PROJECT SUMMARY Cells respond to nutrient shortage by activating autophagy, regulated processes of removing unnecessary or dysfunctional cellular components that allow orderly degradation and recycling of proteins, sugars, and lipids. While it is well known that starvation induces macroautophagy (often simply referred to as just autophagy), a process involving the formation of double-membraned structure called autophagosomes, other autophagic pathways, e.g., endosomal microautophagy, also occur as integral parts of the starvation response to help the cell cope with the stress caused by the nutrient deficiency. As a key step of autophagy, protein degradation in the lysosomes is crucial for regeneration of amino acids needed for the synthesis of essential core proteins that support survival of nutrient-deprived cells. However, how lysosomal degradation is regulated in response to nutrient deprivation is not clear. We have uncovered a novel regulatory pathway of lysosomal degradation centered around glutamine hydrolysis by glutaminases and the production of ammonium. Under fed conditions, the abundance of glutamine supports the ammonium production and in turn alkalization of the lysosome lumen, which slows down protein degradation by keeping lysosomal hydrolases in suboptimal conditions. Upon amino acid or glutamine withdrawal, the loss of ammonium production immediately causes acidification of the lysosomes and acceleration of protein degradation. We further found that this increase in lysosomal degradation following starvation is facilitated by accelerated autolysosome formation through activation of Mixed Lineage Kinase Domain Like Pseudokinase (MLKL), a protein previously mainly known for its role in necroptotic cell death downstream of death receptors and receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1 and RIPK3). We show that starvation activates MLKL through Ca2+-calmodulin-dependent kinase II (CaMKII) independently of RIPK3 and this pathway targets the oligomerized MLKL to autophagosomes, instead of plasma membrane, where it supports phagophore closure, a key step required for the maturation of autophagosomes before they fuse with lysosomes to form autolysosomes where the breakdown of autophagosome cargoes occurs. We aim to define the functional significance of this new pathway in neurons where autophagy, including lysosomal degradation, strongly impacts neuronal cell survival and death (Aim I) and further elucidate how multiple regulatory mechanisms orchestrate the early response of the cells to amino acid shortage in order to cope with the stress of starvation (Aim II). Because of the critical involvement of autophagy and lysosomal dysfunction in many types of neurodegenerative diseases, this exploratory and foundational research, fostering the early and conceptual stages of a novel regulatory mechanism of autophagy and lysosomal regulation, will likely lead to breakthroughs in important areas of neuroscience.