Use of iPSC systems to define roles of microglial TREM2/DAP12 and CR3/DAP12 complexes and their genetic variants in specifying risk for late onset sporadic Alzheimer's disease

项目摘要 小胶质细胞与阿尔茨海默病(AD)的发病机制密切相关，包括迟发性散发性 疾病的形式(负荷)。除了基因研究已经发现小胶质细胞丰富的基因 影响AD风险的变量，最近对来自数百个国家的多尺度组学数据进行了计算分析 我们小组和NIA AMP-AD联盟中的其他人的死后大脑负荷建议大约有一个 与风险相关的基因中有三分之一是在小胶质细胞中丰富或独占表达的。而神经元可能 是产生毒性淀粉样β蛋白的主要细胞类型，小胶质细胞在AD中的作用及其机制 与大脑中其他类型细胞的相互作用导致疾病仍然知之甚少，阿尔茨海默病的病因 仍然难以捉摸。TYROBP/DAP12、TREM2和APOE的关键遗传变异可能患有功能性疾病 改变不同脑细胞类型的影响或在大脑中不同类型的细胞之间相互作用。在此应用程序中，我们 建议系统地识别和表征小胶质细胞对AD相关侮辱的反应。 这些变体的上下文。为了研究这些基因在阿尔茨海默病中的作用及其功能相互作用，我们首先 生成一组CRISPR/Cas9编辑的具有TYROBP/DAP12、TREM2和TREM2等基因突变的IPSC系 所有单等位基因和多等位基因组合中的载脂蛋白E，以及在具有临床单一遗传背景的背景下 病理检查也证实了这一点。然后我们将生成HiPSC衍生的神经共培养系统，然后 从这些等基因系中提取复杂的有机化合物来表征KEY对转录和功能的影响 单细胞和全细胞分析中的遗传变异。单细胞RNA测序数据将 生成以识别多等位基因变体的扰动特征，然后将其映射到亚型 特定网络，为每个变体构建全面的信令地图。功能分析将用于 建立与AD表型相关的证据。我们的总体目标是检验这样一种假设 在TREM2、TYROBP/DAP12和APOE中，IPSC来源的小胶质细胞将产生模仿 小胶质细胞对AD相关侮辱的反应。

Project Summary Microglia are strongly implicated in the pathogenesis of Alzheimer's disease (AD) including late onset sporadic forms of the disease (LOAD). In addition to genetic studies that have identified microglial-enriched genetic variants that influence AD risk, recent computational analysis of multi-scale omics data from hundreds of human LOAD postmortem brains from our group and others in the NIA AMP-AD consortium suggest about one third of the genes associated with risk are enriched or exclusively expressed in microglia. While neurons may be the major cell type generating the toxic amyloid-beta peptide, the functional role of microglia in AD and their interaction with other cell types in the brain to cause disease are still poorly understood and the etiology of AD remains elusive. Key genetic variants in TYROBP/DAP12, TREM2, and APOE may have a functional disease altering impact in distinct brain cell type or interact across cell types in the brain. In this application, we propose to systematically identify and characterize the response of microglia to AD-associated insults in the context of these variants. To study the role of these genes and their functional interaction in AD, we will first generate a panel of CRISPR/Cas9-edited iPSC lines with isogenic mutations in TYROBP/DAP12, TREM2, and APOE in all single and multi-allelic combinations and in the context of a single genetic background with clinical and pathology confirmed LOAD. We will then generate hiPSC-derived neural co-culture systems and then complex organoids from these isogenic lines to characterize the transcriptional and functional impact of key genetic variants in single cell and cell-population-wide analyses. Single cell RNA sequencing data will be generated to identify perturbation signatures for multi-allelic variants that will then be mapped to subtype specific networks to build comprehensive signaling maps for each variant. Functional assays will be used to build evidence for relevance to AD phenotypes. Our overall goal is to test the hypothesis that genetic variants in TREM2, TYROBP/DAP12, and APOE will produce changes in iPSC-derived microglia that mimic the response of microglia to AD-associated insults.