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在于氧化应激、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，因为它们含有高水平的活性氧。甲硫氨酸亚砜还原酶A（MsrA）负责修复甲硫氨酸氧化蛋白和“清除”活性氧。MsrA水平随着年龄的增长而下降，这表明这种酶的活性下降有助于老年生物体的氧化应激增加。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。本申请中描述的工作集中于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期间对氧化应激的敏感性。该假设将通过以下具体目的来解决：（i）确定MsrA是否防止原代多巴胺神经元的选择性死亡;（ii）确定MsrA是否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的MsrA的底物。原代多巴胺神经元的活力将通过用MsrA编码慢病毒感染的胚胎中脑培养物的免疫细胞化学分析来确定。通过测量蛋白羰基（通过斑点印迹分析）、甲硫氨酸氧化蛋白（通过氨基酸分析或质谱法）和核周侵袭体（通过免疫荧光显微术）的丰度，评估MsrA过度产生、永生化多巴胺神经元中氧化蛋白损伤的程度。该项目将推进与衰老和与年龄有关的疾病的发病机制的知识。因此，它与国家老龄问题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杀死提供线索。反过来，这一信息可能会刺激开发新的治疗方法，治疗与年龄有关的大脑疾病。

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.