Myelin oligodendrocyte glycoprotein (MOG) is, quantitatively, a relatively minor component of the myelin membrane. Nevertheless, peritoneal administration of MOG evokes potent cellular and humoral immunoreactivity, resulting in an experimental allergic encephalitis with immunopathology similar to multiple sclerosis. Moreover, antibodies against MOG cause myelin destruction in situ. Therefore, it appears that MOG-related demyelination is dependent on anti-MOG antibody, but the mechanism(s) by which it occurs is unclear. Of potential significance are observations that some proteins are selectively partitioned into specialized plasma membrane microdomains rich in glycosphingolipids and cholesterol (“lipid rafts”). In particular, during ligand or antibody cross-linking, various plasma membrane receptors undergo enhanced partitioning into rafts as an obligatory first step toward participation in early signal transduction events. In contrast to mature myelin, in oligodendrocytes (OLs) in culture MOG is not raft associated [Triton X-100 (TX-100) soluble, 4°C]. However, in this study we show that antibody cross-linking (anti-MOG plus secondary antibody) of MOG on the surface of OLs results in the repartitioning of ∼95% of MOG into the TX-100-insoluble fraction. This repartitioning of MOG is rapid (≤1 min), antibody dose dependent, requires an intact cytoskeleton, leads to phosphorylation or dephosphorylation of tyrosine, serine, and threonine residues in specific proteins (e.g., β-tubulin, Gβ1–2), and invokes a rapid retraction of OL processes. After removal of the cross-linking antibodies, these events are reversed. We hypothesize that antibody-mediated repartitioning of MOG into TX-100-insoluble glycosphingolipid–cholesterol-rich microdomains initiates specific cellular signaling that could be related to initial steps of MOG-mediated demyelination.

Myelin oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of myelin. Nevertheless, MOG evokes potent cellular and humoral immunoreactivity resulting in experimental allergic encephalitis with immunopathology similar to multiple sclerosis; antibodies against MOG cause myelin destruction in situ. Thus, it appears that MOG-related demyelination is dependent on anti-MOG antibody, but the mechanism(s) by which it occurs is unclear. Of potential significance, some proteins are selectively partitioned into specialized plasma membrane microdomains rich in glycosphingolipids and cholesterol (lipid rafts). Upon ligand or antibody crosslinking, many plasma membrane receptors repartition into rafts as an obligatory first step towards crtical signal transduction events. In contrast to mature myelin, in oligodendrocytes (OLs) MOG is not raftassociated (TX-100 soluble, 4 C). However, we have demonstrated that antibody crosslinking of MOG on OLs results in the repartitioning of c. 95% of MOG into the TX-100 insoluble fraction. This repartitioning is rapid (£1 min), antibody dose dependent, requires an intact cytoskeleton, leads to phosphorylation/dephosphorylation of tyrosine, serine and threonine residues in specific proteins (e.g. btubulin, Gb1-s-2) and invokes a rapid retraction of OL processes. Upon removal of the crosslinking antibodies, these events are reversed. We hypothesize that antibody-mediated repartitioning of MOG into glycosphingolipid cholesterol-rich microdomains initiates specific cellular signaling that may be related to initial events in MOG-mediated demyelination. Acknowledgement: The study was supported by grant NIH NS 41078 and NS10861, and NMSS FG1423.

Multiple studies have shown that migration, proliferation, and differentiation of oligodendrocyte (OL) lineage cells are influenced by fibroblast growth factor‐2 (FGF‐2) signaling through its receptors (FGFR) FGFR‐1, FGFR‐2, and FGFR‐3. We report the effectiveness and specificity of a unique inhibitor, PD173074, for inhibiting FGF receptor signaling in OL‐lineage cells. Three FGF‐mediated responses of OL progenitors and two of differentiated OLs were examined by immunofluorescence microscopy and immunoblotting. PD173074 effectively antagonized the effect of FGF‐2 on proliferation and differentiation of OL progenitors in culture. One dose of PD173074 at nanomolar concentrations was sufficient to inhibit ongoing FGF‐2 mediated proliferation for prolonged periods, in a non‐toxic, dose‐dependent manner. In contrast, platelet‐derived growth factor (PDGF)‐induced proliferation was unaffected by PD173074. Similarly, mitogen‐activated protein kinase (MAPK) activation, a downstream event after activation of either FGFR or PDGFR, was also blocked by PD173074 in OL progenitors stimulated with FGF‐2 but not PDGF. A general tyrosine kinase inhibitor (PD166285), however, antagonized both FGF‐2‐ and PDGF‐mediated responses. PD173074 also completely antagonized two phenotypic alterations of differentiated OLs, specifically downregulation of myelin proteins, and their re‐entry into the cell cycle. We conclude that PD173704 is an effective and specific inhibitor for multiple FGF‐2‐mediated responses of both OL progenitors and differentiated OLs. This inhibitor provides a direct approach for identifying the importance of FGF signaling, comparable in effect to a knockout of all FGF receptors and all FGF ligands, while leaving other pathways unaffected. Thus, PD173704 is an excellent tool for investigating the role of FGF signaling in vivo in the context of combinatorial interactions of other signals. © 2003 Wiley‐Liss, Inc.

Oligodendrocyte progenitors originate in the subventricular zone, proliferate, migrate to their final destinations, differentiate, and interact with axons to produce multilamellar myelin sheaths. These processes are regulated by a variety of environmental signals, including growth factors, the extracellular matrix, and adhesion molecules. Heparan sulfate proteoglycans are premier candidates as participants in this regulation by virtue of their structural diversity and their capacity to function as coreceptors for both growth factors and extracellular matrix molecules. Consistently with this, we have previously shown that oligodendrocyte progenitors are unable to proliferate in response to fibroblast growth factor‐2 (FGF‐2) in the absence of sulfated heparan sulfate proteoglycan. Here we show that members of three families of heparan sulfate proteoglycans, syndecan, perlecan, and glypican, are developmentally and posttranscriptionally regulated during oligodendrocyte‐lineage progression: Syndecan‐3 is synthesized by oligodendrocyte progenitors (but not terminally differentiated oligodendrocytes) and is up‐regulated by FGF‐2; perlecan synthesis increases as oligodendrocytes undergo terminal differentiation; glypican‐1 is expressed by both progenitors and differentiated oligodendrocytes. Astrocytes express glypican‐1 and perlecan but not syndecan‐3. All three of these heparan sulfate proteoglycans are shed from the cell surface and bind to specific substrates. The developmentally regulated expression of these heparan sulfate proteoglycans is indicative of their participation in events involving growth factor receptors and the extracellular matrix that may regulate oligodendrocyte progenitor proliferation, migration, and adhesion phenomena. © 2002 Wiley‐Liss, Inc.

Galactocerebroside and sulfatide, major galactosphingolipid components of oligodendrocyte plasma membranes and myelin, are first expressed at a critical point, when progenitors cease to proliferate and commence terminal differentiation. We showed previously that an antibody to galactocerebroside/sulfatide arrested terminal differentiation, suggesting a role for these galactolipids in oligodendrocyte differentiation. We have now investigated the differentiation of oligodendrocytes (1) in response to other anti-galactolipid antibodies, showing that anti-sulfatide O4 but not anti-galactocerebroside O1 blocks terminal differentiation, perhaps by mimicking an endogenous ligand, and (2) in a transgenic mouse unable to synthesize these lipids because of mutation of the gene for ceramide galactosyltransferase, a key enzyme for galactosphingolipid synthesis. We find that galactosyltransferase mRNA expression begins at the late progenitor [pro-oligodendroblast (Pro-OL)] stage of the lineage and that the late progenitor marker pro-oligodendroblast antigen is not synthesized in the absence of galactosyltransferase. The principal outcome of the elimination of these galactolipids is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo. Because the general pattern of differentiation and the level of progenitor proliferation and survival appear to be unaltered in the mutant cultures, we conclude that the increased number of oligodendrocytes is caused by an increased rate and probability of differentiation. In agreement with these two experimental approaches, we present a model in which galactosphingolipids (in particular galactocerebroside and/or sulfatide) act as sensors and/or transmitters of environmental information, interacting with endogenous ligands to function as negative regulators of oligodendrocyte differentiation, monitoring the timely progress of Pro-OLs into terminally differentiating, myelin-producing oligodendrocytes.