Manipulating DNA repair enzymes to examine the interactions between aging and Alzheimers disease with iPSC-derived microglia

项目摘要/摘要 小胶质细胞与阿尔茨海默病(AD)的发病机制和最近的遗传学研究密切相关 已经确定了几个影响阿尔茨海默病风险的小胶质细胞丰富的基因。研究这些基因在阿尔茨海默病中的作用 我们的实验室最近开发了一种完全定义的方法来将诱导多能干细胞(IPSCs)分化为 小胶质细胞。然而，重新编程抹去了许多衰老的关键特征，使得研究 AD基因、病理和衰老与IPSC来源的细胞之间的相互作用。最近的一项研究开发了一种 通过使用Progerin来应对这一挑战的创新方法，Progerin是一种与过早衰老有关的蛋白质 无序。然而，Progerin基因LMNA通常不在人类神经元或神经胶质细胞中表达。在……里面 相比之下，另一种形式的早衰症，Cockayne综合征(CS)，是由破坏DNA的突变引起的 修复，会导致严重的神经缺陷。此外，在CS中突变的两个基因ERCC1 和ERCC5在人小胶质细胞中高表达，它们在小鼠中的缺失模拟了 小胶质细胞老化。鉴于这些发现，我们建议检验ERCC1缺失或突变的假设 ERCC5将产生IPSC来源的小胶质细胞的变化，模拟时序老化和 损害小胶质细胞对AD相关侮辱的反应。为了实现这些目标，我们组建了一个 多学科团队，提供AD IPSC建模、CRISPR介导的基因删除和 小胶质细胞分化和分析、RNA测序和生物信息学以及脑器官培养系统 检验以下三个具体目标和假设：目标1：检查ERCC1/5缺失和 IPSC来源的小胶质细胞功能突变。我们假设ERCC基因敲除和突变的小胶质细胞 表现出与年龄相关的“启动”激活状态，家庭活动受损但加重 炎症反应。目的2：ERCC1/5缺失和突变是否模拟了ERCC1/5的转录效应 在人脑来源的小胶质细胞中观察到的衰老？我们假设这些基因的缺失会产生 小胶质细胞表现出许多与自然脑老化有关的转录变化。目标3：定义 用脑器官培养法研究ERCC缺失对小胶质细胞老化的内在和外在影响。我们 假设内在和外在信号都会影响小胶质细胞的衰老。

Project Summary/Abstract Microglia are strongly implicated in the pathogenesis of Alzheimer's disease (AD) and recent genetic studies have identified several microglial-enriched genes that influence AD risk. To study the role of these genes in AD our lab recently developed a fully-defined approach to differentiate induced pluripotent stem cells (iPSCs) into microglia. However, reprogramming erases many of the key signatures of aging, making it difficult to study the interactions between AD genes, pathology, and aging with iPSC-derived cells. One recent study developed an innovative approach to address this challenge by using Progerin, a protein associated with a premature aging disorder. However, the Progerin gene, LMNA, is not normally expressed in human neurons or glia. In contrast, another form of Progeria, Cockayne Syndrome (CS), which is caused by mutations that impair DNA repair, leads to significant neurological defects. Furthermore, two of the genes that are mutated in CS, ERCC1 and ERCC5, are highly expressed in human microglia and their deletion in mice mimics key aspects of microglial aging. Given these findings, we propose to test the hypothesis that deletion or mutations of ERCC1 and ERCC5 will produce changes in iPSC-derived microglia that mimic the effects of chronological aging and impair the response of microglia to AD-associated insults. To achieve these goals, we have assembled a multidisciplinary team who bring expertise in AD iPSC modeling, CRISPR-mediated gene deletion, and microglial differentiation and analysis, RNA-sequencing and bioinformatics, and brain organoid culture systems to test following three specific aims and hypotheses: Aim 1: Examine the impact of ERCC1/5 deletions and mutations on iPSC-derived microglial function. We hypothesize that ERCC knockout and mutant microglia will exhibit an age-associated `primed' activation state with impaired homestatic activity but exacerbated inflammatory responses. Aim 2: Do ERCC1/5 deletions and mutations model the transcriptional effects of aging observed in human brain-derived microglia? We hypothesize that deletion of these genes will produce microglia that exhibit many of the transcriptional changes associated with natural brain aging. Aim 3: Defining the intrinsic versus extrinsic effects of ERCC deletion on microglial aging with brain organoid cultures. We hypothesis that both intrinsic and extrinsic signals influence microglial aging.