Causal mechanisms of eIF2alpha phosphorylation during brain reperfusion

During early post-ischemic reperfusion, there is a massive and rapid increase in eIF2α phosphorylation. The phosphorylation of eIF2α is responsible for the inhibition of translation initiation that accompanies reperfusion. This study was instrumental in identifying the kinase responsible for this increase in eIF2α phosphorylation seen immediately upon reperfusion following global ischemia. The data presented shows that both the homozygous HRI and the GCN2 mutant deletion mice produced equivalent amounts of eIF2α(P) following BCAO when compared to their cognate wild-types. Western blotting of immunoprecipitated PERK demonstrated the presence of hyperphosphorylated, active PERK in reperfused brains. PERK activation is evidence that an endoplasmic reticulum (ER) stress response termed the unfolded protein response (UPR) occurs during reperfusion, and is responsible for initial eIF2α(P) formation.; eIF2α(P) levels were measured in the following brain regions in control animals, during ischemia, and at 10 and 60 min. reperfusion: (a) brainstem, (b) cerebral cortex + basal ganglia, (c) cerebellum, (d) diencephalon, and (e) hippocampus. My data shows that the brainstem, hippocampus, and thalamus have similar eIF2α(P) levels in control animals. The cerebellum and cortex, respectively have 3-fold and 2-fold higher levels of eIF2α(P). As of present we do not know the functional significance of this regional difference in eIF2α(P) levels. In the ischemic only samples, I did not measure any increase in eIF2α phosphorylation in the different brain regions. My data also shows a relatively consistent ∼30-fold increase in eIF2α(P) at 10 min. reperfusion in all brain regions except thalamus, which had an ∼18-fold increase. The eIF2α(P) persisted in all brain regions at 60 min. reperfusion, except in brainstem which showed a 50% reduction. However, there was a trend to a decrease in cerebellum and thalamus, and an increasing trend in hippocampus and cortex. This suggests that eIF2a(P) is clearing from more resistant regions and staying elevated in vulnerable regions.; Nitric oxide has been implicated in UPR-type stress in neurons. My results show that with brief durations of brain ischemia and reperfusion eIF2α phosphorylation is somewhat attenuated in mice with homozygous knockout of eNOS and in wild-type mice treated with L-NAME. This suggests that NO does contribute to eIF2α(P) formation during reperfusion.