuroprotectionfromglutamateexcitotoxic-
rosci Res 4:243–252.
Chuang DM, Manji HK (2007) In search of the holy grail for the treatment
Chuang DM, Priller J (2006) Potential use of lithium in neurodegenerative
disorders. In: Lithium in neuropsychiatry: the comprehensive guide
(Bauer M, Grof P, Mu ¨ller-Oerlingausen B, eds), pp 381–397. Abington,
UK: Taylor and Francis.
2586 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors
De Sarno P, Li XH, Richard JS (2002) Regulation of Akt and glycogen syn-
Doble BW, Woodgett JR (2003) GSK-3: tricks of the trade for a multi-
tasking kinase. J Cell Sci 116:1175–1186.
Drevets WC (2001) Neuroimaging and neuropathological studies of de-
pression: implications for the cognitive-emotional features of mood dis-
orders. Curr Opin Neurobiol 11:240–249.
Eickholt BJ, Towers GJ, Ryves WJ, Eikel D, Adley K, Ylinen LM, Chadborn
NH, Harwood AJ, Nau H, Williams RS (2005) Effects of valproic acid
derivatives on inositol trisphosphate depletion, teratogenicity, glycogen
for new bipolar disorder drugs derived from the valproic acid core struc-
ture. Mol Pharmacol 67:1426–1433.
Einat H, Yuan P, Gould TD, Li J, Du J, Zhang L, Manji HK, Chen G (2003)
The role of the extracellular signal-regulated kinase signaling pathway in
mood modulation. J Neurosci 23:7311–7316.
Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai LH (2007) Recovery of
learning and memory is associated with chromatin remodelling. Nature
Freeman MP, Stoll AL (1998) Mood stabilizer combinations: a review of
safety and efficacy. Am J Psychiatry 155:12–21.
Go ¨ttlicherM,MinucciS,ZhuP,Kra ¨merOH,SchimpfA,GiavaraS,Sleeman
JP, Lo Coco F, Nervi C, Pelicci PG, Heinzel T (2001) Valproic acid de-
fines a novel class of HDAC inhibitors inducing differentiation of trans-
formed cells. EMBO J 20:6969–6978.
Gould TD, Quiroz JA, Singh J, Zarate CA, Manji HK (2004) Emerging ex-
and cellular actions of current mood stabilizers. Mol Psychiatry
Grimes CA, Jope RS (2001) The multifaceted roles of glycogen synthase
kinase 3? in cellular signaling. Prog Neurobiol 65:391–426.
Hall AC, Brennan A, Goold RG, Cleverley K, Lucas FR, Gordon-Weeks PR,
Salinas PC (2002) Valproate regulates GSK-3-mediated axonal remod-
eling and synapsin I clustering in developing neurons. Mol Cell Neurosci
Hashimoto R, Hough C, Nakazawa T, Yamamoto T, Chuang DM (2002)
Lithium protection against glutamate excitotoxicity in rat cerebral corti-
cal neurons: involvement of NMDA receptor inhibition possibly by de-
creasing NR2B tyrosine phosphorylation. J Neurochem 80:589–597.
Hedgepeth CM, Conrad LJ, Zhang J, Huang HC, Lee VM, Klein PS (1997)
Activation of the Wnt signaling pathway: a molecular mechanism for
lithium action. Dev Biol 185:82–91.
Hong M, Chen DCR, Klein PS, Lee VMY (1997) Lithium reduces tau phos-
phorylation by inhibition of glycogen synthase kinase-3. J Biol Chem
JinN,KovacsAD,SuiZ,DewhurstS,MaggirwarSB (2005) Oppositeeffects
gen synthase kinase-3 activation, c-Jun expression and neuronal cell
death. Neuropharmacology 48:576–583.
Kaidanovich-Beilin O, Milman A, Weizman A, Pick CG, Eldar-Finkelman H
(2004) Rapid antidepressive-like activity of specific glycogen synthase
Kim AJ, Shi Y, Austin RC, Werstuck GH (2005) Valproate protects cells
from ER stress-induced lipid accumulation and apoptosis by inhibiting
glycogen synthase kinase-3. J Cell Sci 118:89–99.
Kim HJ, Rowe M, Ren M, Hong JS, Chen PS, Chuang DM (2007) HDAC
inhibitors exhibit anti-inflammatory and neuroprotective effects in a rat
permanent ischemic model of stroke: multiple mechanisms of action.
J Pharmacol Exp Ther 321:892–901.
Klein PS, Melton DA (1996) Molecular mechanism for the effect of lithium
on development. Proc Natl Acad Sci USA 93:8455–8459.
Kozlovsky N, Amar S, Belmaker RH, Agam G (2006) Psychotropic drugs
affect Ser9-phosphorylated GSK-3? protein levels in rodent frontal cor-
tex. Int J Neuropsychopharmacol 9:337–342.
Langley B, Gensert JM, Beal MF, Ratan RR (2005) Remodeling chromatin
and stress resistance in the central nervous system: histone deacetylase
inhibitors as novel and broadly effective neuroprotective agents. Curr
Drug Targets CNS Neurol Disord 4:41–50.
LengY,ChuangDM (2006) Endogenous?-synucleinispotentlyinducedby
valproate and participates in the neuroprotection against glutamate exci-
totoxicity. J Neurosci 26:7502–7512.
Lenox RH, McNamara RK, Watterson JM, Watson DG (1996) Myristoy-
lated alanine-rich C kinase substrate (MARCKS): a molecular target for
the therapeutic action of mood stabilizers in the brain? J Clin Psychiatry
Li X, Bijur GN, Jope RS (2002) Glycogen synthase kinase-3?, mood stabi-
lizers, and neuroprotection. Bipolar Disord 4:137–144.
Liang MH, Chuang DM (2006) Differential roles of glycogen synthase
kinase-3 isoforms in the regulation of transcriptional activation. J Biol
Liang MH, Chuang DM (2007) Regulation and function of glycogen syn-
Lin D, Mok H, Yatham LN (2006) Polytherapy in bipolar disorder. CNS
Liu F, Iqbal K, Grundke-Iqbal I, Gong CX (2002) Involvement of aberrant
glycosylation in phosphorylation of tau by cdk5 and GSK-3?. FEBS Lett
Manji HK, Drevets WC, Charney DS (2001) The cellular neurobiology of
depression. Nat Med 7:541–547.
Manji HK, Quiroz JA, Sporn J, Payne JL, Denicoff K, Gray NA, Zarate CA,
CharneyDS (2003) Enhancingneuronalplasticityandcellularresilience
to develop novel, improved therapeutics for difficult-to-treat depression.
Biol Psychiatry 53:707–742.
Moore GJ, Bebchuk JM, Wilds IB, Chen G, Manji HK (2000a) Lithium-
induced increase in human brain grey matter. Lancet 356:1241–1242.
Moore GJ, Bebchuk JM, Hasanat K, Chen G, Seraji-Bozorgzad N, Wilds IB,
Faulk MW, Koch S, Glitz DA, Jolkovsky L, Manji HK (2000b) Lithium
increases N-acetyl-aspartate in the human brain: in vivo evidence in sup-
port of bcl-2’s neurotrophic effects? Biol Psychiatry 48:1–8.
Noble W, Planel E, Zehr C, Olm V, Meyerson J, Suleman F, Gaynor K, Wang
L, LaFrancois J, Feinstein B, Burns M, Krishnamurthy P, Wen Y, Bhat R,
Lewis J, Dickson D, Duff K (2005) Inhibition of glycogen synthase
kinase-3 by lithium correlates with reduced tauopathy and degeneration
in vivo. Proc Natl Acad Sci USA 102:6990–6995.
the frontal cortex (A) and cerebellum (B). Each lane represents the result of an individual
Effects of chronic treatment of mice with lithium and/or VPA on levels of
Lengetal.•SynergisticEffectsofLithiumandHDACInhibitorsJ.Neurosci.,March5,2008 • 28(10):2576–2588 • 2587
Nonaka S, Chuang DM (1998) Neuroprotective effects of chronic lithium
on focal cerebral ischemia in rats. NeuroReport 9:2081–2084.
Nonaka S, Hough CJ, Chuang DM (1998) Chronic lithium treatment ro-
bustly protects neurons in the central nervous system against excitotox-
icity by inhibiting N-methyl-D-aspartate receptor-mediated calcium in-
flux. Proc Natl Acad Sci USA 95:2642–2647.
(2004) Glycogen synthase kinase-3? haploinsufficiency mimics the be-
havioral and molecular effects of lithium. J Neurosci 24:6791–6798.
Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS (2001)
sant, mood stabilizer, and teratogen. J Biol Chem 276:36734–36741.
Phiel CJ, Wilson CA, Lee VMY, Klein PS (2003) GSK-3? regulates produc-
tion of Alzheimer’s disease amyloid-? peptides. Nature 423:435–439.
RenM,SenatorovVV,ChenRW,ChuangDM (2003) Post-insulttreatment
with lithium reduces brain damage and facilitates neurological recovery
in a rat ischemia/reperfusion model. Proc Natl Acad Sci USA
Ren M, Leng Y, Jeong MR, Leeds PR, Chuang DM (2004) Valproic acid
potential roles of histone deacetylase inhibition and heat shock protein
induction. J Neurochem 89:1358–1367.
Masliah E (2007) Neuroprotective effects of regulators of the glycogen
synthase kinase-3? signaling pathway in a transgenic model of Alzhei-
mer’s disease are associated with reduced amyloid precursor protein
phosphorylation. J Neurosci 27:1981–1991.
Rowe MK, Wiest C, Chuang DM (2007) GSK-3 is a viable potential target
for therapeutic intervention in bipolar disorder. Neurosci Biobehav Rev
Solomon DA, Keitner GI, Ryan CE, Miller IW (1998) Lithium plus val-
J Clin Psychopharmacol 18:38–49.
Stambolic V, Ruel L, Woodgett JR (1996) Lithium inhibits glycogen syn-
Tariot PN, Loy R, Ryan JM, Porsteinsson A, Ismail S (2002) Mood stabiliz-
ers in Alzheimer’s disease: symptomatic and neuroprotective rationales.
Adv Drug Deliv Rev 54:1567–1577.
Vecsey CG, Hawk JD, Lattal KM, Stein JM, Fabian SA, Attner MA, Cabrera
SM, McDonough CB, Brindle PK, Abel T, Wood MA (2007) Histone
CBP-dependent transcriptional activation. J Neurosci 27:6128–6140.
Xu JH, Culman J, Blume A, Brecht S, Gohlke P (2003) Chronic treatment
with a low dose of lithium protects the brain against ischemic injury by
reducing apoptotic death. Stroke 34:1287–1292.
Yasuda S, Liang MH, Marinova Z, Yahyavi A, Chuang DM (2008) The
brain-derived neurotrophic factor in neurons. Mol Psychiatry, in press.
Zarate CA, Quiroz JA (2003) Combination treatment in bipolar disorder: a
review of controlled trials. Bipolar Disord 5:217–225.
HK (2003) Regulation of cellular plasticity cascades in the pathophysi-
ology and treatment of mood disorders: role of the glutamatergic system.
Ann NY Acad Sci 1003:273–291.
Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS (2003) Inhibitory phos-
phorylation of glycogen synthase kinase-3 (GSK-3) in response to lith-
ium: evidencefor autoregulation
2588 • J.Neurosci.,March5,2008 • 28(10):2576–2588Lengetal.•SynergisticEffectsofLithiumandHDACInhibitors