Jacqueline Trotter

Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.

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.

Channel expression correlates with differentiation stage during the development of Oligodendrocytes from their precursor cells in culture

Membrane currents in cultured murine oligodendrocytes and their precursors were characterized using the patch-clamp technique. Prior to recording, cells were identified by immunofluorescence using monoclonal antibodies characteristic of two types of precursor cells and two differentiation stages of oligodendrocytes. The most immature, A2B5 antigen-positive glial precursors, expressed four types of voltage-activated K+ currents and tetrodotoxin-sensitive Na+ currents. The more differentiated cells, O4 antigen-positive glial precursors, expressed similar K+ currents, but Na+ currents were recorded in only a minority of cells. In differentiated O1 and O10 antigen-positive oligodendrocytes the channels characteristic of precursor cells were no longer observed, but an inwardly rectifying K+ current was apparent. Thus, channel expression by cells of the oligodendrocyte lineage correlates with differentiation stage and is more complex in precursor cells than in oligodendrocytes.

Compartmentation of Fyn Kinase with Glycosylphosphatidylinositol-anchored Molecules in Oligodendrocytes Facilitates Kinase Activation during Myelination

In many cell types, glycosylphosphatidylinositol (GPI)-anchored proteins are sequestered in detergent-resistant membrane rafts. These are plasma membrane microdomains enriched in glycosphingolipids and cholesterol and are suggested to be platforms for cell signaling. Concomitant with the synthesis of myelin glycosphingolipids, maturing oligodendrocytes progressively associate GPI-anchored proteins, including the adhesion molecules NCAM 120 and F3, in rafts. Here we show that these microdomains include Fyn and Lyn kinases. Both kinases are maximally active in myelin prepared from young animals, correlating with early stages of myelination. In the rafts, Fyn kinase is tightly associated with NCAM 120 and F3. In contrast, in oligodendrocyte progenitor cells lacking rafts or in raft-free membrane domains of more mature cells, F3 does not associate with Fyn. The addition of anti-F3 antibodies to oligodendrocytes results in stimulation of Fyn kinase specifically in rafts. Compartmentation of oligodendrocyte GPI-anchored proteins in rafts is thus a prerequisite for association with Fyn, permitting kinase activation. Interaction of oligodendrocyte F3 with axonal ligands such as L1 and ensuing kinase activation may play a crucial role in initiating myelination.

Oligodendrocytes secrete exosomes containing major myelin and stress‐protective proteins: Trophic support for axons?

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.

Lines of Murine Oligodendroglial Precursor Cells Immortalized by an Activated neu Tyrosine Kinase Show Distinct Degrees of Interaction with Axons In Vitro and In Vivo

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.

NG2 cells: properties, progeny and origin

The NG2 proteoglycan is a type 1-transmembrane protein expressed by a range of cell types within and outside the mammalian nervous system. NG2-expressing (NG2) cells are found in grey and white matter tracts of the developing and adult CNS and have previously been assumed to represent oligodendrocyte precursor cells: new work using transgenic mice has shown that NG2 cells generate oligodendrocytes, protoplasmic astrocytes and in some instances neurons in vivo. NG2 cells express GABAA receptors and the AMPA subtype of glutamate receptors. They make intimate contact to neurons prior to myelinating axons and also form electron-dense synaptic specialisations with axons in the cerebellum, cortex and hippocampus and with non-myelinated axons in the corpus callosum. These synaptic NG2 cells respond to neuronal release of glutamate and GABA. This neuron-glia interaction may thus regulate the differentiation and proliferation of NG2 cells. The C-terminal PDZ-binding motif of the NG2 protein binds several PDZ proteins including Mupp1, Syntenin and the Glutamate Receptor Interacting Protein (GRIP). Since GRIP can bind subunits of the AMPA receptors expressed by NG2 cells, the interaction between GRIP and NG2 may orientate the glial AMPA receptors towards sites of neuronal glutamate release. The origin, heterogeneity and function of NG2 cells as modulators of the neuronal network are important incompletely resolved questions.

Wrapping it up: the cell biology of myelination

During nervous system development, oligodendroglia in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) synthesise large amounts of specific proteins and lipids to generate myelin, a specialised membrane that spirally ensheathes axons and facilitates fast conduction of the action potential. Myelination is initiated after glial processes have attached to the axon and polarisation of the plasma membrane has been triggered. Myelin assembly is a multi-step process that occurs in spatially distinct regions of the cell. We propose that assembly of myelin proteins and lipids starts during their transport through the biosynthetic pathway and continues at the plasma membrane aided by myelin-basic protein (MBP). These sequential processes create the special lipid and protein composition necessary for myelin to perform its insulating function during nerve conduction.

Cells positive for the O4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes

Cells expressing the surface antigen O4 were isolated as pure populations from cultures of murine brain or cerebellum using fluorescence activated cell sorting. When these O4-positive cells were further cultured in the presence of fetal calf serum (FCS) many cells expressed both O4 and the astrocyte marker glial fibrillary acidic protein (GFAP) after 4 days of culture. Cells not exposed to FCS expressed O4, but never GFAP. GFAP-positive cells in the presence of fetal calf serum very rarely expressed the myelin associated glycoprotein (MAG) or O1, both of which are expressed on more differentiated oligodendrocytes, and never expressed O10 that is characteristic of even more mature oligodendrocytes. These results show that glial cells expressing O4, but not MAG, O1, O10 or GFAP are bipotential precursor cells able to differentiate into astrocytes or oligodendrocytes depending on the culture conditions and suggest that bipotentiality of glial precursor cells is retained up to a later developmental stage than that of the O2A progenitor cell.

Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease.

Duplications and overexpression of the proteolipid protein (PLP) gene are known to cause the dysmyelinating disorder Pelizaeus-Merzbacher disease (PMD). To understand the cellular response to overexpressed PLP in PMD, we have overexpressed PLP in BHK cells and primary cultures of oligodendrocytes with the Semliki Forest virus expression system. Overexpressed PLP was routed to late endosomes/lysosomes and caused a sequestration of cholesterol in these compartments. Similar results were seen in transgenic mice overexpressing PLP. With time, the endosomal/lysosomal accumulation of cholesterol and PLP led to an increase in the amount of detergent-insoluble cellular cholesterol and PLP. In addition, two fluorescent sphingolipids, BODIPY–lactosylceramide and –galactosylceramide, which under normal conditions are sorted to the Golgi apparatus, were missorted to perinuclear structures. This was also the case for the lipid raft marker glucosylphosphatidylinositol–yellow fluorescence protein, which under normal steady-state conditions is localized on the plasma membrane and to the Golgi complex. Taken together, we show that overexpression of PLP leads to the formation of endosomal/lysosomal accumulations of cholesterol and PLP, accompanied by the mistrafficking of raft components. We propose that these accumulations perturb the process of myelination and impair the viability of oligodendrocytes.

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia.

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell‐surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF‐α‐receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration. GLIA 34:213–228, 2001.