SummaryThe transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigenpresenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia. Journal of Cell Sciencethrough which microglia participate in the degradation of oligodendroglial membrane. Results Internalisation of oligodendroglia-derived exosomes by microgliaTo obtain exosomes in large quantities, we isolated exosomes from the culture medium of the mouse oligodendroglial precursor cell line Oli-neu. Sequential centrifugation steps with increasing centrifugal forces up to 100,000 g yielded a pellet, which we have previously shown to contain small membrane vesicles with a diameter of about 50-100 nm (Trajkovic et al., 2008). Further analysis of the 100,000 g pellet with continuous sucrose density gradients revealed that PLP and myelin oligodendrocyte glycoprotein (MOG) are recovered from fractions with the characteristic density of exosomes that contain the exosomal marker proteins Alix (ALG-2-interacting protein 1), TSG101 and flotillin-2 (Fig. 1A). Hence, we refer to the 100,000 g membrane fractions as exosomes.To follow the fate of exosomes, suspensions of purified exosomes were labelled with the dye PKH67 and added to primary cultures of mouse oligodendrocytes, cortical neurons, astrocytes and microglia. After incubating the exosomes for 2 hours at 37°C, cells were fixed and analysed by confocal microscopy. We observed efficient internalisation of exosomes by microglia, whereas uptake by astrocytes, neurons or oligodendrocytes was almost absent (Fig. 1B). By confocal microscopy, we confirmed that exosomes were inside microglia rather than attached to the cell surface. In addition, we observed that exosomes colocalised with Lamp1, a marker for late endosomes or lysosomes, verifying that exosomes had been endocytosed and transported to endosomal organelles ( Fig. 2A). When exosomes were added to mixed brain cultures that contained all major glial cell types, we again observed that exosomes were almost exclusively internalised by Iba-1-positive microglia (supplementary material Fig. S1). Similar results wer...

Oligodendrocytes make myelin and support axons metabolically with lactate. However, it is unknown how glucose utilization and glycolysis are adapted to the different axonal energy demands. Spiking axons release glutamate and oligodendrocytes express NMDA receptors of unknown function. Here we show that the stimulation of oligodendroglial NMDA receptors mobilizes glucose transporter GLUT1, leading to its incorporation into the myelin compartment in vivo. When myelinated optic nerves from conditional NMDA receptor mutants are challenged with transient oxygen-glucose deprivation, they show a reduced functional recovery when returned to oxygen-glucose but are indistinguishable from wild-type when provided with oxygen-lactate. Moreover, the functional integrity of isolated optic nerves, which are electrically silent, is extended by preincubation with NMDA, mimicking axonal activity, and shortened by NMDA receptor blockers. This reveals a novel aspect of neuronal energy metabolism in which activity-dependent glutamate release enhances oligodendroglial glucose uptake and glycolytic support of fast spiking axons.

We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.

Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity.DOI: http://dx.doi.org/10.7554/eLife.17119.001

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.