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.

项目概要 自噬是许多细胞类型中由环境压力触发的重要细胞降解途径。在 在神经元中，自噬作为维持轴突稳态的组成性活性机制还具有进一步的作用。 在体外和体内，自噬体在轴突末端和突触位点从头产生。一旦形成， 轴突自噬体通过逆行微管运动蛋白细胞质转运回胞体 动力蛋白。自噬体通过与晚期内体和溶酶体融合而成熟。货物 沿轴突运输过程中也会发生降解，导致消化内容物在体内输送 新的生物合成途径中的回收。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发挥着关键作用。符合 根据这一假设，神经元特异性的自噬消融足以引起神经变性。然而，许多 仍然存在必须解决的突出问题：神经元中的自噬是如何调节的？什么控制 自噬体形成和货物吞噬的定位和时间？轴突的功能是什么 自噬——哪些货物是降解的目标，通过什么机制？轴突如何 自噬途径与内溶酶体途径交叉，有效降解货物，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，与生物化学和生物物理相一致 包括蛋白质组分析和计算建模在内的方法，以探究轴突的基本机制 自噬以及这些机制如何受到神经元应激源（包括线粒体功能障碍）的干扰， 蛋白质聚集和溶酶体损伤。我们将实现以下具体目标： 目标 1：如何 神经元中的自噬在空间和时间上受到调节？什么控制轴突自噬的启动 终端或突触前部位？目标 2：哪些货物会被轴突自噬降解？货物被吞没是 选择性过程，还是非特异性过程？是否会优先接收某些货物？如果有，这些货物是什么？ 什么机制控制货物的吸收？目标 3：自噬途径如何与 溶酶体途径？自噬体-溶酶体融合是如何调控的？为什么轴突自噬如此 依赖逆行轴突运输？以及什么机制调节轴突上的溶酶体健康， 因为溶酶体是通过自噬有效清除吞没的货物所必需的。鉴于必要的和 由于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推进我们的研究 了解神经元细胞生物学，为维持轴突的机制提供重要见解。 稳态以及这些机制的扰动如何导致神经退行性变。我们希望这些 进展将为如何最好地干预治疗 ALS 等疾病提供新思路， 亨廷顿氏病和帕金森氏病。