Synucleinopathies include a broad spectrum of neurodegenerative disorders with a common athoanatomical feature, the accumulation of filamentous neuronal and glial a-synuclein (AS) inclusions. Mutations in AS promote biophysical changes that accelerate the formation of filamentous inclusions and mutations of AS have been linked to familial Parkinson's disease (PD). However, Lewy bodies (Lbs) and dystrophic (Lewy) neurites in sporadic PD, dementia with Lbs (DLB), the LB variants of Alzheimer's disease (LBVAD) and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA) contain filaments formed by wild type a-synuclein. The potential biochemical and biophysical alterations of wild type AS that promote the formation of aggregates in these diseases are not known. A potential major cause of neuronal injury in some of these neurodegenerative diseases is oxidative stress. Oxidative stress is the result of overproduction of reactive species that overwhelms the cellular anti-oxidant capacity leading to inactivation of key cellular functions and ultimately to cell death. Proteins are major targets of reactive species and recently we described specific protein modifications induced by reactive species in animal and cellular models of neurodegeneration. These modifications include the nitration of tyrosine residues to form 3-nitrotyrosine and nitrosylation of cysteine to form S-nitrocysteine. AS contains 4 tyrosine but no cysteine residues and contrary to most of the proteins studied to date all four tyrosine residues are highly susceptible to nitration. Preliminary data indicated that AS is specifically modified by nitration in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) mouse model of PD and nitration of AS induces protein oligomerization. Moreover, alpha-synuclein inclusions in PD, DLB and MSA patients stain specifically with antibodies to nitrotyrosine. Based on these data we developed the hypothesis that nitration of AS is a critical post-translational modification responsible for the aggregation and the formation of synuclein inclusions. To test the critical aspects of this hypothesis we propose to: 1) Examine the local and long-range effects of nitration and/or oxidation in the biochemical and biophysical properties of AS. 2) Determine the nature and the molecular mechanism for the aggregation of nitrated and/or oxidatively modified AS. 3) Compare and contrast the kinetics of aggregation and the kinetics of repair and proteolytic removal of modified AS. 4) Investigate the nature of post-translational modifications of AS in PD and other related synucleinopathies. Overall this project is focused on investigating the possible biochemical and biophysical mechanisms responsible for the abnormal function of AS in PD and other related synucleinopathies. Insight into the molecular mechanisms of the apparent abnormal function of the protein may improve strategies for the development of therapeutic interventions in PD and related disorders.
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