Uncovering APOE4 Matrisome endophenotypes using human iPSC-based models

项目摘要 阿尔茨海默病（Alzheimer disease，AD）是最常见的年龄相关性痴呆，以广泛分布的β-淀粉样蛋白为特征 累积、神经元缠结、神经炎症和明显的神经元和突触损失。最近AD GWAS研究已经发现了几个主要在基因内的风险单核苷酸多态性（SNP）， 在胶质细胞中表达。值得注意的是，载脂蛋白E（APOE）ε4等位基因是AD最强的遗传危险因素; 与APOE 33相比，APOE 44/APOE 44（APOE 44）使AD风险增加15倍，并且主要是由APOE 33/APOE 43（APOE 33）引起的。 在星形胶质细胞中表达，但在最近鉴定的疾病相关小胶质细胞表型中也上调。 虽然APOE 4和AD风险之间的关系已经确立，但这种作用的机制 特别是人脑细胞类型尚不完全清楚。全面评估人APOE 4的作用 在人脑细胞类型上，我们表征了四种脑细胞类型中的APOE 4基因型-表型关系： 小胶质细胞、星形胶质细胞、脑微血管内皮细胞和来自人的混合皮质培养物 诱导多能干细胞（iPSC）。全球转录组分析显示，APOE 4驱动增强 与来自iPSCs的星形胶质细胞中的趋化性、胶质细胞活化和脂质代谢相关的基质体基因集- 衍生的混合皮层培养物，并且该特征在AD脑细胞型去卷积转录组中得到证实 数据-当与神经元共培养时来自星形胶质细胞的表型。我们假设， 来源于APOE 4星形胶质细胞的细胞因子诱导神经元活性和小胶质细胞功能的缺陷。我们还假设 含有Siglec受体-唾液酸的糖胺聚糖配体与CD 33风险基因型的相互作用， APOE 4胶质细胞促进斑块形成和神经炎症。为了验证这些假设，人类的“大脑- 由源自同基因iPSC的神经元、星形胶质细胞和小胶质细胞组成的“培养皿”模型系统将被 利用。在目标1中，我们将进行单细胞转录组学分析，以确认二维和三维的基质体信号 模型，并确定APOE 4改变的基质体信号传导如何影响神经元活动和小胶质细胞 在基线和AD神经病理学特征的背景下，在目标2中，我们将确定 APOE 4和Siglec受体CD 33风险在单个细胞中协同作用的分子机制 转录组水平，及其对斑块形成和神经炎症的影响。在目标3中，我们将确定 使用药物筛选平台逆转改变的星形胶质细胞基质体信号传导表型的化合物。的 该项目的目标是评估APOE 4驱动的基质体内表型的功能后果， 了解糖蛋白受体风险与APOE 4基质体信号串扰的机制。推动这一 拟议的研究项目开始发现潜在的治疗药物，以解决APOE 4驱动的基质体 在APOE 4携带者AD患者中发现的内源性表型。

PROJECT SUMMARY Alzheimer disease (AD), the most prevalent age-related dementia, is characterized by widespread β-amyloid accumulation, neurofibrillary tangles, neuroinflammation, and frank neuronal and synaptic loss. Recent AD GWAS studies have uncovered several risk single nucleotide polymorphisms (SNPs) within genes predominantly expressed in glia. Notably, the Apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for AD; APOE ɛ4/ɛ4 (APOE 44) increases AD risk by 15-fold compared to APOE ɛ3/ɛ3 (APOE 33) and is predominantly expressed in astrocytes, but also upregulated in the recently identified disease-associated microglia phenotype. Although the relationship between APOE4 and AD risk is well-established, the mechanisms underlying this effect in particular human brain cell types are not entirely clear. To comprehensively assess the effect of human APOE4 on human brain cell types, we characterized the APOE4 genotype-phenotype relationship in four brain cell types: microglia, astrocytes, brain microvascular endothelial cells and mixed cortical cultures derived from human induced pluripotent stem cells (iPSCs). Global transcriptome analyses reveal that APOE4 drives enhanced Matrisome gene sets associated with chemotaxis, glial activation and lipid metabolism in astrocytes from iPSCs- derived mixed cortical cultures, and this signature is confirmed in AD brain cell-type deconvoluted transcriptome data–a phenotype from astrocytes when co-cultured with neurons. We hypothesize that matrisome signals derived from APOE4 astrocytes induce defects in neuronal activity and microglia function. We also hypothesize that interactions of Siglec receptor-sialic acid containing glycosaminoglycan ligands by CD33 risk genotype and APOE4 glia promote plaque formation and neuroinflammation. To test these hypotheses, human “brain-in-a- dish” model systems, composed of neurons, astrocytes and microglia derived from isogenic iPSCs will be utilized. In aim 1, we will perform single cell transcriptomic analysis to confirm matrisome signals in 2D and 3D models and determine how altered matrisome signaling by APOE4 impacts neuronal activity and microglia phenotype at baseline and in the context of AD neuropathological features. In aim 2, we will determine the molecular mechanism of synergistic effects of APOE4 and Siglec receptor CD33 risk in a single cell transcriptomic level, and its effects on plaque formation and neuroinflammation. In aim 3, we will identify compounds to reverse altered astrocyte matrisome signaling phenotypes using drug screening platforms. The goal of this project is to assess functional consequences of APOE4 driven matrisome endophenotypes and understand the mechanism of glycoprotein receptor risk crosstalk with APOE4 matrisome signals. Further this proposed research project sets out to uncover potential therapeutic drugs to tackle APOE4 driven matrisome endophenotypes found in APOE4 carrier AD patients.