Endocytic trafficking and oligodendroglial exosome secretion in axon-glia communication and myelination

Oligodendrocytes form specialized plasma membrane extensions which spirally enwrap axons, thereby building up the myelin sheath. During myelination, oligodendrocytes produce large amounts of membrane components. Oligodendrocytes can be seen as a complex polarized cell type with two distinct membrane domains, the plasma membrane surrounding the cell body and the myelin membrane. SNARE proteins mediate the fusion of vesicular cargoes with their target membrane. We propose a model in which the major myelin protein PLP is transported by two different pathways. VAMP3 mediates the non-polarized transport of newly synthesized PLP via recycling endosomes to the plasma membrane, while transport of PLP from late endosomes/lysosomes to myelin is controlled by VAMP7. In the second part of the thesis, the role of exosome secretion in glia to axon signaling was studied. Further studies are required to clarify whether VAMP7 also controls exosome secretion. The thesis further focused on putative metabolic effects in the target neurons. Oligodendroglial exosomes showed no obvious influences on neuronal metabolic activity. Analysis of the phosphorylation levels of the neurofilament heavy subunit revealed a decrease in presence of oligodendrocytes, indicating effects of oligodendroglial exosomes on the neuronal cytoskeleton. Finally, candidates for kinases which are possibly activated upon influence of oligodendroglial exosomes and could influence neuronal survival were identified.Oligodendrozyten bilden spezialisierte Ausläufer der Plasmamembran, welche das Axon spiralförmig umwinden und dabei die Myelinscheide aufbauen. Während der Myelinisierung werden große Mengen an Membrankomponenten gebildet. Ein Oligodendrozyt kann als komplexer polarisierter Zelltyp mit zwei verschiedenen Membrandomänen angesehen werden, die den Zellkörper umgebende Plasmamembran und die Myelinmembran. SNARE-Proteine vermitteln die Fusion von Vesikeln mit ihrer Zielmembran. Auf der Basis unserer Studien schlagen wir ein Modell vor, in dem das Hauptmyelinprotein PLP auf zwei unterschiedlichen Transportwegen befördert wird. VAMP3 vermittelt dabei den nicht-polarisierten Transport von neu synthetisiertem PLP über Recycling-Endosomen zur Plasmamembran, während der Transport von PLP von späten Endosomen/Lysosomen zur Myelinmembran von VAMP7 kontrolliert wird. Im zweiten Teil der Arbeit wurde die Rolle der Exosomensekretion bei der Kommunikation zwischen Gliazelle und Axon studiert. Weitere Studien sind erforderlich, um zu klären, ob VAMP7 ebenfalls die Exosomensekretion kontrolliert. Diese Arbeit fokussierte sich darüber hinaus auf die möglichen metabolischen Effekte in den Zielneuronen. Oligodendrogliale Exosomen zeigten keine eindeutigen Effekte auf die Stoffwechselrate der Neuronen. Die Analyse der Phosphorylierung der schweren Untereinheit in Neurofilamenten zeigte eine signifikante Reduzierung durch Oligodendrozyten, was Auswirkungen auf das neuronale Zytoskelett vermuten lässt. Schließlich wurden Kinasen als Kandidaten ermittelt, die durch oligodendrogliale Exosomen aktiviert werden und Einfluss auf die Lebensfähigkeit der Neuronen haben könnten

Transport of the major myelin proteolipid protein is directed by VAMP3 and VAMP7.

International audienceCNS 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 dominant-negative 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-3δ-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 endosome-derived 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 lysosome-related organelles controlled by VAMP7 contributes to myelin biogenesis by delivering cargo to the myelin membrane

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

<div><p>Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.</p></div

Adaxonal localization of MVBs and glutamate-dependent release of exosomes.

<p>(A–C) Immuno-electron microscopy analysis of cross-sections of myelinated axons in the optic nerve or spinal cord of adult mice labeled with antibodies against PLP (scale bar, 250 nm, asterisk marks adaxonal loop). Insets depict enlarged views of a MVB in the oligodendrocyte adaxonal cytoplasmic loop (A) and a fusion profile indicating the release of vesicles with the size corresponding to exosomes (B). (C) Quantification of MVBs located adaxonal, abaxonal, and within compact myelin in optic nerves. MVB number was normalized per axon. (D) Western blot analysis of PLP, Alix, Hsc/Hsp70, and calnexin (CNX) in cell lysates and exosomes pelleted by differential centrifugation from culture supernatants of primary oligodendrocytes (pOL) treated with glutamate (Glu, 100 µM) for 5 h or untreated (untr.). (E) Density gradient analysis of exosomes purified from supernatants of pOL treated with 100 µM glutamate versus untreated control cells. (F) Electron microscopic analysis of 100,000× g exosome pellets derived from supernatants collected over a period of 5 h from untreated (left image) or glutamate-treated cells (100 µM). Scale bar, 100 nm. (G) Transfection of pOL with Rab35- or control-siRNA (Ctrl) and quantification of glutamate-dependent exosome release. Western blot signals of exosomal PLP were normalized to total cellular PLP and defined as relative exosome release (<i>n</i> = 5). (H) Administration of 50 (<i>n</i> = 5), 100 (<i>n</i> = 8), 200 µM (<i>n</i> = 10) glutamate to pOL, and quantitative analysis of exosome release. (I) Incubation of pOL with the Ca²⁺-chelator EDTA and quantification of glutamate-dependent exosome release (<i>n</i> = 3). Error bars, SEM (* <i>p</i><0.05; Wilcoxon test).</p

Oligodendroglial exosome secretion and its role in reciprocal neuron-glia communication.

<p>Electrically active axons release the neurotransmitter glutamate (1) inducing Ca²⁺-entry through oligodendroglial glutamate receptors (2). The rise in intracellular Ca²⁺ triggers exosome release from oligodendrocytes (OL) along internodes or at somas (3). In turn, exosomes are internalized by neurons (N) at axons or cell bodies (4) and their protein and RNA cargo is functionally retrieved (5). Exosome uptake by microglia (M) is also indicated.</p

Functional retrieval of exosomal cargo by neurons.

<p>(A–G) Boyden chamber co-culture of pOL transduced by recombinant AAV/MBP-Cre (pOL-MBP-Cre) and CN transduced by AAV/CBA-floxstop-hrGFP reporter vector. hrGFP expression indicates Cre-mediated recombination. Tuj1 staining (red) of CN exposed to (A) nontransduced pOL, (B) pOL-MBP-Cre, or (C) pOL-MBP-Cre treated with 5 µM GW4869. Scale bar, 100 µm. (D–G) Reporter gene expression (hrGFP) in target neurons detected by Western blotting of neuronal lysates. CN were exposed to (D) pOL-MBP-Cre treated or not with 100 µM glutamate, (E) transfected with Rab35 or control siRNA, or (F) treated or not with 5 µM GW4869. (F, right lane) Exposure of CN to exosome-depleted culture supernatant from pOL-MBP-Cre does not lead to reporter gene expression in neurons. (G) pOL, Oli-neu cells, or HEK cells transduced with AAV/MBP-Cre co-cultured with CN. (H) Cre RT-PCR and (I) Western blot of exosomes derived from pOL-MBP-Cre or control cells. Pgk1 (mRNA), PLP/DM20, and Alix (protein) are shown as standards. (J–L) Stereotactic injection of exosomes derived from pOL-MBP-Cre into the (J, K) hippocampus (HC) and (L) cerebellum of ROSA26-lacZ reporter mice. β-galactosidase positive cells were stained using X-gal and neurons were stained for (K) neuron-specific enolase (NSE) or for (L) GABA-R_α6. Mice (<i>n</i> = 5) were analyzed 14 d after injection.</p

Somatodendritic and axonal uptake of exosomes.

<p>CN were cultured in microfluidic chambers and axons were allowed to grow through the microgrooves for 7 d. Exosomes isolated from PKH67 stained (A, B green) or AAV/MBP-Cre infected pOL (C, D) were applied to either the somatodendritic (A, C) or axonal (B, D) compartment of the device. Cre-containing exosomes were added to AAV/CBA-floxstop-hrGFP infected CN. hrGFP expression indicates Cre-mediated recombination (C, D green). CN were stained with Tuj1 (red) and nuclei with Dapi (blue). Scale bar, 20 µm. (E) Quantification of recombinant neurons located within 100 µm above the microgrooves. Exemplary pictures are depicted above the chart. Asterisk indicates the area of quantification. Error bars, SEM (<i>n</i> = 4).</p

Primary cortical neurons internalize oligodendroglial exosomes.

<p>(A–D) pOL were stained with the lipophilic dye PKH67 (green), washed, and subsequently co-cultured in Boyden chambers for 2 d with mixed neural cultures containing astrocytes (A, B, blue marker GFAP), oligodendrocytes (B, red marker O4), and microglia (A, red marker F4/80, B, arrowheads) or with CN (C, red marker Tuj1). Scale bar, 20 µm. (D) Quantification of exosome uptake by the different types of target cells. Error bars, SEM, (<i>n</i> = 3). (E) Fluorescent exosomes containing SIRT2-EYFP and PLP-EGFP were purified by sucrose density gradient centrifugation from Oli-neu cells and co-incubated with CN for 24 h. The Western blot depicts EGFP and the exosomal marker Tsg101 in gradient fractions. Images show maximum projections of confocal Z-stacks of Tuj1-stained neurons after incubation with exosomes (scale bar, 5 µm). (F) Western blots of purified Oli-neu exosomes (input, left lane) and neuronal lysates after treatment with exosomes (Exo). EGFP/EYFP depicts exosome markers, Tubulin (Tub) is used as normalization standard. Relative exosome uptake reflects normalized signals of SIRT2-EYFP and PLP-EGFP associated with neuronal lysates (<i>n</i> = 8). (G) To remove surface-bound exosomes, neurons were treated with trypsin (Tryp) before lysis (<i>n</i> = 5). (H–K) Boyden chamber co-culture of oligodendroglial cells and CN for 2–3 d and analysis of exosomal PLP and SIRT2 in neurons by Western blot (H, I, and K) or immunostaining (J). (H) PLP-EGFP and SIRT2-EYFP expressing Oli-neu cells were treated or not with 5 µM GW4869 inhibiting exosome release (<i>n</i> = 6). (I) pOL were treated with 100 µM glutamate stimulating exosome release (<i>n</i> = 5). (J) Co-culture of CN with PKH67-labelled (green) pOL and immunostaining of CN with Tuj1 (red) and the late endosomal/lysosomal marker LAMP1 (blue). Maximum projection of a confocal Z-stack. Scale bar, 5 µm. (K) Neurons were pre-treated with Dynasore and co-cultured for 1 d with Oli-neu cells releasing SIRT2-EYFP and PLP-EGFP labeled exosomes (<i>n</i> = 4). Error bars, SEM (* <i>p</i><0.05; Wilcoxon-test).</p