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In our previous study,we showed that Nampt(also called PBEF)is primarily expressed in neurons under normal conditions.Using PBEF heterozygous knockout mice and primary cultured mouse cortical neurons,we also showed that PBEF is neuronal/brain protective in in vivo and in vitro ischemia.Furthermore,overexpression of PBEF can suppress mitochondrial membrane potential depolarization after excitatory glutamate stimulation.In this study,we used in vitro ischemia models to investigate the mechanism of neuroprotective effect of PBEF in ischemia in the primary cultured neurons.We found that overexpression of PBEF can reduce apoptotic neuronal death and inhibit the translocation of apoptosis inducing factor(AIF)from mitochondria to nuclei after glutamate induced excitotoxicity.Over-expression of PBEF can also suppress glutamate-induced neuronal mitochondrial fragmentation.Furthermore,these beneficial effects are dependent on its enzymatic activity of NAD synthesis activity.To further study the mechanism by which PBEF protects neurons and brain in ischemia through mitochondrial pathway,we tested whether neuronal mitochondria express PBEF and NMNAT-3,the other enzyme in NAD+ synthesis pathway using highly purified mitochondrial and cytosolic fractions from primary cultured neurons and adult mouse cortical tissues.Using Western blotting analysis,we found that PBEF and NMNAT-3 are expressed in both mitochondrial and cytosolic fractions,indicating that mitochondria have an own NAD+ biosynthesis pathway.In summary,our study demonstrated that PBEF can protect neurons in ischemia through enhanced energy metabolism and suggests that mitochondrial pathway of NAD+ synthesis might play an important role in brain protection in ischemic stroke.