Deciphering isogenic APOE isoform dependent neurodegenerative response in human glia

项目摘要 载脂蛋白E（apolipoprotein E，APOE）是晚发性阿尔茨海默病（Alzheimer's disease，AD）最重要的危险基因。载脂蛋白E β 4/β 4 纯合性增加AD风险> 14倍。尽管APOE ε 4等位基因与 虽然AD风险增加是公认的，但这种遗传风险的潜在机制仍然难以捉摸。在大脑中， 小胶质细胞和星形胶质细胞诱导炎症并降解（吞噬、消化、储存或再循环）富含脂质的细胞 在衰老和神经退化过程中积累的碎片。我们假设APOE基因型是 对星形胶质细胞和小胶质细胞的细胞自主作用，其影响其他细胞类型。具体来说，我们假设 APOE ε 4/ε 4星形胶质细胞和小胶质细胞对富含脂质的碎片（髓鞘碎片）的反应发生改变， 可检测为整体转录和细胞功能的变化。为了验证这一假设，我们生成了一个 使用CRISPR/Cas9基因组编辑工具，在APOE同种型中唯一不同的独特系列的同基因iPSC。 我们已经建立了一个平台，通过分化人脑细胞， 表达APOE的iPSC衍生的星形胶质细胞和小胶质细胞。我们想把这位病人- 衍生的同基因人类iPSC模型，强大的系统生物学，生物信息学方法和体外 包括神经炎症、吞噬作用和自噬作用在内的代谢试验，以了解其机制 AD的潜在APOE风险。这项拟议的研究项目旨在解开APOE亚型依赖性 对胶质细胞的影响及其对脂质挑战的反应。在目标1中，我们将产生同质种群， 星形胶质细胞和小胶质细胞以及来自同基因APOE同种型和APOE的皮质神经元/胶质细胞的混合培养物 敲除源自患者iPSC。我们将进行差异基因表达分析，以确定 APOE基因型在每种细胞类型中的下游效应。目的2：研究APOE亚型依赖的胶质细胞 对富脂颗粒激发的反应。遵循无偏见的转录组学方法，我们将分析 哪些疾病相关的小胶质细胞特征或AD相关网络是APOE亚型依赖性的 接受挑战在目的3中，我们将研究APOE异构体依赖性对炎症反应的影响， 和脂质颗粒的吞噬/自体溶酶体清除。该项目的目标是确定 转录组网络和细胞功能受APOE基因型的存在或不存在， 疾病相关的环境（髓鞘碎片），以确定最早和潜在的最可治疗的 参与AD发病机制。

PROJECT SUMMARY Apolipoprotein E (APOE) is the most significant risk gene for late-onset Alzheimer's disease (AD). APOE ɛ4/ɛ4 homozygosity increases AD risk by >14-fold. Although an association between the APOE ε4 allele and increased AD risk is well-established, the mechanisms underlying this genetic risk remain elusive. In brain, microglia and astrocytes induce inflammation and degrade (engulf, digest, store or recycle) lipid-rich cellular debris that accumulates during aging and neurodegeneration. We hypothesize that APOE ɛ4/ɛ4 genotype has cell autonomous effects on astrocytes and microglia which affects other cell types. Specifically, we hypothesize that APOE ε4/ε4 astrocytes and microglia exhibit altered response to lipid-rich debris (myelin fragments), detectable as changes in global transcription and cellular function. To test this hypothesis we have generated a unique series of isogenic iPSCs differing solely in APOE isoform using a CRISPR/Cas9 genome-editing tool. We have established a platform to recapitulate cellular systems of human brain in culture by differentiation of iPSC-derived astrocytes and microglia that express APOE. We seek to bring together this established patient- derived isogenic human iPSC model, powerful systems biology, bioinformatic approaches and in vitro metabolic assays including neuroinflammation, phagocytosis and autophagy to understand the mechanism underlying APOE risk for AD. This proposed research project sets out to unravel the APOE isoform-dependent effects in glia and their responses to lipid challenge. In aim 1, we will generate homogenous populations of astrocytes and microglia and mixed cultures of cortical neurons/glia from isogenic APOE isoforms and APOE knockout derived from patient iPSCs. We will perform differential gene expression analysis to determine the downstream effects of APOE genotype in each cell type. In aim 2, we will study APOE isoform-dependent glial responses to challenge with lipid-rich particles. Following an unbiased transcriptomic approach, we will analyze which of the disease associated microglia signatures or AD-associated networks are APOE isoform-dependent upon challenge. In aim 3, we will investigate APOE isoform-dependent effects on the inflammatory response and phagocytic/autolysosomal clearance of lipid particles. The goal of this project is to identify the transcriptomic networks and cellular functions governed by APOE genotype in the presence or absence of a disease relevant environment (myelin debris) to pinpoint the earliest and potentially most treatable mechanisms involved in AD pathogenesis.