RESEARCH ARTICLE

Schwann Cell-Derived Exosomes EnhanceAxonal Regeneration in the Peripheral

Nervous System

Mar�ıa Alejandra Lopez-Verrilli,1 Frederic Picou,1 and Felipe A. Court1,2

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCsdedifferentiate to a progenitor-like state and efficiently guide axons to their original target tissues. Contact and soluble fac-tors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCssecrete nano-vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC-derived exosomesmarkedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift thegrowth cone morphology to a pro-regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growthcone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neigh-boring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by whichthese cells locally support axonal maintenance and regeneration after nerve damage.

GLIA 2013;00:000–000Key words: Schwann cell; exosomes; axonal regeneration; nerve injury

Introduction

Axonal regeneration is the underlying process for func-

tional recovery after nervous system damage due to

mechanical, toxic, or inflammatory processes (Allodi et al.,

2012). In the peripheral nervous system (PNS), Schwann cells

(SCs) greatly contribute to axonal regeneration after nerve

injury (Webber and Zochodne, 2010). Axons do not regener-

ate in the central nervous system (CNS) due to diminished

intrinsic regenerative capacities and lack of glial support. After

peripheral nerve damage, SCs associated to degenerating

axons dedifferentiate and proliferate, remove myelin and axo-

nal debris, and align to support axonal regeneration (Chen

et al., 2007). Regenerating axons, in turn, express adhesion

molecules that promote SC alignment and migration (Yamau-

chi et al., 2008). This bidirectional communication allows

coordination of neuron and SC programs during nerve repair

as well as during development and maintenance of adult

nerve fibers (Chen et al., 2005). The pro-regenerative capaci-

ties of SCs have been exploited to enhance regeneration in

the CNS (Fortun et al., 2009; Toft et al., 2013), an effect

suggested to depend on extracellular matrix proteins and neu-

rotrophic factors expressed by SCs.

Intercellular transfer of macromolecules through vesicles,

known as exosomes, has become the subject of increasing

interest as a novel means for intercellular crosstalk. Exosomes

are 50–100 nm diameter vesicles contained within multivesic-

ular endosomes that are released to the extracellular milieu

constitutively or after specific stimuli (Thery et al., 2009).

Exosomes carrying mRNAs, miRNAs and proteins are

secreted by several cell types in vitro and can be detected in

biological fluids in vivo (Record et al., 2011). In particular,

glial cells from the CNS have been shown to secrete exosomes

(Lopez-Verrilli and Court, 2012). Oligodendrocyte-derived

exosomes inhibit myelin formation (Bakhti et al., 2011),

astrocytes secrete exosomes upon heat shock stress (Taylor

et al., 2007) and microglial cells release exosomes with pro-

teolytic activity (Tamboli et al., 2010). Nevertheless, the

secretion of exosomes by SC has only been associated to a

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22558

Published online in Wiley Online Library (wileyonlinelibrary.com). Received Jan 30, 2013, Accepted for publication July 12, 2013.

Address correspondence to Felipe A. Court, Av. B. O’Higgins 340/Casilla 114-D, Santiago 8331150, Chile. E-mail: fcourt@bio.puc.cl

From the 1Millennium Nucleus for Regenerative Biology, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile; 2Neurounion Bio-

medical Foundation, Santiago, Chile.

Additional Supporting Information may be found in the online version of this article

VC 2013 Wiley Periodicals, Inc. 1

means for the propagation of infective prions (Fevrier et al.,

2004).

Here, we explored the possibility that exosome transfer from

SC to axons regulates intrinsic mechanisms of neuronal regenera-

tion, contributing to the efficient regeneration and functional

recovery characteristic of damaged peripheral nerves. We demon-

strate that SCs secrete exosomes that can be selectively internalized

by axons in vitro and in vivo. We further show that SC-secreted

exosomes, but not fibroblast-derived ones, markedly increase axo-

nal regeneration by acting locally on axons and decreasing RhoA

GTPase activation in growth cones. Finally, we demonstrate invivo that the regenerative capabilities of injured sciatic nerves can

be greatly enhanced by delivering SC-derived exosomes to axons.

In summary, our work shows that vesicular transfer

from SCs to axons constitutes a novel mechanism for increas-

ing axonal regeneration and a potential therapeutic strategy to

promote regeneration in regions with poor regenerative capa-

bilities as is the CNS.

Materials and Methods

AnimalsMale Sprague Dawley (SD) rats were used. Experiments with ani-

mals followed protocols approved by the Institutional Animal Care

and Use Committees and complied with National Institutes of

Health guidelines.

SC and Fibroblast Primary CulturesSC primary cultures were obtained from newborn SD rat sciatic nerves

as previously described (Wilby et al., 1999). The perineurum was

removed and explants were dissociated with 0.05% collagenase for 30

minutes (min) followed by 0.25% trypsin for 5 min at 37�C. Cells

were plated on laminin (40 ng/mL) in Dulbecco’s Modified Eagle

Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine

serum (FBS, Invitrogen) and 10% penicillin–streptomycin (Invitro-

gen). The following day, cells were treated with 10 mM cytosine arabi-

noside (Sigma). After 1 week in culture, contaminant fibroblasts were

eliminated by complement-mediated cell lysis using IgM class anti-Thy

1.1 antibodies (Serotec) and rabbit complement (Sigma). SCs were

maintained in DMEM-10%, FBS-1%, penicillin–streptomycin sup-

plemented with 2 mM forskolin (Millipore) and 20 mg/mL bovine pitu-

itary extract (Invitrogen). These cultures were highly enriched in SC

(98 6 0.4%) based on immunostaining with the SC marker S100 (rab-

bit polyclonal 1:500, Dako). Primary fibroblast cultures were obtained

from newborn SD rats as previously described (Dreesmann et al.,

2009) and characterized by immunofluorescence using an anti-Thy1.1

antibody (rabbit polyclonal 1:500, Serotec) (Supp. Info. Fig. 1).

Exosome Purification and CharacterizationSC primary cultures (�2 3 107 cells) were supplemented with DMEM

containing 2 mM forskolin, 20 mg/mL bovine pituitary extract and 10%

exosome-free FBS (obtained by serum ultracentrifugation at 100,000g

for 12 h). Fibroblast primary cultures (�2 3 107 cells) were supple-

mented with DMEM containing 10% exosome-free FBS in the absence

or presence of forskolin and bovine pituitary extract. After 48 h, the

supernatant was subjected to serial centrifugations (300g for 10 min,

2000g for 10 min, 10,000g for 30 min at 4�C), followed by ultracentri-

fugation at 100,000 g for 70 min at 4�C (T865 rotor, OTD Combi

Sorvall ultracentrifuge, Dupont). The pellet containing exosomes was

washed in cold 0.1M phosphate buffer saline, pH 7.4 (PBS) and ultra-

centrifuged again at 100,000g for 70 min at 4�C. In each exosome prep-

aration, the concentration of total proteins was quantified by Bradford

assay (BioRad). Exosomes were characterized according to their density

over a discontinuous sucrose gradient (0.25–2 M) as described (Thery

et al., 2006). Briefly, exosomes (resuspended in 2 M sucrose) were

poured at the bottom of the centrifuge tube and the sucrose gradient

was laid on top of the exosomes. The gradient was ultracentrifuged over-

night at 100,000g, 4�C (TST60.4 swinging-bucket rotor, OTD Combi

Sorvall ultracentrifuge, Dupont). The next day, twelve fractions (0.3 mL

each) were collected from top to bottom and prepared for Western-blot

analysis as previously described (Thery et al., 2006).

Western-blotSCs and purified exosomes were homogenized in 1% Triton X-100,

50 mM Tris-HCl, pH 6.8, 2 mM EDTA, 1% PMSF and 1% prote-

ase inhibitor cocktail (Sigma). Proteins were quantified by Bradford

assay and 10 mg of SC lysate or 5 mg of exosomes lysate were com-

bined with loading buffer, subjected to 10% SDS-PAGE electropho-

resis, and transferred to polyvinylidene fluoride membranes (Pierce).

The following primary antibodies were used: anti-CD63 (rabbit pol-

yclonal 1:1000; Santa Cruz Biotechnology), anti-p75NTR (rabbit pol-

yclonal 1:1000, Upstate), anti-flotillin-1 (rabbit monoclonal 1:500,

Upstate), anti-heat shock protein 70 (Hsp70, mouse monoclonal

1:1000, Stressgen), anti-heat shock protein 90 (Hsp90, rabbit poly-

clonal 1:1000, Stressgen), anti-TSG101 (goat polyclonal 1:500,

Santa Cruz Biotechnology), anti-Rab 5 (rabbit polyclonal 1:500,

Abcam), anti Na1/K1 ATPase b1 (mouse monoclonal 1:500, Santa

Cruz Biotechnology). Secondary antibodies used were goat anti-

mouse HRP and goat anti-rabbit HRP (BioRad). Western blots were

revealed by enhanced chemiluminescence (Pierce).

Immunoisolation and FACS AnalysisSC exosomes were immunoisolated from conditioned media as pre-

viously described (Ostrowski et al., 2010). Briefly, 4 lm aldehyde-

sulfate beads (Invitrogen) were coupled to 40 lg of mouse anti-

p75NTR or mouse anti-IgG antibodies (the same isotype but directed

against neurofilament medium chain, Sigma) overnight at 4�C. SCs

Abbreviations

CNS Central nervous systemDRG Dorsal root gangliaEM Electron microscopyDMEM Dulbecco’s Modified Eagle MediumFBS Fetal bovine serumGST Glutathione S-transferaseNGF Nerve growth factorPBS Phosphate buffer salinePFA ParaformaldehydePNS Peripheral nervous systemSC Schwann cellSD Sprague Dawley

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(� 2 3 105 cells) were incubated with culture medium during 48

and 120 h, the conditioned media was subjected to serial centrifuga-

tion. After that, the supernatant after the 10,000g step was incubated

with anti-p75NTR or anti-IgG-coupled beads overnight at 4�C. The

next day, beads were washed twice in PBS-2% BSA and incubated

with 1:20 PE-Annexin V (BD Biosciences) as previously described

(Ostrowski et al., 2010). Beads were acquired on a FACSCalibur

flow cytometer (BD) and data were analyzed with FCS Express 4

Plus software (De Novo).

For pull down assay, 4-lm aldehyde-sulfate beads (Invitrogen)

were coupled to 40 lg of anti-CD63 or anti-p75NTR antibody

(goat polyclonal, Santa Cruz Biotechnology). Then, SCs (� 2 3 105

cells) were incubated with culture medium as described above and

the supernatant after the 10,000g step was incubated with anti-

p75NTR or anti-CD63-coupled beads overnight at 4�C. The next

day, beads were washed in PBS, incubated with loading buffer and

analyzed by western-blot using anti-p75NTR (rabbit polyclonal

1:1000, Upstate) or anti-CD63 (rabbit polyclonal 1:1000; Santa

Cruz Biotechnology) antibodies as described above.

DRG Explant CulturesDorsal root ganglia (DRGs) were obtained from day 16 SD rat

embryos. Briefly, DRGs were dissected and placed on coverslips

coated with rat tail collagen (Invitrogen). DRGs were maintained in

Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invi-

trogen), 2 mM L-glutamine, 50 ng/mL human nerve growth factor

(NGF; Invitrogen) and 1% penicillin–streptomycin. In all experi-

ments, DRGs were treated with 10 mM cytosine arabinoside (Sigma)

to eliminate non-neuronal cells. Axonal regeneration was evaluated

by two methods.

METHOD 1. Intact DRGs were plated, and the next day PBS

(control) or exosomes (3 mg resuspended in PBS) were added on a

daily basis for 4 days. Axonal regeneration was measured in mm2

using the Image J software.

METHOD 2. Seven day-old DRG axons were transected and a

bright field picture was taken immediately after injury. Thereafter,

exosomes or PBS supplementation was started and continued daily

for 4 days. Axonal regeneration was measured from the cut edge to

the tip of the longest axon and the length of the 10 longest regener-

ating axons per DRG were measured. Experiments were completed

with 4–5 replicates per condition with a minimum of 10 axons per

coverslip. To test the effect of exosomal membrane proteins or exo-

some content in regeneration experiments, SC exosomes purified by

ultracentrifugation as described above were incubated with 0.25%

trypsin, distilled water or both during 10 min at 37�C. After treat-

ment, exosomes were washed in PBS, ultracentrifuged and resus-

pended in PBS.

Compartmentalized DRG CulturesDRG explants were obtained as described above and then plated in

the proximal compartment of Campenot chambers as previously

described (Campenot et al., 2009). DRG axons were allowed to

growth under high-vacuum grease (Fisher Scientific) barriers and

into left and right distal compartments for 7 days. Then, axons were

axotomized in the distal compartment with distilled water and axo-

nal regeneration was evaluated by adding PBS (control) or exosomes

in each compartment. Axonal regeneration was measured from the

grease barrier to the tip of the longest axon in the track. Regenerat-

ing axons were identified by staining the cell bodies compartment

with the vital dye FM1-43 (2 mM, Invitrogen) and pictures were

taken with a fluorescent microscope. Then DRGs were fixed with

4% paraformaldehyde (PFA) and subjected to immunofluorescence

using a polyclonal rabbit anti-heavy chain neurofilament antibody

(rabbit polyclonal 1:1000, Sigma). Experiments were completed with

five replicates per condition and the length of the 10 longest regen-

erating axons per chamber were measured in the axonal

compartment.

ImmunofluorescenceImmunofluorescence was performed as previously described (Barrien-

tos et al., 2011). Briefly, cells were fixed with 4% PFA in PBS for

10 min, followed by three 10 min washes in PBS and then blocked/

permeabilized in 1% Triton X-100, 5% fish skin gelatin (Sigma) in

PBS for 30 min at room temperature. Cells were incubated in pri-

mary antibodies in blocking/permeabilizing solution overnight at

4�C, washed three times in PBS for 10 min, and incubated with

fluorescent-labeled secondary antibodies (Invitrogen) for 2 h at room

temperature. Cells were washed in PBS and mounted in Vectashield

(Vector Laboratories). To evaluate the morphology of growth cones

in experiments of regeneration, staining with rhodamine-conjugated

phalloidin (1:1000, Invitrogen) was performed. In each experiment,

approximately 150 growth cones were counted per each condition.

The RhoA activity assay on growth cones was performed as

described (Gehler et al., 2004). Regenerating axons were fixed with

4% PFA. Rho-binding domain (RBD) of Rhotekin fused with gluta-

thione S-transferase (GST) was produced as previously described

(Carcamo et al., 2006) and 2 mg/mL were supplemented to DRG

cultures for 4 h. Then DRG cultures were incubated with 1:400

polyclonal rabbit anti-GST (Abcam) in blocking/permeabilizing solu-

tion overnight at 4�C. Cells were rinsed and incubated with

fluorescent-labeled secondary antibody and rhodamine-conjugated

phalloidin (Invitrogen). Activated RhoA was quantified by measuring

fluorescence intensity in each growth cone and activated RhoA was

normalized against growth cone area given by phalloidin-rhodamine

staining. Approximately 200 growth cones were analyzed per condi-

tion in three independent experiments. Exclusion of GST-RBD

resulted in no staining.

Internalization Assays

METHOD 1. Internalization in co-cultures between SCs and

DRGs was evaluated with classical Boyden chambers (BD Falcon,

0.4 mm pore size) as previously described (Schoenmann et al.,

2010). Briefly, SCs were grown on the filter membrane for 2 days,

and transfected with a CD63::eGFP-N3 plasmid and Lipofectamine

2000 reagent (Invitrogen). Seven-day-old DRG explants (on cover-

slips) were added to the bottom chamber one day after SCs transfec-

tion. Co-cultures were maintained for 2 days to allow the secretion

of CD63::eGFP exosomes from SCs and internalization by DRGs

Lopez-Verrilli et al.: Glial exosomes promote axonal regeneration

Month 2013 3

was evaluated by fluorescent microscopy after immunostaining with

a confocal microscope.

METHOD 2. Exosome internalization in DRG axons was

observed using vital dyes, followed by immunofluorescence and con-

focal microscopy. Briefly, exosomes were incubated with PKH67

green fluorescent cell linker kit for cell membrane labeling (Sigma)

as previously described (Lasser et al., 2011). As negative control,

PBS was also incubated with PKH67. Stained exosomes and control

were washed three times with PBS, concentrated using Amicon Ultra

10 kDa tubes (Millipore) and added to DRG explants for 30 min, 2

and 4 h. DRGs were washed with PBS and fixed with 4% PFA.

Immunofluorescence was performed as described above using an

anti-heavy chain neurofilament antibody (rabbit polyclonal 1:1000,

Sigma) and an anti-p75NTR (mouse monoclonal 1:100). The pres-

ence of PKH-stained exosomes in growth cones was quantified over

three separated experiments, n 5 110 growth cones, using a confocal

microscope.

METHOD 3. Ultrastructural analyses of exosome internalization

were performed by electron microscopy (EM). Anti-p75NTR (2 mg,

mouse monoclonal) or anti-acetylated-tubulin (2 mg, mouse mono-

clonal, Sigma) antibodies were conjugated to an anti-mouse gold-

IgG (1:100; 15 nm diameter gold, BB International). Exosomes

were purified from SC cultures by differential centrifugation and

then incubated with p75NTR or acetylated tubulin immunocom-

plexes during 30 min on ice. Then the mix was pelleted by ultracen-

trifugation on a 30% sucrose/D2O density cushion (100,000g for 70

min at 4�C) as described (Thery et al., 2006). In this way, the

immuno-labeled exosomes are purified from the non-bound immu-

nocomplexes. The exosome interface was collected and recovered by

a final ultracentrifugation after dilution with PBS. Finally, the pellet

containing immunogold-labeled exosomes was resuspended in 200

mL PBS and added to DRG explants for 4 h. DRGs were fixed over-

night in 2.5% glutaraldehyde, 0.01% picric acid, 0.1M cacodylate

buffer, pH 7.4. Cells were rinsed in the same buffer, immersed in

1% OsO4 for 1 h followed by incubation with 2% uranyl acetate

for 2 h. Cells were dehydrated with a graded series of ethanol, pro-

pylene oxide, and infiltrated with Epon (Ted Pella). Ultrathin sec-

tions were contrasted with 1% uranyl acetate and lead citrate. In

addition, exosomes incubated with p75NTR-gold and tubulin-

acetylated-gold immunocomplexes were negative-stained as previ-

ously described (Thery et al., 2006). Briefly, exosomes were depos-

ited on Formvar-carbon-coated EM grids, fixed with 2% PFA and

contrasted with uranyl acetate. All the grids were examined with a

Philips Tecnai 12 electron microscope operated at 80 kV. Negative

films were developed and scanned.

Surgical ProceduresIn all experiments, young male SD rats (60 g) were anesthetized

intraperitoneally with ketamine-xylazine (75 mg/kg and 10 mg/kg,

respectively). The sciatic nerve was crushed at the sciatic notch three

times for 10 sec with Dumont #5 forceps. The crush site was

marked with surgical 10-0 nylon monofilament (Ethicon). Exosomes

from SCs were purified by ultracentrifugation and resuspended in

sterile 0.9% NaCl. Two microliters of SC exosomes (2 mg/mL) or

vehicle (0.9% NaCl) were injected daily into the distal part of the

sciatic nerve at 25 mm from the crush site. Four days after crush,

sciatic nerves were fixed in situ in 4% PFA in PBS for 1 h, washed

in PBS and infiltrated with 30% sucrose and frozen at 280�C. Lon-

gitudinal cryostat sections (10 mm) were blocked/permeabilized in

PBS containing 5% fish skin gelatin (Sigma) and 0.2% Triton X-

100 for 1 h at room temperature and incubated overnight in the

same solution with rabbit anti-growth-associated protein 43

(GAP43, rabbit polyclonal 1:1000, Abcam). After washing, the prep-

arations were incubated for 2 h at room temperature with 1:1000

AlexaFluor 488-goat anti-rabbit IgG, washed in PBS, mounted in

Vectashield (Vector Laboratories) and imaged using a confocal

microscope.

For in vivo internalization experiments, SC exosomes were

stained with PKH67 as mentioned above and injected in intact and

crushed sciatic nerves (crush injury was performed 2 days before)

distal to the sciatic notch. As a control of staining, contralateral sci-

atic nerves were injected with PBS previously incubated with

PKH67 (n 5 3 rats per treatment). Twelve hours post-injection,

nerves were fixed, stained with an antibody against neurofilament

heavy chain for intact fibers (rabbit polyclonal 1:1000, Sigma) or

GAP-43 for regenerating fibers and analyzed by confocal microscopy.

The profile of GAP43 positive regenerating fibers was measured

using Matlab, 7.0.1 R14 SP1 version.

Transference and internalization of exosomes in regenerating

fibers was evaluated in vivo in adult male rats. Animals were anaes-

thetized as described above and 1 mg of CD63::eGFP-NS DNA in

PBS containing 0.05% fast green was injected into the distal seg-

ment of an intact or a previously crushed sciatic nerve. Then, seven

pulses (at 60 V, 5 ms long with 50 ms interval) were delivered with

an electric pulse generator (Nepagen NEPA21) using CUY611 elec-

trodes (Nepagen). Seven days after electroporation, transference was

evaluated in intact or in regenerating fibers from intact or crushed

sciatic nerves, respectively. The sciatic nerve was fixed in 4% PFA in

PBS for 1 h and epineurium was removed to obtain teased fibers as

previously described (Court et al., 2008). Immunofluorescence was

performed as described (Court et al., 2008) using an antibody

against neurofilament medium chain (NFM, chicken polyclonal

1:1000, Chemicon) for intact fibers or GAP-43 (rabbit polyclonal

1:1000, Abcam) for regenerating fibers and analyzed by confocal

microscopy. Expression of CD63::eGFP was not observed in axons

proximal to the plasmid injection site and electroporation, indicating

that the only cells that are able to express this fusion protein are SCs

(Supp. Info. Fig. 7A).

Nerve Pinch TestFour days following crush and exosomes or PBS (vehicle) injection,

SD rats were lightly anesthetized with ketamine-xylazine (35 mg/kg

and 10 mg/kg, respectively) and the pinch test was performed

according to (Tapia et al., 1995). Briefly, beginning at 25 mm distal

to the crush site, nerves were pinched with fine forceps, continuing

at successive proximal 0.5-mm intervals until a reflex contraction of

the upper leg took place. The distance between the most distal point

of the nerve that produced a reflex withdrawal response and the

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crush site was measured under a dissecting microscope and scored as

the regeneration distance.

Statistical analysisAll data are presented as means 6 SEM from the indicated number

of experiments. Statistical analysis were performed using Student’s

t-test, linear regression or ANOVA analysis of variance.

Results

SCs secrete Exosomes Carrying thep75-Neurotrophin ReceptorTo investigate whether SCs secrete exosomes, rat sciatic nerve-

derived SCs were cultured at high purity (see Materials and

Methods for details). SC cultures expressed the p75-

neurotrophin receptor (p75NTR), characteristic of a dediffer-

entiated SC phenotype (Fig. 1A). After 48 hours (h), SC con-

ditioned medium (obtained from �2 3 107 cells) was

subjected to differential centrifugation to isolate secreted

vesicles (Thery et al., 2006). In the fraction corresponding to

exosomes (see Materials and Methods for details), the exo-

some markers CD63, Tsg101, Hsp70, Hsp90, and flotillin-1

were detected by Western blot (Fig. 1B) and proteins reported

to be excluded from exosomes, such as the Na1/K1 ATPase

and the early endosome marker Rab5 (Graner et al., 2009;

Kramer-Albers et al., 2007; Potolicchio et al., 2005; Record

et al., 2011), were not found (Fig. 1B). On a discontinuous

sucrose gradient, the presence of CD63 from SC exosomes

was detected in fractions corresponding to the exosome flota-

tion range (Simons and Raposo, 2009) (Fig. 1C). By EM,

SC-derived exosomes appeared as round vesicles with a mean

diameter of 99 6 2 nm (Fig. 1D), which corresponds to the

shape and size of exosomes derived from other cell types and

observed by conventional EM.

Dedifferentiated SCs express p75NTR in vitro and invivo after nerve damage (Fig. 1A) (Ibanez and Simi, 2012).

Since exosomes represent an alternative route for protein recy-

cling, we investigated the presence of p75NTR in these

vesicles. Exosomes isolated from SC culture supernatants con-

tained p75NTR (Fig. 1E). To confirm the presence of p75NTR

in exosomes, SC culture supernatants were obtained after 48

and 120 h and exosomes were precipitated using latex beads

conjugated to an anti-p75NTR antibody that recognizes the

extracellular domain of this protein or anti-neurofilament

medium chain as control. Since exosomes contain phosphati-

dylserine facing the extracellular environment (Fitzner et al.,

2011; Ostrowski et al., 2010), fluorescent annexin-V was

used to analyze exosomes by FACS. We observed secretion of

p75NTR-positive exosomes from SCs, which concentrate in

FIGURE 1: SCs secrete exosomes carrying p75NTR. A, SC primary cultures stained for S100 (green), p75NTR (red) and nuclei with DAPI(blue). Scale bar, 100 mm. B, Characterization of SC exosomes by Western blot. Purified exosomes (Exo, 5 mg per lane) are positive forthe exosomal markers CD63, Tsg101, Hsc70, Hsp90 and flotillin-1 (Flot-1). The non-exosomal proteins Rab5 and Na1/K1 ATPase (Na1/K1) were not detected in exosomes. SC lysate (SCL, 10 mg per lane) was used as a positive control. C, Purified exosomes (30 mg) wereseparated on a discontinuous sucrose gradient and immunoblotted against CD63. Fraction 1 represents the top of the gradient and frac-tions 7 and 8 correspond to 1.12 and 1.14 g/mL sucrose, respectively. D, Distribution of exosome diameters in negatively stained prepa-rations (inset). Scale bar, 50 nm. E, Detection of p75NTR in exosomes (5 mg) purified from SC cultures. SC lysate (SCL, 10 mg) was usedfor comparison. F, Representative histogram showing annexin-V staining of exosomes immunoisolated using anti-p75NTR (blue and redhistograms) or anti-neurofilament-coated beads as control (grey histogram). Blue and red histograms correspond to exosomes purifiedfrom 48 and 120 h conditioned medium, respectively. In bars, the percentages of positive p75NTR-beads are indicated (mean 6 SEMfrom three independent measurements). G, pull down of exosomes using CD63-beads (Lane 2) or p75NTR-beads (Lane 3) and immuno-blotted against CD63 and p75NTR as indicated. Lane 1 shows SC lysate as a positive control. H, SCs expressing CD63::eGFP (green)stained for p75NTR (red) and nuclei with DAPI (blue). Scale bars, 5 mm. [Color figure can be viewed in the online issue, which is availableat wileyonlinelibrary.com.]

Lopez-Verrilli et al.: Glial exosomes promote axonal regeneration

Month 2013 5

the supernatants with time (Fig. 1F). We further confirmed

that exosomes precipitated with p75NTR-beads also express

CD63 by pull down assay (Fig. 1G). When exosomes were

precipitated with CD63-beads, the presence of p75NTR was

also detected by this method, indicating that exosomes carry-

ing p75NTR also contain CD63 exosome marker (Fig. 1G).

In addition, SCs showed intracellular colocalization of the

exosome marker CD63 fused to eGFP (CD63::eGFP) with

p75NTR (Fig. 1H), suggesting the presence of p75NTR within

multivesicular endosomes, sites of exosome biogenesis. Taken

together, these results demonstrate that SCs secrete exosomes

carrying the expected protein repertoire as well as p75NTR, a

protein with diverse functions in the PNS and a novel marker

for SC-derived exosomes.

Exosomes are Internalized by Dorsal Root GangliaAxonsUpon release, exosomes interact with recipient cells by surface

contact, internalization by endocytosis or fusion with the

plasma membrane (Record et al., 2011). We therefore eval-

uated exosome transfer from SCs to axons. First, we

co-cultured SCs and DRG neurons in compartmentalized

Boyden chambers. SCs expressing CD63::eGFP and DRGs

were maintained at different chamber compartments separated

by a 0.4 mm porous membrane. After 2 days, DRGs were

stained against neurofilament and evaluated by confocal

microscopy. A punctate signal of CD63::eGFP colocalizing

with neurofilament-immunostained axons was detected (Fig.

2A and Supp. Info. Fig. 2), suggesting the transfer of

FIGURE 2: Exosomes are specifically internalized by axons. A, SCs and DRGs co-cultured in Boyden chambers. DRG explants were main-tained at the bottom and SCs expressing CD63::eGFP at the top compartment. Axons were stained for neurofilament heavy chain (NFH,red). Scale bar, 2.5 mm. See Supporting Information Fig. 2 for confocal stacks and z-projections. B, Axonal association of PKH67-stainedexosomes from SCs (SC exo-PKH) and fibroblasts (Fibr exo-PKH) after 4 h. Control (Ctrl) represents vehicle incubated with PKH67. Scalebar, 5 mm. C, Quantification of SC-derived PKH67-stained exosomes associated to axons after different times of incubation and com-pared with PKH67-stained vehicle (Ctrl) (mean 6 SEM, n 5 30 pictures/treatment over three separate experiments; * P < 0.05 by linearregression). D, Quantification of PKH67-stained exosomes from SCs and fibroblasts (Fibr) associated to axons after 4 h of incubation(mean 6 SEM, n 5 3 DRG/treatment from three separate experiments; * P < 0.05 by ANOVA test followed by Tukey’s post-test. E,Internalization of PKH67-stained SC exosomes (green) in growth cones from DRGs stained for NFH (red) and p75NTR (blue). Scale bar, 5mm. See Supporting Information Fig. 3 for z-stacks and orthogonal projections. F–I, Representative electron micrographs of SC-derivedexosomes in DRG axons. F, Negatively stained exosomes with p75NTR-gold labeling on their surface. G, Exosomes in close vicinityto the axolemma, H, I, Internalized exosomes within axons. Scale bars, 100 nm. See Supporting Information Fig. 4 for additionalEM images. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

6 Volume 00, No. 00

CD63::eGFP-positive exosomes from SCs to axons. Second,

SC-derived exosomes were purified by ultracentrifugation,

stained with the membrane fluorescent marker PKH67 and

then supplemented to DRG neurons. The number of

PKH67-positive puncta in axons increased progressively with

time of incubation (Fig. 2B,C). Remarkably, the presence of

PKH67 stained exosomes were also observed in 43 6 7% of

the DRG growth cones (Fig. 2E and Supp. Info. Fig. 3).

We then analyzed whether association of SC-derived

exosomes with axons was cell specific. To this end, exosomes

from fibroblast primary cultures were stained with PKH67

and supplemented to DRG explants. Fibroblast-derived

PKH67-positive exosomes from fibroblasts in axons was no

different to control values (Fig. 2B,D), indicating that axons

selectively associate to exosomes secreted by SCs.

Finally, ultrastructural analysis of exosome internalization

by axons was performed. Exosomes were purified from SCs and

surface labeled using gold-conjugated anti-p75NTR antibodies; as

a negative control, exosomes were labeled with a gold-

conjugated anti-tubulin antibody (see Materials and Methods

for a detailed description). After labeling and further purification

(see Materials and Methods for details), only exosomes stained

with p75NTR-gold—and not with gold-acetylated tubulin

immunocomplexes—were decorated by gold particles on their

surface (Fig. 2F). Gold-labeled exosomes were then supple-

mented to DRGs and processed for transmission EM. At the

EM, p75NTR-gold exosomes were found in the extracellular

space associated to the axolemma (Fig. 2G and Supp. Info. Fig.

4A–C). Inside axons, p75NTR-gold exosomes were detected

within bigger vesicles, suggesting that exosomes are endocytosed

by axons (Fig. 2H,I and Supp. Info. Fig. 4D–F). No gold was

observed in axons incubated with exosomes purified after gold-

acetylated tubulin labeling (data not shown). Taken together, our

observations demonstrate that SC-secreted exosomes are specifi-

cally internalized by axons and their growth cones in vitro.

SC-Derived Exosomes Promote AxonalRegeneration After AxotomyAs SCs are essential for efficient axonal regeneration after

nerve injury (Webber and Zochodne, 2010) and can promote

regeneration in the CNS (Fortun et al., 2009), we evaluated

if SC-derived exosomes affect axonal regeneration performing

two different approaches widely used in the literature. First,

we evaluated if SC-derived exosomes might influence axonal

regeneration in DRG explant cultures. One day after DRGs

were explanted, SC-derived exosomes (3 mg of exosomal pro-

tein per DRG) or vehicle solution (PBS) were supplemented

in a daily basis. Impressively, exosome treatment increased the

regeneration rate compared with vehicle-treated explants (41

6 4 vs. 26 6 3 mm2/day compared with vehicle, mean 6

SEM, * P < 0.05, linear regression) (Fig. 3A,B), suggesting

that SC exosomes stimulate axonal regeneration in vitro.

Dose–response experiments demonstrate that this regenerative

effect is observed only when axons are treated with three or

more amount of SC exosomes (Supp. Info. Fig. 5).

Second, we analyzed if SC exosomes modulate axonal

regeneration in DRG explants after mechanical injury. To this

end, axons were allowed to grow for 7 days and then were

mechanically transected. One hour after damage, DRGs were

supplemented with SC-derived exosomes (3 mg per DRG) or

vehicle (PBS) as control. Exosome supplementation was repeated

daily and axonal lengths were measured concomitantly. Impres-

sively, exosome-treated DRGs regenerate faster than vehicle-

treated axons (0.62 6 0.03 vs. 0.44 6 0.02 mm/day compared

with vehicle, mean 6 SEM, * P < 0.05, linear regression) (Fig.

3C,D). Consistently, the distribution of axonal lengths regenerat-

ing for 3 days was shifted toward longer values in exosome-

treated neurons compared with control ones (Fig. 3E).

Notably, the regeneration enhancement by SC-derived

exosomes was dependent on exosome integrity and content,

since digestion of extracellular proteins or exosome lysis before

supplementation, eliminated their pro-regenerating effect (Fig.

3F). We then tested if the exosome-dependent regeneration

enhancement was cell specific. To this end, DRGs were supple-

mented with fibroblast or SC-derived exosomes. Unlike SC-

derived exosomes, fibroblast-derived ones had no effect on axo-

nal regeneration (Fig. 3F), indicating that the composition and

content of exosomes must be cell-specific to enhance axonal

regeneration. No effect on axonal regeneration was observed

when axons were supplemented with exosomes obtained from

fibroblast treated with the same supplements as SCs cultures

(i.e., 2 mM forskolin and 20 mg/mL bovine pituitary extract,

Supp. Info. Fig. 6), reinforcing that the increase of axonal

regeneration is dependent on cell-specific exosomes.

Axonal regeneration can be modulated locally or at the

cell body (Gumy et al., 2010). To assess whether the effect of

exosomes on axonal regeneration was region-dependent, we used

Campenot chambers and supplemented SC-derived exosomes

either to the cell bodies, to the distal axons or to both of them.

DRG neurons were allowed to grow and axons were transected

at the distal compartment (Fig. 3G). Thereafter, SC exosomes

were added to only one compartment and axonal regeneration

was assessed 3 days later. Only when added to the distal (axonal)

compartment, SC-derived exosomes enhanced axonal regenera-

tion compared with the control (Fig. 3H) and exosome supple-

mentation to both cell bodies and distal axons evoked the same

effect as supplementing exosomes only to distal axons (Fig. 3H).

Growth Cone Morphology and Dynamics areModulated by SC-derived ExosomesAs SC-derived exosomes are internalized in both axons and

growth cones (Fig. 2) and enhance the regenerative capability

Lopez-Verrilli et al.: Glial exosomes promote axonal regeneration

Month 2013 7

of DRG neurons (Fig. 3), we asked whether they affected

morphological traits of regenerating growth cones. Axons

were transected, supplemented with SC-derived exosomes and

growth cones were scored into three groups based on their

shapes and number of filopodia: type I (collapsed), type II

(partially collapsed) and type III (extended/fan-shaped) (Fig.

4A). Three days after damage, exosome-treated DRGs showed

three times more type III and half the number of type I

growth cones compared with non-treated DRGs, suggesting

that exosomes promote growth cone extension (Fig. 4B,C).

Considering that Rho GTPases are key regulators of

growth cone dynamics (Hall and Lalli, 2010), we asked

whether exosomes modulate Rho GTPase activity at growth

cones. To this end, we measured endogenous activity of

RhoA, a GTPase which activation inhibits axonal elongation

and promotes growth cone collapse (Zhang et al., 2011).

Three days after transection, exosome, or vehicle-treated

DRGs were fixed and incubated with GST-RBD recombinant

protein which binds to active RhoA-GTP, followed by an

anti-GST antibody and rhodamine-conjugated phalloidin

(Fig. 4F). Quantitative measurement of RhoA activity at

growth cones revealed that GTP-bound RhoA levels decreased

after treatment with SC-derived exosomes (Fig. 4D). Further-

more, exosome-treated DRGs displayed growth cones with

significant more filopodia compared with controls (Fig. 4E),

consistent with the reported consequence of RhoA inhibition

and increased actin polymerization at growth cones (Kuhn

et al., 2000).

FIGURE 3: SC exosomes promote axonal regeneration after injury. A, Axonal regeneration of DRGs after vehicle (Ctrl) or exosome (Exo)treatment during 5 days. DRGs are stained for acetylated tubulin (Ac-Tub, green), phalloidin-rhodamine (Phall, red) and nuclei with DAPI(blue). Scale bar, 50 mm. B, Exosome treatment increased the rate of axonal regeneration compared with vehicle (mean 6 SEM, 41 6 4vs. 26 6 3 mm2/day, respectively; n 5 4 DRG per treatment from three separate experiments; * P < 0.05, linear regression). C, DRG cul-tures stained for acetylated tubulin (green) and nuclei with DAPI (blue). Negative DAPI staining indicates the absence of SCs. After 7days in culture, DRG axons were cut and daily supplemented with SC-derived exosomes (Exo) or vehicle as control (Ctrl). Damage site isindicated by a dotted line in the first image. Scale bar, 200 mm. D, Quantification of the rate of axonal regeneration. Exosome-treatedDRGs regenerate faster than vehicle-treated ones (mean 6 SEM, 0.62 6 0.03 vs. 0.44 6 0.02 mm/day compared with vehicle, n 5 4with four DRGs per treatment,* P < 0.05, linear regression). E, Distribution of axonal lengths 3 days after cut (n 5 200 axons per condi-tion). F, Effect of pre-treated exosomes from SCs and intact exosomes from fibroblasts (Fibr) on axonal regeneration 3 days after axonaldamage. SC exosomes were incubated with trypsin, lysed with water or treated with trypsin followed by lysis with water. PBS repre-sents vehicle treatment (mean 6 SEM, n 5 3 DRG/treatment from three separate experiments; *P < 0.05, ANOVA test followed byTukey’s post-test). G, Schematic representation of Campenot chambers showing the cellular bodies (CB) and axonal (Ax) compartments.After axonal transection, SC exosomes were added either to the CB, Ax or both (C/A) compartments during 3 days. H, Regeneratedaxons stained for acetylated tubulin (Ac-Tub, green) are shown; regeneration front is to the right. Scale bar, 50 mm. The graph showsquantification of axonal regeneration (mean 6 SEM, n 5 5 Campenot chambers per treatment from three separate experiments; *P <0.05 ANOVA test followed by Bonferroni post-test). [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

8 Volume 00, No. 00

Altogether, these results suggest that SC-derived exosomes

mediate axonal regeneration by inhibiting RhoA activity, mod-

ulating growth cone dynamics to promote neurite extension.

SC-Derived Exosomes Enhance AxonalRegeneration in vivoWe then evaluated whether SC-derived exosomes were internal-

ized in vivo and their effect on axonal regeneration after sciatic

nerve crush. Intact or crushed sciatic nerves were injected with

PKH67-stained exosomes derived from SCs, and internaliza-

tion was evaluated 12 h later by confocal microscopy. We

observed exosome internalization in intact fibers as well as in

regenerating ones (Fig. 5A), indicating that SC exosomes are

internalized by intact and regenerating fibers in vivo.

We further evaluated if genetically labelled exosomes were

transferred from SCs to axons. To this end, CD63::eGFP was

expressed in SCs by electroporation into a sciatic nerve segment

in vivo. After 7 days, CD63::eGFP fluorescence appeared in the

axonal compartment as punctate, colocalizing with the axonal

staining in both intact and regenerating axons (Fig. 5B and

Supp. Info. Fig. 7B,C). This data indicate that SCs are able to

transfer CD63::eGFP-labelled exosomes to axons in vivo both

in normal as well as regenerating conditions.

To evaluate the effect of exosomes after nerve injury, sci-

atic nerves were crushed and locally injected with 4 mg of SC-

derived exosomes or vehicle as control. Axonal regeneration

was evaluated 4 days after nerve crush by immunofluorescence

staining with GAP43 and by measuring the length of the

regenerative front with the pinch test. Impressively, GAP43

immunostaining showed two times longer regenerating neurites

in nerves injected with SC exosomes compared with vehicle-

injected nerves (Fig. 5C,D), indicating that exosomes greatly

enhance nerve regeneration after nerve crush. In addition,

nerves injected with exosomes elicited a positive response to

the pinch test at longer distances from the crush site (12 6

0.3 mm vs. 7 6 1.2 mm compared with vehicle) (Fig. 5E).

Altogether, these results indicate that SC-derived exosomes

enhance axonal regeneration after nerve injury in vivo.

Discussion

After nerve injury, SCs dedifferentiate, proliferate and provide

a mechanical substrate and growth factors to regenerating

axons (Chen et al., 2007; Webber et al., 2011). During this

process, the effect of SC secreted factors over axonal regenera-

tion has been well documented (Gordon, 2010), but whether

these glial effector molecules are soluble or delivered to axons

using vesicular vectors has not been evaluated. Here we dem-

onstrate that SCs secrete exosomes, which are selectively inter-

nalized by axons in vitro and in vivo, and increase PNS nerve

regeneration after injury. Therefore, transfer of SC-derived

exosomes to axons constitutes a novel mechanism able to

facilitate regenerative growth of axons.

Exosomes isolated from SC cultures showed the expected

round-shape morphology, size, flotation density, and biochemical

markers, comparable with exosomes from other cell types (Simons

and Raposo, 2009). In addition, SC-derived exosomes carried

p75NTR, a protein expressed by dedifferentiated SCs, but not pre-

viously identified in exosomes or known to be secreted to the

extracellular space. We further demonstrated, using extracellularly

directed antibodies and FACS analysis, that p75NTR in the

FIGURE 4: SC exosomes promote growth cone extension. A, Representative growth cones from DRG cultures classified according totheir shapes and number of filopodia. Growth cones were immunolabeled with Ac-Tub and phalloidin-rhodamine (Phall) and are shownin grayscale. B, Growth cones of DRG regenerating axons treated with vehicle (Ctrl) or exosomes (Exo) for 3 days after cut. Growthcones are stained for Ac-Tub (green) and Phall (red). Scale bar, 10 mm. C, Quantification of growth cone type from control and exosometreated DRGs (mean 6 SEM, n 5 150 growth cones/treatment from three separate experiments; *P <0.05 Two way-ANOVA test fol-lowed by Bonferroni post-test). D, Quantification of RhoA activity in growth cones (mean 6 SEM, n 5 200 growth cones per treatmentobtained from three different experiments; *P < 0.05, Student’s t test). E, Quantification of filopodia number per growth cone 3 daysafter treatment with exosomes or vehicle (Ctrl) (mean 6 SEM, n 5 150 growth cones/treatment from three separate experiments; * P<0.05 Two way-ANOVA test followed by Bonferroni post-test). F, Individual growth cones stained for RhoA GTP (green) and Phall (red)3 days after cut and vehicle (Ctrl) or exosome treatment. Scale bar, 10 mm. [Color figure can be viewed in the online issue, which is avail-able at wileyonlinelibrary.com.]

Lopez-Verrilli et al.: Glial exosomes promote axonal regeneration

Month 2013 9

FIGURE 5: Exosomes promote axonal regeneration in vivo. A, PKH67-stained exosomes (green) were locally injected into intact (toppanel) or previously crushed (48 h) sciatic nerves (bottom panel). Exosomes colocalized with neurofilament heavy chain (NFH, red) orGAP43 (red) staining in intact and regenerating axons, respectively. Scale bar, 5 mm. Right panels correspond to higher magnification ofthe boxed areas. Scale bar, 1 mm. B, Sciatic nerves were electroporated with CD63::eGFP DNA and teased nerve fibers were obtained 7days post-electroporation from intact (top panel) or crushed sciatic nerves (bottom panel). CD63::eGFP particles colocalized with neuro-filament medium chain (NFM, red) or GAP43 (red) staining in intact and regenerating axons, respectively. Scale bar, 10 mm. Right panelscorrespond to higher magnification of the boxed areas. Scale bar, 2.5 mm. See Supporting Information Fig. 7B,C for z-stacks and orthog-onal projections. C, Effect of SC exosomes on axonal regeneration in vivo. Regenerating axons (from left to right) stained for GAP43(green), showed longer extensions after exosome injections. Scale bar, 1 mm. Regeneration starts at the dotted line. D, Quantificationof GAP43-positive neurites at different distances distal from the lesion site 4 days after crush (mean 6 SEM, n 5 4 rats per treatment,*P < 0.05, Two-way ANOVA). E, Pinch test in sciatic nerves treated with vehicle (Ctrl) or exosomes (Exo) 4 days after crush. The distancefrom the crush to the site where a sensitive response was elicited after pinching the nerve was measured (mean 6 SEM, n 5 4 per treat-ment, *P < 0.05, Student’s t test). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

exosomal membrane is oriented at the same topology as in the

plasma membrane (Fig. 1E and see Materials and Methods).

As message conveyors, exosomes must be recognized by

the recipient cell for internalization. In addition to confocal

microscopy, exosome internalization was confirmed by ultra-

structural analysis. Immunolabeled exosomes expressing

p75NTR on their surface were found within larger vesicles in the

axoplasm, suggesting that exosomes are endocytosed and not

fused to the axolemma (Fig. 2H,I, and Supp. Info. Fig. 4). Inter-

estingly, exosome internalization by axons was selective for SCs

but not for fibroblast-derived exosomes (Fig. 2B,D), probably

reflecting the presence of specific molecules on the exosomal

surface allowing their differential recognition by axons. Selective

transfer of exosomes has been previously reported. In the CNS,

oligodendrocytes-derived exosomes are taken by microglial cells

but not by neurons or astrocytes (Fitzner et al., 2011).

Although it has been suggested that exosomes might mod-

ulate neurite outgrowth in the CNS (Wang et al., 2011; Xin

et al., 2012), a direct effect of glial exosomes on neurite growth

or regeneration has not been described yet. In our work, exo-

somes were obtained from p75NTR-positive SCs, a dedifferenti-

ated phenotype that sustains axonal growth and regeneration invivo (Kobayashi et al., 2012). Impressively, we found that SC

exosomes increased the rate of regeneration of cultured DRG

neurons in vitro (Fig. 3) and in vivo (Fig. 5). This pro-

regenerative effect was dependent on both intact proteins on the

exosomal surface -that might mediate exosome internalization-

as well as the exosomal content. In addition, SC exosomes

enhanced axonal regeneration when supplemented to axons and

growth cones, evidencing a local mechanism of action. These

findings reinforce the idea that axons are autonomous for some

processes, including the first stages of growth and regeneration,

but dependent on local regulators (Alvarez et al., 2000; Shaw

and Bray, 1977), such as exosomes derived from associated SCs.

During both axonal pathfinding in development and

regeneration, growth cones project finger-like filopodia that

guide their migration (Hur et al., 2012). We found that SC

exosomes decreased the abundance of collapsed growth cones

while increased the abundance of fan-shaped growth cones

(Fig. 4A–C). In contrast to collapsed ones, fan-shaped growth

cones are highly motile and promote axonal elongation (Endo

et al., 2003; Goswami et al., 2007). Remarkably, SC exosomes

decreased RhoA activity at growth cones, a GTPase that is acti-

vated upon injury and inhibits axonal regeneration (Hall and

Lalli, 2010). Supporting our results, inhibition of RhoA cas-

cade enhances axonal regeneration both in vitro and in vivo

(Cheng et al., 2008). Further experiments will be needed to

determine how exosomes modulates RhoA activation during

axonal regeneration. The detailed analysis of the extensive pro-

tein, mRNA and microRNA content of SC derived exosomes,

will probably help to define the mechanism involved.

In the CNS, the favorable effects of SCs over axonal

regeneration have been mainly attributed to growth factors

and extracellular matrix proteins secreted by SCs (Allodi

et al., 2012; Ganfornina et al., 2010). Our work demon-

strates a novel and unexpected role for SC-derived exosomes

on axonal regeneration after injury and suggests that exosome

content is important for this effect.

We propose that vesicles secreted by SCs and transferred

to axons participate in the processes by which SCs locally sup-

port axonal maintenance and regeneration after nerve damage.

Accordingly, we have shown that SCs transfer ribosomes to

injured and regenerating axons in vivo by an undefined vesicu-

lar mechanism (Court et al., 2008, 2011). Here, we show that

exosomes are transferred from SCs to axons in vitro and in vivo

and that SC exosomes promote axonal regeneration after injury

in the PNS. This exosome-mediated mechanism opens a new

dimension in our understanding of nervous system function

and provides potential therapeutic application to boost the

capabilities of poorly regenerative regions of the nervous system.

Acknowledgment

Grant sponsor: FONDECYT 3110014 (MALV), FONDE-

CYT 1110987, Millennium Nucleus P07-011-F and Anillo

ACT1109 (FAC).

The authors thank M�onica Perez for excellent EM processing,

Jaime Alvarez for his critical opinion, Claudia Escudero for her

advice in the immunogold internalization assay and Jeff Twiss

for critical reading of the manuscript. CD63::eGFP construct

was a kind gift from Dr. Juan Falcon, Parque Tecnol�ogico de

Vizcaya, Spain; p75NTR antibody was kindly provided by Dr

Francisca Bronfman, P. Universidad Cat�olica de Chile.

ReferencesAlvarez J, Giuditta A, Koenig E. 2000. Protein synthesis in axons and termi-nals: Significance for maintenance, plasticity and regulation of phenotype.With a critique of slow transport theory. Prog Neurobiol 62:1–62.

Allodi I, Udina E, Navarro X. 2012. Specificity of peripheral nerve regenera-tion: Interactions at the axon level. Prog Neurobiol 98:16–37.

Bakhti M, Winter C, Simons M. 2011. Inhibition of myelin membrane sheathformation by oligodendrocyte-derived exosome-like vesicles. J Biol Chem286:787–96.

Barrientos SA, Martinez NW, Yoo S, Jara JS, Zamorano S, Hetz C, Twiss JL,Alvarez J, Court FA. 2011. Axonal degeneration is mediated by the mito-chondrial permeability transition pore. J Neurosci 31:966–78.

Campenot RB, Lund K, Mok SA. 2009. Production of compartmented culturesof rat sympathetic neurons. Nat Protoc 4:1869–87.

Carcamo C, Pardo E, Oyanadel C, Bravo-Zehnder M, Bull P, Caceres M,Martinez J, Massardo L, Jacobelli S, Gonzalez A, Soza A. 2006. Galectin-8binds specific beta1 integrins and induces polarized spreading highlightedby asymmetric lamellipodia in Jurkat T cells. Exp Cell Res 312:374–86.

Court FA, Hendriks WT, MacGillavry HD, Alvarez J, van Minnen J. 2008.Schwann cell to axon transfer of ribosomes: Toward a novel understanding ofthe role of glia in the nervous system. J Neurosci 28:11024–9.

Lopez-Verrilli et al.: Glial exosomes promote axonal regeneration

Month 2013 11

Court FA, Midha R, Cisterna BA, Grochmal J, Shakhbazau A, Hendriks WT,Van Minnen J. 2011. Morphological evidence for a transport of ribosomesfrom Schwann cells to regenerating axons. Glia 59:1529–39.

Chen YY, McDonald D, Cheng C, Magnowski B, Durand J, Zochodne DW.2005. Axon and Schwann cell partnership during nerve regrowth. J Neuropa-thol Exp Neurol 64:613–22.

Chen ZL, Yu WM, Strickland S. 2007. Peripheral regeneration. Annu Rev Neu-rosci 30:209–33.

Cheng C, Webber CA, Wang J, Xu Y, Martinez JA, Liu WQ, McDonald D,Guo GF, Nguyen MD, Zochodne DW. 2008. Activated RHOA and peripheralaxon regeneration. Exp Neurol 212:358–69.

Dreesmann L, Mittnacht U, Lietz M, Schlosshauer B. 2009. Nerve fibroblastimpact on Schwann cell behavior. Eur J Cell Biol 88:285–300.

Endo M, Ohashi K, Sasaki Y, Goshima Y, Niwa R, Uemura T, Mizuno K. 2003.Control of growth cone motility and morphology by LIM kinase and Slingshotvia phosphorylation and dephosphorylation of cofilin. J Neurosci 23:2527–37.

Fevrier B, Vilette D, Archer F, Loew D, Faigle W, Vidal M, Laude H, RaposoG. 2004. Cells release prions in association with exosomes. Proc Natl AcadSci U S A 101:9683–8.

Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G, Dibaj P, Bakhti M,Regen T, Hanisch UK, Simons M. 2011. Selective transfer of exosomes fromoligodendrocytes to microglia by macropinocytosis. J Cell Sci 124:447–58.

Fortun J, Hill CE, Bunge MB. 2009. Combinatorial strategies with Schwanncell transplantation to improve repair of the injured spinal cord. Neurosci Lett456:124–32.

Ganfornina MD, Do Carmo S, Martinez E, Tolivia J, Navarro A, Rassart E,Sanchez D. 2010. ApoD, a glia-derived apolipoprotein, is required for periph-eral nerve functional integrity and a timely response to injury. Glia 58:1320–34.

Gehler S, Gallo G, Veien E, Letourneau PC. 2004. p75 neurotrophin receptorsignaling regulates growth cone filopodial dynamics through modulatingRhoA activity. J Neurosci 24:4363–72.

Gordon T. 2010. The physiology of neural injury and regeneration: The roleof neurotrophic factors. J Commun Disord 43:265–73.

Goswami C, Schmidt H, Hucho F. 2007. TRPV1 at nerve endings regulatesgrowth cone morphology and movement through cytoskeleton reorganiza-tion. FEBS J 274:760–72.

Graner MW, Alzate O, Dechkovskaia AM, Keene JD, Sampson JH, MitchellDA, Bigner DD. 2009. Proteomic and immunologic analyses of brain tumorexosomes. Faseb J 23:1541–57.

Gumy LF, Tan CL, Fawcett JW. 2010. The role of local protein synthesis anddegradation in axon regeneration. Exp Neurol 223:28–37.

Hall A, Lalli G. 2010. Rho and Ras GTPases in axon growth, guidance, andbranching. Cold Spring Harb Perspect Biol 2:a001818.

Hur EM, Saijilafu , Zhou FQ. 2012. Growing the growth cone: Remodelingthe cytoskeleton to promote axon regeneration. Trends Neurosci 35:164–74.

Ibanez CF, Simi A. 2012. p75 neurotrophin receptor signaling in nervous systeminjury and degeneration: Paradox and opportunity. Trends Neurosci 35:431–40.

Kobayashi M, Ishibashi S, Tomimitsu H, Yokota T, Mizusawa H. 2012. Prolifer-ating immature schwann cells contribute to nerve regeneration after ischemicperipheral nerve injury. J Neuropathol Exp Neurol 71:511–9.

Kramer-Albers EM, Bretz N, Tenzer S, Winterstein C, Mobius W, Berger H,Nave KA, Schild H, Trotter J. 2007. Oligodendrocytes secrete exosomes con-taining major myelin and stress-protective proteins: Trophic support foraxons? Proteomics Clin Appl 1:1446–61.

Kuhn TB, Meberg PJ, Brown MD, Bernstein BW, Minamide LS, Jensen JR,Okada K, Soda EA, Bamburg JR. 2000. Regulating actin dynamics in neuronalgrowth cones by ADF/cofilin and rho family GTPases. J Neurobiol 44:126–44.

Lasser C, Alikhani VS, Ekstrom K, Eldh M, Paredes PT, Bossios A, SjostrandM, Gabrielsson S, Lotvall J, Valadi H. 2011. Human saliva, plasma and breastmilk exosomes contain RNA: Uptake by macrophages. J Transl Med 9:9.

Lopez-Verrilli MA, Court FA. 2012. Transfer of vesicles from schwann cells toaxons: A novel mechanism of communication in the peripheral nervous sys-tem. Front Physiol 3:205.

Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, MoitaCF, Schauer K, Hume AN, Freitas RP, Goud B, Benaroch P, Hacohen N,Fukuda M, Desnos C, Seabra M, Darchen F, Amigorena S, Moita L, Thery C.2010. Rab27a and Rab27b control different steps of the exosome secretionpathway. Nat Cell Biol 12:19–30

Potolicchio I, Carven GJ, Xu X, Stipp C, Riese RJ, Stern LJ, Santambrogio L.2005. Proteomic analysis of microglia-derived exosomes: Metabolic role ofthe aminopeptidase CD13 in neuropeptide catabolism. J Immunol 175:2237–43.

Record M, Subra C, Silvente-Poirot S, Poirot M. 2011. Exosomes as inter-cellular signalosomes and pharmacological effectors. Biochem Pharmacol 81:1171–82.

Schoenmann Z, Assa-Kunik E, Tiomny S, Minis A, Haklai-Topper L, Arama E,Yaron A. 2010. Axonal degeneration is regulated by the apoptotic machineryor a NAD1-sensitive pathway in insects and mammals. J Neurosci 30:6375–86.

Shaw G, Bray D. 1977. Movement and extension of isolated growth cones.Exp Cell Res 104:55–62.

Simons M, Raposo G. 2009. Exosomes—vesicular carriers for intercellularcommunication. Curr Opin Cell Biol 21:575–81.

Tamboli IY, Barth E, Christian L, Siepmann M, Kumar S, Singh S, Tolksdorf K,Heneka MT, Lutjohann D, Wunderlich P, Walter J. 2010. Statins promote thedegradation of extracellular amyloid {beta}-peptide by microglia via stimula-tion of exosome-associated insulin-degrading enzyme (IDE) secretion. J BiolChem 285:37405–14.

Tapia M, Inestrosa NC, Alvarez J. 1995. Early axonal regeneration: Repres-sion by Schwann cells and a protease? Exp Neurol 131:124–32.

Taylor AR, Robinson MB, Gifondorwa DJ, Tytell M, Milligan CE. 2007. Regu-lation of heat shock protein 70 release in astrocytes: Role of signalingkinases. Dev Neurobiol 67:1815–29.

Thery C, Amigorena S, Raposo G, Clayton A. 2006. Isolation and characteri-zation of exosomes from cell culture supernatants and biological fluids. CurrProtoc Cell Biol Chapter 3:Unit 3 22.

Thery C, Ostrowski M, Segura E. 2009. Membrane vesicles as conveyors ofimmune responses. Nat Rev Immunol 9:581–93.

Toft A, Tome M, Barnett SC, Riddell JS. 2013. A comparative study of glialand non-neural cell properties for transplant-mediated repair of the injuredspinal cord. Glia 61:513–28.

Wang S, Cesca F, Loers G, Schweizer M, Buck F, Benfenati F, Schachner M,Kleene R. 2011. Synapsin I is an oligomannose-carrying glycoprotein, acts asan oligomannose-binding lectin, and promotes neurite outgrowth and neuro-nal survival when released via glia-derived exosomes. J Neurosci 31:7275–90.

Webber C, Zochodne D. 2010. The nerve regenerative microenvironment: Earlybehavior and partnership of axons and Schwann cells. Exp Neurol 223:51–9.

Webber CA, Christie KJ, Cheng C, Martinez JA, Singh B, Singh V, Thomas D,Zochodne DW. 2011. Schwann cells direct peripheral nerve regenerationthrough the Netrin-1 receptors, DCC and Unc5H2. Glia 59:1503–17.

Wilby MJ, Muir EM, Fok-Seang J, Gour BJ, Blaschuk OW, Fawcett JW. 1999. N-Cad-herin inhibits Schwann cell migration on astrocytes. Mol Cell Neurosci 14:66–84.

Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, Shang X, Zhang ZG,Chopp M. 2012. Exosome-mediated transfer of miR-133b from multipotentmesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem Cells 30:1556–64.

Yamauchi J, Miyamoto Y, Chan JR, Tanoue A. 2008. ErbB2 directly activates theexchange factor Dock7 to promote Schwann cell migration. J Cell Biol 181:351–65.

Zhang G, Lehmann HC, Manoharan S, Hashmi M, Shim S, Ming GL, SchnaarRL, Lopez PH, Bogdanova N, Sheikh KA. 2011. Anti-ganglioside antibody-mediated activation of RhoA induces inhibition of neurite outgrowth. J Neu-rosci 31:1664–75.

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