The precise mechanisms underlying the clinical efficacy of lithium and VPA in treating bipolar disorder remain to be defined. We first investigated the neuroprotective effects of lithium against excitotoxicity elicited by glutamate, a major excitatory amino acid neurotransmitter involved in the synaptic plasticity and pathogenesis of neurodegenerative and neuropsychiatric disorders. We found that long-term exposure to lithium chloride dramatically protects cultured rat cerebellar granule cells (CGCs) and cortical neurons, against glutamate-induced excitotoxicity. The lithium-induced neuroprotection against glutamate excitotoxicity involves multiple mechanisms. These include inactivation of NMDA receptors, induction of cytoprotective Bcl-2 but down-regulation of the pro-apoptotic proteins p53 and Bax. In addition, lithium activates the cell survival factor Akt and CREB through their enhanced phosphorylation. The neuroprotective effect of lithium requires the expression of brain-derived neurotrophic factor (BDNF) and activation of its receptor TrkB. The induction of BDNF is preceded by an increase in BDNF exon IV mRNA and its transcriptional promoter activity. We have identified the exon IV promoter region which confers the sensitivity to lithium and VPA for the induction of BDNF.? VPA also induces protection against glutamate excitotoxicity in cultured neurons and the neuroprotection is mimicked by inhibitors of histone deacetylase (HDAC), suggesting the effects are mediated through inhibition of HDAC. Additionally, in CGCs we found that under conditions in which neither lithium nor VPA alone is effective in protecting against glutamate excitotoxicity, combined treatment with lithium and VPA provides a synergy in neuroprotection. We identified the target of the neuoprotective synergy to be on glycogen synthase kinase-3 (GSK-3), a direct target of lithium. Another study showed that VPA induces alpha-synuclein in CGCs and this induction has a neuroprotective role against glutamate excitotoxicity. Moreover, the VPA-induced alpha-synuclein expression is mimicked by other HDAC inhibitors such as butyrate and trichostatin A, and involves an increase in histone H3 acetylation in the alpha-synuclein promoter and a robust activation of alpha-synuclein promoter activity. HDAC inhibitor-induced alpha-synuclein expression has also been shown in the brain of rats treated with these drugs.? GSK-3 has two isoforms termed alpha and beta. Knockdown of GSK-3alpha or GSK-3beta using isoform-specific siRNA or treatment with GSK-3 inhibitors was found to protect against glutamate-induced excitotoxicity, suggesting that both isoforms of GSK-3 are involved in the execution of glutamate excitotoxicity and are targets for lithium-induced neuroprotection. However, during the spontaneous death of cortical neurons resulting from aging of the cultures, GSK-3betaSerine9, but not GSK-3alphaSerine21, is dephosphorylated, indicating activation of GSK-3beta activity. Further evidence for differential roles of GSK-3alpha and GSK-3beta in transcriptional activation is supported by our findings that GSK-3alpha silencing/inhibition is more robust than that of GSK-3beta in causing CRE- and NF-kB-dependent transactivation of transcription. Using protein-DNA array, we identified two novel GSK-3 regulated transcription factors, EGR-1 and Smad3/4, which are oppositely affected by GSK-3alpha or GSK-3beta silencing/inhibition. Thus, our results underscore the importance of developing GSK-3beta isoform-specific inhibitors for therapeutic intervention.? In a collaborative effort with Dr. J-S Hong of NIEHS, NIH, we demonstrated that VPA protects midbrain dopaminergic neurons from LPS-induced inflammation mediated through microglia activation. VPA appears to suppress LPS-induced inflammation, in part, by triggering apoptosis of activated microglia. Additionally, we showed that VPA displays neurotrophic effects on dopaminergic neurons by inducing the expression of BDNF and glial cell line-derived neurotrophic factor (GDNF). We also provided evidence that the neuroprotective and neurotrophic effects of VPA are mediated by HDAC inhibition. Our studies define glial cells as an important target of VPA to induce the above-mentioned effects.? We have extended our in vitro studies by using animal models of neurodegenerative diseases. We investigated the neuroprotective effects of mood stabilizers in a rat model of stroke which is the third leading cause of death in the US. We performed middle cerebral artery occlusion (MCAO) in rats, a procedure which triggers brain infarction and results in neurological deficits. Our results showed that post-treatment with lithium robustly reduces MCAO- induced infarct volume and suppresses the neurological deficits detected in motor, sensory and reflex tests. The lithium-induced neuroprotection is associated with induction of heat shock protein 70 (HSP70) and inhibition of caspase-3. In collaboration with Dr. E. Lo of Harvard Medical School, we found that lithium given 12 hr after the onset of ischemia is still able to increase the somatosensory function measured by fMRI in the rat brain 15 days later. In conjunction with Dr. Y. Qian in China, we found that lithium pretreatment attenuates neuronal apoptosis in the hippocampal CA1 and improves memory performance in gerbils subjected to global ischemia. We also found that VPA has similar neuroprotective actions in the rat MCAO model of stroke: it reduces both the infarct volume and neurological deficit scores, and induces HSP70. The beneficial effects are long-lasting and the protective time window for both drugs is at least three hours after the onset of ischemia. The VPA-induced neuroprotection against ischemic insult is mimicked by other HDAC inhibitors, sodium butyrate and trichostatin A, again suggesting a neuroprotective role of HDAC inhibition. We have succeeded in demonstrating that HDAC inhibitors induce HSP70 and activate its promoter in cortical neurons and human neuroblastoma cells, and we are studying the detailed mechanisms involved. Inflammation plays a prominent role in the pathophysiology of stroke. In collaboration with Dr. Robert Innis of NIMH, we have succeeded in using PET imaging with 11CPBR28 to localize and quantify up-regulated brain peripheral benzodiazepine receptors, a marker of neuroinflammation, after cerebral ischemia in rats. Importantly, in our MCAO studies, we confirmed the anti-inflammatory effects of VPA and other HDAC inhibitors, as revealed by suppression of ischemia-induced microglia activation. Studies on the neurogenesis induced by an HDAC inhibitor in the MCAO model are now underway.? In a rat Huntington's disease model, we injected quinolinic acid (QA), a partial agonist of the NMDA receptor, into the striatum. Our results show that pretreatment with lithium for 16 days or one day decreases the size of striatal lesion by 40-50%. In addition, lithiums neuroprotection effects are associated with over-expression of striatal Bcl-2. The neuroprotection is also correlated with suppression of QA-induced DNA damage and caspase-3 activation. Additionally, lithium induces enhanced cell proliferation in the striatum near the site of QA injection. In corticostriatal organotypic culture, we further demonstrated neuroprotective effects of lithium, which are associated with inhibition of QA-induced caspase-3 activation and intracellular sodium and intracellular calcium increase. The effects of lithium and/or VPA in transgenic mouse models of Huntingtons disease are now under investigation. Thus, our in vitro and in vivo studies raise the possibility that lithium, in addition to its use for bipolar disorder, may have expanded use for the treatment of neurodegenerative diseases, particularly those linked to excitotoxicity, such as stroke, Huntingtons disease and others.
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