GLIA 59:1529–1539 (2011)
Morphological Evidence for a Transport of Ribosomes
from Schwann Cells to Regenerating Axons
FELIPE A. COURT,1,2 RAJIV MIDHA,3 BRUNO A. CISTERNA,1 JOEY GROCHMAL,3 ANTOS SHAKHBAZAU,3
WILLIAM T. HENDRIKS,4 AND JAN VAN MINNEN4,5*
1
Millennium Nucleus for Regenerative Biology, Faculty of Biology, P. Catholic University of Chile, Chile
2
NeuroUnion Biomedical Foundation, Santiago, Chile
3
Department of Clinical Neuroscience and Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary,
Calgary, AB, Canada
4
Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research,
Faculty of Earth and Life Sciences, VU University, Amsterdam, The Netherlands
5
Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary,
Calgary, Alberta, Canada
KEY WORDS
lentiviral transduction; nerve crush; nerve graft; regeneration; intercellular transport; peripheral nervous system
ABSTRACT
Recently, we showed that Schwann cells transfer ribosomes
to injured axons. Here, we demonstrate that Schwann cells
transfer ribosomes to regenerating axons in vivo. For this,
we used lentiviral vector-mediated expression of ribosomal
protein L4 and eGFP to label ribosomes in Schwann cells.
Two approaches were followed. First, we transduced
Schwann cells in vivo in the distal trunk of the sciatic
nerve after a nerve crush. Seven days after the crush, 12%
of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was
inserted into the sciatic nerve. Fluorescent ribosomes were
detected in regenerating axons up to 8 weeks after graft
insertion. Together, these data indicate that regenerating
axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein
synthesis by transferring protein synthetic machinery and
mRNAs to these axons. V 2011 Wiley-Liss, Inc.
C
INTRODUCTION
Schwann cells have a variety of functions in neuronal
development, maintenance, and regeneration after nerve
injury. They form intimate contacts with axons, which
secrete molecules that are essential for survival of
Schwann cell precursors and their differentiation into
mature Schwann cells (Nave and Salzer, 2006).
One of the most striking features of Schwann cells is
their plasticity, which is especially evident following a
nerve crush. Axons in the distal stump undergo Wallerian degeneration, and although the mechanism is currently unknown, increase in intracellular calcium and
activation of axonal proteases have been implicated in
the final stages of axonal degeneration (Coleman, 2005).
After axonal degeneration, Schwann cells dedifferentiate
and proliferate, and together with macrophages, they
clear endoneurial myelin and axonal debris. Schwann
C 2011
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Wiley-Liss, Inc.
cells line up to form the bands of B€
ungner, characterized
by a continuous basal lamina that creates an outgrowthpermissive environment. In addition, Schwann cells
secrete molecules that aid peripheral nerve regeneration
in a direct or indirect way. These molecules include
nerve growth factor (NGF), brain-derived neurotrophic
factor (BDNF), neurotrophin (NT)-3, and NT-4/5. Following receptor binding and retrograde transport, they
induce the expression of regeneration-associated genes
[RAGs; (Raivich and Makwana, 2007; Raivich et al.,
2004; Stam et al., 2007)]. In addition, these molecules
signal locally to regenerating axons, where they affect
axon guidance and extension (Leung et al., 2006; Lin
and Holt, 2007; Wu et al., 2005; Zhang et al., 1999),
mRNA translocation, and local protein synthesis (Willis
et al., 2007).
From the above it is evident that the interplay
between Schwann cells and axons is essential during peripheral nerve regeneration, although the exact mechanisms are only partially elucidated. Recently, we
described a novel and unexpected relationship between
Schwann cells and peripheral axons. We showed that
Schwann cells are able to provide polyribosomes to
axons, and that this process is strongly upregulated in
injured axons of both wild type mice and those belonging
to the Wallerian degeneration slow (Wlds) strain. We
surmised that the transferred polyribosomes support the
Additional Supporting Information may be found in the online version of this article.
Grant sponsor: FONDECYT; Grant number: 1070377; Grant sponsor: Millennium Nucleus; Grant number: P-07-011-F (to F.A.C.); Grant sponsor: Natural Sciences and Engineering Research Council of Canada; Grant number: 355356 (to
J.V.M.); Grant sponsor: University of Calgary; Grant number: 1011660
(to J.V.M.); Grant sponsor: European Union grant; Grant number: STREP 12702
(to J.V.M. and W.T.J.H.); Grant sponsor: Canadian Institutes for Health Research;
Grant number: MOP 82726 (to R.M.); Grant sponsors: Hotchkiss Brain Institute
Fellowship award (to J.G.) and AHFMR PDF award (to A.S.).
William T. Hendriks is currently at Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
*Correspondence to: Jan van Minnen, Department of Cell Biology and Anatomy,
Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary, Calgary,
Alberta, Canada T2N 4N1. E-mail: [email protected]
Received 19 May 2010; Accepted 16 May 2011
DOI 10.1002/glia.21196
Published online 8 June 2011 in Wiley Online Library (wileyonlinelibrary.com).
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COURT ET AL.
long-term survival of desomatized axons of the Wlds
strain and may also be involved in plastic responses of
adult axons to local stimuli (Court et al., 2008). We wondered whether regenerating axons also receive polyribosomes from Schwann cells to support local translation
and regeneration after a nerve crush. To investigate this
we used a lentiviral (LV) vector to express fluorescent
ribosomes in Schwann cells, using ribosomal protein L4
fused to eGFP (L4-eGFP). This vector was injected into
the distal nerve trunk of a crushed sciatic nerve. In a
second experiment, a decellularized nerve graft was
inserted into a sciatic nerve, after which the graft was
injected with Schwann cells expressing fluorescent ribosomes. The experiments showed that regenerating axons
harbor fluorescent ribosomes up to 8 weeks following
nerve injury, indicating that regenerating axons receive
polyribosomes from their accompanying Schwann cells.
MATERIALS AND METHODS
Lentivirus Production
A LV vector was constructed containing a fusion reporter gene comprising ribosomal protein L4 and
enhanced green fluorescent protein (eGFP). The L4 fragment was amplified with PCR from a rat cDNA library
with a forward primer (50 ) containing a NheI restriction
site; AAG-CTA-GCC-CGC-CAC-CAT-GGC-TTG-TGC-CCGTCC-CC and reverse primer (30 ) containing a SalI site;
GAG-TCG-ACT-GCA-GCA-GAC-TTT-TTT-TCT-TCT-G.
The L4 fragment was cloned into the multiple cloning
site (MCS) of pEGFP-N2 (BD Biosciences Clontech, CA)
between NheI (50 ) and SalI (30 ). The fragment was cut
from pL4-eGFP-N2 with NheI (50 ) and XbaI (30 ) and
cloned into the MCS at the XbaI restriction site of the
vector p156RRLsin-PPThCMVMCS-pre (kindly provided
by Dr. L. Naldini). This vector contains a CMV promoter
along with the HIV-1-Sin 18 LTR and the HIV-1 genomic
RNA packaging signal, a Rev response element (RRE), a
central polypurine tract (PPT), the human CMV promoter (hCMV) followed by the fusion construct L4-eGFP
and the woodchuck posttranscriptional regulatory element (WPRE), flanked by two LTRs. The LV fusion construct was sequenced to verify its insert and correct orientation. To produce viral vectors, the LV-L4-eGFP
transfer plasmid was co-transfected with the viral core
packaging construct pCMVdeltaR8.74 and the VSV-G
envelope protein vector pMD.G.2 into 293T cells as previously described (Court et al., 2008; Naldini et al.,
1996a,b). The titer (1.5 3 109 TU/mL) was determined
by counting eGFP positive cells.
kg). The sciatic nerve was crushed twice for 10 s with
Dumont #5 forceps. The crush site was marked with
surgical 10-0 nylon monofilament (Ethicon). One to 2 lL
Lentivirus in PBS containing 0.1% fast green was
injected into distal part of the sciatic nerve, near its trifurcation (Supp. Info. Figs. 1 and 2A).
Fixation and Immunocytochemistry
Seven days following the crush and LV injection,
nerves were fixed in situ in 4% paraformaldehyde in 0.1
M phosphate buffer, pH 7.4 for 20 min, and after dissection for an additional 40 min at room temperature,
washed in PBS and infiltrated with 30% sucrose and frozen at 280°C. Cryostat sections 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 the following
primary antibodies; human anti-ribosome antiserum,
1:1,000 [from a patient carrying a systemic lupus erythematosus characterized by Massardo et al. (2002), a
kind gift of S. Jacobelli]; mouse anti-neurofilament (clone
N52, Sigma), 1:1,000; rabbit anti-S100 (Dako, Glostrup,
Denmark), 1:200; rabbit anti-peripheral myelin glycoprotein P0 (a kind gift from J.P. Brockes), 1:600; rabbit antiGFP (AbCam, Cambridge, UK), 1:500. After washing,
the preparations were incubated for 2.5 h at room temperature with secondary antisera; FITC-donkey antimouse IgG1, 1:200; TRITC-goat anti-human IgG, 1:200
(both from Jackson ImmunoResearch, West Grove, PA);
AlexaFluor 647-goat anti-rabbit IgG, 1:400; AlexaFluor
647-goat anti-mouse IgG1, 1:400 (both from Molecular
Probes), in PBS, mounted in Vectashield (Vector laboratories, Burlingame, CA) and viewed with a Bio-Rad radiance 2000 confocal microscope (Cambridge, MA).
Quantification of eGFP-labeled ribosomes
Nerves (n 5 4) were crushed and injected with the
LV-L4-GFP and fixed after 7 days as described above.
Fourteen micrometer cryostat cross-sections from segments distal to the crush site were stained with human
anti-ribosome antiserum and neurofilament antibody
and imaged with confocal microscopy. Neurofilamentpositive axons (n 5 5,509) were analyzed for the presence of colocalized ribosomal and GFP signal using
ImageJ.
Nerve Graft Experiments
Nerve Crush Experiments
Animals and surgical procedures
Animal care and surgical procedures complied with
NIH guidelines. C57BL/6J mice weighing about 20 to 25
g were anaesthetized with sodium pentobarbital (36 mg/
GLIA
Animals
Male Lewis rats weighing 225 to 250 g (Charles River,
QC, Canada) were used. Surgical interventions were
carried out under inhalation anaesthetic (Isofluroane,
99.9% Halocarbon Laboratories, River Edge, NJ) and
pain control was provided by means of intraperitoneal or
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INTERCELLULAR RIBOSOME TRANSFER
oral administration of buprinorphene. Surgical procedures were carried out aseptically, and standard microsurgical techniques were used with an operating microscope (Wild M651; Wild Leitz, Willowdale, ON, Canada).
Animals were sacrificed at endpoint under deep anaesthesia using an overdose of intracardiac Euthanol
(Bimeda-MTC, Cambridge, ON, Canada). All efforts
were made to minimize suffering and animal numbers
by using appropriate protocols. The protocol was
approved and monitored by the University of Calgary
animal care committee and adhered strictly to guidelines set by the Canadian Council on Animal Care.
Preparation of acellular grafts
Nerve graft material was obtained from 16 deeply
anaesthetized adult Lewis rats by exposing the sciatic
nerve bilaterally and excising a 30-mm segment from
each side, following which animals were euthanized.
Harvested nerves were rinsed in sterile saline and divided into two 15 mm segments, yielding a total of four
grafts obtained from each animal. Segments were placed
in sterile cryotubes and subjected to five cycles of freezing in liquid nitrogen (2 min) and thawing in 37°C water
bath (2 min), a process which completely decellularizes
the nerve (Hall, 1986; Ide et al., 1983; Walsh et al.,
2009). The segments were subsequently frozen at 280°C
until use.
In vitro transduction of fluorescent Schwann cells
Schwann cells, isolated from P2 neonatal rat sciatic
nerve were cultured in Schwann cell medium to 50%
confluence in poly-L-lysine coated culture dishes. For a
comprehensive description of the isolation and culture
procedure see (Walsh et al., 2009). One ll of the LV-L4eGFP stock solution was added to the culture dish and
incubated for 24 h. After this, the medium was changed
and Schwann cells were cultured for another 3 days
until 95% to 100% confluence. Next, the cells were
sorted on a fluorescence activated cell sorter after which
the percentage of cells expressing the L4-eGFP fusion
protein was >95%. The culture was expanded, without
apparent loss of the intensity of fluorescence or the percentage of cells expressing fluorescent ribosomes. Cells
were dissociated, counted, and the percentage of cells
containing fluorescent ribosomes was determined. Only
cultures that had a percentage of fluorescent Schwann
cells >95% were used for the nerve graft experiments
(Supp. Info. Fig. 3).
Surgical methods
The sciatic nerve was exposed unilaterally and transected at mid-thigh level with a small segment resected
proximal to the trifurcation to create 10 to 12 mm injury
gaps. These were repaired with the freeze-thawed nerve
graft (previously thawed at 37°C and trimmed to 13
mm), using standard microsurgical techniques, as previously described (Walsh et al., 2009). Following repair, 5
3 105 transduced SCs in 5 lL DMEM was injected into
the grafts (Supp. Info. Fig. 2B).
Immunofluorescence
At 9 (n 5 2) and 16 days (n 5 4), 3 (n 5 2), 6 (n 5 4),
and 8 weeks (n 5 2) the graft plus 3 mm of attached
host nerve were removed and fixed overnight in 4%
paraformaldehyde in phosphate-buffered saline (PBS).
Samples were washed in PBS, cryoprotected in 30% sucrose and embedded in optimal cutting temperature
(OCT) compound (Sakura Fine technical Co., Torrance,
CA) and frozen at 280°C. Fourteen lm sections were
cut with a cryostat (Leica Microsystems Inc., Richmond
Hill, ON, Canada) at 220°C and mounted on Superfrost
slides (Fisher Scientific). Antigen retrieval was performed by incubating sections in 0.1 M citric acid and
0.1 M sodium citrate at 80°C for 20 min. Next, sections
were blocked in incubation medium (10% goat serum
and 0.5% Triton X100 in PBS) for 10 min. followed by
an overnight incubation of the primary antibodies in
incubation medium. Antibodies used were chicken-anti160 kD neurofilament medium (Abcam, 1:1,000),
human-anti-ribosomal P-protein (Immunovision, 1:500),
mouse-anti-ribosomal RNA (Lerner et al., 1981), rabbitanti-GFP (In Vitrogen, 1:500) and anti-myelin basic protein (MBP, 1:500, Stem Cell Technologies). Following
washing with PBS, slides were incubated with the Alexa
Fluor conjugated secondary antibodies, diluted 1:400
(Molecular Probes, Inc.) for 2 h. and viewed under a confocal microscope (Zeiss CLSM 510, Oberkochen, Germany). Substitution of primary antibodies with nonimmune serum was used as negative control for the
staining process. No specific staining was observed when
the primary antibodies were substituted by non-immune
sera (data not shown).
Electron Microscopy
Distal trunks of crushed sciatic nerves, nerve grafts
and control sciatic nerves were quickly dissected and
fixed by overnight immersion in 2.5% glutaraldehyde in
phosphate buffer (0.1 M, pH 7.4). The tissues were postfixed in 1% osmium tetroxide for 2 h, dehydrated with
ethanol and embedded in Epon. Ultrathin sections (70
nm) were contrasted with uranyl acetate and lead citrate (Reynolds, 1963).
RESULTS
Ribosomal Transfer to Regenerating Axons
We recently demonstrated that Schwann cells transfer
ribosomes to injured peripheral axons (Court et al.,
2008). To explore whether transfer is restricted to
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COURT ET AL.
Fig. 1. Ribosomal transfer in distal segments of a sciatic nerve one
week after crush and LV-L4-eGFP injection. A, A regenerating axon
(arrow), distal to nerve crush, outlined by neurofilament staining,
shows co-localized L4-eGFP as well as ribosomal immunoreactivity,
which indicates Schwann cell to axon transfer of ribosomes. Insets,
high magnification of area pointed at by arrow. B, Proximal to lesion,
the absence of L4-eGFP immunoreactivity indicates that the fusion pro-
tein is not expressed in this region. C, A small regenerating axon outlined by neurofilament staining, contains co-localized L4-eGFP and
ribosome immunoreactivity (arrows). Rib, ribosome, NF, neurofilament,
GFP, L4-eGFP immunoreactivity. Ax, axon. Scale bar A, B: 10 lm; C:
1 lm. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
injured axons, or may occur during other physiological
conditions, we investigated ribosome transfer to regenerating axons after nerve crush and nerve transection in
mice and rats, respectively. We reasoned that during
regeneration, ribosomal transfer might be enhanced as it
is known that growth cones contain ribosomes (Tennyson,
1970), and that regenerating axons require local protein
synthesis for elongation and growth cone steering (Brittis
et al., 2002; Gaete et al., 1998; Lin and Holt, 2007, 2008;
Merianda et al., 2008; Zheng et al., 2001). We used two
different models of regeneration. First, we used a crush
model and transduced Schwann cells with L4-eGFP
exclusively in the distal segment of the sciatic nerve. Second, we transected the nerve, after which it was repaired
with a nerve graft containing in vitro transduced
Schwann cells expressing L4-eGFP tagged ribosomes.
crush was dissected and processed for immunocytochemistry. The immunofluorescence data show that many
cells in the segment distal to the crush express the
fusion protein (Fig. 1A). In addition, we dissected the
segment proximal to the crush, and dorsal root ganglia
L4 and L5 and investigated whether neurons in the dorsal root ganglia and cells in the proximal stump of the
sciatic nerve contained fluorescent ribosomes. Confocal
microscopy revealed that neither neurons in the dorsal
root ganglia (data not shown) nor cells (e.g. Schwann
cells, fibroblasts) in the sciatic nerve immediately proximal to the lesion expressed L4-eGFP labeled fluorescent
ribosomes (Fig. 1B). These data demonstrate that the
LV vector does not transduce cells including neurons
proximal to the lesion. In contrast, in the segment distal
to the crush, nucleated cells, including Schwann cells
expressed fluorescent ribosomes (Fig. 1A). In addition to
these cells, within 12% 6 1% of regenerating axons
(5,509 axons from four sciatic nerves), eGFP and antiribosomal signals co-localized. (Fig. 1A,C, and insets).
Since the LV-vector did not transduce neurons or
Schwann cells in regions proximal to the nerve crush,
these data indicate that Schwann cells distal to the
nerve crush are the source of fluorescent ribosomes
found within regenerating axons.
Ribosome Transfer After Nerve Crush
For the first series of experiments we used mice. Immediately following a sciatic nerve crush, we injected a
LV vector to express the L4-eGFP fusion protein (LV-L4eGFP). After 7 days, the nerve segment distal to the
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INTERCELLULAR RIBOSOME TRANSFER
Ribosome Transfer in Nerve Grafts
It has been shown that direct injection into the peripheral nerve of LV vectors encoding reporter genes
such as GFP can result in an immunological response to
the fusion protein, thereby severely attenuating the
expression of the reporter protein (Hendriks et al.,
2007). In agreement with this, we noticed a significant
reduction in the number of transduced Schwann cells
and the intensity of the eGFP signal 3 weeks after LV
infection (data not shown). To allow study of ribosome
transfer for periods up to 8 weeks, we used a nerve
graft that was repopulated with Schwann cells that
were transduced in vitro to express fluorescent ribosomes, using the L4-eGFP construct (Supp. Info. Figs.
2B and 3). Furthermore, this strategy completely abolishes the possibility of LV transduction of other cells in
the nerve, rendering Schwann cells the only source of
fluorescently labeled ribosomes. Nerve grafts were
injected with 5 3 105 transduced Schwann cells. Sixteen
days and 3, 6, and 8 weeks after injection, nerve grafts
were harvested and investigated by means of confocal
microscopy and transmission electron microscopy. The
immunofluorescence data of 16-day nerve grafts showed
that axons had regenerated through the complete length
of the graft, since at the most distal end of the graft,
NF-positive axon profiles were present (data not shown).
This is in line with previous studies in which Schwann
cell-filled nerve grafts were used (Walsh et al., 2009),
and indicates that transduced Schwann cells do not negatively influence regeneration. After 16 days, L4-eGFP
expressing cells were found throughout the nerve graft,
many of which were associated with regenerating axons
(Fig. 2A). In the 3, 6, and 8 week groups the eGFP fluorescent Schwann cells were found in island-like structures (Fig. 2B,C) reminiscent of mini-fascicles previously
described (Morris et al., 1972). In between these minifascicles few cells were present, as can be concluded
from the lack of ribosomal staining (Fig. 2B). Electron
microscopical analysis of these grafts confirmed the
presence of minifascicles, each surrounded by a perineural cell, containing several myelinated as well as
unmyelinated axons (Fig. 2C). Furthermore, the tissue
in between mini-fascicles consisted mainly of collagen
fibers.
Co-localized immunoreactivity for L4-eGFP and ribosomes was detected in a subpopulation of regenerating
axons at all time-points assessed, as is illustrated for the
16 day, 3- and 8-week-old grafts (Fig. 3A–C; Supp. Info.
Fig. 4). Analyses of confocal stacks demonstrated that L4eGFP/ribosome
immunoreactivity
was
completely
immersed in the axoplasm, and showed no connection to
the adaxonal Schwann cell, which makes it unlikely that
the observed immunoreactivities are due to invaginations
of the Schwann cell cytoplasm into the axon (Supp. Info.
Fig. 5). In addition, we observed ribosome immunofluorescence in axons that was not co-localized with L4-eGFP immunoreactivity (Fig. 3A), suggesting that these ribosomes
originated in non-transduced Schwann cells, or, alterna-
1533
tively, were transported from the neuronal cell body into
regenerating axons.
Electron Microscopy Identifies Polyribosomes
in Regenerating Axons in Crushed Nerves
and Nerve Grafts
At the ultrastrucural level, we detected polyribosomes
in the axonal compartment at all time points, both in myelinated and unmyelinated axons (Fig. 4A–E). Interestingly, we observed Schwann cell membranes that invaginated into the axonal space, akin to the axon-Schwann
cell networks described previously (Gatzinsky, 1996; Gatzinsky et al., 1997, 2003) and vesicles, surrounded by
double or multiple membranes, containing ribosomes
(Fig. 4F). However, we cannot exclude the possibility that
these ‘‘vesicles’’ are still connected to the Schwann cell
protrusions by connection that are outside the plane of
sectioning. Although we cannot distinguish at the EM
level which Schwann cells express L4-eGFP, and which
have migrated into the distal segment or nerve graft, the
large number of Schwann cells expressing the fusion protein (see Fig. 2) indicates that the expression of the fusion
protein did not negatively affect Schwann cell functions
such as myelination. Moreover, the presence of myelin
proteins such as myelin basic protein and protein zero
were confirmed by immunocytochemistry in myelin
sheaths produced by L4-eGFP expressing Schwann cells
(data not shown).
Transfer of Schwann Cell Cytoplasmic
Constituents
To investigate whether cytoplasmic constituents of
Schwann cells are transferred to regenerating axons, we
used Schwann cells that were transduced with LV coding for eGFP alone which, after translation, remains in
the cytosol (Bl€
omer et al., 1996). These cells were
injected into a nerve graft, harvested after 4 weeks and
processed for confocal immunofluorescence as described
above. The injected Schwann cells lined up with regenerating axons. In these axons, ribosome immunoreactivity was found co-localized with eGFP fluorescence (Fig.
5; Supp. Info. Fig. 6), whereas eGFP-only fluorescence in
axons was also observed. Analysis of the confocal stacks
showed that these vesicles were completely immersed in
the axoplasm and had no connection with the adaxonal
Schwann cell cytoplasm (Supp. Info. Figs. 7 and 8). In
addition, we observed eGFP and anti-ribosome positive
cytoplasmic Schwann cell protrusions, directed towards
the NF space of the axon (Supp. Info. Fig. 9), suggestive
for transfer of material from Schwann cells to axons. Together, these data indicate that Schwann cell cytoplasm
can be transferred together with ribosomes, most likely
via ribosome-containing vesicles (RVs) described previously (Court et al., 2008). Using electron microscopy,
we investigated whether ribosome-containing vesicles
contained other structures, in addition to ribosomes. DeGLIA
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COURT ET AL.
Fig. 2. L4-eGFP expressing Schwann cells in nerve graft. A, Sixteen
days, B, 6 weeks postgraft insertion. L4-eGFP expressing Schwann
cells (green) are abundant in nerve grafts and are arranged together
with regenerating axons in so-called mini-fascicles, which are clearly
discernible in the 6-week graft (B, arrows); Ribo, ribosome, NF, neurofilament and eGFP, L4-eGFP immunoreactivity. C, electron microscope
image, 8 week postinsertion showing a minifascicle containing myelinated (arrows) and unmyelinated (arrowheads) axons. The fascicle is
surrounded by a perineural cell (P), separating the fascicle from the
connective tissue. Co, collagen fibers, Sc, Schwann cell. Scale bars,
50 lm in A, B and 1 lm in C. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
spite a very thorough search, we were unable to detect
such structures as e.g. mitochondria, lysosomes, or
microtubules in these vesicles, which suggests that ribosomes are the only organelles transferred to regenerating axons.
The present data strongly suggest that also regenerating
axons receive polyribosomes from periaxonal Schwann
cells. Using Schwann cells that were transduced in vitro
with a lentiviral vector encoding ribosomal protein L4
fused to eGFP and subsequently injected into a nerve
graft, we excluded the possibility that neuronal cell
bodies contribute L4-eGFP labeled ribosomes to regenerating axons. Also, in the nerve crush experiments it is
highly unlikely that neuronal cell bodies are a source of
L4-eGFP positive axonal ribosomes since neither the
dorsal root ganglia, nor cells or axons in the nerve segment immediately proximal to the crush expressed L4eGFP fluorescent ribosomes. In addition, the electron
microscopical data support a Schwann cell origin of the
DISCUSSION
Regenerating Axons Harbor Polyribosomes that
have Originated in Schwann Cells
We previously demonstrated that Schwann cells transfer ribosomes to desomatized axons (i.e. axons no longer
connected to their cell body) of Wlds and wild-type mice.
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INTERCELLULAR RIBOSOME TRANSFER
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Fig. 3. Schwann cells transfer L4-eGFP fluorescent ribosomes to
regenerating axons. A, Sixteen-day graft, the arrow shows an L4-eGFP/
ribosome immunopositive punctum in the axon, arrowhead shows immunoreactivity in periaxonal Schwann cell cytoplasm, but not in the
axon. Small arrow points at ribosome immunoreactivity in axon that
does not co-localize with L4-eGFP immunoreactivity. B, Three-week-old
graft, showing a small axon containing L4-eGFP-positive ribosomes
(arrows). C, Eight-week-old graft. Arrows point to co-localized ribosome
and L4-eGFP immunoreactivity in two separate and adjacent axons.
Ribo, ribosome, NF, neurofilament, and eGFP, L4-eGFP immunoreactivity. Scale bars, 2 lm in A, B and 5 lm in C. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
axonal ribosomes. We identified double or multiple
walled vesicles, comparable with those described in our
previous study (Court et al., 2008). Similar ribosomecontaining vesicles were identified in axons in the spinal
cord at the myelin axon interface (Li et al., 2005). These
vesicles are considered to be instrumental in the process
whereby glial cells transfer polyribosomes to axons
(Court et al., 2008; Li et al., 2005).
These observations add to previous studies on the
interplay between Schwann cells and axons, in which
the nodes of Ranvier have been described as specific
sites of exchange between Schwann cells and axons. Socalled axon-Schwann cell networks in large alpha motor
axons function as structures to dispose of axonal organelles. These networks consist of protrusions of ad-axonal
Schwann cell cytoplasm into axons. Here, retrogradely
transported degenerating organelles and organelles carrying waste products and foreign materials are segregated from the axons, engulfed by and in degraded in
the Schwann cell cytoplasm (Gatzinsky, 1996; Gatzinsky
et al., 1997). It has been suggested that this process is
essential for the long-term maintenance of axons that
would otherwise be clogged by cellular waste products.
It is unlikely that these axon-Schwann cell networks
are involved in sequestering ribosomes from axons,
since eGFP-tagged ribosomes of Schwann cell origin reside in regenerating axons. Furthermore, ribosomes
occur in Schwann cell derived eGFP laden vesicles.
These vesicles are completely surrounded by axoplasm,
suggesting a route from Schwann cell to axons, in
which ribosome-laden vesicles are budded of from
adaxonal Schwann cell cytoplasm, and not vice versa.
An interesting possibility is that these axon-Schwann
cell networks are involved in delivering ribosomes to
axons, although our present data do not provide an answer to this question. Together, these previous studies
on axon-Schwann cell networks, and our studies on
Schwann cell to axon transfer of ribosomes suggest that
there is a two-way cellular trafficking between axons
and Schwann cells, one that is involved in removing
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COURT ET AL.
Fig. 4. Electron microscopy; ribosomes in regenerating axons. A,
electron microscopical image of a regenerating axon, outlined in purple,
in distal nerve segment 1 week after crush. Arrowheads point at the
Schwann cell cytoplasm surrounding the axon. In the dashed box a
polyribosome in the axoplasm is present, further detailed in B, arrow.
C, unmyelinated axon 16 days postgraft insertion; D, myelinated axon
3 weeks postinsertion; E, myelinated axon 6 weeks postinsertion. Inset
shows a polyribosome in which two ribosomes are connected by a thin
thread (arrowhead), most likely representing an mRNA molecule. At all
time points, polyribosomes were detected in axons (arrows). F, myelinated axon in a 6-week-old graft, containing a double walled vesicle filled
with ribosomes (arrow); asterisks denote vesicles with two to multiple
membranes, most likely consisting of myelin membranes. Arrowheads
point at invagination of the adaxonal myelin, suggestive for how these
vesicles are formed. This particular axon did not contain polyribosomes
in the axoplasm. Ax, axon, M, myelin, Sc, Schwann cell. Scale bars,
1 lm in A, 100 nm in B–F; inset 50 nm. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
harmful material from axons, whereas the other supplies polyribosomes to regenerating and injured axons.
This further illustrates the intimate communication
that exists between axons and their periaxonal
Schwann cells.
Functional Implications of Ribosome Transfer
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Schwann cells play an essential role in peripheral
nerve regeneration, which is underscored by observations that successful regeneration largely fails when
INTERCELLULAR RIBOSOME TRANSFER
1537
Fig. 5. Schwann cells transfer cytosolic eGFP to regenerating axons.
Four weeks postgraft insertion. A thin layer of periaxonal Schwann cell
cytoplasm expressing eGFP (arrowheads) envelops regenerating axon
outlined by neurofilament staining (blue). In the axon a small structure
is indicated by an arrow that shows partly overlapping fluorescence for
anti-ribosome (red) and eGFP (green), suggesting that ribosomes are
transferred in structures that contain both Schwann cell cytoplasm and
ribosomes. Scale bar, 5 lm. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Schwann cells are absent or prevented from migrating
into a regenerating nerve (Chen et al., 2005). Despite the
identification of many molecules that are produced by
denervated and dedifferentiated Schwann cells such as
neurotrophic factors, regeneration promoting cell adhesion molecules, and receptors (including neurotrophin-,
neurotransmitter-, and purinergic receptors; Vrbova
et al., 2009), the full extent of their supportive role
remains to be elucidated. Our data point to a novel mechanism that might be involved in the neuronal regeneration-supporting role of Schwann cells and indicate that
Schwann cells transfer ribosomes to regenerating axons,
which suggests that they support local protein synthesis
in these axons. Given that polyribosomes are transferred, and that polyribosomes consist of multiple ribosomes simultaneously translating a mRNA molecule
(Alberts et al., 2002), our findings suggest that mRNA is
also transferred to and subsequently translated in
regenerating axons. As has been demonstrated previously, mainly in in vitro experiments, regenerating
axons contain a large and diverse population of mRNAs,
tRNAs, and rRNAs, components of the post-translation
protein synthesis machinery, resident endoplasmic reticulum and Golgi proteins (Capano et al., 1987; Crispino
et al., 1997; Giuditta et al., 1977, 1980; Kun et al., 2007;
Merianda et al., 2008; Twiss and van Minnen, 2006;
Zheng et al., 2001). In addition, many proteins are synthesized in regenerating axons, including cytoskeletal,
resident ER, heat shock and integral membrane proteins
(Willis and Twiss, 2006; Willis et al., 2005; Zheng et al.,
2001). Most likely, these locally translated proteins play
important roles in axon elongation and pathfinding,
since inhibition of protein synthesis in vitro causes retraction of growth cones (Zheng et al., 2001) whereas in
vivo it severely delays axonal regeneration (Gaete et al.,
1998). In addition, in vitro experiments demonstrated
that locally synthesized cytoskeletal proteins such as
actin, peripherin, and thymosin play a role in growth
cone turning and elongation (Leung et al., 2006; Lin and
Holt, 2007; Piper and Holt, 2004; van Kesteren et al.,
2006). Delivery of ribosomes and associated mRNAs
from nearby glial cells might prove an advantageous
mechanism to supply regeneration-associated molecules
to regenerating axons, especially those that are located
at a long distance from their cell body. Our results however, do not exclude the possibility that neuronal cell
body-derived ribosomes and mRNAs are also transported
into distal domains of regenerating axons, since we
observed ribosome staining that did not co-localize with
L4-eGFP immunoreactivity. We anticipate that both neuron-derived (via anterograde transport) and Schwann
cell-derived ribosomes and mRNAs contribute to axonal
protein synthesis for successful nerve regeneration.
An important questions concerns the signaling mechanism that controls transfer of ribosomes from Schwann
cells to axons. Schwann cells have been shown to express
cholinergic (Rousse and Robitaille, 2006) and purinergic
receptors (Abbracchio et al., 2009). Recently, it was shown
that blocking nicotinic AchR and purinergic P2Y receptors in the distal stump of a cut sciatic nerve reduced the
number of motor neurons regenerating into this stump
(Vrbova et al., 2009). These receptors are likely to be
stimulated by acetylcholine and ATP released from the
growth cones of the regenerating axons. However, how
activation of these receptors results in the stimulation of
regeneration is largely unknown. It is attractive to speculate that activation of one or both of these receptors may
be involved in the control of ribosomal transfer from
Schwann cells to regenerating axons, but future experiments will be necessary to substantiate such a role.
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1538
COURT ET AL.
CONCLUSIONS
Our present data extend previous findings, which proposed a novel aspect of axon-Schwann cell communication: Schwann cells transfer polyribosomes, albeit at a
very low level, to intact axons, and this process is
strongly upregulated in injured axons (Court et al.,
2008). Our present data indicate that regenerating
mouse and rat axons also receive ribosomes from
Schwann cells. These data point to a hitherto unknown
feature of Schwann cell physiology, in that Schwann cells
provide translational machinery to axons in order to
meet their local demands, both under physiological conditions but perhaps even more under conditions related
to injury, re-growth and repair. Manipulating ribosomal
transfer may have far reaching consequences to stimulate regeneration, or in cases of diseases involving axonal
degeneration, to attenuate the degenerative process.
ACKNOWLEDGMENTS
The authors thank Joost Verhaagen and Ruben
Eggers for help with the lentivirus experiments. The
authors thank Monica Perez for excellent EM processing, Jean Kawasoe, Joanne Forden, and Yvonne Gouwenberg for technical expertise.
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