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

操纵 DNA 修复酶以检查衰老和阿尔茨海默病与 iPSC 衍生的小胶质细胞之间的相互作用

• 批准号: 9924476
• 负责人: Mathew Mark Blurton-Jones
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9924476
• 关键词: 3-Dimensional; Address; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease risk; Apoptosis; Astrocytes; Autopsy; Bioinformatics; Blood Vessels; Brain; Cell Differentiation process; Cells; Chronology; Clustered Regularly Interspaced Short Palindromic Repeats; Cockayne Syndrome; DNA; DNA Repair; DNA Repair Enzymes; Data; Defect; Deletion Mutation; Development; Disease; ERCC1 gene; Exhibits; Fibroblasts; Gene Deletion; Gene Expression; Generations; Genes; Genetic Transcription; Genetic study; Goals; Human; Impaired cognition; Impairment; Individual; Inflammatory; Inflammatory Response; Knock-out; Knockout Mice; Mediating; Methods; Microglia; Modeling; Mus; Mutate; Mutation; Neuroglia; Neurologic; Neurologic Signs; Neurologic Symptoms; Neurons; Oligodendroglia; Organoids; Pathogenesis; Pathology; Pathway Analysis; Patients; Phagocytosis; Phenotype; Premature aging syndrome; Production; Progeria; Proteins; Protocols documentation; Reactive Oxygen Species; Research Personnel; Resources; Risk Factors; Role; Signal Transduction; Source; Stimulus; Stroke; Superoxides; Syndrome; System; Testing; Tissues; Transcript; Transgenic Organisms; age effect; aged; aging brain; base; cytokine; dopaminergic neuron; gene function; genetic manipulation; induced pluripotent stem cell; innovation; migration; multidisciplinary; mutant; nerve stem cell; neuropathology; neuroregulation; novel; overexpression; relating to nervous system; response; stem cell model; transcriptome; transcriptome sequencing; young adult

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.

项目总结/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AD风险的小胶质细胞富集基因。研究这些基因在AD中的作用 我们的实验室最近开发了一种完全定义的方法，将诱导多能干细胞（iPSC）分化为 小胶质细胞然而，重编程消除了许多衰老的关键特征，使得研究衰老的过程变得困难。 AD基因、病理学和衰老与iPSC衍生细胞之间的相互作用。最近的一项研究发现， 通过使用与过早衰老相关的蛋白质Progerin来应对这一挑战的创新方法 disorder.然而，早老蛋白基因，LMNA，在人类神经元或神经胶质中通常不表达。在 相反，另一种形式的早衰症，Cockayne综合征（CS），这是由DNA损伤突变引起的 修复，导致严重的神经缺陷。此外，CS中的两个突变基因ERCC 1 和ERCC 5在人类小胶质细胞中高度表达，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ERCC 1基因缺失或突变可能导致癌症的假设。 ERCC 5将在iPSC衍生的小胶质细胞中产生变化，模拟时间老化的影响， 削弱小胶质细胞对AD相关损伤的反应。为了实现这些目标，我们组织了一个 多学科团队，他们在AD iPSC建模，CRISPR介导的基因缺失， 小胶质细胞分化和分析，RNA测序和生物信息学，以及脑类器官培养系统 目的1：检测ERCC 1/5缺失的影响， iPSC衍生的小胶质细胞功能的突变。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，具有受损的静态活动，但加重 炎症反应。目的2：ERCC 1/5缺失和突变是否模拟了 在人脑衍生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大脑衰老相关的转录变化。目标3：界定 ERCC缺失对脑类器官培养的小胶质细胞老化的内在与外在影响。我们 假设内在和外在信号都影响小胶质细胞老化。