Experiments evidenced have implicated oxidative stress as a contributing element in the neuropathology of degenerative disorders of late life such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Oxidative stress is the result of over production of reactive species which can overwhelm cellular neuroprotective mechanisms and inactivate critical processes for preserving cellular integrity in function. While much of the existing data has concentrated on the contribution of oxygen-derived reactive species, recent evidence has also implicated nitric oxide derived oxidants in the pathogenesis of neuronal injury. The toxicity of reactive oxidant species is coupled with the reactivity of nitric oxide. Since both nitric oxide and superoxide or free radicals they react in a near diffusion limited rate to form peroxynitrite. Recent data has revealed that peroxynitrite is a major oxidant generated in biological systems under pathologic conditions demonstrating that the proposed chemistry is possible in vivo. Furthermore, peroxynitrite reacts selectively with nitrogen residues of protein to form nitrotyrosine. Protein tyrosine nitration results in the inactivation of protein function and interferes with tyrosine phosphorylation a key event in cellular signal transduction. Previously this group of collaborative investigators has shown that exposure of PC12 cells to low levels of peroxynitrite resulted in significant and irreversible inhibition of DOPA synthesis. Preliminary data indicates that peroxynitrite is mediated nitration of tyrosine residues in the active site of tyrosine hydroxylase could account for the inhibition of DOPA synthesis. Moreover, tyrosine hydroxylase is detected after exposure of PC12 lysates to peroxynitrite or after induction of endogenous peroxynitrite production. The major goal of this proposed grant proposal is to elucidate the role of peroxynitrite-mediated tyrosine nitration in the inactivation of tyrosine hydroxylase in vitro as well as cellular and animal models of aging and Parkinson's disease. The investigators propose two major specific aims. 1) to determine if tyrosine nitration is responsible for the peroxynitrite inactivation of tyrosine hydroxylase. 2) characterize the peroxynitrite mediated modifications present in tyrosine hydroxylase in three models 1) PC12 cells stimulate to generate peroxynitrite, 2) brains of aged rats and 3) mouse brains following administration of MPTP.
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