Stroke is the third leading cause of death and disability in the United States. Glutamate is a major mediator of neuronal damage in focal ischemia models of stroke. This excitotoxicity is mediated in part by mitochondrial dysfunction and free radical induced oxidative damage. The culture and animal models, glutamate mediates most of its toxicity through activation of NMDA receptors. The increase in intracellular Ca2+ concentration leads to an activation of Ca2+ dependent enzymes, including neuronal nitric oxide synthase (nNOS) which produces nitric oxide (NO). Additionally the increase in intracellular Ca2+ results in increased superoxide anion which can react with NO to from peroxynitrite. Increased peroxynitrite can induce DNA nicks and breaks which activates the nuclear enzyme, poly (ADP-ribose) polymerase (PARP) which catalyzes the transfer of ADP-ribose from NAD to nuclear proteins. For every model of NAD consumed, four free energy equivalents of ATP are required to restored NAD levels. It is possible that this consumption of NAD and subsequently ATP in an injured cell leads to cell death, however, the mechanism(s) by which PARP activation mediates neuronal death are not known. Consistent with pharmacologic inhibition of PARP, primary cortical cultures from PARP knockout mutant mice are resistant to NMDA and NO neurotoxicity and combined oxygen-glucose deprivation induced neuronal damage. Consistent with these in vitro findings, PARP knockout mutant mice have remarkably reduced infarct volumes following transient focal ischemia. Protection in the PARP knockout cultures and mice exceed protection observed with nNOS knockout mice or following treatment with the NMDA receptor antagonist, MK801 suggesting that other cell death signals may converge on PARP to elicit toxicity in ischemia-reperfusion injury. PARP is a complex protein. The biochemistry and cell biology of PARP in post-mitotic neurons is not known. The mechanism of activation of PARP in post-mitotic neurons is assumed to be DNA nicks but this is not known. The mechanism of PARP mediated neurotoxicity are not known. The following specific aims are proposed to investigate PARP neurobiology and discover the actions of PARP that result in neuronal cell death: 1. What is the role of PARP in excitotoxic versus apoptotic neuronal death? 2. What is the role of reactive oxygen species and DNA damage in the activation of PARP? 3. What is the interaction between caspase activation and PARP-mediated neurotoxicity? 4. What is the role of energy impairment in PARP mediated neuronal death? 5. What is the role of protein ribosylation in PARP mediated neuronal death?
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