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

摘要 迟发性阿尔茨海默病(LOAD)是人类最常见的神经退行性疾病。 老年人口，影响到近600万65岁以上的美国成年人。尽管是第六大事业 在美国，仍然没有有效的治疗方法来减缓或阻止疾病的进展。这个 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斑块的细胞外聚集 (AB1-42)在大脑中。作为回应 为了产生AB1-42，小胶质细胞，中枢神经系统(CNS)的驻留巨噬细胞激活和 迁移到斑块聚集的地方，然后分解和吞噬斑块，同时还 分泌促炎细胞因子以刺激先天免疫反应。坚持不懈地生产这些 细胞因子降低了小胶质细胞在负反馈环路中清除AB1-42的能力，结果增加了形成 神经元中的纤维间缠结加剧了神经退化。大的全基因组关联 研究已经确定了几个单核苷酸多态(SNPs)，它们与LOAD和 驻留在具有已知的影响小胶质细胞功能的罕见编码变体的基因附近，进一步强调了 小胶质细胞在负载病理中的重要性。然而，尽管GWAS已经成功地确定了许多 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原因SNP，作为 Gwas前哨SNP代表了SNPs的整个单倍型。此外，其中大部分 GWASSNP位于基因组的非编码区，并不一定与最近的基因有关 作为因果关系。相反，这些SNP可能通过调节 远端调控元件的活性，如增强子，进而调节LOAD基因的表达。 因此，我推测GWASNPs的负载导致了炎症和炎症的失调。 通过改变小胶质细胞增强剂的调节活性和血管紧张素转换酶在LOAD患者脑内的吞噬作用 它们的连锁效应基因的表达。在目标1中，我将利用“变异到基因图谱”的方法来识别 通过识别位于开放染色质内的SNPs，推测小胶质细胞中存在GWASSNPs 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基因座 模特们。我将通过小胶质细胞模型中的荧光素酶分析来验证这些增强剂的活性， 我还将确定可能的效应基因，它们的表达受这些增强子通过 我们实验室的以启动子为中心的捕获-C试验。在目标2中，我将从功能上验证 在小胶质细胞模型中通过敲除负载相关的小胶质细胞增强子 CRISPR，然后评估这些敲除如何影响全球基因表达、炎症和 吞噬作用。综上所述，这些目标将提供对负荷的小胶质细胞遗传机制的洞察，以及 可能会导致可以治愈或预防这种疾病的新疗法的开发。

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.