Aging and Parkinson's disease are characterized by oxidative stress, proteasome dysfunction, and protein aggregation. Oxidative stress triggers protein damage in aged cells, including the oxidation of methionine residues to methionine sulfoxide. Dopamine neurons are highly vulnerable to oxidative stress in aging and Parkinson's disease because they contain high basal levels of reactive oxygen species. The enzyme methionine sulfoxide reductase A (MsrA) is responsible for repairing methionine-oxidized proteins and for 'scavenging' reactive oxygen species. MsrA levels decrease with age, suggesting that a decline in the activity of this enzyme contributes to increased oxidative stress in older organisms. The long-term objective of the proposed research is to understand how antioxidant proteins protect neurons from toxic phenomena associated with aging and Parkinson's disease. The work described in this application is focused on the role of MsrA in dopamine neurons. It is hypothesized that MsrA protects dopamine neurons from oxidative damage, and that a loss of MsrA activity increases the sensitivity of dopamine neurons to oxidative stress during aging. This hypothesis will be addressed with the following specific aims: (i) to determine whether MsrA prevents the selective death of primary dopamine neurons; (ii) to determine whether MsrA prevents oxidative protein damage in dopamine neurons; and (iii) to assess whether methionine-oxidized alpha- synuclein is a substrate of MsrA in dopamine neurons. The viability of primary dopamine neurons will be determined via immunocytochemical analysis of embryonic midbrain cultures infected with MsrA-encoding lentivirus. The extent of oxidative protein damage in MsrA-overproducing, immortalized dopamine neurons will be assessed by measuring the abundance of protein carbonyls (via dot-blot analysis), methionine- oxidized proteins (via amino-acid analysis or mass spectrometry), and perinuclear aggresomes (via immunofluorescence microscopy). This project will advance knowledge of pathogenetic mechanisms involved with aging and age-related diseases. Accordingly, it is directly relevant to the mission of the National Institute on Aging. The results of these studies will provide clues as to why dopamine neurons are selectively killed in aging and Parkinson's disease. In turn, this information may stimulate the development of new therapeutic approaches in the treatment of age-related diseases of the brain. ? ?
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