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

项目总结 阿尔茨海默病(AD)是一种进行性神经退行性疾病，记忆严重受损， 认知和执行功能具有巨大的健康和经济负担。缺乏有效的 阻止AD病理的治疗需要开发人脑微生理系统来 了解疾病的发病机制，开发潜在的治疗方法。神经血管单位与血脑 屏障(BBB)功能障碍在AD进展中起着关键的病因作用；然而，遗传和 脑微血管损伤的环境风险因素尚不清楚。针对RFA(#PAR-20-055)， 该项目将产生经过验证的人类多能干细胞(PSC)来源的NVU细胞，开发一种灌流的NVU 青年和老年患者血浆或血液的三维微生理系统及其转录组的比较 与人类AD脑血管组的图谱，以剖析遗传因素与年龄相关因素在AD中的作用 广告。在初步研究中，我们使用CRISPR/Cas9方法来敲入FAD或加载突变 控制PSC细胞系，开发将PSC分化为脑微血管内皮细胞的策略 并利用NVU细胞和Flow建立单芯片脑模型。此外，我们的 24例正常人和阿尔茨海默病患者脑的初步单核RNA-SEQ鉴定出4种不同的BMEC 在转录组水平上的群体，其中一个与认知障碍呈正相关，Ab和 牛磺酸的积累。我们假设遗传和环境风险因素之间的协同作用 通过改变关键的细胞通路或转录因子来损害NVU功能和BBB特性。我们会 用三个目标来解释这一假设。在目标1中，我们将优化协议以通过以下方式生成BMEC 从内皮祖细胞或内皮细胞转分化，验证其与单细胞的分子同一性 RNA-seq，并使用细胞、生化、成像和功能方法验证其生物学功能。 我们将把BMEC与hPSC来源的周细胞一起整合到NVU 3D微生理系统中，并 并比较NVU之间的细胞生物学、转录组、成像和功能屏障特性 携带AD相关风险基因和同基因对照。在目标2中，我们将利用来自AMP-AD的数据 识别与认知障碍、抗体和tau相关的AD脑血管组特异性特征的数据库 在NVU 3D中是否存在体内AD相关脑血管改变的蓄积和评估 从携带AD相关风险基因的hPSC系衍生的微生理系统。最后，在目标3中，我们将 评估是否使用蛋白酶体抑制剂来模拟蛋白凝集素的丢失，这些试剂会产生 晚期糖基化终末产物，或老年血液的动态流动改变形态和转录组 NVU细胞的轮廓和血脑屏障转运。拟议的研究将建立一种新的灌流血液/NVU 3D 微生理系统，使我们能够利用它与AD脑血管和 研究遗传和环境因素之间的相互作用对AD血管病理的影响。

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.