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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 associates with rafts in primary cultures of oligodendrocytes only after increasing expression times. Oligodendrocytes were infected with SFV-PLP–myc or were left uninfected (endogenous). 8 or 16 h after infection, the cells were extracted with 20 mM CHAPS for 30 min at 4°C and floated in a density gradient. Six fractions were collected from the top and proteins were resolved on SDS-PAGE. After Western blotting, proteins were detected with anti-PLP (endogenous) or anti-myc (SFV-PLP–myc, 8 or 16 h) antibodies. The top fraction represents the low-density, CHAPS-insoluble membrane fraction, whereas the fractions of high density contain CHAPS-soluble membrane.
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fig8: Exogenously expressed PLP associates with rafts in primary cultures of oligodendrocytes only after increasing expression times. Oligodendrocytes were infected with SFV-PLP–myc or were left uninfected (endogenous). 8 or 16 h after infection, the cells were extracted with 20 mM CHAPS for 30 min at 4°C and floated in a density gradient. Six fractions were collected from the top and proteins were resolved on SDS-PAGE. After Western blotting, proteins were detected with anti-PLP (endogenous) or anti-myc (SFV-PLP–myc, 8 or 16 h) antibodies. The top fraction represents the low-density, CHAPS-insoluble membrane fraction, whereas the fractions of high density contain CHAPS-soluble membrane.

Mentions: Normally, the majority of PLP is incorporated into myelin rafts and is subsequently transported to and retained in myelin. We therefore determined the association of overexpressed PLP with myelin rafts. Oligodendrocytes were infected with SFV-PLP–myc, and 8 h after infection, the cells were extracted with 20 mM CHAPS followed by density gradient centrifugation. PLP–myc was mainly recovered from the fractions of higher density, indicating that exogenous PLP does not associate with myelin rafts (Fig. 8). In contrast, and as shown previously, endogenous PLP and its smaller isoform DM20 were predominantly found in the low-density, detergent-insoluble membrane fraction (Fig. 8). It is possible that exogenous overexpressed PLP does not associate with rafts during biosynthetic transport, because rafts are saturated with endogenous PLP. To investigate this possibility, we expressed PLP and PLP–myc using the SFV expression system in immortalized oligodendroglial precursor cells. These cells do not synthesize endogenous PLP, but they express significant amounts of myelin lipid (Jung et al., 1995). We found that at 8 h after infection, PLP and PLP–myc were incorporated to the same extent (∼40–50%, n = 3) into myelin rafts in oligodendroglial precursor cells (unpublished data). These data rule out the possibility that the viral expression system or the myc tag interferes with raft incorporation and suggest that raft association is reduced in primary cultures of oligodendrocytes because the protein is overexpressed.


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 associates with rafts in primary cultures of oligodendrocytes only after increasing expression times. Oligodendrocytes were infected with SFV-PLP–myc or were left uninfected (endogenous). 8 or 16 h after infection, the cells were extracted with 20 mM CHAPS for 30 min at 4°C and floated in a density gradient. Six fractions were collected from the top and proteins were resolved on SDS-PAGE. After Western blotting, proteins were detected with anti-PLP (endogenous) or anti-myc (SFV-PLP–myc, 8 or 16 h) antibodies. The top fraction represents the low-density, CHAPS-insoluble membrane fraction, whereas the fractions of high density contain CHAPS-soluble membrane.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199249&req=5

fig8: Exogenously expressed PLP associates with rafts in primary cultures of oligodendrocytes only after increasing expression times. Oligodendrocytes were infected with SFV-PLP–myc or were left uninfected (endogenous). 8 or 16 h after infection, the cells were extracted with 20 mM CHAPS for 30 min at 4°C and floated in a density gradient. Six fractions were collected from the top and proteins were resolved on SDS-PAGE. After Western blotting, proteins were detected with anti-PLP (endogenous) or anti-myc (SFV-PLP–myc, 8 or 16 h) antibodies. The top fraction represents the low-density, CHAPS-insoluble membrane fraction, whereas the fractions of high density contain CHAPS-soluble membrane.
Mentions: Normally, the majority of PLP is incorporated into myelin rafts and is subsequently transported to and retained in myelin. We therefore determined the association of overexpressed PLP with myelin rafts. Oligodendrocytes were infected with SFV-PLP–myc, and 8 h after infection, the cells were extracted with 20 mM CHAPS followed by density gradient centrifugation. PLP–myc was mainly recovered from the fractions of higher density, indicating that exogenous PLP does not associate with myelin rafts (Fig. 8). In contrast, and as shown previously, endogenous PLP and its smaller isoform DM20 were predominantly found in the low-density, detergent-insoluble membrane fraction (Fig. 8). It is possible that exogenous overexpressed PLP does not associate with rafts during biosynthetic transport, because rafts are saturated with endogenous PLP. To investigate this possibility, we expressed PLP and PLP–myc using the SFV expression system in immortalized oligodendroglial precursor cells. These cells do not synthesize endogenous PLP, but they express significant amounts of myelin lipid (Jung et al., 1995). We found that at 8 h after infection, PLP and PLP–myc were incorporated to the same extent (∼40–50%, n = 3) into myelin rafts in oligodendroglial precursor cells (unpublished data). These data rule out the possibility that the viral expression system or the myc tag interferes with raft incorporation and suggest that raft association is reduced in primary cultures of oligodendrocytes because the protein is overexpressed.

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