Dysregulated intracellular calcium in microglia during Alzheimer's disease

项目摘要 小胶质细胞是大脑的免疫细胞，执行基本的体内平衡功能，包括通过以下方式清除碎片 通过释放生长因子和其他分子来吞噬和维持神经元。然而，在 阿尔茨海默病(AD)，小胶质细胞聚集在淀粉样斑块附近，在那里它们获得了激活的 表型，失去其动态平衡表型，并呈现一种激活的表型，可以诱导 神经毒性。小胶质细胞的细胞内钙调节它们从这种动态平衡静息状态的转化 状态变为激活的免疫效应器状态。小胶质细胞胞内钙协调以下功能 吞噬、增殖、迁移、神经营养因子的产生、细胞因子的产生和释放 形成神经毒性的活性氧物种。细胞内钙离子失衡出现在激活的、非 AD小鼠模型中淀粉样蛋白β(Aβ)斑块附近的动态平衡小胶质细胞。我们的总体假设是 阿尔茨海默病患者细胞内钙离子失衡改变小胶质细胞表型。我们的初步数据显示，阻止 L型电压依赖性钙通道(L-VDCCs)可使小胶质细胞向内环境更稳定的方向移动 表型。建议的实验验证了L-VDCCs可以驱动小胶质细胞钙的假设 AD病理过程中的调节失调。我们将利用L-VDCC的药理拮抗作用来验证这一点 结合有AD病理存在的小胶质细胞特异性L-VDCC的遗传消融，以确定 L-VDCCs对小胶质细胞表型和功能的影响对于本提案的培训方面，我们将使用1)中 用于评估小胶质细胞功能和钙动力学的活体多光子成像和2)单核RNA 测序以确定L-血管内皮生长因子受体拮抗作用的体内细胞特异性效应。如果我们的假设是正确的，L- VDCC功能将是连接小胶质细胞钙离子失衡和小胶质细胞功能表型的机制纽带 在AD期间。这一发现将与许多与年龄相关的神经退行性疾病相关 观察到小胶质细胞表型的变化。

Project Summary Microglia, the immune cells of the brain, perform essential homeostatic functions including debris clearance by phagocytosis and maintenance of neurons by release of growth factors and other molecules. However, during Alzheimer’s disease (AD), microglia accumulate near amyloid plaques where they acquire an activated phenotype, lose their homeostatic phenotype, and take on an activated phenotype that can induce neurotoxicity. Intracellular calcium in microglia regulates their transformation from this homeostatic resting state to an activated immune-effector state. Microglia intracellular calcium orchestrates functions such as phagocytosis, proliferation, migration, generation of neurotrophic factors, cytokine production and release, and formation of neurotoxic reactive oxygen species. Intracellular calcium dysregulation appears in activated, non- homeostatic microglia near amyloid β (Aβ) plaques in mouse models of AD. Our overall hypothesis that dysregulated intracellular calcium shifts microglia phenotype in AD. Our preliminary data suggest that blocking L-type voltage dependent calcium channels (L-VDCCs) can shift microglia towards a more homeostatic phenotype. The proposed experiments test the hypothesis that L-VDCCs can drive microglia calcium dysregulation during AD pathology. We will test this by using pharmacological antagonism of L-VDCCs combined with genetic ablation of microglia-specific L-VDCCs in the presence of AD pathology to define the role of L-VDCCs on microglia phenotype and function. For the training aspect of this proposal, we will use 1) in vivo multiphoton imaging to assess microglia function and calcium dynamics and 2) single-nuclei RNA sequencing to define the cell-specific effects of L-VDCC antagonism in vivo. If our hypothesis is correct, L- VDCC function will be a mechanistic link microglia calcium dysregulation and microglia functional phenotype during AD. This finding would be relevant to numerous age-associated neurodegenerative disorders where changes in microglia phenotype are observed.