Role of Microglia in Ethanol-induced Oxidative Stress

描述（由申请方提供）：乙醇暴露诱导与神经炎症相关的神经毒性。小胶质细胞作为中枢神经系统（CNS）中的免疫细胞，并已被牵连作为对神经元的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知之甚少，特别是在神经元-小胶质细胞相互作用的背景下。在这个建议中，我们将重点阐明亚硝化应激在酒精诱导的小胶质细胞活化中的作用， 导致神经元损伤。我们将采用新的质谱和蛋白质组学方法，以确定和量化差异蛋白质表达和亚硝化应激相关蛋白质修饰的变化，以测试我们的工作假设，即酒精诱导的神经毒性是介导的，至少部分是由亚硝化损伤从小胶质细胞活化，这反过来又调制与周围神经元的相互作用。我们提出了两个具体的目标，以确定乙醇暴露的小胶质细胞的反应，以及如何通过与神经元的相互作用来修改该反应，并定义亚硝化蛋白修饰在乙醇暴露诱导的细胞功能障碍中的作用。在SA 1中，我们将采用稳定同位素标记细胞培养物中的氨基酸（SILAC），然后进行定量质谱分析，以分析这些细胞培养系统中的差异蛋白表达。在SA 2中，新的基于活动的 蛋白质谱分析（ABPP）探针将用于在SA 1中描述的细胞培养模型中准确测量（通过亲和纯化，然后进行半定量质谱分析或荧光可视化）促炎蛋白标志物，诱导型一氧化氮合酶。我们提出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退行性变中的作用，并确定新的治疗策略的潜在靶点，用于治疗或预防过度和长期饮酒引起的神经元损伤。

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.