Replication-defective retroviruses expressing the t-neu oncogene, or a hybrid protein with the neu tyrosine kinase linked to the external region of the human epidermal growth factor receptor (egfr-neu), were used to establish lines of murine oligodendroglial precursor cells. Differentiation of the t-neu lines into myelin-associated glycoprotein (MAG)-positive oligodendrocytes was induced by dibutyryl cAMP, and the egfr-neu line showed limited differentiation in vitro upon withdrawal of epidermal growth factor. Cerebellar granule cell neurons expressed mitogens for the cell lines. Upon transplantation into demyelinated lesions, t-neu line cells engaged with the demyelinated axons whereas the egfr-neu line cells differentiated further and ensheathed the axons. These cell lines thus interact with neurons in vitro and in vivo and can be used as tools to define the molecules involved in different stages of neuron-glia interaction.

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid–protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid–protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.

Fyn kinase plays an important role during myelination and has been shown to promote morphological differentiation of cultured oligodendrocytes. We analyzed the downstream targets of Fyn kinase in oligodendrocytes. Because process outgrowth and wrapping of axons involve cytoskeletal rearrangement, we focused on cytoskeletal proteins linked to Fyn. Here we demonstrate that Fyn binds to the cytoskeletal proteins Tau and ␣-Tubulin in oligodendrocytes. Tau interacts with the Fyn SH3 domain whereas ␣-Tubulin binds to the Fyn SH2 and SH3 domains. To study the function of the Fyn-Tau interaction in oligodendrocytes, we designed a Tau deletion mutant that would compete with endogenous Tau-Fyn binding in transfected cells. The mutant Tau protein binds to the Fyn SH3 domain but lacks the microtubuli interaction domain and thus cannot bind to microtubuli. In the presence of the mutant Tau protein, a reduction of the process number and process length in oligodendroglial cells was observed. This effect is likely to be caused by interference with the Fyn-Tau-microtubuli cascade rather than inactivation of the kinase, because Fyn bound to the mutant Tau retains activity. A similar inhibition of process outgrowth was observed when oliogodendroglial cells were cultured in the presence of Fumonisin B1, an inhibitor of sphingolipid synthesis that prevents the formation of rafts. Because ligation of the cell adhesion molecule F3 on oligodendrocytes leads to activation of Fyn kinase localized in rafts, these findings suggest that recruitment of Tau and Tubulin to activated Fyn kinase in rafts is an important step in the initiation of myelination. Key words: myelination; oligodendrocytes; Fyn kinase; cytoskeleton; Tau; glycosphingolipid-rich raftsThe myelination of axons by oligodendrocytes involves the coordinated recognition of the axonal surface, ensheathment of the axonal process, and ultimately compaction of the wrappings of oligodendroglial membrane to generate the myelin sheath. The interplay of the adhesion molecules expressed by oligodendrocytes and axons as well as the downstream signal transduction cascades mediating these complex cellular interactions still remain primarily unelucidated (Pedraza et al., 2001).Substantial evidence exists that Fyn expressed by oligodendrocytes plays an important role in myelination. Mice deficient for Fyn kinase are hypomyelinated (Umemori et al., 1994;). In addition, knock-in mice expressing a kinase-defective Fyn protein that cannot be activated because of a mutation in the ATP-binding site of Fyn show severe myelination defects . Inhibition of the Fyn activity in cultured oligodendrocytes using kinase inhibitors or dominant negative Fyn constructs inhibited the process outgrowth of the cells (Osterhout et al., 1999). We have shown that in oligodendrocytes [as in neurons (Zisch et al., 1995)] the F3 adhesion molecule is complexed to the Src-family tyrosine kinase Fyn and that this association takes place within glycosphingolipid (gsl)/cholesterol-rich microdomains (Kramer et al., 1999), ge...

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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.