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

描述（由申请人提供）：本项目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完整的阿尔茨海默病（AD）小鼠模型神经功能中的基本作用。研究使用携带星形细胞特异性启动子Gfa2的腺相关病毒载体（AAV）靶向野生型和APP/PS1小鼠星形细胞中的蛋白磷酸酶钙调磷酸酶（CN）和NFAT转录因子。AAV-Gfa2载体在不同年龄/疾病阶段被双侧递送到海马，并对小鼠进行不同AD生物标志物的评估。在Aim 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和突触蛋白的Western blot测量来评估突触功能。在Aim 2中，使用基于陶瓷酶的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调节。在Aim 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胶质细胞激活和神经炎症水平。在目标4中，免疫组化用于确定A？而elisa用于定量A？40和A？42在可溶性和不可溶性海马组织中，western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，因此将决定这种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。这些研究为活化星形胶质细胞的研究提供了一种高度新颖的方法，并可能对阿尔茨海默病和其他神经退行性疾病的治疗策略的发展产生重大影响。

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.