A quantitative framework for understanding endosomal trafficking networks in Alzheimer's disease

了解阿尔茨海默氏病内体运输网络的定量框架

基本信息

批准号：
10470286
负责人：
JEFFREY W HARPER
金额：
$ 45.94万
依托单位：
HARVARD MEDICAL SCHOOL
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：
2018-09-30 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10470286
关键词：
Abeta clearance Abeta synthesis Affect Alzheimer&apos s Disease Amyloid beta-Protein Architecture Autophagocytosis Biochemical Biological Biological Models Biotinylation CRISPR screen Candidate Disease Gene Cell Line Cell membrane Cells Defect Degradation Pathway Dependence Disease Event Excision Genes Genetic Goals Golgi Apparatus Image Individual Laboratories Link Lysosomes Maps Membrane Proteins Methodology Mitochondria Molecular Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Organelles PINK1 gene Pathogenesis Pathway Analysis Pathway interactions Predisposition Prevention Process Proteins Proteomics Quality Control Recycling Reporter Role Sorting - Cell Movement Specificity System Testing Therapeutic Time Toxic effect Ubiquitin Validation Work abeta accumulation abeta toxicity arm base cell type design experimental study gain of function mutation gene network genetic analysis human embryonic stem cell insight late endosome misfolded protein multicatalytic endopeptidase complex mutant neuropathology novel protein degradation protein misfolding protein transport proteostasis repaired screening tissue/cell culture trafficking

项目摘要

PROJECT SUMMARY Accumulating evidence suggest that neurodegenerative diseases including Alzheimer’s disease (AD) are frequently the result of alterations in endosomal trafficking and proteostasis pathways, one consequence of which can be the accumulation of aggregation-prone proteins. Numerous familial and sporadic loss or gain-of- function mutations have been identified in such pathways, illuminating potential drivers of disease pathogenesis. In addition, excess protein mis-folding due to altered trafficking could affect proteostasis pathways by blocking protein turnover or trafficking. Protein and organelle trafficking within cells is a highly dynamic and interconnected process, and defects in one arm of the system can affect other aspects of the network in unpredictable ways including reduced flux in turnover pathways. Indeed, it is conceivable that many seemingly unrelated mutations across the trafficking landscape in various neurodegenerative diseases reveal common mechanistic vulnerabilities downstream but with distinct cell-type sensitivities reflective of the identity of mis-trafficked proteins. As such, understanding the global architecture of trafficking systems and the key machinery that controls the directionality and efficiency of trafficking, particularly of aggregation-prone neurodegenerative proteins such as APP and its aggregation-prone form A, represents a central goal of the field. A aggregation as a toxic driver of AD neuropathology has been a dominant hypothesis in the field. However, thus far therapeutics directed at aggregate prevention or removal have not been successful, and alternative hypotheses including alterations in intracellular trafficking as an important event in neuropathology have emerged. Here, we seek to combine powerful genetic and proteomic approaches to develop a quantitative framework for understanding how disruption of major endosomal trafficking systems – retromer and retriever, found defective in neurodegenerative diseases – alter global membrane protein trafficking, and specific trafficking and processing of APP proteoforms. These studies make use of an extensive tool-kit of mutant tissue culture cell lines and induced neurons derived from human embryonic stem cell (hESC), in combination with targeted and unbiased proteomics of individual organelles linked with endosomal trafficking, to assemble a global map of cargo and trafficking dependencies. In parallel, we will employ novel flux-based screening strategies to search for genes controlling APP/A trafficking to the lysosome and the plasma membrane, and will examine the extent to which A accumulation within the endo-lysosomal system alters selective autophagic flux using new cargo-specific reporters. The central hypothesis being tested is that specific defects in protein trafficking networks underlies the susceptibility of neurons to A and other aggregation prone proteins and that these defects can be molecularly unmasked through systematic network and genetic analysis.

项目摘要积累的证据表明，包括阿尔茨海默氏病（AD）在内的神经退行性疾病是经常导致内体贩运和蛋白质途径改变的结果，这是许多家庭和零星的损失或零星损失可能是易于聚集的蛋白质的积累。在这种途径中已经确定了功能突变，阐明了潜在的疾病驱动因素发病。此外，由于运输的改变而导致的过量蛋白质折叠可能会影响蛋白质通过阻止蛋白质周转或贩运来通过途径。细胞内的蛋白质和细胞器运输是一种高度动态和互连的过程以及系统的一个部门的缺陷会影响该系统的其他方面网络以不可预测的方式进行网络，包括流动途径中的通量减少。确实，可以想象很多揭示了各种神经退行性疾病的贩运局势的似乎无关的突变显示下游的常见机械脆弱性，但具有不同的细胞类型灵敏度反映了身份贩运蛋白质不良的蛋白质。因此，了解贩运系统的全球体系结构和关键控制贩运的方向性和效率的机械，特别是容易汇总的机械神经退行性蛋白（例如应用程序及其易于聚集的A）代表了该蛋白质的核心目标场地。作为AD神经病理学的有毒驱动因素，A聚集在该领域是一个主要的假设。但是，到目前为止，针对预防或清除的疗法尚未成功，并且替代假设，包括改变细胞内贩运作为神经病理学的重要事件出现了。在这里，我们试图结合强大的遗传和蛋白质组学方法来开发定量框架，用于了解主要内体贩运系统的破坏 - 逆转录器和检索器，发现神经退行性疾病有缺陷 - 改变全球膜蛋白运输和 APP蛋白质成型的特定贩运和处理。这些研究利用了广泛的工具套件突变组织培养细胞系和源自人类胚胎干细胞（HESC）的神经元在与内体贩运相关的单个细胞器的靶向和公正蛋白质组学结合，组装全球货物和贩运依赖地图。同时，我们将采用基于新颖的助剂筛选搜索控制App/A运输溶酶体和等离子体的基因的策略膜，并将检查在内部溶酶体系统中A积累的程度选择性自噬通量使用新的货物特异性记者。测试的中心假设是蛋白质运输网络中的特定缺陷是神经元对A和其他的敏感性的基础聚集易于蛋白质，并且这些缺陷可以通过系统网络分子揭示和遗传分析。