Peter Leeds

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti‐convulsant and mood‐stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia‐induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia‐induced neuronal caspase‐3 activation in the cerebral cortex. VPA treatments resulted in a time‐dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non‐surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time‐dependently up‐regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.

Synergistic Neuroprotective Effects of Lithium and Valproic Acid or Other Histone Deacetylase Inhibitors in Neurons: Roles of Glycogen Synthase Kinase-3 Inhibition

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 α and β isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3α small interfering RNA (siRNA) and/or GSK-3β 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 β-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.

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non‐neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post‐insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo‐2′‐deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho‐cAMP response element‐binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co‐localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up‐regulated protein levels of BDNF, phospho‐CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB‐induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB‐induced nestin expression and CREB activation, and attenuated the long‐lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor‐induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long‐term beneficial effects of SB after ischemic injury.

Valproic Acid Attenuates Blood–Brain Barrier Disruption in a Rat Model of Transient Focal Cerebral Ischemia: The Roles of HDAC and MMP-9 Inhibition

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is known to protect against cerebral ischemia. The effects of VPA on blood–brain barrier (BBB) disruption were investigated in rats subjected to transient middle cerebral artery occlusion (MCAO). Postischemic VPA treatment remarkably attenuated MCAO-induced BBB disruption and brain edema. Meanwhile, VPA significantly reduced MCAO-induced elevation of matrix metalloproteinase-9 (MMP-9), degradation of tight junction proteins, and nuclear translocation of nuclear factor-κB (NF-κB). Sodium butyrate, another HDAC inhibitor, mimicked these effects of VPA. Our findings suggest that BBB protection by VPA involves HDAC inhibition-mediated suppression of NF-κB activation, MMP-9 induction, and tight junction degradation.

Regulation of c-Jun N-terminal kinase, p38 kinase and AP-1 DNA binding in cultured brain neurons: roles in glutamate excitotoxicity and lithium neuroprotection.

In rat cerebellar granule cells, glutamate induced rapid activation of c‐Jun N‐terminal kinase (JNK) and p38 kinase to phosphorylate c‐Jun (at Ser63) and p53 (at Ser15), respectively, and a subsequent marked increase in activator protein‐1 (AP‐1) binding that preceded apoptotic death. These glutamate‐induced effects and apoptosis could largely be prevented by long‐term (7 days) pretreatment with 0.5–2 mm lithium, an antibipolar drug. Glutamates actions could also be prevented by known blockers of this pathway, MK‐801 (an NMDA receptor blocker), SB 203580 (a p38 kinase inhibitor) and curcumin (an AP‐1 binding inhibitor). The concentration‐ and time‐dependent suppression of glutamates effects by lithium and curcumin correlated well with their neuroprotective effects. These results suggest a prominent role of JNK and p38, as well as their downstream AP‐1 binding activation and p53 phosphorylation in mediating glutamate excitotoxicity. Moreover, the neuroprotective effects of lithium are mediated, at least in part, by suppressing NMDA receptor‐mediated activation of the mitogen‐activated protein kinase pathway.

β-Amyloid peptide-induced death of PC 12 cells and cerebellar granule cell neurons is inhibited by long-term lithium treatment

Treatment of rat pheochromocytoma cells (PC 12) cells with beta-amyloid peptide-(1-42) for 24 h induced a concentration-dependent decrease in cellular redox activity in the dose range of 1 to 20 microM. These effects were markedly attenuated by pretreatment with 2 mM LiCl for 7 days, whereas 1-day pretreatment was ineffective. Measurements of live and dead cells by double-staining with fluorescein diacetate and propidium iodide, respectively revealed that protracted lithium pretreatment attenuated PC 12 cell death induced by beta-amyloid-(1-42) and cerebellar granule cell death induced by beta-amyloid-(25-35). Preceding PC 12 cell death, beta-amyloid peptide elicited a slight decrease in protein levels of Bcl-2. Conversely, 7-day pretreatment with lithium resulted in an approximate doubling of Bcl-2 protein levels in cells treated with or without beta-amyloid peptide-(1-42). Lithium-induced Bcl-2 upregulation was temporally associated with the cytoprotective effects of this drug. Thus, lithium protection against beta-amyloid peptide neurotoxicity might involve Bcl-2 overexpression, and lithium treatment for Alzheimers disease should be reexamined.

Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation.

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. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up‐regulated 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. Pre‐treatment with phosphatidylinositol 3‐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 phosphatidylinositol 3‐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.

Valproic acid inhibits histone deacetylase activity and suppresses excitotoxicity-induced GAPDH nuclear accumulation and apoptotic death in neurons.

ABSTRACTValproic acid (VPA), used to treat bipolar mood disorder and seizures, also inhibits histone deacetylase (HDAC). Here, we found that VPA and other HDAC inhibitors, butyrate and trichostatin A, robustly protected mature cerebellar granule cell cultures from excitotoxicity induced by SYM 2081 ((2S, 4R)-4-methylglutamate), an inhibitor of excitatory amino-acid transporters and an agonist of low-affinity kainate receptors. These neuroprotective effects required protracted treatment and were correlated with enhanced acetylated histone levels, indicating HDAC inhibition. SYM-induced excitotoxicity was blocked by MK-801 ((5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate), supporting that the toxicity was largely N-methyl-D-aspartate receptor dependent. SYM excitotoxicity had apoptotic characteristics and was prevented by a caspase inhibitor. SYM-induced apoptosis was associated with a rapid and robust nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene previously shown to be proapoptotic. VPA pretreatment suppressed SYM 2081-induced GAPDH nuclear accumulation, concurrent with its neuroprotective effects. Chromatin immunoprecipitation (ChIP) revealed that GAPDH is copresent with acetylated histone H3, including Lys9-acetylated histone, and that VPA treatment caused a time-dependent decrease in the levels of nuclear GAPDH with a concomitant increase in acetylated histones in the ChIP complex. Our results strongly suggest that VPA protects neurons from excitotoxicity through inhibition of HDAC activity and that this protective effect may involve suppression of excitotoxicity-induced accumulation of GAPDH protein in the nucleus.

Combined Treatment with the Mood Stabilizers Lithium and Valproate Produces Multiple Beneficial Effects in Transgenic Mouse Models of Huntington's Disease

Emerging evidence suggests that the mood stabilizers lithium and valproate (VPA) have broad neuroprotective and neurotrophic properties, and that these occur via inhibition of glycogen synthase kinase 3 (GSK-3) and histone deacetylases (HDACs), respectively. Huntingtons disease (HD) is an inherited neurodegenerative disorder characterized by impaired movement, cognitive and psychiatric disturbances, and premature death. We treated N171-82Q and YAC128 mice, two mouse models of HD varying in genetic backgrounds and pathological progressions, with a diet containing therapeutic doses of lithium, VPA, or both. Untreated, these transgenic mice displayed a decrease in levels of GSK-3β serine 9 phosphorylation and histone H3 acetylation in the striatum and cerebral cortex around the onset of behavioral deficits, indicating a hyperactivity of GSK-3β and HDACs. Using multiple well-validated behavioral tests, we found that co-treatment with lithium and VPA more effectively alleviated spontaneous locomotor deficits and depressive-like behaviors in both models of HD mice. Furthermore, compared with monotherapy with either drug alone, co-treatment more successfully improved motor skill learning and coordination in N171-82Q mice, and suppressed anxiety-like behaviors in YAC128 mice. This combined treatment consistently inhibited GSK-3β and HDACs, and caused a sustained elevation in striatal as well as cortical brain-derived neurotrophic factor and heat shock protein 70. Importantly, co-treatment markedly prolonged median survival of N171-82Q mice from 31.6 to 41.6 weeks. Given that there is presently no proven treatment for HD, our results suggest that combined treatment with lithium and VPA, two mood stabilizers with a long history of safe use in humans, may have important therapeutic potential for HD patients.

Neuronal apoptosis induced by pharmacological concentrations of 3-hydroxykynurenine: characterization and protection by dantrolene and Bcl-2 overexpression.

Abstract : We have studied neurotoxicity induced by pharmacological concentrations of 3‐hydroxykynurenine (3‐HK), an endogenous toxin implicated in certain neurodegenerative diseases, in cerebellar granule cells, PC12 pheochromocytoma cells, and GT1‐7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose‐dependent manner by 3‐HK at high micromolar concentrations and had features characteristic of apoptosis, including chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3‐HK neurotoxicity was unaffected by xanthine oxidase inhibitors but markedly potentiated by superoxide dismutase and its hemelike mimetic, MnTBAP [manganese(III) tetrakis(benzoic acid)porphyrin chloride]. Catalase blocked 3‐HK neurotoxicity in the absence and presence of superoxide dismutase or MnTBAP. The formation of H2O2 was demonstrated in PC12 and GT1‐7 cells treated with 3‐HK, by measuring the increase in the fluorescent product, 2′,7′‐dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral amino acid transporter that mediates the uptake of 3‐HK failed to block 3‐HK toxicity. However, their toxicity was slightly potentiated by the iron chelator, deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3‐HK in these cell types is mediated primarily by H2O2, which is formed most likely by auto‐oxidation of 3‐HK in extracellular compartments. 3‐HK‐induced death of PC12 and GT1‐7 cells was protected by dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. The protection by dantrolene was associated with a marked increase in the protein level of Bcl‐2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl‐2 in GT1‐7 cells elicited by gene transfection suppressed 3‐HK toxicity. Thus, dantrolene may elicit its neuroprotective effects by mechanisms involving up‐regulation of the level and function of Bcl‐2 protein.