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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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Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.
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fig5: Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.

Mentions: Having shown that PLP induces redistribution of cellular cholesterol to late endosomes/lysosomes, we tested whether this affects the sorting of other (raft) lipids. To monitor the trafficking of sphingolipids, we added BODIPY-labeled lipids to the cells. It has been shown previously that BODIPY–lactosylceramide or –galactosylceramide are taken up by cells and transferred to the Golgi apparatus (Puri et al., 1999). In several lysosomal storage diseases, however, these lipids are rerouted to the late endosomes/lysosomes (Puri et al., 1999). We therefore analyzed how these lipids are transported in cells that express PLP. BHK cells were infected with SFV-PLP–myc, and 16–20 h after infection, BODIPY-labeled lipids were added to the cells as BSA complexes for 1 h at 37°C, and internalization was allowed to proceed for 90 min. After “back exchange” (see Materials and methods) of the lipids at 12°C, the distribution of the labeled lipid was monitored. As previously shown, both BODIPY–galactosylceramide and –lactosylceramide distributed to the Golgi region in uninfected cells (Fig. 4, a and c). In cells infected with SFV-PLP–myc, however, both lipids were found in a perinuclear location in enlarged vesicles within the cell (Fig. 4, b and d). These results demonstrate that expression of PLP in BHK cells alters the trafficking of both cholesterol and fluorescent sphingolipids. One consequence of this might be an altered trafficking of lipid rafts. As a marker for lipid rafts, we used yellow fluorescence protein (YFP) fused to a glucosylphosphatidylinositol (GPI) anchor (GPI–YFP; Keller et al., 2001). In BHK cells, GPI–YFP is mainly found at the plasma membrane (Fig. 5 a). When GPI–YFP and PLP were coexpressed in BHK cells, the localization of GPI–YFP was completely altered and both proteins showed a complete colocalization (Fig. 5 c). A similar redistribution of GPI–YFP was observed when GPI–YFP-expressing cells were treated with the drug U18666A, a hydrophobic amine, which induces late endosomal/lysosomal cholesterol accumulation by inhibition of cholesterol mobilization (Liscum, 2000; Fig. 5 b). Taken together, we conclude that the trapping of cholesterol by overexpressed PLP in the endosomal–lysosomal system induces the missorting of raft membrane components.


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)

Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199249&req=5

fig5: Expression of PLP in BHK cells affects the sorting of GPI–YFP. (a) BHK cells were transfected with GPI–YFP and, after 24 h, were fixed and analyzed by immunofluorescence. (b) Cells were transfected with GPI–YFP, incubated for 24 h in the presence of 10 μg/ml U18666A, fixed, and analyzed by immunofluorescence. (c) BHK cells were cotransfected with PLP and GPI–YFP and analyzed after 24 h by immunofluorescence for PLP (red) and GPI–YFP (green). In the merged image, yellow indicates colocalization of PLP and GPI–YFP. Bars, 10 μm.
Mentions: Having shown that PLP induces redistribution of cellular cholesterol to late endosomes/lysosomes, we tested whether this affects the sorting of other (raft) lipids. To monitor the trafficking of sphingolipids, we added BODIPY-labeled lipids to the cells. It has been shown previously that BODIPY–lactosylceramide or –galactosylceramide are taken up by cells and transferred to the Golgi apparatus (Puri et al., 1999). In several lysosomal storage diseases, however, these lipids are rerouted to the late endosomes/lysosomes (Puri et al., 1999). We therefore analyzed how these lipids are transported in cells that express PLP. BHK cells were infected with SFV-PLP–myc, and 16–20 h after infection, BODIPY-labeled lipids were added to the cells as BSA complexes for 1 h at 37°C, and internalization was allowed to proceed for 90 min. After “back exchange” (see Materials and methods) of the lipids at 12°C, the distribution of the labeled lipid was monitored. As previously shown, both BODIPY–galactosylceramide and –lactosylceramide distributed to the Golgi region in uninfected cells (Fig. 4, a and c). In cells infected with SFV-PLP–myc, however, both lipids were found in a perinuclear location in enlarged vesicles within the cell (Fig. 4, b and d). These results demonstrate that expression of PLP in BHK cells alters the trafficking of both cholesterol and fluorescent sphingolipids. One consequence of this might be an altered trafficking of lipid rafts. As a marker for lipid rafts, we used yellow fluorescence protein (YFP) fused to a glucosylphosphatidylinositol (GPI) anchor (GPI–YFP; Keller et al., 2001). In BHK cells, GPI–YFP is mainly found at the plasma membrane (Fig. 5 a). When GPI–YFP and PLP were coexpressed in BHK cells, the localization of GPI–YFP was completely altered and both proteins showed a complete colocalization (Fig. 5 c). A similar redistribution of GPI–YFP was observed when GPI–YFP-expressing cells were treated with the drug U18666A, a hydrophobic amine, which induces late endosomal/lysosomal cholesterol accumulation by inhibition of cholesterol mobilization (Liscum, 2000; Fig. 5 b). Taken together, we conclude that the trapping of cholesterol by overexpressed PLP in the endosomal–lysosomal system induces the missorting of raft membrane components.

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