Role of Microglia in Ethanol-induced Oxidative Stress

描述(申请人提供)：酒精暴露会导致与神经炎症有关的神经毒性。小胶质细胞是中枢神经系统(CNS)中的免疫细胞，被认为是神经元氧化和亚硝化应激的主要贡献者；然而，关于小胶质细胞在酒精诱导的神经元功能障碍中的作用，尤其是在神经元-小胶质细胞相互作用的背景下，人们知之甚少。在这项提案中，我们将重点阐明亚硝化应激在酒精诱导的小胶质细胞激活中的作用，最终 会导致随后的神经元损伤。我们将使用新的质谱学和蛋白质组学方法来鉴定和量化亚硝化应激相关蛋白修饰的差异蛋白表达和变化，以验证我们的工作假设，即酒精诱导的神经毒性至少部分是由小胶质细胞激活引起的亚硝化性损伤介导的，而小胶质细胞激活反过来又通过与周围神经元的相互作用来调节。我们提出了两个特定的目标来确定小胶质细胞对酒精暴露的反应以及这种反应是如何通过与神经元的相互作用来改变的，并确定了亚硝化蛋白修饰在乙醇暴露引起的细胞功能障碍中的作用。在SA1中，我们将使用细胞培养中氨基酸的稳定同位素标记(SILAC)，然后进行定量质谱分析来分析这些细胞培养系统中差异蛋白质的表达。在SA2中，新的基于活动的 在SA1中描述的细胞培养模型中，蛋白质图谱(ABPP)探针将被用来准确地测量(通过亲和纯化，然后进行半定量质谱分析或荧光可视化)促炎蛋白标记物-诱导型一氧化氮合酶。我们的研究结果将进一步深入了解小胶质细胞在乙醇诱导的神经变性中的作用，并确定新的治疗策略的潜在靶点，以治疗或预防长期过量饮酒带来的神经元损伤。

DESCRIPTION (provided by applicant): Ethanol exposure induces neurotoxicity that is related to neuroinflammation. Microglia serve as immune cells in the central nervous system (CNS) and have been implicated as the primary contributor to oxidative and nitrosative stress on neurons; however, little is known regarding the role of microglia in alcohol-induced neuronal dysfunction, especially in the context of neuron-microglia interplay. In this proposal, we will focus on elucidating the role of nitrosative stress in alcohol-induced microglial activation that ultimately leads to subsequent neuronal injury. We will employ novel mass spectrometric and proteomic methods to identify and quantify differential protein expression and changes in nitrosative stress-related protein modifications to test our working hypothesis that alcohol-induced neurotoxicity is mediated, at least in part, by nitrosative injury from microglial activation whichis in turn modulated by interaction with surrounding neurons. We propose two specific aims to determine the response of microglia to ethanol exposure and how that response is modified by interaction with neurons, and define the role of nitrosative protein modification in cellular dysfunction induced by ethanol exposure. In SA1, we will employ stable isotope labeling with amino acids in cell culture (SILAC) followed by quantitative mass spectrometric analysis to profile differential protein expression in these cell culture systems. In SA2, novel activity-based protein profiling (ABPP) probes will be utilized to accurately measure (through affinity purification followed by semi-quantitative mass spectrometry or fluorescence visualization) a pro-inflammatory protein marker, inducible nitric oxide synthase, in the cell culture models described in SA1. The results from our proposed studies will provide further insight into the role of microglia in ethanol-induced neurodegeneration and identify potential targets for new therapeutic strategies for the treatment or prevention of neuronal injury brought about by excessive and long-term alcohol consumption.