Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 ␣ and ␤ 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, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated ␤-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. The present study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly upregulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors -sodium butyrate, trichostatin A and Class I HDAC-specific inhibitors MS-275 and apicidin, -all mimicked the ability of VPA to induce HSP70. Pretreatment with PI3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the PI3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.

Glycogen synthase kinase-3 (GSK-3) exists as two structurally similar isoforms, ␣ and ␤, whose activities are negatively regulated by serine phosphorylation but positively controlled by tyrosine phosphorylation. We used GSK-3 isoform-specific small interfering RNAs, dominant negative mutants, and pharmacological inhibitors to search for the differential roles for both GSK-3 isoforms in regulating transcriptional activation in cultured rat cerebral cortical neurons. GSK-3␣ and GSK-3␤ were shown to have differentially regulated transactivation such that GSK-3␣ silencing/inhibition was more robust than GSK-3␤ silencing/inhibition in causing cAMP-responsive element-and NF-B-dependent transactivation. Moreover, protein-DNA array studies identified two novel GSK-3-regulated transcription factors, early growth response 1 and Smad3/4, which were oppositely affected by GSK-3␣ or GSK-3␤ silencing or inhibition. Taken together, our results underscore critical variations in the function and regulation of GSK-3␣ and GSK-3␤. The development of GSK-3 isoform-specific inhibitors is thus crucial for therapeutic intervention of GSK-3-related neuropathological conditions.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase consisting of two isoforms, ␣ and ␤. ⌻he activities of GSK-3 are regulated negatively by serine phosphorylation but positively by tyrosine phosphorylation. GSK-3 inactivation has been proposed as a mechanism to promote neuronal survival. We used GSK-3 isoform-specific small interfering RNAs, dominant-negative mutants, or pharmacological inhibitors to search for functions of the two GSK-3 isoforms in regulating neuronal survival in cultured cortical neurons in response to glutamate insult or during neuronal maturation/aging. Surprisingly, RNA interference-induced depletion of either isoform was sufficient to block glutamate-induced excitotoxicity, and the resulting neuroprotection was associated with enhanced N-terminal serine phosphorylation in both GSK-3 isoforms. However, GSK-3␤ depletion was more effective than GSK-3␣ depletion in suppressing spontaneous neuronal death in extended culture. This phenomenon is likely due to selective and robust inhibition of GSK-3␤ activation resulting from GSK-3␤ Ser 9 dephosphorylation during the course of spontaneous neuronal death. GSK-3␣ silencing resulted in reduced tyrosine phosphorylation of GSK-3␤, suggesting that tyrosine phosphorylation is also a critical autoregulatory event. Interestingly, GSK-3 inhibitors caused a rapid and long-lasting increase in GSK-3␣ Ser 21 phosphorylation levels, followed by a delayed increase in GSK-3␤ Ser 9 phosphorylation and a decrease in GSK-3␣ Tyr 279 and GSK-3␤ Tyr 216 phosphorylation, thus implying additional levels of GSK-3 autoregulation. Taken together, our results underscore important similarities and dissimilarities of GSK-3␣ and GSK-3␤ in the roles of cell survival as well as their distinct modes of regulation. The development of GSK-3 isoform-specific inhibitors seems to be warranted for treating GSK-3-mediated pathology.

Smad proteins are intracellular transducers for transforming growth factor-β (TGF-β signaling and play a critical role in differentiation, tissue repair and apoptosis of the central nervous system. Both TGF-β and its regulated gene, plasminogen activator inhibitor type-1 (PAI-1), have been implicated in the etiology and progression of neurodegenerative diseases and mood disorders. We previously reported that GSK-3β protein depletion suppresses Smad3/4-dependent gene transcription and causes a reduction in PAI-1 expression. Here, we provide evidence that lithium, the drug for the treatment and prophylaxis of bipolar disorder, inhibits Smad-dependent signaling by regulating cAMP-protein kinase A (PKA), AKT-glycogen synthase kinase-3β (GSK-3β), and CRE-dependent signaling pathways in neuron-enriched cerebral cortical cultures of rats. We demonstrate that lithium-induced activation of these pathways inhibits Smad3/4-dependent gene transcription through an increase in pCREB Ser133 protein levels, an enhanced interaction between pCREB Ser133 and p300/CBP, which causes Smad3/4-p300/CBP complex disruption and transcriptional suppression of Smad3/4-dependent genes. Therapeutic implications of our findings are discussed.