From the Section on Molecular Neurobiology, Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20892-1272
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
This study was undertaken to investigate the
molecular mechanisms underlying the neuroprotective actions of lithium
against glutamate excitotoxicity with a focus on the role of
proapoptotic and antiapoptotic genes. Long term, but not acute,
treatment of cultured cerebellar granule cells with LiCl induces a
concentration-dependent decrease in mRNA and protein
levels of proapoptotic p53 and Bax; conversely, mRNA and protein
levels of cytoprotective Bcl-2 are remarkably increased. The ratios of
Bcl-2/Bax protein levels increase by approximately 5-fold after lithium
treatment for 5-7 days. Exposure of cerebellar granule cells to
glutamate induces a rapid increase in p53 and Bax mRNA and protein
levels with no apparent effect on Bcl-2 expression. Pretreatment
with LiCl for 7 days prevents glutamate-induced increase in p53 and Bax
expression and maintains Bcl-2 in an elevated state. Glutamate exposure
also triggers the release of cytochrome c from the
mitochondria into the cytosol. Lithium pretreatment blocks
glutamate-induced cytochrome c release and cleavage of
lamin B1, a nuclear substrate for caspase-3. These results strongly
suggest that lithium-induced Bcl-2 up-regulation and p53 and Bax
down-regulation play a prominent role in neuroprotection against
excitotoxicity. Our results further suggest that lithium, in addition
to its use in the treatment of bipolar depressive illness, may have an
expanded use in the intervention of neurodegeneration.
For almost half a century, lithium has been the most widely used
treatment for bipolar depressive illness, although its therapeutic mechanism of action remains obscure. Among the many biochemical actions
of long term lithium treatment in vivo and in
vitro are its ability to inhibit phosphoinoside turnover and
stimulated levels of calcium, influence signal transduction by
modulating G-proteins and protein kinase C, and regulate gene
expression involving AP-1 DNA binding activity (1-7). Increasing
evidence supports the notion that lithium has neuroprotective effects. We have recently reported that long term treatment of cultured neurons
from the central nervous system with therapeutically relevant concentrations of lithium robustly protects against apoptotic cell
death associated with glutamate excitotoxicity via
N-methyl-D-aspartate (NMDA)1 receptors (8). In
cerebellar granule cells (CGCs), the protection afforded by lithium is
accompanied by an inhibition of NMDA receptor-mediated calcium influx
that is independent of a loss of receptor binding activity or subunit
expression (8). In a rat model of ischemia that involves glutamate
excitotoxicity, chronic lithium administration was found to markedly
reduce brain infarction and neurological deficits induced by occlusion
of the middle cerebral artery (9).
In an attempt to elucidate the molecular mechanisms underlying
lithium-induced neuroprotection in cultured CGCs, we examined the long
term effects of lithium on the expression of key genes involved in
proapoptosis and cytoprotection, notably p53, Bax, and Bcl-2. p53, a
nuclear protein that binds to specific DNA sequences and functions as a
transcriptional activator, promotes the expression of the proapoptotic
gene, Bax, but suppresses the expression of the antiapoptotic gene,
Bcl-2 (10). Bax has been shown to bind to mitochondrial membranes and
thereby to cause the release of cytochrome c, which in turn
leads to activation of caspases and degradation of specific protein
substrates (11-13). The antiapoptotic action of Bcl-2 is mediated, at
least in part, by blocking Bax-induced cytochrome c release
and caspase activation (14-16). Thus, it has been proposed that the
relative amounts of Bcl-2 and Bax determine the fate of a living cell
(17, 18). In this study, we determined the levels of these proapoptotic
and antiapoptotic gene products in CGCs pretreated with lithium and/or
exposed to glutamate.
Primary Cultures of CGCs and Treatment Conditions--
CGCs were
prepared from 8-day-old Sprague-Dawley rat pups as described previously
(8). Cultures were routinely pretreated with different concentrations
(0.5-5 mM) of LiCl for 7 days, starting from the first day
in vitro. Glutamate (50-100 µM) was added to the cultures to trigger neuronal apoptosis. Cell viability was determined by measuring the mitochondrial dehydrogenase activity that
cleaves 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(8).
Northern Blotting--
Total RNA was extracted from CGCs with
guanidine thiocyanate and isolated by cesium chloride gradient
centrifugation as described previously (19). After electrophoresis on
1% agarose gel containing formaldehyde, RNA was transferred to a
Duralose membrane (Stratagene, La Jolla, CA) and hybridized with
cDNA probes for p53, Bcl-2, and Bax cDNA probes, separately.
The cDNA probes were labeled with [ Western Blotting--
CGCs were detached by scraping and
sonicated for 30 s in lysis buffer containing 10 mM
Tris·HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.28 unit/ml aprotinin, 50 µg/ml leupeptin, 1 mM benzamidine, and 7 µg/ml pepstatin A. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis
and then transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Blots were incubated with antibodies against p53 (Pab240), Bcl-2
(N-19), Bax (P-19) (Santa Cruz Biotechnology, Santa Cruz, CA), and
lamin B1 (Serotec LTD, Oxford, UK) and then probed with a horseradish
peroxidase-conjugated second antibody. Detection was performed by using
enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech).
Measurement of Cytochrome c Levels--
For measurement of
cytochrome c, the cytosol and mitochondrial fractions were
prepared as previously reported (15). A monoclonal antibody to
cytochrome c (Pharmingen, San Diego, CA) was used for
Western blotting as described above.
Result Analysis--
Data are expressed as the means ± S.E. from multiple experiments. Statistical significance was analyzed
by Student's t test (Statview 4.01).
Treatment of CGCs with LiCl induces a
concentration-dependent decrease in the levels of p53
mRNA and protein determined by Northern and Western blotting,
respectively (Fig. 1, a and
b). These effects were detected at therapeutic
concentrations of lithium (0.5-1.0 mM), although a more
robust decrease was observed at higher concentrations. The lithium (3 mM)-induced decrease in p53 mRNA and protein levels was
time-dependent (Fig. 1, c and d).
Loss of p53 was observed after treatment for 1 day and reached a
maximum after 5-7 days. Similar to the effects on p53, LiCl treatment
induced a dose- and time-dependent decrease in Bax mRNA and protein levels (Fig. 2). In contrast,
Bcl-2 mRNA and protein levels were markedly increased by LiCl
treatment (Fig. 3). The effect was also
dose- and time-dependent; 3 days or longer was necessary to
observe a significant increase.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]dCTP by
the random priming method. Hybridizations were performed at 42 °C
for 16 h, followed by two washes at room temperature with 2 × SSC/0.1% SDS and then two additional washes at 50 °C in 0.1×
SSC/0.1% SDS. Northern blots were quantified using a Betascope model
603 Blot Analyzer (Betagen Corp., Waltham, MA).
RESULTS
View larger version (78K):
[in a new window]
Fig. 1.
Effects of lithium on p53 mRNA and
protein levels in CGCs. Cells were treated with the indicated
concentration of LiCl for 7 days (a and b) or
with 3 mM LiCl for the indicated length of time
(c and d). Cells were then harvested for the
measurement of p53 mRNA levels (a and c) or
protein levels (b and d). Northern or Western
blots shown at the top of each panel are from a
typical experiment. Quantified results are the means ± S.E. from
two independent experiments in a and c and the
means ± S.E. from three independent experiments in b
and d. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with the untreated
control (CTL).
View larger version (90K):
[in a new window]
Fig. 2.
Effects of lithium on Bax mRNA and
protein levels. CGCs were treated with the indicated concentration
of LiCl for 7 days (a and b) or with 3 mM LiCl for the indicated time (c and
d). Bax mRNA and protein levels were then determined.
Northern or Western blots shown at the top of each
panel are from a typical experiment. Quantified results are
the means ± S.E. from two independent experiments in a
and c and the means ± S.E. from three independent
experiments in b and d. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control (CTL).
View larger version (90K):
[in a new window]
Fig. 3.
Effects of lithium on Bcl-2 mRNA and
protein levels. CGCs were treated with the indicated concentration
of LiCl for 7 days (a and b) or with 3 mM LiCl for the indicated time (c and
d). Levels of Bcl-2 mRNA and protein were then
determined. Northern or Western blots shown at the top of
each panel are from a typical experiment. Quantified results
are the means ± S.E. from two independent experiments in
a and c and the means ± S.E. from three
independent experiments in b and d. *,
p < 0.05; **, p < 0.01, compared with
control (CTL).
In contrast, exposure to 100 µM glutamate for 4 or 8 h markedly increased the mRNA levels of p53 and Bax but had little or no effect on Bcl-2 mRNA (Fig. 4, a, c, and e). Pretreatment with LiCl (3 mM) for 7 days abolished glutamate-induced increase in p53 and Bax mRNA levels, while maintaining elevated levels of Bcl-2 mRNA even in cells exposed to glutamate. Similar effects on protein levels of p53, Bax, and Bcl-2 were observed in CGCs exposed to 50 or 100 µM glutamate for 24 h in the absence or presence of lithium pretreatment (Fig. 4, b, d, and f). Lithium treatment for 5-7 days was found to cause a 5-fold increase in the Bcl-2/Bax protein ratio (Fig. 4g). Conversely, the Bcl-2/Bax ratio was decreased with glutamate exposure for 24 h, and this decrease was prevented by lithium pretreatment for 7 days. Under these experimental conditions, lithium pretreatment effectively protected cells from excitotoxicity (Fig. 4h). It should be mentioned that the effects of glutamate on cell viability and levels of p53, Bax, and Bcl-2 were completely blocked by MK-801 (10 µM), a selective NMDA receptor antagonist (data not shown), confirming our previous report that glutamate-induced apoptosis is entirely mediated by NMDA receptors (8, 20).
|
Because Bax and Bcl-2 modulate the release of cytochrome c from mitochondria (11, 14, 15), we studied the effects of glutamate and lithium on cytochrome c protein levels in the cytosolic and mitochondrial fractions. Glutamate exposure for 6 or 12 h caused a time-dependent increase in the levels of cytochrome c in the cytosol, with a concomitant decrease of cytochrome c level in mitochondria (Fig. 5, a and b). Lithium pretreatment completely antagonized glutamate-induced changes in cytochrome c levels in both subcellular fractions. Additionally, lithium pretreatment suppressed the degradation of lamin B1 (Fig. 5c), a nuclear membrane protein and a substrate of caspase-3. This effect is consistent with the finding that NMDA receptor-mediated excitotoxicity in CGCs involves the activation of caspase-3 (21).
|
![]() |
DISCUSSION |
---|
Bcl-2 family plays a prominent antiapoptotic role by acting upstream of caspase activation. Specifically, Bcl-2 may inhibit the Bax-mediated release of cytochrome c or AIF (apoptosis-inducing factor) from mitochondria, thereby preventing caspase activation and cell death (14, 22). In addition, Bcl-2 may physically interact with Apaf-1 (apoptotic protease-activating factor-1) to prevent Apaf-1-mediated caspase activation (23, 24). Lithium-induced neuroprotection against glutamate excitotoxicity is likely mediated in part by Bcl-2 up-regulation, whereas glutamate-induced apoptosis involves the overexpression of p53 and Bax. In support of the role of p53 in mediating glutamate excitotoxicity, we found that cultured CGCs derived from p53-deficient mice are resistant to the glutamate insult.2 We also found that glutamate triggers the release of cytochrome c from mitochondria and the cleavage of lamin B1, a nuclear membrane and substrate of caspase-3. Moreover, these glutamate-induced effects are almost completely blocked by long term lithium pretreatment.
Other actions of Bcl-2 may also contribute to lithium-induced
neuroprotection against excitotoxicity. For example, Bcl-2
down-regulates the activity of transcription factor NF-B induced
during apoptosis (25) and inhibits abnormal release of calcium from the
endoplasmic reticulum, thus reducing calcium loading to mitochondria
and subsequent oxidative stress (26, 27). Bcl-2 overexpression could
also be related to the lithium protection against apoptosis induced by
anticonvulsants (carbamazepine and phenytoin) in CGCs (28) and by brain
damage resulting from focal ischemia in rats (9).
This study shows for the first time that lithium influences the
expression of p53 and some Bcl-2 family members. Moreover, we show that
these changes in gene expression are associated with neuronal survival.
In this context, Bcl-2 levels appear to be increased in the frontal
cortex of rats chronically treated with lithium and valproate, another
drug used to treat manic depressive illness.3
It is well established that p53 positively regulates Bax but negatively
regulates Bcl-2 expression (10). Thus, lithium-elicited changes in the
mRNA and protein levels of Bax and Bcl-2 are likely the results of
the inhibition of p53 expression. Consistent with this possibility, we
found that only 1 day of lithium treatment is needed to observe a
decrease in p53 levels but 3 days are needed to observe a change in Bax
and Bcl-2 levels. However, it is also possible that lithium has a
direct regulatory role in the expression of Bcl-2 and/or Bax. Lithium
has been shown to increase basal transcription factor binding to AP-1
and CRE sites (6, 7) but decreases stimulated binding to the AP-1 site
in cultured cells and the brain (5). These lithium-induced changes in
transcription factor binding may be related to the ability of lithium
to inhibit the activity of glycogen synthase kinase (29), which
phosphorylates transcription factors such as c-Jun, causing a loss of
its DNA binding activity (30). Lithium modulation of transcription
factor DNA binding activity may underlie the effects of this drug on the expression of genes involved in apoptosis. Regardless of the mechanisms involved in lithium-induced gene expression, our results suggest that lithium, in addition to treating bipolar disorder, may
also be useful in the treatment of neurodegenerative diseases, particularly those involving glutamate excitotoxicity.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Section on Molecular
Neurobiology, BPB/NIMH/NIH, Bldg. 10, Rm. 3N212, 10 Center Dr. MSC
1272, Bethesda, MD 20892-1272. Tel.: 301-496-4915; Fax: 301-480-9290;
E-mail: chuang{at}helix.nih.gov.
2 R.-W. Chen and D.-M. Chuang, unpublished results.
3 H. K. Manji, W. Z. Zeng, and G. Chen, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NMDA, N-methyl-D-aspartate; CGC, cerebellar granule cell.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|