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Endolysosomal defects in secretory autophagy and microglial toxicity in FTD

FTD 中分泌性自噬和小胶质细胞毒性的内溶酶体缺陷

基本信息

批准号：
10623233
负责人：
Jayanta Debnath
金额：
$ 71.39万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10623233
关键词：
Affect Amyloid beta-Protein Autophagocytosis Autophagosome Brain Catabolism Cell secretion Coculture Techniques Defect Degradation Pathway Disease Equilibrium Exocytosis Follow-Up Studies Frontotemporal Dementia Functional disorder Funding Genetic Gliosis Homeostasis Inflammation Mediators Inflammatory Response Intracellular Transport Laboratories Lipids Lysosomes MYO5A gene Mediating Microglia Modeling Mus Myosin ATPase Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurons Organelles PGRN gene Pathogenesis Pathology Pathway interactions Phenotype Phospholipids Process Proteins Proteomics RNA-Binding Proteins Regulation Role Signal Transduction Testing Toxic effect Up-Regulation Vesicle Work age related age related neurodegeneration aging brain cell type extracellular extracellular vesicles glial activation hyperphosphorylated tau insight lipid metabolism lipid transport loss of function misfolded protein nanoparticle neuron loss neurotoxic neurotoxicity particle protein TDP-43 proteostasis receptor trafficking transcriptomics

项目摘要

PROJECT SUMMARY Glial pathology is increasingly recognized as a key feature in neurodegenerative diseases. However, the exact mechanisms that promotes glial pathology remain unclear. Several studies implicate the autophagy machinery, a highly conserved lysosomal degradation pathway, in maintaining protein homeostasis in neurons during brain aging and neurodegeneration. In addition, autophagy regulates the check-and-balance of microglia-mediated inflammatory response to misfolded proteins, including β-amyloid peptides, hyperphosphorylated Tau, and TDP- 43, which accumulate during neurodegeneration. Collectively, these results underscore the critical and cell type- specific role of the autophagy machinery in maintaining neuron-glia homeostasis by regulating the trafficking of intracellular organelles and cargoes, such as misfolded proteins. In the previous funding period, we uncovered new autophagy-dependent secretory processes in non-neuronal and glial cells, including most notably, LC3- dependent extracellular vesicle loading and secretion (LDELS). LDELS targets include RNA binding proteins and extracellular vesicle (EV) cargoes that have been implicated in the pathogenesis of frontotemporal dementia (FTD). In addition, our results showed that loss of function in FTD gene Progranulin (Grn) leads to age-dependent endolysosomal defects and hypersecretory phenotypes in microglia that promotes neuronal loss and abnormal accumulation of the RNA binding protein TDP-43 in Grn-/- mouse brain. Together, these results underscore the important roles of autophagy and endolysosomal trafficking in microglial activation in FTD. In follow up studies, we have further expanded the scope of our work and identified a highly regulated mechanism, Secretory Autophagy during Lysosome Inhibition (SALI), which is critically dependent on multiple ATGs for autophagosome formation and on Rab27a for vesicle exocytosis. Using TMT-based quantitative proteomics, we uncovered that loss of Progranulin leads to a marked increase in unconventional myosin Myo5a, a known Rab27a interacting partner. Given the well-documented role of Rab27a and Myo5a in the transport of intracellular organelles, these results broach the hypothesis that blockage of endolysosomal trafficking disrupts microglial homeostasis and promotes secretory autophagy and neurodegeneration in Grn-/- mice. To test this hypothesis, we will: 1) Delineate how FTD-associated endolysosomal defects influence secretory autophagy and dissect the effects of SALI on intracellular homeostasis; 2) Scrutinize how secretory autophagy and endolysosomal defects in microglia impact lipid metabolism and microglial toxicity; and 3) Determine whether genetic autophagy loss mitigates microglial toxicity and neurodegeneration in Progranulin-deficient mice. Together, the studies proposed in this renewal harness the unique expertise in Drs. Huang and Debnath’s laboratories and provide critical insights on how SALI controls the delicate balance of secretion and intracellular trafficking of disease-relevant proteins in microglia to promote neurodegeneration in FTD.

项目摘要神经胶质病理学越来越被认为是神经退行性疾病的一个关键特征。但具体促进神经胶质病理学的机制仍不清楚。几项研究涉及自噬机制，一种高度保守的溶酶体降解途径，在脑损伤期间维持神经元中的蛋白质稳态，老化和神经退化。此外，自噬还调节小胶质细胞介导的对错误折叠的蛋白质的炎症反应，包括β-淀粉样肽、过度磷酸化的Tau和TDP- 43，在神经变性过程中积累。总的来说，这些结果强调了关键和细胞类型- 自噬机制在通过调节神经元-胶质细胞的运输来维持神经元-胶质细胞稳态中的特定作用细胞内的细胞器和货物，如错误折叠的蛋白质。在上一个融资期，我们发现非神经元和神经胶质细胞中新的自噬依赖性分泌过程，包括最值得注意的LC 3- 依赖性细胞外囊泡装载和分泌（LDELS）。LDELS靶标包括RNA结合蛋白和细胞外囊泡（EV）货物已牵连在发病机制的额颞叶痴呆症（FTD）。此外，我们的研究结果表明，FTD基因颗粒蛋白原（Grn）的功能丧失导致年龄依赖性的小胶质细胞内溶酶体缺陷和过度分泌表型促进神经元丢失和异常 RNA结合蛋白TDP-43在Grn-/-小鼠脑中的积累。总之，这些结果强调了 FTD中自噬和内溶酶体运输在小胶质细胞活化中的重要作用。在后续研究中，我们进一步扩大了我们的工作范围，并确定了一个高度管制的机制，即秘密机制，溶酶体抑制过程中的自噬（SALI），自噬体严重依赖于多种ATG Rab 27 a对囊泡胞吐作用的影响。使用基于TMT的定量蛋白质组学，我们发现，颗粒蛋白前体的缺失导致非常规肌球蛋白Myo 5a的显著增加，Myo 5a是一种已知的Rab 27 a相互作用蛋白，搭档考虑到Rab 27 a和Myo 5a在细胞内细胞器转运中的作用，这些结果提出了这样的假设，即内溶酶体运输的阻断破坏了小胶质细胞的稳态，促进Grn-/-小鼠的分泌性自噬和神经变性。为了验证这一假设，我们将：1）描述 FTD相关的内溶酶体缺陷如何影响分泌性自噬，并分析SALI对细胞内稳态; 2）仔细研究小胶质细胞中的分泌性自噬和内溶酶体缺陷如何影响脂质代谢和小胶质细胞毒性;以及3）确定遗传自噬损失是否减轻小胶质细胞毒性。前粒蛋白缺陷小鼠的毒性和神经变性。总之，在这次更新中提出的研究利用黄博士和Debnath实验室的独特专业知识，并提供关于SALI如何控制小胶质细胞中疾病相关蛋白质的分泌和细胞内运输的微妙平衡，促进FTD的神经变性。