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.
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