NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.     

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2^−/− and npc1^−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.     

Promotion of tau phosphorylation by MAP kinase Erk1/2 is accompanied by reduced cholesterol level in detergent-insoluble membrane fraction in Niemann-Pick C1-deficient cells

Niemann-Pick type C (NPC) disease is a cholesterol-storage disease accompanied by neurodegeneration with the formation of neurofibrillary tangles, the major component of which is the hyperphosphorylated tau. Here, we examined the mechanism underlying hyperphosphorylation of tau using mutant Chinese hamster ovary (CHO) cell line defective in NPC1 (CT43) as a tool. Immunoblot analysis revealed that tau was hyperphosphorylated at multiple sites in CT43 cells, but not in their parental cells (25RA) or the wild-type CHO cells. In CT43 cells, mitogen-activated protein (MAP) kinase Erk1/2 was activated and the specific MAPK inhibitor, PD98059, attenuated the hyperphosphorylation of tau. The amount of protein phosphatase 2A not bound to microtubules was decreased in CT43 cells. CT43 cells but not 25RA cells were amphotericin B-resistant, indicating that cholesterol level in the plasma membrane of CT43 is decreased. In addition, the level of cholesterol in the detergent-insoluble, low-density membrane (LDM) fraction of CT43 cells was markedly reduced compared with the other two types of CHO cells. As LDM domain plays critical role in signaling pathways, these results suggest that the reduced cholesterol level in LDM domain due to the lack of NPC1 may activate MAPK, which subsequently promotes tau phosphorylation in NPC1-deficient cells.     

Use of BODIPY‐Cholesterol (TF‐Chol) for Visualizing Lysosomal Cholesterol Accumulation

Dipyrromethene difluoride‐cholesterol (TopFluor‐Cholesterol, TF‐Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF‐Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time‐point both affect the membrane distribution and labeling pattern of TF‐Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low‐density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes.      

Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse,a model for cholesteryl ester storage disease

Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal^−/− mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3 ± 3.4) was only 56% of that in those not given ezetimibe (133.5 ± 6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD.     

Endosomal lipid accumulation in NPC1 leads to inhibition of PKC, hypophosphorylation of vimentin and Rab9 entrapment

Background information. Within the group of lysosomal storage diseases, NPC1 [NPC (Niemann‐Pick type C) 1] disease is a lipidosis characterized by excessive accumulation of free cholesterol as well as gangliosides, glycosphingolipids and fatty acids in the late E/L (endosomal/lysosomal) system (Chen et al., 2005 ) due to a defect in late endosome lipid egress. We have previously demonstrated that expression of the small GTPase Rab9 in NPC1 cells can rescue the lipid transport block phenotype (Walter et al., 2003 ), albeit by an undefined mechanism. Results. To investigate further the mechanism by which Rab9 facilitates lipid movement from late endosomes we sought to identify novel Rab9 binding/interacting proteins. In the present study, we report that Rab9 interacts with the intermediate filament phosphoprotein vimentin and this interaction is altered by lipid accumulation in late endosomes, which results in inhibition of PKC (protein kinase C) and hypophosphorylation of vimentin, leading to late endosome dysfunction. Intermediate filament hypophosphorylation, aggregation and entrapment of Rab9 ultimately leads to transport defects and inhibition of lipid egress from late endosomes. Conclusions. These results reveal a previously unappreciated interaction between Rab proteins and intermediate filaments in regulating intracellular lipid transport.     

Telocytes: a potential defender in the spleen of Npc1 mutant mice

Niemann–Pick disease, type C1 (Npc1), is an atypical lysosomal storage disorder caused by autosomal recessive inheritance of mutations in Npc1 gene. In the Npc1 mutant mice (Npc1^?/?), the initial manifestation is enlarged spleen, concomitant with free cholesterol accumulation. Telocytes (TCs), a novel type of interstitial cell, exist in a variety of tissues including spleen, presumably thought to be involved in many biological processes such as nursing stem cells and recruiting inflammatory cells. In this study, we found that the spleen is significantly enlarged in Npc1^?/? mice, and the results from transmission electron microscopy examination and immunostaining using three different TCs markers, c‐Kit, CD34 and Vimentin revealed significantly increased splenic TCs in Npc1^?/? mice. Furthermore, hematopoietic stem cells and macrophages were also elevated in Npc1^?/? spleen. Taken together, our data indicate that splenic TCs might alleviate the progress of splenic malfunction via recruiting hematopoietic stem cells and macrophages.     

Improvement in Lipid and Protein Trafficking in Niemann‐Pick C1 Cells by Correction of a Secondary Enzyme Defect

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1‐deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis‐(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

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

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

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

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

GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

Sphingolipids and lysosomal pathologies

Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates, including cytotoxic lysosphingolipids, such as galactosylsphingosine and glucosylsphingosine, and lead to a sphingolipidosis. Analysis of patients with prosaposin deficiency revealed the accumulation of intra-endolysosmal vesicles and membrane structures (IM). Feeding of prosaposin reverses the storage, suggesting inner membrane structures as platforms of sphingolipid degradation. Water soluble enzymes can hardly attack sphingolipids embedded in the membrane of inner endolysosomal vesicles. The degradation of sphingolipids with few sugar residues therefore requires the help of the SAPs, and is strongly stimulated by anionic membrane lipids. IMs are rich in anionic bis(monoacylglycero)phosphate (BMP). This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal^−/−:Soat2^+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal^+/+:Soat2^+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal^−/−:Soat2^+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal^−/−:Soat2^−/− littermates. The level of EC accumulation in the SI of the Lal^−/−:Soat2^−/− mice was also much less than in their Lal^−/−:Soat2^+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal^−/−:Soat2^−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.     

Lipidomic Analysis Reveals Altered Fatty Acid Metabolism in the Liver of the Symptomatic Niemann–Pick,Type C1 Mouse Model

Niemann–Pick disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result of the genetic defect, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system causing both visceral and neurological defects. These manifest clinically as hepatosplenomegaly, liver dysfunction, and neurodegeneration. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive these pathologies remains less understood. In this study, it is sought to investigate free fatty acid levels in Npc1^?/? mice with focus on the polyunsaturated ω‐3 and ω‐6 fatty acids. Since fatty acids are the main constituents of numerous lipids species, a discovery based lipidomic study of liver tissue in Npc1^?/? mice is also performed. To this end, alterations in fatty acid synthesis, including the ω‐3 and 6 fatty acids, are reported. Further, alterations in enzymes that regulate the synthesis of ω‐3 and 6 fatty acids are reported. Analysis of the liver lipidome reveals alterations in both storage and membrane lipids including ceramides, fatty acids, phosphatidylcholamines, phosphatidylglycerols, phosphatidylethanolamines, sphingomyelins, and triacylglycerols in Npc1^?/? mice at a late stage of disease.