1. Volume 20, Issue 1, Pages 99-111 (July 2017)
Exosomes Mediate Mobilization of Autocrine Wnt10b to Promote Axonal Regeneration in the Injured CNS 
Nardos G. Tassew, Jason Charish, Alireza P. Shabanzadeh, Valbona Luga, Hidekiyo Harada, Nahal Farhani, Philippe D’Onofrio, Brian Choi, Ahmad Ellabban, Philip E.B. Nickerson, Valerie A. Wallace, Paulo D. Koeberle, Jeffrey L. Wrana, Philippe P. Monnier 
Cell Reports 
Volume 20, Issue 1, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 99-111DOI: (10.1016/j.celrep.2017.06.009)
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3. Figure 1 Exosomes Promote Axonal Growth on an Inhibitory Substrate
(A) Exosomes (Exo) derived from L-cell media conditioned for 3 days in vitro were purified. Western blot was performed on purified exosomes using the endoplasmic reticulum marker calnexin and two exosomal markers Cd81 and Flotillin-1. As expected exosomes were negative for calnexin and positive for Cd81 and Flotillin-1.
(B and C) Cortical neurons cultured on myelin for 2 days in the presence of PBS (Ctrl), exosomes (Exo; 50 ng/mL), or exosomes pretreated with 10 mM MβCD (Exo + MβCD). Representative images of neurites visualized by βIII tubulin staining and immunofluorescence microscopy are shown. Scale bars, 50 μm. (C) Axonal lengths were quantified and plotted as mean length per neuron ± SEM of three independent experiments. ∗p <
(D) Representative images of embryonic retinal explants cultured on myelin after the addition of PBS (Ctrl), exosomes (Exo; 50 ng/mL), or exosomes pretreated with 10 mM MβCD (Exo + MβCD). Scale bars, 100 μm.
(E) Exosomes significantly increased outgrowth on myelin, and this effect was suppressed by MβCD. Error bars indicate SEM; n = 3 independent experiments. ∗p <
(F) Representative images of embryonic retinal explants cultured on myelin after the addition of FD exosomes (FD-exo, 50 ng/mL), HEK293-cell-derived exosomes (HEK-exo; 50 ng/mL), and COS-7-cell-derived exosomes (COS-Exo; 50 ng/mL). Scale bars, 100 μm.
(G) HEK293- and COS-7-derived exosomes did not increase outgrowth on myelin. Error bars indicate SEM; n = 3 independent experiments. ∗p <
(H) Representative images of retinal explants cultured on myelin after the addition of PBS plus increasing exosome concentrations (0, 25, and 100 ng/mL). Scale bars, 100 μm.
(I) Exosomes show a concentration-dependent effect on axons growing on myelin. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05.
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4. Figure 2
Exosomes Promote Axonal Growth of Multiple Axons on Both Myelin and CSPGs
(A) Representative images of adult rat DRG neurons cultured on myelin (50 μg/mL) for 24 hr after the addition of PBS (Ctrl) or exosomes (Exo; 50 ng/mL). Scale bars, 50 μm.
(B) Exosomes significantly increased DRG-axon outgrowth on myelin. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05.
(C) Representative images of postnatal mouse retinal neurons cultured on myelin for 24 hr after the addition of PBS (Ctrl) or exosomes (Exo; 50 ng/mL). Scale bars, 50 μm.
(D) Exosomes significantly increased mouse RGC-axon outgrowth on myelin. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05.
(E) Representative images of embryonic retinal explants cultured on CSPGs (10 μg/mL) after the addition of PBS (Ctrl) or exosomes (Exo; 50 ng/mL).
(F) Exosomes significantly increased outgrowth on CSPGs. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05.
(G) Cortical neurons pre-incubated with exosomes expressing Cd81-EYFP show internalization of the eYFP marker. βIII-tubulin staining confirm that these cells are neurons. The rightmost panel presents the inset of the Merge panel. Scale bars, 25 μm.
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5. Figure 3
Exosome-Mediated Recruitment of Wnt10b Promotes Axonal Growth on Myelin
(A–D) Cortical neurons co-transfected with TRITC-labeled siRNAs (red) plus different siRNAs and were plated on myelin and treated with exosomes (Exo). After fixation, staining for βIII tubulin was performed (green). Overlay of the staining is presented. (A) siRNA for Porcupine (Porc.) altered outgrowth stimulation by exosomes. Scale bars, 50 μm. (B) Silencing of Porcupine significantly suppressed the growth stimulation. n = 3 independent experiments. ∗p < (C) An siRNA for Wnt10b altered outgrowth stimulation by exosomes. Scale bars, 75 μm. (D) Wnt10b silencing significantly suppressed growth stimulation by exosomes. n = 3 independent experiments. ∗p < 0.05.
(E) Immunostaining for Wnt10b showed expression in axonal growth cones.
(F) Wnt10b was expressed by cortical cells but not by exosome-producing L cells.
(G) Cortical neurons from wild-type (wt) and Wnt10b−/− animals were plated on myelin and treated with exosomes. Scale bars, 50 μm.
(H) Wnt10b knockdown significantly suppressed exosome-stimulated outgrowth. n = 3 independent experiments. ∗p < 0.05.
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6. Figure 4 Exosome-Mediated Recruitment of Wnt10b into Lipid Rafts
(A) PC12 cells expressing Wnt10b were incubated with or without FD exosomes (Exo.) for 1 hr. After collecting the cells, membranes were prepared, and western blotting analysis was performed. In four independent experiments, Wnt10b was not visible in the membrane of FD-exosome-treated cells. Wnt10b was observed in the membranes of PBS-treated cultures. Transferrin receptor (TfR) was used to ensure equal loading.
(B) PC12 cells expressing Wnt10b were incubated with PBS or exosomes for 1 hr. Whole-cell extracts were collected, and western blotting analysis was performed. The addition of exosomes induced a decrease of Wnt10b levels when compared to PBS-treated controls.
(C) Quantification of the Wnt10b levels shows that, after 1 hr of FD exosome treatment, levels for Wnt10b were significantly reduced. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05.
(D) Cortical neurons were grown on myelin and treated for 10 min with exosomes (+exo.; 50 ng/mL) and PBS (−exo.), and staining for βIII tubulin and Wnt10b was performed. Incubation with the Wnt10b antibody was performed before the addition of Triton (no Triton) to stain for cell-surface Wnt10b.
(E) FD exosomes (Exo.) significantly decreased the levels of cell-surface Wnt10b when compared to PBS treatment. To visualize total Wnt10b, we performed Triton X-100 treatment before the addition of the Wnt10b antibody. Levels of Total Wnt10b were not altered by 10-min treatment with FD exosomes. ∗p < 0.05, ∗∗p < 0.01; ns, not significant.
(F) PC12 cells expressing Wnt10b were incubated with PBS or exosomes for 1 hr. Cells were collected, and membrane fractionations were performed. In control experiments, Wnt10b localizes to the Transferrin receptor (TfR) containing heavy membrane fraction. In FD-exosome-treated cells, Wnt10b was not present in any fraction.
(G) PC12 cells expressing Wnt10b were incubated with chloroquine (200 μM) for 30 min, followed by 1-hr treatment with either PBS (control) or FD exosomes. Cells were collected and membrane fractionations were performed. In control chloroquine + PBS experiments, Wnt10b localizes to the TfR-containing heavy membrane fraction. In chloroquine + exosome experiments, Wnt10b mostly localizes to the Flotillin-containing lipid raft fraction (red lines).
(H) Quantification of the percentage of Wnt10b in lipid rafts and heavy membrane fractions in three independent repeats of experiments performed in (E) and (G). In chloroquine + exosomes, experiments show a significant re-localization of Wnt10b toward lipid rafts. Error bars indicate SEM; n = 3 independent experiments. ∗p < 0.05, ∗∗p < 0.01.
(I and J) Shown here: (I) immunolabeling of primary cortical neurons for Wnt10b (green) and Flotillin-1 (red) following 10-min treatment with either PBS or FD exosomes. Co-localization map (yellow) was generated from pixel-by-pixel calculation of co-localization coefficients. (J) Data indicate means ± SE of co-localization coefficients derived from single-cell, region-of-interest measurements from at least 60 cells from three independent experiments. ∗p < 0.05.
(K and L) Shown here: (K) immunolabeling of primary cortical neurons for Flotillin-1 (green) and Flotillin-2 (red) following 10-min treatment with either PBS or FD exosomes. Co-localization map (yellow) was generated from pixel-by-pixel calculation of co-localization coefficients. (L) Data indicate means ± SE of co-localization coefficients derived from single-cell, region-of-interest measurements from at least 60 cells from three independent experiments. ∗p < 0.05.
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7. Figure 5 Pathway by which Exosomes Promote mTOR Activation
(A) Cortical neurons transfected with plasmids for GFP, GSK3β+ GFP, TSC2+ GFP, and DN-TCF4+GFP were plated on myelin (50 μg/mL) and treated with FD exosomes (Exo.; 50 ng/mL). Scale bar, 100 μm.
(B) Expression of GSK3β and TSC2 significantly reduced outgrowth stimulation by FD exosomes (n = 3 independent experiments, ∗p <
(C) Retinal explants were grown on myelin plus FD exosomes (50 ng/mL) and PBS or rapamycin (Rapa). Scale bars, 100 μm. Ctrl, control.
(D) Rapamycin significantly abolished FD-exosome-mediated neurite growth. ∗p < 0.05.
(E) pS6K staining of growth cones in PBS- or FD-exosome-treated cortical neuron cultures.
(F) Presence of FD exosomes increased pS6K levels by ∼5-fold (n = 3 independent experiments). ∗p < 0.05.
(G) Cortical neuron cultures treated with PBS or exosomes (50 ng/mL) were submitted to western blotting analysis.
(H) In western blots, exosomes increased pS6K levels by ∼2-fold. ∗p < 0.05.
(I) βIII tubulin (β-Tub.) and pS6K staining of cortical neuron growth cones treated with (1) exosomes plus PBS, DKK1, and Wnt10b-antibody or (2) soluble (sol.) Wnt10b protein.
(J) Levels of pS6K were significantly reduced when DKK1 and Wnt10b antibody were given together with exosomes, when compared to exosomes + PBS. Soluble Wnt10b did not increase pS6K levels (n = 3 independent experiments). ∗p < 0.05.
(K) Schematic representation of the pathway by which Wnt10b stimulates mTOR.
Error bars indicate SEM.
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8. Figure 6
Exosomes Promote Axonal Regeneration following Optic Nerve Injury
(A) 4 μL PBS (Control) or exosomes (50 ng/μL) were injected in the vitreous of the rat eye, and DAPI and pS6K staining were performed. In retinal sections, staining appears predominant in the RGC layer. Scale bars, 150 μm.
(B) Exosome (Exo) injection significantly increases pS6K levels in the RGC layer. ∗p < Ctrl, control.
(C) Optic nerve injury was performed in adult rats, and two injections (at 3 and 10 days following injury) of 4 μL PBS (Control) or exosomes were injected in the vitreous of the eye (nasal side). Three weeks after injection, GAP-43 staining was performed to visualize regenerating fibers. Panels on the right show magnifications of the insets presented in the figures. Scale bars, 300 μm.
(D) The total number of regenerating axons was quantified within bins of the optic nerve, starting at the crush site, as follows: 0–250 μm, 250–500 μm, and >500 μm. Exosomes (n = 10) significantly promote axonal regeneration following optic nerve crush when compared to PBS (n = 10). ∗p < 0.01.
Error bars indicate SEM.
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9. Figure 7 Exosome-Mediated Axonal Regeneration and Cell Survival
(A) Optic nerve injury was performed in Wnt10b−/− and Wnt10b+/+ adult mice, and 2 μL of exosomes (50 ng/μL) were injected in the vitreous of the eye. Injections were performed at 3 and 9 days following injury on the temporal side and nasal side (1 μL each side). Three weeks after injection, GAP-43 staining was performed to visualize regenerating fibers. Panels on the right show magnifications of the insets presented in the figures. Scale bars, 250 μm.
(B) The total number of regenerating axons was quantified within bins of the optic nerve, starting at the crush site, as follows: 0–250 μm, 250–500 μm, and >500 μm. Following exosome injections, regeneration was significantly higher in Wnt10b+/+ (n = 7) when compared to Wnt10b−/− mice (n = 6). ∗p < 0.01.
(C) Optic nerve injury was performed in adult mice, and two injections (at 3 and 9 days following injury) of 2 μL of exosomes (40 ng/μL or 200 ng/μL) were injected in the vitreous of the mouse eye. Three weeks after injection, GAP-43 staining was performed to visualize regenerating fibers. Scale bars, 300 μm.
(D) The total number of regenerating axons were quantified within bins of the optic nerve, starting at the crush site, as follows: 0-250 μm,  μm,  μm,  μm > 1000 μm. High Exosome concentration (200ng/μl, n = 6) significantly promotes axonal regeneration when compared to lower concentration (40ng/μl, n = 6). ∗p < 0.01.
(E–G) FD exosomes increase cell survival following optic nerve injury. (E) Representative confocal micrographs of RGCs in flat-mounted retinas from adult rats. Panels show RGCs retrograde labeled with Fluorogold by injecting it into the superior colliculus 7 days before axotomy. Control retinas, injected with saline at 3 and 7 days after axotomy, had very few RGCs remaining at 14 days after axotomy, whereas exosome-injected retinas had many surviving RGCs. (F) Representation of the retinal areas selected in our analyses. (G) Quantification of the density of surviving RGCs at 14 days post-axotomy showing that FD exosomes significantly increased RGC survival.
Data are average ± SEM (n = 6 eyes per condition). Significant differences were determined by ANOVA, followed by Tukey’s test. ∗p < 0.01.
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