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.

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The central nervous system myelin sheath is a multilayered specialized membrane with compacted and non-compacted domains of defined protein composition. How oligodendrocytes regulate myelin membrane trafficking and establish membrane domains during myelination is largely unknown. Oligodendroglial cells respond to neuronal signals by adjusting the relative levels of endocytosis and exocytosis of the major myelin protein, proteolipid protein (PLP). We investigated whether endocytic trafficking is common to myelin proteins and analyzed the endocytic fates of proteins with distinct myelin subdomain localization. Interestingly, we found that PLP, myelin-associated glycoprotein (MAG) and myelin-oligodendrocyte glycoprotein (MOG), which localize to compact myelin, periaxonal loops and abaxonal loops, respectively, exhibit distinct endocytic fates. PLP was internalized via clathrin-independent endocytosis, whereas MAG was endocytosed by a clathrin-dependent pathway, although both proteins were targeted to the late-endosomal/lysosomal compartment. MOG was also endocytosed by a clathrin-dependent pathway, but in contrast to MAG, trafficked to the recycling endosome. Endocytic recycling resulted in the association of PLP, MAG and MOG with oligodendroglial membrane domains mimicking the biochemical characteristics of myelin domains. Our results suggest that endocytic sorting and recycling of myelin proteins may assist plasma membrane remodeling, which is necessary for the morphogenesis of myelin subdomains.

CNS myelination by oligodendrocytes requires directed transport of myelin membrane components and a timely and spatially controlled membrane expansion. In this study, we show the functional involvement of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) proteins VAMP3/cellubrevin and VAMP7/TI-VAMP in myelin membrane trafficking. VAMP3 and VAMP7 colocalize with the major myelin proteolipid protein (PLP) in recycling endosomes and late endosomes/lysosomes, respectively. Interference with VAMP3 or VAMP7 function using small interfering RNA-mediated silencing and exogenous expression of dominantnegative proteins diminished transport of PLP to the oligodendroglial cell surface. In addition, the association of PLP with myelin-like membranes produced by oligodendrocytes cocultured with cortical neurons was reduced. We furthermore identified Syntaxin-4 and Syntaxin-3 as prime acceptor Q-SNAREs of VAMP3 and VAMP7, respectively. Analysis of VAMP3-deficient mice revealed no myelination defects. Interestingly, AP-3␦-deficient mocha mice, which suffer from impaired secretion of lysosome-related organelles and missorting of VAMP7, exhibit a mild dysmyelination characterized by reduced levels of select myelin proteins, including PLP. We conclude that PLP reaches the cell surface via at least two trafficking pathways with distinct regulations: (1) VAMP3 mediates fusion of recycling endosomederived vesicles with the oligodendroglial plasma membrane in the course of the secretory pathway; (2) VAMP7 controls exocytosis of PLP from late endosomal/lysosomal organelles as part of a transcytosis pathway. Our in vivo data suggest that exocytosis of lysosomerelated organelles controlled by VAMP7 contributes to myelin biogenesis by delivering cargo to the myelin membrane.

Oligodendrocytes form the central nervous system myelin sheath by spiral wrapping of their plasma membrane around axons, necessitating a high rate of exocytic membrane addition to the growing myelin membrane. Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs), which act by specific pairing of vesicle (R)- and target (Q)-SNAREs. To characterize oligodendroglial SNAREs and their trafficking pathways, we performed a detailed expression analysis of SNAREs in differentiating cultured oligodendrocytes and myelin and determined their subcellular localization. Expression of the plasma membrane Q-SNAREs syntaxin 3, syntaxin 4, SNAP23, and the endosomal R-SNARE VAMP3/cellubrevin increased with oligodendroglial maturation, while the expression of SNAP29 decreased. Interestingly, syntaxin 3, syntaxin 4, and VAMP7/tetanustoxin-insensitive VAMP accumulated in myelin during development, suggesting a role in myelin membrane fusion. Coimmunoprecipitation from oligodendroglial cell lysates elucidated interactions between SNAREs: for example, Golgi-localized VAMP4 associated with syntaxin 6 and SNAP29. Furthermore, we identified a cognate core complex composed of VAMP3, syntaxin 4, and SNAP23, which may mediate fusion of endosome-derived vesicles with the plasma membrane. This study provides a comprehensive analysis of SNARE proteins in oligodendrocytes and assigns defined SNAREs to putative vesicle trafficking pathways in myelinating oligodendrocytes, thus facilitating future functional analysis of distinct SNAREs in oligodendroglial membrane traffic and myelination.