Autophagy-dependent exosome loading and biogenesis in AD and FTD

项目总结 阿尔茨海默病(AD)的特征是异常裂解的A淀粉样蛋白进行性积累 多肽和过度磷酸化的tau蛋白，导致淀粉样斑块和神经原纤维缠结， 分别进行了分析。虽然目前还不清楚是什么触发了这些蛋白病变，但有几条证据表明 细胞内转运的缺陷可能调节AD的发病。重要的是，新出现的证据表明 分泌与AD相关的蛋白，包括tau、淀粉样前体蛋白(APP)和A淀粉样多肽 通过外显体。尽管有这些发现，但目前仍不清楚是什么因素控制着 外切体，外切体的生物发生是否在功能上与疾病相关的细胞内转运有关 蛋白质，是否神经元和胶质细胞形成不同的机制来处理这些蛋白质，如果是的话，是如何处理的 神经元和神经胶质细胞中的蛋白平衡异常协同促进了神经退化。 我们发现了一种新的途径，在这种途径中，自噬机制指定包装和分泌 外切体内的蛋白质。传统上被认为是一种促进细胞存活的自动消化途径 应激，自噬也促进缺乏N-末端信号序列的蛋白质的非常规分泌。 使用基于邻近的生物素化(BioID)蛋白质组学策略，我们发现了~90个新的假定 依赖自噬的分泌的靶标，包括在外体中释放的大量蛋白质。这些 蛋白质与哺乳动物ATG8亚型和自噬调节因子MAP1LC3B相互作用 用于货物扣押。根据这些结果，我们假设自噬机制介导了 依赖于LC3的募集和包装特定的细胞内货物，以通过外体分泌它们。 此外，我们假设自噬控制着分泌和细胞内的微妙平衡。 疾病相关蛋白在神经元和神经胶质细胞中的运输促进AD和FTD的神经退变。至 检验这些预测，我们将：1)剖析自噬是否以及如何指定外体包装和 正常和阿尔茨海默病神经细胞群体的分泌物；以及2)描绘了阿尔茨海默病溶酶体功能障碍 FTD影响依赖于Lc3的外切体包装和蛋白平衡。这些研究是独一无二的 定义自噬机制在外体的生物发生和分泌中的新功能，并 阐明其在AD发病机制中的作用。这一多PI R01应用程序协同合并了独特的 Jayanta Debnath博士在自噬细胞生物学方面的专长和Eric Huang博士在分子生物学方面的专长 神经退行性疾病的机制通过发现新的发现来解决RFA-AG-17-051的目标 AD中指导外体生物发生和外体货物分子分泌的机制。

PROJECT SUMMARY Alzheimer's Disease (AD) is characterized by the progressive accumulation of abnormally cleaved A amyloid peptides and hyperphosphorylated tau proteins, which lead to amyloid plaques and neurofibrillary tangles, respectively. While it remains unclear what triggers these proteinopathies, several lines of evidence indicate that defects in intracellular trafficking may regulate AD pathogenesis. Importantly, emerging evidence suggests that AD-related proteins, including tau, amyloid precursor protein (APP) and A amyloid peptides are secreted via exosomes. Despite these findings, it remains unclear what regulates the formation and packaging of exosomes, whether exosome biogenesis is functionally connected to intracellular trafficking of disease-related proteins, whether neurons and glia develop different mechanism to process these proteins, and if so, how abnormal proteostasis in neurons and glia cooperatively promotes neurodegeneration. We have discovered a new pathway in which the autophagy machinery specifies packaging and secretion of proteins within exosomes. Traditionally studied as an autodigestive pathway that promotes cell survival during stress, autophagy also promotes the unconventional secretion of proteins lacking N-terminal signal sequences. Using a proximity-based biotinylation (BioID) proteomics strategy, we have uncovered ~90 novel putative targets of autophagy-dependent secretion, including numerous proteins released within exosomes. These proteins biochemically interact with MAP1LC3B, a mammalian ATG8 isoform and autophagy regulator crucial for cargo sequestration. Based on these results, we hypothesize that the autophagy machinery mediates the LC3-dependent recruitment and packaging of specific intracellular cargo for their secretion via exosomes. Furthermore, we hypothesize that autophagy controls a delicate balance of secretion and intracellular trafficking of disease-relevant proteins in neurons and glia to promote neurodegeneration in AD and FTD. To test these predictions, we will: 1) Dissect whether and how autophagy specifies exosome packaging and secretion in normal and Alzheimer neural cell populations; and 2) Delineate how lysosomal dysfunction in AD and FTD impacts LC3-dependent exosome packaging and proteostasis. These studies are uniquely poised to define new functions for the autophagy machinery in the biogenesis and secretion of exosomes and to delineate its contributions to AD pathogenesis. This multi-PI R01 application synergistically merges the unique expertise of Dr. Jayanta Debnath in the cell biology of autophagy and Dr. Eric Huang in the molecular mechanisms of neurodegenerative diseases to address the goals of RFA-AG-17-051 by uncovering new machineries directing exosome biogenesis and the secretion of exosomal cargo molecules in AD.