Neuronal activity provokes myelinating oligodendrocytes to release exosomes by stimulation of ionotropic glutamate receptors, and that once released, these vesicles are internalized by neurons conveying neuroprotection.

Oligodendrocytes synthesize the CNS myelin sheath by enwrapping axonal segments with elongations of their plasma membrane. Spatial and temporal control of membrane traffic is a prerequisite for proper myelin formation. The major myelin proteolipid protein (PLP) accumulates in late endosomal storage compartments and multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in the release of the intralumenal vesicles, termed exosomes, into the extracellular space. Here, we show that cultured oligodendrocytes secrete exosomes carrying major amounts of PLP and 2'3'-cyclic-nucleotide-phosphodiesterase (CNP). These exosomes migrated at the characteristic density of 1.10-1.14 g/mL in sucrose density gradients. Treatment of primary oligodendrocytes with the calcium-ionophore ionomycin markedly increased the release of PLP-containing exosomes, indicating that oligodendroglial exosome secretion is regulated by cytosolic calcium levels. A proteomic analysis of the exosomal fraction isolated by sucrose density centrifugation revealed in addition to PLP and CNP, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) as constituents of oligodendroglial exosomes, together with a striking group of proteins with proposed functions in the relief of cell stress. Oligodendroglial exosome secretion may contribute to balanced production of myelin proteins and lipids, but in addition exosomes may embody a signaling moiety involved in glia-mediated trophic support to axons.

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.