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，包括迟发性散发性阿尔茨海默病（AD）。 疾病类型（LOAD）。除了遗传学研究已经确定小胶质细胞富集的遗传 影响AD风险的变异，最近对来自数百个 来自我们小组和NIA AMP-AD联盟的其他人的人类LOAD死后大脑表明， 三分之一与风险相关的基因在小胶质细胞中富集或专门表达。虽然神经元可能 是产生毒性淀粉样β肽的主要细胞类型，小胶质细胞在AD中的功能作用及其 与大脑中其他细胞类型的相互作用导致疾病仍然知之甚少， 仍然难以捉摸TYROBP/DAP 12，TREM 2和APOE中的关键遗传变异可能具有功能性疾病 改变不同脑细胞类型的影响或在大脑中跨细胞类型相互作用。在本申请中，我们 建议系统地识别和表征小胶质细胞对AD相关损伤的反应， 这些变种的背景。为了研究这些基因的作用及其在AD中的功能相互作用，我们将首先 产生一组CRISPR/Cas9编辑的iPSC系，其在TYROBP/DAP 12、TREM 2和 在所有单和多等位基因组合中以及在具有临床意义的单一遗传背景背景下， 病理学证实了负载。然后，我们将产生hiPSC衍生的神经共培养系统， 从这些等基因系的复杂类器官，以表征关键的转录和功能的影响， 单细胞和细胞群体范围内的遗传变异分析。单细胞RNA测序数据将在 产生以鉴定多等位基因变体的扰动特征，然后将其映射到亚型 特定的网络，为每个变体构建全面的信令映射。功能测定将用于 建立与AD表型相关的证据。我们的总体目标是检验遗传变异 在TREM 2中，TYROBP/DAP 12和APOE将在iPSC衍生的小胶质细胞中产生变化， 小胶质细胞对AD相关损伤的反应。

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.