Antioxidant function of MsrA in dopaminergic neurons

描述(申请人提供)：衰老和帕金森病的特征是氧化应激、蛋白酶体功能障碍和蛋白质聚集。氧化应激在衰老细胞中触发蛋白质损伤，包括将蛋氨酸残基氧化为甲硫氨酸亚砜。在衰老和帕金森氏病中，多巴胺神经元非常容易受到氧化应激的影响，因为它们含有高水平的基础活性氧物种。蛋氨酸亚硫氧化物还原酶A(MSRA)负责修复蛋氨酸氧化的蛋白质和清除活性氧。MSRA水平随着年龄的增长而下降，这表明该酶活性的下降导致了老年生物体中氧化应激的增加。这项拟议研究的长期目标是了解抗氧化蛋白如何保护神经元免受与衰老和帕金森病相关的毒性现象的影响。本申请中描述的工作重点是MSRA在多巴胺神经元中的作用。假设MSRA保护多巴胺神经元免受氧化损伤，并且MSRA活性的丧失增加了多巴胺神经元在衰老过程中对氧化应激的敏感性。这一假说的具体目的如下：(I)确定MSRA是否可以阻止原代多巴胺神经元的选择性死亡；(Ii)确定MSRA是否可以防止多巴胺神经元中的氧化蛋白损伤；以及(Iii)评估甲硫氨酸氧化的α-突触核蛋白是否是多巴胺神经元中MSRA的底物。原代多巴胺神经元的活性将通过感染编码MSRA慢病毒的胚胎中脑培养物的免疫细胞化学分析来确定。MSRA过度产生、永生化的多巴胺神经元的氧化蛋白质损伤程度将通过测量蛋白质羰基(通过斑点印迹分析)、蛋氨酸氧化蛋白质(通过氨基酸分析或质谱学)和核周侵袭体(通过免疫荧光显微镜)的丰度来评估。这个项目将促进对衰老和与年龄相关的疾病的致病机制的了解。因此，它与国家老龄研究所的使命直接相关。这些研究的结果将为为什么多巴胺神经元在衰老和帕金森病中被选择性地杀死提供线索。反过来，这些信息可能会刺激治疗与年龄相关的大脑疾病的新治疗方法的发展。

DESCRIPTION (provided by applicant): 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.