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

基本信息

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
项目类别：
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&apos s disease brain Alzheimer&apos s disease pathology Alzheimer&apos s disease patient Alzheimer&apos 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

项目摘要

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.

抽象的晚期的阿尔茨海默氏病（负载）是最常见的神经退行性疾病老年人口影响了65岁以上的近600万美国成年人。尽管是第六个主要原因在美国的死亡中，仍然没有有效的疗法可以减慢或停止疾病的进展。盛行的分子特征将负载与其他类型的神经退行性痴呆区分开细胞外裂解的淀粉样蛋白斑块的细胞外聚集（AB1-42）在大脑中。作为回应至AB1-42生产，小胶质细胞，中枢神经系统（CNS）的巨噬细胞激活和迁移到斑块积累的地点，然后分解并吞噬斑块，同时也分泌促炎性细胞因子以刺激先天免疫反应。这些的持续生产细胞因子降低了小胶质细胞在负反馈循环中清除AB1-42的能力，结果增加了形成加剧神经变性的神经元中的纤维缠结。大基因组关联研究（GWAS）已经确定了几种与负载和负载和驻留在具有影响小胶质功能的已知稀有编码变体的附近，进一步强调了小胶质细胞在负荷病理学中的重要性。但是，尽管GWAS成功地确定了许多与负载相关的遗传局部性，它无法直接识别这些地方实施的因果SNP，作为一个 GWAS Sentinel SNP代表了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域内，不一定暗示最近的基因作为因果。相反，这些SNP可能通过调节负载相关基因的表达来调节不同的调节元件（例如增强子）的活性，这些元素又调节载荷基因表达。因此，我假设负载GWAS SNP导致注射失调和通过改变小胶质增强剂的调节活性和其链接效应基因的表达。在AIM 1中，我将使用一种“变体对基因映射”方法来识别通过识别位于开放染色质中的SNP的指定因果负载GWAS SNP，是富含活性增强子的标记，并充当小胶质细胞中的表达定量性状基因座型号。我将通过小胶质细胞模型中的荧光素酶测定来验证这些增强子的活性，我还将确定可能通过这些增强子调节的可能效应子基因我们实验室以促进者为重点的Capture-C测定法。在AIM 2中，我将在功能上验证由通过使用小胶质细胞模型中的这些增强子来淘汰这些增强子，使用负载相关的小胶质细胞增强剂 CRISPR，然后评估这些敲除如何影响全球基因表达，注射和吞噬作用。综上可能导致开发可以治愈或预防疾病的新疗法。