Role of Microglia in Ethanol-induced Oxidative Stress

小胶质细胞在乙醇诱导的氧化应激中的作用

基本信息

批准号：
8445822
负责人：
BIN LIU
金额：
$ 22.48万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-05 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8445822
关键词：
3-nitrotyrosine Acute Affinity Affinity Chromatography Alcohol consumption Alcohol-Induced Neurotoxicity Alcoholic Intoxication Alcoholism Alcohols Amino Acids Brain Cell Communication Cell Culture System Cell Culture Techniques Cells Chronic Coculture Techniques Consumption Detection Ethanol Evaluation Fluorescence Functional disorder Goals Imagery Immune Immune response Immunoprecipitation Inflammatory Inflammatory Response Injury Label Lead Life Mass Spectrum Analysis Measures Mediating Methodology Methods Microglia Modeling Modification Molecular Nerve Degeneration Neuraxis Neuroglia Neuronal Dysfunction Neuronal Injury Neurons Nitrogen Organ Oxidative Stress Oxygen Peripheral Play Post-Translational Protein Processing Prevention Process Production Proteins Proteomics Rattus Research Role Stable Isotope Labeling Stress Surveys Technology Testing Therapeutic Intervention Tissues Work activity-based protein profiling alcohol abuse therapy alcohol effect alcohol exposure base cell type chronic alcohol ingestion human NOS2A protein innovation insight neuroinflammation neurotoxicity new therapeutic target nitrosative stress novel novel therapeutics protein expression public health relevance research study response stable isotope treatment strategy

项目摘要

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.

描述（由申请人提供）：乙醇暴露会引起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，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引起的神经元功能障碍中的作用，特别是在神经元与小胶质细胞相互作用的背景下，人们知之甚少。在本提案中，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，最终导致随后的神经元损伤。我们将采用新颖的质谱和蛋白质组学方法来识别和量化差异蛋白表达以及亚硝化应激相关蛋白修饰的变化，以检验我们的工作假设，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损伤介导的，这是反过来又通过与周围神经元的相互作用进行调节。我们提出了两个具体目标，以确定小胶质细胞对乙醇暴露的反应以及如何通过与神经元的相互作用来改变该反应，并确定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，我们将在细胞培养物 (SILAC) 中采用稳定同位素标记氨基酸，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质表达。在 SA2 中，基于新颖的活动蛋白质分析 (ABPP) 探针将用于准确测量（通过亲和纯化，然后进行半定量质谱或荧光可视化）SA1 中描述的细胞培养模型中的促炎蛋白标记物、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引起的神经变性中的作用，并确定治疗或预防长期过量饮酒引起的神经元损伤的新治疗策略的潜在靶点。