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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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ARF6-GTP expression restores sphingolipid targeting to the Golgi.A, CtxB-AlexaFluor555 internalization in control (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) was performed as described in Methods. To localize the Golgi, cells were fixed and immuno-labeled for GM130 with a mouse monoclonal antibody and a Cy5-conjugated anti-mouse secondary antibody (pseudo-colored green). B, Control and NPC mutant fibroblasts were transfected with ARF6(Q67L)-pIRES-GFP (monochrome, third row from left), subjected to CtxB-AlexaFluor555 internalization 20 h post-transfection, and immuno-labeled for GM130 (pseudo-colored green) as described in A. Transfected cells are marked with asterisks in merged images. See Table 1 for quantification of Golgi targeting of Ctx in NPC fibroblasts transfected with ARF6(Q67L)-pIRES-GFP.
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pone-0005193-g004: ARF6-GTP expression restores sphingolipid targeting to the Golgi.A, CtxB-AlexaFluor555 internalization in control (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) was performed as described in Methods. To localize the Golgi, cells were fixed and immuno-labeled for GM130 with a mouse monoclonal antibody and a Cy5-conjugated anti-mouse secondary antibody (pseudo-colored green). B, Control and NPC mutant fibroblasts were transfected with ARF6(Q67L)-pIRES-GFP (monochrome, third row from left), subjected to CtxB-AlexaFluor555 internalization 20 h post-transfection, and immuno-labeled for GM130 (pseudo-colored green) as described in A. Transfected cells are marked with asterisks in merged images. See Table 1 for quantification of Golgi targeting of Ctx in NPC fibroblasts transfected with ARF6(Q67L)-pIRES-GFP.

Mentions: We also investigated the impact of ARF6 activation on the distribution of other lipids, such as glycosphingolipids, that traffic inappropriately and accumulate with cholesterol in LE/LY compartments in NPC [14], [17]. We analyzed the distribution of cholera toxin (Ctx) that binds to GM1 ganglioside at the PM and serves as a marker for its localization. In normal cells, after internalization Ctx targets to the Golgi via retrograde traffic, whereas in NPC cells, Ctx accumulates instead in endosomal vesicles throughout the cytoplasm. Cholesterol depletion using methyl-β-cyclodextrin effectively restores normal Golgi targeting of Ctx [17]. Thus we determined if the reduction in cholesterol accumulation induced by sustained ARF6 activation is sufficient to restore Golgi targeting of internalized Ctx. As seen in Figure 4A, the efficiency of Ctx targeting to the Golgi in NPC fibroblasts was noticeably reduced as evidenced by increased labeling of Ctx in vesicles throughout the cytoplasm and in marked contrast with control cells that showed efficient targeting of Ctx to the Golgi. Next, we examined the effect of exogenous ARF6-GTP on Ctx trafficking in NPC fibroblasts. We observed increased targeting of Ctx to the Golgi, as evidenced by reduced Ctx label outside of the Golgi (Figure 4B). We quantitated the percentage of Ctx label at the Golgi compared to the total Ctx label in transfected and non-transfected NPC fibroblasts. We found expression of constitutively active ARF6 increased targeting of Ctx to the Golgi by 80–90% (see Table 1). NPC cells expressing dominant negative ARF6, ARF6(T27N), exhibited no change in Ctx targeting (Figure S4). These findings suggest that the reduction in cholesterol storage induced by sustained ARF6 activation allows for restoration of normal lipid trafficking. Alternatively, ARF6 itself may be involved in the trafficking of Ctx to the Golgi and expression of ARF6-GTP may stimulate its transport to the Golgi.


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)

ARF6-GTP expression restores sphingolipid targeting to the Golgi.A, CtxB-AlexaFluor555 internalization in control (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) was performed as described in Methods. To localize the Golgi, cells were fixed and immuno-labeled for GM130 with a mouse monoclonal antibody and a Cy5-conjugated anti-mouse secondary antibody (pseudo-colored green). B, Control and NPC mutant fibroblasts were transfected with ARF6(Q67L)-pIRES-GFP (monochrome, third row from left), subjected to CtxB-AlexaFluor555 internalization 20 h post-transfection, and immuno-labeled for GM130 (pseudo-colored green) as described in A. Transfected cells are marked with asterisks in merged images. See Table 1 for quantification of Golgi targeting of Ctx in NPC fibroblasts transfected with ARF6(Q67L)-pIRES-GFP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664925&req=5

pone-0005193-g004: ARF6-GTP expression restores sphingolipid targeting to the Golgi.A, CtxB-AlexaFluor555 internalization in control (GM05659) and NPC mutant fibroblasts (GM03123, GM17923, and GM18436) was performed as described in Methods. To localize the Golgi, cells were fixed and immuno-labeled for GM130 with a mouse monoclonal antibody and a Cy5-conjugated anti-mouse secondary antibody (pseudo-colored green). B, Control and NPC mutant fibroblasts were transfected with ARF6(Q67L)-pIRES-GFP (monochrome, third row from left), subjected to CtxB-AlexaFluor555 internalization 20 h post-transfection, and immuno-labeled for GM130 (pseudo-colored green) as described in A. Transfected cells are marked with asterisks in merged images. See Table 1 for quantification of Golgi targeting of Ctx in NPC fibroblasts transfected with ARF6(Q67L)-pIRES-GFP.
Mentions: We also investigated the impact of ARF6 activation on the distribution of other lipids, such as glycosphingolipids, that traffic inappropriately and accumulate with cholesterol in LE/LY compartments in NPC [14], [17]. We analyzed the distribution of cholera toxin (Ctx) that binds to GM1 ganglioside at the PM and serves as a marker for its localization. In normal cells, after internalization Ctx targets to the Golgi via retrograde traffic, whereas in NPC cells, Ctx accumulates instead in endosomal vesicles throughout the cytoplasm. Cholesterol depletion using methyl-β-cyclodextrin effectively restores normal Golgi targeting of Ctx [17]. Thus we determined if the reduction in cholesterol accumulation induced by sustained ARF6 activation is sufficient to restore Golgi targeting of internalized Ctx. As seen in Figure 4A, the efficiency of Ctx targeting to the Golgi in NPC fibroblasts was noticeably reduced as evidenced by increased labeling of Ctx in vesicles throughout the cytoplasm and in marked contrast with control cells that showed efficient targeting of Ctx to the Golgi. Next, we examined the effect of exogenous ARF6-GTP on Ctx trafficking in NPC fibroblasts. We observed increased targeting of Ctx to the Golgi, as evidenced by reduced Ctx label outside of the Golgi (Figure 4B). We quantitated the percentage of Ctx label at the Golgi compared to the total Ctx label in transfected and non-transfected NPC fibroblasts. We found expression of constitutively active ARF6 increased targeting of Ctx to the Golgi by 80–90% (see Table 1). NPC cells expressing dominant negative ARF6, ARF6(T27N), exhibited no change in Ctx targeting (Figure S4). These findings suggest that the reduction in cholesterol storage induced by sustained ARF6 activation allows for restoration of normal lipid trafficking. Alternatively, ARF6 itself may be involved in the trafficking of Ctx to the Golgi and expression of ARF6-GTP may stimulate its transport to the Golgi.

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