Dynamics of Axonal Autophagy in Neurons

项目摘要 自噬是许多细胞在环境胁迫下发生的一种重要的细胞降解途径。在 在神经元中，自噬作为维持轴突稳态的组成性活性机制具有进一步的作用。 在体外和体内，自噬体在轴突终末和突触部位重新产生。一旦形成， 轴突自噬体通过逆行微管马达蛋白质运输回索马 动力蛋白自噬体在途中通过与晚期内体和溶酶体融合而成熟。货物 降解也发生在沿着轴突的运输过程中，导致消化内容物的体传递， 在新的生物合成途径中循环利用。轴突自噬降解线粒体片段和疾病- 相关的蛋白质聚集体，这表明在维持轴突稳态的关键作用。符合 这种假设，神经元特异性的自噬消融足以引起神经变性。但不少 尚未解决的问题仍然必须解决：自噬是如何调节神经元？什么控制 自噬体形成和货物吞噬的位置和时间？轴突的功能是什么 自噬-降解的目标是什么货物，通过什么机制？轴突是如何 自噬途径与内溶酶体途径交叉，以有效地降解货物， 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技术， 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学相一致， 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神经元应激源的干扰，包括线粒体功能障碍， 蛋白质聚集和溶酶体损伤。我们将讨论以下具体目标：目标1：如何 自噬在神经元中的时空调控是什么控制了轴突自噬的启动 末端或突触前部位目的2：轴突自噬降解哪些物质？货物吞没是一种 选择性过程还是非特异性过程？是否对某些货物有优先吸收，如果有，这些货物是什么？ 什么机制控制货物摄取？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节的？为什么轴突自噬如此 依赖于逆向轴突运输是什么机制调节着轴突上的溶酶体健康沿着， 因为溶酶体是通过自噬有效清除吞噬的货物所必需的。鉴于基本的和 自噬在神经元中发挥的保守作用，我们预计这些研究将大大推进我们的研究。 了解神经元细胞生物学，提供重要的见解维持轴突的机制。 内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 这些进展将为如何最好地进行治疗干预以治疗ALS等疾病提供新的想法， 亨廷顿舞蹈症和帕金森氏症。

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.