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Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease.

Simons M, Kramer EM, Macchi P, Rathke-Hartlieb S, Trotter J, Nave KA, Schulz JB - J. Cell Biol. (2002)

Bottom Line: This was also the case for the lipid raft marker glucosylphosphatidylinositol-yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex.Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components.We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Tübingen, 72076 Tübingen, Germany. mika.simons@uni-tuebingen.de

ABSTRACT
Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY-lactosylceramide and -galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol-yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

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Targeting of PLP to late endosomes/lysosomes leads to cholesterol accumulation. (a) BHK cells were transfected with PLP–myc and, after 24 h, incubated with rhodamine–dextran (red) for 2 h at 37°C. Immunofluorescence was performed to analyze for PLP (green). Cholesterol was labeled with filipin (blue). (b–d) BHK cells were infected with SFV-PLP–myc (b and c) or SFV-PLP (d), fixed 10 (b) or 20 h (c and d) after infection, and analyzed by immunofluorescence for PLP (red) and cholesterol (blue). Insets represent a magnification of the region indicated. In the merged image, pink indicates colocalization of PLP and cholesterol. Bars, 10 μm.
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fig2: Targeting of PLP to late endosomes/lysosomes leads to cholesterol accumulation. (a) BHK cells were transfected with PLP–myc and, after 24 h, incubated with rhodamine–dextran (red) for 2 h at 37°C. Immunofluorescence was performed to analyze for PLP (green). Cholesterol was labeled with filipin (blue). (b–d) BHK cells were infected with SFV-PLP–myc (b and c) or SFV-PLP (d), fixed 10 (b) or 20 h (c and d) after infection, and analyzed by immunofluorescence for PLP (red) and cholesterol (blue). Insets represent a magnification of the region indicated. In the merged image, pink indicates colocalization of PLP and cholesterol. Bars, 10 μm.

Mentions: Cholesterol is not uniformly distributed within the endosomal–lysosomal system. It is enriched within early endosomes and recycling endosomes and relatively depleted from late endosomes and lysosomes (Brotherus and Renkonen, 1977; Hornick et al., 1997; Mukherjee et al., 1998; Gagescu et al., 2000; Kobayashi et al., 2001; Lusa et al., 2001; Nichols et al., 2001). We have previously shown that PLP specifically associates with cholesterol (Simons et al., 2000). We therefore examined whether the accumulation of PLP within late endosomes/lysosomes causes a redistribution of cellular cholesterol. To test this hypothesis, BHK cells were transfected with PLP–myc and the subcellular distribution of cholesterol was monitored with filipin, an antibiotic that specifically binds to free cholesterol. We found that in BHK cells, PLP–myc showed a remarkable colocalization with cholesterol (Fig. 2 a). Co-localization of PLP–myc and cholesterol was also observed in cells that had been incubated during the time of transfection with medium containing lipoprotein-deficient serum, indicating that the cholesterol was from a cellular source (unpublished data). After a 2-h uptake of dextran, the cholesterol-loaded vesicles were identified as endosomes/lysosomes (Fig. 2 a). To analyze whether the redistribution of cellular cholesterol correlates with PLP expression levels, we took advantage of the Semliki Forest virus (SFV) vector. The SFV expression system is ideally suited for this purpose, as infected cells synthesize a synchronized wave of protein that can be followed at various times after infection (Liljeström and Garoff, 1991). Infection of BHK cells with SFV-PLP–myc or SFV-PLP resulted in the infection of >80% of cells. When cells infected with SFV-PLP–myc were fixed 5 h after infection, PLP was observed within the Golgi region and at the plasma membrane in most cells (unpublished data). 10 h after infection, PLP was distributed in intracellular vesicles throughout the cell (Fig. 2 b). 20 h after infection, the vesicles appeared enlarged and many of them exhibited a perinuclear localization (Fig. 2 c). These vesicles were labeled with rhodamine–dextran that was allowed to endocytose for 2 h, but did not colocalize with dextran that had been internalized for only 10 min, thus identifying the labeled structures as late endosomal/lysosomal organelles (unpublished data). Filipin staining demonstrated a striking colocalization of PLP with cholesterol (Fig. 2 c). Quantitative analysis indicated that 78% of the cells that were infected with SFV-PLP–myc for 20 h exhibited intracellular accumulation of cholesterol and PLP (>100 cells examined). Similar results were observed in cells that had been infected with SFV-PLP lacking a myc tag (Fig. 2 d). In contrast, cells that had been infected by a control virus expressing green fluorescence protein (SFV-GFP) or a virus expressing the myelin oligodendrocyte glycoprotein (SFV-MOG) did not accumulate cholesterol (unpublished data). This result shows that overexpression of PLP induces an accumulation of cholesterol in late endosomes/lysosomes of BHK cells.


Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease.

Simons M, Kramer EM, Macchi P, Rathke-Hartlieb S, Trotter J, Nave KA, Schulz JB - J. Cell Biol. (2002)

Targeting of PLP to late endosomes/lysosomes leads to cholesterol accumulation. (a) BHK cells were transfected with PLP–myc and, after 24 h, incubated with rhodamine–dextran (red) for 2 h at 37°C. Immunofluorescence was performed to analyze for PLP (green). Cholesterol was labeled with filipin (blue). (b–d) BHK cells were infected with SFV-PLP–myc (b and c) or SFV-PLP (d), fixed 10 (b) or 20 h (c and d) after infection, and analyzed by immunofluorescence for PLP (red) and cholesterol (blue). Insets represent a magnification of the region indicated. In the merged image, pink indicates colocalization of PLP and cholesterol. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199249&req=5

fig2: Targeting of PLP to late endosomes/lysosomes leads to cholesterol accumulation. (a) BHK cells were transfected with PLP–myc and, after 24 h, incubated with rhodamine–dextran (red) for 2 h at 37°C. Immunofluorescence was performed to analyze for PLP (green). Cholesterol was labeled with filipin (blue). (b–d) BHK cells were infected with SFV-PLP–myc (b and c) or SFV-PLP (d), fixed 10 (b) or 20 h (c and d) after infection, and analyzed by immunofluorescence for PLP (red) and cholesterol (blue). Insets represent a magnification of the region indicated. In the merged image, pink indicates colocalization of PLP and cholesterol. Bars, 10 μm.
Mentions: Cholesterol is not uniformly distributed within the endosomal–lysosomal system. It is enriched within early endosomes and recycling endosomes and relatively depleted from late endosomes and lysosomes (Brotherus and Renkonen, 1977; Hornick et al., 1997; Mukherjee et al., 1998; Gagescu et al., 2000; Kobayashi et al., 2001; Lusa et al., 2001; Nichols et al., 2001). We have previously shown that PLP specifically associates with cholesterol (Simons et al., 2000). We therefore examined whether the accumulation of PLP within late endosomes/lysosomes causes a redistribution of cellular cholesterol. To test this hypothesis, BHK cells were transfected with PLP–myc and the subcellular distribution of cholesterol was monitored with filipin, an antibiotic that specifically binds to free cholesterol. We found that in BHK cells, PLP–myc showed a remarkable colocalization with cholesterol (Fig. 2 a). Co-localization of PLP–myc and cholesterol was also observed in cells that had been incubated during the time of transfection with medium containing lipoprotein-deficient serum, indicating that the cholesterol was from a cellular source (unpublished data). After a 2-h uptake of dextran, the cholesterol-loaded vesicles were identified as endosomes/lysosomes (Fig. 2 a). To analyze whether the redistribution of cellular cholesterol correlates with PLP expression levels, we took advantage of the Semliki Forest virus (SFV) vector. The SFV expression system is ideally suited for this purpose, as infected cells synthesize a synchronized wave of protein that can be followed at various times after infection (Liljeström and Garoff, 1991). Infection of BHK cells with SFV-PLP–myc or SFV-PLP resulted in the infection of >80% of cells. When cells infected with SFV-PLP–myc were fixed 5 h after infection, PLP was observed within the Golgi region and at the plasma membrane in most cells (unpublished data). 10 h after infection, PLP was distributed in intracellular vesicles throughout the cell (Fig. 2 b). 20 h after infection, the vesicles appeared enlarged and many of them exhibited a perinuclear localization (Fig. 2 c). These vesicles were labeled with rhodamine–dextran that was allowed to endocytose for 2 h, but did not colocalize with dextran that had been internalized for only 10 min, thus identifying the labeled structures as late endosomal/lysosomal organelles (unpublished data). Filipin staining demonstrated a striking colocalization of PLP with cholesterol (Fig. 2 c). Quantitative analysis indicated that 78% of the cells that were infected with SFV-PLP–myc for 20 h exhibited intracellular accumulation of cholesterol and PLP (>100 cells examined). Similar results were observed in cells that had been infected with SFV-PLP lacking a myc tag (Fig. 2 d). In contrast, cells that had been infected by a control virus expressing green fluorescence protein (SFV-GFP) or a virus expressing the myelin oligodendrocyte glycoprotein (SFV-MOG) did not accumulate cholesterol (unpublished data). This result shows that overexpression of PLP induces an accumulation of cholesterol in late endosomes/lysosomes of BHK cells.

Bottom Line: This was also the case for the lipid raft marker glucosylphosphatidylinositol-yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex.Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components.We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Tübingen, 72076 Tübingen, Germany. mika.simons@uni-tuebingen.de

ABSTRACT
Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY-lactosylceramide and -galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol-yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

Show MeSH
Related in: MedlinePlus