Lundbeckfonden - Transport and function of Niemann-Pick C2 protein in maintenance of intracellular cholostorol homeostasis

Niemann-Pick disease type C is a neurodegenerative disorder characterised by abnormal
accumulation of cholesterol in lysosomes. Lysosomes can be considered as the stomach of
a cell, where most ingested macromolecules are degraded. If this degradation process is
disturbed, lipids and proteins accumulate with fatal consequences for the cellular
machinery. Affected patients develop liver disease and suffer from progressive neuronal
degeneration, leading to dementia and death in childhood. The Niemann-Pick type C2
disease associated NPC2 gene encodes a small cholesterol binding protein, NPC2 that is
pivotal in intracellular cholesterol transport from ingested lipoproteins. The exact function
of the protein, however, remains unknown and no therapeutic treatment of the disease
exists. NPC2 is also found in milk, epididymal fluid as well as in bile secreted from
hepatocytes. We will investigate intracellular transport routes of NPC2 and monitor its
interaction with sterols in various cell types using quantitative fluorescence microscopy and
digital image analysis. Cultured skin fibroblasts from unaffected persons and Niemann-Pick
disease type C2 patients will be compared with respect to 1) the uptake mechanism and
transport of NPC2 protein and 2) the function of NPC2 in transport of lipoprotein-derived
fluorescent cholesterol analogues. Using polarized human hepatocyte-like cells the
transport of NPC2 and its function in hepatic cholesterol metabolism will be determined by
parallel live-cell imaging of protein and sterol-ligand. Molecular mechanisms underlying
membrane interaction and sterol-transport activity of NPC2 will be studied by cutting edge
biophysical techniques including atomic force microscopy, fluorescence nanoscopy and
mass spectrometry. Outcome of this project will provide new insight into molecular
mechanisms underlying protein-mediated sterol transport and will set the stage for
development of therapies against Niemann Pick disease.