Investigating mechanisms activating the selective autophagy of lysosomes

摘要 溶酶体是主要的降解细胞器。溶酶体功能障碍已经被认为是几个 神经退行性疾病，包括帕金森病。在帕金森氏病中，神经毒性聚集体是 运输到溶酶体并可导致溶酶体破裂。溶酶体破裂威胁神经元健康。因此，在本发明中， 神经元依赖于质量控制机制，拯救溶酶体的完整性或保护细胞免受溶酶体的伤害， 介导的细胞死亡。溶酶体质量控制始于试图修复受损的溶酶体。溶酶体 修复需要运输所需的内体分选复合物（ESCRT）机制。如果修复失败， 破裂的溶酶体然后被自噬体选择性地隔离，并通过一种选择性的 自噬作用称为噬血作用。溶噬发生在一个刻板的过程，开始于添加泛素 受损的溶酶体这种泛素与选择性自噬受体相互作用，将自噬货物连接到 新形成的自噬体在神经元中，噬血和修复阶段是如何协调的尚不清楚。 在修复阶段清除溶酶体泛素可以防止过早的噬血。泛素去除 去泛素化酶（DUBs）的作用。在人类中有两种与ESCRT相关的DUB。我 初步数据表明，在人iPSC衍生的诱导型神经元（i3神经元）中，受损的溶酶体募集了 DUB AMSH和AMSH在HeLa细胞中的表达足以减少受损细胞上的泛素化。 溶酶体然而，AMSH在噬血中的作用尚不清楚。此外，噬血需要招募 选择性自噬受体。我的初步数据表明，i3神经元和HeLa细胞招募了 选择性自噬受体p62。p62在饥饿诱导的自噬中具有确定的作用，但是p62的作用在饥饿诱导的自噬中没有被证实。 p62在噬菌体中的作用尚不清楚。p62被认为是从细胞溶质中通过细胞膜螯合细胞毒性物质。 形成液体状冷凝物。体外重建试验表明，p62浓缩物可以 结合了自噬机制。因此，p62浓缩物可以通过增加局部的细胞凋亡来促进噬血作用。 自噬蛋白的浓度。然而，p62缩合物在选择性自噬中的重要性， 没有被证明。我假设噬血行为受到严格的控制，首先是由 ESCRT相关DUB AMSH和第二，自噬受体p62的正调控。在目标1中，我将 研究AMSH和修复相在i3神经元和HeLa细胞中的噬血调节中的作用。我会 使用定量活细胞成像以及无细胞的体外实验来实现这一点。在目标2中，我将研究 p62在HeLa细胞和i3神经元中的作用，使用免疫细胞化学和超分辨率显微镜。我 目的是研究溶酶体质量控制中的保守机制。成功完成这些 具体目标将确定噬血的机制。定义噬血的机制将提供必要的 深入了解我们对神经退行性疾病中溶酶体功能障碍的理解， 用于治疗神经退行性疾病。

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.