Defining microglial and endothelial cell-type specific proteomic alterations of APOE risk in Alzheimer's disease

摘要(项目摘要) 迟发性阿尔茨海默病的全基因组关联研究已经确定了20多个基因与之相关 有疾病风险，其中载脂蛋白E(APOE)基因变异是最大的风险因素。APOE是一种 已知的在胆固醇代谢中有功能的脂蛋白，但它传递的机制 广告风险仍然知之甚少。载脂蛋白E有三种亚型ε2(E2) ，ε3(E3) ，和ε4(E4)，其中有 ε4等位基因患阿尔茨海默病的可能性是其他人的3-12倍，而ε2等位基因则降低了罹患AD的风险 广告。载脂蛋白E4影响AD和AD患者的淀粉样β蛋白(Aβ)聚集、代谢和斑块负荷 阿尔茨海默病小鼠模型。载脂蛋白E4也不同地影响脑内小胶质细胞介导的炎症反应 以及内皮介导的Aβ在脑血管中的清除，提示APOE相关 AD风险可能在一定程度上是由对Aβ病理的功能失调的炎症和血管反应驱动的。目前， 我们对小胶质细胞和/或血管内皮细胞的分子组成的了解有很大差距。 载脂蛋白E相关AD的发病机制。对这些机制的思考有助于我们理解 可能共同作用影响疾病状态或保护的蛋白质，为 疾病的病理生理学，并指导对治疗靶点的探索。 这项研究的长期目标是更好地了解载脂蛋白E基因对细胞的影响 脑部的表型。为了实现这一目标，我将使用数据驱动的蛋白质组和系统生物学方法来 整合人和小鼠模型研究，解决载脂蛋白E基因对AD风险的影响。具体来说， 这项提案中概述的实验将检验中心假设：小胶质细胞和内皮细胞 阿尔茨海默病的细胞表型是由APOE基因引起的。这一假设得到了我令人兴奋的支持 初步数据显示，APOE基因影响全球蛋白质组改变、小胶质细胞和内皮细胞 人类阿尔茨海默病大脑中的丰度和细胞类型。这些蛋白质组学特征暗示了 阿尔茨海默病脑内小胶质细胞、内皮细胞和载脂蛋白E介导的病理生理，但由于它们是从 在整个脑组织中，细胞类型对蛋白质组改变的特定贡献可能被掩盖。鉴于此， 我们最近优化了基于细胞分离和质谱学(MS)的技术，以全面 首次同时鉴定小鼠脑内皮细胞和小胶质细胞的蛋白质组学特征 大脑。我将鉴定从EFAD转基因小鼠脑中分离的细胞的蛋白质组学特征，以揭开 载脂蛋白E基因对小胶质细胞和血管内皮细胞分子表型以及年龄特征的影响 这些细胞的相关变化。重要的是，这些数据将提供对融合APOE基因驱动的洞察 小鼠和人脑中小胶质细胞和内皮细胞的机制。

ABSTRACT (PROJECT SUMMARY) Genome wide association studies (GWAS) of late-onset AD have identified more than 20 genes to be associated with disease risk, with variation in the apolipoprotein E (APOE) gene being the strongest risk factor. APOE is a lipoprotein that is known to have functions in cholesterol metabolism, but the mechanisms by which it imparts AD risk remain poorly understood. APOE has three isoforms ε2 (E2) , ε3 (E3) , and ε4 (E4) where individuals with the ε4 allele are 3-12 times more likely to develop AD and those with ε2 allele have reduced risk of developing AD. APOE4 affects amyloid beta (Aβ) aggregation, metabolism, and plaque load in both individuals with AD and mouse models of AD. APOE4 also differentially affects microglial-mediated inflammatory responses in the brain as well as endothelial-mediated clearance of Aβ in the cerebrovasculature, suggesting that APOE-associated AD risk may in part be driven by dysfunctional inflammatory and vascular responses to Aβ pathology. At present, there is a significant gap in our knowledge of what molecular constituents mediate microglial and/or endothelial disease mechanisms in APOE-linked AD. The consideration of these mechanisms can aid our understanding of proteins that may work together to affect the disease state or protection, create a framework for the pathophysiology of the disease, and guide exploration into therapeutic targets. The long-term goal of this research is to better understand how APOE genotype effects cellular phenotypes in brain. To achieve this goal, I will use data-driven proteomic and systems biology approaches to integrate human and mouse model studies and resolve the impact of APOE genotype on AD risk. Specifically, the experiments outlined in this proposal will test the central hypothesis that microglial and endothelial cellular phenotypes in AD are caused by APOE genotype. This hypothesis is supported by my exciting preliminary data that show APOE genotype impacts global proteomic alterations, microglial and endothelial cell abundance, and cell-type profiles in human AD brains. These proteomic profiles imply a linkage between the microglia, endothelia, and APOE-mediated pathophysiologies in AD brain, but since they were obtained from whole brain tissue, the cell-type specific contributions to the proteomic alterations may be masked. Given this, we have recently optimized cell isolation and mass-spectrometry (MS)-based techniques to comprehensively characterize, for the first time, proteomic profiles of brain endothelial cells in parallel with microglia from mouse brain. I will characterize the proteomic profile of isolated cells from the brains of EFAD transgenic mice to unravel the impact of APOE genotype on microglial and endothelial molecular phenotypes as well as characterize age- related changes in these cells. Importantly, these data will provide insight into convergent APOE genotype driven mechanisms in microglial and endothelial cells in both mice and human brain.