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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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Increased expression of PLP does not disturb endosomal compartmentalization. BHK cells were infected with SFV-PLP–myc. 20 h after infection, cells were fixed and double labeled with a monoclonal antibody against LBPA (green), to label late endosomes, and a polyclonal antibody against EEA1 (a, red) or M6PR (b, red). Neither marker colocalizes with late endosomes. Bars, 10 μm.
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fig6: Increased expression of PLP does not disturb endosomal compartmentalization. BHK cells were infected with SFV-PLP–myc. 20 h after infection, cells were fixed and double labeled with a monoclonal antibody against LBPA (green), to label late endosomes, and a polyclonal antibody against EEA1 (a, red) or M6PR (b, red). Neither marker colocalizes with late endosomes. Bars, 10 μm.

Mentions: This could possibly lead to a perturbation of endosomal compartments. To assess whether early and late endosomal compartments mix, cells were infected with SFV-PLP–myc, and 20 h after infection, double immunofluorescence with EEA1 and LBPA was performed. Both markers remained separated, indicating that early and late endosomes did not mix (Fig. 6 a). Next, we analyzed whether the mannose-6-phosphate receptor (M6PR), which mostly resides in the TGN in BHK cells, is displaced into late endosomes, as it is in Niemann-Pick type C (NPC) cells or upon treatment with U18666A (Kobayashi et al., 1999). Cells were infected and the distribution of the M6PR was compared with LBPA. Likewise, immunofluorescence microscopy revealed that M6PR and LBPA remained largely separated (Fig. 6 b). Our results suggest that the separation of late endosomes/lysosomes from early endosomes and the TGN is not altered by the expression of PLP in 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)

Increased expression of PLP does not disturb endosomal compartmentalization. BHK cells were infected with SFV-PLP–myc. 20 h after infection, cells were fixed and double labeled with a monoclonal antibody against LBPA (green), to label late endosomes, and a polyclonal antibody against EEA1 (a, red) or M6PR (b, red). Neither marker colocalizes with late endosomes. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199249&req=5

fig6: Increased expression of PLP does not disturb endosomal compartmentalization. BHK cells were infected with SFV-PLP–myc. 20 h after infection, cells were fixed and double labeled with a monoclonal antibody against LBPA (green), to label late endosomes, and a polyclonal antibody against EEA1 (a, red) or M6PR (b, red). Neither marker colocalizes with late endosomes. Bars, 10 μm.
Mentions: This could possibly lead to a perturbation of endosomal compartments. To assess whether early and late endosomal compartments mix, cells were infected with SFV-PLP–myc, and 20 h after infection, double immunofluorescence with EEA1 and LBPA was performed. Both markers remained separated, indicating that early and late endosomes did not mix (Fig. 6 a). Next, we analyzed whether the mannose-6-phosphate receptor (M6PR), which mostly resides in the TGN in BHK cells, is displaced into late endosomes, as it is in Niemann-Pick type C (NPC) cells or upon treatment with U18666A (Kobayashi et al., 1999). Cells were infected and the distribution of the M6PR was compared with LBPA. Likewise, immunofluorescence microscopy revealed that M6PR and LBPA remained largely separated (Fig. 6 b). Our results suggest that the separation of late endosomes/lysosomes from early endosomes and the TGN is not altered by the expression of PLP in 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