We investigated the hypothesis that glycogen synthase kinase-3 (GSK-3) and histone deacetylases (HDACs) are initial targets of lithium and valproic acid (VPA), respectively, that trigger diverse neurobiological events including neuroprotective and neurotrophic effects, anti-inflammatory effects, antidepressant effects, promotion of stem cell migration, regulation of microRNA expression, and modulation of behavioral phenotypes. Our studies identified a number of novel signaling pathways and neuroprotective and neurotrophic genes targeted by lithium and VPA, and underlying mechanisms were elucidated in both in vitro and in vivo experimental settings. Studies on the effects of mood stabilizers in the rat ischemic stoke model and animal models of Huntingtons disease (HD) were markedly expanded, and additional animal models of CNS disorders were also explored. Taken together, the results of these studies identified previously unknown neurobiological actions induced by these mood stabilizers. In primary cortical neurons, we found that lithium and VPA selectively upregulated brain-derived neurotrophic factor (BDNF) promoter IV activity and exon IV-containing mRNA, and provided evidence that these effects are mediated by inhibition of GSK-3 and HDACs, respectively (Yasuda et al. Mol Psychiatry 14: 51-59, 2009). In aging primary neurons, we identified a previously unknown function of fibroblast growth factor-21, namely its ability to mediate the synergistic neuroprotective effects of lithium and VPA against glutamate excitotoxicity (Leng et al. Neurosci Meeting abstr 62.07, 2012). We demonstrated that VPA inhibition of class I HDACs induced functional heat shock protein 70 (HSP70) by enhancing its promoter activity via increased histone H3 acetylation and histone H3 lys4 methylation at the promoter in primary neurons and astrocytes (Marinova et al., J Neurochem 111: 976-987, 2009;2011). We also advanced our understanding of the beneficial effects of mood stabilizers using experimental models of brain disorders. We expanded our studies using middle cerebral artery occlusion (MCAO) in rats as a model of focal cerebral ischemia, and made a number of original findings. Similar to the effects of lithium, we found that post-insult treatment with VPA reduced infarct volume and improved behavioral outcomes in rats that underwent MCAO. For the first time, we demonstrated that VPA robustly reduced blood-brain barrier (BBB) disruption induced by MCAO, and that this reduction was largely due to decreases in ischemia-induced matrix metalloproteinase 9 (MMP-9) overexpression and tight junction degradation (Wang et al., 2011a, b). We also showed that long-term VPA treatment enhanced post-ischemic angiogenesis by increasing microvessel density, facilitating endothelial cell proliferation, and augmenting relative cerebral blood flow in the ipsilateral cortex by upregulating hypoxia inducible factor-1alpha (HIF-1alpha) and its downstream targets, pro-angiogenic vascular endothelial growth factor (VEGF) and MMP-2/9 (Wang et al., 2012). In addition, tail-vein injection of mesenchymal stem cells (MSCs) into MCAO rats was found to be highly beneficial when these MSCs were primed with both lithium and VPA to induce MMP-9 and CXC chemokine receptor 4 (CXCR4), respectively, thereby promoting MSC migration to the infarct region (Tsai et al., Neuropsychopharm 35: 2225-2237, 2010;2011). Furthermore, we reported that HDAC inhibition by a VPA analog sodium butyrate enhanced post-MCAO-induced neurogenesis in multiple ischemic brain regions, and this effect required activation of BDNF-TrkB signaling (Kim et al., J Neurochem 110: 1226-1240, 2009). Finally, our very recent work reported microRNA (miRNA) regulation following ischemic stroke (e.g. miR-446f, miR-446h, miR-155, miR-1224, and miR-297a) and their potential for underlying the benefits of post-insult VPA treatment (e.g. miR-885-3p and miR-331) in a rat model of cerebral ischemia (Hunsberger et al., 2012), suggesting that miRNAs may underlie disease processes that contribute to numerous neurological disorders. Additionally, we found miR34a to be involved in regulating the life and death of neuronal cells in an in vitro study (Hunsberger et al., 2012, Neuroscience Lett, submitted). HD is an inherited, fatal neurodegenerative/neuropsychiatric disorder with no available treatment to halt symptom progression. We assessed the therapeutic potential of dietary treatment with lithium and/or VPA in two transgenic mouse models of HD, N171-82Q and YAC128, which reproduce the development of HD by expressing mutant huntingtin protein (mHtt), but differ in symptom progression and lifespan. We detected hyperactivity of GSK-3 and HDACs in the brains of untreated HD mice, which correlated with the onset of behavioral symptoms. We then found that co-treatment with lithium and VPA more effectively alleviated impaired locomotion and depressive-like behaviors than mono-treatment in both models of HD mice (Chiu et al., 2011). In addition, this co-treatment was more effective for improving motor skill learning and coordination in N171-82Q mice, and suppressed anxiety-like behaviors in YAC128 mice. Levels of BDNF and HSP70 in the brains of both strains were also more consistently elevated by lithium-VPA co-treatment. Finally, the lifespan of N171-82Q mice was markedly prolonged by co-treatment with these mood stabilizers (Chiu et al., 2011). In a related study, we found that injection of shRNA specific for GSK-3beta into the hippocampal dentate gyrus of stressed rats induced antidepressant-like behaviors (Omata et al., 2011), reaffirming the role of GSK-3 in regulating the mood state. We also launched three projects into new areas of research. (1) We are studying the regulation of miRNAs by mood stabilizers in order to identify novel miRNA-mediated signatures and mechanisms in patient-derived lymphoblastoid cell lines from BD patients who were lithium responders and non-responders. Several prominent miRNAs were identified and confirmed by Real-Time PCR, and their function and targets are currently being investigated (Hunsberger et al., Neurosci Meeting abstr 521.02, 2012). (2) Another new study evaluates the effects of lithium treatment in an experimental mouse model of traumatic brain injury (TBI). We recently reported that post-insult treatment with lithium ameliorated TBI-induced lesion size, neurodegeneration, neuroinflammation, and functional impairments, in association with GSK-3 inhibition (Yu et al., 2012a). Moreover, for the first time we showed that lithium blocked TBI-induced beta-secretase overexpression, thereby decreasing beta-amyloid burden, and Tau protein hyperphosphorylation, as well as improving spatial memory performance (Yu et al., 2012b). (3) We examined the neuroprotective properties of a third mood stabilizer, lamotrigine, which is also an anticonvulsant. Our results showed that lamotrigine robustly protected primary brain neurons from glutamate excitotoxicity and this protection required the induction of cytoprotective Bcl-2 (Leng et al., 2012). We also showed for the first time that lamotrigine indirectly inhibited HDACs, thereby increasing Bcl-2 promoter activity by histone hyperacetylation. Interestingly, a sub-effective dose of lamotrigine showed synergistic neuroprotective effects when used in combination with lithium or VPA (Leng et al., 2012). Our recent work has markedly contributed to our understanding of molecular and cellular actions of mood stabilizers, and substantially advanced our knowledge of their effects in a number of experimental models of neurodegenerative and neuropsychiatric diseases. With the completion of future studies, we expect to provide further mechanistic insights and set the stage for clinical investigations into the use of mood stabilizers to intervene in certain CNS disorders.
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