Single and double strand DNA breaks activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) which catalyzes the cleavage of NAD+ into nicotinamide and ADP-ribose and synthesizes branched polymers of ADP-ribose (PAR) on nuclear acceptor proteins. Although this process ordinarily assists DNA repair and long-term DNA stabilization, severe oxidative damage seen in models of stroke and NMDA excitotoxicity is thought to lead to over-activation of PARP and subsequent neuronal cell death by a mechanism that does not produce classical apoptotic morphology. Previous work demonstrates that NMDA excitotoxicity can produce a novel form of programmed cell death that is independent of caspase activation and that involves the translocation of apoptosis inducing factor (AIF) from mitochondria to the nucleus. This translocation requires PARP activation and may depend on a PAR signaling mechanism in oxidatively stressed cells. Preliminary data shows that PARP2 gene deletion, like PARP1 gene deletion, confers robust ischemic protection. The overall goal of this project is to investigate the potential molecular mechanisms by which activation of PARP promotes cell death in experimental stroke. The time course ofAIF translocation to the nucleus will be contrasted after different durations of transient focal cerebral ischemia in ischemic core and border regions. The dependency of AIF translocation on neuronal generation of nitric oxide and on subsequent activation of PARP1 and PARP2 will be determined with appropriate knockout animals and pharmacological inhibitors. The role of AIF in focal ischemic injury will be determined using conditional AIF knockout and stable AIF knockdown animal strains. Because AIF mediated cell death in C. elegans involves DNA fragmentation by endonuclease G, the role of this endonuclease in ischemic injury will be assessed in endonuclease G knockout animals. Finally, the role of PAR in signaling AIF translocation will be evaluated by manipulating PAR catabolism in animals with increased and decreased expression of PAR glycohydrolase (PARG). Preliminary evidence showing a decrease in infarct volume with PARG over-expression and an increase in infarct volume with PARG knockdown supports a role of PAR in the signaling process. Therefore, the proposed experiments will generate new insights into the potential role of a novel form of programmed cell death in vivo that may be critical during the evolution of injury from stroke.
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