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

项目摘要 阿尔茨海默病（AD）是美国十大杀手中唯一没有疾病修饰的疾病。 疗法遗传学研究提供了一种强有力的手段，以确定基因和途径，是因果关系， 疾病病因学技术进步大大降低了基因组分析的成本， 生成可集成以执行多尺度分析的大型公开数据集。 从这些数据中产生的假设可以在细胞和动物模型中得到验证。一个主要问题 在全基因组研究中遇到的最大困难是力量，特别是在寻找罕见变异时。的一种方法 这个问题是执行基于基因或基于基因组的分析。在过去的三年里， 显然AD风险基因座（常见和罕见变体）富含骨髓细胞表达的基因， 包括APOE、TREM 2、CD 33、SORL 1、ABCA 7。小胶质细胞是大脑的常驻吞噬细胞， 与外周髓样细胞共享共同的胚胎谱系。我们建议使用基因组和功能 小胶质细胞功能受AD风险和保护性调节这一假设的检验方法 基因中的等位基因在特定的功能网络中富集。该提案将使用公开可用的 由阿尔茨海默病测序计划（ADSP）生成的全基因组/外显子组序列数据， 全基因组关联研究（GWAS）数据来自国际阿尔茨海默病基因组学项目（IGAP） 以及其他人与来自纯化的巨噬细胞和单核细胞的基因表达数据一起鉴定髓系 表达的基因携带影响AD风险的罕见或常见变异（Aim 1）。通过整合这些数据 在单核细胞和巨噬细胞的共表达网络中，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）。为了验证这些网络并确定其功能后果， 风险/保护等位基因，我们将进行全球转录组学和ATACseq与功能测定平行 在源自同基因人iPSC细胞系和小鼠BV 2小胶质细胞的小胶质细胞中，其中 敲低候选基因表达或敲入突变（Aim 2）。最后，我们将在体内使用 在成年小胶质细胞中特异性敲低基因表达，以测试 破坏AD相关的功能网络（Aim 3）。为了进行这些研究，我们开发了一种新的 小鼠模型，可用于分析大脑中小胶质细胞的核糖体结合转录组， 还条件性地和特异性地下调感兴趣的基因如MS 4A 6A的表达， 小胶质细胞使用与AD小鼠模型交叉的相同模型，我们将研究AD相关的结果 如在小胶质细胞中MS 4A 6A表达减少的情况下的小胶质细胞增生和β-淀粉样蛋白沉积。 这些研究不仅将进一步加深我们对AD遗传结构的理解， 关于分子机制的关键信息，为新的治疗开发奠定基础。

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.