Cell and Molecular Consequences of Alzheimer's Disease Genetic Variants on BBB Integrity and Function

血脑屏障(BBB)功能障碍已被证明在早发性和晚发性中都起到了因果作用。 阿尔茨海默病(Eoad，Load)。虽然关于血脑屏障的分子机制已经了解了很多， 阿尔茨海默病的功能和功能障碍来自小鼠模型系统，许多重要的悬而未决的问题仍然存在 AD相关突变和基因变异如何影响人类血脑屏障的完整性和功能。 需要改进的人体实验系统来补充现有的动物模型。开拓性 合作者Pi Ingber的研究已经产生了微流控3D芯片上器官模型，包括BBB-on-a-Chip(BBB-on-a-Chip) 芯片)，越来越能代表人体的活体生理学。我们已经调整了这个系统，以创建 正常受试者和Eoad和Load受试者的等基因IPSC衍生的BBB芯片模型。 Young-Pearse实验室已经产生了一组捕捉到不同遗传风险的IPSC系 AD的因素包括：APP的Eoad突变和校正的对照，DS和DS的副本去除 高影响的chr21基因，包括apoE 2/2、3/3、4/4和KO的等基因apoE系列，以及 我们从ROS/MAP队列中的50多个人那里生成的线条代表了临床和 病理性荷载谱。在这里，我们建议将BBB-Chip模型与IPSC系列相结合 收集研究早发和晚发的遗传变异对血脑屏障功能的影响，并确定 受这些变异体影响的分子通路。在第一个目标中，我们解决了神经元 表达eoad突变分泌一种β物种，该物种通过毒性作用对血脑屏障完整性产生负面影响 脑微血管内皮细胞。我们将使用人类血脑屏障体外模型来检测β- 21三体和FAD突变通过a)对血脑屏障完整性和功能的依赖和独立影响 跨内皮细胞电阻(TEER)的测量，b)通透性分析，和c) 血脑屏障细胞的免疫细胞化学和形态分析。此外，我们还将鉴定分子 通过RNA测序和无偏倚研究Eoad在BMVEC、周细胞和星形胶质细胞中的作用途径 蛋白质组学。在目标2中，我们确定了Aβ聚集体的组成改变对清除量的功能影响 穿过血脑屏障的病理性Aβ。为此，我们还将使用各种定义良好的合成Aβ物种 作为人类神经元和大脑来源的β系统地定义β的组成和聚集 状态影响：1)血脑屏障对Aβ的摄取和跨细胞作用；2)血脑屏障的完整性和健康 周细胞、星形胶质细胞和骨髓微血管内皮细胞组成血脑屏障。最后，在第三个目标中，我们解决了这样的假设 负荷风险基因SORL1和Clu与载脂蛋白E协同工作，调节血脑屏障对Aβ的清除。在这 目的，我们将确定APOE、CLU和SORL1的变体对血脑屏障完整性的功能后果 还有一份β许可。最后，我们将确定APOE、CLU和 人血脑屏障实验系统中BMVEC、周细胞和星形胶质细胞中SORL1的表达。

Blood-brain barrier (BBB) dysfunction has been shown to play a causal role in both early- and late-onset Alzheimer's disease (EOAD, LOAD). While much has been learned about the molecular mechanisms of BBB function and dysfunction in AD from mouse model systems, many important unanswered questions remain regarding how AD-associated mutations and genetic variants affect human BBB integrity and function. Improved human experimental systems are required to complement existing animal models. Pioneering studies by co-PI Ingber have produced microfluidic 3D organ-on-chip models, including a BBB-on-a-chip (BBB- Chip), increasingly representative of human in vivo physiology. We have adapted this system to create isogenic iPSC-derived BBB-Chip models of normal human subjects and of subjects with EOAD and LOAD. The Young-Pearse lab has generated a collection of iPSC lines that capture the diverse set of genetic risk factors for AD including: EOAD mutation of APP and corrected controls, DS and DS with removal of copies of high impact chr21 genes, an isogenic APOE series including APOE 2/2, 3/3, 4/4 and KO, and a collection of lines that we've generated from over 50 individuals in the ROS/MAP cohorts that represent the clinical and pathological spectrum of LOAD. Here, we propose to combine the BBB-Chip model with the iPSC line collection to examine the impact of early- and late-onset genetic variants on BBB function, and to define the molecular pathways impacted by these variants. In the first aim, we address the hypothesis that neurons expressing EOAD mutations secrete Aβ species that negatively affect BBB integrity through toxic effects on brain microvasculature endothelial cells (BMVECs). We will use human BBB in vitro models to examine Aβ- dependent and independent impacts of trisomy 21 and fAD mutation on BBB integrity and function via a) measurements of transendothelial electrical resistance (TEER), b) permeability assays, and c) immunocytochemistry and morphological analyses of BBB cells. In addition, we will identify the molecular pathways affected in EOAD in BMVECs, pericytes and astrocytes via RNA sequencing and unbiased proteomics. In aim 2, we determine the functional impact of altered composition of Aβ aggregates on clearance of pathologic Aβ across the BBB. To this end, we will use a variety of well defined synthetic Aβ species as well as human neuron-derived and brain-derived Aβ to systematically define how Aβ composition and aggregation state affects: 1) uptake and transcytosis of Aβ across the BBB and 2) integrity of the BBB and health of the pericytes, astrocytes and BMVECs composing the BBB. Finally, in the third aim we address the hypothesis that the LOAD risk genes SORL1 and CLU work in concert with APOE to mediate Aβ clearance by the BBB. In this aim, we will determine the functional consequences of variants of APOE, CLU and SORL1 on BBB integrity and Aβ clearance. Finally, we will determine the molecular consequences of modulation of APOE, CLU and SORL1 in BMVECs, pericytes and astrocytes in our human BBB experimental system.