Shutdown of Translation and Ischemia/Stroke-Induced Cell Death

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DESCRIPTION (provided by applicant): Ischemia/stroke presents a major health problem worldwide. A prominent feature of ischemic cell death is an irreversible suppression of protein synthesis in vulnerable cells. Impairment of endoplasmic reticulum (ER) function has been identified as the mechanism underlying the shutdown of translation induced by ischemia. Our hypothesis has been that the post-ischemic impairment of ER function and subsequent long-lasting suppression of protein synthesis found in vulnerable neurons play an important role in the pathological process triggered by transient cerebral ischemia and culminating in neuronal cell death. To date this hypothesis has been based only on correlative evidence: a proof of the hypothesis is still lacking. Our Specific Aim is to test the hypothesis that the extent of ischemic cell death can be reduced by facilitating a recovery of protein syn- thesis and restoration of ER function. To this end, we will take advantage of the metabolic pattern found in vulnerable neurons after transient ischemia where protein synthesis is severely suppressed and transcription of stress genes is activated. We will perform experiments with neuronal cells transfected with genetic constructs exhibiting three distinct components that will guarantee that the protein required to restore function is synthesized specifically after ischemia. The constructs will contain the gene coding for a protein that is believed to facilitate recovery of protein synthesis and restoration of ER function, a sequence that will activate translation of the respective mRNA under conditions associated with suppression of protein synthesis, and a promoter with a consensus sequence for the binding of transcription factors known to be activated after ischemia. The genes coding for a potentially protective protein will include Bcl-2 targeted to the ER, GADD34, and GRP78, and we will use promoters with binding sequences for heat shock factors. Stably transfected cells will then be transiently exposed to oxygen/glucose deprivation, a severe form of stress causing metabolic disturbances that mimic those induced by transient ischemia. We will then investigate whether recovery of protein synthesis and restoration of ER function do indeed help cells to withstand the metabolic stress conditions. The proposed project will allow us to establish the conditional gene expression platform needed to confirm, in future animal experiments, the role of ischemia-induced impairment of ER function and subsequent shutdown of translation in the pathological process resulting in neuronal cell death. Furthermore, the conditional gene expression approach will enable us to elucidate mechanisms of neuronal cell death in various pathological states of the brain and other organs associated with suppression of global protein synthesis and activation of transcription factors. PUBLIC HEALTH RELEVANCE: Stroke is a major cause of morbidity and mortality, which according to the American Heart Association affects more than 700,000 citizens in the United States, resulting in more than 160,000 deaths per year and $55 billion direct and indirect annual costs. The proposed project is designed to elucidate the mechanisms underlying neuronal cell death caused by ischemia/stroke. The project will thus help to establish new avenues of therapeutic intervention to improving public health in the United States and therefore reducing the costs associated with stroke treatments.