Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking.

UNLABELLED: The cellular location of Niemann-Pick C2 protein (NPC2) in cultured human fibroblasts and Chinese hamster ovary cells was examined immunocytochemically and in living cells by expression of a functional red fluorescent protein chimeric analogue. RESULTS: NPC2 is present in the lysosomes of both cholesterol-depleted and -replenished cells, unlike Niemann-Pick C1 protein (NPC1) which is recruited to late endosomes only upon uptake of low-density lipoprotein. With mobilization of cholesterol from lysosomes, immunocytochemical detection of NPC2 in lysosomes is greatly diminished, whereas NPC1 remains in the late endosomal compartment. We found a partial overlap in the trafficking and organellar sites of accumulation of NPC2 and NPC1. In living cells, NPC2 traffics with NPC1 in late endosomal tubules. However, in contrast to NPC1, which remains either in late endosomal vesicles and tubules or at the peripheries of cholesterol-laden lysosomes, NPC2 moves into the central core of lysosomes. Glycolipid analysis reveals that, in contrast to null mutant NPC1 cells, which accumulate GM2 ganglioside only at the plasma membrane, with no endocytic storage, absence of NPC2 protein in null mutant NPC2 cells does not block internalization of GM2 into endocytic vesicles. This difference in the cellular distribution of GM2 in NPC1 and NPC2 null mutants is the first report of a variation in the phenotypic expression of these genotypically distinct lesions. CONCLUSION: We speculate that while NPC1 may play a major role in the sorting of glycolipids as well as cholesterol within the late endosomes, NPC2 primarily plays a role in the egress of cholesterol and, potentially, glycolipids from lysosomes. These proteins appear not to be integrated into a tightly bound biological complex, but rather represent separate functional entities that complement each other.

Cellular localization and trafficking of the human ABCA1 transporter.

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

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.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Cholesterol deprivation affects the fluorescence properties of a ceramide analog at the Golgi apparatus of living cells.

Previous studies have established that a fluorescent analog of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] -6-aminohexanoyl-D-erythro-sphingosine (C6-NBD-Cer), is a vital stain for the Golgi apparatus and a useful tool for studying the sorting and transport of sphingolipids along the secretory pathway in animal cells. Here, we examine the effects of various culture conditions on labeling of the Golgi apparatus of human skin fibroblasts by C6-NBD-Cer and demonstrate that cholesterol deprivation affects the fluorescence properties of the probe at this organelle. Labeling of the Golgi apparatus by C6-NBD-Cer was dramatically reduced in cells grown in medium containing lipoprotein-deficient serum compared to cells grown in medium containing normal serum. Quantitative fluorescence microscopy showed that this apparent reduction in labeling resulted from accelerated photo-bleaching of the fluorescent analog. C6-NBD-Cer labeling of the Golgi apparatus was restored in cholesterol-deprived cells by stimulating endogenous cholesterol biosynthesis with mevalonic acid or by adding exogenous nonlipoprotein cholesterol or low density lipoprotein to the culture medium. In addition, when cells grown in medium containing normal serum were perforated and treated with cholesterol oxidase, an apparent reduction in labeling resulted, further implicating an intracellular pool of cholesterol in the potentiation of C6-NBD-Cer fluorescence. These results demonstrate that cytological studies using C6-NBD-Cer are affected by cholesterol deprivation and suggest that this fluorescent lipid may be used to monitor cholesterol at the Golgi apparatus of living 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.

Clinical, pathologic, and biochemical features of a cholesterol lipidosis accompanied by hyperlipidemia and xanthomas.

We describe the unique clinical and histopathologic features of a child with biochemical and immunocytochemical features of Niemann-Pick disease type C (NPC). Clinically, she was found to have multiple xanthomas of the upper aerodigestive tract with dysphagia and expressive language delay, splenomegaly, bony infarcts, and type IIb hyperlipidemia. Neurologic examination was otherwise normal. Microscopy revealed foam cells in her bone marrow, liver, tongue, tonsils, glottis, and in normal-appearing peritonsillar mucosa. Lipid analysis of a liver biopsy specimen showed a small increase in phospholipids, a twofold increase in sphingomyelin, a fivefold increase in cholesterol, and a marked (25-fold) increase in bis(monoacylglycerol) phosphate. Lysosomal acid hydrolase activities in cultured skin fibroblasts were nondiagnostic. Biochemical and immunocytochemical studies of cultured fibroblasts demonstrated lysosomal accumulation of unesterified LDL-derived cholesterol as well as delayed induction of homeostatic responses to endogenous cholesterol consistent with a diagnosis of NPC. Based upon these observations, we speculate that this patient could have a new phenotypic expression of NPC or represents a new cholesterol lipidosis biochemically resembling NPC. The chance occurrence of two separate lipid disorders seems less likely.

Niemann-Pick type-C disease: deficient intracellular transport of exogenously derived cholesterol.

NPC disease is an autosomal recessive neurovisceral storage disorder. A pleiotropic array of secondary enzymatic and storage abnormalities has in the past obscured a cohesive understanding of the underlying metabolic basis of this disorder. Recent findings, reviewed in this report, demonstrate that NPC disease is a cholesterol lipidosis resulting from defective intracellular cholesterol transport. The sequence of cellular events characteristic of NPC is 1) deficient intracellular transport of exogenously derived cholesterol resulting in retarded induction of cellular cholesterol homeostatic regulation; 2) accumulation of cholesterol in lysosomes; and 3) secondary cellular effects. Retarded esterification of exogenous cholesterol and accumulation of unesterified cholesterol in lysosomes is tightly coupled to the primary defect and serves as the basis for biochemical diagnosis of NPC.

Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol.

A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification.

Type C Niemann-Pick disease: use of hydrophobic amines to study defective cholesterol transport.

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.

Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes.

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (+/- 5%) and 45% (+/- 16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.

Type C Niemann-Pick disease: dimethyl sulfoxide moderates abnormal LDL-cholesterol processing in mutant fibroblasts.

Biochemical and cytochemical studies have revealed that abnormal processing of low-density-lipoprotein (LDL) cholesterol can be reversed in mutant Niemann-Pick C (NP-C) fibroblasts when 2% dimethyl sulfoxide (DMSO) is added to the culture medium. Both the excessive lysosomal accumulation of LDL cholesterol and the delayed induction of cellular homeostatic responses associated with the uptake of LDL by the mutant cells were substantially reversed by DMSO. DMSO appears to accelerate the intracellular mobilization of LDL-derived cholesterol through effects that may reflect enhanced membrane permeability or cholesterol solubilization.

Clinical spectrum of Niemann-Pick disease type C.

Analysis of the neurologic symptomatology in 22 patients with Niemann-Pick disease type C revealed 3 phenotypes: (1) an early-onset, rapidly progressive form associated with severe hepatic dysfunction and psychomotor delay during infancy and later with supranuclear vertical gaze paresis, ataxia, marked spasticity, and dementia; (2) a delayed-onset, slowly progressive form heralded by the appearance, usually in early childhood, of mild intellectual impairment, supranuclear vertical gaze paresis, and ataxia, and later associated with dementia and, variably, seizures and extrapyramidal deficits; (3) a late-onset slowly progressive form distinguished from the 2nd pattern by later age of onset (adolescence or adulthood) and a much slower rate of progression. The existence of the 1st and 2nd phenotypes within the same sibship suggests that they are variant expressions of the same clinicopathologic disorder. Niemann-Pick disease type C should be considered not only in infants and children who present with organomegaly and a progressive neurodegenerative course, but also in adolescents and adults who have insidiously progressive neurologic dysfunction and only slight organomegaly. Associated with the disease is a marked deficiency in the ability of cultured fibroblasts to esterify exogenously supplied cholesterol. Assay of this deficiency is particularly useful for confirming the diagnosis in patients with atypical presentation.

Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes.

Incubation of fibroblasts derived from patients with type-C Niemann-Pick disease with low density lipoprotein results in excessive intracellular accumulation of unesterified cholesterol. Cytochemical techniques revealed that this abnormal cholesterol accumulation is associated not only with a massive storage of cholesterol in lysosomes but also with a premature cholesterol enrichment of the Golgi complex. Cholesterol appeared also in the Golgi complex of some normal fibroblasts after 24 hr of low density lipoprotein loading. These findings indicate that components of the Golgi complex play a role in the intracellular translocation of exogenously derived cholesterol and that disruptions of the cholesterol transport pathway at the Golgi may, in part, be responsible for the deficiency in cholesterol utilization in type-C Niemann-Pick fibroblasts.

Cholesteryl ester storage disease: a patient with massive splenomegaly and splenic abscess.

Cholesteryl ester storage disease (CESD), a rare lysosomal storage disorder characterized by functional deficiency of acid lipase activity, classically features hepatomegaly in conjunction with lipid-laden macrophages containing excessive quantities of cholesteryl esters. We present a patient whose clinical course was complicated by massive, symptomatic splenomegaly, and an unsuspected splenic abscess. Computed tomographic and magnetic resonance imaging are correlated. Histologic, electron microscopic, and biochemical features are presented. To our knowledge, this is the first report of splenic abscess in CESD.

Niemann-Pick disease group C: clinical variability and diagnosis based on defective cholesterol esterification. A collaborative study on 70 patients.

Seventy patients were selected to cover the range of variability in clinical expression of Niemann-Pick disease group C (NP-C). Their individual main clinical features and course of the disease (age at discovery and type of visceromegaly, age at onset and first neurological manifestation, later neurological symptoms) are schematically described. In cultured skin fibroblasts from these patients, sphingomyelinase activities measured in vitro showed decreased values only in approximately half of the cases, and when the metabolic fate of [14C]-sphingomyelin was studied in living cell cultures, still 20% of the cases had a normal hydrolysis rate. Esterification of exogenous cholesterol was investigated in cell lines from these and 5 additional patients and in 21 of their parents. Using a non-lipoprotein [3H]cholesterol source, very low esterification rates were obtained in more than 90% of the cases. All the numerous other pathological conditions studied, including Niemann-Pick disease types A and B, gave normal results. A more sensitive method was elaborated, in which the cells were challenged with pure human low density lipoproteins (LDL) and the early rate of esterification studied. With the latter procedure, a pronounced deficiency could also be demonstrated in the few cases which had shown a milder impairment using a [3H]cholesterol source, and intermediate rates of esterification were obtained in heterozygotes. Discrimination of these difficult cases and of heterozygotes could also be achieved replacing LDL with total unfrozen human serum. Correlations were established between given clinical phenotypes and the severity of the biochemical lesion. Defective intracellular cholesterol esterification is further established as an intrinsic feature of NP-C, and demonstration of this metabolic alteration appears as a major advance in diagnosing the condition.

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol.

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.

Group C Niemann-Pick disease: faulty regulation of low-density lipoprotein uptake and cholesterol storage in cultured fibroblasts.

Incubation of mutant Niemann-Pick C fibroblasts with low-density lipoprotein (LDL) resulted in excessive internalization of lipoprotein and extensive cellular over-accumulation of unesterified cholesterol. The uptake of LDL by the mutant cells appeared to occur through the classic LDL receptor pathway and internalized lipoprotein was processed in lysosomes. Lipoprotein uptake into mutant cells was associated with delays in the initiation of established cellular cholesterol homeostatic responses. Subcellular fractionation of mutant Niemann-Pick C fibroblasts accumulating LDL-cholesterol showed excess unesterified sterol to be localized in the light lysosome-light membrane region of a Percoll gradient, and revealed that cholesterol storage was associated with a specific alteration in the normal profiles of lysosomal marker enzymes.