Oligodendrocytes Direct Glycosyl Phosphatidylinositol-anchored Proteins to the Myelin Sheath in Glycosphingolipid-rich Complexes

Abstract

The myelin sheath synthesized by oligodendrocytes insulates central nervous system axons and is a specialized subdomain of the plasma membrane, containing a restricted pattern of proteins and lipids. Myelin is enriched in glycosphingolipids and cholesterol, a lipid environment favored by glycosylphosphatidylinositol (GPI)-anchored proteins, which associate with these lipids in detergent-insoluble complexes in many cell types. Since proteins regulating oligodendroglia-neuron interaction are largely unknown and GPI-anchored proteins are often involved in cell-cell interactions, we examined oligodendrocytes and myelin for their expression of these proteins. Oligodendrocyte precursors and maturing oligodendrocytes express a similar pattern of GPIanchored proteins, which unlike the majority of oligodendrocyte plasma membrane proteins, accumulate in myelin. To elucidate mechanisms underlying the expression of GPI-anchored proteins in myelin, we analyzed detergent-insoluble complexes from cells and myelin using TX-100 extraction and sucrose density gradients. In precursor cells, the GPI-anchored proteins are not incorporated in detergent-insoluble complexes. In contrast, GPI-anchored proteins from maturing oligodendrocytes and from myelin were isolated as complexes associated with glycosphingolipids and cholesterol. These results show a specific association of GPI-anchored proteins with glycosphingolipids and cholesterol during oligodendrocyte maturation and suggest sorting of these macromolecular complexes to myelin.

An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

“…The present report, as well as those by Kim et al (1995) and Krämer et al (1997), indicates that an apical-type transport machinery, reflected by detergent-insoluble rafts as described for model polarized cells, is present in the mature OLGs as well. The evidence presented here was derived from experiments in which the fate of the viral glycoproteins HA and VSVG was examined.…”

“…In addition, evidence was presented demonstrating that the presence or absence of galactosylceramide or sulfatide was of no consequence to PLP transport to the plasma membrane of Chinese hamster ovary cells transfected with PLP. Yet it has also been reported that the MAL proteolipid (Kim et al, 1995) and GPI-anchored proteins (Krämer et al, 1997) are present in detergent-insoluble, sphingolipid-containing vesicles in myelinating OLGs.…”

“…However, it is difficult to define an apical membrane in OLGs because no membrane area could be found that is specific for HA. Nevertheless, the MAL proteolipid (Kim et al, 1995) and GPI-anchored proteins (Krämer et al, 1997) are present in detergent-insoluble complexes, at least in mature OLGs, and apparently end up in myelin. Because VSVG also arrives in the myelin sheet, as shown in the present work, it appears that raft-mediated transport does not represent the exclusive transport pathway that leads to the myelin sheet.…”

Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes

“…Concomitant with the cross-linking-dependent redistribution of MAG into the detergentinsoluble fraction, there are changes in the phosphorylation of specific proteins, including Fyn and Gβ1-2, which have been shown previously to reside in lipid rafts (Kramer et al, 1997;Kramer et al, 1999;Melkonian et al, 1999;Marta et al, 2003). Fyn is a member of the Src family of kinases and is associated with cell adhesion, migration, proliferation, differentiation, apoptosis and cytoskeletal rearrangements (Osterhout et al, 1999).…”

“…Lipid rafts have an important role as platforms for the initiation of signal transduction by favoring specific protein-protein interactions (Simons and Toomre, 2000). Using biochemical criteria to identify proteins in rafts, it has been shown that in myelin the GPI-linked proteins NCAM-120 and contactin, the doubly acylated proteins Fyn and Lyn kinases, 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) and myelin oligodendrocyte glycoprotein (MOG) partition into rafts (Kim et al, 1995;Kramer et al, 1997;Kim and Pfeiffer, 1999;Kramer et al, 1999;Taylor et al, 2002), whereas MAG does not. Cross-linking of some proteins to either ligand or antibody can result in their enhanced partitioning into rafts and participation in early signaltransduction events (Simons and Toomre, 2000).…”