Niemann-Pick Disease Type C Cells Research Articles

Analysis of Metabolic Changes in Endogenous Metabolites and Diagnostic Biomarkers for Various Diseases Using Liquid Chromatography and Mass Spectrometry.

Analysis of endogenous metabolites in various diseases is useful for searching diagnostic biomarkers and elucidating the molecular mechanisms of pathophysiology. The author and collaborators have developed some LC/tandem mass spectrometry (LC/MS/MS) methods for metabolites and applied them to disease-related samples. First, we identified urinary conjugated cholesterol metabolites and serum N-palmitoyl-O-phosphocholine serine as useful biomarkers for Niemann-Pick disease type C (NPC). For the purpose of intraoperative diagnosis of glioma patients, we developed the LC/MS/MS analysis methods for 2-hydroxyglutaric acid or cystine and found that they could be good differential biomarkers. For renal cell carcinoma, we searched for various biomarkers for early diagnosis, malignancy evaluation and recurrence prediction by global metabolome analysis and targeted LC/MS/MS analysis. In pathological analysis, we developed a simultaneous LC/MS/MS analysis method for 13 steroid hormones and applied it to NPC cells, we found 6 types of reductions in NPC model cells. For non-alcoholic steatohepatitis (NASH), model mice were prepared with special diet and plasma bile acids were measured, and as a result, hydrophilic bile acids were significantly increased. In addition, we developed an LC/MS/MS method for 17 sterols and analyzed liver cholesterol metabolites and found a decrease in phytosterols and cholesterol synthetic markers and an increase in non-enzymatic oxidative sterols in the pre-onset stage of NASH. We will continue to challenge themselves to add value to clinical practice based on cutting-edge analytical chemistry methodology.

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

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

Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

ABSTRACTThe drug 2-hydroxypropyl-β-cyclodextrin (HPβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-β-cyclodextrin (MβCD), a potent analog of HPβCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MβCD binding to its β-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK β1 or β2 subunit) expression and an AMPK inhibitor abolished MβCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MβCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, includingMycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. Results. Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

Cholesterol-dependent increases in glucosylceramide synthase activity in Niemann-Pick disease type C model cells: Abnormal trafficking of endogenously formed ceramide metabolites by inhibition of the enzyme

Sphingolipids such as sphingomyelin and glycosphingolipids (GSLs) derived from glucosylceramide (GlcCer), in addition to cholesterol, accumulate in cells/neurons in Niemann-Pick disease type C (NPC). The activities of acid sphingomyelinase and lysosomal glucocerebrosidase (GCase), which degrade sphingomyelin and GlcCer, respectively, are down-regulated in NPC cells, however, changes in GlcCer synthase activity have not yet been elucidated. We herein demonstrated for the first time that GlcCer synthase activity for the fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide) increased in intact NPC1(−/−) cells and cell lysates without affecting the protein levels. In NBD-ceramide-labeled NPC1(−/−) cells, NBD-fluorescence preferentially accumulated in the Golgi complex and vesicular specks in the cytoplasm 40 and 150 min, respectively, after labeling, while a treatment for 48 h with the GlcCer synthase inhibitors, N-butyldeoxynojirimycin (NB-DNJ) and 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, accelerated the appearance of vesicular specks emitting NBD-fluorescence within 40 min. The treatment of NPC1(−/−) cells with NB-DNJ for 48 h additionally increased the levels of cholesterol, but not those of sphingomyelin. Increases in the activity of GlcCer synthase and formation of vesicular specks emitting NBD-fluorescence in NPC1(−/−) cells were dependent on cholesterol. LacCer taken up by endocytosis, which accumulated in the Golgi complex in normal cells, accumulated in vesicular specks after 10 and 40 min in NPC1(−/−) cells, and this response was not accelerated by the NB-DNJ treatment, but was restored by the depletion of cholesterol. The cellular roles for enhanced GlcCer synthesis and increased levels of cholesterol in the trafficking of NBD-ceramide metabolites in NPC1(−/−) cells have been discussed.

Cholesterol-Lowering Effect of Octaarginine-Appended β-Cyclodextrin in Npc1-Trap-CHO Cells.

Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disorder, which is an inherited disease characterized by the accumulation of unesterified cholesterol in endolysosomes. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has been used for the treatment of NPC, and ameliorated a hepatosplenomegaly in the patients. However, to obtain the treatment efficacy, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by using active intracellular delivery system of β-CyD to NPC cells is expected. In this study, to efficiently deliver β-CyD to NPC-like cells, we newly synthesized octaarginine (R8)-appended β-CyD with a spacer of γ-aminobutyric acid (R8-β-CyD) and evaluated its cytotoxicity, intracellular distribution, endocytosis pathway and cholesterol-lowering effect in Npc1-trap-Chinese hamster ovary (CHO) cells, cholesterol-accumulated cells through the impairment of NPC1 function. R8-β-CyD did not show cytotoxicity in the cells. In addition, Alexa568-labeled R8-β-CyD was actively internalized into Npc1-trap-CHO cells, possibly through micropinocytosis. Notably, R8-β-CyD significantly decreased intracellular cholesterol content compared with HP-β-CyD. These results suggest that R8-β-CyD may be a promising therapeutic agent for ameliorating cholesterol accumulation in NPC.

The proteasome inhibitor bortezomib reduced cholesterol accumulation in fibroblasts from Niemann-Pick type C patients carrying missense mutations.

Niemann-Pick disease type C (NPC) is a lipid storage disorder mainly caused by mutations in the NPC1 gene. Approximately 60% of these mutations are missense changes that may induce reduced NPC1 protein levels by increased degradation via ubiquitin-proteasome. This is the case for the most prevalent worldwide mutation, p.Ile1061Thr, as well as for other three missense changes. In the present study, we analyzed the NPC1 levels in fibroblasts from eighteen NPC patients presenting missense mutations. We found that fourteen of these cells lines showed decreased levels of NPC1. Six of these cell lines were homozygous, whereas the other eight were associated with a frame shifting mutation. We focused our attention in the NPC homozygous samples and demonstrated that, in most of the cases, NPC1 reduction was a consequence of a decrease of its half-life. NPC cells were treated not only with the proteasome inhibitors carbobenzoxy-l-leucyl-l-leucyl-l-leucinal or N-acetyl-leucyl-leucyl-norleucinal, both widely used as a research tools, but also with bortezomib, the first proteasome inhibitor to reach clinical applications, although it has never been used in NPC disease. We observed that, after treatment, the mutant NPC1 protein levels were partially recovered in most of the cell lines. Importantly, these mutant proteins partially recovered their activity and substantially reduced free cholesterol levels. These results suggest that by enhancing the NPC1 protein stability with the use of proteasome inhibitors, their functionality might be recovered and this might represent a therapeutical approach for future treatments of NPC disease resulting from specific missense mutations.

Modulation of Arachidonic Acid Metabolism 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. The mechanism of neuro-toxicity induced by the lipid storage has not been well elucidated. Here, we report that NPC1-deficient Chinese hamster ovary cells exhibit increased release of arachidonic acid (AA) and synthesis of prostaglandin E2 compared with wild-type cells. The enhanced release of AA was inhibited by both treatment with the selective inhibitor of cytosolic phospholipase A2α (cPLA2α) 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, and induced cell death and cell cycle delay/arrest in L929 cells. Interestingly, these responses induced by U18666A were much weaker in cPLA2α knockdown L929 cells. These results suggest that cPLA2α-AA pathway plays important roles in the cytotoxicity and the reactive oxygen species formation in NPC cells. Keywords: arachidonic acid, cytosolic phospholipase A2α, cytotoxicity, Niemann-Pick disease type C, reactive oxygen species, cell cycle, cholesterol

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca(2+) response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.

Arachidonic acid metabolism via cytosolic phospholipase A2α 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.

A High-Content RNAi-Screening Assay to Identify Modulators of Cholesterol Accumulation in Niemann–Pick Type C Cells

Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder characterized by a defect in intracellular trafficking of exogenous cholesterol and glycosphingolipids. A goal for therapeutic treatment of NPC is to decrease/normalize cholesterol accumulation. We developed a functional genomics-based assay, combining high-throughput RNA interference (HT-RNAi) screening with high-content fluorescence imaging to identify specific genes in NPC cells that will result in more normal cholesterol levels in the diseased cells. Conditions for siRNA tranfections were optimized for 2 NPC fibroblast cell lines (GM03123, GM18453) and a normal fibroblast cell line (GM05659). RNAi screening was done using a focused-set siRNA library targeting 40 cholesterol trafficking-associated genes, knowledge mined from the existing literature on NPC disease, and/or their association with NPC1/NPC2 genes. We utilized filipin staining as a measure of cholesterol accumulation in fixed NPC cells. Data analysis of these screens confirmed several genes including LDLR and RAB9A that reduced cholesterol content in NPC cells. Nine genes were validated using filipin staining to detect unesterified cholesterol as well as cholesteryl BODIPY esters to study lipid trafficking. Gene silencing was also confirmed using qRT-PCR. Our results show that this technology can be applied to larger screens to identify genes responsible for lipid accumulation and/or trafficking in NPC disease, which could be instrumental in developing innovative therapies for individuals afflicted with NPC disease.

Association of autophagy with cholesterol-accumulated compartments in Niemann-Pick disease type C cells

Niemann-Pick disease type C (NPC) is an autosomal recessive disease most commonly caused by a mutation of NPC1, resulting in the accumulation of cholesterol in late endosomes or lysosomes. In this study, we examined whether an abnormality of autophagy is involved in the pathogenesis of NPC and how cholesterol accumulation participates in this process, using both a U18666A-induced NPC model and NPC1-deficient Chinese hamster ovary cells. In these cells, an increase in the level of the microtubule-associated protein 1 light chain 3 (LC3-II) was demonstrated by Western blotting. An increase in the number of granular LC3-positive structures that colocalized with filipin-labeled accumulated cholesterol was also observed in morphological studies. Cholesterol depletion inhibited the formation of granular LC3-positive structures that colocalized with filipin-labeled cholesterol, and instead promoted the formation of ring-shaped LC3-positive filipin-negative structures in U18666A-treated cells. These results demonstrate the close association of the accumulation of LC3 with accumulated cholesterol in NPC cells.

Protein transduction of Rab9 in Niemann‐Pick C cells reduces cholesterol storage

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9-mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22-Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 microM. A fraction of the VP22-Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22-Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY-lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL-receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22-Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

Pathological cholesterol metabolism fails to modify electrophysiological properties of afflicted neurones in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a recessive inherited neurovisceral lipid storage disease characterized by progressive motor impairment and a loss of neurones including those integrated into the motor system. One of the key neuropathological findings is the intracellular accumulation of lysosomes enriched with free cholesterol. This accumulation is due to impaired transport proteins named NPC1 (approx. 95% of the cases) or NPC2 (approx. 5%) responsible for the transport of endocytosed cholesterol from lysomes to plasma membranes. The perturbed lipid-transport in NPC cells leads to an altered lipid composition of the plasma membrane. Available evidence suggests that the lipid matrix influences the electrophysical properties of ion channels in membranes. We therefore evaluated whether electrophysiological properties of NPC neurones differ from healthy neurones. Both, acute brain slices and primary neuronal cell cultures from wildtype and NPC mice, a well-established mouse model for the Niemann-Pick type C disease, were used for a comparison of electrophysiological properties like resting membrane potential, input resistance, action potential amplitudes and synaptic properties of the neurones. In addition we optically recorded the changes of intraneuronal calcium levels elicited by depolarization. Our results show that the characteristics of ion channels in NPC neurones do not differ significantly from wildtype neurones. We therefore conclude that gross alterations of the electrophysiological properties of neurones will probably not initiate or substantially contribute to the development of the motor impairment or other neurological signs of NPC.

Quantified increases of cholesterol, total lipid and globotriaosylceramide in filipin-positive Niemann-Pick type C fibroblasts

Background: Niemann-Pick disease type C (NPC) is a neurovisceral lysosomal lipidosis caused in most cases by mutations in the NPC1 gene that codes for the cholesterol regulating NPC1 protein. Methods: Cultured skin fibroblasts from 11 NPC patients aged 0.25 to 34 years at diagnosis with different severity of neurologic and visceral involvement, diagnosed by the cytochemical filipin test for lysosomally stored cholesterol, were analyzed for lipid composition. Cholesterol and other lipids were separated on thin-layer chromatography from fibroblast total lipid extracts, quantified by densitometry and compared with the total cell lipid mass. Results: Cholesterol concentration in the patient cells was 1.5 to 5-fold higher than normal and total lipids up to 2.4-fold normal. Cholesterol and total lipids were particularly high in cells from NPC patients aged less than about 6 years, and for the whole patient series the abundance of fibroblast cholesterol was correlated with the tentatively assessed clinical disease severity. The findings in NPC suggested that NPC1 protein has a role not only in the balance of cholesterol but also the distribution of the total cell lipid mass. Another increase found in the NPC cells was that of a minor lipid fraction, globotriaosylceramide (Gb3, known as a cell signalling glycolipid). Gb3, in the average of its very variable individual concentrations, was about 2.5-fold higher in the NPC cell group as compared to normal or pathologic control group, but there was no correlation of Gb3 with the other lipid concentrations studied. Conclusions: For NPC diagnosis, the fibroblast cholesterol and total lipid quantification can be used as an alternative to the usual filipin test for lysosomal cholesterol, but both test methods are prone to equivocal results in cells from a small fraction of atypical NPC patients, where chemical testing in organ biopsies or mutational analysis of the NPC1 gene should be tried.

Altered sensitivities to potential inhibitors of cholesterol biosynthesis in Niemann-Pick type C fibroblasts.

Cultured fibroblasts from patients with Niemann-Pick disease type C (NP-C) are characterized by the lysosomal accumulation of unesterified cholesterol and the inability of low-density lipoprotein (LDL) to stimulate cholesterol esterification, in addition to impaired LDL-mediated down-regulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in the transport of cholesterol from lysosomes to other intracellular membrane sites has been suggested, it is unclear how cells regulate the levels of cellular sterols and whether their membrane cholesterol requirements are satisfied or not. We studied the esterification of exogenously added cholesterol, total levels of cellular cholesterol and cholesteryl ester, cholesterol synthesis from a two-carbon precursor, and sensitivities to potential inhibitors of cholesterol biosynthesis in proliferating NP-C cells. We observed the following: (a) esterification of [3H]cholesterol was decreased but the total amount of cellular cholesteryl ester was not decreased; (b) synthesis of cholesterol from [3H]acetate was increased; and (c) cells were hypersensitive to cholecalciferol, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, and were resistant to filipin, which binds to membrane sterols and presumably damages the membrane. The results indicate that NP-C cells depend on the cellular cholesterol synthetic pathway for their proliferation, but the plasma membrane sterols are presumably decreased. The altered sensitivities to potential inhibitors of cholesterol biosynthesis should be a useful marker for diagnosis and genetic studies.

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.