Investigating mechanisms of NPC1 proteostasis in human neurons

Abstract Niemann-Pick disease type C is a childhood-onset, autosomal recessive, lysosomal storage disease characterized by the accumulation of unesterified cholesterol. Clinical phenotypes are heterogeneous, but typically include progressive neurodegeneration, seizures, and early death. Niemann-Pick C is commonly (~95% of cases) caused by loss-of-function mutations in the NPC1 gene, which encodes a transmembrane glycoprotein required for exporting cholesterol from late endosomes and lysosomes. Most commonly, Niemann-Pick C patients have an isoleucine to threonine missense mutation at position 1061 (I1061T NPC1). In fibroblasts from these patients, we showed that I1061T NPC1 misfolds in the endoplasmic reticulum (ER) and is rapidly degraded by ER-associated degradation (ERAD) and by ER-autophagy (ER-phagy). Importantly, escape from these pathways by genetic or pharmacologic manipulations enables I1061T NPC1 to reach the lysosome, where it is still functional. As such, NPC1 protein homeostasis (proteostasis) pathways have emerged as prime targets for therapeutic intervention. While modulators of these pathways have shown success in patient fibroblasts, thus far they failed to improve neurological phenotypes when tested in vivo. Critically, the extent to which pathways identified in fibroblasts also function in neurons, a critical disease target cell, is unknown. The overall objective of this application is to determine the effectors mediating NPC1 folding, trafficking, and degradation in neurons. Our central hypothesis is that cholesterol acts as a pharmacological chaperone to promote folding of NPC1 in the ER, and that folding and trafficking of newly synthesized NPC1 to the lysosome are regulated by neuron- specific pathways. To address this hypothesis, we will use inducible human stem cell-derived neurons (iNeurons) with WT or I1061T NPC1 to measure NPC1 trafficking, function, and half-life after manipulation of cellular cholesterol or use of therapeutic cholesterol analogs (Aim 1). We will also use iNeurons expressing fluorescently tagged WT or I1061T NPC1 to find proteostasis regulators via a targeted CRISPR interference (CRISPRi) screen of proteostasis-related genes (Aim 2). The rationale for this work is that characterizing the machinery that regulates NPC1 folding and trafficking will deepen our mechanistic understanding of the NPC1 protein and identify new targets for development of Niemann-Pick C disease therapeutics. This research plan will not only deepen our understanding of neuronal NPC1 proteostasis but will provide learning opportunities that will further my development during my doctoral training. Using the University of Michigan’s vast resources for scientific investigation, I will gain new skills in live cell and high-content imaging, CRISPR-based techniques, and in written and oral scientific communication. This new training will position me well for success as a post-doctoral trainee and long-term as an independent investigator.