The principal objective of this research proposal is to test the hypothesis that cerebral hypoxia causes immediate, measurable damage to mitochondrial and nuclear DNA, and activates DNA repair in the rat brain. In humans, cerebral hypoxia is a component of severe brain insults, including trauma, stroke and perinatal asphyxia. These insults may cause long term neuropathological and developmental deficits. We propose to address the premise that DNA damage and efficiency of DNA repair are among the early determinants of cellular survival in the brain, after hypoxic/ischemic injury. To date, DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Likewise, activation of genes involved in DNA repair and correlation of their expression with cell survival or cell death in the brain has not been examined. Our preliminary findings show that hypoxia induces immediate DNA damage, AP-endonuclease (APE), DNA polymerase b and the mammalian homologue of MutY (MYH) glycosylase are activated. Dissimilar repair kinetics for nuclear and mitochondrial DNA are also observed following hypoxia. Furthermore, with hypoxic insult, MYH appears to translocate into the mitochondria. This is the first evidence, to our knowledge, suggestive of activation of DNA repair in the mitochondria after cerebral hypoxia.
Our Specific Aims are: 1) To measure hypoxia-induced damage and repair in nuclear and mitochondrial DNA and to determine the dose response relationship between severity of hypoxia and the extent and distribution of DNA damage. 2) To map in situ, in specific cells populations, distribution of hypoxia-induced DNA damage. 3) To characterize the hypoxia-induced DNA damage response: mechanisms of activation of DNA damage repair enzymes and sensing molecules. Two sensitive Quantitative PCR (QPCR) assays are used to measure DNA damage. One measures the integrity of the mitochondrial genome and the other measures the integrity of nuclear DNA. The later is a novel assay, developed in our laboratory, which utilizes the high copy number of Short Interspersed DNA Elements (SINEs) mammalian genomes, to simultaneously amplify long random segments of nuclear DNA. Due to the high magnitude of attainable signal, we have adapted this assay for in situ detection of DNA damage and repair in specific cell populations in the brain. Our studies are intended to help understand the relevance of DNA damage to neuronal survival and, whether the DNA repair response is consequential to preservation and survival of neuronal cells. This information will augment strategies seeking to reduce neuropathologies of brain hypoxia in humans.
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