Lysosomal Cholesterol Research Articles

Scavenger receptor class B type I localizes to a late endosomal compartment

Scavenger receptor class B type I (SR-BI) has an established role in mediating the selective uptake of cholesterol from HDL in hepatocytes, steroidogenic cells, and other tissues. SR-BI is present on the plasma membrane but also localizes to stable intracellular compartments of unknown function. Using indirect immunofluorescence and subcellular fractionation, we have investigated the subcellular distribution of SR-BI. We report that red fluorescent protein-tagged mouse SR-BI (RFP-mSR-BI) colocalizes with the late endosomal and lysosomal markers, Rab7, LBPA, and Rab9. In addition, endogenous SR-BI is also found on lysosomes and colocalizes with LAMP-2 in primary hepatocytes. Furthermore, we demonstrate that the trafficking of SR-BI through these compartments is Rab7 dependent. Interestingly, filipin staining indicates accumulation of lysosomal cholesterol in SR-BI-deficient ((-/-)) as compared with wild-type hepatocytes. In addition to its role as a plasma membrane receptor, SR-BI may function in cholesterol trafficking from late endosomes/lysosomes.

Adulte Form des M. Niemann-Pick Typ C

We report the case of a 43-year-old male patient, who presented with a slowly progressive gait disorder since the age of 27. In the neurological examination he showed a supranuclear vertical gaze palsy, cognitive impairment, limb and gait ataxia and dystonia. The MRI scan showed marked temporal und cerebellar atrophy, an ultrasound of the abdomen revealed splenomegaly. Bone marrow cytology revealed seablue histiocytes and foamy storage macrophages. Fibroblast cultures were assayed with the Filipin method and revealed positive lysosomal cholesterol accumulation. Genetic testing demonstrated two heterozygous mutations of defect alleles in the NPC1 gene (compound heterozygousity). Niemann-Pick disease type C is a rare autosomal recessive lipid storage disorder with variable clinical presentation, mostly beginning in early childhood or adolescence. Late manifestations have only rarely been reported. With oral Miglustat, an inhibitor of ceramide-specific glycosyltransferase, a first therapeutic strategy with possible benefits is currently under investigation.

Lysosomal Cholesterol Accumulation Inhibits Subsequent Hydrolysis of Lipoprotein Cholesteryl Ester

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.

Cholesterol regulates the loading of foreign antigens onto MHC class II in dendritic cells

In immature DCs, MHCII resides in cholesterol‐rich intracellular lysosomal compartments. Upon activation, intracellular MHCII binds antigenic peptides and traffics to the plasma membrane. In this study, we have investigated the role of membrane cholesterol in MHCII transport through the use of the cholesterol transport inhibitor U18666A. Pretreatment of immature DCs with U18666A had no effect on the LPS‐induced expression of either the DC maturation marker CD40 or cellular levels of total MHCII and only a slight effect on CD86. However, U18666A treated DCs had a reduced capacity to stimulate naïve antigen‐specific T cells. Similar results were found using DCs from mice lacking NPC1, a protein involved in lysosomal cholesterol egress. Pretreatment of DCs with U18666A resulted in a 4‐fold reduction in the surface and cellular expression of MHCII‐peptide complexes in immature DCs and almost completely inhibited the LPS‐induced appearance of MHCII‐peptide at the plasma membrane of mature DCs. Microscopic analysis of U18666A treated DCs shows that MHCII‐peptide complexes are not found in cholesterol engorged lysosomal antigen processing compartments, suggesting that there are fewer compartments that are competent for antigen loading. We conclude that lysosomal cholesterol regulates MHCII‐peptide loading but not MHCII transport from lysosomes to the plasma membrane in DCs.This work is supported by the NCI.

An Anesthetic Experience in a Patient with Niemann-Pick Disease - A case report -

Niemann-Pick disease (NPD) is an autosomal recessive, lipid storage disorder caused by the deficiency of the lysosomal enzyme sphingomyelinase or defective cholesterol transport from lysosome to cytosol. The clinical symptoms and signs include dysphagia, loss of motor function, hepatosplenomegaly, recurred respiratory infections, seizure, mental retardation, spasticity, myoclonic jerks and ataxia, but vary depending on the type of this disease. We report a successful anesthetic experience, including endotracheal intubation with Glidescope under propofol and remifentanil infu sion without neuromuscular blockade, in a 21-year old woman with Niemann-Pick disease for wound revision of gastrostomy site. (Korean J Anesthesiol 2008; 54: 109∼12)

Acid sphingomyelinase‐deficient Niemann–Pick disease: Novel findings in a Greek child

Niemann-Pick Disease (NPD) is a heterogeneous group of autosomal recessive disorders characterized by progressive accumulation of sphingomyelin and cholesterol in lysosomes. Six types of NPD have been described based on clinical presentation and involved organs. The primary defect in NPD types A and B is a deficiency of lysosomal acid sphingomyelinase (ASM). We present a case of a 5-year-old boy with type B NPD who had severe clinical manifestations, including heart involvement. He was first admitted to the hospital at 2 months because of vomiting, refusal to feed, lethargy, hepatomegaly and mild transaminasaemia. Liver biopsy at 12 months showed lipid accumulation and fibrosis. Investigations for lysosomal storage disorders revealed increased plasma chitotriosidase (549 nmol/h per ml, normal value 0-150). At 18 months, no detectable ASM activity was observed in cultured fibroblasts (normal range 23-226 nmol/h per mg protein) confirming NPD B. Pulmonary involvement was detected with high-resolution computerized tomography which revealed reticulonodular infiltrations and thickening of the interlobular septa. At 2 years growth retardation and kyphosis were noted. At 2.5 years he manifested neurodevelopment regression, indicating CNS involvement. Cardiac involvement (grade III mitral valve insufficiency) developed at 4 years and heart failure at 5 years. Genetic analysis revealed two mutations: a H421Y mutation that is common in Saudi Arabian and Turkish patients, and a W32X mutation, which has been found in other Mediterranean patients.

Structural Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in Niemann-Pick Type C2 Disease

NPC2 is a small lysosomal glycoprotein that binds cholesterol with submicromolar affinity. Deficiency in NPC2 is the cause of Niemann-Pick type C2 disease, a fatal neurovisceral disorder characterized by accumulation of cholesterol in lysosomes. Here we report the crystal structure of bovine NPC2 bound to cholesterol-3-O-sulfate, an analog that binds with greater apparent affinity than cholesterol. Structures of both apo-bound and sterol-bound NPC2 were observed within the same crystal lattice, with an asymmetric unit containing one molecule of apoNPC2 and two molecules of sterol-bound NPC2. As predicted from a previously determined structure of apoNPC2, the sterol binds in a deep hydrophobic pocket sandwiched between the two beta-sheets of NPC2, with only the sulfate substituent of the ligand exposed to solvent. In the two available structures of apoNPC2, the incipient ligand-binding pocket, which ranges from a loosely packed hydrophobic core to a small tunnel, is too small to accommodate cholesterol. In the presence of sterol, the pocket expands, facilitated by a slight separation of the beta-strands and substantial reorientation of some side chains, resulting in a perfect molding of the pocket around the hydrocarbon portion of cholesterol. A notable feature is the repositioning of two aromatic residues at the tunnel entrance that are essential for NPC2 function. The NPC2 structures provide evidence of a malleable binding site, consistent with the previously documented broad range of sterol ligand specificity.

Lysosomal acid lipase over‐expression disrupts lamellar body genesis and alveolar structure in the lung

The functional role of neutral lipids in the lung is poorly understood. Lysosomal acid lipase (LAL) is a critical enzyme in hydrolysis of cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Human LAL was over-expressed in a doxycycline-controlled system in mouse respiratory epithelial cells to accelerate intracellular neutral lipid degradation and perturb the surfactant homeostasis in the lung. In this animal system, neutral lipid concentrations of pulmonary surfactant were reduced in bronchoalveolar lavage fluid (BALF) in association with decrease of surfactant protein C (SP-C) gene expression. The size and the number of lamellar bodies in alveolar type II epithelial cells (AT II cells) were significantly reduced accordingly. The number of macrophages required for surfactant recycling in BALF was also significantly reduced. As a result of these combinatory effects, emphysema of the alveolar structure was observed. Taken together, neutral lipid homeostasis is essential for maintenance of lamellar body genesis and the alveolar structure in the lung.

Intraparenchymal injections of acid sphingomyelinase results in regional correction of lysosomal storage pathology in the Niemann–Pick A mouse

Niemann–Pick A disease (NPD-A) is caused by a deficiency of acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. We evaluated the effects of direct intraparenchymal brain injections of purified recombinant human ASM (hASM) at correcting the storage pathology in a mouse model of NPD-A (ASMKO). Different doses (0.1 ng to 10 μg of hASM) were injected into the right hemisphere of the hippocampus and thalamus of 12- to 14-week-old ASMKO mice. Immunohistochemical analysis after 1 week indicated that animals treated with greater than 1 μg hASM/site showed detectable levels of enzyme around the injected regions. However, localized clearance of sphingomyelin and cholesterol storage were observed in animals administered lower doses of enzyme, starting at 100 ng hASM/site. Areas of correction were also noted at distal sites such as in the contralateral hemispheres. Indications of storage re-accumulation were seen after 2 weeks post-injection. Injections of hASM did not cause any significant cell infiltration, astrogliosis, or microglial activation. These results indicate that intraparenchymal injection of hASM is associated with minimal toxicity and can lead to regional reductions in storage pathology in the ASMKO mouse.

Effects of cellular cholesterol loading on macrophage foam cell lysosome acidification

Macrophages incubated with mildly oxidized low density lipoprotein (OxLDL), aggregated low density lipoprotein (AggLDL), or cholesteryl ester-rich lipid dispersions (DISPs) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis. The variety of cholesterol-containing particles producing inhibition of hydrolysis suggests that inhibition may relate to general changes in lysosomes. Lysosome pH is a key mediator of activity and thus is a potential mechanism for lipid-induced inhibition. We investigated the effects of cholesterol accumulation on THP-1 macrophage lysosome pH. Treatment with OxLDL, AggLDL, and DISPs resulted in inhibition of the lysosome's ability to maintain an active pH and concomitant decreases in CE hydrolysis. Consistent with an overall disruption of lysosome function, exposure to OxLDL or AggLDL reduced lysosomal apolipoprotein B degradation. The lysosomal cholesterol sequestration and inactivation are not observed in cholesterol-equivalent cells loaded using acetylated low density lipoprotein (AcLDL). However, AcLDL-derived cholesterol in the presence of progesterone (to block cholesterol egression from lysosomes) inhibited lysosome acidification. Lysosome inhibition was not attributable to a decrease in the overall levels of vacuolar ATPase. However, augmentation of membrane cholesterol in isolated lysosomes inhibited vacuolar ATPase-dependent pumping of H+ ions into lysosomes. These data indicate that lysosomal cholesterol accumulation alters lysosomes in ways that could exacerbate foam cell formation and influence atherosclerotic lesion development.

Lysosomal unesterified cholesterol content correlates with liver cell death in murine Niemann-Pick type C disease

Niemann-Pick type C (NPC) disease is a multisystem disorder resulting from mutations in the NPC1 gene that encodes a protein involved in intracellular cholesterol trafficking. Significant liver dysfunction is frequently seen in patients with this disease. The current studies used npc1 mutant mice to investigate the association between liver dysfunction and unesterified cholesterol accumulation, a hallmark of NPC disease. Data from 92 npc1(-/-) mice (age range, 9-56 days) revealed a significant positive correlation between the plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and whole liver cholesterol content. In 56 day old npc1(-/-) mice that had been fed from 35 days of age a rodent diet or the same diet containing either cholesterol (1.0%, w/w) or ezetimibe (a sterol absorption inhibitor; 0.0125%, w/w), whole liver cholesterol content averaged 33.5 +/- 1.1, 87.9 +/- 1.7, and 20.8 +/- 0.9 mg, respectively. Again, plasma ALT and AST activities were positively correlated with hepatic cholesterol content. In contrast, plasma transaminase levels remained in the normal range in npc1(+/+) mice, in which hepatic esterified cholesterol content had been increased by 72-fold by feeding a high-cholesterol, high-fat diet. These studies suggest that the late endosomal/lysosomal content of unesterified cholesterol correlates with cell damage in NPC disease.

Mechanism of Cholesterol Transfer from the Niemann-Pick Type C2 Protein to Model Membranes Supports a Role in Lysosomal Cholesterol Transport

Mechanism of Cholesterol Transfer from the Niemann-Pick Type C2 Protein to Model Membranes Supports a Role in Lysosomal Cholesterol Transport

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.

Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells

Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.

Mechanism of Cholesterol Transfer from the Niemann-Pick Type C2 Protein to Model Membranes Supports a Role in Lysosomal Cholesterol Transport

Cells acquire cholesterol either by de novo synthesis in the endoplasmic reticulum or by internalization of cholesterol-containing lipoproteins, particularly low density lipoprotein (LDL), via receptor-mediated endocytosis. The inherited disorder Niemann-Pick type C (NPC), in which abnormal LDL-cholesterol trafficking from the endo/lysosomal compartment leads to substantial cholesterol and glycolipid accumulation in lysosomes, is caused by defects in either of two genes that encode for proteins designated as NPC1 and NPC2. NPC2 is a small intralysosomal protein that has been characterized biochemically as a cholesterol binding protein. We determined the rate and mechanism by which NPC2 delivers cholesterol to model phospholipid membranes. A fluorescence dequenching assay was used to monitor the kinetics of cholesterol transfer from the protein to membranes. The endogenous tryptophan fluorescence of the NPC2 was quenched upon binding of cholesterol, and the subsequent addition of acceptor vesicles resulted in dequenching of the tryptophan signal, enabling the monitoring of cholesterol transfer to membranes. The rates of cholesterol transfer were evaluated as a function of acceptor vesicle concentration, acceptor vesicle phospholipid headgroup composition, and aqueous phase properties. The results suggest that NPC2 rapidly transports cholesterol to phospholipid vesicles via a collisional mechanism which involves a direct interaction with the acceptor membrane. Transfer of cholesterol to membranes is faster in an acidic environment and is greatly enhanced by the presence of the unique lysosomal/late endosomal phospholipid lyso-bisphosphatidic acid (LBPA) (also known as bismonoacylglycerol phosphate). Finally, we found that the rate of transfer of cholesterol from vesicles to NPC2 was dramatically increased by the presence of lyso-bisphosphatidic acid in the donor vesicles. These results support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.

The Niemann-Pick C1 protein in recycling endosomes of presynaptic nerve terminals

Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative disorder caused in 95% of cases by loss of function of NPC1, a ubiquitous endosomal transmembrane protein. A biochemical hallmark of NPC deficiency is cholesterol accumulation in the endocytic pathway. Although cholesterol trafficking defects are observed in all cell types, neurons are the most vulnerable to NPC1 deficiency, suggesting a specialized function for NPC1 in neurons. We investigated the subcellular localization of NPC1 in neurons to gain insight into the mechanism of action of NPC1 in neuronal metabolism. We show that NPC1 is abundant in axons of sympathetic neurons and is present in recycling endosomes in presynaptic nerve terminals. NPC1 deficiency causes morphological and biochemical changes in the presynaptic nerve terminal. Synaptic vesicles from Npc1(-/-) mice have normal cholesterol content but altered protein composition. We propose that NPC1 plays a previously unrecognized role in the presynaptic nerve terminal and that NPC1 deficiency at this site might contribute to the progressive neurological impairment in NPC disease.

Changes in Cholesterol Metabolism are Associated With PS1 and PS2 Gene Regulation in SK-N-BE

Several lines of evidence suggest that the cholesterol content of neuronal membranes influences amyloid precursor protein (APP) processing; however, its role in transcriptional regulation of the cofactors for gamma-secretase, the key enzyme for the production of the Abeta peptide, is poorly understood. This study investigates whether the changes in cellular cholesterol metabolism modulate the expression of genes involved in the gamma-secretase complex function. The abundance of mRNA transcripts for presenilin 1 and 2 (PS1 and PS2), APP, and nicastrin were evaluated in neuroblastoma cells exposed either to serum-depleted medium or to low-density lipoproteins (LDL). Cholesterol esterification was markedly inhibited by mevinolin and U18666A, but was not significantly affected by any other of the tested treatments. gamma-Secretase genes and cofactors were not co-regulated and were not influenced by statin inhibition of cholesterol synthesis. Nicastrin and the APP isoforms showed constitutive expression. In the absence of exogenous lipids, cell PS1 and PS2 expression was induced by LDL and by lysosomal sequestration of cholesterol. However, a different pattern of induction of presenilin gene expression was observed in the latter condition, suggesting that lysosomal cholesterol levels are strong inducers of PS2 transcription. Taken together, these results indicate that lipid metabolism has a complex influence on gamma-secretase transcriptional pathways and, in particular, exogenous cholesterol and compartmentalization in neuroblastoma cells play a relevant role in regulating the transcription of presenilins, while modulation of the cholesterol biosynthesis pathway seems to exert a minor influence on the expression of gamma-secretase genes and cofactors.

Niemann–Pick type C disease: Novel NPC1 mutations and characterization of the concomitant acid sphingomyelinase deficiency

Niemann–Pick type C (NPC) disease is an inherited lipid storage disorder characterized by the lysosomal accumulation of free cholesterol in affected cells. Three novel mutations in the NPC1 gene (c.3615delA, c.2000C > T, and c.2240delT) were detected in two unrelated patients with the severe phenotype of NPC. The analyses showed that the c.2240delT mutation, which causes a premature stop at codon 748, resulted in nonsense-mediated decay of the mutant transcripts. Immunoblotting analyses for the NPC1 protein did not detect the mutant proteins in COS-1 cells transiently transfected with the two mutant NPC1 cDNA constructs (c.3615delA and c.2000C > T). In NPC cells, sphingomyelin accumulates with cholesterol, leading to an identical subcellular distribution of both lipids. Acid sphingomyelinase (ASM), which is responsible for the lysosomal hydrolysis of sphingomyelin, is partially reduced in NPC fibroblasts. Therefore, NPC fibroblasts were studied to determine if ASM activity was perturbed due to the accumulation of cholesterol. However, these studies demonstrated that the subcellular localization of ASM was preserved, suggesting that the high content of lysosomal cholesterol was not responsible for the decreased ASM activity.

Aggregated LDL and Lipid Dispersions Induce Lysosomal Cholesterol Accumulation in Macrophage Foam Cells

Aggregated LDL and Lipid Dispersions Induce Lysosomal Cholesterol Accumulation in Macrophage Foam Cells

Gene expression profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish

Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.