IDENTIFICATION OF DEATH REGULATORY GENES IN ISCHEMIA

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Project 1: Identification of Death-Regulatory Genes in Ischemia A growing body of evidences supports the concept that death regulatory genes modulate the fate of neurons in the setting of ischemic injury. The hypothesis of these studies is that survival os ischemic neurons is determined, at least in part, by the expression of death-promoter and death-suppressor genes. Thus, blockade of the expression of the effects of death promoter genes or overexpression of death suppressor genes or mimicking their effects could result in novel therapies for stroke. The broad long-term objectives of project 1 are to identify and clone known and novel death-promoter or death-suppressor genes whose expression is increased in rat brain after global ischemia. Cloning these genes will allow for the development of techniques by which expression of these genes may be selectively altered and provide insights into the mechanism of ischemic neuronal death. The following specific aims will be addressed: 1) Identify, fully sequence, and characterize the expression of the rat brain homologues of cysteine protease death promoter genes that are induced in CA1 neurons prior to their death from global ischemia. Candidate genes include cpp 32, ich-2, ich-2 and mch-2. 2) Identify, fully sequence and characterize the expression of the rat brain homologues of bcl-2-related death-promoter or death-suppressor genes, that are induced in CA1 neurons prior to their death from global ischemia. Candidate genes include Bax, bik, and bak, mcl-l, bfl-1 bag-l. 3) Clone novel genes induced in ischemic rat brain identified by subtractive hybridization and differential screening. Candidate death- promoter genes will be identified by their expression in CA1 neurons prior to death. Candidate death-suppressor genes will be identified by their expression in neurons that survive ischemia, such as those in CA3 or dentate gyrus. In this project we will determine which of the known death-regulatory genes are expressed in rat brain following global ischemia and identify novel genes whole expression is induced by global ischemia. The effect of these genes upon the survival of the neuron will be tested in vitro (project 2) and selected genes will be examined in ischemia in vivo (project 3).