Sterols and intracellular vesicular trafficking: lessons from the study of NPC1

Cholesterol is an important structural component of membranes as well as a precursor for steroid hormone, bile acid and regulatory oxysterol biosynthesis. Recent observations revealed that cholesterol plays an important role in signaling and the regulation of intracellular vesicular trafficking. Studies on Niemann-Pick type C disease, a fatal neuro-visceral cholesterol storage disorder, led to the elucidation of a sterol-modulated vesicular trafficking pathway. Mutations in the NPC1 gene, which cause the majority of cases of Niemann-Pick type C disease, result in the accumulation of free cholesterol in lysosomes and associated defects in glycolipid sorting. NPC1 has a sterol-sensing domain that presumably recognizes free sterols in the protein's environment and participates in the movement of cholesterol out of lysosomes. The compartment containing NPC1 is a subset of late endosomes; it is highly mobile, travels along microtubules, emitting flexible tubules. The movements of this compartment require an intact NPC1 sterol-sensing domain and are dramatically suppressed when free cholesterol accumulates in the late endosomes. Two other proteins involved in sterol trafficking enter into the NPC1 compartment, NPC2 also known as HE1, a secreted sterol-binding glycoprotein, and MLN64, a StAR-related lipid transfer (START) domain protein, which can bind cholesterol and promote its movement from donor to acceptor membranes. Mutations in NPC2 cause a rarer form of Niemann-Pick type C disease, establishing its importance in intracellular sterol movement. NPC2, NPC1 and MLN64 may act in an ordered sequence to sense cholesterol, effect sterol movement, and consequently, influence the process of vesicular trafficking.     

Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.     

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.     

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor delta (PPARdelta) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARdelta. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARdelta. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.     

Niemann-pick type C1 (NPC1) overexpression alters cellular cholesterol homeostasis

The Niemann-Pick type C1 (NPC1) protein is a key participant in intracellular trafficking of low density lipoprotein cholesterol, but its role in regulation of sterol homeostasis is not well understood. To characterize further the function of NPC1, we generated stable Chinese hamster ovary (CHO) cell lines overexpressing the human NPC1 protein (CHO/NPC1). NPC1 overexpression increases the rate of trafficking of low density lipoprotein cholesterol to the endoplasmic reticulum and the rate of delivery of endosomal cholesterol to the plasma membrane (PM). CHO/NPC1 cells exhibit a 1.5-fold increase in total cellular cholesterol and up to a 2.9-fold increase in PM cholesterol. This increase in PM cholesterol is closely paralleled by a 3-fold increase in de novo cholesterol synthesis. Inhibition of cholesterol synthesis results in marked redistribution of PM cholesterol to intracellular sites, suggesting an unsuspected role for NPC1 in internalization of PM cholesterol. Despite elevated total cellular cholesterol, CHO/NPC1 cells exhibit increased cholesterol synthesis, which may be attributable to both resistance to oxysterol suppression of sterol-regulated gene expression and to reduced endoplasmic reticulum cholesterol levels under basal conditions. Taken together, these studies provide important new insights into the role of NPC1 in the determination of the levels and distribution of cellular cholesterol.     

Mechanisms and consequences of impaired lipid trafficking in Niemann–Pick type C1-deficient mammalian cells

Niemann–Pick C disease is a fatal progressive neurodegenerative disorder caused in 95% of cases by mutations in the NPC1 gene; the remaining 5% of cases result from mutations in the NPC2 gene. The major biochemical manifestation of NPC1 deficiency is an abnormal sequestration of lipids, including cholesterol and glycosphingolipids, in late endosomes/lysosomes (LE/L) of all cells. In this review, we summarize the current knowledge of the NPC1 protein in mammalian cells with particular focus on how defects in NPC1 alter lipid trafficking and neuronal functions. NPC1 is a protein of LE/L and is predicted to contain thirteen transmembrane domains, five of which constitute a sterol-sensing domain. The precise function of NPC1, and the mechanism by which NPC1 and NPC2 (both cholesterol binding proteins) act together to promote the movement of cholesterol and other lipids out of the LE/L, have not yet been established. Recent evidence suggests that the sequestration of cholesterol in LE/L of cells of the brain (neurons and glial cells) contributes to the widespread death and dysfunction of neurons in the brain. Potential therapies include treatments that promote the removal of cholesterol and glycosphingolipids from LE/L. Currently, the most promising approach for extending life-span and improving the quality of life for NPC patients is a combination of several treatments each of which individually modestly slows disease progression.     

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.     

Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.     

Accumulation of sphingomyelin in Niemann-Pick disease type C cells disrupts Rab9-dependent vesicular trafficking of cholesterol

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9-dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9-dependent vesicular trafficking defect.     

Sphingolipid storage induces accumulation of intracellular cholesterol by stimulating SREBP-1 cleavage

We showed previously that the intracellular transport of sphingolipids (SLs) is altered in SL storage disease fibroblasts, due in part to the secondary accumulation of free cholesterol. In the present study we examined the mechanism of cholesterol elevation in normal human skin fibroblasts induced by treatment with SLs. When cells were incubated with various natural SLs for 44 h, cholesterol levels increased 25-35%, and cholesterol esterification was reduced. Catabolism of the exogenous SLs was not required for elevation of cholesterol because (i) a non-hydrolyzable and a degradable SL analog elevated cellular cholesterol to similar extents, and (ii) incubation of cells with various SL catabolites, including ceramide, had no effect on cholesterol levels. Elevated cholesterol was derived primarily from low density lipoproteins (LDL) and resulted from up-regulation of LDL receptors induced by cleavage of the sterol regulatory element-binding protein-1. Upon SL treatment, cholesterol accumulated with exogenous SLs in late endosomes and lysosomes. These results suggest a model in which excess SLs present in endocytic compartments serve as a "molecular trap" for cholesterol, leading to a reduction in cholesterol at the endoplasmic reticulum, induction of sterol regulatory element-binding protein-1 cleavage, and up-regulation of LDL receptors.     

Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine

The importance of Niemann-Pick C1 Like-1 (NPC1L1) protein in intestinal absorption of dietary sterols, including both cholesterol and phytosterols, is well documented. However, the exact mechanism by which NPC1L1 facilitates cholesterol transport remains controversial. This study administered 22-(N(-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol) and [(3)H]cholesterol to Npc1l1(+/+) and Npc1l1(-/-) mice to determine whether NPC1L1 facilitates dietary sterol uptake by enterocytes and/or participates in intracellular sterol delivery to the endoplasmic reticulum (ER) for lipoprotein assembly before secretion into plasma circulation. Results showed that [(3)H]cholesterol absorption was reduced but not abolished in Npc1l1(-/-) mice compared with Npc1l1(+/+) mice. In the presence of Pluronic L-81 to block pre-chylomicron exit from the ER, significant amounts of [(3)H]cholesterol were found to be associated with lipid droplets in the intestinal mucosa of both Npc1l1(+/+) and Npc1l1(-/-) mice, and the intracellular [(3)H]cholesterol can be esterified to cholesteryl esters. These results provided evidence indicating that the main function of NPC1L1 is to promote cholesterol uptake from the intestinal lumen but that it is not necessary for intracellular cholesterol transport to the ER. Surprisingly, NBD-cholesterol was taken up by intestinal mucosa, esterified to NBD-cholesteryl esters, and transported to plasma circulation to similar extent between Npc1l1(+/+) and Npc1l1(-/-) mice. Ezetimibe treatment also had no impact on NBD-cholesterol absorption by Npc1l1(+/+) mice. Thus, NBD-cholesterol absorption proceeds through an NPC1L1-independent and ezetimibe-insensitive sterol absorption mechanism. Taken together, these results indicate that NBD-cholesterol can be used to trace the alternative cholesterol absorption pathway but is not suitable for tracking NPC1L1-mediated cholesterol absorption.     

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.     

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.     

Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain

Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.     

Purified NPC1 protein. I. Binding of cholesterol and oxysterols to a 1278-amino acid membrane protein

The Niemann-Pick, Type C1 protein (NPC1) is required for the transport of lipoprotein-derived cholesterol from lysosomes to endoplasmic reticulum. The 1278-amino acid, polytopic membrane protein has not been purified, and its mechanism of action is unknown. Unexpectedly, we encountered NPC1 in a search for a membrane protein that binds 25-hydroxycholesterol (25-HC) and other oxysterols. A 25-HC-binding protein was purified more than 14,000-fold from rabbit liver membranes and identified as NPC1 by mass spectroscopy. We prepared recombinant human NPC1 and confirmed its ability to bind oxysterols, including those with a hydroxyl group on the 24, 25, or 27 positions. Hydroxyl groups on the 7, 19, or 20 positions failed to confer binding. Recombinant human NPC1 also bound [(3)H]cholesterol in a reaction inhibited by Nonidet P-40 above its critical micellar concentration. Low concentrations of unlabeled 25-HC abolished binding of [(3)H]cholesterol, but the converse was not true, i.e. unlabeled cholesterol, even at high concentrations, did not abolish binding of [(3)H]25-HC. NPC1 is not required for the known regulatory actions of oxysterols. Thus, in NPC1-deficient fibroblasts 25-HC blocked the processing of sterol regulatory element-binding proteins and activated acyl-CoA:cholesterol acyltransferase in a normal fashion. The availability of assays to measure NPC1 binding in vitro may further the understanding of ways in which oxysterols regulate intracellular lipid transport.     

Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells

Mammalian cells obtain cholesterol via two pathways: endogenous synthesis in the endoplasmic reticulum and exogenous sources mainly through the low density lipoprotein (LDL) receptor pathway. We performed pulse-chase experiments to monitor the fate of endogenously synthesized cholesterol and showed that, after reaching the plasma membrane from the endoplasmic reticulum, the newly synthesized cholesterol eventually accumulates in an internal compartment in Niemann-Pick type C1 (NPC1) cells. Thus, the ultimate fate of endogenously synthesized cholesterol in NPC1 cells is the same as LDL-derived cholesterol. However, the time required for endogenous cholesterol to accumulate internally is much slower than LDL-derived cholesterol. Different pathways thus govern the post-plasma membrane trafficking of endogenous cholesterol and LDL-derived cholesterol to the internal compartment. Results using the inhibitor N-butyldeoxynojirimycin, which depletes cellular complex glycosphingolipids, demonstrates that the cholesterol trafficking defect in NPC1 cells is not caused by ganglioside accumulation. The ultimate cause of death in NPC disease is progressive neurological deterioration in the central nervous system, where the major source of cholesterol is derived from endogenous synthesis. Our current study provides a plausible link between defects in intracellular trafficking of endogenous cholesterol and the etiology of Niemann-Pick type C disease.