Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

The Niemann‐Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein‐derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl‐CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element‐binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low‐density lipoprotein (LDL)‐derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein‐deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL‐derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL‐derived cholesterol‐dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL‐derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL‐derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis. J. Cell. Biochem. 108: 1102–1116, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of liver disease and lipid metabolism in the Niemann-Pick C1 mouse

Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive cholesterol-storage disorder characterized by liver dysfunction, hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is expressed in every tissue of the body, with liver expressing the highest amounts of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1 protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in maintaining intracellular cholesterol homeostasis. The present study characterizes liver disease and lipid metabolism in NPC1 mice at 35 days of age before the development of weight loss and neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were characterized with mild hepatomegaly, an elevation of liver enzymes, and an accumulation of liver cholesterol approximately four times that measured in normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without hepatomegaly and an elevation of liver enzymes, but the livers had a significant accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein metabolism, the results indicated only minor alterations in NPC1(-/-) mouse serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers, suggesting a relative deficiency of cholesterol at the metabolically active pool of cholesterol located at the endoplasmic reticulum. The results from this study further support the hypothesis that an accumulation of lipoprotein-derived cholesterol within late endosomes/lysosomes, in addition to altered intracellular cholesterol homeostasis, has a key role in the biochemical and cellular pathophysiology associated with NPC1 liver disease.     

Niemann-Pick type C mutations cause lipid traffic jam

The Niemann–Pick C protein (NPC1) is required for cholesterol transport from late endosomes and lysosomes to other cellular membranes. Mutations in NPC1 cause lysosomal lipid storage and progressive neurological degeneration. Cloning of the NPC1 gene has given us tools with which to investigate the function of this putative cholesterol transporter. Here, we discuss recent studies indicating that NPC1 is not a cholesterol-specific transport molecule. Instead, NPC1 appears to be required for the vesicular shuttling of both lipids and fluid-phase constituents from multivesicular late endosomes to destinations such as the trans -Golgi network.     

The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1(-/-) fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1(-/-) fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2(-/-) cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1(-/-) and NPC2(-/-) fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1(-/-) fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2(-/-) fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1(-/-) cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells

Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc (nih) mouse model of NPC disease. A library of 44240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells. One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels.     

Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

Niemann-Pick C1 expression is not regulated by the amount of cholesterol flowing through cells in the mouse

The Niemann-Pick C1 (NPC1) protein functions to regulate the transport of cholesterol from late endosomes/lysosomes to other cellular compartments after lipoprotein uptake through the coated-pit pathway. The present study examines the relative expression of NPC1 mRNA and NPC1 protein in different tissues of the mouse in relation to the uptake of total cholesterol carried in chylomicron remnants (CMr-TC), low density lipoproteins (LDL-TC), cholesteryl ester carried in high density lipoproteins (HDL-CE), and cholesterol synthesis. Results from this study demonstrate that the highest relative expression of NPC1 is in the liver, which is also the tissue with the highest uptake of CMr-TC, LDL-TC, HDL-CE, and cholesterol synthesis. However, there was no similar relation in the remaining tissues. To examine the relative expression of NPC1 in relation to the amount of cholesterol that flowed through the coated-pit pathway, mice were fed a diet supplemented with increasing amounts of cholesterol or cholestyramine. The results from this study demonstrated that there was no relation between the relative expression of NPC1 and the amount of cholesterol that flowed through the coated-pit pathway. We conclude that the relative expression of NPC1 is not regulated by the flow of cholesterol through cells in the mouse and is therefore constitutive.     

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.     

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact “later” ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum.     

Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells.

Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1^−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1^−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2^−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1^−/− and NPC2^−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1^−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2^−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1^−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Intracellular trafficking of Niemann-Pick C proteins 1 and 2: obligate components of subcellular lipid transport

Niemann-Pick C 1 (NPC1) is a large integral membrane glycoprotein that resides in late endosomes, whereas NPC2 is a small soluble protein found in the lumen of lysosomes. Mutations in either NPC1 or NPC2 result in aberrant lipid transport from endocytic compartments, which results in lysosomal storage of a complex mixture of lipids, primarily cholesterol and glycosphingolipids. What are the biological functions of the NPC1 and NPC2 proteins? Here we review what is known about the intracellular itinerary of these two proteins as they facilitate lipid transport. We propose that the intracellular trafficking patterns of these proteins will provide clues about their function.     

Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.     

The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

Adenovirus RID-α activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C

Host–pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-α that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-α mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-α rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-α induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-α regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-α also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.     

Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

Niemann–Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.     

The transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in NPC1 cells

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.