Implicating novel microglial mechanisms of late-onset Alzheimer's disease with variant-to-gene mapping methods

用变异到基因作图方法揭示晚发性阿尔茨海默病的新小胶质细胞机制

• 批准号: 10672240
• 负责人: ELIZABETH Anne BURTON
• 金额: $ 3.5万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10672240
• 关键词: 3-Dimensional; ATAC-seq; Address; Adult; Affect; Alleles; Alzheimer' s disease brain; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; American; Amyloid beta-Protein; Area; Biological Assay; Brain; CRISPR/Cas technology; Cause of Death; Cell Line; Cell model; Cells; Central Nervous System; ChIP-seq; Characteristics; Chromatin; Chromosome Mapping; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Development; Disease; Disease Progression; Distal; Economic Burden; Elderly; Enhancers; Epigenetic Process; Excretory function; Feedback; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Haplotypes; Healthcare Systems; Heritability; Human; Immune; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Response; Ingestion; Innate Immune Response; Interferon Type II; Knock-out; Late Onset Alzheimer Disease; Link; Location; Luciferases; Macrophage; Maps; Measures; Methods; Microglia; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Output; Pathogenesis; Pathway interactions; Phagocytes; Phagocytosis; Phenotype; Play; Population; Production; Proteins; Quantitative Trait Loci; Regulator Genes; Regulatory Element; Role; Senile Plaques; Sentinel; Single Nucleotide Polymorphism; Site; Tissues; Untranslated RNA; Validation; Variant; abeta deposition; causal variant; cell type; cytokine; effective therapy; epigenetic marker; extracellular; gene interaction; genome wide association study; genome-wide; genomic locus; glial activation; human old age (65+); induced pluripotent stem cell; insight; interest; migration; misfolded protein; neurodegenerative dementia; novel; novel therapeutics; prevent; promoter; response; targeted treatment; therapeutic development; therapeutic gene; transcriptome; transcriptome sequencing

ABSTRACT Late-onset Alzheimer’s disease (LOAD) is the most common neurodegenerative disease among the elderly population, affecting nearly 6 million US adults over the age of 65. Despite being the 6th leading cause of death in the US, there are still no effective therapies that can slow or halt disease progression. The prevailing molecular feature that differentiates LOAD from other types of neurodegenerative dementia is the extracellular aggregation of inappropriately cleaved amyloid-b protein plaques (Ab1-42) in the brain. In response to Ab1-42 production, microglia, the resident macrophages of the central nervous system (CNS) activate and migrate to the site of plaque accumulation, and then break down and phagocytose the plaques, while also secreting pro-inflammatory cytokines to stimulate the innate immune response. Persistent production of these cytokines reduces microglial ability to clear Ab1-42 in a negative feedback loop, and results increased formation of interfibrillary tangles in the neurons that exacerbates neurodegeneration. Large genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) that associate with LOAD and reside near genes with known rare coding variants that affect microglial function, further emphasizing the importance of microglia in LOAD pathology. However, while GWAS has successfully identified numerous genetic loci associated with LOAD, it cannot directly identify the causal SNP implicated by these loci, as a GWAS sentinel SNP is representative of an entire haplotype of SNPs. Additionally, the majority of these GWAS SNPs lie within non-coding regions of the genome, and may not necessarily implicate the nearest gene as causal. Instead, these SNPs likely regulate the expression of LOAD-associated genes by modulating the activity of distal regulatory elements, such as enhancers, which in turn regulate LOAD gene expression. Therefore, I hypothesize that LOAD GWAS SNPs contribute to the dysregulated inflammation and phagocytosis in the brains of LOAD patients by altering the regulatory activity of microglial enhancers and the expression of their linked effector genes. In Aim 1, I will utilize a “variant-to-gene mapping” approach to identify putatively causal LOAD GWAS SNPs in the microglia by identifying SNPs that lie within open chromatin, are enriched in marks of active enhancers, and function as expression quantitative trait loci in microglial cell models. I will validate the activity of these enhancers through luciferase assays in the microglial cell models, and I will also identify the likely effector genes whose expression are modulated by these enhancers through our lab’s promoter-focused Capture-C assay. In Aim 2, I will functionally validate the phenotype conferred by LOAD-associated microglial enhancers by knocking out these enhancers in microglial cell models using CRISPR, and then assessing how these knockouts impact global gene expression, inflammation, and phagocytosis. Taken together, these aims will provide insight into microglial genetic mechanisms of LOAD, and may lead to the development of new therapies that can cure or prevent the disease.

摘要 晚发性阿尔茨海默病（LOAD）是老年人中最常见的神经退行性疾病， 老年人口，影响近600万65岁以上的美国成年人。尽管是第六大原因， 在美国，死亡率高达100%，但仍然没有有效的治疗方法可以减缓或阻止疾病的进展。的 将LOAD与其他类型的神经退行性痴呆区分开来的主要分子特征是 不适当切割的淀粉样蛋白-B蛋白斑的细胞外聚集 （Ab 1 -42）在脑中。响应 对于Ab 1 -42的产生，小胶质细胞，中枢神经系统（CNS）的常驻巨噬细胞活化， 迁移到斑块积聚的部位，然后分解并吞噬斑块，同时还 分泌促炎细胞因子以刺激先天免疫反应。持续生产这些 细胞因子在负反馈回路中降低小胶质细胞清除Ab 1 -42的能力，并导致形成增加 神经元间的缠结加剧了神经退化。大基因组关联 研究（GWAS）已经鉴定了几种与LOAD相关的单核苷酸多态性（SNP）， 存在于具有影响小胶质细胞功能的已知罕见编码变体的基因附近，进一步强调了 小胶质细胞在LOAD病理中的重要性。然而，尽管GWAS成功地识别了许多 与LOAD相关的遗传基因座，它不能直接识别这些基因座所涉及的因果SNP，作为一种 GWAS哨兵SNP代表SNP的整个单倍型。此外，其中大多数 GWAS SNP位于基因组的非编码区，可能不一定涉及最近的基因 作为因果。相反，这些SNPs可能通过调节LOAD相关基因的表达来调节LOAD相关基因的表达。 远端调控元件如增强子的活性，其反过来调控LOAD基因表达。 因此，我假设LOAD GWAS SNP导致炎症失调， 通过改变小胶质细胞增强子的调节活性， 其连锁效应基因的表达。在目标1中，我将利用“变体-基因作图”方法来识别 通过鉴定位于开放染色质内的SNP，在小胶质细胞中检测致病性LOAD GWAS SNP， 富含活性增强子的标记，在小胶质细胞中作为表达数量性状位点 模型我将在小胶质细胞模型中通过荧光素酶测定来验证这些增强子的活性， 我还将通过以下方式来识别可能的效应基因，其表达受到这些增强子的调节： 我们实验室的启动子聚焦Capture-C检测在目标2中，我将在功能上验证以下所赋予的表型： LOAD相关的小胶质细胞增强子，通过在小胶质细胞模型中使用 CRISPR，然后评估这些敲除如何影响全球基因表达，炎症， 吞噬作用总之，这些目标将提供对LOAD的小胶质细胞遗传机制的深入了解， 可能导致新疗法的发展，可以治愈或预防这种疾病。