The broad long-term objective of the proposed work is to understand the role of environmental toxins in Parkinson's Disease. This disorder affects 1% of people over age 40 and 50,000 new cases are diagnosed per year in the United States. Exposure to the herbicide paraquat is associated with an increased risk of Parkinson's Disease. Lewy bodies, the pathological marker of the disease, are abnormal protein aggregates found in cells of the substantia nigra. The main hypothesis is that paraquat-induced reactive oxygen species lead to Lewy body formation. The proposed in vitro studies focus on the reactive oxygen species, hydrogen peroxide, and two proteins found in Lewy bodies, cytochrome C and alpha-synuclein. The specific hypothesis to be tested is that four steps link the formation of hydrogen peroxide to the formation of Lewy bodies: 1) hydrogen peroxide induces tyrosine free radicals on cytochrome C; 2) the cytochrome C radicals are transferred to tyrosine residues on alpha-synuclein; 3) these radicals react to form covalent crosslinks; and, 4) the covalent crosslinks accelerate alpha-synuclein aggregation. Each step is a specific aim.
Aim 1 is to quantify the hydrogen peroxide-induced radicals on the surface of cytochrome C.
Aim 2 is to quantify the tyrosine radicals transferred from cytochrome c to alpha- synuclein.
Aim 3 is to show that the cytochrome C and alpha-synuclein radicals react to form covalent crosslinks.
Aim 4 is to show that crosslinking accelerates alpha-synuclein aggregation. Spin trapping will be used to stabilize the radicals. Mass spectrometry will be used to count the radical sites. Electron paramagnetic resonance will be used to identify the type of radical. Site-specific variants of both proteins will used to locate the radicals in the primary structure. Mass spectrometry and amino acid analysis will be used to demonstrate crosslinking, and SDS PAGE will be used to assess aggregation.
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