REGULATION OF NCX3 EXPRESSION BY HISTONE DEACETYLASES (HDACS) IN CORTICAL NEURONS AND IN BRAIN ISCHEMIA

Na+- Ca2+ exchanger isoform 3 (NCX3) plays a fundamental role in the pathogenesis of stroke damage. Indeed its ablation worsens the experimentally-induced ischemic damage. Interestingly it has been found that NCX3 mRNA and protein are both reduced after stroke. However, the mechanism by which stroke-induced ncx3 gene reduction is still unclear. Notably, in the last decades it has been found that histone deacetylases (HDACs) inhibition by regulating specific neuroprotective genes ameliorates the neurodegeneration that occurs in brain ischemia. Interestingly, we found that neurons treated with Trichostatin A (TSA), a pan HDACs inhibitor (HDACi), and MC1568, a class II HDACs inhibitor, significantly increased ncx3 promoter activity, whereas MS-275 (class I HDACs inhibitor) had no significant effect. Notably, among the HDACs class II A, we found that when the HDAC4 and HDAC5 isoforms were overexpressed by construct transfection or knocked-down by small interfering RNA (siRNA) transfection, NCX3 mRNA and protein levels were downregulated or increased, respectively. Moreover, experiments of site direct mutagenesis of DREAM (downstream regulatory element antagonist modulator) consensus sequence on ncx3 promoter in MC1568 treated neurons, corroborated that NCX3 downregulation induced by HDACs is achieved by DREAM. Notably, Chromatin Immunoprecipitation (ChIP) assay demonstrated that HDAC4 and HDAC5 binding on ncx3 promoter was significantly increased after transient middle cerebral artery occlusion (tMCAO). Our findings identify a new epigenetic regulatory mechanism that controls NCX3 gene transcription and demonstrated that HDAC class II A inhibition, by blocking HDAC4 and HDAC5 and modulating the acetylation of ncx3 gene promoter sequence, could be a new therapeutic strategy in stroke treatment.