To Study The Underlying Protective Function And Mechanism Of 14-3-3gamma In Ischemic Neurons

14-3-3 proteins are abundant in brain tissues and the gamma isoform is mainly expressed in neurons. We have previously reported that 14-3-3gamma could be up-regulated in astrocytes by in vitro ischemia and attenuated ischemia-induced astrocytic death by binding to phosphorylated Bad. In this study, we studied the expression, function and underlying mechanism of 14-3-3gamma in ischemic neurons. The selective up-regulation of 14-3-3gamma in primary cultures of mouse cerebral cortical neurons exposed to oxygen-glucose deprivation (OGD) was demonstrated by Western blot analysis, immunostaining and co-immunoprecipitation methods. Other 14-3-3 isoforms (β,ε,ηandζwere not altered significantly by OGD treatment. Blocking the interaction of 14-3-3 proteins with their ligands by over-expression diforpein (DFP) exacerbated cell death in primary mouse cortical neurons and N2a neuroblastoma cells. Over-expression of 14-3-3gamma enhanced cell survival in OGD-treated neurons and N2a cells significantly while other 14-3-3 isoforms were less effective. Moreover, suppressing 14-3-3gamma expression by siRNA technique facilitated cell death in neurons and N2a cells upon OGD treatment. To study the underlying protective mechanism of 14-3-3gamma, we investigated the interaction of 14-3-3gamma with beta-catenin, Bad, p53 and Ask-1. We found that 14-3-3gamma bound more phospho-s37-beta-catenin in neurons upon OGD incubation while the binding of 14-3-3gamma with Bad, p53 and Ask-1 did not altered evidently. In addition, the expression of Bax was reduced in N2a cells overexpressed 14-3-3gamma while 14-3-3gamma-siRNA increased the Bax level in N2a cells. Finally, in the model of anoxia preconditioning induced by cobalt chloride, we found that the expression of 14-3-3 gamma but not other 14-3-3 isoform was increased in cerebral cortex compared with control. Taken together, these data suggested that 14-3-3gamma-beta-catenin-Bax pathway was an important part of endogenous protective machinery in ischemic neurons. The induction of 14-3-3 gamma may be a protective mechanism of preconditioning.