Calcineurin and inflammatory signaling processes in aging and Alzheimer's Disease

描述(申请人提供)：本项目中的实验使用创新的基因传递技术和多管齐下的方法来评估星形胶质细胞激活在阿尔茨海默病(AD)完整小鼠模型神经功能中的基础作用。研究使用携带星形胶质细胞特异性启动子Gfa2的腺相关病毒载体(AAV)靶向野生型和APP/PS1小鼠星形胶质细胞中的蛋白磷酸酶钙调神经磷酸酶(CN)和NFAT转录因子。AAV-Gfa2载体在不同年龄/疾病阶段双边输送到海马区，并对小鼠进行不同AD生物标志物的评估。在目标1中，认知状态通过主动回避任务来评估，而突触功能则通过海马片电生理学和突触蛋白的Western印迹测量来评估。在目标2中，使用陶瓷酶微电极阵列和谷氨酸转运体水平的测量来研究海马谷氨酸的调节。在目标3中，通过免疫组织化学(IHC)分析和使用多重ELISA评估细胞因子水平来确定神经胶质细胞激活和神经炎症的水平。在目标4中，使用IHC来确定A？ELISA用于定量可溶和不可溶的海马区组织中A？40和A？42的水平，Western ns用于评估BACE蛋白的表达。AAV-Gfa2载体编码CN/NFAT信号的有效抑制剂或激活剂，因此将决定这一星形细胞通路在驱动和/或维持AD小鼠神经功能障碍中的必要性和充分性。这些研究为研究激活的星形胶质细胞提供了一种非常新颖的方法，并可能对AD和其他神经退行性疾病的治疗策略的发展产生重大影响。 公共卫生相关性：越来越多的证据表明，激活的星形胶质细胞与各种神经退行性疾病有关，包括阿尔茨海默病(AD)。然而，这些细胞很难通过治疗选择性地靶向。在这个项目中，我们使用尖端的腺相关病毒载体选择性地阻止星形胶质细胞的激活，并改善完整的AD模型小鼠的神经功能。这种方法可能成为AD和其他神经退行性疾病的一种新的治疗策略。

DESCRIPTION (provided by applicant): The experiments in this project use innovative gene delivery techniques and a multipronged approach to assess the fundamental role of astrocyte activation in neurologic function in an intact mouse model of Alzheimer's disease (AD). Studies use adeno-associated virus vectors (AAV) bearing the astrocyte-specific promoter Gfa2 to target the protein phosphatase calcineurin (CN) and NFAT transcription factors in astrocytes of wild-type and APP/PS1 mice. AAV-Gfa2 vectors are bilaterally delivered to the hippocampus at different ages/disease stages and mice are assessed on different AD biomarkers. In Aim 1, cognitive status is assessed using the active avoidance task, while synaptic function is evaluated using hippocampal slice electrophysiology and Western blot measures of synaptic proteins. In Aim 2, hippocampal glutamate regulation is investigated using ceramic enzyme-based microelectrode arrays and measures of glutamate transporter levels. In Aim 3, levels of glial activation and neuroinflammation are determined with immunohistochemical (IHC) analyses and assessment of cytokine levels using Multiplex ELISAs. In Aim 4, IHC is used to determine the extent of A? deposition, while ELISAs are used to quantify levels of A?40 and A?42 in soluble and insoluble hippocampal tissue fractions, and Westerns used to assess BACE protein expression. AAV-Gfa2 vectors encode either potent inhibitors or activators of CN/NFAT signaling and therefore will determine the necessity and sufficiency of this astrocytic pathway in driving and/or maintaining neurologic dysfunction in AD mice. These studies provide a highly novel approach to the study of activated astrocytes and could have a major impact on the development of treatment strategies for AD and other neurodegenerative conditions. PUBLIC HEALTH RELEVANCE: Increasing evidence implicates activated astrocytes in a variety of neurodegenerative conditions, including Alzheimer's disease (AD). However, these cells are difficult to target selectively with therapeutics. In this project, we use cutting-edge adeno associated virus vectors to selectively prevent astrocyte activation and improve neurologic function in intact AD model mice. This approach could emerge as a new treatment strategy for AD and other neurodegenerative disorders.