The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one

In mammalian cells, low density lipoprotein (LDL) is bound, internalized, and delivered to lysosomes where LDL-cholesteryl esters are hydrolyzed to unesterified cholesterol. The mechanisms of intracellular transport of LDL-cholesterol from lysosomes to other cellular sites and LDL-mediated regulation of cellular cholesterol metabolism are unknown. We have identified a pharmacological agent, U18666A (3-beta-[2-diethyl-amino)ethoxy]androst-5-en-17-one), which impairs the intracellular transport of LDL-derived cholesterol in cultured Chinese hamster ovary (CHO) cells. U18666A blocks the ability of LDL-derived cholesterol to stimulate cholesterol esterification, and to suppress 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL receptor activities. However, U18666A does not impair 25-hydroxycholesterol-mediated regulation of these processes. In addition, U18666A impedes the ability of LDL-derived cholesterol to support the growth of CHO cells. However, U18666A has only moderate effects on growth supported by non-lipoprotein cholesterol. LDL binding, internalization, and lysosomal hydrolysis of LDL-cholesteryl esters are not affected by the presence of U18666A. Analysis of intracellular cholesterol transport reveals that LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated CHO cells which results in impaired movement of LDL-derived cholesterol to other cell membranes.     

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.     

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.     

Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum

Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol-robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol-binding membrane protein, Niemann-Pick type C1 (NPC1)-bearing vesicles devoid of lysosomal markers were upregulated in HL-60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low-density lipoprotein (LDL)-derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans-Golgi network-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptors, vesicle-associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL-derived cholesterol vesicular transport were recruited to A. phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.     

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol. Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.     

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.     

Low density lipoprotein (LDL)-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type C fibroblasts

One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.     

Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells

The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage.     

Cholesterol efflux via HDL resecretion occurs when cholesterol transport out of the lysosome is impaired

Recently, we showed that holo HDL particle uptake and resecretion occur in physiologically relevant cell lines and that HDL uptake is mediated by scavenger receptor class B type I (SR-BI). Furthermore, we established that HDL resecretion is accompanied by [(3)H]cholesterol efflux. This study shows that HDL uptake and resecretion occur even when LDL uptake and cholesterol trafficking are disturbed. First, we used a set of inhibitors that block cholesterol transport out of the lysosome: chloroquine, imipramine, U18666A, and monensin. In all cases, HDL retroendocytosis occurred and HDL resecretion mediated [(3)H]cholesterol efflux, although to a lesser extent. Second, cell lines carrying somatic mutations in intracellular cholesterol transport were used: CHO 2-2 and CHO 3-6 cells accumulated LDL-derived lipid in the lysosome but showed all components of HDL retroendocytosis. SR-BI overexpression increased HDL uptake and resecretion and [(3)H]cholesterol efflux in these mutant cells. Finally, we used Niemann-Pick type C (NPC) patient fibroblast cells, which carry a defect in cholesterol transfer out of the lysosome. NPC fibroblast cells accumulate cholesterol in the lysosome as a result of a mutation in the NPC1 gene. Despite disturbed intracellular cholesterol transfer, NPC fibroblast cells exhibited HDL retroendocytosis and [(3)H]cholesterol efflux via HDL resecretion, although to a lesser extent. Thus, [(3)H]cholesterol efflux via HDL resecretion is independent of the cholesterol uptake pathway via the LDL receptor and may be an alternative way to remove excess cholesterol.     

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.     

Role for LAMP‐2 in endosomal cholesterol transport

The mechanisms of endosomal and lysosomal cholesterol traffic are still poorly understood. We showed previously that unesterified cholesterol accumulates in the late endosomes and lysosomes of fibroblasts deficient in both lysosome associated membrane protein-2 (LAMP-2) and LAMP-1, two abundant membrane proteins of late endosomes and lysosomes. In this study we show that in cells deficient in both LAMP-1 and LAMP-2 (LAMP^−/−), low-density lipoprotein (LDL) receptor levels and LDL uptake are increased as compared to wild-type cells. However, there is a defect in esterification of both endogenous and LDL cholesterol. These results suggest that LAMP^−/− cells have a defect in cholesterol transport to the site of esterification in the endoplasmic reticulum, likely due to defective export of cholesterol out of late endosomes or lysosomes. We also show that cholesterol accumulates in LAMP-2 deficient liver and that overexpression of LAMP-2 retards the lysosomal cholesterol accumulation induced by U18666A. These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export. Moreover, the late endosomal/lysosomal cholesterol accumulation in LAMP^−/− cells was diminished by overexpression of any of the three isoforms of LAMP-2, but not by LAMP-1. The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect. Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.     

Sterol-modulated glycolipid sorting occurs in niemann-pick C1 late endosomes

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterol-containing vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4Glcbeta1-1'-ceramide (G(M2)) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterol-modulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.     

Arachidonic acid metabolism via cytosolic phospholipase A2 α induces cytotoxicity in niemann-pick disease type C cells

Niemann-Pick disease type C (NPC) is a neurodegenerative lipid storage disorder caused by mutations in NPC1 or NPC2 genes. Loss of function of either protein results in the endosomal accumulation of cholesterol and other lipids. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E(2) compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A(2) α (cPLA(2) α) and cultivation in lipoprotein-deficient medium. There was no difference in the expression of both cyclooxygenase-1 and -2 between NPC cells and wild-type cells. U18666A, a cholesterol transport-inhibiting agent commonly used to mimic NPC, also increased the release of AA in L929 mouse fibrosarcoma cells. Furthermore, U18666A-induced formation of reactive oxygen species (ROS) resulted in the induction of cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA(2) α knockdown L929 cells. These results suggest that cPLA(2) α-AA pathway plays important roles in the cytotoxicity and the ROS formation in NPC cells.     

Progesterone blocks cholesterol translocation from lysosomes.

Fluorescent microscopic examination of fibroblasts cultured with low density lipoprotein (LDL) and progesterone (10 micrograms/ml) for 24 h revealed extensive filipin-cholesterol staining of perinuclear lysosomes. Levels of unesterified cholesterol were 2-fold greater than in fibroblasts cultured with LDL alone. Progesterone strongly blocked cholesteryl ester synthesis. When cellular uptake of LDL was monitored in the presence of 58035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase, excess unesterified cholesterol was not stored in lysosomes. Discontinuation of LDL uptake in conjunction with progesterone washout markedly reversed the filipin-cholesterol staining of lysosomes. Reversal of the lysosomal cholesterol lipidosis was associated with a rapid burst of cholesteryl ester synthesis and a normalization of the cellular levels of free and esterified cholesterol. In contrast to normal cells, progesterone removal from Niemann-Pick C fibroblasts did not reverse the lysosomal cholesterol accumulation of these mutant cultures. The metabolic precursor of progesterone, pregnenolone, also induced extensive accumulation of cholesterol in lysosomes. Other steroids induced less vacuolar cholesterol accumulation in the following decreasing order: corticosterone and testosterone, promegestone, RU 486. The relative inhibition of cellular cholesterol esterification by the steroids paralleled their respective abilities to sequester cholesterol in lysosomes rather than their inhibition of acyl-CoA:cholesterol acyltransferase activity in cell-free extracts. The progesterone-related inhibition and restoration of lysosomal cholesterol trafficking is a useful experimental means of studying intracellular cholesterol transport. A particularly important feature of its utility is the facile reversibility of the steroid-induced block. The lysosomal cholesterol lipidosis established with a hydrophobic amine, U18666A, was not as readily reversed.     

Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles

The effect of dexamethasone on LH-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450(scc)) enzyme. Treatment of preovulatory follicles dissected from ovaries of cyclic adult rats on the morning of proestrus with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 10 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of the follicles with increasing concentrations (1-1000 ng/ml) of dexamethasone suppressed LH (10 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450(scc) was not affected by this dexamethasone treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of P450(scc). Dexamethasone also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analogue 8-Br-cAMP (0.5 mM). The effects of dexamethasone on 8-Br-cAMP-induced StAR protein levels and progesterone production were blocked by cotreatment of the follicles with glucocorticoid receptor antagonist RU-486. These results demonstrate that dexamethasone inhibits the LH-induced StAR protein levels and that the effects of dexamethasone are mediated by the glucocorticoid receptor.     

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.     

Primary cilium alterations and expression changes of Patched1 proteins in niemann‐pick type C disease

Niemann‐Pick type C disease (NPC) is a disorder characterized by abnormal intracellular accumulation of unesterified cholesterol and glycolipids. Two distinct disease‐causing genes have been isolated, NPC1 and NPC2. The NPC1 protein is involved in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. It has extensive homology with the Patched1 (Ptc1) receptor, a transmembrane protein localized in the primary cilium, and involved in the Hedgehog signaling (Shh) pathway. We assessed the presence of NPC1 and Ptc1 proteins and evaluated the relative distribution and morphology of primary cilia in fibroblasts from five NPC1 patients and controls, and in normal fibroblasts treated with 3‐ß‐[2‐(diethylamino)ethoxy]androst‐5‐en‐17‐one (U18666A), a cholesterol transport‐inhibiting drug that is widely used to mimic NPC. Immunofluorescence and western blot analyses showed a significant decrease in expression of NPC1 and Ptc1 in NPC1 fibroblasts, while they were normally expressed in U18666A‐treated fibroblasts. Moreover, fibroblasts from NPC1 patients and U18666A‐treated cells showed a lower percentage distribution of primary cilia and a significant reduction in median cilia length with respect to controls. These are the first results demonstrating altered cytoplasmic expression of Ptc1 and reduced number and length of primary cilia, where Ptc1 is located, in fibroblasts from NPC1 patients. We suggest that the alterations in Ptc1 expression in cells from NPC1 patients are closely related to NPC1 expression deficit, while the primary cilia alterations observed in NPC1 and U18666A‐treated fibroblasts may represent a secondary event derived from a defective metabolic pathway.     

Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.     

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.