The nigrostriatal dopaminergic neurons show prominent degeneration in normal aging and are lost at a greater rate in Parkinson's disease. The level of protein carbonyl modifications in human substantial nigra is approximately 2-fold higher than in basal ganglia or other regions, indicating that oxidative stress is higher in the nigra. Protein carbonyls have been associated with aging in many animal systems, but their role in nigral neurodegeneration remains unclear.. Three series of experiments on the identify and function of protein carbonyls are proposed. 1) The types and amounts of carbonyls in soluble proteins from nigrostriatal regions of post-mortem human brain (substantial nigra and putamen) and control regions (prefrontal cortex and cerebellum) of subjects of a range of ages and from Parkinson's disease patients will be determined for the first time. Carbonyls will be quantitated either by LC/MS with selected- ion monitoring or by labeling with carbonyl-specific reagents (sodium borotritide or fluoresceinamine) and amino acid analysis. In parallel with these experiments we will study an abundant Schiff base-related oxidation product, epsilon-N-methyllysine, identified recently in this laboratory. This compound may result from reaction of formaldehyde with lysine residues. (2) Cellular mechanisms of oxidative stress underlying observed changes in carbonyl/Schiff base composition will be analyzed in the rat PC12 cell culture model. Cells will be subjected to oxidative stresses of anti-oxidant treatments and the resulting carbonyl and Schiff base compositions of cell proteins compared with those of human brain proteins. This comparative approach aims to gain insight into the modes of oxidative stress that exist in human brain in vivo. (3) The chemical structures of carbonyl-modified amino and subsequent impact on protein function. Functional consequences of carbonyl modifications Functional consequences of carbonyl modifications will be assessed by determining their locations in the structure of the calcium- regulated protein phosphatase, calcineurin, from rat and bovine brain. Structural analysis and activity assays of calcineurin modified by [`4C] formaldehyde will be performed to assess the effects of lysine Schiff base formation. It is hoped that this research will lead to new neuroprotective strategies to preserve the vulnerable dopaminergic neurons of the substantia nigra.
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