Protein ubiquitination based neuroprotection for stroke

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Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Classic tolerance is gene-mediated, protein synthesis-dependent and requires at least 24 hours to occur. In contrast, rapid ischemic tolerance occurs within 1 hour of the preconditioning ischemia and is not dependent on the synthesis of new proteins. As such understanding the endogenous mechanism of neuroprotection which are involved in rapid ischemic tolerance, may identify novel fast acting therapeutic strategies to treat acute stroke. Our preliminary data suggests that cell death promoting proteins are rapidly down regulated, following preconditioning, which may account for the neuroprotective effects observed in rapid ischemic tolerance. Our central hypothesis is that brief ischemia stimulates the generation of rapid ischemic tolerance by acute alterations in the levels of cell death promoting proteins by the process of ubiquitination, which targets the proteins for degradation by the proteasome. The specific aims are:-1. We will test our hypothesis using a candidate cell death promoting protein, Bim. Experiments are designed to investigate Bim ubiquitination and degradation in the 1 hour period following ischemic preconditioning. 2. We will further test our hypothesis broadly with immunoprecipitation and mass spectrographic analysis to identify novel proteins that are ubiquitinated following preconditioning ischemia. Experiments will then further validate this data, and identify potential new targets for regulating cell death following ischemia. These studies will support our hypothesis that protein down regulation is a powerful endogenous neuroprotective mechanism in the brain. We will identify the molecular mechanisms that favor the degradation of pro-apoptotic proteins, which may offer new acute therapeutic targets for the treatment of stroke and other neurological insults, which would not be available via gene modulation strategies based on classic tolerance, requiring over 24h to exert a neuroprotective effect.