The Transport of Low Density Lipoprotein-derived Cholesterol to the Plasma Membrane Is Defective in NPC1 Cells

Abstract

Niemann-Pick disease type C (NPC) is characterized by lysosomal storage of cholesterol and gangliosides, which results from defects in intracellular lipid trafficking. Most studies of NPC1 have focused on its role in intracellular cholesterol movement. Our hypothesis is that NPC1 facilitates the egress of cholesterol from late endosomes, which are where active NPC1 is located. When NPC1 is defective, cholesterol does not exit late endosomes; instead, it is carried along to lysosomal storage bodies, where it accumulates. In this study, we addressed whether cholesterol is transported from endosomes to the plasma membrane before reaching NPC1-containing late endosomes. Our study was conducted in Chinese hamster ovary cell lines that display the classical NPC biochemical phenotype and belong to the NPC1 complementation group. We used three approaches to test whether low density lipoprotein (LDL)-derived cholesterol en route to NPC1-containing organelles passes through the plasma membrane. First, we used cyclodextrins to measure the arrival of LDL cholesterol at the plasma membrane and found that the arrival of LDL cholesterol in a cyclodextrin-accessible pool was significantly delayed in NPC1 cells. Second, the movement of LDL cholesterol to NPC1-containing late endosomes was assessed and found to be normal in Chinese hamster ovary mutant 3-6, which exhibits defective movement of plasma membrane cholesterol to intracellular membranes. Third, we examined the movement of plasma membrane cholesterol to the endoplasmic reticulum and found that this pathway is intact in NPC1 cells, i.e. it does not pass through NPC1-containing late endosomes. Our data suggest that in NPC1 cells LDL cholesterol traffics directly through endosomes to lysosomes, bypassing the plasma membrane, and is trapped there because of dysfunctional NPC1. Niemann-Pick disease type C (NPC)1 is an autosomal recessive lysosomal storage disease that is caused by mutations in either the NPC1 or NPC2 genes and leads to progressive neurodegeneration and premature death (1). Fibroblasts from NPC1 patients exhibit massive storage of unesterified cholesterol and gangliosides in lysosomes, which results from defects in intracellular lipid trafficking (reviewed in Refs. 2 and 3). Most studies of NPC1 have focused on its role in intracellular cholesterol movement. In normal cells, low density lipoproteins (LDL) are internalized and transported through the endocytic pathway where the cholesteryl esters are hydrolyzed. The cholesterol that is released is transported to the plasma membrane and endoplasmic reticulum (4). Our working hypothesis has been that NPC1 facilitates the egress of free cholesterol from late endosomes, which are the location of functional NPC1 (5, 6). When NPC1 is defective, the free cholesterol does not exit the late endosomes; instead, it is carried along the endocytic pathway to lysosomal storage bodies where it accumulates along with mutant NPC1 protein.In this study, we addressed whether cholesterol released by hydrolysis of LDL-derived chol...

Transport of LDL-derived cholesterol from the NPC1 compartment to the ER involves the trans-Golgi network and the SNARE protein complex

“…S1C Top). These results confirm previous studies (2,3) and indicate that in cells lacking functional NPC1, the movement of LDL-CHOL from the NPC1 compartment to the ER and to the PM is defective. Using the pulse-chase protocol, we tested the effects of several compounds (cytochalasin D, wortmannin, latrunculin A, nocodazole, BFA, etc.)…”

“…The results (Fig. 1B Left) show that at 0 time, in both the 25RA (blue) and the CT43 cell extracts (red), 3 H-LDL-CHOL was enriched in fractions 4 and 5. At 20-min chase, in 25RA cells, fraction 5, rich in the Golgi and the early endosome (EE), was enriched in 3 H-LDL-CHOL.…”

“…The endocytosed CE undergoes hydrolysis in an early endosomal compartment enriched in acid lipase (2); the cholesterol released then emerges in endosomes containing the protein Niemann-Pick type C1 (NPC1); from the NPC1 compartment(s), the LDL-derived cholesterol (LDL-CHOL) moves to various destinations including the plasma membrane (PM) and the endoplasmic reticulum (ER), etc. (2)(3)(4). At the ER, the LDL-CHOL can be re-esterified to CE by acyl-CoA:cholesterol acyltransferase (ACAT).…”

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

“…Inhibition of cholesterol export from late endosomes reduces cholesterol delivery back to the PM (Neufeld et al , 1996; Wojtanik and Liscum, 2003; Cubells et al , 2007). This could contribute to reduced cell surface association of SNAP23 and syntaxin-4 in CHO-A6 cells.…”

Pregnane X receptor (PXR) activation: A mechanism for neuroprotection in a mouse model of Niemann–Pick C disease

“…Mutations in the NPC1 gene are responsible for Ϸ95% of human NPC disease. NPC1 loss-of-function mutants exhibit marked impairment of low-density lipoprotein (LDL) cholesterol esterification and mobilization of newly hydrolyzed LDL cholesterol to the plasma membrane (2)(3)(4), resulting in lysosomal sequestration of LDL cholesterol, delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis, and impaired ABCA1-mediated cholesterol efflux (5)(6)(7). Despite recent progress in characterizing the biochemical and genetic defects in NPC disease, the mechanisms underlying the neurodegenerative phenotype are not well understood.…”