Elucidating cellular pathways controlling the reactive state of astrocytes in Alzheimer's Disease

项目摘要/摘要 星形胶质细胞在大脑中执行基本的动态平衡功能。为了应对局部组织损伤，星形胶质细胞 变成“反应性”，这是一个典型的以形态肥大和GFAP上调为特征的过程。 反应性星形胶质细胞是阿尔茨海默病(AD)神经病理的一个重要特征， 与阿尔茨海默病认知功能减退相关。然而，控制星形胶质细胞反应的机制并不是很好。 明白了。最近的研究发现了一种由炎症激活诱导的反应性星形胶质细胞。 (“炎症反应”，又名。“A1”反应性)，这可能在AD中起作用。炎性反应性星形胶质细胞 以丧失正常的体内平衡功能为特征的，如吞噬中枢神经系统底物和增益 具有神经毒性的特性。重要的是，在AD的小鼠模型和正常衰老的小鼠中都发现了它们。 此外，人类死后阿尔茨海默病的大脑富含表达炎症标志物的星形胶质细胞 反应性。阐明人类炎症反应及其功能产物的控制机制 星形胶质细胞，在我的前期工作中，我实现了在人IPSC中进行池CRISPRI功能缺失筛查- 衍生星形胶质细胞(IAstros)。从初步筛选中，我确定肌球蛋白磷酸酶是一种潜在的调节因子。 炎性星形胶质细胞吞噬活性降低。此外，我利用现有的星形细胞 推断炎症反应的转录调控因子的rna-seq数据集。在我的第一个目标中，我建议 确定肌球蛋白磷酸酶调节如何导致炎症反应中的吞噬功能减少 星形胶质细胞。在我的第二个目标中，我建议整合全基因组CRISPRi筛选和共表达分析 对现有的星形胶质细胞rna-seq数据集进行分析，以发现控制炎症反应的细胞通路。 通过将这些途径与星形胶质细胞反应性的功能输出联系起来。我的赞助人马丁·坎普曼博士 世卫组织共同开发了CRISPRi筛查技术，我的联合发起人塞尔吉奥·巴兰齐尼博士是 神经炎症和转录数据集的整合和分析，是支持 我提议的研究。此外，我的合作者埃里克·乌利安博士是一位高分辩学的专家。 从人类IPSCs中分离出高质量的星形胶质细胞，这进一步支持了拟议工作的可行性。除了……之外 我的两个赞助商和我的合作者Ullian博士，我还将接受医生高爱梅的指导- 科学家和执业神经科医生，我将与他一起接受神经学方面的纵向临床培训。 布鲁斯·康克林，基于IPSC的技术和基因组手术的全球领导者。总体而言，建议的工作 这种合作应该有助于药物的开发，这种药物可以选择性地调节不同的 治疗阿尔茨海默病的星形胶质细胞反应性功能输出。此外，通过这项工作，我将发展 在用IPSC衍生模型和功能基因组学揭示疾病机制方面的专业知识 加强我作为内科科学家的培训。

PROJECT SUMMARY/ABSTRACT Astrocytes perform essential homeostatic functions in the brain. In response to local tissue injury, astrocytes become “reactive”, a process classically characterized by morphological hypertrophy and upregulation of GFAP. Reactive astrocytes are a defining feature of Alzheimer's disease (AD) neuropathology and are strongly correlated with cognitive decline in AD. However, mechanisms controlling astrocyte reactivity are not well understood. Recent studies have identified a form of reactive astrocyte induced by inflammatory activation (“inflammatory reactivity”, a.k.a. “A1” reactivity) that may play a role in AD. Inflammatory reactive astrocytes are characterized by loss of normal homeostatic functions such as phagocytosis of CNS substrates as well as gain of neurotoxic properties. Importantly, they are found in a mouse model of AD as well as normal aged mice. Furthermore, human post-mortem AD brains are enriched for astrocytes expressing a marker of inflammatory reactivity. To elucidate mechanisms controlling inflammatory reactivity and its functional outputs in human astrocytes, in my preliminary work I implemented pooled CRISPRi loss-of-function screening in human iPSC- derived astrocytes (iAstros). From a preliminary screen, I identified myosin phosphatase as a potential regulator of the decreased phagocytic activity in inflammatory astrocytes. Furthermore, I leveraged existing astrocyte RNA-seq datasets to infer transcriptional regulators of inflammatory reactivity. In my first aim, I propose to determine how regulation of myosin phosphatase leads to decreased phagocytosis in inflammatory reactive astrocytes. In my second aim, I propose integrate genome-wide CRISPRi screening and coexpression analysis of existing astrocyte RNA-seq datasets to discover cellular pathways controlling inflammatory reactivity, followed by connecting these pathways to functional outputs of astrocyte reactivity. My sponsor Dr. Martin Kampmann, who co-developed the CRISPRi screening technology, and my co-sponsor Dr. Sergio Baranzini, an expert in neuroinflammation and the integration and analysis of transcriptomic datasets, are ideally positioned to support my proposed research. Furthermore, my collaborator Dr. Erik Ullian is an expert on the differentiation of high- quality astrocytes from human iPSCs, which further supports the feasibility of the proposed work. In addition to my two sponsors and my collaborator Dr. Ullian, I will also receive mentorship from Dr. Aimee Kao, a physician- scientist and practicing neurologist with whom I will undergo longitudinal clinical training in neurology, and Dr. Bruce Conklin, a global leader in iPSC-based technologies and genome surgery. Overall, the work proposed in this fellowship should contribute towards the development of drugs that can selectively modulate different functional outputs of astrocyte reactivity for the treatment of AD. Furthermore, through this work I will develop expertise in uncovering disease mechanisms with iPSC-derived models and functional genomics which will strengthen my training as a physician-scientist.