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

• 批准号: 10241471
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241471
• 关键词: Abeta clearance; Abeta synthesis; Affect; Alzheimer' 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; genetic analysis; human embryonic stem cell; insight; late endosome; multicatalytic endopeptidase complex; mutant; neuropathology; novel; protein degradation; protein misfolding; protein transport; proteostasis; repaired; screening; tissue/cell culture; trafficking

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 越来越多的证据表明，神经退行性疾病，包括阿尔茨海默病（AD）， 通常是内体运输和蛋白质稳态途径改变的结果， 这可能是聚集倾向蛋白质的积累。许多家族性和散发性的丧失或获得- 在这些途径中已经发现了功能突变，阐明了疾病的潜在驱动因素 发病机制此外，由于运输改变而导致的过量蛋白质错误折叠可能影响蛋白质稳态 通过阻断蛋白质周转或运输途径。蛋白质和细胞器在细胞内的运输是一个高度 动态和相互关联的过程，并且系统的一个臂中的缺陷可以影响系统的其他方面。 网络以不可预测的方式，包括减少流动的周转途径。事实上，可以想象，许多 在各种神经退行性疾病的贩运景观中，看似无关的突变揭示了 常见的下游机制漏洞，但具有反映身份的不同细胞类型敏感性 错误运输的蛋白质。因此，了解贩运系统的全球结构和 控制贩运方向和效率的机制，特别是易于聚集的贩运 神经退行性蛋白质如APP及其聚集倾向形式A β，代表了本发明的中心目标。 领域作为AD神经病理学的毒性驱动因素的聚集一直是该领域的主导假设。 然而，迄今为止，针对聚集体预防或去除的治疗尚未成功， 包括细胞内运输的改变作为神经病理学中的重要事件的替代假设 涌现在这里，我们寻求联合收割机强大的遗传和蛋白质组学的方法来开发一个 定量框架，了解如何破坏主要的内体运输系统- retromer 和寻回犬，发现有缺陷的神经退行性疾病-改变全球膜蛋白运输， APP蛋白形式的特异性运输和加工。这些研究利用了一个广泛的工具包， 从人胚胎干细胞（hESC）衍生的突变组织培养细胞系和诱导的神经元， 结合与内体运输相关的单个细胞器的靶向和无偏倚蛋白质组学， 收集全球货运和贩运依赖地图。同时，我们将采用新的通量为基础的 筛选策略以寻找控制APP/A β运输到溶酶体和血浆的基因 膜，并将检查在何种程度上A β积累内溶酶体系统改变 使用新的货物特异性报告基因的选择性自噬通量。正在测试的中心假设是， 蛋白质运输网络中的特定缺陷是神经元对A β和其他神经元敏感性的基础。 聚集倾向的蛋白质，这些缺陷可以通过系统的网络分子揭露 基因分析。