PEROXYNITRITE IN NEURODEGENERATIVE DISEASES OF AGING

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DESCRIPTION (Adapted from the Applicant's Abstract): The activity of Tyrosine Hydroxylase (TH) is essential for the production of catecholamines. During the progression of Parkinson's disease (PD) distinct changes in TH activity and concentration have been described. A decrease in dopamine levels without a loss of either TH immunoreactivity or dopaminergic neurons has been described during the early phase of the disease. The middle stage of the disease is characterized by a loss in dopamine and immunoreactive TH without a loss of dopaminergic neurons. Loss of dopamine, TH and dopaminergic neurons characterize late phase of the disease. These distinct events in PD are faithfully reproduced in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of PD. However, the biochemical basis to explain the changes in TH, activity and content prior to the death of dopaminergic neurons are not clearly understood. Our published data generated during the last two years of funding has provided a reasonable biochemical explanation for the changes in TH during the early phase of MPTP neurotoxicity. The data revealed that TH is a selective target for nitration. Nitration of tyrosine residues represents a post-translational protein modification that results from the reaction of nitrating agents with proteins. Nitrating agents such as peroxynitrite are formed during oxidative stress. Oxidative stress has been implicated in the pathogenesis of PD and in the MTPT neurotoxicity. The published data showed that for the first 6 hours post MPTP injection, nitration of a single tyrosine in TH results in the inactivation of the enzyme. The inactivation of TH paralleled the decline in dopamine levels in the mouse striatum whereas the levels of TH protein remain unchanged. However, 12 hours after the last MPTP injection, preliminary data indicated that an apparent non proteolytic, enzymatic process has repaired nitrated TH and this is reflected by an increase in the catalytic activity of the protein and in brain dopamine levels. At the same time the protein levels of TH have declined to nearly 50 percent of control. The loss of protein appears to be mediated by the ubiquitin-proteosome pathway. Based on these preliminary data we formulated the following working hypothesis: Protein nitration (specifically TH) represents a pathophysiology stimulus that is managed by two processes; non-proteolytic repair involving a unique denitrase, and/or protein degradation. Critical aspects of this working hypothesis will be examined by: 1) determining the kinetics of repair and degradation of TH in the mouse MPTP and in the PC12 cell models, 2) purifying and characterizing the brain denitrase activity, and 3) investigating the molecular mechanisms for the proteolytic degration of TH. This application is a natural extension of our previous work that elucidated the biochemical mechanism for the inactivation of tyrosine hydroxylase during the early stages of MPTP toxicity. The proposed experiments will elucidate biochemical, cellular and molecular changes in TH during the middle stages of MPTP toxicity and PD by integrating our experiences with protein nitration chemistry and biological chemistry of reactive species, with Dr. Horwitz's PC12 cell model and Dr. Przedborski's MPTP mouse model. The collaboration between the three different laboratories has been productive. Understanding the basic biochemical and molecular changes in TH during the progression of MPTP and PD will facilitate the development of approaches to correct the functional deficit in dopamine production in PD.