Developing a microfluidic human neurovascular unit system to investigate genetic and age-related risk factors in Alzheimer's disease

开发微流体人类神经血管单元系统来研究阿尔茨海默病的遗传和年龄相关危险因素

• 批准号: 10504196
• 负责人: Dritan Agalliu
• 金额: $ 120.15万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10504196
• 关键词: 3-Dimensional; Address; Advanced Glycosylation End Products; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease pathology; Alzheimer' s disease risk; Amyloid; Astrocytes; Automobile Driving; Autopsy; Biochemical; Biological; Biological Process; Blood; Blood - brain barrier anatomy; Blood Vessels; Brain; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cells; Cellular biology; Cerebrovascular Disorders; Cerebrum; Cognition; Data; Data Set; Databases; Development; Disease; Disease Progression; Early Onset Familial Alzheimer' s Disease; Economic Burden; Environment; Environmental Risk Factor; Etiology; Extracellular Matrix; Functional disorder; Future; Genetic; Genetic Risk; Genetic Transcription; Hand; Human; Image; Immune; Impaired cognition; Impairment; In Vitro; Knock-in; Knowledge Portal; Label; Late Onset Alzheimer Disease; Maintenance; Mediating; Memory impairment; Methodology; Microfluidics; Modeling; Molecular; Morphology; Mutation; Neurodegenerative Disorders; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Perfusion; Pericytes; Permeability; Persons; Phenotype; Plasma; Plasma Proteins; Play; Pluripotent Stem Cells; Population; Property; Proteasome Inhibitor; Prothrombin; Protocols documentation; Risk; Risk Factors; Role; Source; Structure; System; Therapeutic; Vascular Dementia; Vascular Diseases; Viral; age related; aged; blood-brain barrier function; brain endothelial cell; brain tissue; cell type; cerebrovascular; cohort; disease-causing mutation; effective therapy; efficacy evaluation; endothelial stem cell; executive function; familial Alzheimer disease; genetic risk factor; health economics; human data; human pluripotent stem cell; in vivo; innovation; microphysiology system; mutant; neurovascular unit; novel; response; risk variant; single-cell RNA sequencing; tau aggregation; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; transdifferentiation

PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with severe impairment of memory, cognition and executive functions that has a tremendous health and economic burden. The lack of effective treatments to halt AD pathology necessitates development of human brain microphysiological systems to understand disease pathogenesis and develop potential therapeutics. Neurovascular unit (NVU) and blood-brain barrier (BBB) dysfunction play a key etiological role in AD progression; yet the contribution of genetic versus environmental risk factors for brain microvascular damage is unclear. In response to RFA (# PAR-20-055) this project will generate validated human pluripotent stem cells (PSC)-derived NVU cells, develop a perfused NVU 3D microphysiological system with plasma or blood from young or aged patients and compare its transcriptome profiles with the human AD brain vasculome to dissect the contribution of genetic versus age-related factors in AD. In preliminary studies, we have used CRISPR/Cas9 methodology to knock-in FAD or LOAD mutations in control PSC cell lines, developed strategies to differentiate PSC into brain microvascular endothelial cells (BMECs), pericytes or astrocytes and build brain-on-a-chip models with NVU cells and flow. In addition, our preliminary single nucleus RNA-seq of 24 human control and AD brains has identified 4 distinct BMEC populations, at the transcriptome level, one of which is positively correlated with cognitive impairment, Ab and tau accumulation. We hypothesize that synergistic interactions between genetic and environmental risk factors impair NVU function and BBB properties by altering key cellular pathways or transcription factors. We will address this hypothesis with three aims. In Aim 1, we will optimize protocols to generate BMECs via transdifferentiation from either endothelial progenitor cells or ECs, verify their molecular identity with single cell RNA-seq and validate their biological function using cellular, biochemical, imaging and functional approaches. We will incorporate BMECs into an NVU 3D microphysiological system along with hPSC-derived pericytes and astrocytes and compare cell biological, transcriptome, imaging and functional barrier properties between NVUs carrying AD-associated risk genes and isogenic controls. In Aim 2, we will leverage the data from the AMP-AD database to identify the AD brain vasculome-specific profiles associated with cognitive impairment, Ab and tau accumulation and evaluate whether AD-associated brain vasculome changes in vivo are present in the NVU 3D microphysiological system derived from hPSC lines carrying AD-associated risk genes. Finally in Aim 3, we will assess whether treatment with proteasome inhibitors that mimick loss of proteostatis, agents that produce advanced glycation end products, or dynamic flow of aged blood alters the morphology and transcriptome profiles of NVU cells and BBB transport. The proposed studies will establish a novel perfused blood/NVU 3D microphysiological system that will allow us to leverage its relevance to changes in the AD brain vasculome and examine interactions between genetic and environment factors for AD vascular pathology.

项目摘要 阿尔茨海默氏病（AD）是一种进行性神经退行性疾病，记忆严重损害， 认知和行政职能具有巨大的健康和经济负担。缺乏有效 阻止AD病理学的治疗方法必须开发人脑微生物生理系统 了解疾病发病机理并发展潜在的治疗。神经血管单元（NVU）和血脑 障碍（BBB）功能障碍在AD进展中起关键的病因作用；然而遗传与 脑微血管损伤的环境风险因素尚不清楚。响应RFA（＃PAR-20-055） 项目将生成经过验证的人多能干细胞（PSC）衍生的NVU细胞，发展灌注NVU 3D微生物生理系统，具有血浆或年轻患者的血液或血液，并比较其转录组 与人类AD脑血管组的轮廓，以剖析遗传与年龄相关因素的贡献 广告。在初步研究中，我们已经使用CRISPR/CAS9方法来敲入时尚或负载突变 控制PSC细胞系，开发了将PSC区分为脑微血管内皮细胞的策略 （BMEC），周细胞或星形胶质细胞，并用NVU细胞和流动构建脑线模型。另外，我们的 24人类对照和AD大脑的初步单核RNA-SEQ已鉴定出4个不同的BMEC 在转录组级别的种群，其中一个与认知障碍，AB和 tau积累。我们假设遗传和环境风险因素之间的协同互动 通过改变关键细胞途径或转录因子来损害NVU功能和BBB性质。我们将 以三个目标解决这一假设。在AIM 1中，我们将优化协议以通过 从内皮祖细胞或EC的转差，验证其分子身份用单细胞验证 RNA-Seq并使用细胞，生化，成像和功能方法来验证其生物学功能。 我们将将BMEC与HPSC衍生的周细胞一起纳入NVU 3D微生物生理系统和 星形胶质细胞和比较NVU之间的细胞生物学，转录组，成像和功能性屏障特性 携带广告相关的风险基因和同源控制。在AIM 2中，我们将利用AMP-AD的数据 数据库以识别与认知障碍，AB和TAU相关的AD脑血管特异性概况 积累并评估NVU 3D中是否存在与AD相关的脑血管变化 源自携带AD相关风险基因的HPSC系的微生物生理系统。终于在AIM 3中，我们将 评估用蛋白酶体抑制剂处理模仿蛋白质抑制剂的蛋白质抑制剂，即产生的药物 高级糖基化最终产物或衰老血液的动态流动改变形态和转录组 NVU细胞和BBB转运的轮廓。拟议的研究将建立一种新型的灌注血/NVU 3D 微生物生理系统将使我们能够利用其与广告大脑血管群变化的相关性和 检查AD血管病理学的遗传因素与环境因素之间的相互作用。