Autophagy-dependent exosome loading and biogenesis in AD and FTD

项目摘要 阿尔茨海默病（Alzheimer's Disease，AD）的特征在于异常裂解的A β淀粉样蛋白的进行性积累， 肽和过度磷酸化的tau蛋白，导致淀粉样斑块和神经纤维缠结， 分别虽然目前还不清楚是什么触发了这些蛋白质病，但有几条证据表明， 细胞内运输的缺陷可能调节AD的发病机制。重要的是，新出现的证据表明， AD相关蛋白，包括tau蛋白、淀粉样前体蛋白（APP）和A β淀粉样肽， 通过外泌体。尽管有这些发现，但仍不清楚是什么调节了蛋白质的形成和包装。 外泌体，外泌体生物发生是否在功能上与疾病相关的细胞内运输有关， 蛋白质，神经元和神经胶质细胞是否发展了不同的机制来处理这些蛋白质，如果是这样， 神经元和神经胶质中的异常蛋白质稳态协同促进神经变性。 我们已经发现了一种新的途径，其中自噬机制指定包装和分泌 外泌体中的蛋白质。传统上被研究为一种促进细胞存活的自我消化途径， 在应激时，自噬也促进缺乏N-末端信号序列的蛋白质的非常规分泌。 使用基于邻近性的生物素化（BioID）蛋白质组学策略，我们发现了约90种新的推定的 自噬依赖性分泌的靶点，包括外泌体内释放的许多蛋白质。这些 蛋白质与MAP 1 LC 3B发生生物化学相互作用，MAP 1 LC 3B是哺乳动物ATG 8亚型和自噬调节因子， 用于货物封存基于这些结果，我们假设自噬机制介导了细胞的凋亡。 LC 3依赖性募集和包装特定的细胞内货物，用于通过外泌体分泌。 此外，我们假设自噬控制着细胞内分泌和细胞内分泌的微妙平衡。 神经元和神经胶质细胞中疾病相关蛋白的运输，促进AD和FTD的神经变性。到 测试这些预测，我们将：1）解剖自噬是否以及如何指定外泌体包装， 正常和阿尔茨海默氏症神经细胞群中的溶酶体分泌;和2）描述AD中溶酶体功能障碍如何 FTD影响LC 3依赖性外泌体包装和蛋白质稳态。这些研究是独一无二的， 定义自噬机制在外来体的生物发生和分泌中的新功能， 描述其在AD发病机制中的作用。这种多PI R 01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业知识和Eric Huang博士在分子生物学方面的专业知识。 神经退行性疾病的机制，通过揭示新的 在AD中指导外泌体生物发生和外泌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.