The survival of neuronal cells critically depends on the correct function of their lysosomes, catabolic organelles
that play key roles in disposing damaged and harmful cellular components. Niemann-Pick type C (NPC) is a
neurodegenerative and metabolic disease triggered by mutations of the NPC1 gene, which encodes a lysosomal
membrane protein involved in the trafficking and partitioning of cholesterol. In cells lacking NPC1, cholesterol
accumulates aberrantly inside the lysosome, triggering a cascade of downstream events that impact
mitochondria, ultimately leading to cellular dysfunction and death. Understanding how the molecular details of
the primary lysosomal dysfunction, and the downstream processes that impact mitochondrial homeostasis and
overall cell metabolism is key to the design of more effective and targeted strategies to restore neuronal cell
homeostasis in NPC. Recently, the PI (RZ) and co-Investigator (PO) laboratories have made foundational
discoveries that deepened the current understanding of NPC pathogenesis. In particular, through organelle
immunoisolation and profiling we uncovered a profound impairment in lysosomal proteolysis, hydrolase content
and membrane stability, coupled with defective delivery of damaged mitochondria to the lysosome during
autophagy. Additionally, through initial studies in iPSC-derived patient-specific and NPC1-deleted neurons, we
uncovered a direct connection between alterations of lysosomal function, mitochondrial metabolism and neuronal
failure. Finally, we established that mTORC1, a nutrient-sensing pathway based at the lysosome, becomes
dysregulated in NPC, and that its pharmacological manipulation corrects multiple organelle defects. Combined,
these findings provide a rich and detailed understanding of molecular aspects of NPC pathology. Specifically,
they lead us to hypothesize that the lysosomal cholesterol-mTORC1 pathway we discovered drives loss of
neuronal cell homeostasis and may be a therapeutic target in NPC. In order to effectively target the cholesterol-
mTORC1 axis for neuronal resilience and NPC therapy, we propose to i) mechanistically and structurally
elucidate the molecular mechanisms connecting cholesterol levels to mTORC1 regulation, ii) determine how
selectively disabling the cholesterol-mTORC1 sensing pathway impacts mitochondrial metabolism and neuronal
survival in NPC disease models iii) discover new pathways that control organelle function and cellular
metabolism, and evaluate their role as candidate NPC disease modifiers. To accomplish these goals, we will
combine biochemical, cellular and unbiased approaches, including functional genomics and metabolomics in
both neuronal and non-neuronal models of NPC disease. We will rigorously prioritize and validate the most
promising targets, and elucidate their role within the framework of organelle function and neuronal cell
homeostasis. This proposal will deepen our understanding of NPC drivers and disease modifiers, with relevance
to neurodegenerative conditions linked to cholesterol imbalance, such as Alzheimer Disease.