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

• 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
• 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' s Disease; Alzheimer' 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

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）功能障碍已显示出在早期和晚期发挥因果作用 阿尔茨海默氏病（EOAD，负载）。尽管已经了解了BBB的分子机制 鼠标模型系统的广告功能和功能障碍，许多重要的未解决问题仍然存在 关于广告相关的突变和遗传变异如何影响人BBB的完整性和功能。 需要改进的人类实验系统以完成现有的动物模型。开创性 共同研究的研究产生了微流体3D器官芯片模型，包括BBB-A-A-Chip（BBB-- 芯片），越来越多地代表人体内生理学。我们已经适应了这个系统来创建 正常人受试者和具有EOAD和负载的受试者的同源IPSC衍生的BBB芯片模型。 年轻的pearse实验室产生了一系列IPSC线，可捕获各种遗传风险 AD的因素包括：APP的EOAD突变和校正对照，DS和DS，并取消副本 高影响CHR21基因，一个ISEGENIC APOE系列，包括APOE 2/2、3/3、4/4和KO，以及集合 我们从ROS/MAP同伙中的50多个人产生的线，代表临床和 负载的病理光谱。在这里，我们建议将BBB-ChIP模型与IPSC线相结合 收集以检查早期和晚期遗传变异对BBB功能的影响，并定义 这些变体影响的分子途径。在第一个目标中，我们解决了神经元的假设 表达EOAD突变秘密Aβ物种，通过对BBB完整性对毒性影响负面影响 脑微举行内皮细胞（BMVEC）。我们将使用人类BBB体外模型检查Aβ- 通过A的依赖和独立影响对BBB的完整性和功能的依赖性和独立影响） 跨内皮电阻（TEER），b）渗透率评估的测量，C） BBB细胞的免疫细胞化学和形态分析。另外，我们将确定分子 通过RNA测序和无偏见 蛋白质组学。在AIM 2中，我们确定了Aβ聚集物改变组成对清除的功能影响 跨BBB的病理Aβ。为此，我们还将使用各种定义明确的合成Aβ物种 随着人神经衍生和脑衍生的Aβ的系统定义Aβ组成和聚集 状态影响：1）Aβ在BBB中的摄取和跨经细胞增多和2）BBB的完整性和健康状况 构成BBB的周细胞，星形胶质细胞和BMVEC。最后，在第三个目的中，我们解决了一个假设 负载风险基因SORL1和CLU与APOE协同工作，以介导BBB的Aβ清除率。在这个 目的，我们将确定APOE，CLU和SORL1变体对BBB完整性的功能后果 和Aβ清除率。最后，我们将确定APOE，CLU和 BMVEC，周细胞和星形胶质细胞中的SORL1中，我们的人类BBB实验系统中的SORL1。