Stroke is the third leading cause of death and disability in the United States. Glutamate excitotoxicity is a major mediator of neuronal damage in focal ischemia models of stroke. In culture and animal models, glutamate mediates most of its toxicity through activation of NMDA receptors, activation of Ca2+ dependent enzymes, including neuronal nitric oxide synthase (nNOS). The increase in intracellular Ca 2+ results in increased superoxide anion which can react with NO to from peroxynitrite that can induce DNA nicks and breaks subsequently activating the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Pharmacologic inhibition or genetic knockout of PARP- 1 provides significant neuroprotection against experimental models of stroke both in vitro and in vivo. However, the mechanism(s) by which PARP-1 activation mediates neuronal cell death are not known. Recently, apoptosis inducing factor (AIF) has been identified as key mediator of neurotoxicity triggered by glutamate, reactive oxygen species and DNA damage. AIF resides in the mitochondria in normal healthy cells but moves to the nucleus following a lethal stimulus in a PARP-1 dependent manner. Blocking AIF from entering the nucleus can spare cells from death. The proteins that are involved in facilitating the release of AIF from the mitochondria and the proteins that AIF activates in the nucleus to execute the cell are not known. The following aims are proposed to explore the actions of AIF in neurotoxicity: 1) What is the role of AIF in excitotoxic (NMDA) mediated neuronal injury? 2) What is the role of caspase-dependent versus caspaase-independent mediated cell death in reactive oxygen species induced cellular injury and excitotoxic (NMDA versus Kainate/AMPA) brain injury? 3) What is the relationship and is there an interaction between AIF and EndoG in excitotoxic (NMDA versus Kainate/AMPA) brain injury? 4) Identification of casapase-independent cell death effectors and proteins that participate in the AIF cell death signal cascade. We hope that understanding the activation of AIF and the identification of additional components of the AIF signaling complex will lead to new methods to terminate its toxic actions.
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