Hypoxic cell death in the form of stroke and myocardial infarction is the largest single cause of death in the United States. The fundamental molecular mechanisms of hypoxic cell death are incompletely understood. Studies in genetically tractable model organisms such as Caenorhabditis elegans and Drosophila have made major advances in the fields of cell death, hypoxia sensing, and adaptation. However, direct screens for mutations that alter hypoxia-induced cell death have not been reported in C. elegans and results in Drosophila have been limited. The proposed work will utilize the powerful genetic tools in C. elegans to identify determinants of hypoxic cell death.
The specific aims are: 1) Identify genes regulating hypoxic death in C. elegans. This will be accomplished by screening for mutant animals that are resistant to hypoxic death. In pilot mutagenesis screens, several mutants have been identified that are hypoxia resistant. One of the mutant genes, daf-2, codes for an insulin/IGF receptor, previously shown to regulate lifespan in C. elegans. The proposed work will similarly identify other genes regulating hypoxic death. 2) Define the mechanisms whereby the identified genes regulate hypoxic death. Once a number of hypoxic death genes are found, the question will become how do they perform this function. The sequence of the gene product will likely suggest potential mechanisms and pathways through which the genes function. The cell types protected from cell death and the cell types in which the genes are expressed will be determined. For each identified gene, specific hypoxic protective mechanisms will be tested including: reduction in superoxide levels, alterations in oxygen consumption, enhancement of hypoxic preconditioning, and regulation of apoptotic or necrotic death. 3) Use DNA microarrays to identify gene products controlling hypoxic response/survival. The hypoxic death genes will likely control the expression levels, either directly or indirectly, of a number of other genes mediating their function. To find these """"""""downstream"""""""" genes, the RNA expression levels of the whole C. elegans genome will be measured in mutant and wild type animals in normal and hypoxic conditions using DNA microarrays. The role of outstanding candidates implicated by the microarrays in the control of hypoxic cell death will be tested by reducing and increasing their function through RNA-mediated interference and over-expression experiments. The combination of genetic, and genomic tools in C. elegans should provide novel directions for vertebrate studies aimed at ameliorating damage from hypoxic insults.
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