Our current studies explore the phenotypic homogeneity of lysosomal storage diseases relative to their respective pathology in organelle function. The present studies were undertaken to visualize the dynamics of intracellular endocytic membranes in sphingolipid storage disease cells including, living Tays Sachs, Gaucher and Fabry fibroblasts using fluorescent Ad?NPC I ?GFP as a membrane marker and fluorescent dextran as a bulk flow marker for the late endocytic pathway. Cultured cells from several sphingolipid storage diseases have an abnormal cellular location of internalized fluorescent glycosphingolipid analogues traffickmg from the plasma membrane. In normal cells exogeneously supplied fluorescent GSL's internalized via caveolae are transported to the Golgi complex whereas in SLSD cells, internalized GSL's are transported through the late endocytic pathway where they accumulate in endosomes and lysosomes. We examined the organellar accumulations of GM2 in Tay Sachs fibroblasts, CTH in Fabry fibroblasts and glucocerebrosidase in Gaucher fibroblasts as indicators of endocytic membrane trafficking modulated by cellular cholesterol content. Tay Sachs: Tay Sachs disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme hexosaminidase A. Both Tay Sachs cell strains examined in this study have reduced ?hexosaminidase A activity resulting in the cellular accumulation of the ganglioside GM2 in cultured fibroblasts grown in FBS and with LDL. LDL uptake, which increases the cholesterol concentration in Tay Sachs cells, recruits endogencous NPC I into the late endosomal Rab 7 containing compartment separate from the cholesterol laden lysosomes. The GM2 that accumulates upon uptake of LDL locates also to the NPC I containing late endosomal compartment, Thus in mutant Tay Sachs cells, the accumulations of GM2 and NPC I visualized with immunocytochemistry, are components of the late endosomal compartment which is the identical location of these proteins in both normal and NPC I mutant fibroblasts. Ad?NPC I ?GFP expressed in Tay Sachs cells traffics in the late endosomal compartment, similar to its trafficking in normal and NPC fibroblasts, and as a membrane spanning protein becomes a fluorescent marker for membranes of late endosomal tubules. Tay Sachs cells grown on FBS with and without LDL contain long immobile late endosomal tubules visualized in living cells by Ad-PC1-GFP fluorescence or fluorescent dextran. Cellular cholesterol depletion, either by transduction of ad-NPC1-GFP or incubation in lipid depleted serum, restores mobility of the late endosomal tubules concomitant with egress of cholesterol and GM2 from the late endocytic pathway visualized as a decrease in GM2 immunofluorescence from late endosomes and cholesterol-filipin fluorescence from lysosomes. A second approach, other than GM2 immunostaining, was used to demonstrate cellular trafficking of GM2. We visualized fluorescent GM2 in Tay Sachs fibroblasts by endocytic uptake of Bodipy GM1. In normal fibroblasts GM1 travels in the late endocytic pathway to lysosomes where it is catabolized to GM2 and GM3. However GM1 is degraded only to GM2 in Tay Sachs fibroblasts, due to diminished hexosaminidase A activity and the bodipy fluorescence present in cells after uptake of bodipy-GM1 co-localizes with immunofluorescent staining for GM2. Using this approach we visualized decreased Bodipy-GM2 fluorescence and filipin-cholesterol fluorescence in the late endocytic pathway concomitant with re-establishment of late endosomal tubular mobility in living Tay Sachs fibroblasts grown in either lipid depleted serum or after expression of Ad-NPC1-GFP. To associate further decreased GM2 in late endosomes with cholesterol mobilization we incubated cells with cyclodextrin. Cyclodextrins result in rapid depletion of cholesterol from tissue culture cells. In Tay Sachs fibroblasts the rapid cyclodextrin induced decrease in cholesterol was accompanied by a decrease in GM2 from the endocytic pathway. We measured cellular cholesterol and GM2 content in Tay Sachs cells incubated in LPDS and after uptake of LDL, biochemically. Cellular unesterified cholesterol increased in Tay Sachs cells after 24 hr LDL uptake and decreased in cells grown subsequently in LPDS. In depleted cells there was no increased accumulation of cholesterol ester, indicating that free cholesterol was mobilized from the fibroblasts. In contrast the GM2 concentration remained constant in cells grown in LPDS even though GM2 was visually reduced in the late endocytic pathway as visualized with bodipy or immunofluorescence. This indicates that GM2 may relocate to other cellular sites such as the plasma membrane, by trafficking with cholesterol in mobile late endosomal tubules to the cell surface. An increase in GM2 fluorescence in the plasma membrane or other cellular membranes could not be resolved possibly due to dispersion of the GM2 in these membranes. Fabry Disease: Fabry cells are deficient in ceramidetrihexosidase activity resulting in the cellular accumulation of ceramidetrihexoside (CTH). CTH accumulates in cholesterol-rich lysosomes in Fabry fibroblasts as it does in normal fibroblasts incubated with LDL. Tay Sachs cells grown in FBS with and without LDL contain long immobile late endosomal tubules visualized in living cells by Ad-PC1-GFP fluorescence or fluorescent dextran. Cholesterol depletion, either by transduction of Ad-NPC1-GFP or incubation in lipid depleted serum, restores mobility of the late endosomal tubules. Within 48 h, expression of Ad NPC1-GFP affects restoration of the dynamic tubulo-vesicular motion of late endosomes which is accompanied by a decrease in lysosomal CTH and cholesterol content. Quantitative analysis showed that lysosomal CTH immunofluorescence decreased 40% in transduced cells in FBS and lysosomal CTH immunofluorescence present in Fabry cells after LDL uptake decreased 80% after 24 h in LPDS and an additional 70% after another 24 h in LPDS. Thus, depletion of cellular and lysosomal free cholesterol in Fabry fibroblsts correlates with re-establishment of late endosomal tubular trafficking and resultant mobilization of CTH from lysosomes. Gaucher Disease: Gaucher's disease is characterized by the accumulation of glucosylceramide (GlcCer) in cells of the reticuloendothelial system resulting from impaired glucocerebrosidase activity. However, Gaucher fibroblasts apparently do not accumulate GlcCer and only small increases of GlcCer occur in some Gaucher fibroblasts but not in others . Thus in this study on Gaucher fibroblasts we monitered immunocytochemically the intracellular location of glucocerebrosidase as an indicater of membrane trafficking in the endocytic pathway. In Gaucher cells endogeneous glucocerebrosidase is present in the Rab 7 positive, NPC1 containing late endosomal compartment which is in contrast to normal fibroblasts where the enzyme is found in a non-NPC1 containing late endocytic organelle. Gaucher cells grown in FBS with and without LDL contain long immobile late endosomal tubules visualized in living cells by Ad-NPC1-GFP fluorescence or fluorescent dextran. Cellular cholesterol depletion, either by transduction of Ad-NPC1-GFP or incubation in lipid depleted serum, restores mobility of the late endosomal tubules. Concomitant with late endosomal mobility is the egress of lysosomal cholesterol and late endosomal glucocerebrosidase. From these results it appears that, in Gaucher fibroblasts, cholesterol enrichment without GlcCer accumulation in the endocytic pathway effects stasis of late endosomal tubules and accumulation of glucocerebrosidase in late endosdomes while cholesterol depletion allows egress of glucocerebrosidase from late endosomes via re-establishment of late endosomal tubular trafficking.
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