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

阿尔茨海默病遗传变异对血脑屏障完整性和功能的细胞和分子影响

基本信息

批准号：
10037760
负责人：
Tracy L YOUNG-PEARSE
金额：
$ 359.85万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10037760
关键词：
3-Dimensional Abeta clearance Address Affect Aging Alzheimer&apos s Disease Alzheimer&apos s disease risk Amyloid beta-Protein Amyloid beta-Protein Precursor Animal Model Apolipoprotein E Archives Astrocytes Binding Biological Assay Biological Models Blood Blood - brain barrier anatomy Brain Cells ChIP-on-chip Clinical Collaborations Collection Complement Development Dose Down Syndrome Electrical Resistance Embryo Endothelial Cells Endothelium Excision Failure Family Functional disorder Generations Genes Genetic Genetic study Health Human Hypoxia Impaired cognition Impairment Individual Late Onset Alzheimer Disease Link Measurement Mediating Memory Metabolic Clearance Rate Microfluidics Modeling Molecular Morphology Mus Mutation Neurons Organ Pathogenesis Pathologic Pathway interactions Patients Pericytes Permeability Physiological Physiology Play Presenile Alzheimer Dementia Process Proteomics Protocols documentation Risk Role Route Series Signal Pathway System Technology Toxic effect Variant Work apolipoprotein E-2 base blood-brain barrier function brain cell brain endothelial cell cerebral microvasculature cohort genetic risk factor genetic variant human subject immunocytochemistry improved in vitro Model in vivo induced pluripotent stem cell insight member mouse model negative affect organ on a chip receptor religious order study risk variant shear stress transcriptome sequencing transcytosis uptake

项目摘要

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.

血脑屏障（BBB）功能障碍已被证明在早发型和晚发型发病中发挥着因果作用阿尔茨海默氏病（EOAD、LOAD）虽然对 BBB 的分子机制已经了解很多。小鼠模型系统中 AD 的功能和功能障碍，许多重要的未解答问题仍然存在关于 AD 相关突变和基因变异如何影响人类 BBB 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。联合 PI Ingber 的研究已经产生了微流控 3D 器官芯片模型，包括 BBB 芯片 (BBB- Chip），越来越代表人体体内生理学，我们已经适应了这个系统来创建。正常人类受试者以及 EOAD 和 LOAD 受试者的同基因 iPSC 衍生 BBB 芯片模型。 Young-Pearse 实验室已经生成了一系列 iPSC 细胞系，可以捕获不同的遗传风险 AD 的因素包括： APP 的 EOAD 突变和校正对照、DS 和删除 DNA 拷贝的 DS 高影响 chr21 基因，同基因 APOE 系列，包括 APOE 2/2、3/3、4/4 和 KO，以及一系列我们从 ROS/MAP 队列中的 50 多个个体中生成了代表临床和在这里，我们建议将 BBB-Chip 模型与 iPSC 系结合起来。收集以检查早发和晚发遗传变异对 BBB 功能的影响，并定义受这些变异影响的分子途径在第一个目标中，我们提出了神经元的假设。表达 EOAD 突变会分泌 Aβ 物质，通过对 BBB 的毒性作用对 BBB 完整性产生负面影响。我们将使用人类 BBB 体外模型来检查 Aβ-。 21 三体和 fAD 突变对 BBB 完整性和功能的依赖性和独立影响通过 a) 跨内皮电阻 (TEER) 测量，b) 渗透性测定，以及 c) 此外，我们还将对 BBB 细胞进行免疫细胞化学和形态学分析。通过 RNA 测序和无偏性影响 BMVEC、周细胞和星形胶质细胞中 EOAD 的通路在目标 2 中，我们确定 Aβ 聚集体组成改变对清除的功能影响。为此，我们还将使用各种明确定义的合成 Aβ 物种。作为人类神经元来源和脑来源的 Aβ，系统地定义 Aβ 的组成和聚集方式状态影响：1) Aβ 跨 BBB 的摄取和转胞吞作用，以及 2) BBB 的完整性和 BBB 的健康最后，在第三个目标中，我们提出了以下假设： LOAD 风险基因 SORL1 和 CLU 与 APOE 协同作用，介导 BBB 清除 Aβ。目标，我们将确定 APOE、CLU 和 SORL1 变体对 BBB 完整性的功能影响最后，我们将确定 APOE、CLU 和 Aβ 调节的分子后果。我们的人类 BBB 实验系统中 BMVEC、周细胞和星形胶质细胞中的 SORL1。