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

摘要（项目总结） 晚发性AD的全基因组关联研究（GWAS）已经确定了20多个相关基因 载脂蛋白E（APOE）基因变异是最强的风险因素。APOE是一种 已知脂蛋白在胆固醇代谢中具有功能，但它赋予胆固醇代谢的机制 AD风险仍然知之甚少。APOE有三种亚型ε2（E2） 、ε3（E3） ，和ε4（E4），其中具有 ε4等位基因发生AD可能性是3-12倍，而具有ε2等位基因的人发生AD的风险降低 AD. APOE 4影响AD和AD患者的β淀粉样蛋白（Aβ）聚集、代谢和斑块负荷， AD小鼠模型。APOE 4也不同程度地影响脑中小胶质细胞介导的炎症反应 以及血管内皮介导的Aβ清除，表明APOE相关的 AD风险可能部分由对Aβ病理的功能失调性炎症和血管反应驱动。目前， 我们对什么样的分子成分介导小胶质细胞和/或内皮细胞 APOE相关AD的发病机制考虑这些机制可以帮助我们理解 这些蛋白质可能共同作用以影响疾病状态或保护作用，从而为疾病的发生创造了一个框架。 疾病的病理生理学，并指导治疗靶点的探索。 这项研究的长期目标是更好地了解APOE基因型如何影响细胞凋亡。 大脑中的表型为了实现这一目标，我将使用数据驱动的蛋白质组学和系统生物学方法， 整合人类和小鼠模型研究，并解决APOE基因型对AD风险的影响。具体地说， 本提案中概述的实验将测试小胶质细胞和内皮细胞的中心假设 AD的细胞表型是由APOE基因型引起的。这个假设得到了我令人兴奋的 初步数据显示，APOE基因型影响整体蛋白质组学改变，小胶质细胞和内皮细胞 丰度和人类AD大脑中的细胞类型谱。这些蛋白质组学图谱暗示了 小胶质细胞，内皮细胞，和APOE介导的病理生理学在AD脑，但因为他们是从 在整个脑组织中，细胞类型特异性对蛋白质组学改变的贡献可能被掩盖。鉴于此， 我们最近优化了细胞分离和基于质谱（MS）的技术， 首次描述了小鼠脑内皮细胞与小胶质细胞的蛋白质组学特征 个脑袋我将描述EFAD转基因小鼠脑中分离细胞的蛋白质组学特征， APOE基因型对小胶质细胞和内皮细胞分子表型的影响以及年龄特征， 这些细胞的变化。重要的是，这些数据将为APOE基因型驱动的聚合提供深入了解。 在小鼠和人脑中的小胶质细胞和内皮细胞中的机制。

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.