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ARF6-mediated endosome recycling reverses lipid accumulation defects in Niemann-Pick Type C disease.

Schweitzer JK, Pietrini SD, D'Souza-Schorey C - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and the Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, Indiana, United States of America.

ABSTRACT
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.

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Depletion of ARF6 promotes cholesterol accumulation in normal cells.HeLa cells were mock-treated with transfection reagent alone or transfected with siRNA directed against ARF6 alone or plus pIRES-GFP-ARF6si-res (“Rescue”) and analyzed 48 h later. A, ARF6 levels in cell lysates were determined using Western Blot analysis and probing for mouse anti-α-tubulin antibody (loading control) and a monoclonal antibody against ARF6. B, Cholesterol localization in fixed cells was assessed using BCθ toxin and streptavidin conjugated to Alexa Fluor 594. All three images were acquired in the same experiment using identical acquisition parameters. Contrast was enhanced using identical parameters in Adobe Photoshop 7.0. Inset in last image displays GFP expression indicating cells transfected with pIRES-GFP-ARF6si-res. Bars, 20 µm. C, Quantitation of cholesterol concentration using colorometric assay (see Methods). Values shown are percent of control. For each condition, the average of four independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6 siRNA vs. control, p = 0.056 and ARF6 siRNA vs. Rescue, p = 0.012, using a two-tailed t-test.
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pone-0005193-g002: Depletion of ARF6 promotes cholesterol accumulation in normal cells.HeLa cells were mock-treated with transfection reagent alone or transfected with siRNA directed against ARF6 alone or plus pIRES-GFP-ARF6si-res (“Rescue”) and analyzed 48 h later. A, ARF6 levels in cell lysates were determined using Western Blot analysis and probing for mouse anti-α-tubulin antibody (loading control) and a monoclonal antibody against ARF6. B, Cholesterol localization in fixed cells was assessed using BCθ toxin and streptavidin conjugated to Alexa Fluor 594. All three images were acquired in the same experiment using identical acquisition parameters. Contrast was enhanced using identical parameters in Adobe Photoshop 7.0. Inset in last image displays GFP expression indicating cells transfected with pIRES-GFP-ARF6si-res. Bars, 20 µm. C, Quantitation of cholesterol concentration using colorometric assay (see Methods). Values shown are percent of control. For each condition, the average of four independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6 siRNA vs. control, p = 0.056 and ARF6 siRNA vs. Rescue, p = 0.012, using a two-tailed t-test.

Mentions: Next, we investigated whether ARF6 is required for cholesterol homeostasis under normal conditions. We depleted ARF6 protein levels by transfecting cells with ARF6 siRNA and 48 h after transfection, we examined cholesterol distribution. As shown in Figure 2B, we found that siRNA-treated cells exhibited significant cholesterol accumulation inside the cell as visualized by staining with BCθ (biotinylated θ-toxin). Like filipin, BCθ binds to cholesterol-rich domains and is visualized using fluorescently labeled streptavidin [15]. Compared to filipin staining, use of BCθ leads to less labeling of the cell surface, less perturbation of membrane-lipid organization and more distinct labeling of internal cholesterol membranes [15]. When co-transfected with siRNA and a wild-type ARF6 construct bearing silent mutations in the region targeted by RNAi, no cholesterol accumulation was observed (Figure 2B, right panel). Using a biochemical assay to measure cholesterol levels, we found that ARF6-depleted cells exhibited greater than 65% increase in cholesterol after 48 h of siRNA treatment (Figure 2C). These results show that ARF6 is important for normal cholesterol homeostasis. These findings support the idea that perturbations in membrane recycling—in this case through the depletion of ARF6—can lead to cholesterol accumulation.


ARF6-mediated endosome recycling reverses lipid accumulation defects in Niemann-Pick Type C disease.

Schweitzer JK, Pietrini SD, D'Souza-Schorey C - PLoS ONE (2009)

Depletion of ARF6 promotes cholesterol accumulation in normal cells.HeLa cells were mock-treated with transfection reagent alone or transfected with siRNA directed against ARF6 alone or plus pIRES-GFP-ARF6si-res (“Rescue”) and analyzed 48 h later. A, ARF6 levels in cell lysates were determined using Western Blot analysis and probing for mouse anti-α-tubulin antibody (loading control) and a monoclonal antibody against ARF6. B, Cholesterol localization in fixed cells was assessed using BCθ toxin and streptavidin conjugated to Alexa Fluor 594. All three images were acquired in the same experiment using identical acquisition parameters. Contrast was enhanced using identical parameters in Adobe Photoshop 7.0. Inset in last image displays GFP expression indicating cells transfected with pIRES-GFP-ARF6si-res. Bars, 20 µm. C, Quantitation of cholesterol concentration using colorometric assay (see Methods). Values shown are percent of control. For each condition, the average of four independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6 siRNA vs. control, p = 0.056 and ARF6 siRNA vs. Rescue, p = 0.012, using a two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664925&req=5

pone-0005193-g002: Depletion of ARF6 promotes cholesterol accumulation in normal cells.HeLa cells were mock-treated with transfection reagent alone or transfected with siRNA directed against ARF6 alone or plus pIRES-GFP-ARF6si-res (“Rescue”) and analyzed 48 h later. A, ARF6 levels in cell lysates were determined using Western Blot analysis and probing for mouse anti-α-tubulin antibody (loading control) and a monoclonal antibody against ARF6. B, Cholesterol localization in fixed cells was assessed using BCθ toxin and streptavidin conjugated to Alexa Fluor 594. All three images were acquired in the same experiment using identical acquisition parameters. Contrast was enhanced using identical parameters in Adobe Photoshop 7.0. Inset in last image displays GFP expression indicating cells transfected with pIRES-GFP-ARF6si-res. Bars, 20 µm. C, Quantitation of cholesterol concentration using colorometric assay (see Methods). Values shown are percent of control. For each condition, the average of four independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6 siRNA vs. control, p = 0.056 and ARF6 siRNA vs. Rescue, p = 0.012, using a two-tailed t-test.
Mentions: Next, we investigated whether ARF6 is required for cholesterol homeostasis under normal conditions. We depleted ARF6 protein levels by transfecting cells with ARF6 siRNA and 48 h after transfection, we examined cholesterol distribution. As shown in Figure 2B, we found that siRNA-treated cells exhibited significant cholesterol accumulation inside the cell as visualized by staining with BCθ (biotinylated θ-toxin). Like filipin, BCθ binds to cholesterol-rich domains and is visualized using fluorescently labeled streptavidin [15]. Compared to filipin staining, use of BCθ leads to less labeling of the cell surface, less perturbation of membrane-lipid organization and more distinct labeling of internal cholesterol membranes [15]. When co-transfected with siRNA and a wild-type ARF6 construct bearing silent mutations in the region targeted by RNAi, no cholesterol accumulation was observed (Figure 2B, right panel). Using a biochemical assay to measure cholesterol levels, we found that ARF6-depleted cells exhibited greater than 65% increase in cholesterol after 48 h of siRNA treatment (Figure 2C). These results show that ARF6 is important for normal cholesterol homeostasis. These findings support the idea that perturbations in membrane recycling—in this case through the depletion of ARF6—can lead to cholesterol accumulation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and the Center for Rare and Neglected Diseases, University of Notre Dame, Notre Dame, Indiana, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus