Nuclear Bcl-2 Promoted Apoptosis: Relevance to Stroke

University Of Texas Medical Br Galveston, Galveston TX

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Abstract

DESCRIPTION (provided by applicant): Ischemic stroke is an especially grievous disease due to the functional deficits inflicted on victims following initial insult. One proposed treatment regimen is Bcl-2 overexpression following initial ischemia. While Bcl-2 is a known antiapoptotic protein when stably expressed at the mitochondria, its function at extra-mitochondrial sites (nuclear membrane and ER) is uncharacterized. Therefore, generalized Bcl-2 overexpression following initial ischemia could result in Bcl-2 at multiple cellular locations (mitochondria, ER and nuclear membrane) and even increase rather than prevent cell death. This project will investigate Bcl-2's localization following differential expression and determine localization specific changes in apoptosis regulation in the well characterized, neuron-like, PC12 cell line. The techniques of subcellular fractionation, western blot, flow cytometry (nuclei and whole cells) and confocal calcium imaging will be used to test the following hypotheses: 1.) Short term, induced Bcl-2 expression promotes nuclear Bcl-2 localization an event that promotes apoptosis. 2.) Bcl-2 localized at the nuclear membrane can be removed following elevation in FKBP38 (chaperone protein) expression. 3) The mechanism nuclear Bcl-2 utilizes to induce apoptosis is acted through elevating intra-nuclear calcium concentrations. These results will provide important new information on the cellular localization and function of Bcl-2 following rapid overexpression (endogenous or gene-therapy). They will also provide valuable insight into potential therapeutic interventions that would invoke proper Bcl-2 localization which in turn would induce anti-apoptotic Bcl-2 function, ultimately saving neurons and decreasing functional loss following ischemic stroke.