u-18666a and Niemann-Pick-Diseases

Topics: Androstenes; Animals; Anticholesteremic Agents; Cells, Cultured; Cholesterol; Cholesterol Esters; Golgi Apparatus; Humans; Lipoproteins; Lysosomes; Mutation; Niemann-Pick Diseases

Topics: Androstenes; Animals; Anticholesteremic Agents; Biological Transport; Carrier Proteins; Cells; Cholesterol; Cytoplasm; Endocytosis; Humans; Liposomes; Mitochondria; Niemann-Pick Diseases; Pancreatic Elastase

Niemann-Pick Type C (NPC) disease is a cholesterol lipidosis resulting from defective postlysosomal cholesterol transport. In normal cells this segment of cholesterol trafficking is inhibited by treatment with either U18666A or imipramine. Other compounds are also capable of blocking postlysosomal cholesterol transport: stearylamine, RV-538, and sphinganine inhibit low-density lipoprotein-induced esterification of cholesterol and cause unesterified cholesterol to accumulate in perinuclear vesicles. These vesicles can be stained with filipin to give a staining pattern indistinguishable from that seen in NPC fibroblasts. Because all of these compounds are hydrophobic amines, we conclude that most, if not all, hydrophobic amines block the postlysosomal transport of cholesterol. These results also raise the possibility that an endogenous amine, e.g., sphinganine, may inhibit cholesterol transport in NPC.

Topics: Amines; Androstenes; Animals; Biological Transport; Cells, Cultured; Cholesterol; Cholesterol Esters; Disease Models, Animal; Fibroblasts; Filipin; Humans; Imipramine; Lysosomes; Mice; Morpholines; Niemann-Pick Diseases; Sphingosine; Structure-Activity Relationship

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3-4 hours, ∼4-5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease.

Topics: Androstenes; Biological Transport; Cells, Cultured; Chlorpromazine; Cholesterol; Cholesterol Oxidase; Endosomes; Fibroblasts; Humans; Lysosomes; Niemann-Pick Diseases

Niemann-Pick type C disease (NPC) is a hereditary neurovisceral atypical lipid storage disorder produced by mutations in the NPC1 and NPC2 genes. The disease is characterized by unesterified cholesterol accumulation in late endosomal/lysosomal compartments and oxidative stress. The most affected tissues are the cerebellum and the liver. The lysotropic drug U18666A (U18) has been widely used as a pharmacological model to induce the NPC phenotype in several cell culture lines. It has already been reported that there is an increase in copper content in hepatoma Hu7 cells treated with U18. We confirmed this result with another human hepatoma cell line, HepG2, treated with U18 and supplemented with copper in the media. However, in mouse hippocampal primary cultures treated under similar conditions, we did not find alterations in copper content. We previously reported increased copper content in the liver of Npc1 (-/-) mice compared to control animals. Here, we extended the analysis to the copper content in the cerebella, the plasma and the bile of NPC1 deficient mice. We did not observe a significant change in copper content in the cerebella, whereas we found increased copper content in the plasma and decreased copper levels in the bile of Npc1(-/-) mice. Finally, we also evaluated the plasma content of ceruloplasmin, and we found an increase in this primary copper-binding protein in Npc1 (-/-) mice. These results indicate cell-type dependence of copper accumulation in NPC disease and suggest that copper transport imbalance may be relevant to the liver pathology observed in NPC disease.

Topics: Androstenes; Animals; Blotting, Western; Cell Line, Tumor; Cells, Cultured; Ceruloplasmin; Cholesterol; Copper; Hep G2 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Proteins; Rats

Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Topics: Amiodarone; Androstenes; Animals; Anti-Arrhythmia Agents; Cells, Cultured; Cholesterol; Dose-Response Relationship, Drug; Dronedarone; Endosomes; Humans; Lysophospholipids; Molecular Structure; Monoglycerides; Niemann-Pick Diseases; Nocodazole

Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function of the npc1 gene leads to abnormal accumulation of free cholesterol and sphingolipids within the late endosomal and lysosomal compartments resulting in progressive neurodegeneration and dysmyelination. Here, we show that oligodendroglial cells secrete cholesterol by exosomes when challenged with cholesterol or U18666A, which induces late endosomal cholesterol accumulation. Up-regulation of exosomal cholesterol release was also observed after siRNA-mediated knockdown of NPC1 and in fibroblasts derived from NPC1 patients and could be reversed by expression of wild-type NPC1. We provide evidence that exosomal cholesterol secretion depends on the presence of flotillin. Our findings indicate that exosomal release of cholesterol may serve as a cellular mechanism to partially bypass the traffic block that results in the toxic lysosomal cholesterol accumulation in Niemann-Pick type C1 disease. Furthermore, we suggest that secretion of cholesterol by exosomes contributes to maintain cellular cholesterol homeostasis.

Topics: Androstenes; Biological Transport; Carrier Proteins; Cells, Cultured; Cholesterol; Exosomes; Fibroblasts; Homeostasis; Humans; Intracellular Signaling Peptides and Proteins; Lysosomes; Membrane Glycoproteins; Membrane Proteins; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Oligodendroglia

U18666A is a cholesterol transport-inhibiting agent that is used widely to mimic Niemann-Pick type C disease. The effect of U18666A on tumour necrosis factor (TNF)-alpha production in mouse macrophage cell line, RAW 264.7 cells and peritoneal macrophages was examined. U18666A induced TNF-alpha mRNA expression 48 h after the treatment, and TNF-alpha production 48 and 72 h after stimulation in RAW 264.7 cells. U18666A accumulated intracellular free cholesterol in the culture of normal medium but not cholesterol-free medium. U18666A also induced reactive oxygen species (ROS) generation in normal medium but much less in cholesterol-free medium. Anti-oxidant N-acetyl-L-cysteine (NAC) abolished U18666A-induced TNF-alpha production. U18666A led to the phosphorylation of p38 mitogen-activated protein kinase 24 and 48 h after the stimulation and the p38 activation was inhibited in presence of cholesterol-free medium or NAC. A p38 inhibitor reduced U18666A-induced TNF-alpha production. Taken together, U18666A was suggested to induce TNF-alpha production in RAW 264.7 cells via free cholesterol accumulation-mediated ROS generation.

Topics: Androstenes; Animals; Anticholesteremic Agents; Cell Line; Cholesterol; Enzyme Activation; Macrophages; Mice; Niemann-Pick Diseases; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.

Topics: Androstenes; Animals; Biological Transport; Carrier Proteins; Cell Survival; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Filipin; Heterocyclic Compounds; Heterocyclic Compounds, 2-Ring; Heterocyclic Compounds, 3-Ring; Heterocyclic Compounds, 4 or More Rings; Humans; Image Processing, Computer-Assisted; Intracellular Signaling Peptides and Proteins; Lysosomes; Membrane Glycoproteins; Microscopy; Microscopy, Fluorescence; Molecular Structure; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Staining and Labeling

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.

Topics: Androstenes; Animals; Antioxidants; Ascorbic Acid; Cell Line; Enzyme Inhibitors; Fibroblasts; Heparan Sulfate Proteoglycans; Humans; Lung; Mice; Models, Biological; Neuroblastoma; Niemann-Pick Diseases; Nitric Oxide; Protein Structure, Secondary; RNA Interference; Suramin

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.

Topics: Androstenes; Animals; Biological Transport; CCAAT-Enhancer-Binding Proteins; Cholesterol; Cholesterol, LDL; COS Cells; Cricetinae; DNA-Binding Proteins; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Luciferases; Mutation; Niemann-Pick Diseases; Promoter Regions, Genetic; Receptors, LDL; Recombinant Proteins; Signal Transduction; Steroids; Sterol Regulatory Element Binding Protein 1; Transcription Factors

Atheroma macrophages internalize large quantities of lipoprotein-derived lipids. While most emphasis has been placed on cholesterol, lipoprotein-derived fatty acids may also play important roles in lesional macrophage biology. Little is known, however, about the trafficking or metabolism of these fatty acids. In this study, we first show that the cholesterol-fatty acyl esterification reaction, catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT), competes for the incorporation of lipoprotein-derived fatty acids into cellular phospholipids. Furthermore, conditions that inhibit trafficking of cholesterol from late endosomes/lysosomes to the endoplasmic reticulum (ER), such as the amphipathic amine U18666A and the Npc1+/- mutation, also inhibit incorporation of lipoprotein-derived fatty acids into phospholipids. The biological relevance of these findings was investigated by studying the suppression of agonist-induced prostaglandin E(2) (PGE(2)) and leukotriene C(4)/D(4)/E(4) production during lipoprotein uptake by macrophages, which has been postulated to involve enrichment of cellular phospholipids with non-arachidonic fatty acids (NAAFAs). We found that eicosanoid suppression was markedly enhanced when ACAT was inhibited and prevented when late endosomal/lysosomal lipid trafficking was blocked. Moreover, PGE(2) suppression depended entirely on acetyl-LDL-derived NAAFAs, not on acetyl-LDL-cholesterol, and was not due to decreased cPLA(2) activity per se. These data support the following model: lipoprotein-derived NAAFAs traffic via the NPC1 pathway from late endosomes/lysosomes to a critical pool of phospholipids. In competing reactions, these NAAFAs can be either esterified to cholesterol or incorporated into phospholipids, resulting in suppression of eicosanoid biosynthesis. In view of recent evidence suggesting dysfunctional cholesterol esterification in late lesional macrophages, these data predict that such cells would have highly suppressed eicosanoid synthesis, thus affecting eicosanoid-mediated cell signaling in advanced atherosclerosis.

Topics: Androstenes; Animals; Arteriosclerosis; Calcimycin; Cholesterol; Dinoprostone; Eicosanoids; Endoplasmic Reticulum; Endosomes; Enzyme Inhibitors; Esterification; Fatty Acids; Humans; Intracellular Signaling Peptides and Proteins; Leukotrienes; Lipoproteins; Lipoproteins, LDL; Lysosomes; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Phospholipases A; Phospholipids; Proteins; Recombinant Proteins; Signal Transduction; Sterol O-Acyltransferase

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.

Topics: Adolescent; Adult; Amyloid beta-Protein Precursor; Androstenes; Animals; Animals, Newborn; Blotting, Western; Cathepsin D; Cells, Cultured; Cerebral Cortex; Child; Child, Preschool; Endosomes; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Inclusion Bodies; Infant; Mice; Middle Aged; Neurons; Niemann-Pick Diseases; Peptide Fragments; Protein Transport

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus but are mistargeted to lysosomes in sphingolipid storage disorders. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Niemann-Pick type C (NPC) disease is a disorder of intracellular transport where glycosphingolipids (GSLs) and cholesterol accumulate in endosomal compartments. The mechanisms of altered intracellular trafficking are not known but may involve the mistargeting and disrupted function of proteins associated with GSL membrane microdomains. Membrane microdomains were isolated by Triton X-100 and sucrose density gradient ultracentrifugation. High pressure liquid chromatography and mass spectrometric analysis of NPC1(-/-) mouse brain revealed large increases in GSL. Sphingosine was also found to be a component of membrane microdomains, and in NPC liver and spleen, large increases in cholesterol and sphingosine were found. GSL and cholesterol levels were increased in mutant NPC1-null Chinese hamster ovary cells as well as U18666A and progesterone induced NPC cell culture models. However, inhibition of GSL synthesis in NPC cells with N-butyldeoxygalactonojirimycin led to marked decreases in GSL but only small decreases in cholesterol levels. Both annexin 2 and 6, membrane-associated proteins that are important in endocytic trafficking, show distorted distributions in NPC cells. Altered BODIPY lactosylceramide targeting, decreased endocytic uptake of a fluid phase marker, and mistargeting of annexin 2 (phenotypes associated with NPC) are reversed by inhibition of GSL synthesis. It is suggested that accumulating GSL is part of a mislocalized membrane microdomain and is responsible for the deficit in endocytic trafficking found in NPC disease.

Topics: 1-Deoxynojirimycin; Androstenes; Animals; Antigens, CD; Biological Transport; Boron Compounds; Brain; Cell Line; Centrifugation, Density Gradient; CHO Cells; Cholesterol; Chromatography, High Pressure Liquid; Cricetinae; Detergents; Endosomes; Enzyme Inhibitors; Glucosyltransferases; Glycosphingolipids; Golgi Apparatus; Lactosylceramides; Mass Spectrometry; Membrane Microdomains; Mice; Mice, Transgenic; Models, Biological; Niemann-Pick Diseases; Octoxynol; Phenotype; Progesterone; Sucrose; Ultracentrifugation

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann-Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts.

Topics: Androstenes; Animals; ATP-Binding Cassette Transporters; Carcinoma, Hepatocellular; CHO Cells; Cholesterol; Cricetinae; Down-Regulation; Esterification; Fibroblasts; Humans; Hydroxymethylglutaryl-CoA Synthase; Hypercholesterolemia; Liver Neoplasms; Niemann-Pick Diseases; Progesterone; Receptors, LDL; RNA, Messenger; Tumor Cells, Cultured; Up-Regulation

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.

Topics: Androstenes; Animals; Axons; Biological Transport; Cells, Cultured; Cholesterol; Immunohistochemistry; Mice; Microscopy, Confocal; Neurons; Niemann-Pick Diseases; Progesterone

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.

Topics: Amphotericin B; Androstenes; Anti-Bacterial Agents; Anticholesteremic Agents; Antineoplastic Agents; Biological Transport; Cell Membrane; Cholesterol; Cyclodextrins; Drug Resistance; Fibroblasts; Humans; Kinetics; Lysosomes; Macrolides; Niemann-Pick Diseases; Nocodazole

We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.

Topics: Androstenes; Animals; Anticholesteremic Agents; Antigens, CD; Cell Compartmentation; Cell Line; Cholesterol; Cricetinae; Dynactin Complex; Endocytosis; Endosomes; Green Fluorescent Proteins; Guanine Nucleotide Dissociation Inhibitors; HeLa Cells; Humans; Kinesins; Luminescent Proteins; Lysophospholipids; Microtubule-Associated Proteins; Microtubules; Monoglycerides; Niemann-Pick Diseases; Platelet Membrane Glycoproteins; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Recombinant Fusion Proteins; Tetraspanin 30

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.

Topics: Amyloid beta-Peptides; Androstenes; Animals; Anticholesteremic Agents; CHO Cells; Cholesterol; Cricetinae; Endocytosis; Endosomes; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Niemann-Pick Diseases

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.

Topics: Androstenes; Animals; Carrier Proteins; CHO Cells; Cricetinae; Endocytosis; Endoplasmic Reticulum; Humans; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Membrane Glycoproteins; Microscopy, Fluorescence; Movement; Mutation; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Organelles; Recombinant Fusion Proteins

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Topics: Androstenes; Animals; Anticholesteremic Agents; Carrier Proteins; Cell Membrane; CHO Cells; Cholesterol, LDL; Cricetinae; Detergents; Endosomes; Extracellular Space; Fibroblasts; Glycolipids; Humans; Hydrolysis; Intracellular Signaling Peptides and Proteins; Lysosomes; Membrane Glycoproteins; Membrane Microdomains; Niemann-Pick C1 Protein; Niemann-Pick Diseases; Tritium

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.

Topics: Androstenes; Animals; CHO Cells; Cholesterol; Cholesterol, LDL; Cholinergic Antagonists; Cricetinae; Fibroblasts; Golgi Apparatus; Humans; Mutagenesis, Site-Directed; Niemann-Pick Diseases; Phosphorylation; Protein Transport; Receptors, Steroid

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.

Topics: Androstenes; Anticholesteremic Agents; Cell Line; Cell Membrane; Cells, Cultured; Cholesterol; Culture Media; Endoplasmic Reticulum; Fibroblasts; Homeostasis; Humans; Imipramine; Kinetics; Lipoproteins; Lysosomes; Membrane Lipids; Monensin; Niemann-Pick Diseases; Skin; Trifluoperazine

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.

Topics: Androstenes; Anticholesteremic Agents; Cells, Cultured; Cholesterol; Cholesterol Esters; Corticosterone; Fibroblasts; Humans; Lipoproteins, LDL; Lysosomes; Microscopy, Fluorescence; Mifepristone; Niemann-Pick Diseases; Oleic Acid; Oleic Acids; Progesterone; Reference Values; Testosterone