Many neurodegenerative diseases, including Alzheimer's disease, huntington's disease, stroke and epilepsy have been linked to derangements in glutamate neurotransmission. This neurotoxicity is mediated in part through the generation of nitric oxide (NO). While inhibition of NO formation can be neuroprotective, the mechanism of NO-induced neurotoxicity has yet to be elucidated. One potentially important pathway may involve activation of poly (ADP-ribose) synthetase (PARS) activity. To address the potential role of PARS in mediating glutamate-induced free radical neurotoxicity the following specific aims are proposed. Experiments will be designed and performed to evaluate the role of PARS in neurotoxicity induced by exposure of neurons to neurotoxic concentrations of glutamate or free radicals. The effect of PARS inhibitors on neurotoxicity elicited by glutamate and its analogs as well as by NO, superoxide anion and peroxynitrite will be examined. The time course of these effects and the specificity of PARS inhibitors will be evaluated to determine whether the neuroprotective effects of PARS inhibitors are due to pharmacologic inhibition of the enzyme or due to interference with other cellular functions. To address conflicting reports regarding DNA damage following NMDA or NO neurotoxicity, cultures will be examined for both single and double stranded DNA breaks, the nuclear morphology will be examined with bisbenzimide and by electron microscopy, and blots of agarose gels will be examined for laddering. The activation of PARS under similar conditions will be determined by Western blot analysis and polyacrylamide gel autoradiography. In thymocytes, PARS elicited cytotoxicity corresponds with cellular depletion of NAD and ATP. In in vitro nuclear preparations and in tumor cell lines, PARS is involved in apoptotic events following cleavage to its 85 kd form form the full length 120 kd form. These two events may be complimentary or may occur differentially in different cell types. To evaluate if either event contributes to the development of neurotoxicity, primary neuronal cortical cultures will be exposed to neurotoxic agents and the cellular levels of NAD and ATP will be determined and sister cultures will be examined for PARS cleavage. If cleavage occurs then inhibitors of the protease which cleaves PARS will be evaluated for potential neuroprotective effects.
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