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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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PLP localizes to late endosomes/lysosomes in BHK cells. (a) BHK cells were transfected with PLP or myc-tagged PLP for 24 h and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of transfection. Cells were fixed and labeled with monoclonal anti-myc or polyclonal anti-PLP antibodies (left) to visualize PLP. Rhodamine–dextran is shown in the middle. In the merged image (right), yellow indicates colocalization of PLP and rhodamine–dextran. (b) BHK cells were transfected with myc-tagged PLP, fixed, and double labeled with monoclonal anti-myc antibody to visualize PLP (left) and polyclonal anti-EEA1 to resolve early endosomes (middle). (c) PLP-transfected BHK cells were incubated with rhodamine–transferrin (middle) for 1 h at 37°C as a marker for recycling endosomes and analyzed by immunofluorescence for PLP (left). (d) PLP-transfected BHK cells were fixed and double labeled with polyclonal anti-PLP antibody to visualize PLP (left) and with a monoclonal antibody against LBPA to resolve late endosomes (middle). In the merged image (right), yellow indicates colocalization of PLP and LBPA. Bars, 10 μm.
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fig1: PLP localizes to late endosomes/lysosomes in BHK cells. (a) BHK cells were transfected with PLP or myc-tagged PLP for 24 h and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of transfection. Cells were fixed and labeled with monoclonal anti-myc or polyclonal anti-PLP antibodies (left) to visualize PLP. Rhodamine–dextran is shown in the middle. In the merged image (right), yellow indicates colocalization of PLP and rhodamine–dextran. (b) BHK cells were transfected with myc-tagged PLP, fixed, and double labeled with monoclonal anti-myc antibody to visualize PLP (left) and polyclonal anti-EEA1 to resolve early endosomes (middle). (c) PLP-transfected BHK cells were incubated with rhodamine–transferrin (middle) for 1 h at 37°C as a marker for recycling endosomes and analyzed by immunofluorescence for PLP (left). (d) PLP-transfected BHK cells were fixed and double labeled with polyclonal anti-PLP antibody to visualize PLP (left) and with a monoclonal antibody against LBPA to resolve late endosomes (middle). In the merged image (right), yellow indicates colocalization of PLP and LBPA. Bars, 10 μm.

Mentions: To analyze the general trafficking behavior of newly synthesized PLP, we used BHK cells, which do not contain endogenous PLP but are often used in trafficking studies. We transiently transfected the cDNA encoding for PLP or PLP containing a myc tag at its COOH terminus into BHK cells and analyzed its distribution by immunofluorescence microscopy at various times after transfection. We found that the protein was exported from the ER (unpublished data) and, at time points early after transfection (up to ∼14 h), was mainly found in the Golgi region and at the plasma membrane (unpublished data). At later time points after transfection, PLP accumulated in the endosomal–lysosomal system, as shown by colocalization with the fluid-phase marker rhodamine–dextran (Fig. 1 a). Cells were labeled with different markers of the endosomal–lysosomal system to determine the exact subcellular localization of PLP. We found no overlap between PLP–myc and early endosomal antigen 1 (EEA1), a marker of early endosomes (Fig. 1 b), or between PLP and transferrin–rhodamine, a marker of recycling endosomes (Fig. 1 c). In contrast, PLP colocalized with lysobisphosphatidic acid (LBPA), which localizes to late endosomes (Fig. 1 d). The presence of PLP within late endosomes/lysosomes is consistent with earlier reports (Gow et al., 1994; Sinoway et al., 1994; Kalway et al., 1997; Krämer et al., 2001).


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)

PLP localizes to late endosomes/lysosomes in BHK cells. (a) BHK cells were transfected with PLP or myc-tagged PLP for 24 h and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of transfection. Cells were fixed and labeled with monoclonal anti-myc or polyclonal anti-PLP antibodies (left) to visualize PLP. Rhodamine–dextran is shown in the middle. In the merged image (right), yellow indicates colocalization of PLP and rhodamine–dextran. (b) BHK cells were transfected with myc-tagged PLP, fixed, and double labeled with monoclonal anti-myc antibody to visualize PLP (left) and polyclonal anti-EEA1 to resolve early endosomes (middle). (c) PLP-transfected BHK cells were incubated with rhodamine–transferrin (middle) for 1 h at 37°C as a marker for recycling endosomes and analyzed by immunofluorescence for PLP (left). (d) PLP-transfected BHK cells were fixed and double labeled with polyclonal anti-PLP antibody to visualize PLP (left) and with a monoclonal antibody against LBPA to resolve late endosomes (middle). In the merged image (right), yellow indicates colocalization of PLP and LBPA. Bars, 10 μm.
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Related In: Results  -  Collection

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fig1: PLP localizes to late endosomes/lysosomes in BHK cells. (a) BHK cells were transfected with PLP or myc-tagged PLP for 24 h and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of transfection. Cells were fixed and labeled with monoclonal anti-myc or polyclonal anti-PLP antibodies (left) to visualize PLP. Rhodamine–dextran is shown in the middle. In the merged image (right), yellow indicates colocalization of PLP and rhodamine–dextran. (b) BHK cells were transfected with myc-tagged PLP, fixed, and double labeled with monoclonal anti-myc antibody to visualize PLP (left) and polyclonal anti-EEA1 to resolve early endosomes (middle). (c) PLP-transfected BHK cells were incubated with rhodamine–transferrin (middle) for 1 h at 37°C as a marker for recycling endosomes and analyzed by immunofluorescence for PLP (left). (d) PLP-transfected BHK cells were fixed and double labeled with polyclonal anti-PLP antibody to visualize PLP (left) and with a monoclonal antibody against LBPA to resolve late endosomes (middle). In the merged image (right), yellow indicates colocalization of PLP and LBPA. Bars, 10 μm.
Mentions: To analyze the general trafficking behavior of newly synthesized PLP, we used BHK cells, which do not contain endogenous PLP but are often used in trafficking studies. We transiently transfected the cDNA encoding for PLP or PLP containing a myc tag at its COOH terminus into BHK cells and analyzed its distribution by immunofluorescence microscopy at various times after transfection. We found that the protein was exported from the ER (unpublished data) and, at time points early after transfection (up to ∼14 h), was mainly found in the Golgi region and at the plasma membrane (unpublished data). At later time points after transfection, PLP accumulated in the endosomal–lysosomal system, as shown by colocalization with the fluid-phase marker rhodamine–dextran (Fig. 1 a). Cells were labeled with different markers of the endosomal–lysosomal system to determine the exact subcellular localization of PLP. We found no overlap between PLP–myc and early endosomal antigen 1 (EEA1), a marker of early endosomes (Fig. 1 b), or between PLP and transferrin–rhodamine, a marker of recycling endosomes (Fig. 1 c). In contrast, PLP colocalized with lysobisphosphatidic acid (LBPA), which localizes to late endosomes (Fig. 1 d). The presence of PLP within late endosomes/lysosomes is consistent with earlier reports (Gow et al., 1994; Sinoway et al., 1994; Kalway et al., 1997; Krämer et al., 2001).

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