Genomic approach to identification of microglial networks involved in Alzheimer’s disease risk

项目摘要 阿尔茨海默病(AD)是美国十大杀手中唯一一种没有变化的疾病 心理治疗。基因研究提供了一种强大的手段来识别与疾病相关的基因和途径 疾病病因学。技术进步大大降低了基因组分析的成本，使 生成可集成以执行多尺度分析的大型公开可用的数据集。 根据这些数据产生的假设随后可以在细胞和动物模型中得到验证。一个大问题 全基因组研究遇到的是力量，特别是在搜索稀有变异时。一种方法是 这个问题是执行基于基因或基于基因集的分析。在过去的三年里，它已经成为 显然，AD危险基因(包括常见和罕见的变种)富含髓系细胞表达的基因， 包括APOE、TREM2、CD33、SORL1、ABCA7。小胶质细胞是大脑中常驻的吞噬细胞， 与外周髓系细胞有共同的胚胎谱系。我们建议使用基因组和功能 小胶质细胞功能受阿尔茨海默病风险和保护性调节假说的检验方法 在特定功能网络中丰富的基因中的等位基因。这项提案将使用公开提供的 由阿尔茨海默病测序项目(ADSP)和 来自国际阿尔茨海默病基因组学计划(IGAP)的全基因组关联研究(GWAS)数据 以及其他结合来自纯化的巨噬细胞和单核细胞的基因表达数据来鉴定髓系 携带影响阿尔茨海默病风险的罕见或常见变异的表达基因(目标1)。通过集成这些数据 进入单核细胞和巨噬细胞的共表达网络，我们将确定AD基因座是否位于一个 或更多的监管网络(目标1)。验证这些网络并确定功能后果 对于风险/保护等位基因，我们将在进行功能分析的同时进行全局转录和ATACseq 在来自同基因的人iPSC细胞系和小鼠BV2小胶质细胞系的小胶质细胞中，其中 候选基因表达被敲除或突变被敲入(目标2)。最后，我们将在体内使用 敲除成年小胶质细胞特异性基因表达以测试其生理后果 破坏与AD相关的功能网络(目标3)。为了能够进行这些研究，我们开发了一种新的 小鼠模型，可用于描述脑中小胶质细胞的核糖体结合转录组 也有条件地和特定地下调目的基因的表达，如MS4A6A 小胶质细胞。使用相同的模型与AD小鼠模型杂交，我们将研究与AD相关的结果 在小胶质细胞中MS4A6A表达减少的背景下，如小胶质细胞的小胶质细胞增生和β-淀粉样蛋白沉积。 总之，这些研究不仅将加深我们对阿尔茨海默病遗传结构的理解，而且还将为我们提供 关于分子机制的关键信息，为新的治疗开发奠定了基础。

Project Summary Alzheimer's disease (AD) is the only disease among the top ten killers in the U.S. without a disease modifying therapy. Genetic studies provide a powerful means to identify genes and pathways that are causally linked to disease etiology. Technological advances have substantially reduced the cost of genomic analyses enabling the generation of large publicly available datasets that can be integrated to perform multi-scale analyses. Hypotheses generated from these data can then be validated in cell and animal models. A major problem encountered by genome-wide studies is power, particularly when searching for rare variants. One approach to this problem is to perform gene-based or gene-set-based analyses. Over the last three years it has become apparent that AD risk loci (both common and rare variants) are enriched for myeloid cell expressed genes, including APOE, TREM2, CD33, SORL1, ABCA7. Microglia are the resident phagocytic cells of the brain and share a common embryonic lineage with peripheral myeloid cells. We propose to use genomic and functional approaches to test the hypothesis that microglial function is modulated by AD risk and protective alleles in genes that are enriched within specific functional networks. This proposal will use publicly available whole genome/exome sequence data generated by the Alzheimer's Disease Sequencing Project (ADSP) and genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) and others together with gene expression data from purified macrophages and monocytes to identify myeloid expressed genes that carry rare or common variants that influence risk for AD (Aim 1). By integrating this data into co-expression networks in monocytes and macrophages we will determine whether AD loci lie within one or more regulatory networks (Aim 1). To validate these networks and determine the functional consequences of risk/protective alleles we will perform global transcriptomics and ATACseq in parallel with functional assays in microglial cells derived from isogenic human iPSC cell lines and mouse BV2 microglial cells, in which candidate gene expression is knocked-down or mutations are knock-in (Aim 2). Finally, we will use in vivo knock-down of gene expression specifically in adult microglia to test the physiological consequences of disrupting an AD-linked functional network (Aim 3). To enable these studies, we have developed a novel mouse model that can be used to profile the ribosome-bound transcriptome of microglial cells in the brain while also conditionally and specifically down-regulating the expression of a gene of interest like MS4A6A in microglia. Using the same model crossed with an AD mouse model, we will investigate AD-related outcomes like micro-gliosis and ß-amyloid deposition in the context of reduced MS4A6A expression in microglia. Together these studies will not only further our understanding of the genetic architecture of AD but also provide key information regarding the molecular mechanisms, setting the stage for novel therapeutic development.