Deciphering isogenic APOE isoform dependent neurodegenerative response in human glia

项目总结 载脂蛋白E(ApoE)是晚发性阿尔茨海默病(AD)最重要的危险基因。APOEɛ4/ɛ4 纯合子使患AD的风险增加14倍。尽管载脂蛋白Eε4等位基因与 AD风险增加是公认的，但这种遗传风险背后的机制仍然难以捉摸。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、储存或循环)富含脂肪的细胞。 在老化和神经退化过程中积累的碎片。我们假设载脂蛋白Eɛ4/ɛ4基因有 对星形胶质细胞和小胶质细胞的细胞自主作用，从而影响其他类型的细胞。具体来说，我们假设 载脂蛋白ε4/ε4星形胶质细胞和小胶质细胞对富含脂质的碎片(髓鞘片段)的反应发生了变化， 可检测到整体转录和细胞功能的变化。为了测试这一假设，我们生成了一个 使用CRISPR/Cas9基因组编辑工具，仅在APOE亚型上不同的独特的等基因IPSCs系列。 我们建立了一个平台，通过分化人脑细胞系统来概括培养中的人脑细胞系统 IPSC来源的星形胶质细胞和表达APOE的小胶质细胞。我们寻求将这位病患聚集在一起- 衍生的等基因人IPSC模型、强大的系统生物学、生物信息学方法和体外 代谢检测包括神经炎症、吞噬和自噬，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载脂蛋白E的异构体依赖 对神经胶质细胞的影响及其对脂质挑战的反应。在目标1中，我们将生成同质种群 星形胶质细胞和小胶质细胞以及来自同基因载脂蛋白E亚型和载脂蛋白E的皮质神经元/胶质细胞的混合培养 来自患者ipscs的基因敲除。我们将进行差异基因表达分析，以确定 APOE基因在每种细胞类型中的下游效应。在目标2中，我们将研究载脂蛋白E亚型依赖的神经胶质细胞 对富含脂质颗粒的挑战的反应。遵循公正的转录切分方法，我们将分析 哪种疾病相关的小胶质细胞特征或AD相关网络是APOE异构体依赖的 在接受挑战时。在目标3中，我们将研究载脂蛋白E异构体依赖对炎症反应的影响。 和脂粒的吞噬/自溶酶体清除。该项目的目标是确定 在存在或不存在ApoE基因的情况下，转录转录网络和细胞功能由APOE基因决定 与疾病相关的环境(髓鞘碎片)，以确定最早和最可能最可治疗的 阿尔茨海默病发病机制。

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.