Limits...
Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Show MeSH

Related in: MedlinePlus

RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3814149&req=5

Figure 1: RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Mentions: We previously showed that RIDα rescued the LSO cholesterol storage phenotype when transiently expressed in NPC1-mutant fibroblasts upon LDL loading (Figure 1A; Cianciola and Carlin, 2009). LSOs that accumulate in NPC disease cells can be visualized by costaining with LAMP1 to identify aberrant LEs and filipin, a fluorescent antibiotic that selectively binds free cholesterol. However, because of low transfection efficiency of patient fibroblasts, we are unable to perform biochemical assays in these cells. To further address the mechanism of how RIDα rescues the NPC1 cholesterol storage phenotype, we used two NPC1-deficient model systems with stable RIDα expression (Table 1): 1) CT43 cells, which are Chinese hamster ovary cells isolated after a chemical mutagenesis screen that harbor a mutation in NPC1 causing premature translational termination after 933 amino acids, producing a nonfunctional protein (Cruz et al., 2000), and 2) shNPC1 cells, which are immortalized human hepatocytes (IHHs) stably expressing shRNA to NPC1 (Ulatowski et al., 2011). CT43 cells and shNPC1 cells both displayed enlarged LAMP1/filipin-positive LEs indicative of LSOs upon LDL loading (Figure 1, B and C). Stable RIDα expression in either cell line rescued the NPC1-deficient cholesterol storage phenotype, as LSOs no longer persisted (Figure 1, B and C). We also tested whether RIDα expression could rescue the cholesterol storage phenotype in NPC2-mutant fibroblasts. NPC2-mutant fibroblasts displayed enlarged LAMP1/filipin-positive LEs upon LDL loading (Figure 1D); however, the LSO phenotype of NPC2-mutant fibroblasts was not affected by FLAG-RIDα expression (Figure 1D). Although we previously showed that RIDα colocalized with the atypical LE lipid LBPA in NPC1-mutant fibroblasts (Figure 1E; Cianciola and Carlin, 2009), in contrast, we found that RIDα did not localize with an LBPA-positive compartment in NPC2-mutant fibroblasts (Figure 1E). Collectively these data imply that the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells with different genetic defects depends on NPC2 and that RIDα function may involve an LBPA-positive compartment.


Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

Cianciola NL, Greene DJ, Morton RE, Carlin CR - Mol. Biol. Cell (2013)

RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814149&req=5

Figure 1: RIDα rescues cholesterol storage phenotype in NPC1-deficient cells but not in NPC2-mutant fibroblasts. (A) Confocal images of NPC1-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (B) Confocal images of CT43 and CT43-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Confocal images of shNPC1 and shNPC1-RIDα cells stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (D) Confocal images of NPC2-mutant fibroblasts mock transfected or transfected with FLAG-RIDα and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (E) Confocal images of NPC1- or NPC2-mutant fibroblasts transfected with FLAG-RIDα and stained with antibodies to LBPA and FLAG-RIDα. Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
Mentions: We previously showed that RIDα rescued the LSO cholesterol storage phenotype when transiently expressed in NPC1-mutant fibroblasts upon LDL loading (Figure 1A; Cianciola and Carlin, 2009). LSOs that accumulate in NPC disease cells can be visualized by costaining with LAMP1 to identify aberrant LEs and filipin, a fluorescent antibiotic that selectively binds free cholesterol. However, because of low transfection efficiency of patient fibroblasts, we are unable to perform biochemical assays in these cells. To further address the mechanism of how RIDα rescues the NPC1 cholesterol storage phenotype, we used two NPC1-deficient model systems with stable RIDα expression (Table 1): 1) CT43 cells, which are Chinese hamster ovary cells isolated after a chemical mutagenesis screen that harbor a mutation in NPC1 causing premature translational termination after 933 amino acids, producing a nonfunctional protein (Cruz et al., 2000), and 2) shNPC1 cells, which are immortalized human hepatocytes (IHHs) stably expressing shRNA to NPC1 (Ulatowski et al., 2011). CT43 cells and shNPC1 cells both displayed enlarged LAMP1/filipin-positive LEs indicative of LSOs upon LDL loading (Figure 1, B and C). Stable RIDα expression in either cell line rescued the NPC1-deficient cholesterol storage phenotype, as LSOs no longer persisted (Figure 1, B and C). We also tested whether RIDα expression could rescue the cholesterol storage phenotype in NPC2-mutant fibroblasts. NPC2-mutant fibroblasts displayed enlarged LAMP1/filipin-positive LEs upon LDL loading (Figure 1D); however, the LSO phenotype of NPC2-mutant fibroblasts was not affected by FLAG-RIDα expression (Figure 1D). Although we previously showed that RIDα colocalized with the atypical LE lipid LBPA in NPC1-mutant fibroblasts (Figure 1E; Cianciola and Carlin, 2009), in contrast, we found that RIDα did not localize with an LBPA-positive compartment in NPC2-mutant fibroblasts (Figure 1E). Collectively these data imply that the ability of RIDα to rescue the cholesterol storage phenotype in NPC1-deficient cells with different genetic defects depends on NPC2 and that RIDα function may involve an LBPA-positive compartment.

Bottom Line: Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7.The molecular identity of putative alternative pathways, however, is poorly characterized.We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.

ABSTRACT
Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Show MeSH
Related in: MedlinePlus