ROLE OF NEURONAL NOS AND SUPEROXIDE IN NEURODEGENERATION

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The long-term goal of this proposed research is to understand the role of ROS and RNS in the onset of age-related neurodegenerative diseases. The general hypothesis to be tested is that nNOS can generate both superoxide and nitric oxide in a ratio that is regulated by flavin cofactors, redox active compounds, L-arginine and tetrahydrobiopterin (BH4). The specific hypothesis to be tested is that nNOS plays a crucial role in the neurotoxicity of MPP+, a mitochondrial neurotoxin that induces Parkinsonian symptoms. Specific aims: 1) the kinetic of superoxide and NO formation from purified nNOS in the presence and absence of MPP+ will be determined. 2) The role of nNOS in the cytotoxic and apoptotic effects of MPP+ in neuronal cells will be determined. Similar experiments will be determined in BH4-deficient neuronal cells that do not synthesize NO. In these systems, ROS and RNS formation will be measured. 3) The effect of MPP+ toxicity and apoptosis in neuronal cells isolated from nNOS knockout mice will be determined. Methods: Extracellular generation of superoxide will be measured by spin-trapping and intracellular formation of superoxide will be determined by monitoring mitochondrial aconitase activity. Aconitase carbonyls and nitrated protein will be determined by immunochemical methods. significance results from this study should provide new mechanistic insight into ROS and RNS formation from nNOS. Neuronal NOS has been implicated in the onset of several age-related neurodegeneration disorder including Parkinson's, Huntington's, and Lou Gehrig's diseases. Inhibition of nNOS and scavenger of ROS have been shown to exert neuroprotection in animal model mimicking these pathologies. New therapeutic strategies for treating age-associated neurodegenerative diseases may emerge from these studies. Novelty: Novel aspect of this proposal is the ability to measure simultaneously both NO and superoxide from purified nNOS in response to MPP+ and other co-factors. The superoxide-mediated oxidation of 4Fe-4S clusters in aconitase inactivates the enzyme and increases oxidative stress. The availability of novel superoxide dismutase mimetics has made it possible to prove more thoroughly regarding the nature of ROS causing the inactivation of aconitase in the intracellular milieu.