Dynamics of Axonal Autophagy in Neurons

神经元轴突自噬的动力学

• 批准号: 10223588
• 负责人: Erika L Holzbaur
• 金额: $ 42.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10223588
• 关键词: Ablation; Address; Aging; Autophagocytosis; Autophagosome; Axon; Axonal Transport; Back; Biochemical; Caenorhabditis elegans; Cells; Cellular biology; Complex; Computer Models; Cytoplasmic Protein; Defect; Degradation Pathway; Disease; Drosophila genus; Dynein ATPase; Eating; Ensure; Genes; Health; Homeostasis; Human; Huntington Disease; Huntington gene; In Vitro; Intracellular Transport; Lead; Length; Longevity; Lysosomes; Maintenance; Microscopy; Microtubules; Mitochondria; Modeling; Molecular; Motor; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Parkinson Disease; Pathway interactions; Patients; Play; Presynaptic Terminals; Process; Proteomics; Recycling; Resolution; Role; Route; Site; Stress; Synapses; Therapeutic; Work; axonal degeneration; biophysical techniques; cell type; dynactin; in vitro Assay; in vivo; induced pluripotent stem cell; insight; late endosome; live cell imaging; meter; mitochondrial dysfunction; mouse model; mutant; neuronal cell body; novel therapeutic intervention; presynaptic; protein aggregation; retrograde transport; stressor; superoxide dismutase 1; uptake

Project Summary Autophagy is an essential cellular degradative pathway triggered by environmental stress in many cell types. In neurons, autophagy has a further role as a constitutively active mechanism that maintains axonal homeostasis. In vitro and in vivo, autophagosomes are generated de novo at axon terminals and synaptic sites. Once formed, axonal autophagosomes are trafficked back to the soma by the retrograde microtubule motor protein cytoplasmic dynein. Autophagosomes mature en route through fusion with late endosomes and lysosomes. Cargo degradation also occurs during transport along the axon, leading to the somal delivery of digested contents for recycling in new biosynthetic pathways. Axonal autophagy degrades mitochondrial fragments and disease- associated protein aggregates, suggesting a key role in the maintenance of axonal homeostasis. Consistent with this hypothesis, neuron-specific ablation of autophagy is sufficient to cause neurodegeneration. However, many outstanding questions remain that must be addressed: How is autophagy regulated in neurons? What controls the localization and timing of autophagosome formation and cargo engulfment? What is the function of axonal autophagy – what cargos are targeted for degradation, and by what mechanisms? And how does the axonal autophagy pathway intersect with the endolysosomal pathway to effectively degrade cargos such as dysfunctional organelles and aggregated proteins? To address these questions, we will use live cell imaging in primary neurons and gene-edited iPSC-derived human neurons, in concert with biochemical and biophysical approaches including proteomic analysis and computational modeling, to query the basic mechanisms of axonal autophagy and how these mechanisms are perturbed by neuronal stressors including mitochondrial dysfunction, protein aggregation, and lysosomal damage. We will address the following specific aims: Aim 1: How is autophagy spatially and temporally regulated in neurons? What controls the initiation of autophagy at the axon terminal or presynaptic sites? Aim 2: What cargos are degraded by axonal autophagy? Is cargo engulfment a selective process, or nonspecific? Is there preferential uptake of some cargos, and if so, what are these cargos? What mechanisms control cargo uptake? And Aim 3: How does the autophagy pathway intersect with the lysosomal pathway? How is autophagosome-lysosome fusion regulated? Why is axonal autophagy so dependent on retrograde axonal transport? And what mechanisms regulate lysosomal health along the axon, as lysosomes are required for the effective clearance of engulfed cargos by autophagy. Given the essential and conserved role that autophagy plays in neurons, we anticipate that these studies will significantly advance our understanding of neuronal cell biology, providing important insights into the mechanisms maintaining axonal. homeostasis and how the perturbation of these mechanisms may lead to neurodegeneration. We hope that these advances will provide new ideas on how to best intervene therapeutically to treat diseases such as ALS, Huntington's, and Parkinson's disease.

项目摘要 自噬是一种必不可少的细胞降解途径，在许多细胞类型的环境应力下触发。在 神经元，自噬是维持轴突稳态的组成型活跃机制的进一步作用。 在体外和体内，自噬体是在轴突末端和突触部位产生的。一旦形成， 通过逆行微管蛋白质细胞质将轴突自噬体运回SOMA 动力蛋白。自噬体成熟在与晚期内体和溶酶体的融合中。货物 沿轴突运输期间也会发生降解，导致消化内容物的som缩 在新的生物合成途径中回收。轴突自噬降解线粒体碎片和疾病 - 相关的蛋白质聚集体，表明在维持轴突稳态中起关键作用。与 自噬的神经特异性消融的假设足以引起神经退行性。但是，很多 仍然需要解决的杰出问题仍然存在：神经元中的自噬如何受到调节？什么控制 自噬体形成和货物吞噬的本地化和时机？轴突的功能是什么 自噬 - 哪些货物是降解的目标，以及哪种机制？以及轴突如何 自噬途径与内溶血途径相交，以有效降解嘉戈斯（例如） 功能失调的细胞器和聚集的蛋白质？为了解决这些问题，我们将使用活细胞成像 原代神经元和基因编辑的IPSC衍生的人类神经元，与生化和生物物理合作 包括蛋白质组学分析和计算建模的方法，以查询轴突的基本机制 自噬以及这些机制如何受到包括线粒体功能障碍在内的神经元压力源的扰动， 蛋白质聚集和溶酶体损伤。我们将解决以下具体目标：目标1：如何 自噬在神经元中受到空间和临时调节？是什么控制了轴突自噬的主动性 末端还是突触前的位点？ AIM 2：轴突自噬降解了哪些货物？货物吞没了吗 选择性过程，还是非特异性的？是否有一些嘉戈斯的优先吸收，如果是，这些货物是什么？ 哪些机制控制货物的吸收？和目标3：自噬途径如何与 溶酶体途径？如何调节自噬体 - 散糖体融合？为什么轴突自噬如此 取决于逆行轴突运输？哪些机制调节轴突沿轴突的溶酶体健康， 由于自动噬菌体有效清除吞噬的碳，因此需要溶酶体。鉴于必不可少的 自噬在神经元中发挥的保守作用，我们预计这些研究将显着提高我们的 了解神经元细胞生物学，为维持轴突的机制提供重要的见解。 稳态以及这些机制的扰动如何导致神经变性。我们希望这些 进步将提供有关如何最好地干预热治疗ALS等疾病的新想法 亨廷顿和帕金森氏病。