ABSTRACT
The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer's disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk.
Subject(s)
Alzheimer Disease , Induced Pluripotent Stem Cells , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Apolipoprotein E3/genetics , Apolipoprotein E3/metabolism , Apolipoprotein E4/genetics , Apolipoprotein E4/metabolism , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Astrocytes/metabolism , Cholesterol/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Microglia/metabolismABSTRACT
Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.
Subject(s)
Cholesterol/metabolism , Fibroblasts/metabolism , HSP90 Heat-Shock Proteins/metabolism , Niemann-Pick C1 Protein/metabolism , Cells, Cultured , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , MutationABSTRACT
Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1 I1061T , the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.
ABSTRACT
Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.
Subject(s)
2-Hydroxypropyl-beta-cyclodextrin/therapeutic use , Histone Deacetylase Inhibitors/therapeutic use , Niemann-Pick Disease, Type C/drug therapy , Polyethylene Glycols/therapeutic use , Vorinostat/therapeutic use , Animals , Cells, Cultured , Drug Combinations , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/metabolismABSTRACT
This corrects the article DOI: 10.1038/nm.4355.
ABSTRACT
Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, that responds to lipid accumulation via modulation of the nanotube's optical band gap. The engineered nanomaterial, composed of short, single-stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis, was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned subcellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone-marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers additional capabilities for drug development processes and the investigation of lipid-linked diseases.
Subject(s)
Atherosclerosis/blood , DNA, Single-Stranded/chemistry , Lipids/chemistry , Nanotubes, Carbon/chemistry , Atherosclerosis/pathology , DNA, Single-Stranded/blood , Endosomes/chemistry , Humans , Luminescent Measurements , Lysosomes/chemistry , Lysosomes/metabolism , Macrophages/chemistry , Macrophages/metabolism , Monocytes/chemistry , Monocytes/metabolism , Niemann-Pick Disease, Type C , Optics and Photonics/instrumentation , Single-Cell Analysis/methods , Transport Vesicles/chemistry , Transport Vesicles/metabolismABSTRACT
With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.
Subject(s)
Adenoma/genetics , Adenomatous Polyposis Coli/genetics , Antibiotics, Antineoplastic/pharmacology , Cell Proliferation/drug effects , Colon/drug effects , Colorectal Neoplasms/genetics , Human Embryonic Stem Cells , Organoids/drug effects , Adenoma/pathology , Adenomatous Polyposis Coli Protein/genetics , Blotting, Western , Cell Differentiation , Colon/cytology , Colon/metabolism , Colorectal Neoplasms/pathology , Drug Screening Assays, Antitumor , Enteroendocrine Cells/cytology , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Profiling , Gentamicins/pharmacology , Germ-Line Mutation , Goblet Cells/cytology , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Immunohistochemistry , Induced Pluripotent Stem Cells , Microscopy, Confocal , Mutation , Organoids/cytology , Organoids/metabolism , Organoids/pathology , Real-Time Polymerase Chain Reaction , Sirolimus/pharmacology , Wnt Signaling PathwayABSTRACT
Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat.
Subject(s)
Carrier Proteins/genetics , Cholesterol/metabolism , Histone Deacetylase Inhibitors/administration & dosage , Membrane Glycoproteins/genetics , Niemann-Pick Disease, Type C/drug therapy , Carrier Proteins/antagonists & inhibitors , Cell Line , Endoplasmic Reticulum-Associated Degradation/drug effects , Endosomes/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Hydroxamic Acids/administration & dosage , Indoles/administration & dosage , Intracellular Signaling Peptides and Proteins , Lysosomes/metabolism , Membrane Glycoproteins/antagonists & inhibitors , Mutation , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Niemann-Pick Disease, Type C/pathology , Panobinostat , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Transfection , VorinostatABSTRACT
Cholesterol plays an important role in determining the biophysical properties of membranes in mammalian cells, and the concentration of cholesterol in membranes is tightly regulated. Cholesterol moves among membrane organelles by a combination of vesicular and nonvesicular transport pathways, but the details of these transport pathways are not well understood. In this review, we discuss the mechanisms for nonvesicular sterol transport with an emphasis on the role of STARD4, a small, soluble, cytoplasmic sterol transport protein. STARD4 can rapidly equilibrate sterol between membranes, especially membranes with anionic lipid headgroups. We also discuss the sterol transport in late endosomes and lysosomes, which is mediated by a soluble protein, NPC2, and a membrane protein, NPC1. Homozygous mutations in these proteins lead to a lysosomal lipid storage disorder, Niemann-Pick disease type C. Many of the disease-causing mutations in NPC1 are associated with degradation of the mutant NPC1 proteins in the endoplasmic reticulum. Several histone deacetylase inhibitors have been found to rescue the premature degradation of the mutant NPC1 proteins, and one of these is now in a small clinical trial.
Subject(s)
Carrier Proteins/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/metabolism , Sterols/metabolism , Biological Transport , Humans , Intracellular Signaling Peptides and Proteins , Niemann-Pick C1 ProteinABSTRACT
Niemann-Pick Type C1 (NPC1) disease is a rare neurovisceral, cholesterol-sphingolipid lysosomal storage disorder characterized by ataxia, motor impairment, progressive intellectual decline, and dementia. The most prevalent mutation, NPC1(I1061T), encodes a misfolded protein with a reduced half-life caused by ER-associated degradation. Therapies directed at stabilization of the mutant NPC1 protein reduce cholesterol storage in fibroblasts but have not been tested in vivo because of lack of a suitable animal model. Whereas the prominent features of human NPC1 disease are replicated in the null Npc1(-/-) mouse, this model is not amenable to examining proteostatic therapies. The objective of the present study was to develop an NPC1 I1061T knock-in mouse in which to test proteostatic therapies. Compared with the Npc1(-/-) mouse, this Npc1(tm(I1061T)Dso) model displays a less severe, delayed form of NPC1 disease with respect to weight loss, decreased motor coordination, Purkinje cell death, lipid storage, and premature death. The murine NPC1(I1061T) protein has a reduced half-life in vivo, consistent with protein misfolding and rapid ER-associated degradation, and can be stabilized by histone deacetylase inhibition. This novel mouse model faithfully recapitulates human NPC1 disease and provides a powerful tool for preclinical evaluation of therapies targeting NPC1 protein variants with compromised stability.
Subject(s)
Alleles , Carrier Proteins/genetics , Disease Models, Animal , Gene Knock-In Techniques , Membrane Glycoproteins/genetics , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/pathology , Animals , Cells, Cultured , Female , Gene Knock-In Techniques/methods , Humans , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Niemann-Pick C1 Protein , PrevalenceABSTRACT
There is extensive evidence that cholesterol and membrane lipids play a key role in Alzheimer disease (AD) pathogenesis. Cyclodextrins (CD) are cyclic oligosaccharide compounds widely used to bind cholesterol. Because CD exerts significant beneficial effects in Niemann-Pick type C disease, which shares neuropathological features with AD, we examined the effects of hydroxypropyl-ß-CD (HP-ß-CD) in cell and mouse models of AD. Cell membrane cholesterol accumulation was detected in N2a cells overexpressing Swedish mutant APP (SwN2a), and the level of membrane cholesterol was reduced by HP-ß-CD treatment. HP-ß-CD dramatically lowered the levels of Aß42 in SwN2a cells, and the effects were persistent for 24 h after withdrawal. 4 mo of subcutaneous HP-ß-CD administration significantly improved spatial learning and memory deficits in Tg19959 mice, diminished Aß plaque deposition, and reduced tau immunoreactive dystrophic neurites. HP-ß-CD lowered levels of Aß42 in part by reducing ß cleavage of the APP protein, and it also up-regulated the expression of genes involved in cholesterol transport and Aß clearance. This is the first study to show neuroprotective effects of HP-ß-CD in a transgenic mouse model of AD, both by reducing Aß production and enhancing clearance mechanisms, which suggests a novel therapeutic strategy for AD.
Subject(s)
Alzheimer Disease/prevention & control , Neuroprotective Agents/pharmacology , beta-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/psychology , Amyloid beta-Peptides/metabolism , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cell Line , Cholesterol/metabolism , Disease Models, Animal , Humans , Learning/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Memory/drug effects , Mice , Mice, 129 Strain , Mice, Transgenic , Peptide Fragments/metabolism , Plaque, Amyloid/pathology , Plaque, Amyloid/prevention & control , tau Proteins/metabolismABSTRACT
Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F(v))-PE38), are proposed to traffic to the trans-Golgi network (TGN) and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO) cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity - presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.
Subject(s)
Furin/metabolism , Immunotoxins/metabolism , Membrane Glycoproteins/metabolism , Animals , Antibodies, Monoclonal/metabolism , CHO Cells , Cell Survival/genetics , Cell Survival/physiology , Cricetinae , Exotoxins/metabolism , Fluorescent Antibody Technique, Indirect , Furin/genetics , Humans , Immunoblotting , Immunotoxins/genetics , Membrane Glycoproteins/genetics , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolismABSTRACT
Nonvesicular transport of cholesterol plays an essential role in the distribution and regulation of cholesterol within cells, but it has been difficult to identify the key intracellular cholesterol transporters. The steroidogenic acute regulatory-related lipid-transfer (START) family of proteins is involved in several pathways of nonvesicular trafficking of sterols. Among them, STARD4 has been shown to increase intracellular cholesteryl ester formation and is controlled at the transcriptional level by sterol levels in cells. We found that STARD4 is very efficient in transporting sterol between membranes in vitro. Cholesterol levels are increased in STARD4-silenced cells, while sterol transport to the endocytic recycling compartment (ERC) and to the endoplasmic reticulum (ER) are enhanced upon STARD4 overexpression. STARD4 silencing attenuates cholesterol-mediated regulation of SREBP-2 activation, while its overexpression amplifies sterol sensing by SCAP/SREBP-2. To analyze STARD4's mode of action, we compared sterol transport mediated by STARD4 with that of a simple sterol carrier, methyl-ß-cyclodextrin (MCD), when STARD4 and MCD were overexpressed or injected into cells. Interestingly, STARD4 and cytosolic MCD act similarly by increasing the rate of transfer of sterol to the ERC and to the ER. Our results suggest that cholesterol transport mediated by STARD4 is an important component of the cholesterol homeostasis regulatory machinery.
Subject(s)
Cholesterol/metabolism , Membrane Transport Proteins/metabolism , Amino Acid Motifs , Cell Line, Tumor , Cell Membrane/metabolism , Cholesterol Esters/biosynthesis , Endoplasmic Reticulum/metabolism , Ergosterol/analogs & derivatives , Ergosterol/metabolism , Esterification , Fluorescence Recovery After Photobleaching , Fluorescent Dyes/metabolism , Gene Knockdown Techniques , Homeostasis , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Kinetics , Liposomes/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Protein Structure, Tertiary , RNA Interference , Sterol O-Acyltransferase/antagonists & inhibitors , Sterol O-Acyltransferase/metabolism , Sterol Regulatory Element Binding Protein 2/metabolism , Time-Lapse Imaging , Transferrin/metabolism , Transport Vesicles/metabolism , beta-Cyclodextrins/pharmacologyABSTRACT
Niemann-Pick type C (NPC) disease is predominantly caused by mutations in the NPC1 protein that affect intracellular cholesterol trafficking and cause accumulation of unesterified cholesterol and other lipids in lysosomal storage organelles. We report the use of a series of small molecule histone deacetylase (HDAC) inhibitors in tissue culture models of NPC human fibroblasts. Some HDAC inhibitors lead to a dramatic correction in the NPC phenotype in cells with either one or two copies of the NPC1(I1061T) mutation, and for several of the inhibitors, correction is associated with increased expression of NPC1 protein. Increased NPC1(I1061T) protein levels may partially account for the correction of the phenotype, because this mutant can promote cholesterol efflux if it is delivered to late endosomes and lysosomes. The HDAC inhibitor treatment is ineffective in an NPC2 mutant human fibroblast line. Analysis of the isoform selectivity of the compounds used implicates HDAC1 and/or HDAC2 as likely targets for the observed correction, although other HDACs may also play a role. LBH589 (panobinostat) is an orally available HDAC inhibitor that crosses the blood-brain barrier and is currently in phase III clinical trials for several types of cancer. It restores cholesterol homeostasis in cultured NPC1 mutant fibroblasts to almost normal levels within 72 h when used at 40 nM. The findings that HDAC inhibitors can correct cholesterol storage defects in human NPC1 mutant cells provide the potential basis for treatment options for NPC disease.
Subject(s)
Cholesterol/metabolism , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Niemann-Pick Disease, Type C/blood , Blotting, Western , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line , Dose-Response Relationship, Drug , Humans , Image Processing, Computer-Assisted , Indoles , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Microscopy, Fluorescence , Mutation/genetics , Niemann-Pick C1 Protein , Panobinostat , Time FactorsABSTRACT
BACKGROUND: Niemann-Pick type C (NPC) disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles. METHODOLOGY/PRINCIPAL FINDINGS: The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds. CONCLUSIONS/SIGNIFICANCE: Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165).
Subject(s)
Anticholesteremic Agents/pharmacology , Cholesterol/metabolism , Mutation , Niemann-Pick Diseases/metabolism , Small Molecule Libraries/pharmacology , Sterols/metabolism , Animals , CHO Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cricetinae , Cricetulus , Drug Evaluation, Preclinical , Humans , Intracellular Signaling Peptides and Proteins , Lipoproteins/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Niemann-Pick C1 Protein , Niemann-Pick Diseases/geneticsABSTRACT
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.
Subject(s)
Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/metabolism , Proteins/genetics , Proteins/metabolism , Animals , Antigens, CD , CHO Cells , Cholesterol/genetics , Cholesterol/metabolism , Cricetinae , Cricetulus , Fibroblasts/metabolism , Humans , Lipids/genetics , Lysosomes/genetics , Lysosomes/metabolism , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/metabolism , Protein Transport/genetics , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , TransfectionABSTRACT
The bundling of the N-terminal, partial domain helix (Helix C') of human erythroid alpha-spectrin (alphaI) with the C-terminal, partial domain helices (Helices A' and B') of erythroid beta-spectrin (betaI) to give a spectrin pseudo structural domain (triple helical bundle A'B'C') has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern-Volmer quenching constants of Helix C' of alphaI bound to Helices A' and B' of betaI, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and alphaI mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C' was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation-induced blood disorder.
Subject(s)
Point Mutation , Spectrin/genetics , Spectrin/metabolism , Humans , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrin/chemistryABSTRACT
We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.
Subject(s)
Carbocyanines/metabolism , Ergosterol/analogs & derivatives , Lysophospholipids/metabolism , Monoglycerides/metabolism , Niemann-Pick Disease, Type C/metabolism , Receptors, Transferrin/metabolism , Animals , Biological Transport , CHO Cells , Cell Membrane/metabolism , Cricetinae , Cricetulus , Endosomes/metabolism , Ergosterol/metabolism , Fluorescent Dyes , Kinetics , Lysosomes/metabolism , Microscopy, Fluorescence , Protein TransportABSTRACT
The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.
Subject(s)
Mutation , Oncogene Protein pp60(v-src)/physiology , Transfection , Cell Line , Fluorescent Antibody Technique , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Oncogene Protein pp60(v-src)/genetics , PlasmidsABSTRACT
Niemann-Pick disease type C (NPC) is an autosomal recessive genetic disorder manifested by abnormal accumulation of unesterified cholesterol and other lipids. We screened combinatorially synthesized chemical libraries to identify compounds that would partially revert cholesterol accumulation. Cultured CHO cells with NPC phenotypes (CT60 and CT43) were used for screening along with normal CHO cells as a control. We developed an automated microscopy assay based on imaging of filipin fluorescence for estimating cholesterol accumulation in lysosomal storage organelles. Our primary screen of 14,956 compounds identified 14 hit compounds that caused significant reduction in cellular cholesterol accumulation at 10 microM. We then screened a secondary library of 3,962 compounds selected based on chemical similarity to the initial hits and identified 7 compounds that demonstrated greater efficacy and lower toxicity than the original hits. These compounds are effective at concentrations of 123 nM to 3 microM in reducing the cholesterol accumulation in cells with a NPC1 phenotype.