Niemann-Pick type C disease (NPC) is a fatal rare inherited lysosomal storage disorder that mainly affects children. It is caused by mutations in NPC1 or NPC2 gene leading to accumulation of free cholesterol and other lipids in late endosomes/lysosomes. The molecular details of NPC are still not fully understood and current treatments can only alleviate the symptoms. Here, we will test the hypothesis that lipid dyshomeostasis in NPC alters biophysical properties of the membranes causing defective protein/lipid sorting and trafficking of endocytic vesicles. The goal of this project is to identify pathological changes in endosome biogenesis in NPC that contribute to the phenotype and could instruct us on key lipid/protein signals needed for this process. Firstly, using our recently developed fluorescent probes we will measure membrane tension in living NPC1/2-KO vs. wt HeLa cells by fluorescence-lifetime imaging microscopy (FLIM). Secondly, we will perform proteomics and lipidomics to identify key molecules of endosome biogenesis that are altered in NPC1/2-KO cells and are regulated by altered membrane tension. Thirdly, since NPC has a strong CNS component with profound neurodegeneration and neuroinflammation, we will translate the obtained results to NPC1/2-mouse neurons, microglia and astrocytes – the brain cells that are mainly affected in NPC. Lastly, we will validate the obtained findings in NPC1/2 patient's fibroblasts and in NPC1 human brain cells derived from CRISPR/Cas9 edited human induced pluripotent stem cells. These studies will identify the biologically relevant lipids/proteins involved in deregulation of membrane tension and endosome biogenesis in NPC. These molecules could be further employed to design an early diagnosis and/or effective therapy against NPC. The knowledge gained through this project could have a broader impact since defects of endolysosomal pathway are shared by a number of other rare as well as more common neurodegenerative disorders.