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. Defin...