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

阿尔茨海默病中受体介导的转胞吞途径的分析

基本信息

批准号：
10285761
负责人：
Casim Sarkar
金额：
$ 22.63万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-04-30
项目状态：
已结题

项目摘要

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.

项目摘要血脑屏障（BBB）是脑微血管内皮细胞的高度选择性屏障（BMEC）提出了用于治疗诸如阿尔茨海默氏病（AD）的疾病的主要瓶颈。除了防止治疗进入大脑外，最早观察到BBB失调 AD的阶段，导致清除淀粉样β（Aβ）的能力下降，这在很大程度上有助于积累大脑中的Aβ。研究BBB中的受体介导的转胞胞病症对于发现新型途径都是至关重要的将治疗剂传递到大脑中，以治疗AD并发展对机械的理解 BBB的早期病理变化最终导致Aβ积累。父母的赠款专注于开发新型的高通量筛查技术，以识别在用于口服药物输送的肠上皮。拟议补充工作的目的是将这些技术应用于BBB的干细胞衍生模型。人类诱导的多能茎的使用细胞来源的BMEC将使用具有不同拷贝数的细胞系对AD BBB的转胞细胞增多 APOE-ε4等位基因，这是迟到AD的最大遗传危险因素。而精度连接 APOE-ε4等位基因和BBB功能障碍之间的表征没有很好，已经表明表达由APOE-ε4编码的APOE4的of ApoE4，改变了BBB的Aβ跨经细胞增多症，抑制了其适当的清除率。这拟议的研究由两个目的组成：（1）鉴定转胞胞菌病介导的药物的新受体输送到大脑进行AD治疗，（2）BMEC中Aβ受体的鉴定。在第一个目标中，bmecs 将与包含零，一两个副本APOE-ε4等位基因的HIPSC线区分开基于经胞吞作用的定向进化法（父母赠款）将用于发现新的蛋白质能够转移BMEC单层的配体。介导这些配体的跨胞症的接收器然后，将被确定为向AD BBB递送治疗剂的潜在靶标。在第二个目标中将利用交联的质谱法（CLM）来识别BMEC中Aβ受体的完整曲目与具有不同apoE-ε4状态的IPSC线区分开。这些研究将对任何基因型进行分析 - 已知Aβ受体的表达表达的特定差异，例如LRP1和RAGE，以及新发现不同BMEC之间的Aβ受体。这些试点研究将纳入一个新的R01提案中阐明与Aβ清除率有关的转胞胞菌病的新机制，以及将热有效载荷传递到广告患者的大脑。