Antioxidant function of MsrA in dopaminergic neurons

描述（由申请人提供）：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。氧化应激触发衰老细胞中的蛋白质损伤，包括蛋氨酸残基氧化为蛋氨酸亚砜。多巴胺神经元在衰老和帕金森病中极易受到氧化应激的影响，因为它们含有高基础水平的活性氧。蛋氨酸亚砜还原酶A （MsrA）负责修复蛋氨酸氧化蛋白和“清除”活性氧。MsrA水平随着年龄的增长而下降，表明这种酶活性的下降有助于老年生物体氧化应激的增加。这项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。本应用程序中描述的工作主要集中在MsrA在多巴胺神经元中的作用。据推测，MsrA保护多巴胺神经元免受氧化损伤，并且MsrA活性的丧失增加了衰老过程中多巴胺神经元对氧化应激的敏感性。这一假设将以以下具体目标来解决：(i)确定MsrA是否阻止初级多巴胺神经元的选择性死亡；（ii）确定MsrA是否能阻止多巴胺神经元氧化蛋白损伤；（iii）评估蛋氨酸氧化的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力将通过免疫细胞化学分析确定感染msra编码慢病毒的胚胎中脑培养物。通过测量蛋白质羰基（通过点印迹分析）、蛋氨酸氧化蛋白（通过氨基酸分析或质谱分析）和核周聚合体（通过免疫荧光显微镜）的丰度，可以评估msra过量产生的永生化多巴胺神经元的氧化蛋白损伤程度。该项目将推进与衰老和年龄相关疾病有关的发病机制的知识。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氏症中被选择性杀死提供线索。反过来，这些信息可能会刺激治疗与年龄有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.