NITRIC OXIDE AS A MEDIATOR OF NEUROTOXICITY

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In primary neuronal cultures, N-methyl-D-aspartate neurotoxicity is mediated in part by nitric oxide (NO). The mechanisms involved in NO neurotoxicity, as well as the source of NO is not known. Accordingly, experiments will be performed and designed to further elucidate the role of NO in neurotoxicity. Conditions for the selective removal or inactivation of NO synthase (NADPH diaphorase) neurons will be developed. this will be followed by a detailed analysis of excitatory amino acid neurotoxicity in these cultures. It will be determined whether an inducible NO synthase is formed after excitatory amino acid administration, and whether it plays a role in NMDA neurotoxicity. NO synthase (NADPH diaphorase) neurons are known to be relatively resistant to NMDA neurotoxicity. Experiments will be performed to determine whether NO synthase is involved in this protection. Conditions for transient expression of NO synthase in neurons will be developed and excitatory amino acid neurotoxicity will be studied in detail. In addition, the role that antioxidant enzymes, such as copper/zinc-superoxide dismutase, manganese- superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase play in neurotoxicity and in neuroprotection will be studied. The role of the superoxide anion in mediating NO cell death will be investigated by exploring NMDA neurotoxicity, as well as exogenously applied NO in the presence of various inhibitors of superoxide dismutase and liberators of the superoxide anion. Furthermore, cell lines over and under-expressing manganese-superoxide dismutase will be developed. Employing these cell lines, studies on exogenously applied NO and its subsequent toxicity will be investigated. Finally, the functional consequences of phosphorylation or dephosphorylation of NO synthase will be investigated in primary neuronal cultures after excitatory amino acid administration. In addition, determination of the subtype(s) of glutamate receptor responsible for NO synthase activation will be identified by co-transfection studies.