Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

Abstract

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [ 3 H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

“…Few other studies have identifi ed biochemical differences between cells lacking NPC1 and cells lacking NPC2. Using dextran to follow membrane-impermeable lysosomal cargo, Goldman and Krise found that NPC1 was involved in the initial late endosome/lysosome fusion, whereas NPC2 was required for membrane fi ssion from the nascent hybrid endosome/lysosomes and the release of lysosomal cargo-containing vesicles ( 37 ). Other studies have implicated NPC2 in the regulation of cell differentiation in adipocytes and fi broblasts, a role that has not been described for NPC1 ( 38,39 ).…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…Thus, although cholesterol accumulates in LEs/Ls, there is a defi cit of cholesterol in the ER so that the sterol response element-binding protein (SREBP) pathway ( 33 ) is enhanced, and the synthesis and uptake of cholesterol are increased ( 9,34 ). The impaired traffi cking of cholesterol in NPC-defi cient cells profoundly affects multiple cellular functions such as regulation of lysosomal calcium homeostasis ( 35 ), oxidative stress ( 36,37 ), and vesicle traffi cking pathways mediated by Rab proteins ( 28,29 ), as well as fusion of LEs/Ls ( 38 ). NPC1 defi ciency is particularly detrimental for the functioning of LEs/Ls in the brain ( 39 ).…”

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…Third, in addition to extracellular recycling, NPC1 has also been shown to be necessary for the efficient fusion of lysosomes with late endosomes. 18 Continuous fusion of lysosomes with late endosomes creates hybrid organelles in which the bulk of the lysosomal cargo is degraded. 18,19 NPC1 dysfunction caused accumulation of lysososmal cargo, 18 again consistent with the results presented here which show that more LNP colocalize with late endosomes/lysosomes in the presence of NP3.47.…”

“…18 Continuous fusion of lysosomes with late endosomes creates hybrid organelles in which the bulk of the lysosomal cargo is degraded. 18,19 NPC1 dysfunction caused accumulation of lysososmal cargo, 18 again consistent with the results presented here which show that more LNP colocalize with late endosomes/lysosomes in the presence of NP3.47. An additional question concerning the mechanism of NP3.47 is whether it applies to other forms of endocytosis.…”