11024 • The Journal of Neuroscience, October 22, 2008 • 28(43):11024 –11029

Brief Communications

Schwann Cell to Axon Transfer of Ribosomes: Toward a

Novel Understanding of the Role of Glia in the Nervous
System
Felipe A. Court,1 William T. J. Hendriks,2,3 Harold D. MacGillavry,3 Jaime Alvarez,1 and Jan van Minnen3,4
1Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica, 114-D Santiago, Chile, 2Laboratory for Neuroregeneration,

Netherlands Institute for Neuroscience, an Institute of the Royal Academy of Arts and Sciences, 1105 BA, Amsterdam, the Netherlands, 3Department of
Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Faculty of Earth and Life Sciences, VU University, 1081 HV,
Amsterdam, the Netherlands, and 4Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary,
Alberta, Canada T2N 4N1

Schwann cells play pivotal roles in the development and maintenance of the peripheral nervous system. Here, we show that intact sciatic
nerve axons of mice contain a small population of ribosomes, which increases by several orders of magnitude when axons are desoma-
tized (severed from their cell bodies). We furthermore demonstrate, using the Wallerian degeneration slow mouse as a model, that
Schwann cells transfer polyribosomes to desomatized axons. These data indicate that Schwann cells have the propensity to control axonal
protein synthesis by supplying ribosomes on local basis.
Key words: Schwann cell; ribosomes; Wallerian degeneration; axons; glia; intercellular transport

Introduction Along the same line, Eyman et al. (2007) proposed that mRNAs
Schwann cells, the glia of the peripheral nervous system (PNS), transcribed in squid glial cells are transferred to, and translated
play several pivotal roles in the development and maintenance of in, the axoplasm. Together, these data suggest that the axon re-
the PNS. In addition to forming the myelin sheath, Schwann cells ceives on a local basis transcripts and components of the machin-
are involved in neuronal survival where they also provide support ery for protein synthesis, an unsuspected form of neuron– glia
to axons during development and throughout adulthood. For interaction. However, unequivocal evidence for the existence of
example, Schwann cells regulate the caliber and microtubules of such interaction is yet to be obtained.
axons (Windebank et al., 1985; Hernandez et al., 1989; Bustos et Here, we provide evidence that Schwann cells transfer polyribo-
al., 1991), orchestrate the formation and maintenance of the somes to the axoplasm. For this, we tagged Schwann cell ribosomes
nodes of Ranvier, provide trophic support to the axon after injury with enhanced green fluorescent protein (eGFP) and showed that
and during regeneration (Fields and Stevens-Graham, 2002), and tagged ribosomes were translocated to desomatized axons.
in mature fibers, the differentiated Schwann cells impair the out-
growth and ingrowth of axons (Court and Alvarez, 2000, 2005).
Materials and Methods
Animals. Mice of the C57BL/6J/Wld s strain were supplied by Biosonda or
The ability of Schwann cells to regulate axons and their func-
Harlan, and wild-type (C57BL/6J) mice were supplied by the local animal
tions relies on reciprocal communications (Stevens and Fields, house (Facultad de Ciencias Biológicas, Santiago, Chile). Animal care and
2000). Recently, in the field of neuron– glia interaction, a novel surgical procedures complied with National Institutes of Health guidelines.
concept is emerging. Specifically, Schwann cells have been pro- Mice weighing ⬃20 g were anesthetized with Nembutal (36 mg/kg) or
posed to supply ribosomes and mRNA to desomatized axons of xylasine-ketamine (6.7– 66.7 mg/kg, respectively). The nerve was crushed at
the Wallerian degeneration slow (Wld s) mice (Alvarez, 2001). the sciatic notch or at midthigh with fine forceps. The crush site was marked
with surgical 10-0 nylon monofilament (Ethicon). If applicable, 1–2 ␮l len-
tivirus in PBS containing 0.1% fast green was injected into the distal segment
Received May 23, 2008; revised Aug. 5, 2008; accepted Sept. 10, 2008. of a previously crushed sciatic nerve. In some animals, sciatic nerves were
This work was supported by Fondecyt (1980973, 1070377), Chile; J.v.M. and W.T.J.H. were supported by a transected at the sciatic notch in addition.
European Union STREP Grant (12702). We thank Joost Verhaagen and Ruben Eggers for help with the Lentivirus Electron microscopy. Sciatic nerves were processed and sectioned for
experiments; Angelo Quattrini for EM expertise; Mónica Pérez, Gloria Méndez, Adrian Thomson, Yvonne Gouwen- electron microscopy as described previously (Court and Alvarez, 2000).
berg, and Jean Kawasoe for technical assistance; and Guus Smit, Naweed Syed, and Lawrence Wrabetz for critical Immunocytochemistry. Teased fibers were blocked/permeabilized in
discussion.
PBS containing 5% fish skin gelatin (Sigma) and 0.2% Triton X-100 for
Correspondence should be addressed to either of the following: Felipe A. Court, Departamento de Fisiología,
Facultad de Ciencias Biológicas, Pontificia Universidad Católica, 114-D Santiago, Chile, E-mail: fcourt@bio.puc.cl; or
1 h at room temperature and incubated overnight in the same solution
Jan van Minnen, Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, Faculty of Medicine, University with the following primary antibodies: human anti-ribosome antiserum,
of Calgary, Calgary, Alberta, Canada T2N 4N1, E-mail: jvanminn@ucalgary.ca. 1:1000 [from a patient carrying a systemic lupus erythematosus character-
DOI:10.1523/JNEUROSCI.2429-08.2008 ized by Massardo et al. (2002), a kind gift from S. Jacobelli, Pontificia Uni-
Copyright © 2008 Society for Neuroscience 0270-6474/08/2811024-06$15.00/0 versidad Católica, Santiago, Chile]; mouse anti-neurofilament (clone N52;
Court et al. • Schwann Cell to Axon Transfer of Ribosomes J. Neurosci., October 22, 2008 • 28(43):11024 –11029 • 11025

Figure 1. Ribosomes and polyribosomes in axons. A, B, Intact (A) and Wld s (B) desomatized for 7 d. A, Ribosomes are scarce (arrowhead). B, A field of polyribosomes stands out in the axoplasm,
which has not degenerated despite its protracted disconnection from the cell body. Inset, High magnification of an axonal polyribosome. M, Myelin. Scale bars: 200 nm; inset, 50 nm. C, Quantification
of ribosomes in wild-type intact (WT int), Wld s intact (Wld s int) axons, and in desomatized Wld s axons crushed 7 d before (Wld s Crush). Ordinates: scale is logarithmic. Notice the impressive increase
in ribosomal content, both in ribosomes per volume of axoplasm (*p ⬍ 0.03), as well as the number of axons containing ribosomes. (*p ⬍ 0.01). Student’s t test.

Sigma), 1:1000; rabbit anti-S100 (Dako), 1:200; rabbit anti-peripheral mye- constructed containing a fusion reporter gene comprising the ribosomal
lin glycoprotein P0 (a kind gift from J. P. Brockes, Ludwig Institute for protein L4 and eGFP. For construction of the fusion protein L4-eGFP, the L4
Cancer Research, London, UK), 1:600; rabbit anti-myelin basic protein fragment was amplified with PCR from a rat cDNA library with the following
(MBP; Stem Cell Technologies), 1:200; rabbit anti-GFP (AbCam ab290), primers: forward primer (5⬘) containing an NheI restriction site: AAG-CTA-
1:500; and human anti-ribosomal P antigen (ImmunoVision), 1:100. After GCC-CGC-CAC-CAT-GGC-TTG-TGC-CCG-TCC-CC; reverse primer
washing, the preparations were incubated for 2.5 h at room temperature (3⬘) containing a SalI site: GAG-TCG-ACT-GCA-GCA-GAC-TTT-TTT-
with the appropriate secondary antisera; FITC-donkey anti-mouse IgG1, TCT-TCT-G. The L4 fragment was subsequently cloned into the multiple
1:200; TRITC-goat anti-human IgG, 1:200 (both from Jackson ImmunoRe- cloning site (MCS) of pEGFP-N2 (BD Biosciences; Clontech) between NheI
search); AlexaFluor 647-goat anti-rabbit IgG, 1:400; AlexaFluor 647-goat (5⬘) and SALI (3⬘). The L4-eGFP fragment was then cut from pL4-eGFP-N2
anti-mouse IgG1, 1:400 (both from Molecular Probes), washed three times with NheI (5⬘) and XbaI (3⬘) and cloned into the MCS at the XbaI restriction
in PBS, mounted in Vectashield (Vector Laboratories) and viewed with a site of the LV vector p156RRLsin-PPThCMVMCS-eGFP-wpre (kindly pro-
Bio-Rad radiance 2000 confocal microscope. vided by Dr. L. Naldini, San Raffaele Institute, Milan, Italy). This LV vector
Quantitative immunocytochemistry. From five (p ⬍ 0.01) mice and five contains a cytomegalovirus (CMV) promoter along with the HIV-1-Sin 18
wild-type mice, sciatic nerves were dissected. The nerves were divided into LTR and the HIV-1 genomic RNA packaging signal, a Rev response element
four equal parts. One part was immediately frozen and stored at ⫺80°C. The (RRE), a central polypurine tract (PPT), the human CMV promoter
other three parts were transferred to culture medium (see above). At 18, 24, (hCMV) followed by the fusion construct L4-eGFP and the woodchuck
and 48 h, nerves were frozen and stored at ⫺80°C. Cryostat sections (10 ␮m) posttranscriptional regulatory element (WPRE), flanked by two long-
were collected on Superfrost slides, air-dried, and stored at ⫺80°C. After terminal repeats (LTRs). The LV fusion construct was finally sequenced to
thawing, slides were fixed in 4% paraformaldehyde for 30 min, rinsed in PBS verify its insert and checked for right orientation. To produce viral vectors,
(3⫻), and incubated overnight at 4°C with human anti-ribosome antibody, the LV-L4-eGFP or the LV-eGFP (Blömer et al., 1997) transfer plasmids
dilution 1:1000 as described above. Next, sections were washed (3⫻ in PBS) were co-transfected with the viral core packaging construct pCM-
and incubated for 1 h in 1:200 diluted goat anti-human IgG, conjugated to VdeltaR8.74 and the VSV-G envelope protein vector pMD.G.2 into 293T
horse-radish peroxidase (Dako). After washing (3⫻ in PBS), the bound cells as previously described (Naldini et al., 1996b). Briefly, 5 ⫻ 10 6 293T
antibodies were visualized with 0.01% diaminobenzidine (Merck). As a con- cells were seeded in 10 cm dishes 24 h before transfection in complete
trol for the immunocytochemical reaction, we used normal human serum. Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal calf
Staining was observed neither in the axoplasm nor in the cytoplasm of resi- serum (FCS), 1⫻ glutamine (Gln) and 1⫻ penicillin/streptomycin (PS) and
dent cells of the nerve (data not shown). grown in a 5% CO2 incubator. Two hours before transfection, the culture
To quantify the immunoreactive axons, only cross-sectioned fibers medium was changed. For each 10 cm dish, 10 ␮g of transfer vector plasmid,
with a complete myelin sheath were considered. From each nerve, at least 6.5 ␮g of envelope plasmid, and 3.5 ␮g of packaging plasmid were co-
250 fibers were evaluated double blindly. For statistical analysis, the Stu- transfected using the calcium phosphate method. The medium was replaced
dent’s t test was performed to determine statistical differences. with IMDM containing 2% FCS, 1⫻ Gln and 1⫻ PS after 14 –16 h, and the
Quantification of ribosomes. Quantification of ribosomal content in LV particle containing conditioned medium was collected 24 h later, cleared
axons was performed on intact Wld s and wild-type axons, and in Wld s by low-speed centrifugation (176 g for 5 min) and filtered through a 0.22 ␮m
axons transected for 7 d (four nerves for each condition). At the electron cellulose acetate filters. The supernatant was concentrated ⬃100-fold by ultra
microscopical (EM) level, ribosomes were counted in 250 axons ran- centrifugation (53,000 g for 2.5 h). The pellet was resuspended in PBS and ali-
domly chosen in two sections 100 ␮m apart; the thickness of EM sections quots of LV-L4-eGFP stored at ⫺80°C. The titer (transducing units/ml; TU/ml)
was 80 nm. To estimate the volume of the axoplasm sampled, axons were was determined by transducing 293T cells with serial dilutions of the LV-L4-
assumed to be cylinders. In semithin Epon sections, the diameter of 100 eGFP vector in complete IMDM. Transduced cells were incubated for 24 h and
axons randomly chosen was measured with an ocular micrometer. Val- thereafter medium was replaced with fresh IMDM and cells were incubated for
ues are expressed as ribosome/␮m 3 of axoplasm [(average ribosome/ another 24 h. The titer was determined by counting GFP positive cells under a
axon)/(average volume of axoplasm surveyed)]. fluorescence microscope and was 1.5 ⫻ 10 9 TU/ml.
Ribosome purification. The supernatant of the ribosomal pellet was
dialyzed and concentrated. The ribosomal pellet (see Fig. 2 A, inset) was
resuspended in sample buffer. Twenty micrograms of each sample
Results
was loaded on a 10% acrylamide gel. As a control, histidine-eGFP (20 ␮g) Axonal desomatization increases content of axonal ribosomes
was loaded also. After blotting, the PDVF membrane was incubated with Desomatized axons of Wld s mice survive for several weeks (Lunn
rabbit anti-GFP (1:10,000) followed by anti-rabbit HRP (1:10,000), and et al., 1989). Our previous study on Wld s mice demonstrated that
immunoreactive bands were visualized with an ECL kit. desomatized axons contain many ribosomes compared with their
Lentivirus production. For ribosomal labeling, an lentiviral (LV) vector was intact axon counterparts (Court and Alvarez, 2005). To follow up
11026 • J. Neurosci., October 22, 2008 • 28(43):11024 –11029 Court et al. • Schwann Cell to Axon Transfer of Ribosomes

this finding, we studied ribosomes of axons in three conditions: antibody. In axons, the ribosomal staining appeared as diffuse
wild-type intact, Wld s intact, and Wld s desomatized for 7 d. In intact patches and as puncta, in keeping with the EM data, and the L4-
axons of wild-type or Wld s mice, ribosomes, mostly in the form of eGFP fluorescence colocalized with the ribosomal marker (Fig. 2B;
single ribosomes, were scarce (Fig. 1A). In desomatized Wld s axons, supplemental Fig. 2A, available at www.jneurosci.org as supplemen-
a dramatic increase occurred and ribosomes were often arranged in tal material). All axons showing the L4-eGFP label were enveloped
polysomal clusters (Fig. 1B). These polyribosomes were embedded by labeled Schwann cells. Central to the crush, a zone adjacent to that
in the neurofilament space and also packed in multimembrane ves- infiltrated with the viral suspension, neither resident cells of the
icles [ribosome-containing vesicles (RVs)] (Fig. 3C). To quantify the nerve nor axons showed L4-eGFP signal (supplemental Fig. 3, avail-
magnitude of this phenomenon, in this study we determined the able at www.jneurosci.org as supplemental material).
number of polyribosomes in the cytosol. In intact axons of wild-type To quantify the above data, Schwann cells and their associated
and Wld s mice, the average number of polyribosomes per volume of axons were scored for the LV-L4-eGFP labeling. A total of 236 axons
axoplasm was ⬃0.02/␮m 3, whereas during desomatization of Wld s from three animals were surveyed. In desomatized Wld s axons, 79%
axons, ribosomes increased by two orders of magnitude. The frac- of myelinating Schwann cells exhibited L4-eGFP signal, and ⬃60%
tion of axons that exhibited ribosomes in a cross-section of the nerve of axons associated with these cells showed a diffuse and/or punctate
(⬃1% in intact axons) increased by one order of magnitude during L4-eGFP signal in the neurofilament space (compare Fig. 2B; sup-
desomatization (Fig. 1C). In desomatized axons of wild-type mice, plemental Fig. 2A, available at www.jneurosci.org as supplemental
before their degeneration, ribosomes also increased. For this, nerve material). Conversely, uninfected Schwann cells, i.e., L4-eGFP neg-
explants of Wld s and wild-type mice were kept in vitro for 18 – 48 h. ative, encompassed L4-eGFP negative axons (supplemental Fig. 2B,
At time 0, ⬃4% of axons showed anti-ribosome immunoreactivity. available at www.jneurosci.org as supplemental material). The
At 18 h, ⬃15% were positive, whereas up to 48 h, there was no L4-eGFP signal largely colocalized with the immunostaining of ribo-
significant increase. Values for wild-type and Wld s nerves were sim- somes in both Schwann cells and axons (Fig. 2B, supplemental Fig.
ilar at all time points (supplemental Fig. 1, available at www. 2A, available at www.jneurosci.org as supplemental material): in
jneurosci.org as supplemental material). Because the nerve explants 84% of puncta in the axoplasm, signals colocalized. In summary, our
deteriorate with time, the early arrest of the ribosomal increase may data point positively to a Schwann cell-to-axon transfer of ribo-
not apply to in vivo severed axons. These results indicate that deso- somes, whereas the cell body as an alternative supplier has been
matization triggers a fast increase of axonal ribosomes, as most of it excluded by desomatization.
occurs in the first hours and that the increase of ribosomes in axons We studied next whether ribosomes were transferred selectively,
and the underlying mechanism are phenomena that belong to the or along with other Schwann cell proteins. For this, we focused on
species rather than to the Wld s mouse strain. the Schwann cell specific protein S100, which resides in the Schwann
cell cytosol (Moore, 1972). In desomatized Wld s fibers, Schwann
Schwann cells transfer ribosomes to desomatized axons cells were stained strongly with an antiserum to S100. In axons, S100
Ribosomal subunits are assembled in the nucleus and exported to was undetectable, whereas ribosomes showed a clear immunostain-
the cytoplasm. When the small subunit recruits the initiation ing (supplemental Fig. 4, available at www.jneurosci.org as supple-
complex and binds a mRNA, the large subunit is subsequently mental material). As an additional control for unspecific transfer of
bound to form a ribosome (Alberts et al., 2002). In a desomatized Schwann cell cytoplasmic components, we used the LV coding for
axon, the parent cell body cannot supply extra ribosomes to the eGFP which, after translation, remains in the cytosol of infected cells
axoplasm, and the axoplasm is unable to synthesize RNA. There- (Blömer et al., 1997). The same experimental protocol described
fore, desomatized axons must receive their ribosomes from an above was used to express the LV-eGFP vector in the nerve. The
extraneuronal source. Because glial cells were reported to tran- Schwann cell cytoplasm exhibited a strong eGFP fluorescence and
scribe axonal RNAs in a squid model system (Eyman et al., 2007), anti-ribosome immunofluorescence. In the neurofilament space of
we surmised that Schwann cells supply ribosomes to mammalian the encompassed axon, ribosome immunoreactivity was also de-
axons. To test this conjecture, Schwann cell ribosomes were tected whereas eGFP fluorescence was absent (Fig. 2C). Together,
tagged with eGFP. For this, we constructed an LV vector to ex- these data indicate that transcellular transfer of ribosomes is selec-
press the ribosomal protein L4 fused to eGFP (LV-L4-eGFP). tive, as at least the two cytosolic proteins we investigated were ex-
Upon infection, the host cell produces DNA strands from the cluded from transfer.
viral RNA, which are integrated into its genome for subsequent
transcription and translation (Naldini et al., 1996a). Thus, if the Intercellular route of Schwann cell to axon transfer
vector is injected into severed nerves and afterward axons exhibit of ribosomes
L4-eGFP-tagged ribosomes, they should have originated in in- To investigate how Schwann cell-derived ribosomes gain access
fected cells associated with the desomatized axons. to the axoplasm, we applied confocal microscopy and electron
First, to assess whether L4-eGFP fusion protein does incorpo- microscopy. Immunofluorescence revealed that protrusions of
rate into ribosomes, HEK cells were infected with the lentivirus. Schwann cells carrying anti-ribosome signal occasionally invagi-
Abundant L4-eGFP was recovered in the ribosomal fraction of nated into the axon both in internodes (Fig. 3A) and paranodes
these cells, whereas it was undetectable in the cytosolic fraction (supplemental Fig. 5, available at www.jneurosci.org as supple-
(Fig. 2 A). mental material). In close vicinity to these invaginations, in the
Next, the sciatic nerve of Wld s mice was crushed at mid-thigh, axoplasm, anti-ribosome positive puncta were detected. Some of
and LV-L4-eGFP was injected into the nerve distal to the crush; in these puncta showed complete or partial colocalization of anti-
addition, the nerve was cut at the sciatic notch to introduce a gap of ribosome and anti-MBP immunoreactivity (supplemental Fig.
⬃2 mm to insure that axons are indeed severed from their cell bod- 6 A–C, available at www.jneurosci.org as supplemental material),
ies. Seven days later, nerves were processed, and cryostat section and which suggests that these puncta were former Schwann cell in-
teased fibers were investigated with the relevant antisera. In the in- vaginations that during detachment became part of the axo-
jected nerve, i.e., distal to the crush, Schwann cells exhibited L4- plasm. The partial colocalization may be indicative of the release
eGFP fluorescence, which colocalized with a specific anti-ribosomal of ribosomes from the puncta into the axoplasm (supplemental
Court et al. • Schwann Cell to Axon Transfer of Ribosomes J. Neurosci., October 22, 2008 • 28(43):11024 –11029 • 11027

Figure 2. Transfer of ribosomes from Schwann cell to axon. A, Western blot showing incorporation of the L4-eGFP fusion protein into ribosomes. HEK cells were infected with LV-L4-eGFP,
fractionated, after which the blot was stained with anti-eGFP. First lane, Histidine-tagged eGFP; second lane, coomassie-stained supernatant of pellet fraction; third lane, supernatant of pellet
fraction; fourth lane, sucrose gradient of purified ribosomal fraction. The antibody recognizes a single band that corresponds to the molecular weight of His-eGFP (first lane) and the fusion protein
(fourth lane), respectively. In the supernatant (third lane), no signal is detected, indicating that the cytosol contains little if any L4-eGFP. Bottom image, The ribosomal pellet shows intense eGFP
fluorescence after sucrose gradient purification. B, C, Wld s teased fibers, 7 d after crush; the distal segment of the nerve was injected immediately after the crush with LV-L4-eGFP (B), or with
LV-eGFP (C). Color codes indicate the antibodies used. B, Top, Triple immunostaining and Z-projections at the corresponding numbers; in the neurofilament space, puncta are seen in which ribosomal
and L4-eGFP fluorescent markers colocalize. Bottom, Higher magnification of boxed area shows in detail colocalization of signals in both Schwann cell cytoplasm and axoplasm (merged signals, left),
ribosome signal (middle), and L4-eGFP signal (right). C, as in B, except that the encoded protein lacked the ribosomal protein L4. Ribosomal signals are present in Schwann cells and axoplasm, but
eGFP is present only in the Schwann cell. These results suggest that the transfer mechanism between Schwann cells and axons is selective. Scale bars, 10 ␮m.

Fig. 6C, available at www.jneurosci.org as supplemental material) vesicles and the axoplasm (Fig. 3D; supplemental Fig. 7E–H,
(see also Fig. 3D; supplemental Fig. 7E–H, available at www. available at www.jneurosci.org as supplemental material). Com-
jneurosci.org as supplemental material). In intact nerves, anti- bining the LM and EM data we propose the pathway depicted in
ribosome positive puncta could also be observed, although they Figure 4 for the transit of ribosomes from the outer cytoplasm of
were detected much less frequently (supplemental Fig. 8 A, avail- the Schwann cell to the axoplasm.
able at www.jneurosci.org as supplemental material). In intact axons, in addition to ribosomes (see above) also RVs
At the EM level, in 7 d desomatized Wld s axons, we identified were occasionally detected. (supplemental Fig. 8 B, available at
polyribosomes in one to several enlarged layers of adaxonal www.jneurosci.org as supplemental material), suggesting that in
Schwann cell cytoplasm and also in cytoplasmic protrusions of intact axons ribosomes may also be derived from glial cells.
Schwann cell into the axoplasm (Fig. 3B). In addition, we ob-
served polyribosomes in vesicles surrounded by two or multiple
membranes, giving rise to RVs. The morphology of these struc- Discussion
tures can be quite complex with ribosomes present between mul- We here demonstrate that mature axons harbor a small popula-
tiple layers of the Schwann cell membranes (Fig. 3C; supplemen- tion of ribosomes, confirming and extending the report of Koe-
tal Fig. 7, available at www.jneurosci.org as supplemental nig et al. (2000), and in addition, we show that their numbers
material). From some of these RVs the outer membrane was increase dramatically on desomatization of axons. Moreover, us-
discontinuous, creating a continuum between the content of the ing the Wld s mouse as a model, our data point to a novel notion
11028 • J. Neurosci., October 22, 2008 • 28(43):11024 –11029 Court et al. • Schwann Cell to Axon Transfer of Ribosomes

Figure 3. Putative intercellular pathway of ribosomal transfer. Wld s axons desomatized 7 d before. A, Teased fiber: triple immunostaining using anti-ribosome, neurofilament, and P0 antisera.
Top, Ribosome immunoreactive puncta are clearly seen in the axoplasmic space outlined by the neurofilament staining; the arrow points to a structure penetrating through the myelin marked by
the P0 staining, akin to a Schmidt-Lanterman incisure; bottom, Z-projections of the corresponding numbers of the top show ribosomal signal from the Schwann cell penetrating into the axoplasm
(arrows in 2 and 3). Scale bar, 10 ␮m. B–D, EM micrographs of Wld s axons desomatized for 7 d. B, Polyribosomes (arrowheads) can be seen in enlarged adaxonal pockets of Schwann cell cytoplasm.
At the arrow, a Schwann cell protrusion loaded with ribosomes appears to be in the process of disengaging itself from the Schwann cell. C, Vesicle with multiple membranes contains several
polyribosomes (arrow), whereas many polyribosomes are present in the axoplasm (arrowhead). D, Vesicle with ruptured membranes, creating a continuum between the vesicular space and
axoplasm [note a polyribosome at the interface between vesicle and axoplasm (arrow) and polyribosomes in the axoplasm (arrowheads)]. Scale bars, 300 nm. B1–D2 are high magnification images
of areas indicated by arrows and arrowheads in B–D. Together, these images suggest the pathway followed by ribosomes from the Schwann cell to the axoplasm.

in the relationship between Schwann cells and axons in that somes consist of several ribosomes translating simultaneously a
polyribosomes in desomatized axons originate in the Schwann strand of mRNA (Alberts et al., 2002), our data imply that
cell. However, we cannot exclude the possibility that mRNA en- Schwann cells have the ability to transfer mRNA as well. This
coding the L4-eGFP fusion protein is also transferred from creates the possibility for Schwann cells to modify the repertoire
Schwann cells, translated in the axon and added to partially as- of gene expression and protein production of the recipient axon.
sembled ribosomes or exchanged with L4-protein on preexisting Several authors have raised the possibility that glial cells modulate
ribosomes. This latter possibility was suggested for synaptically gene expression in axons. For instance, Li et al. (2005) suggested in
translated Aplysia ribosomal proteins (Moccia et al., 2003). Fur- an EM study that CNS glia transfers ribosomes to spinal cord axons.
thermore, our data do not exclude the neuronal cell body as In this study, they showed the presence of double-walled vesicles at
another supplier of axonal ribosomes in intact axons. the axonal–myelin sheath interface, which often contained
The transfer of polyribosomes between two cell types of the ribosome-like particles (Li et al., 2005). More recently, Eyman et al.
nervous system is surprising and unsuspected. Because polyribo- (2007) proposed, for a squid nerve fiber model, that glial cells tran-
Court et al. • Schwann Cell to Axon Transfer of Ribosomes J. Neurosci., October 22, 2008 • 28(43):11024 –11029 • 11029

that the phenotype of axons can be controlled on a local basis and

relies on genetic programs of the associated glial cells. We anticipate
that this notion will suggest new avenues of research in the organi-
zation of the nervous system as well as neuropathology.
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