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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: Similar results were seen in transgenic mice overexpressing PLP.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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Exogenously expressed PLP is not transported to the processes but accumulates cholesterol in primary cultures of oligodendrocytes. (a) Oligodendrocytes were infected with SFV containing myc-tagged PLP and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of infection. 8 h after infection, cells were fixed and processed for immunofluorescence. Exogenous PLP (green) is mainly found in the cell body. The panels at the right show the cell body at lower exposure. Colocalization of PLP (green) and rhodamine–dextran (red) is shown in the merged image as yellow. (b) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 8 h after infection, cells were fixed and processed for immunofluorescence for PLP (green) and calnexin (red). (c) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 16 h after infection, cells were fixed and processed for immunofluorescence for PLP (red) and cholesterol (blue). The region indicated in the upper panel is shown below at a higher magnification. Colocalization of PLP and cholesterol is shown in the merged image as pink. (d) Oligodendrocytes were infected with SFV containing MOG, and 16 h after infection, cells were fixed and processed for immunofluorescence for MOG (red) and cholesterol (blue). Bars, 10 μm.
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fig7: Exogenously expressed PLP is not transported to the processes but accumulates cholesterol in primary cultures of oligodendrocytes. (a) Oligodendrocytes were infected with SFV containing myc-tagged PLP and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of infection. 8 h after infection, cells were fixed and processed for immunofluorescence. Exogenous PLP (green) is mainly found in the cell body. The panels at the right show the cell body at lower exposure. Colocalization of PLP (green) and rhodamine–dextran (red) is shown in the merged image as yellow. (b) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 8 h after infection, cells were fixed and processed for immunofluorescence for PLP (green) and calnexin (red). (c) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 16 h after infection, cells were fixed and processed for immunofluorescence for PLP (red) and cholesterol (blue). The region indicated in the upper panel is shown below at a higher magnification. Colocalization of PLP and cholesterol is shown in the merged image as pink. (d) Oligodendrocytes were infected with SFV containing MOG, and 16 h after infection, cells were fixed and processed for immunofluorescence for MOG (red) and cholesterol (blue). Bars, 10 μm.

Mentions: Because the pathology of PLP overexpression is mediated by oligodendrocytes in the CNS, it is important to analyze the trafficking of PLP in these cells. In primary cultures of oligodendrocytes, endogenous PLP is incorporated into cholesterol- and galactosylceramide-enriched membrane domains (myelin rafts) and is transported to the plasma membrane (Simons et al., 2000). As shown previously, a fraction of PLP colocalizes with lysosome-associated membrane protein 1 (LAMP-1), a marker for late endosomes/lysosomes (Krämer et al., 2001; unpublished data). To investigate the fate of overexpressed PLP, primary cultures of oligodendrocytes were infected with SFV-PLP–myc to distinguish it from endogenous protein. We found that 8 h after infection, the myc-tagged PLP was not transported out to the distal part of the processes, but was found in vesicles in the cell body and proximal part of the processes (Fig. 7 a). When oligodendrocytes were double labeled for PLP–myc and calnexin, no colocalization was observed (Fig. 7 b), ruling out the possibility that overexpressed PLP was trapped in the ER. Instead PLP–myc colocalized with rhodamine–dextran that had been internalized for 2 h, identifying the vesicles as part of the endosomal–lysosomal system (Fig. 7 a). Labeling of SFV-PLP–myc-infected oligodendrocytes with filipin demonstrated colocalization of overexpressed PLP and cholesterol, which was drastically pronounced 16 h after infection (Fig. 7 c). Quantitative analysis indicated that 82% of the cells infected with SFV-PLP–myc for 16 h exhibited colocalization of cholesterol and PLP (>100 cells examined). To analyze whether the myc tag interfered with the normal trafficking behavior of PLP, we infected oligodendrocytes with SFV-PLP. To minimize the staining from endogenous protein, we used a 10-fold higher dilution than usual of the PLP antibody for immunofluorescence analysis. 16 h after infection, accumulation of cholesterol and PLP was observed as in the cells expressing PLP–myc (unpublished data). To analyze whether overexpression of any myelin protein leads to endosomal/lysosomal accumulation, oligodendrocytes were infected with SFV-MOG for 16 h. We found that MOG was transported to the distal processes and the cells did not accumulate intracellular cholesterol (Fig. 7 d). These results suggest that overexpression of PLP in oligodendrocytes causes accumulation of PLP and sequestration of cholesterol in the endosomal–lysosomal system as observed 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)

Exogenously expressed PLP is not transported to the processes but accumulates cholesterol in primary cultures of oligodendrocytes. (a) Oligodendrocytes were infected with SFV containing myc-tagged PLP and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of infection. 8 h after infection, cells were fixed and processed for immunofluorescence. Exogenous PLP (green) is mainly found in the cell body. The panels at the right show the cell body at lower exposure. Colocalization of PLP (green) and rhodamine–dextran (red) is shown in the merged image as yellow. (b) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 8 h after infection, cells were fixed and processed for immunofluorescence for PLP (green) and calnexin (red). (c) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 16 h after infection, cells were fixed and processed for immunofluorescence for PLP (red) and cholesterol (blue). The region indicated in the upper panel is shown below at a higher magnification. Colocalization of PLP and cholesterol is shown in the merged image as pink. (d) Oligodendrocytes were infected with SFV containing MOG, and 16 h after infection, cells were fixed and processed for immunofluorescence for MOG (red) and cholesterol (blue). Bars, 10 μm.
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Related In: Results  -  Collection

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fig7: Exogenously expressed PLP is not transported to the processes but accumulates cholesterol in primary cultures of oligodendrocytes. (a) Oligodendrocytes were infected with SFV containing myc-tagged PLP and incubated with rhodamine–dextran for 2 h at 37°C during the last 2 h of infection. 8 h after infection, cells were fixed and processed for immunofluorescence. Exogenous PLP (green) is mainly found in the cell body. The panels at the right show the cell body at lower exposure. Colocalization of PLP (green) and rhodamine–dextran (red) is shown in the merged image as yellow. (b) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 8 h after infection, cells were fixed and processed for immunofluorescence for PLP (green) and calnexin (red). (c) Oligodendrocytes were infected with SFV containing myc-tagged PLP, and 16 h after infection, cells were fixed and processed for immunofluorescence for PLP (red) and cholesterol (blue). The region indicated in the upper panel is shown below at a higher magnification. Colocalization of PLP and cholesterol is shown in the merged image as pink. (d) Oligodendrocytes were infected with SFV containing MOG, and 16 h after infection, cells were fixed and processed for immunofluorescence for MOG (red) and cholesterol (blue). Bars, 10 μm.
Mentions: Because the pathology of PLP overexpression is mediated by oligodendrocytes in the CNS, it is important to analyze the trafficking of PLP in these cells. In primary cultures of oligodendrocytes, endogenous PLP is incorporated into cholesterol- and galactosylceramide-enriched membrane domains (myelin rafts) and is transported to the plasma membrane (Simons et al., 2000). As shown previously, a fraction of PLP colocalizes with lysosome-associated membrane protein 1 (LAMP-1), a marker for late endosomes/lysosomes (Krämer et al., 2001; unpublished data). To investigate the fate of overexpressed PLP, primary cultures of oligodendrocytes were infected with SFV-PLP–myc to distinguish it from endogenous protein. We found that 8 h after infection, the myc-tagged PLP was not transported out to the distal part of the processes, but was found in vesicles in the cell body and proximal part of the processes (Fig. 7 a). When oligodendrocytes were double labeled for PLP–myc and calnexin, no colocalization was observed (Fig. 7 b), ruling out the possibility that overexpressed PLP was trapped in the ER. Instead PLP–myc colocalized with rhodamine–dextran that had been internalized for 2 h, identifying the vesicles as part of the endosomal–lysosomal system (Fig. 7 a). Labeling of SFV-PLP–myc-infected oligodendrocytes with filipin demonstrated colocalization of overexpressed PLP and cholesterol, which was drastically pronounced 16 h after infection (Fig. 7 c). Quantitative analysis indicated that 82% of the cells infected with SFV-PLP–myc for 16 h exhibited colocalization of cholesterol and PLP (>100 cells examined). To analyze whether the myc tag interfered with the normal trafficking behavior of PLP, we infected oligodendrocytes with SFV-PLP. To minimize the staining from endogenous protein, we used a 10-fold higher dilution than usual of the PLP antibody for immunofluorescence analysis. 16 h after infection, accumulation of cholesterol and PLP was observed as in the cells expressing PLP–myc (unpublished data). To analyze whether overexpression of any myelin protein leads to endosomal/lysosomal accumulation, oligodendrocytes were infected with SFV-MOG for 16 h. We found that MOG was transported to the distal processes and the cells did not accumulate intracellular cholesterol (Fig. 7 d). These results suggest that overexpression of PLP in oligodendrocytes causes accumulation of PLP and sequestration of cholesterol in the endosomal–lysosomal system as observed in BHK cells.

Bottom Line: Similar results were seen in transgenic mice overexpressing PLP.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