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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.

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Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
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Figure 3: Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.

Mentions: To further explore the effect of LD formation in RIDα LSO rescue, we treated NPC1-deficient cells with the prototypical ACAT inhibitor Sandoz 58-035 (S58-035; Ross et al., 1984). The LSO phenotype of mock-transfected NPC1-mutant fibroblasts was unaffected by vehicle treatment or 12-h treatment with S58-035 during LDL loading (Figure 3A); however, ACAT inhibitor treatment completely blocked the ability of RIDα to rescue the cholesterol storage phenotype in FLAG-RIDα–transfected NPC1-mutant fibroblasts (Figure 3B). We quantified the peak area of LAMP1/filipin-positive LSOs in mock-transfected and FLAG-RIDα–transfected NPC1-mutant fibroblasts upon LDL loading with vehicle and S58-035 treatment. RIDα expression induced a statistically significant decrease in peak LSO area in vehicle-treated cells compared with mock-transfected cells, whereas S58-035 treatment caused LSOs to persist in FLAG-RIDα–transfected cells (Figure 3C). As expected, S58-035 treatment also blocked LD formation in FLAG-RIDα–transfected NPC1-mutant fibroblasts stimulated with LDL (Figure 3D and Supplemental Figure S1D). To corroborate the results obtained in NPC1-mutant fibroblasts, we similarly loaded CT43 cells with LDL, treated them with vehicle or ACAT inhibitor for 12 h, and stained them for LAMP1 and filipin. The CT43 LSO phenotype was again unaffected by vehicle or S58-035 treatment (Figure 3E), and LSOs persisted in CT43-RIDα cells treated with S58-035 (Figure 3F). Similarly, quantification of confocal microscopy results showed a significant decrease in peak LSO area in vehicle treated CT43-RIDα cells compared with CT43 cells, and LSOs again persisted in CT43-RIDα cells treated with ACAT inhibitor (Figure 3G). Taken together these data indicate that RIDα promotes transport of free cholesterol to the ER, where it can be esterified by ACAT and stored in LDs in three different NPC1 mutant backgrounds.


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)

Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
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Figure 3: Inhibition of ACAT blocks RIDα rescue of cholesterol storage phenotype in NPC1-deficient cells. (A, B) Confocal images of NPC1-mutant fibroblasts mock transfected (A) or transfected with RIDα (B) treated with dimethyl sulfoxide (DMSO) vehicle (left) or S58-035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (C) Quantification of peak LSO area per cell in cells treated similarly to cells in A and B as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). (D) Confocal image of NPC1-mutant fibroblasts transfected with RIDα treated with S58-035 for 12 h and stained with antibody to FLAG-RIDα and with BODIPY 493/503 and DAPI. Mock-transfected cell is shown in the same field as designated by an asterisk. (E, F) CT43 (E) and CT43-RIDα cells (F) treated with DMSO vehicle (left) or S58–035 (right) for 12 h and stained with antibodies to LAMP1 and FLAG-RIDα and with filipin. (G) Quantification of peak LSO area per cell in cells treated similarly to cells in E and F as described in Materials and Methods. Data are presented as mean ± SEM (*p < 0.001). Boxed areas, regions of the image that were magnified. Bars, 10 μm. Nu, nucleus.
Mentions: To further explore the effect of LD formation in RIDα LSO rescue, we treated NPC1-deficient cells with the prototypical ACAT inhibitor Sandoz 58-035 (S58-035; Ross et al., 1984). The LSO phenotype of mock-transfected NPC1-mutant fibroblasts was unaffected by vehicle treatment or 12-h treatment with S58-035 during LDL loading (Figure 3A); however, ACAT inhibitor treatment completely blocked the ability of RIDα to rescue the cholesterol storage phenotype in FLAG-RIDα–transfected NPC1-mutant fibroblasts (Figure 3B). We quantified the peak area of LAMP1/filipin-positive LSOs in mock-transfected and FLAG-RIDα–transfected NPC1-mutant fibroblasts upon LDL loading with vehicle and S58-035 treatment. RIDα expression induced a statistically significant decrease in peak LSO area in vehicle-treated cells compared with mock-transfected cells, whereas S58-035 treatment caused LSOs to persist in FLAG-RIDα–transfected cells (Figure 3C). As expected, S58-035 treatment also blocked LD formation in FLAG-RIDα–transfected NPC1-mutant fibroblasts stimulated with LDL (Figure 3D and Supplemental Figure S1D). To corroborate the results obtained in NPC1-mutant fibroblasts, we similarly loaded CT43 cells with LDL, treated them with vehicle or ACAT inhibitor for 12 h, and stained them for LAMP1 and filipin. The CT43 LSO phenotype was again unaffected by vehicle or S58-035 treatment (Figure 3E), and LSOs persisted in CT43-RIDα cells treated with S58-035 (Figure 3F). Similarly, quantification of confocal microscopy results showed a significant decrease in peak LSO area in vehicle treated CT43-RIDα cells compared with CT43 cells, and LSOs again persisted in CT43-RIDα cells treated with ACAT inhibitor (Figure 3G). Taken together these data indicate that RIDα promotes transport of free cholesterol to the ER, where it can be esterified by ACAT and stored in LDs in three different NPC1 mutant backgrounds.

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