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

描述（由申请人提供）：该项目中的实验使用创新的基因递送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）完整小鼠模型中的神经功能中的基本作用。研究使用携带星形胶质细胞特异性启动子Gfa 2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磷酸酶（CN）和NFAT转录因子。将AAV-Gfa 2载体双侧递送至不同年龄/疾病阶段的海马体，并评估小鼠的不同AD生物标志物。在目标1中，使用主动回避任务评估认知状态，而使用海马切片电生理学和突触蛋白的Western印迹测量评估突触功能。在目的2中，海马谷氨酸调节研究使用陶瓷酶为基础的微电极阵列和谷氨酸转运体水平的措施。在目的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活化和神经炎症的水平。在目标4中，免疫组化用于确定A？沉积，而ELISA是用来量化水平的A？40和A？42的可溶性和不溶性海马组织级分中，以及用于评估BACE蛋白表达的蛋白质印迹。AAV-Gfa 2载体编码CN/NFAT信号传导的有效抑制剂或激活剂，因此将决定该星形胶质细胞途径在驱动和/或维持AD小鼠神经功能障碍中的必要性和充分性。这些研究为活化星形胶质细胞的研究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，活化的星形胶质细胞在各种神经退行性疾病，包括阿尔茨海默病（AD）。然而，这些细胞难以用治疗剂选择性地靶向。在这个项目中，我们使用最先进的腺相关病毒载体来选择性地阻止星形胶质细胞激活并改善完整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新治疗策略。

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.