The Cellular, Molecular, and Developmental Impact of Mutations in NPC1

Niemann Pick Type C (NPC) disease is a pediatric neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in late endosomes and lysosomes. 95% of patients harbor a mutation in the endosomal/lysosomal transmembrane protein NPC1. Live-cell imaging of NPC1 revealed that it incorporates into large spherical vesicles (LSV) and tubulo-vesicular membranes (TVM) which transport cholesterol. Mutations in NPC1, specifically at I1061T, do not change the endosomal localization of the protein, but a decrease in tubulation is observed. Microtubule based motility and the tip-tracking protein p150-Glued are required for TVM formation. Small molecule inhibitors were utilized to increase the tubulation of NPC1 mutant membranes and improve cholesterol trafficking, indicating the mutant protein is not functionally dead. To gain a better understanding of how membrane tubules transport cholesterol we investigated proteins implicated in tubulation. Several proteins were identified as being involved in NPC1 membrane tubule formation such as Rab8a, EHD1, and SNX4. Over-expression of these proteins in NPC patient fibroblasts improve cholesterol trafficking, further supporting a role for these proteins in cholesterol transport. In an effort to identify other proteins involved in NPC1 function, mass spectrometry was employed. Differences in the protein and phosphotidylinositol (PI) composition of wild type and mutant NPC1 membranes were identified. Changes in PI composition could lead to changes in the recruitment of tubule-inducing proteins to NPC1 membranes. Proteins identified as absent from NPC1 mutant membranes include cytoplasmic dynein, dynactin, and STARD9, indicating a loss of microtubule based motility. To address the developmental impact of loss of NPC1 function, zebrafish was developed as a new model system. Morpholino-mediated knockdown of NPC1 led to cholesterol accumulations, increased cell death, and delayed development. Differentiation of neuronal cell types was also impaired in the morphant embryos, specifically purkinje neurons which are the cell type affected in human disease and the knockout mouse. NPC1 function is required for the normal development of purkinje neurons in the developing zebrafish. Taken together, these studies provide important information about the cellular, molecular, and developmental consequences on Niemann Pick Type C disease.