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1.  Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments▿  
Journal of Virology  2009;83(16):7982-7995.
Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.
PMCID: PMC2715784  PMID: 19474101
2.  Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic EventsV⃞ 
Molecular Biology of the Cell  2001;12(3):601-614.
People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.
PMCID: PMC30967  PMID: 11251074
3.  IFITM3 Restricts Influenza A Virus Entry by Blocking the Formation of Fusion Pores following Virus-Endosome Hemifusion 
PLoS Pathogens  2014;10(4):e1004048.
Interferon-induced transmembrane proteins (IFITMs) inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV) which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.
Author Summary
Interferon-induced transmembrane proteins (IFITMs) block infection of many enveloped viruses, including the influenza A virus (IAV) that enters from late endosomes. IFITMs are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. Here we performed single IAV imaging and found that IFITM3 did not interfere with hemifusion, but prevented complete fusion. Also, contrary to a current view that excess cholesterol in late endosomes of IFITM3-expressing cells inhibits IAV entry, we show that cholesterol-laden endosomes are permissive for virus fusion. The ability of IFITM3 to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. IFITM3 may also redirect IAV to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane.
PMCID: PMC3974867  PMID: 24699674
4.  Cholinergic Abnormalities, Endosomal Alterations and Up-Regulation of Nerve Growth Factor Signaling in Niemann-Pick Type C Disease 
Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC) disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i) NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF) after axotomy and ii) PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF.
NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT), whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A.
Our results suggest that the NPC cellular phenotype causes neuronal dysfunction through the abnormal up-regulation of survival pathways, which causes the perturbation of signaling cascades and anomalous phosphorylation of the cytoskeleton.
PMCID: PMC3395862  PMID: 22458984
NGF; Endosomes; Cholesterol; Niemann-Pick type C1; cholinergic system; PC12
5.  Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O 
Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O.
To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO–positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells.
GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-β-cyclodextrin.
Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.
PMCID: PMC4005833  PMID: 24775609
Niemann-Pick C disease; Lipid storage diseases; Cholesterol-binding proteins; Filipin; Perfringolysin O
6.  Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells* 
The Journal of biological chemistry  2006;281(26):17890-17899.
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 accompaniedby 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 man-nose 6-phosphate receptorswere 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.
PMCID: PMC3650718  PMID: 16644737
7.  Hrs and SNX3 Functions in Sorting and Membrane Invagination within Multivesicular Bodies  
PLoS Biology  2008;6(9):e214.
After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.
Author Summary
The cell's genetic program is modulated by extracellular signals that activate cell surface receptors and, in turn, intracellular effectors, to regulate transcription. For cells to function normally, these signals must be turned off to avoid permanent activation—a situation often associated with cancer. For many receptors, signaling is repressed, or down-regulated, in a process that first internalizes and then degrades the receptors. After receptors are removed from the cell surface into structures called early endosomes, they are selectively incorporated within vesicles that form inside the endosome. During this process, endosomal membranes are pulled away from the cytoplasm towards the endosome lumen, against the flow of intracellular membrane traffic, eventually resulting in the formation of a “multivesicular body” (vesicles within vesicles). The common view is that these intralumenal vesicles are then delivered to lysosomes, where they are degraded along with their receptor cargo. We have investigated the mechanisms responsible for the biogenesis of intralumenal vesicles in multivesicular bodies. We find that the small protein SNX3, which binds the signaling lipid phosphatidyl inositol-3-phosphate, is necessary for the formation of intralumenal vesicles, but is not involved in the degradation of the cell surface receptor for EGF. Conversely, we find that Hrs, which also binds phosphatidyl inositol-3-phosphate and mediates receptor sorting into intralumenal vesicles, is essential for lysosomal targeting but dispensable for multivesicular body biogenesis. Phosphatidyl inositol-3-phosphate thus controls the complementary functions of Hrs and SNX3 in the sorting of signaling receptors and multivesicular body biogenesis.
SNX3 plays a direct role in the formation of intralumenal vesicles of multivesicular bodies (MVBs) but is not involved in EGF receptor degradation, whereas Hrs is essential for lysosomal targeting but dispensable for MVB biogenesis. Hence, intralumenal vesicle formation in MVB biogenesis can be uncoupled from lysosomal targeting.
PMCID: PMC2528051  PMID: 18767904
8.  Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function 
Molecular Biology of the Cell  2011;22(21):4108-4123.
This study shows that impaired cholesterol egress from late endosomes in cells with high annexin A6 levels is associated with altered soluble N-ethylmaleimide–sensitive fusion protein 23 (SNAP23) and syntaxin-4 cellular distribution and assembly and accumulation in Golgi membranes. This correlates with reduced secretion of cargo along the constitutive and SNAP23/syntaxin-4–dependent secretory pathway.
Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4–dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway.
PMCID: PMC3204072  PMID: 22039070
9.  Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization 
PLoS ONE  2008;3(7):e2758.
The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abundance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.
PMCID: PMC2447177  PMID: 18648502
10.  A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking 
The Journal of Cell Biology  2011;192(1):121-135.
ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes.
Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.
PMCID: PMC3019559  PMID: 21220512
11.  Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11 
Molecular Biology of the Cell  2002;13(9):3107-3122.
To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate–labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis.
PMCID: PMC124146  PMID: 12221119
12.  Deficiency of a Niemann-Pick, Type C1-related Protein in Toxoplasma Is Associated with Multiple Lipidoses and Increased Pathogenicity 
PLoS Pathogens  2011;7(12):e1002410.
Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved ‘sterol-sensing domain’ (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma.
Author Summary
The intracellular parasite Toxoplasma is auxotrophic for several lipids that it scavenges from host organelles. Several studies focused on deciphering the pathways implicated in host lipid delivery to the parasite, but less effort has been devoted to understand how lipids are regulated in Toxoplasma. The ‘sterol-sensing domain’ (SSD) is conserved across phyla and present in several membrane proteins involved in cholesterol homeostasis, cell signaling and cytokinesis. We studied the role of a SSD-containing protein in Toxoplasma which shows significant similarity with Niemann-Pick type C1 proteins (NPC1). Human NPC disease is typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of the parasite NPC1-related protein (named TgNCR1) in mammalian NPC1 mutant cells suppresses lipid accumulation in lysosomes. However, Toxoplasma never internalizes host cholesterol into lysosomes, which predicts a function for TgNCR1 unrelated to exogenous sterol transport. Indeed, genomic deletion of NCR1 does not result in abnormal levels of cholesterol in the parasites but in the overaccumulation of cholesteryl esters and sphingolipids. TgNCR1-deficient parasites form abundant storage lipid bodies and multiple parasites per cycle of division. This suggests that TgNCR1 functions in monitoring the levels of various lipids within Toxoplasma, which in turn impacts the parasite's lipid homeostasis and growth rate.
PMCID: PMC3234224  PMID: 22174676
13.  Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection 
PLoS ONE  2013;8(2):e56265.
Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC50 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.
PMCID: PMC3575416  PMID: 23441171
14.  Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin 
PLoS Genetics  2013;9(4):e1003462.
Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.
Author Summary
The myelin sheath in the central nervous system is a specialized extension of the oligodendrocyte plasma membrane that serves as an electrical insulator to ensure proper nerve conduction. To accomplish this, myelin is enriched in lipids, particularly unesterified cholesterol, which is an essential and limiting component for myelin formation. Here we determine the contribution of exogenously derived cholesterol to myelination by using a conditional null mutant of the mouse Npc1 gene. Npc1 encodes a transmembrane protein critical for mobilizing exogenously derived cholesterol from late endosomes and lysosomes, and is mutated in patients with Niemann-Pick type C disease, a degenerative disorder caused by impaired intracellular lipid trafficking. We show that mice lacking Npc1 in either neurons or oligodendrocytes exhibit a defect in myelin formation in selected brain regions, with an arrest in oligodendrocyte maturation. In addition, mice with Npc1 deficiency in oligodendrocytes, when aged, show progressive motor dysfunction with myelin breakdown and secondary Purkinje neuron loss. Taken together, our findings demonstrate the role of Npc1 in mediating reciprocal signaling between neurons and glia, and highlight the importance of exogenous cholesterol for CNS myelin formation and maintenance.
PMCID: PMC3623760  PMID: 23593041
15.  Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments 
PLoS Pathogens  2012;8(5):e1002694.
Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.
Author Summary
Endosomal trafficking and regulation of retrovirus fusion is poorly understood, due in part to heterogeneity of viral particles and their asynchronous entry into an endocytic pathway. Here, we used an avian retrovirus that enters host cells in a receptor- and low pH-dependent manner. This feature allowed capturing the virus in intracellular compartments through raising the endosomal pH. Virus fusion was synchronously initiated upon permitting endosome acidification and visualized in real-time by single particle imaging. We found that different receptor isoforms directed virus into distinct sub-cellular compartments supporting quick release of the viral core. Through tracking individual sub-viral particles released from endosomes, we found that the full length receptor mediated core delivery into the cytoplasm. By contrast, fusion mediated by the GPI-anchored receptor released the core into another endosomal compartment, from which the core entered the cytosol through an additional temperature-dependent step. These findings demonstrate different permissiveness of endosomal compartments to viral fusion and the existence of a post-fusion step leading to the cytosolic release of cores and initiation of infection.
PMCID: PMC3349758  PMID: 22589725
16.  ACAT1-associated Late Endosomes/Lysosomes Significantly Improve Impaired Intracellular Cholesterol Metabolism and the Survival of Niemann-Pick Type C Mice 
We previously demonstrated that macrophages exhibit endoplasmic reticulum fragmentation under cholesterol-rich conditions, which results in the generation of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1)-associated late endosomes/lysosomes (ACAT1-LE). ACAT1-LE efficiently esterify free cholesterol in loco, even with abnormal egress of free cholesterol from late endosomes. Because impaired free cholesterol transport from late endosomes results in Niemann-Pick type C disease (NPC), the induction of ACAT1-LE is a potential therapeutic intervention for NPC. To examine the effects of ACAT1-LE induction on intracellular cholesterol metabolism, we incubated bone marrow-derived macrophages possessing NPC phenotype (npc1–/–) with methyl-β-cyclodextrin-cholesterol complex (mβCD-cho), a cholesterol donor. Immunofluorescence confocal microscopy revealed that mβCD-cho treatment of npc1–/– macrophages resulted in significant colocalization of signals from ACAT1 and lysosome-associated membrane protein 2, a late endosome/lysosome marker. npc1–/– macrophages contained significant amounts of free cholesterol with negligible amounts of cholesteryl ester, while wild-type macrophages possessed the same amounts of both cholesterols. mβCD-cho treatment also induced marked restoration of cholesterol esterification activity. mβCD-cho administration in neonate npc1–/– mice improved survival. These results indicate that ACAT1-LE induction in npc1–/– mice corrects impaired intracellular cholesterol metabolism and that restoring cholesterol esterification improves prognosis of npc1–/–. These data suggest that ACAT1-LE induction is a potential alternative therapeutic strategy for NPC.
PMCID: PMC4105798  PMID: 25221362
Niemann-Pick type C disease; acyl-coenzyme A: cholesterol acyltransferase 1; late endosomes; cholesterol; methyl-β-cyclodextrin
17.  Inhibition of cholesterol recycling impairs cellular PrPSc propagation 
Cellular and Molecular Life Sciences   2009;66(24):3979-3991.
The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrPSc formation is not impaired, suggesting that PrPSc degradation is increased by intracellular cholesterol accumulation. Interestingly, PrPSc propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrPSc to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrPSc content, indicating that PrPSc production is highly sensitive to alterations in dynamics of vesicle trafficking.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-009-0158-4) contains supplementary material, which is available to authorized users.
PMCID: PMC2777232  PMID: 19823766
Cholesterol; Recycling; Prion; PrP; NPC-1; Rab 9
18.  Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content 
Molecular Biology of the Cell  2012;23(5):864-880.
Caveolin-1 traffics to late endosomal/lysosomal membranes in response to manipulations of the cholesterol content of cells, suggesting that caveolin functions in the egress of cholesterol from this organelle. Cavicles associate with the periphery of the lysosome as they do with caveosomes, but these are separate organelles.
Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.
PMCID: PMC3290645  PMID: 22238363
19.  Adenovirus RID-α activates an autonomous cholesterol regulatory mechanism that rescues defects linked to Niemann-Pick disease type C 
The Journal of Cell Biology  2009;187(4):537-552.
Viral subversion of cholesterol homeostasis provides insights into sterol trafficking, autophagy, and lysosomal storage diseases.
Host–pathogen interactions are important model systems for understanding fundamental cell biological processes. In this study, we describe a cholesterol-trafficking pathway induced by the adenovirus membrane protein RID-α that also subverts the cellular autophagy pathway during early stages of an acute infection. A palmitoylation-defective RID-α mutant deregulates cholesterol homeostasis and elicits lysosomal storage abnormalities similar to mutations associated with Niemann-Pick type C (NPC) disease. Wild-type RID-α rescues lipid-sorting defects in cells from patients with this disease by a mechanism involving a class III phosphatidylinositol-3-kinase. In contrast to NPC disease gene products that are localized to late endosomes/lysosomes, RID-α induces the accumulation of autophagy-like vesicles with a unique molecular composition. Ectopic RID-α regulates intracellular cholesterol trafficking at two distinct levels: the egress from endosomes and transport to the endoplasmic reticulum necessary for homeostatic gene regulation. However, RID-α also induces a novel cellular phenotype, suggesting that it activates an autonomous cholesterol regulatory mechanism distinct from NPC disease gene products.
PMCID: PMC2779231  PMID: 19948501
20.  In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101 
Molecular Biology of the Cell  2008;19(11):4942-4955.
Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively.
PMCID: PMC2575168  PMID: 18768755
21.  Treatment of Niemann–Pick Type C Disease by Histone Deacetylase Inhibitors 
Neurotherapeutics  2013;10(4):688-697.
Niemann–Pick type C disease (NPC) is a devastating, recessive, inherited disorder that causes accumulation of cholesterol and other lipids in late endosomes and lysosomes. Mutations in 2 genes, NPC1 and NPC2, are responsible for the disease, which affects about 1 in 120,000 live births. About 95 % of patients have mutations in NPC1, a large polytopic membrane protein that is normally found in late endosomes. More than 200 missense mutations in NPC1 have been found in NPC patients. The disease is progressive, typically leading to death before the age of 20 years, although some affected individuals live well into adulthood. The disease affects peripheral organs, including the liver, spleen, and lungs, but the most severe symptoms are associated with neurological disease. There are some palliative treatments that slow progression of NPC disease. Recently, it was found that histone deacetylase (HDAC) inhibitors that are effective against HDACs 1, 2, and 3 can reduce the cholesterol accumulation in fibroblasts derived from NPC patients with mutations in NPC1. One example is vorinostat. As vorinostat is a Food and Drug Administration–approved drug for treatment of cutaneous T-cell lymphoma, this opens up the possibility that HDAC inhibitors could be repurposed for treatment of this rare disease. The mechanism of action of the HDAC inhibitors requires further study, but these drugs increase the level of the NPC1 protein. This may be due to post-translational stabilization of the NPC1 protein, allowing it to be transported out of the endoplasmic reticulum.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0217-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3805865  PMID: 24048860
Histone deacetylase inhibitors; Cholesterol; Lysosomes; Lipids; Proteostasis
22.  Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza A Replication and Propagation 
mBio  2013;4(6):e00608-13.
Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca2+-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis.
Influenza A virus (IAV) is a major public health concern, and yet, major host-pathogen interactions regulating IAV replication still remain poorly understood. It is known that host cell cholesterol is a critical factor in the influenza virus life cycle. The viral envelope is derived from the host cell membrane during the process of budding and, hence, equips the virus with a special lipid-protein mixture which is high in cholesterol. However, the influence of host cell cholesterol homeostasis on IAV infection is largely unknown. We show that IAV infection success critically depends on host cell cholesterol distribution. Cholesterol sequestration in the endosomal compartment impairs progeny titer and infectivity and is associated with reduced cholesterol content in the viral envelope.
PMCID: PMC3892785  PMID: 24194536
23.  Rab8-dependent Recycling Promotes Endosomal Cholesterol Removal in Normal and Sphingolipidosis Cells 
Molecular Biology of the Cell  2007;18(1):47-56.
The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann–Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.
PMCID: PMC1751315  PMID: 17050734
24.  ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease 
PLoS ONE  2009;4(4):e5193.
In human Niemann-Pick Type C (NPC) disease, endosomal trafficking defects lead to an accumulation of free cholesterol and other lipids in late endosome/lysosome (LE/LY) compartments, a subsequent block in cholesterol esterification and significantly reduced cholesterol efflux out of the cell. Here we report that nucleotide cycling or cellular knockdown of the small GTP-binding protein, ARF6, markedly impacts cholesterol homeostasis. Unregulated ARF6 activation attenuates the NPC phenotype at least in part by decreasing cholesterol accumulation and restoring normal sphingolipid trafficking. These effects depend on ARF6-stimulated cholesterol efflux out of the endosomal recycling compartment, a major cell repository for free cholesterol. We also show that fibroblasts derived from different NPC patients displayed varying levels of ARF6 that is GTP-bound, which correlate with their response to sustained ARF6 activation. These studies support emerging evidence that early endocytic defects impact NPC disease and suggest that such heterogeneity in NPC disease could result in diverse responses to therapeutic interventions aimed at modulating the trafficking of lipids.
PMCID: PMC2664925  PMID: 19365558
25.  Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation 
The Journal of Cell Biology  2010;191(3):615-629.
Identification of the pathway by which caveolin-1 is degraded when caveolae assembly is compromised suggests that “caveosomes” may be endosomal accumulations of the protein awaiting degradation.
Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that “caveosomes,” previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.
PMCID: PMC3003328  PMID: 21041450

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