The pathophysiological processes responsible for the development of Parkinson's disease has been identified as a loss of dopaminergic neurons in the pars compacta of the substantia nigra. Despite intensive study, the molecular mechanisms responsible for the selective loss of these neurons remains unknown. An intriguing feature of these neurons is the presence of neuromelanin. The biological function of neuromelanin in these cells is unknown and the current understanding of its synthesis is based on that of melanin biosynthesis. Studies in our laboratory have demonstrated that the precursor to neuromelanin, dopamine can be enzymatically and non-enzymatically oxidized to form both DNA adducts and oxidative base damage. Based on these observations and the unique association of the presence of neuromelanin and specific neuronal cell loss we propose to determine whether the process of neuromelanin synthesis leads to the production of DNA damage. In order to achieve this goal we propose to: 1a) Optimize the 32P-postlabeling procedure for detection of stable-DNA adducts and HPLC with electrochemical detection for the quantification of both unstable adducts and oxidative base damage formed by dopamine. 1b) Identify the structure(s) of the DNA adducts formed by dopamine using a combination of spectroscopic techniques. 2) Insert the human gene for tyrosine into an expressed plasmid. The expressed protein will be affinity purified and characterized. This human enzyme will be used to study oxidation of dopamine and will provide information as to the enzymatic mechanisms for production of dopamine induced DNA damage. 3) We will engineer PC12 cells to express tyrosine under transcriptional control. These cells, PC12/tyr, will be used to determine if DNA damage occurs during neuromelanin synthesis. Parallel studies with these cells will investigate the induction of cellular cytotoxicity during neuromelanin synthesis. We believe that these Specific Aims will allow us to test our hypothesis that DNA damage can occur during the synthesis of neuromelanin. In addition, the results of these studies will provide unique molecular markers that will be used in future studies to evaluate whether this process is occurring in the substantia nigra of human brain.
