Protective Role And Mechanism Of 14-3-3 Protein In Neuronal Survival And Ischemic Injury

The 14-3-3 proteins are a group highly conserved regulatory molecules found in alleukaryotes. 14-3-3 proteins are highly expressed in the brain and seven mammalianisoforms (β,γ,ε,η,ζ,σ,τ) are identified. 14-3-3 proteins are involved in manybiologically important processes, including cell differentiation, proliferation,metabolism control, apoptosis. However the exact physiological function of the 14-3-3proteins in neurons remains unclear. We have previously reported that 14-3-3γprotectedischemic astrocytes from apoptosis. Here, we studied the role of 14-3-3 on neuronalsurvival and the protective effects of 14-3-3γon ischemic neurons in vitro and in vivo.First, we studied the role of endogenous 14-3-3 proteins in neuronal survival by usingdifopein (dimeric fourteen-three-three peptide inhibitor), which specifically blocks theinteraction of 14-3-3 and its ligands. Expression of difopein in N2a cells and primaryculture of cerebral cortical neurons caused severe cell death as measured by Hoechststaining, MTT and Fluorescence microplate assays, suggesting that 14-3-3 proteins wereessential for neuronal survival in normal condition. Then, the protective effects of 14-3-3 proteins on ischemic neuronal cells were studied by over-expressing difopein and 14-3-3 isoforms (β,γ,ζ,η,ε,τ,σ) in N2a cells, Hoechst staining and MTT assaydemonstrated that blocking 14-3-3 function exacerbated cell death evidently underOGD-treatment and the gamma isoform of 14-3-3 was the most effective in protectingN2a cell from OGD-induced death. We further investigated the protective mechanism of14-3-3 proteins on neuronal apoptosis under ischemia. Caspase 3 was activated afterblocking 14-3-3 function and inhibiting endogenous 14-3-3γby siRNA techniquedemonstrated that caspase 3 was activated and the expression of Bax was increased in N2a cells under OGD condition, suggesting that 14-3-3 protects ischemic neurons fromapoptosis through Bax-caspase pathway. In MACO rats, immunohistofluorescenceanalysis showed that the expression of 14-3-3γwas separated from those of P53 and Baxin cortical neurons, suggesting that 14-3-3 suppresses mitochondrial-mediated apoptoticpathway by down-regulating P53 and Bax expression. Moreover, we found thatcytoplasmic 14-3-3γtranslocated into nucleus in cortical neurons at the penumbra area,indicating that 14-3-3 works as a transcription co-factor. Finally, we studied theprotective role of 14-3-3γin ischemic neuron in vivo by viral-transfection. Takentogether, our data demonstrated that 14-3-3 proteins were required for neuronal survivaland were protective in ischemic neurons. Theγisoform was the most effective inproviding protection to ischemic neurons. 14-3-3γexerts its anti-apoptotic effectthrough P53-Bax-caspase 3 pathway.