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 Ca 2+ 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.

Brain function largely depends on the communication between electrically excitable neurons and surrounding glial cells. Myelinating oligodendrocytes are a type of brain cell that insulate major neuronal processes (axons) and help to sustainably maintain axonal health, which is poorly understood in molecular terms. Several cell types release microvesicles termed exosomes that include genetic information (primarily RNA) and can act as vehicles transferring specific cargo to target cells. Here, we demonstrate that exosomes secreted by oligodendrocytes in response to neuronal signals enter neurons to make their cargo functionally available to the neuronal metabolism. We revealed in cultured cells that exosome release from oligodendrocytes is triggered by the neurotransmitter glutamate through activation of ionotropic glutamate receptors. We also show that glial exosomes are internalized by neurons via an endocytic pathway. By modifying oligodendroglial exosomes with a reporter enzyme, we could demonstrate that the exosome cargo is recovered by target neurons in culture as well as in vivo after injection of exosomes into the mouse brain. Neurons challenged with stressful growth conditions were protected when treated with oligodendroglial exosomes. The study introduces a new concept of reciprocal cell communication in the nervous system and identifies the signal-mediated transfer of exosomes from oligodendrocytes to neurons contributing to the preservation of axonal health.

Funding: The study was supported by grants of ELA (2007-027C1) and DFG (KR 3668/1-1) to EMKA, DFG SFB 894 (A12) to FK, DFG Research Center Molecular Physiology of the Brain (CNMPB), and ERC Advanced Grant to KAN. CF received internal research funding for early career researchers from JGU Mainz. MK is an Australian Research Council (ARC) Future Fellow. DF received fellowships from the Stipendienstiftung Rheinland Pfalz and the DFG GRK 1044. WPK is a fellow of the Focus Program Translational Neuroscience JGU Mainz. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we analyze the role of exosomes in axon-glia communication. We demonstrate that neuronal activity-mediated release of the neurotransmitter glutamate regulates oligodendroglial exosome secretion by activation of glial ionotropic glutamate receptors. In turn, neurons internalize exosomes released from oligodendrocytes and retrieve their cargo. Furthermore, our results indicate that oligodendrocyte-derived exosomes mediate neuroprotective functions. These findings reveal a novel mode of cell communication among cells of the CNS that may be employed by oligodendrocytes to support axons.

Oligodendrocytes release membrane vesicles with the characteristics of exosomes, which include specific myelin proteins such as proteolipid protein (PLP) [10] , [11] . Since exosomes have the capacity to affect neighboring cells, they have been generally implicated in intercellular communication [12] , [13] Exosomes of 50–100 nm in size are generated within the endosomal system and secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane. The exosomal membrane exhibits the topology of the plasma membrane and encloses cytoplasmic cargo. Most if not all cell types secrete exosomes and other microvesicles, budding from the plasma membrane. Consequently, body fluids such as serum, urine, and CSF contain significant amounts of mixed microvesicles, including exosomes [14] . Exosomes carry cell-type-specific components as well as common cargo, including proteins involved in MVB biogenesis, heat shock proteins, and integral membrane proteins such as integrins and tetraspanins. Furthermore, exosomes contain mRNA and miRNA, which upon horizontal transfer can alter protein expression, thus modulating the properties of recipient cells [15] – [17] . They have been described to contribute to immune responses, to spread pathogens such as viruses and prions, to modulate the tumor cell micro-environment, and furthermore to educate the phenotype of bone marrow cells [18] – [20] . Although cells exhibit a basal level of release, secretion of exosomes is a regulated process. Increase in cytoplasmic Ca 2+ triggers exosome release from several cell types, including neurons and oligodendrocytes [10] , [21] , [22] .

In the CNS, oligodendrocytes insulate axons with a multilayered myelin sheath enabling rapid impulse conduction. Formation of functional axon-myelin units depends on bidirectional axon-glia interaction [1] , [2] . During nervous system development neuronal signals including activity-dependent neurotransmitter release control the differentiation of oligodendrocytes and myelination [3] – [5] . Axon-glia communication remains important throughout life. In addition to axon ensheathment, oligodendrocytes provide trophic support to neurons critical for long-term axonal integrity [6] . Glial support has been suggested to represent an ancestral function independent of myelination [7] . The mechanisms of neuron-glia communication essential to sustainably maintain and protect the highly specialized axon-glial entity over a lifetime are not well understood. Recent studies indicate that glycolytic oligodendrocytes provide axons with external energy substrates such as lactate [8] , [9] . These studies reveal new insights into axonal energy supply, although it remains still open how other resources (such as enzymes of a certain half-life) reach distal sites of axons.

Results

Oligodendroglial Cre Driver Mice Exhibit Reporter Gene Recombination in Neurons Expression of Cre recombinase under control of a cell-type-specific promoter is utilized to drive the recombination of floxed target genes in a defined subset of cells within a tissue. MOGi-Cre mice carry Cre as a knock-in allele under control of the endogenous MOG promoter, which is described to be specifically active in the late stage of oligodendrocyte maturation [23] driving Cre expression in oligodendrocytes exclusively [24],[25]. However, analysis of double transgenic MOGi-Cre/Rosa26-lacZ mice revealed reporter gene expression not only in oligodendrocytes but also in a subset of neurons in several brain regions (Figure 1). In the cerebellar granule cell layer, 17% of NeuN-labeled cells were positive for LacZ, while a lower number of recombined cells carrying neuronal markers were present in the cortex (3.8%), hippocampus (1.2%), and brainstem (2.9%). This finding may be explained by (1) activity of the MOG promoter in individual neurons or their precursors or (2) the horizontal transfer of Cre recombinase from oligodendrocytes to neurons. By q-PCR, MOG transcripts were either undetectable or at the detection limit in the embryonic brain and turned up during the first postnatal week coinciding with the appearance of mature oligodendrocytes (Figure 1E). Therefore, it is unlikely that MOG-promoter activity in early embryonic progenitor cells is responsible for recombination persisting through neuronal differentiation into adulthood. The present study explores the possibility of horizontal transfer of molecules from oligodendrocytes to neurons by vesicles secreted from MVBs via the exosome pathway. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Neuronal recombination in MOGi-Cre/lacZ reporter mice. (A–D) Immunohistochemical analysis of brain sections derived from MOGi-Cre/Rosa26-LacZ reporter mice. β-galactosidase positive cells were stained using X-gal as substrate and neurons were labeled with antibodies against NeuN. Recombined cells were detected in the cortex (Ctx, A, C), cerebellum (Cb, B), and brainstem (Bst, D). Scale bar, 50 µm. (E) MOG and NG2 expression analysis by q-PCR of E10, E14, P0, P7, and adult total brains (n = 3). The maximal expression was set to 1. NG2 is shown as an example of a gene expressed in progenitor cells. https://doi.org/10.1371/journal.pbio.1001604.g001

Oligodendrocyte MVBs Are Present at Periaxonal Sites Exosomes are generated by inward budding of the endosomal limiting membrane and stored in MVBs before release. Ultrastructural examination of optic nerve or spinal cord myelinated fibers by electron microscopy revealed that MVBs are present in the cytoplasm of oligodendrocytes, including the innermost uncompacted wrapping of the myelin membrane (adaxonal loop) in close proximity to the axon (Figure 2A). In rare cases, we detected fusion profiles of MVBs indicating the release of intraluminal vesicles (exosomes) into the periaxonal space (Figure 2B). The MVBs occasionally carried immunolabeling of LAMP1 (not shown) or PLP in the MVB limiting membrane and the intraluminal vesicles. The quantification revealed that MVBs are most prominent in the adaxonal loop at periaxonal sites, compared to their localization in the outer abaxonal loop or in channels between myelin lamellae (Figure 2C). Among adaxonal MVBs, 29±8.8% carried immunolabeling for PLP. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Adaxonal localization of MVBs and glutamate-dependent release of exosomes. (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 (n = 5). (H) Administration of 50 (n = 5), 100 (n = 8), 200 µM (n = 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 (n = 3). Error bars, SEM (* p<0.05; Wilcoxon test). https://doi.org/10.1371/journal.pbio.1001604.g002 Taken together, these in vivo observations support the concept of exosomes playing a role in oligodendrocyte-neuron communication.

Glutamate Stimulates Oligodendroglial Exosome Release We first asked whether oligodendroglial exosome secretion is regulated by neuronal signals. Our previous work revealed that elevation of cytosolic Ca2+ levels stimulates exosome release [10]. Since neurotransmitters such as glutamate mediate Ca2+ signaling in oligodendrocytes [26], we investigated whether glutamate triggers oligodendroglial exosome release. Primary cultured oligodendrocytes were treated with glutamate for 5 h and exosomes were isolated from the supernatant by differential centrifugation. Oligodendroglial exosomes are known to include the myelin tetraspan protein PLP and its splice variant DM20 [10], which was utilized to identify oligodendrocyte-derived exosomes by Western blotting. The amount of PLP/DM20 detected in 100,000× g pellets obtained from supernatants of glutamate-treated cells was significantly increased compared to untreated cells, while total PLP expression in the cells was unaffected. Consistently, we observed higher levels of the exosomal marker proteins Alix and Hsc/Hsp70 in the 100,000× g pellet obtained from glutamate stimulated cells, indicating that more exosomes were secreted (Figure 2D). Previous studies showed that oligodendrocyte viability is affected by glutamate-mediated toxicity depending on intensity and duration of glutamate exposure as well as other side effects such as oxidative stress [27],[28]. We observed no apparent damage of cultured oligodendrocytes 5 h after glutamate administration (Figure S1A). To finally rule out that membrane fragments released from dying cells contaminated the exosome preparation, we investigated the integrity of the plasma membrane by evaluating lactate dehydrogenase (LDH) release and propidium iodide exclusion and found no detrimental influence of glutamate within the exosome collection period (Figure S1B and S1C). Moreover, we utilized the ER-localized protein calnexin to determine contamination with nonexosomal membranes, which were virtually absent in most preparations (Figure 2D). We further purified exosomes by sucrose density gradient centrifugation and detected PLP, Alix, and Hsc/Hsp70 in fractions of the typical density range reported for exosomes. Compared to untreated cells, the amounts of PLP, Alix, and Hsc/Hsp70 were increased, indicating that more exosomes had been isolated from glutamate-treated cultures (Figure 2E). The slightly different position of the exosomal marker proteins in the gradient may reflect different exosome subpopulations, consistent with the current view of exosome heterogeneity [12]. Nanoparticle tracking analysis revealed that glutamate-stimulated cells release significantly more particles with a mean size of 95 nm, reflecting the expected size of exosomes (Figure S1D). Notably, the size distribution of the released particles was not influenced by glutamate treatment. Examination of 100,000× g pellets derived from supernatants of glutamate-stimulated cells by electron microscopy identified larger aggregates of vesicles with the characteristic size and structure of exosomes as compared to pellets obtained from control cells, which released only few particles within the 5 h collection period (Figure 2F). To obtain further proof that glutamate acts on exosome secretion, we performed siRNA silencing of the small GTPase Rab35. Previous work has demonstrated that Rab35 regulates exosome secretion from oligodendrocytes [29]. Silencing of Rab35 in primary oligodendrocytes (by 60±6.8% on the protein level) interfered with the glutamate-dependent release of PLP and Alix demonstrating a specific effect of glutamate on exosome secretion (Figures 2G and S1E). Thus, the particles released from oligodendrocytes in response to glutamate are released in a Rab35-dependent manner, and match the marker profile, size, and the density of exosomes. Next, we determined the dose dependence of glutamate-mediated exosome release. A concentration of 50 µM was sufficient to stimulate exosome release and saturation was reached at the dose of 100 µM (Figure 2H). Moreover, we asked if extracellular Ca2+ is mobilized to stimulate glutamate-dependent exosome release and pre-incubated primary oligodendrocytes with EDTA, a nonmembrane permeable chelator of divalent cations, before exposure to glutamate. Depletion of divalent ions from the medium completely prevented stimulation of exosome release by glutamate, indicating that entry of extracellular Ca2+ through ionotropic glutamate channels is essential to trigger exosome release (Figure 2I). Analysis of intracellular Ca2+ levels using a fluorescent calcium indicator showed that the cells responded to glutamate with a rise in intracellular calcium (Figure S1F). Taken together, these results demonstrate that the neurotransmitter glutamate triggers Ca2+-dependent exosome release from oligodendrocytes.

Glial NMDA and AMPA Receptors Mediate Exosome Release Glutamate-mediated Ca2+ influx across the oligodendroglial plasma membrane occurs through ligand-operated Ca2+ channels, such as NMDA and AMPA receptors [26]. Expression of functional receptors and their subunits by mature oligodendrocytes and their precursors has been described previously [30]–[33]. We confirmed their presence in differentiated oligodendrocytes in vitro by immunocytochemistry, with the expected localization of NMDA receptors in the membrane sheets, while AMPA receptors appear preferentially on cell bodies and primary processes (Figure S2A, B) [34]. To investigate if these receptors regulate oligodendroglial exosome release, we stimulated cells with receptor-specific agonists to provoke Ca2+ entry. NMDA as well as AMPA application induced a significant increase in exosome secretion. Moreover, administration of both agonists had a synergistic effect (Figure 3A). Next, we co-applied glutamate with selective NMDA and AMPA receptor antagonists. MK801 and NBQX block oligodendroglial NMDA and AMPA receptors, respectively [32]. Both antagonists impaired glutamate-dependent exosome release (Figure 3B), though NMDA receptor inhibition affected exosome release more potently. When we applied the known NMDA receptor co-agonist D-serine [35] together with glutamate, D-serine potentiated glutamate-dependent exosome release over glutamate alone (Figure 3C). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. NMDA and AMPA receptors mediate exosome release. Quantification of exosome release upon (A) stimulation with glutamate receptor agonists NMDA (100 µM, n = 7), AMPA (100 µM, n = 5), or NMDA and AMPA (n = 4), (B) application of NMDA and AMPA receptor antagonists MK801 (5 µM, n = 9) or NBQX (25 µM, n = 9), or (C) the NMDA receptor co-agonist D-Serine (100 µM, n = 5). (D) Glutamate-stimulated exosome release from NMDA receptor-deficient oligodendrocytes obtained from CNPCre/+*NR1flox/flox mice (n = 6) compared to CNPCre/+ control mice (n = 9). Representative Western blots of PLP in exosome pellets are depicted. Error bars, SEM (n.s., not significant ; * p<0.05; ** p<0.01; Wilcoxon test). https://doi.org/10.1371/journal.pbio.1001604.g003 To obtain further evidence for the role of NMDA receptors in oligodendroglial exosome secretion, we used conditional knock-out mice lacking the essential NMDA receptor subunit NR1 selectively in oligodendrocytes (CNP+/Cre*NR1flox/flox) [36],[37]. The floxed NR1-locus is recombined by Cre, which is expressed under control of the CNP promoter. Conditional NR1-null mice were heterozygous for ROSA26-flox-stop-EYFP, which we used as reporter for Cre expressing cells. As expected, primary cultured oligodendrocytes derived from these mice expressed EYFP confirming efficient recombination in oligodendrocytes (unpublished data). Furthermore, NR1 was absent from mature oligodendrocytes expressing PLP (Figure S2A). We compared the relative exosome release in response to glutamate in cells lacking NR1 (CNP+/Cre*NR1flox/flox) and in control cells (CNP+/Cre*NR1+/+) and found that exosome release was not stimulated by glutamate in the absence of NR1 (Figure 3D). These results suggest that NMDA receptors are essential for the glutamate-dependent secretion of exosomes from oligodendrocytes.

Neuronal Activity Stimulates Glial Exosome Release Electrically active axons releasing glutamate evoke oligodendroglial Ca2+ signals [38]–[40]. To test if oligodendroglial exosome secretion is linked to neuronal electrical activity, we made use of a transwell device (Boyden chamber), allowing contact-free co-culture of cells and exchange of metabolites by diffusion through pores (Figure 4A). Cortical neurons grown for 7 d in vitro and placed on top of oligodendrocytes were depolarized with 20 mM potassium to trigger neurotransmitter release. Depolarization induced a significant increase in exosome secretion from oligodendrocytes (Figure 4B). Exposure of oligodendrocytes to potassium in the absence of neurons was ineffective. In addition, we treated cortical neurons grown for 14 d to allow synapse formation with the GABA A -receptor antagonist bicuculline to block inhibitory activity and enhance spontaneous glutamatergic activity [22]. Again, oligodendroglial exosome release was strongly increased in response to enhanced neuronal electrical activity (Figure 4C). Although bicuculline provoked a response of isolated oligodendrocytes, the stimulation of exosome release was 3 times more potent in the presence of neurons. To investigate if glutamate released by neurons acts on oligodendroglial NMDA receptors, we interfered with NR1 expression using RNAi. Silencing of NR1 in oligodendrocytes reduced the amount of secreted exosomes upon neuronal depolarization (Figures 4D and S2C). We obtained similar results using oligodendrocytes derived from conditional NR1 null mice (Figure S2D). These findings suggest that glutamate released by neurons in response to depolarization activates oligodendroglial glutamate receptors (mainly of the NMDA receptor subtype) stimulating exosome secretion. Thus, oligodendroglial exosome release is linked to neuronal electrical activity. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Neuronal activity stimulates exosome release from oligodendrocytes. (A) Cartoon illustrating the Boyden chamber co-culture of cortical neurons (CN) and pOL separated by a membrane permitting exchange of metabolites and small particles (<1 µm). (B) Application of 20 mM KCl (n = 9) or (C) 60 µM bicuculline (n = 6) to CN in the top well and analysis of relative exosome release from pOL. KCl or bicuculline applied to pOL is depicted as control (n = 5). (D) Boyden chamber co-culture of CN and pOL transfected with siRNA against NR1 or control siRNA. Depolarization of CN with 20 mM KCl and quantification of oligodendroglial exosome release (n = 3). Error bars, SEM (* p<0.05; ** p<0.01; Wilcoxon test). https://doi.org/10.1371/journal.pbio.1001604.g004

Neurons Internalize Glial Exosomes We further studied the fate of the released exosomes and asked whether exosomes can be internalized by other neural cells. We utilized the transwell co-culture system and cultured oligodendrocytes labeled with the fluorescent dye PKH67 on porous filters, allowing the passage of exosomes. PKH67 is a lipophilic dye that is released in association with exosomes [41]. Cortical neurons or glial cultures were placed in the bottom chamber and imaged to visualize the uptake of fluorescent membrane particles in different neural cell types. We observed internalization of oligodendroglia-derived particles by 20.7±7% of the cortical neurons and by 93.4±3.5% of the microglia, while uptake by astrocytes and oligodendrocytes was detected only in 3±1% and 2.2±0.4% of the cells, respectively (Figure 5A–D, see Figure S3 for overview). These data suggest that extracellular vesicles including exosomes released by oligodendrocytes are internalized preferentially by microglia and neurons. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Primary cortical neurons internalize oligodendroglial exosomes. (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, (n = 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 (n = 8). (G) To remove surface-bound exosomes, neurons were treated with trypsin (Tryp) before lysis (n = 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 (n = 6). (I) pOL were treated with 100 µM glutamate stimulating exosome release (n = 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 (n = 4). Error bars, SEM (* p<0.05; Wilcoxon-test). https://doi.org/10.1371/journal.pbio.1001604.g005 To confirm exosome uptake by neurons, we exposed primary cortical neurons to density gradient purified exosomes derived from the oligodendroglial cell line Oli-neu ectopically expressing SIRT2-EYFP and PLP-EGFP. Both proteins are sorted to oligodendroglial exosomes (Figure 5E) [10]. PLP-EGFP is located in the exosomal membrane, while SIRT2-EYFP is intraluminal. Incubation of neurons with these exosomes led to an uptake of fluorescent particles (Figure 5E). Furthermore, neurons incubated with purified exosomes accumulated glial-specific, exosome-associated PLP and SIRT2 with time (Figure 5F). Exosomes were indeed internalized, since removal of surface-bound exosomes by protease treatment before the lysis did not prevent the detection of PLP-EGFP/SIRT2-EYFP in neurons (Figure 5G). Transfer of exosomes can also be visualized after co-culture of primary cortical neurons with Oli-neu cells in Boyden chambers. We utilized the neutral sphingomyelinase inhibitor GW4869, which inhibits the release of exosomes [42]. Application of GW4869 to Oli-neu cells reduced the amount of secreted and thus internalized exosomes significantly, verifying that exosomes were responsible for the horizontal transfer of proteins and not other cell-derived particles (Figure 5H). Moreover, the transfer appears to be selective, since untagged EGFP overexpressed in Oli-neu cells is not delivered to neurons (Figure S4). We further employed primary oligodendrocytes to examine exosome transfer to neurons. Exosome-associated PLP/DM20 as well as SIRT2 were detectable in neurons co-cultured with oligodendrocytes in Boyden chambers (Figure 5I). Stimulation of oligodendroglial exosome release with glutamate significantly increased the level of PLP/DM20 and SIRT2 associated with neurons, demonstrating that neuronal uptake correlates with the amount of exosomes. To identify the subcellular destination of internalized exosomes, we performed double-labeling experiments of PKH67-stained glial exosomes together with endocytic markers. PKH67-stained exosomes accumulated within the neurons in endosome-like structures partly overlapping with LAMP1-positive late endosomes/lysosomes (Figure 5J). A 3D reconstruction of confocal images confirmed that exosomes were located inside the neurons (Figure S5A). To exclude that excess dye instead of stained exosomes was taken up by neurons, we pelleted PKH67-labelled exosomes and incubated neurons with exosomes and with exosome-depleted supernatant, respectively. PKH67-positive particles only associated with neurons when the exosome pellet was used, ruling out a transfer of excess dye (unpublished data). To determine whether neurons internalize oligodendroglial exosomes by endocytosis, we pretreated neurons with Dynasore and Pitstop-2 inhibiting dynamin and clathrin-dependent endocytosis, respectively [43],[44]. Dynasore and Pitstop2 application, as well as inhibition of actin polymerization by Cytochalasin D treatment, reduced the neuronal uptake of oligodendroglial exosomes (Figures 5K and S5B,C). Impeding cholesterol-dependent (clathrin-independent) endocytosis by Methyl-β-Cyclodextrin application, which sequesters cholesterol from the plasma membrane, did not affect the uptake (Figure S5C). Importantly, oligodendroglial cells secreted normal levels of exosomes in the presence of the inhibitors (Figure S5D). Similar results were obtained utilizing the neuronal cell line HT22 (Figure S5E–G). Expression of dominant negative dynamin K44A in HT22 cells inhibited endocytosis of Transferrin-Alexa568 and uptake of exosomes (Figure S5H–J). These results indicate that neurons internalize oligodendroglial exosomes via an endocytic pathway that requires dynamin, clathrin, and actin polymerization.

Retrieval of Exosome Cargo by Neurons Targeting of oligodendroglial exosomes to endosomes and late endosomes in neurons may result in lysosomal degradation or the recovery of exosomal components. In addition to protein cargo, oligodendroglial exosomes include distinct RNA species (Figure S6). To analyze, if the exosomal cargo is functionally retrieved by neurons, we employed Cre-mediated recombination as a reporter. Primary oligodendrocytes were infected with a replication-deficient recombinant Adeno-associated virus (AAV) vector encoding Cre-recombinase under control of the oligodendrocyte-specific MBP promoter (AAV/MBP-Cre) prior to co-culture with neurons in transwells [45]. These neurons had been transduced with reporter virus delivering the ubiquitous chicken β-actin promoter, followed by a floxed transcriptional termination element and the GFP coding region (AAV/CBA-floxstop-GFP). Reporter protein expression in neurons is specifically induced upon Cre-mediated excision of the floxed sequence [46]. Indeed, neurons acquired GFP reporter expression upon co-culture with AAV/MBP-Cre–infected oligodendrocytes (Figure 6A,B). Inhibition of glial exosome secretion by the sphingomyelinase inhibitor GW4869 (Figure 6C,F) or siRNA-mediated knockdown of Rab35 (Figure 6E) interfered with neuronal GFP expression, while stimulation of oligodendroglial exosome release with glutamate enhanced reporter expression (Figure 6D). Neurons incubated with oligodendroglial culture supernatant depleted from exosomes by 100,000× g centrifugation did not acquire GFP expression (Figure 6F). Furthermore, co-culture of reporter-virus–infected neurons and AAV/MBP-Cre–infected HEK cells or Oli-neu cells, which do not synthesize MBP promoter-driven Cre, did not result in GFP expression (Figure 6G), demonstrating that Cre expression in the donor cells is required and excluding a potential leakiness of the viral expression system. Consistent with these results, Cre protein, as well as Cre mRNA, were detectable in exosomes pelleted from supernatants of AAV/MBP-Cre–infected oligodendrocytes (Figure 6H,I). Taken together, these results demonstrate that the cargo of glial exosomes internalized by neurons is functionally retrieved within the target cells. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Functional retrieval of exosomal cargo by neurons. (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 (n = 5) were analyzed 14 d after injection. https://doi.org/10.1371/journal.pbio.1001604.g006 To provide the proof of principle that exosome transfer to neurons can occur in vivo, exosomes derived from AAV/MBP-Cre–infected oligodendrocytes were stereotactically injected into the cerebellum and hippocampus of adult Rosa26-lacZ reporter mice. Exosome internalization and cargo retrieval labels target cells as β-galactosidase-positive cells. In the injected hippocampus, individual neurons within the pyramidal cell layer of the CA3 region were positive for β-gal and neuron-specific enolase (eight cells per injection, n = 5, Figure 6J,K). In the cerebellum, we observed single recombined cells, which carried the GABA-R α6 subunit, a marker of cerebellar granule cells (five cells per injection, n = 5, Figure 6L). The number of detected recombined neurons may be limited by spatial restraints of the injected exosomes and by the fact that the retrieval of Cre from exosomes requires several steps before recombination can take place. Recombined cells were only present in animals injected with Cre-exosomes and never detected in brains of control mice injected with glial exosomes lacking Cre or at the contra-lateral side of injection. These experiments demonstrate that oligodendroglial exosomes can be internalized by neurons in vivo and validate the concept that exosome-mediated transfer of Cre from oligodendrocytes to neurons may underlie the neuronal reporter gene recombination observed in oligodendrocyte-specific Cre-driver mice (Figure 1).

Exosome Uptake Occurs at Axonal and Somatodendritic Sites We utilized microfluidic chambers to examine whether exosomes are internalized at the somatodendritic or the axonal domain. Cortical neurons cultured with their cell bodies in one compartment of the chamber grow axons through microgrooves into the other compartment of the chamber. Isolated exosomes labeled with PKH67 dye or containing Cre recombinase were added either to the somatodendritic or the axonal compartment and neuronal exosome uptake was monitored by imaging of exosomes or Cre reporter detection (Figure 7). Internalization of fluorescent exosomes was visible at the somatodendritic as well as at the axonal domain of the neurons. After application of Cre-bearing exosomes, we quantified the number of recombined neurons located within an area 100 µm from the microgrooves, thus focusing on neurons having the chance to project axons through the microgrooves into the axonal compartment (not all neuronal cell bodies in this area will successfully grow axons through the microgrooves). The number of recombined neurons did not differ significantly upon somatodendritic or axonal addition of exosomes. Thus, uptake of exosomes is possible at both sites. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. Somatodendritic and axonal uptake of exosomes. 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 (n = 4). https://doi.org/10.1371/journal.pbio.1001604.g007