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）功能障碍已被证明在早发性和迟发性脑梗死中发挥因果作用。 阿尔茨海默病（EOAD，LOAD）。虽然对血脑屏障的分子机制已经有了很多了解， 从小鼠模型系统中，AD的功能和功能障碍，许多重要的未回答的问题仍然存在 关于AD相关突变和遗传变异如何影响人类BBB的完整性和功能。 需要改进的人类实验系统来补充现有的动物模型。开创性 共同PI Ingber的研究已经产生了微流体3D器官芯片模型，包括BBB芯片（BBB- 芯片），越来越多地代表人类体内生理学。我们改造了这个系统 正常人受试者和患有EOAD和LOAD的受试者的等基因iPSC衍生的BBB-芯片模型。 Young-Pearse实验室已经产生了一系列iPSC系，这些系捕获了不同的遗传风险 AD的因素包括：APP和校正对照的EOAD突变，DS和DS的拷贝去除 高影响chr 21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多名个体中生成的线条代表了临床和 LOAD的病理谱。在这里，我们建议将联合收割机的BBB芯片模型与iPSC线 收集，以检查早发性和迟发性遗传变异对BBB功能的影响，并确定 分子途径受到这些变体的影响。在第一个目标中，我们提出了神经元 表达EOAD突变的人分泌Aβ物质，通过对血脑屏障的毒性作用对血脑屏障的完整性产生负面影响。 脑微血管内皮细胞（BMVECs）。我们将使用人血脑屏障体外模型来检查Aβ- 21三体和fAD突变对BBB完整性和功能的依赖性和独立性影响，通过a） 跨内皮电阻（TEER）的测量，B）渗透性测定，和c） BBB细胞的免疫细胞化学和形态学分析。此外，我们还将鉴定 通过RNA测序和无偏倚分析， 蛋白质组学在目标2中，我们确定Aβ聚集体组成改变对清除率的功能影响 病理性Aβ穿过血脑屏障为此，我们也将使用各种定义明确的合成Aβ物质 作为人类神经元源性和脑源性Aβ，系统地定义Aβ的组成和聚集 状态影响：1）Aβ穿过BBB的摄取和转胞吞作用，以及2）BBB的完整性和 周细胞、星形胶质细胞和BMVEC构成BBB。最后，在第三个目标中，我们提出了一个假设， LOAD风险基因SORL 1和CLU与APOE协同作用，介导Aβ被BBB清除。在这 我们的目的是确定APOE、CLU和SORL 1变异体对血脑屏障完整性的功能影响 和Aβ清除率。最后，我们将确定APOE、CLU和 SORL 1在我们的人BBB实验系统中的BMVECs、周细胞和星形胶质细胞中的表达。