Analysis of receptor-mediated transcytosis pathways implicated in Alzheimer's disease

项目总结 血脑屏障(BBB)是由脑微血管内皮细胞组成的高度选择性屏障。 (BMECs)，这是治疗阿尔茨海默病(AD)等疾病的药物输送的主要瓶颈。 除了阻止治疗药物进入大脑外，最早观察到的是血脑屏障失调。 阿尔茨海默病的各个阶段，导致清除淀粉样β蛋白(Aβ)的能力降低，这是导致堆积的主要原因 大脑中的β。研究受体在血脑屏障中介导的跨细胞作用是两种新途径发现的核心 将治疗药物输送到大脑治疗阿尔茨海默病，并发展对 血脑屏障的早期病理改变，最终导致Aβ积聚。家长助学金的重点是 发展新的高通量筛选技术以确定全套跨细胞受体 肠道上皮在口服给药中的应用。拟议补充工作的目标是 将这些技术应用于干细胞衍生的血脑屏障模型。人诱导多能干细胞的应用 细胞来源的BMECs将利用不同拷贝数的细胞系研究AD BBB中的跨细胞作用 ApoE-ε4等位基因是晚发性AD的最大遗传危险因素。而精确的连接 载脂蛋白E-ε4等位基因和血脑屏障功能障碍之间的关系尚未得到很好的描述，已有研究表明， 由apoE-ε4编码的apoE4改变Aβ跨血脑屏障的细胞转运，抑制其适当的清除。这个 建议的研究包括两个目标：(1)鉴定跨细胞介导性药物的新受体 (2)骨髓内皮细胞中A-β受体的鉴定。在第一个目标中，BMEC 将与含有APOE-ε4等位基因零拷贝、一拷贝或两拷贝的HiPSC系和一种新的 基于跨细胞分裂的定向进化方法将被用来发现新的蛋白质，这在父母的资助中有详细说明。 能够跨细胞连接BMEC单层的配体。介导这些配体跨细胞作用的受体 然后将被确定为向AD BBB提供治疗药物的潜在目标。在第二个目标中， 将利用交联质谱鉴定BMEC中Aβ受体的全部谱系 与不同APOE-ε4状态的IPSC系不同。这些研究将使任何基因分析成为可能- 已知的Aβ受体LRP1和RAGE表达的特异性差异以及新的发现 Aβ受体在不同的BMEC中的表达。这些试验性研究将被纳入新的R01提案，以 阐明与Aβ清除以及治疗有效载荷输送相关的细胞转运的新机制 阿尔茨海默病患者的大脑。

PROJECT SUMMARY The blood-brain barrier (BBB) is a highly selective barrier comprised of brain microvascular endothelial cells (BMECs) that presents a major bottleneck for drug delivery to treat disorders such as Alzheimer's disease (AD). In addition to preventing therapeutics from entering the brain, BBB dysregulation is observed in the earliest stages of AD, leading to a decreased ability to clear amyloid beta (Aβ) that largely contributes to the accumulation of Aβ in the brain. Studying receptor-mediated transcytosis in the BBB is central to both discovering novel routes for delivery of therapeutics into the brain for treatment of AD and developing a mechanistic understanding of early pathological changes to the BBB that lead to eventual Aβ accumulation. The parent grant is focused on developing novel high-throughput screening technologies to identify the full set of transcytosis receptors in the intestinal epithelium for applications in oral drug delivery. The objective of the proposed supplemental work is to apply these technologies to a stem cell-derived model of the BBB. The use of human induced pluripotent stem cell-derived BMECs will enable studies of transcytosis in the AD BBB using cell lines with varying copy numbers of the APOE-ε4 allele, which is the greatest genetic risk factor for late-onset AD. While the precise connections between the APOE-ε4 allele and BBB dysfunction are not well characterized, it has been shown that expression of apoE4, encoded by APOE-ε4, alters Aβ transcytosis across the BBB, inhibiting its proper clearance. The proposed research consists of two aims: (1) identification of novel receptors for transcytosis-mediated drug delivery to the brain for AD treatment and (2) identification of Aβ receptors in BMECs. In the first aim, BMECs will be differentiated from hiPSC lines containing zero, one or two copies of the APOE-ε4 allele, and a novel transcytosis-based directed evolution method, as detailed in the parent grant, will be used to discover new protein ligands that are able to transcytose the BMEC monolayer. The receptors mediating transcytosis of these ligands will then be identified as potential targets for delivery of therapeutics to the AD BBB. In the second aim, crosslinking mass spectrometry (CLMS) will be utilized to identify the full repertoire of Aβ receptors in BMECs differentiated from iPSC lines with varying APOE-ε4 status. These studies will enable analysis of any genotype- specific differences in expression of known Aβ receptors, such as LRP1 and RAGE, as well as discovery of new Aβ receptors among the different BMECs. These pilot studies will be incorporated into a new R01 proposal to elucidate new mechanisms of transcytosis related to Aβ clearance as well as delivery of therapeutic payloads to the brains of AD patients.