DNA Adducts Formed by Dopamine

描述(摘自申请者的摘要)：导致帕金森病发展的病理生理过程一直是 被确认为视网膜部致密部的多巴胺能神经元的丢失 黑质。尽管进行了深入的研究，但相关的分子机制 因为这些神经元的选择性丢失仍不清楚。耐人寻味的特点 这些神经元中有神经黑色素的存在。它的生物学功能 这些细胞中的神经黑色素是未知的，目前对其 黑色素的合成是基于黑色素的生物合成。我们实验室里的研究 已经证明了神经黑色素的前体多巴胺可以 酶氧化和非酶氧化形成DNA加合物和 氧化碱基损伤。基于这些观察和独特的联系 神经黑色素的存在和特定的神经细胞丢失，我们建议 确定神经黑色素的合成过程是否会导致 DNA损伤的可能性。为了实现这一目标，我们建议：1)优化 ~(32)P-后标记方法检测稳定DNA加合物 电化学检测法测定不稳定加合物和 由多巴胺形成的氧化性碱基损伤。1b)确定结构(S) 使用组合光谱技术的多巴胺形成的DNA加合物。 2)将人类酪氨酸基因插入到表达载体中。所表达的 蛋白质将进行亲和纯化和鉴定。这种人体酶将会是 用于研究多巴胺的氧化，并将提供关于 产生多巴胺引起DNA损伤的酶机制。3)我们会 使PC12细胞在转录调控下表达酪氨酸。这些 细胞，PC12/Tyr，将被用来确定在 神经黑色素合成。对这些细胞的平行研究将调查 神经黑素合成过程中细胞毒性的诱导。我们相信 这些特定的目标将使我们能够检验我们的假设DNA损伤 可发生在神经黑色素的合成过程中。此外，结果显示， 这些研究将提供独特的分子标记，将在未来使用 评估这一过程是否发生在黑质的研究 人类大脑的。

DESCRIPTION (Adapted from applicant's abstract): 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.