Uncovering APOE4 Matrisome endophenotypes using human iPSC-based models

项目总结 阿尔茨海默病(AD)是最常见的老年性痴呆，其特征是广泛存在的β-淀粉样蛋白 堆积，神经原纤维缠结，神经炎症，以及坦率的神经元和突触丢失。最近的AD Gwas的研究发现了基因中几个危险的单核苷酸多态(SNPs)，主要是 在神经胶质细胞中表达。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； 与载脂蛋白Eɛ3/ɛ3(载脂蛋白E 33)相比，载脂蛋白Eɛ4/ɛ4(载脂蛋白44)使AD风险增加15倍，并且主要是 在星形胶质细胞中表达，但在最近发现的与疾病相关的小胶质细胞表型中也上调。 尽管APOE4和AD风险之间的关系已经确立，但这种效应背后的机制 特别是，人类的脑细胞类型并不完全清楚。全面评估人载脂蛋白4的作用 关于人脑细胞类型，我们描述了四种脑细胞类型的APOE4基因-表型关系： 人类来源的小胶质细胞、星形胶质细胞、脑微血管内皮细胞和混合皮质培养 诱导多能干细胞(IPSCs)。全球转录组分析显示APOE4驱动功能增强 与星形胶质细胞趋化、胶质激活和脂代谢相关的Matrisome基因组 来源于混合皮质培养，这一特征在AD脑细胞型去卷曲转录组中得到证实 数据-星形胶质细胞与神经元共培养时的一种表型。我们假设母体信号 来源于APOE4的星形胶质细胞会导致神经元活动和小胶质细胞功能的缺陷。我们还假设 CD33危险基因与Siglec受体-含唾液酸糖胺聚糖配体的相互作用 ApoE4胶质细胞促进斑块形成和神经炎症。为了检验这些假说，人类的“脑中之脑-- 由来自同基因IPSCs的神经元、星形胶质细胞和小胶质细胞组成的DISH模型系统将被 被利用了。在目标1中，我们将进行单细胞转录分析，以确认2D和3D中的矩阵信号 建立模型并确定APOE4改变的基质信号如何影响神经元活动和小胶质细胞 在基线和AD神经病理特征的背景下的表型。在目标2中，我们将确定 APOE4和Siglec受体CD33风险在单细胞中协同作用的分子机制 转录水平，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信号表型的化合物。这个 该项目的目标是评估APOE4驱动的母体内表型和 了解糖蛋白受体风险与APOE4信号串扰的机制。再往前走 拟议的研究项目旨在发现潜在的治疗药物来应对APOE4驱动的母体 载脂蛋白4携带者AD患者的内表型。

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.