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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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Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts.A, NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B, NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A. Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A) were scored (see Methods). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C, GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D, Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E, Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.
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pone-0005193-g003: Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts.A, NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B, NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A. Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A) were scored (see Methods). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C, GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D, Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E, Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.

Mentions: Next we determined how sustained ARF6 activation impacts cholesterol accumulation observed in fibroblasts derived from NPC patients. Normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with the ARF6-GTP mutant, ARF6(Q67L). In each case, sustained ARF6 activation led to a reduction in filipin intensity (Figure 3A, top row, and Figure S2). Notably, the extent of response to ARF6-GTP expression varied among NPC fibroblasts derived from different patients (Figure 3B). In GM03123 cells, we observed reduced filipin intensity in 31% of transfected cells compared to neighboring, non-transfected cells. However, less than 18% of transfected GM17923 cells exhibited reduced filipin intensity and even fewer GM18436 cells responded to sustained ARF6 activation with a reduction in filipin intensity (9%; Figure 3B). In NPC cells, cholesterol-rich LE/LY compartments stain positive for lysosome markers, such as Lamp1. We observed that approximately 25% of the NPC cells expressing constitutively active ARF6 display a pool of filipin staining with significantly reduced overlap with Lamp1 (see arrows in Figure 3C and Figure S3), consistent with the hypothesis that sustained activation of ARF6 can lead to exit of cholesterol from the Lamp1-positive LE/LY compartments. We found no change in filipin intensity or overlap with Lamp1 in cells transfected with dominant negative ARF6, ARF6(T27N) (Figure 3C, bottom row). To investigate whether the varied response to ARF6 activation in NPC cells was coupled to endogenous ARF6-GTP levels, we examined the levels of endogenous ARF6-GTP using a GST-MT2 pull-down assay [16]. In each case, we compared the ratio of ARF6-GTP to total ARF6. Of the NPC fibroblasts we examined, GM03123 exhibited greater than 30% reduction in the level of ARF6-GTP compared to control cells, GM17923 had the same level as control cells, and GM18436 had a higher level of ARF6-GTP (Figure 3C,D). Thus, sustained ARF6 activation induced the most significant reduction in cholesterol accumulation in the NPC fibroblasts with the lowest endogenous levels of ARF6-GTP (GM03123).


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)

Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts.A, NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B, NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A. Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A) were scored (see Methods). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C, GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D, Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E, Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.
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pone-0005193-g003: Reduction in cholesterol accumulation induced by ARF6-GTP expression is coupled to endogenous ARF6-GTP levels in NPC mutant fibroblasts.A, NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with pIRES-GFP bearing ARF6(Q67L). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored blue in merged image) and counterstained with rhodamine phalloidin (red in merged image). Transfected cells are marked with asterisks in filipin images. Note decreased filipin staining in GFP-positive cells. B, NPC mutant fibroblasts (GM03123, GM17923 and GM18436) were treated as in A. Transfected cells with filipin intensity that was lower than neighboring non-transfected cells (as depicted in A) were scored (see Methods). 80–100 transfected cells of each cell line were counted in each of three independent experiments. The average percentage of transfected cells displaying reduced filipin intensity is graphed. Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM17923, p = 0.0117 and GM03123 vs. GM18436, p = 0.0114. C, GM03123 cells were transfected with pIRES-GFP bearing ARF6(Q67L) (top row) or ARF6(T27N) (bottom row). Cells were fixed approximately 24 h post-transfection and stained with filipin (pseudo-colored green in merged image) and immunolabeled for Lamp1 (red in merged image). Bars, 20 µm. Arrow points to region where filipin staining no longer overlaps with Lamp1. D, Lysates were prepared from normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, GM18436) and subjected to the GST-MT-2 pull-down assay and probed for ARF6. Top row is ARF6 precipitated with GST-MT-2 beads and bottom row is ARF6 from the total cell lysate. E, Immunoblots from three independent experiments were subjected to densitometric analysis and the ARF6-GTP/total ARF6 ratios were calculated and normalized to control levels (GM05659). Standard error bars are shown. Statistically significant comparisons: GM03123 vs. GM05659, p = 0.011 and GM18436 vs. GM05659, p = 0.022.
Mentions: Next we determined how sustained ARF6 activation impacts cholesterol accumulation observed in fibroblasts derived from NPC patients. Normal (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) were transfected with the ARF6-GTP mutant, ARF6(Q67L). In each case, sustained ARF6 activation led to a reduction in filipin intensity (Figure 3A, top row, and Figure S2). Notably, the extent of response to ARF6-GTP expression varied among NPC fibroblasts derived from different patients (Figure 3B). In GM03123 cells, we observed reduced filipin intensity in 31% of transfected cells compared to neighboring, non-transfected cells. However, less than 18% of transfected GM17923 cells exhibited reduced filipin intensity and even fewer GM18436 cells responded to sustained ARF6 activation with a reduction in filipin intensity (9%; Figure 3B). In NPC cells, cholesterol-rich LE/LY compartments stain positive for lysosome markers, such as Lamp1. We observed that approximately 25% of the NPC cells expressing constitutively active ARF6 display a pool of filipin staining with significantly reduced overlap with Lamp1 (see arrows in Figure 3C and Figure S3), consistent with the hypothesis that sustained activation of ARF6 can lead to exit of cholesterol from the Lamp1-positive LE/LY compartments. We found no change in filipin intensity or overlap with Lamp1 in cells transfected with dominant negative ARF6, ARF6(T27N) (Figure 3C, bottom row). To investigate whether the varied response to ARF6 activation in NPC cells was coupled to endogenous ARF6-GTP levels, we examined the levels of endogenous ARF6-GTP using a GST-MT2 pull-down assay [16]. In each case, we compared the ratio of ARF6-GTP to total ARF6. Of the NPC fibroblasts we examined, GM03123 exhibited greater than 30% reduction in the level of ARF6-GTP compared to control cells, GM17923 had the same level as control cells, and GM18436 had a higher level of ARF6-GTP (Figure 3C,D). Thus, sustained ARF6 activation induced the most significant reduction in cholesterol accumulation in the NPC fibroblasts with the lowest endogenous levels of ARF6-GTP (GM03123).

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