Investigating mechanisms activating the selective autophagy of lysosomes

摘要 溶酶体是主要的降解细胞器。溶酶体功能障碍一直被认为是几个 神经退行性疾病，包括帕金森氏症。在帕金森氏症中，神经毒性聚集体是 被贩卖到溶酶体，并可能导致溶酶体破裂。溶酶体破裂威胁神经元健康。因此， 神经元依靠质量控制机制来挽救溶酶体的完整性或保护细胞不受溶酶体的影响- 介导的细胞死亡。溶酶体质量控制从尝试修复受损的溶酶体开始。溶酶体 维修需要运输(ESCRT)机械所需的内体分拣复合体。如果修复失败， 破裂的溶酶体随后被自噬小体选择性地隔离，并通过一种选择性的 自噬被称为溶血性。溶血症发生在一个刻板的过程中，这个过程始于泛素的添加 到受损的溶酶体。这种泛素与选择性的自噬受体相互作用，将自噬货物连接到 新形成的自噬小体。神经元的溶血和修复阶段是如何协调的尚不清楚。 在修复阶段去除溶酶体泛素可以防止过早溶血。泛素去除 是由脱泛素酶(DUBS)促进的。在人类中有两个与ESCRT相关的配音。我的 初步数据表明，在人类IPSC来源的诱导性神经元(I3Neurons)中，受损的溶酶体招募 在HeLa细胞中AMSH的表达足以减少泛素对损伤的影响 溶酶体。然而，AMSH在溶血中的作用尚不清楚。此外，溶血还需要招募 选择性自噬受体。我的初步数据显示，i3Neurons和HeLa细胞招募 选择性自噬受体p62。P62在饥饿诱导的自噬中具有既定的作用，但p62的作用 P62在溶血性中的作用尚不清楚。P62被认为是通过细胞内的 形成类似液体的冷凝物。体外重建分析表明，p62凝聚体可以 整合自噬机器。因此，P62凝集物可能通过增加局部的 自噬蛋白的浓度。然而，p62凝聚体在选择性自噬中的意义 没有被演示过。我假设溶血是受到严格控制的，首先是由 第二，受自噬受体p62的正调控。在《目标1》中，我将 探讨AMSH及其修复期在调节I3Neurons和HeLa细胞溶血作用中的作用。这就做 要做到这一点，可以使用定量活细胞成像和无细胞体外实验。在《目标2》中，我将调查 用免疫细胞化学和超分辨显微镜观察p62在HeLa细胞和i3神经元中的作用。我的 目的是研究溶酶体质量控制中的保守机制。成功完成这些任务 特定的目标将确定溶血的机制。定义溶血的机制将提供必要的 洞察我们对神经退行性疾病溶酶体功能障碍的理解，提供关键靶点 用于治疗神经退行性疾病的疗法。

Abstract Lysosomes are the primary degradative cellular organelle. Lysosomal dysfunction has been liked to several neurodegenerative diseases, including Parkinson’s Disease. In Parkinson’s Disease, neurotoxic aggregates are trafficked to lysosomes and can result in lysosomal rupture. Lysosomal rupture threatens neuronal health. Thus, neurons rely on quality control mechanisms that rescue lysosomal integrity or protect the cell from lysosome- mediated cell death. Lysosomal quality control begins with an attempt to repair damaged lysosomes. Lysosomal repair requires the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery. If repair fails, ruptured lysosomes are then selectively sequestered by autophagosomes and degraded via a form of selective autophagy termed lysophagy. Lysophagy occurs in a stereotypic process that begins with the addition of ubiquitin to damaged lysosomes. This ubiquitin interacts with selective autophagy receptors, linking autophagy cargo to the newly formed autophagosome. How lysophagy and the repair phase are coordinated in neurons is unknown. Removal of lysosomal ubiquitin during the repair phase could prevent premature lysophagy. Ubiquitin removal is facilitated by deubiquitinating enzymes (DUBs). There are two ESCRT-associated DUBs in humans. My preliminary data suggest that in human iPSC-derived inducible neurons (i3Neurons), damaged lysosomes recruit the DUB AMSH, and expression of AMSH in HeLa cells is sufficient to decrease ubiquitin on damaged lysosomes. However, the role of AMSH in lysophagy is unclear. In addition, lysophagy requires the recruitment of selective autophagy receptors. My preliminary data demonstrate that i3Neurons and HeLa cells recruit the selective autophagy receptor p62. p62 has an established role in starvation-induced autophagy, but the role of p62 in lysophagy remains unclear. p62 is suggested to sequester cytotoxic material from the cytosol through the formation of liquid-like condensates. In vitro reconstitution assays demonstrate that p62 condensates can incorporate autophagy machinery. Thus, p62 condensates may facilitate lysophagy by increasing the local concentration of autophagy proteins. However, the significance of p62 condensates in selective autophagy has not been demonstrated. I hypothesize that lysophagy is tightly controlled, first negatively regulated by the ESCRT-associated DUB AMSH and second, positively regulated by the autophagy receptor p62. In Aim 1, I will investigate the role AMSH and the repair phase in the regulation of lysophagy in i3Neurons and HeLa cells. I will do this using quantitative live-cell imaging as well as cell-free in vitro experiments. In Aim 2, I will investigate the role of p62 in both HeLa cells and i3Neurons, using immunocytochemistry and super-resolution microscopy. My goal is to investigate conserved mechanisms within lysosomal quality control. Successful completion of these specific aims will identify mechanisms of lysophagy. Defining mechanisms of lysophagy will provide essential insight into our understanding of lysosomal dysfunction in neurodegenerative disease, providing crucial targets for therapies to treat neurodegenerative disease.