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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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Constitutively active ARF6 increases cholesterol removal in NPC-like cells.A, HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B, HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C, Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).
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pone-0005193-g001: Constitutively active ARF6 increases cholesterol removal in NPC-like cells.A, HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B, HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C, Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).

Mentions: Previous results suggest that cholesterol cycles, in part, through vesicles containing the GTP-binding protein, ARF6, a potent regulator of early endosomal membrane internalization and recycling [10], [11]. Consistent with these findings, we find significant overlap of cholesterol with ARF6 in HeLa cells (Figure S1A). To mimic abnormal cholesterol accumulation observed in a variety of lipid storage diseases, we treated HeLa cells with the hydrophobic polyamine U18666A. Treatment with U18666A induces acute cholesterol accumulation that is indistinguishable from that seen in NPC (Figure 1A) [12]. U18666A blocks cholesterol exit from LE/LY likely by binding negatively charged phospholipids in late endosomal membranes, such as LBPA [13], [14]. Since ARF6 activation is coupled to the efflux of recycling endosomal membrane [10], we examined the impact of constitutive ARF6 activation on the lipid accumulation phenotype induced by U18666A. To this end, HeLa cells were transfected with a GTPase defective, constitutively activated ARF6 mutant, ARF6(Q67L), after incubation with U18666A for 6–8 h to induce cholesterol accumulation. Cells were stained with filipin to visualize cholesterol. Sustained ARF6 activation induced a marked decrease in filipin staining compared to non-transfected cells in the same experiment (Figure 1B, top row, and Figure S1B). To quantitate the reduction in cholesterol accumulation induced by sustained ARF6 activation, we measured the filipin intensity in transfected and untransfected cells and found that approximately 60% of the cells expressing exogenous ARF6-GTP exhibited reduced filipin intensity compared to non-transfected cells (Figure 1C). In contrast, when cells were transfected with ARF6(T27N), the dominant negative ARF6 mutant, no significant change in filipin intensity was observed compared to untransfected cells (Figure 1B, bottom row, and 1C).


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)

Constitutively active ARF6 increases cholesterol removal in NPC-like cells.A, HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B, HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C, Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).
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pone-0005193-g001: Constitutively active ARF6 increases cholesterol removal in NPC-like cells.A, HeLa cells (with or without 1 µg/ml U18666A treatment for 24 h) were fixed and stained with filipin. Note cholesterol accumulation in cells treated with U18666A. Bars, 20 µm. B, HeLa cells were treated with U18666A to induce cholesterol accumulation and then transfected with pIRES-GFP encoding ARF6(Q67L), the ARF6-GTP mutant, or ARF6(T27N), the ARF6-GDP mutant, and fixed approximately 24 h post-transfection. Left panels show GFP expression and right panels show filipin staining. Transfected cells are marked with asterisks in filipin images. Bars, 20 µm. C, Quantitation of the percentage of transfected cells with reduced filipin intensity (see Methods). For each condition, the average of three independent experiments is shown with standard error bars. The difference between control cells and ARF6(Q67L)-expressing cells is statistically significant (p = 0.021), using a two-tailed t-test. D, Relative cholesterol efflux from HeLa cells treated with U18666A and then transfected with pIRES-GFP (EV) or pIRES-GFP encoding ARF6(Q67L) or ARF6(T27N). The average of three independent experiments is shown with standard error bars. Statistically significant comparisons: ARF6(Q67L) vs. EV, p = 0.024 and ARF6(Q67L) vs. ARF6(T27N), p = 0.037. The actual percentage of cellular cholesterol effluxed in each case: 2.41% for EV control, 2.56% for ARF6(T27N), and 3.18% for ARF6(Q67L).
Mentions: Previous results suggest that cholesterol cycles, in part, through vesicles containing the GTP-binding protein, ARF6, a potent regulator of early endosomal membrane internalization and recycling [10], [11]. Consistent with these findings, we find significant overlap of cholesterol with ARF6 in HeLa cells (Figure S1A). To mimic abnormal cholesterol accumulation observed in a variety of lipid storage diseases, we treated HeLa cells with the hydrophobic polyamine U18666A. Treatment with U18666A induces acute cholesterol accumulation that is indistinguishable from that seen in NPC (Figure 1A) [12]. U18666A blocks cholesterol exit from LE/LY likely by binding negatively charged phospholipids in late endosomal membranes, such as LBPA [13], [14]. Since ARF6 activation is coupled to the efflux of recycling endosomal membrane [10], we examined the impact of constitutive ARF6 activation on the lipid accumulation phenotype induced by U18666A. To this end, HeLa cells were transfected with a GTPase defective, constitutively activated ARF6 mutant, ARF6(Q67L), after incubation with U18666A for 6–8 h to induce cholesterol accumulation. Cells were stained with filipin to visualize cholesterol. Sustained ARF6 activation induced a marked decrease in filipin staining compared to non-transfected cells in the same experiment (Figure 1B, top row, and Figure S1B). To quantitate the reduction in cholesterol accumulation induced by sustained ARF6 activation, we measured the filipin intensity in transfected and untransfected cells and found that approximately 60% of the cells expressing exogenous ARF6-GTP exhibited reduced filipin intensity compared to non-transfected cells (Figure 1C). In contrast, when cells were transfected with ARF6(T27N), the dominant negative ARF6 mutant, no significant change in filipin intensity was observed compared to untransfected cells (Figure 1B, bottom row, and 1C).

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