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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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Perinuclear PLP/DM20 localizes to late endosomes/lysosomes and colocalizes with cholesterol in PLP-overexpressing transgenic mice. 3-wk-old homozygous PLP-overexpressing transgenic mice were perfused, and sagittal sections of the brain were prepared and stained with antibodies against PLP/DM20 and LAMP-1. Immunofluorescence staining is shown in red for PLP/DM20 and green for LAMP-1 (a–c). Nuclei are visualized with DAPI and shown in blue in the merged image (a). In the merged image (a), yellow indicates colocalization of PLP/DM20 and LAMP-1. The inset represents a magnification of the region indicated. Cholesterol was labeled with filipin and shown in blue (b and c). Several cells are shown in the overview (b), whereas a single cell is shown in the image of higher magnification (c). Bars, 8 μm.
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fig9: Perinuclear PLP/DM20 localizes to late endosomes/lysosomes and colocalizes with cholesterol in PLP-overexpressing transgenic mice. 3-wk-old homozygous PLP-overexpressing transgenic mice were perfused, and sagittal sections of the brain were prepared and stained with antibodies against PLP/DM20 and LAMP-1. Immunofluorescence staining is shown in red for PLP/DM20 and green for LAMP-1 (a–c). Nuclei are visualized with DAPI and shown in blue in the merged image (a). In the merged image (a), yellow indicates colocalization of PLP/DM20 and LAMP-1. The inset represents a magnification of the region indicated. Cholesterol was labeled with filipin and shown in blue (b and c). Several cells are shown in the overview (b), whereas a single cell is shown in the image of higher magnification (c). Bars, 8 μm.

Mentions: Transgenic mice with increased dosage of the PLP gene have been useful models for PMD (Kagawa et al., 1994; Readhead et al., 1994). These mice develop a dys- and demyelinating disease, the severity of which is related to the level of PLP overexpression. Immunocytochemistry with antibodies against PLP/DM20 has shown that PLP/DM20 distributes differently in transgenic mice compared with wild type (Gow et al., 1998). PLP/DM20 is found in myelin tracts that can be followed over a long distance through brain sections in wild-type mice, whereas PLP/DM20 localizes to short and swollen myelin segments in transgenic mice. Furthermore, PLP/DM20 was found to accumulate at a perinuclear location in many oligodendrocytes of the transgenic animal (Macklin et al., 1995; Gow et al., 1998). The subcellular compartment of these perinuclear accumulations has not yet been determined. To analyze whether PLP/DM20 accumulates in late endosomes/lysosomes in PLP-overexpressing oligodendrocytes, we performed immunolocalization of PLP in situ. Sagittal sections of the brain of 3-wk-old homozygous PLP-overexpressing mice (line no. 66 in Readhead et al., 1994) were stained with antibodies against PLP/DM20 and LAMP-1. We found that PLP/DM20 accumulated in the perinuclear region and colocalized with LAMP-1 (Fig. 9 a). Quantitative analysis of the sections indicated that colocalization of PLP and LAMP-1 was increased >10-fold in PLP-overexpressing mice (line no. 66) compared with age-matched wild-type control mice. When tissue sections were labeled with filipin, we found that a significant amount (∼40%) of LAMP-1– and PLP-positive structures were also labeled with filipin (Fig. 9, b and c). These results strongly suggest that PLP overexpression in vivo also causes the proteins to accumulate in late endosomes/lysosomes; thus, our in vitro results are also relevant for the pathogenesis of PMD.


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)

Perinuclear PLP/DM20 localizes to late endosomes/lysosomes and colocalizes with cholesterol in PLP-overexpressing transgenic mice. 3-wk-old homozygous PLP-overexpressing transgenic mice were perfused, and sagittal sections of the brain were prepared and stained with antibodies against PLP/DM20 and LAMP-1. Immunofluorescence staining is shown in red for PLP/DM20 and green for LAMP-1 (a–c). Nuclei are visualized with DAPI and shown in blue in the merged image (a). In the merged image (a), yellow indicates colocalization of PLP/DM20 and LAMP-1. The inset represents a magnification of the region indicated. Cholesterol was labeled with filipin and shown in blue (b and c). Several cells are shown in the overview (b), whereas a single cell is shown in the image of higher magnification (c). Bars, 8 μm.
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Related In: Results  -  Collection

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fig9: Perinuclear PLP/DM20 localizes to late endosomes/lysosomes and colocalizes with cholesterol in PLP-overexpressing transgenic mice. 3-wk-old homozygous PLP-overexpressing transgenic mice were perfused, and sagittal sections of the brain were prepared and stained with antibodies against PLP/DM20 and LAMP-1. Immunofluorescence staining is shown in red for PLP/DM20 and green for LAMP-1 (a–c). Nuclei are visualized with DAPI and shown in blue in the merged image (a). In the merged image (a), yellow indicates colocalization of PLP/DM20 and LAMP-1. The inset represents a magnification of the region indicated. Cholesterol was labeled with filipin and shown in blue (b and c). Several cells are shown in the overview (b), whereas a single cell is shown in the image of higher magnification (c). Bars, 8 μm.
Mentions: Transgenic mice with increased dosage of the PLP gene have been useful models for PMD (Kagawa et al., 1994; Readhead et al., 1994). These mice develop a dys- and demyelinating disease, the severity of which is related to the level of PLP overexpression. Immunocytochemistry with antibodies against PLP/DM20 has shown that PLP/DM20 distributes differently in transgenic mice compared with wild type (Gow et al., 1998). PLP/DM20 is found in myelin tracts that can be followed over a long distance through brain sections in wild-type mice, whereas PLP/DM20 localizes to short and swollen myelin segments in transgenic mice. Furthermore, PLP/DM20 was found to accumulate at a perinuclear location in many oligodendrocytes of the transgenic animal (Macklin et al., 1995; Gow et al., 1998). The subcellular compartment of these perinuclear accumulations has not yet been determined. To analyze whether PLP/DM20 accumulates in late endosomes/lysosomes in PLP-overexpressing oligodendrocytes, we performed immunolocalization of PLP in situ. Sagittal sections of the brain of 3-wk-old homozygous PLP-overexpressing mice (line no. 66 in Readhead et al., 1994) were stained with antibodies against PLP/DM20 and LAMP-1. We found that PLP/DM20 accumulated in the perinuclear region and colocalized with LAMP-1 (Fig. 9 a). Quantitative analysis of the sections indicated that colocalization of PLP and LAMP-1 was increased >10-fold in PLP-overexpressing mice (line no. 66) compared with age-matched wild-type control mice. When tissue sections were labeled with filipin, we found that a significant amount (∼40%) of LAMP-1– and PLP-positive structures were also labeled with filipin (Fig. 9, b and c). These results strongly suggest that PLP overexpression in vivo also causes the proteins to accumulate in late endosomes/lysosomes; thus, our in vitro results are also relevant for the pathogenesis of PMD.

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