Axonal Regeneration and Lack of Astrocytic Gliosis in EphA4-Deficient Mice

Article (PDF Available)inThe Journal of Neuroscience : The Official Journal of the Society for Neuroscience 24(45):10064-73 · November 2004with18 Reads
DOI: 10.1523/JNEUROSCI.2981-04.2004 · Source: PubMed
Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that adult mice lacking EphA4 (-/-), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4-/- mice recovered stride length, the ability to walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1-3 months of injury. EphA4 expression was upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned EphA4-/- spinal cords. EphA4-/- astrocytes failed to respond to the inflammatory cytokines, interferon-gamma or leukemia inhibitory factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4-/- astrocytes, but longer neurites when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury, axonal inhibition, and astrocytic gliosis.
Axonal Regeneration and Lack of Astrocytic Gliosis in
EphA4-Deficient Mice
Yona Goldshmit,
Mary P. Galea,
* Graham Wise,
Perry F. Bartlett,
* and Ann M. Turnley
Center for Neuroscience and
School of Physiotherapy, University of Melbourne, Melbourne, Victoria 3010, Australia,
School of Biomedical Sciences and
The Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia, and
Development and Neurobiology Group, Walter
and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, Victoria 3050, Australia
Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that
adult mice lacking EphA4 (/), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal
regeneration and functional recovery after spinal cord hemisection. Anterograde and retrograde tracing showed that axons from multiple
pathways, including corticospinal and rubrospinal tracts, crossed the lesion site. EphA4/mice recovered stride length, the ability to
walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1–3 months of injury. EphA4 expression was
upregulated on astrocytes at the lesion site in wild-type mice, whereas astrocytic gliosis and the glial scar were greatly reduced in lesioned
EphA4/spinal cords. EphA4/astrocytes failed to respond to the inflammatory cytokines, interferon-
or leukemia inhibitory
factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4/astrocytes, but longer neurites
when the astrocyte EphA4 was blocked by monomeric EphrinA5-Fc. Thus, EphA4 regulates two important features of spinal cord injury,
axonal inhibition, and astrocytic gliosis.
Key words: spinal cord injury; astrocytic gliosis; anterograde; retrograde; neurite outgrowth; cytokine
Injury to the CNS usually results in very limited, if any, regener-
ation of lesioned axons, with subsequent permanent impairment
of function. Although some CNS neurons appear to lose the in-
trinsic ability to regenerate neurites postnatally (Goldberg et al.,
2002), many others, such as corticospinal tract (CST) neurons,
appear able to regenerate, but are inhibited from doing so by the
environment of the injury site. Major impediments to CNS re-
generation are the presence of myelin inhibitors and astrocytic
Myelin proteins that inhibit axonal regeneration include
Nogo (Caroni and Schwab, 1988; Schnell and Schwab, 1990,
1993; Bandtlow and Schwab, 2000), myelin-associated glycopro-
tein (MAG) (McKerracher et al., 1994; Mukhopadhyay et al.,
1994; DeBellard et al., 1996; Schafer et al., 1996), and
oligodendrocyte-myelin glycoprotein (OMgp) (Wang et al.,
2002b). Each of these proteins appears to inhibit regeneration by
the same mechanism. Nogo, MAG, and OMgp bind to the Nogo
receptor (Domeniconi et al., 2002; Liu et al., 2002; Wang et al.,
2002a), which interacts with the p75 neurotrophin receptor on
the axon (Wang et al., 2002a). This leads to activation of the small
GTPase RhoA (Wang et al., 2002a; Yamashita et al., 2002), which
inhibits neurite outgrowth. However, although blocking individ-
ual myelin proteins or their common receptor in vivo after spinal
cord injury results in partial regeneration and improves func-
tional recovery, at best only a small percentage of axons regrow
(Bregman et al., 1995; GrandPre et al., 2002; Kim et al., 2003;
Simonen et al., 2003; Zheng et al., 2003). Therefore, other imped-
iments to regeneration still need to be overcome before robust
regeneration can occur.
The other major barrier to axonal regeneration is glial scar-
ring, the main component of which is astrocytic gliosis (Stichel
and Muller, 1998). Normally quiescent astrocytes in the adult
show a vigorous response to injury. They become hypertrophic,
proliferative, upregulate expression of glial fibrillary acidic pro-
tein (GFAP), and form a dense network of glial processes both at
and extending from the lesion site. Accompanying these mor-
phological changes are a range of physiological changes, includ-
ing secretion of a variety of cytokines and production of cell
adhesion and extracellular matrix molecules. Some of these prod-
ucts are inhibitory to regeneration, such as chondroitin sulfate
proteoglycan (CSPG) (McKeon et al., 1991) and collagen IV
(Stichel et al., 1999), and if their deposition is inhibited, axonal
regeneration is promoted (Stichel et al., 1999).
Other factors that may be implicated in inhibition of axonal
regeneration, but which have received little attention in this re-
gard, are cell surface molecules that are involved in axon guidance
Received July 21, 2004; revised Sept. 27, 2004; accepted Sept. 27, 2004.
This work was supported by The BHP Community Trust, the National Health and Medical Research Council of
Australia, and SpinalCure Australia. Y.G. was supported by an International Postgraduate Research Scholarship from
the University of Melbourne. We thank Drs. David Wilkinson and Jason Coonan for anti-EphA4 antibodies and Prof.
room staff at the Walter and Eliza Hall Institute and the University of Melbourne for excellent care of the mice.
*M.P.G., P.F.B., and A.M.T. contributed equally to this work.
Correspondence should be addressed to either of the following: Ann Turnley, Center for Neuroscience, University
of Melbourne, Melbourne, Victoria 3010, Australia, E-mail:; or Perry Bartlett, The
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia. E-mail:
Copyright © 2004 Society for Neuroscience 0270-6474/04/2410064-10$15.00/0
10064 The Journal of Neuroscience, November 10, 2004 24(45):10064 –10073
during development. One such family of molecules is the Eph
receptor tyrosine kinase family, which, together with their li-
gands, the Ephrins, are implicated in the formation of a variety of
neural structures, including the corticospinal tract and anterior
commissure (Henkemeyer et al., 1996; Dottori et al., 1998;
Coonan et al., 2001; Kullander et al., 2001a; Leighton et al., 2001).
Both the Eph receptors and the Ephrins are membrane-bound,
and cell– cell contact is required for signaling. In addition, some
of the Ephrins are able to transduce signals, so reverse signaling
can also occur (for review, see Kullander and Klein, 2002). Be-
cause Eph–Ephrin signaling appears to regulate axon guidance
through contact repulsion, inducing the collapse of neuronal
growth cones (Wahl et al., 2000; Kullander et al., 2001b), and
members of this family are upregulated in the adult after neural
injury (Moreno-Flores and Wandosell, 1999; Rodger et al., 2001;
Willson et al., 2002), the aberrant expression or absence of Eph
receptors could prove pivotal in determining the outcome of
injury in the adult CNS. This possibility was investigated in this
study by comparing neural regeneration after spinal cord he-
misection in wild-type and EphA4/mice.
Materials and Methods
Adult EphA4/and C57BL/6 mice, 3–12 months old, and maintained
as previously described (Coonan et al., 2001), were used in this study.
Experiments were approved by the Royal Melbourne Hospital and the
University of Melbourne Animal Ethics Committees in accordance with
the Australian Code of Practice for the Care and Use of Animals for
Scientific Purposes.
Spinal cord lesions
Mice were anesthetized with a mixture of ketamine and xylazine (100
mg/kg and 16 mg/kg, respectively). The spinal cord was exposed via a
laminectomy, in which two or three vertebral arches were removed at
levels T12–L1, corresponding to the level of the lumbar enlargement. A
spinal left hemisection at T12 was performed using a fine corneal blade
(cut twice in the same place to ensure complete section), and the overly-
ing muscle and skin were then sutured. A total of 71 wild-type and 83
EphA4/mice underwent the spinal hemisection surgery. EphA4/
mice were more sensitive to the anesthetic than wild-type mice, as a result
of which 4% of wild-type mice and 12% of EphA4/mice died. Mice
of both genotypes that recovered from surgery showed good survival,
with no spontaneous deaths recorded, however three wild-type and four
EphA4/mice were killed because of infection, and two EphA4/
mice because of full left and right hindlimb paralysis. The success rate of
the hemisection model, as assessed by total paralysis of the left hindlimb
at 24 hr after surgery, was 96% for wild-type mice and 95% for
EphA4/mice. Mice that showed only partial paralysis at this time
point, indicative of incomplete lesion, were excluded from analysis. Spi-
nal cords of animals that were included in the analysis were also exam-
ined histochemically to ensure complete hemisection.
Anterograde tracing
Five weeks after spinal cord lesion, tetramethylrhodamine dextran
(“Fluoro-Ruby”; molecular weight, 10,000 kDa) was injected as two 0.2
l injections into the midlateral aspect of the spinal cord at the level of the
cervical enlargement, ipsilateral to the lesion, via a glass pipette attached
to a Hamilton syringe. After an additional 7 d survival period, the animals
were perfused with 4% paraformaldehyde. Longitudinal serial sections of
spinal cord were cut at 50
m on a freezing microtome, and sections were
mounted on gelatinized slides and examined using fluorescence and con-
focal microscopy. This technique labeled many but not all axons in path-
ways ipsilateral to the injection site (estimated to be 25%) but none
contralateral to the injection site (supplemental material, available at
The number of labeled axons running rostrally to caudally through a
m-wide box placed in the white matter, at the border of the gray
matter, of all intact serial sections (8 –10 per spinal cord) was counted at
400by focusing up and down through the sections at 2.5 mm and
50 –100
m proximal to the lesion site and 50 –100
distal to the hemisection. Axons at the lateral edge of the white matter
and near the pial surface were therefore excluded from these counts, as
were sections in which the entire length of the labeled spinal cord was not
available. The lumbar site of the lesion precluded analysis of regrowth
longer than 5 mm because of termination of the fibers and commence-
ment of the Cauda Equina. Significance of results was analyzed using the
Student’s ttest.
Retrograde tracing
The lumbar spinal cord below the lesion was exposed via a lower lumbar
laminectomy. Fast Blue [2% (w/v), 0.3
l per injection; EMS-POLYLOY;
GmBH, Gross-Umstadt, Germany], which labels the neuronal soma of
axons damaged by the injection, was injected into two sites of the spinal
cord ipsilateral to the lesion site with a glass micropipette attached to a
Hamilton syringe. Aftera5dsurvival period, the animals were perfused
with 4% paraformaldehyde in PBS. The brain and spinal cord were re-
moved and postfixed for 24 hr in 20% sucrose in fixative before being
serially sectioned at 50
m on a freezing microtome in the coronal–
transverse plane. Injections were considered successful by confirmation
of a unilateral injection site in the operated spinal cord longitudinal
sections. Qualitative and quantitative comparisons of labeled neurons
were made by mapping the locations of labeled cells in every fourth
section of a series using a computer-linked digitizing system (MD3 mi-
croscope digitizer and MD-plot software; Minnesota Datametrics
Behavioral analysis
The hopping gait of the EphA4/mice precluded use of commonly
used behavioral assessments, such as the Basso, Beattie, Bresnahan scale.
The behavioral assessments chosen, and especially the grip strength test
and climbing on an angled grid, allow direct comparison of EphA4/
mice and wild-type mice, independent of hopping or reciprocal gait.
Both the grid walking and grip test rely on sensory as well as motor input.
Stride length. Before and after hemisection, mice were footprinted by
painting their hindpaws with nontoxic ink and placing them in a tunnel
on blotting paper (wild type, n7; EphA4/,n9 mice). Stride
length was determined by measurement of multiple successive steps and
results were expressed as a percentage of each animal’s own baseline
stride length.
Grid walking. The ability of wild-type (n5) and EphA4/(n7)
mice to walk on a horizontal or angled (75° from horizontal) wire grid
(1.2 1.2 cm grid spaces; 35 45 cm total area) was determined to assess
their locomotion (Ma et al., 2001). The mice were tested 1, 2, and 3
months after the spinal cord hemisection and compared with nonle-
sioned mice from each group. On the horizontal grid each mouse was
allowed to walk freely around the grid for 5 min, during which a mini-
mum 2 min of walking time was required. On the angled grid, each
mouse was measured over 10 climbs. If the left hindpaw protruded en-
tirely through the grid, with all toes and heel extended below the wire
surface, it was counted as a misstep. The total number of steps taken with
the left hindlimb was also counted. The results were expressed as the
percentage of accurate footsteps and significance was analyzed using the
Student’s ttest.
Sensory and motor ability-grasp test
The ability of hemisected and nonlesioned wild-type (n5) and
EphA4/(n7) mice to graspa2mmdiameter rod was tested on the
left hindlimb. The hindlimbs of the mice were lifted 2 cm from the table
top while allowing the forelimbs to remain in contact with the table.
Grasp ability was tested by lightly touching the left footpad with the rod
and assessing the response based on a scale from 0 to 4: 0, no movement
of paw and toes; 1, partial movement of the paw, no movement of the
toes; 2, partial grasp, slight movement of toes and paw; 3, weak full grasp,
not maintained with gentle rod movement; 4, strong grasp, maintained
with gentle rod movement. Mice were graded at least three times in
parallel with the grid tests described. Results were expressed as the
mean SEM of the score of each group, and significance was analyzed
using the Student’s ttest.
Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice J. Neurosci., November 10, 2004 24(45):10064 –10073 • 10065
Immunohistochemistry and astrocyte counts
Standard immunohistochemical procedures, using rabbit anti-GFAP (1:
500; Dako, Carpenteria, CA), mouse anti-CSPG (1:200; Sigma, St. Louis,
MO), and rabbit anti-EphA4 were followed. The rabbit anti-EphA4 an-
tibody, obtained from Dr. J. Coonan (Walter and Eliza Hall Institute,
Melbourne, Australia), was prepared against a peptide corresponding to
amino acids 938 –953 of the intracellular SAM domain of EphA4 (Gen-
Bank accession number NM007936) using standard procedures (Cooper
and Paterson, 2000). The number of hypertrophic astrocytes, as well as
the total number of GFAP-expressing astrocytes, were counted in a 0.25
grid at and 2.5 mm proximal to the lesion site, in every third serial
longitudinal 8
m section. Hypertrophic astrocytes were defined as in-
tensely stained GFAP-positive cells with a large cell body and multiple
thick long processes. Nonhypertrophic astrocytes stained less intensely
for GFAP and had a small cell body with thin, less complex processes.
Hypertrophic astrocytes were more than twice the size of nonhypertro-
phic astrocytes.
Astrocyte and neuronal cultures and neurite length measurement
Purified astrocyte and neuronal cultures were prepared as previously
described (Turnley et al., 2002). For analysis of neurite length, E16 cor-
tical neurons were plated at 5000 per well in chamber slides (Falcon;
Becton Dickinson, Franklin Lakes, NJ) containing wild-type or
EphA4/astrocyte monolayers or which were poly-DL-ornithine—
laminin coated. In some experiments, astrocytes were pretreated for 1 hr
with monomeric EphrinA5-Fc (0.15, 1.5, 10
g/ml) or complexed
EphrinA5-Fc (1.5
g/ml complexed with 0.15
g/ml anti-human IgG
(Vector Laboratories, Burlingame, CA) for 30 min at room temperature
before addition). After 22 hr, cells were fixed and immunostained for the
neuronal marker
III-tubulin (1:2000; Promega, Madison, WI). Neurite
length was measured using image analysis as previously described (Turn-
ley and Bartlett, 1998). Significance of differences in the mean neurite
lengths was analyzed using the Student’s ttest.
For biochemical analysis of astrocytes, factors as indicated were added
to 80% confluent monolayers in 10 cm plates (Falcon) for the times
indicated. EphrinA5-Fc (a gift from Prof. Andrew Boyd, Queensland
Institute of Medical Research, Brisbane, Australia) was precomplexed as
Immunoprecipitation and Western analysis
Cells were lysed in lysis buffer (50 mMHEPES, pH 7.5, 150 mMNaCl, 10%
glycerol, 1% Triton X-100, 1 mMEDTA, 1 mMEGTA, pH 8.0, and 1.5 mM
MgCl) containing protease inhibitors [Complete protease inhibitor
cocktail tablet (Roche), 200
Msodium vanadate, and 2 mMPMSF] and
a sample kept aside for analysis of total protein levels. The remainder of
the lysate was used for immunoprecipitation in EphA4 or Rho activation
assays. For Western analysis, gel sample reducing buffer was added to cell
lysates and boiled. For other experiments, an aliquot of lysates was taken
for Western analysis and the remainder of the lysate was used for immu-
noprecipitation with mouse anti-phosphotyrosine antibody (1:100; Cell
Signaling Technology, Beverly, MA). Antibodies were first coupled to
anti-mouse IgG agarose (Pierce, Rockford, IL) by 1 hr preincubation
then proteins in the lysate supernatant were immunoprecipitated with
aliquots of the antibody–agarose complex for 2 hr at 4°C. The immuno-
precipitates were washed three times with HNTG buffer (20 mMHEPES,
pH 7.5, 0.15 mMNaCl, 0.1% Triton X-100, and 10% glycerol), resolved
by SDS-PAGE gel electrophoresis through 12 or 8 –16% gels (Gradipore,
Sydney, Australia) and electrophoretically transferred to a nitrocellulose
membrane (Bio-Rad, Hercules, CA). Membranes were blocked for 2 hr
in TBS containing 0.05% Tween 20 and 6% skim milk powder then
incubated with rabbit anti-EphA4 antibody (kindly provided by Dr. D.
Wilkinson, National Institute for Medical Research, London, UK). Total
EphA4 and
-actin expression levels were determined in nonlesioned
and 7 d postlesioned spinal cords by Western analysis using rabbit anti-
EphA4 antibody as above and mouse anti-
-actin antibody (Sigma).
Densitometry was performed on the autoradiographs using NIH Image
software to determine relative levels of the EphA4 bands and normalized
-actin levels.
Rho activation assay
Cells were exposed to indicated stimuli, and after treatment, cells were
lysed in lysis buffer as above. Rho activation assays were performed using
the Rhotekin RBD assay, according to the manufacturer’s instructions
(Upstate Biotechnology, Lake Placid, NY). Endogenous Rho-GTP was
precipitated from cell lysates at 4°C for 1 hr, using a GST-tagged fusion
protein, corresponding to residues 7– 89 of mouse Rhotekin Rho-
binding domain. Beads were collected by centrifugation, washed with
lysis buffer and resuspended in sample buffer. The eluted protein samples
were resolved on 12% SDS-PAGE and electrophoretically transferred to
nitrocellulose membrane. Rho was detected using mouse anti-Rho anti-
body (1:500; Pierce) overnight, followed by anti-mouse secondary anti-
body linked to horseradish peroxidase (1:40,000; Cell Signaling). Immu-
noreactive bands were detected with ECL as above. Densitometry was
performed on the autoradiographs using NIH Image software to deter-
mine relative levels of activated compared with total Rho.
Cell proliferation assay
The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide
(MTT) assay, which determines mitochondrial activity in living cells, is
commonly used as a proliferation assay (Mosmann, 1983). Living cells
transform the tetrazolium ring into dark blue formazan crystals, which
can be quantified by reading the optical density (OD); an increase in OD
correlates with an increase in cell number over time. Wild-type and
EphA4/astrocytes were plated on 96 well plates (Falcon) at 3 10
cells/well in DMEM supplemented with 10% calf serum in the presence
or absence of either leukemia inhibitory factor (LIF; 1000 U/ml; Chemi-
con, Temecula, CA) or interferon-
; 100 U/ml; Becton Dickin-
son). The MTT assay was performed at 2, 24, 48, and 72 hr after plating.
MTT (0.25 mg/ml; Sigma) was incubated with the cells at each time point
for 2 hr at 37°C, the cells were then lysed with an equal volume of acidic
isopropanol (0.04 MHCl in absolute isopropanol), and the OD of the
formazan product was measured at 550 – 650 nm.
Astrocyte scratch wound assay
Wild-type and EphA4/astrocytes were plated in DMEM (Invitrogen,
San Diego, CA) supplemented with 10% FCS on poly-DL-ornithine-
coated 8 well chamber slides (Falcon) and grown to confluence. The
medium was then removed, and the monolayer was scratched with a
sterile 20 –200
l plastic pipette tip. The cells were washed twice with
sterile PBS, and medium was replaced, with or without LIF or IFN
. After
3 d of incubation, cells were fixed and immunostained for
-actin, GFAP,
and DAPI, and the number of astrocytes entering the wound area was
determined by counting the number of nuclei per square millimeter.
Tracing of lesioned axons indicates extensive regeneration by
6 weeks
An anterograde tracing technique was used whereby the tracer,
tetramethylrhodamine dextran, was injected into the cervical spi-
nal cord, well above the lumbar lesion site. This allowed us to
assess general regeneration of individual axons from multiple
different tracts without making any assumptions about the effect
of EphA4 on particular pathways. Use of this technique in unle-
sioned wild-type and EphA4/mice showed equivalent label-
ing of descending axonal pathways ipsilateral to the injection site
but none contralateral to the injection site (supplemental mate-
rial, available at
At 6 d after lesion, in both wild-type and EphA4/mice,
anterograde labeling revealed no labeled fibers within the lesion
site, although there were axons with growth cones near the lesion
site in the EphA4/mice (Fig. 1a,b). By 6 weeks after spinal
hemisection, however, many anterogradely labeled axons crossed
the EphA4/lesion site (Fig. 2a) (supplemental material,
available at, unlike in the wild-type lesion
(Fig. 2b). Only sections in which the entire length of spinal cord
was intact were included in the results and axons that were close
10066 J. Neurosci., November 10, 2004 24(45):10064 –10073 Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice
to the pial surface were excluded from the counts. Of the antero-
gradely labeled axons counted that reached the lesion site (sum
total 133 16.8 in EphA4/mice compared with 58 5.3 in
wild-type, in a 250-
m-wide box over the white matter bordering
the gray matter; p0.05), 70% of
EphA4/axons crossed it (as measured
m distally) and, of these, 75% were
maintained at 1 mm and 15% at 5 mm
distal to the injury (Fig. 2a,c). In contrast,
in wild-type mice 4% of the fibers
crossed the lesion site and virtually none
of these was detected at 1 mm and 5 mm
distal. Furthermore, in lesioned mice 2.5
mm upstream of the lesion site, there was
no significant difference in the number of
labeled axons between wild-type and
EphA4/animals (25.4 2.7 and
31.7 2.2 axons per section, respectively;
p0.13); it was only close to the lesion
site that the difference between wild-type
and EphA4/numbers became signifi-
cant. Because considerably more axons in
the EphA4/mice reached the lesion
site, the magnitude of the difference is
even more pronounced and demonstrates
that there is considerable inhibition of re-
growth of wild-type axons upstream of the
lesion. Although the regenerating axons
that crossed the lesion site at 6 weeks ap-
peared “wavy” (Fig. 2av), as is typical dur-
ing regeneration, the vast majority could
be traced as running in an unhindered ros-
tral to caudal line (Fig. 2a). Although
some fibers showed branching or devia-
tion, particularly after the lesion site (Fig.
2aiii, aiv), a montage of confocal micro-
graphs covering the entire lesion area and
across the midline (Fig. 2a) (supplemental
material, available at
revealed that no fibers crossed from the
unlesioned side and contributed to the la-
beled fiber bundle running through or dis-
tal to the lesion. Thus, the large number of
labeled axons passing through and beyond
the lesion site in EphA4/mice can
only be attributed to genuine regrowth of
severed axons.
Because the anterograde tracing used
in this study labeled many axons in multi-
ple descending spinal pathways, retro-
grade tracing was used to identify which
specific axonal tracts had regenerated.
This revealed that in the EphA4/but
not the wild-type mice multiple axonal
tracts showed regeneration. Labeled
neurons were present in motor cortex
(corticospinal tract) and the red nucleus
(rubrospinal tract), as well as in the hy-
pothalamus, the vestibular and reticular
nuclei, and the periaqueductal gray mat-
ter (Fig. 3a,b), the same regions that
were labeled in the nonlesioned control
EphA4/and wild-type mice (Fig.
3c). In the wild-type mice, only a small number of bilaterally
projecting reticulospinal neurons were labeled after lesion.
Interestingly, unlike wild-type mice, unlesioned EphA4/
mice showed a bilateral projection of the rubrospinal pathway
Figure 1. At 6 d after injury, EphA4/axons approach but do not cross the lesion site. Anterograde tracing and confocal
analysis of lesioned EphA4/spinal cords 6 d after hemisection ( a) show large numbers of labeled axons 2.5 mm proximal to
the lesion (ia) and a small number of axons with growth cones (aiii, arrows) approaching the lesion site, which is indicated by the
dotted line ( i) and shown more clearly in a hematoxylin and eosin (H&E)-stained section (ii). b, Wild-type spinal cord also shows
veryfew axons approaching thelesion site. biashows labeling2.5 mmupstream ofthe lesionsite. biii,An enlargement ofbi shows
few axons upstream of the lesion site. In both panels, rostral is to the right and caudal to the left, and the lesion site is indicated by
dotted lines. Enlarged areas are indicated by boxed areas and arrows. Scale bars: i, 250
m; ii, 200
m; iii, 50
Figure2. Extensive axonal regeneration in EphA4/mice at 6weeks after injury. Anterogradetracing and confocal analysis
of lesioned EphA4/spinal cords 6 weeks after hemisection showed that a large percentage of EphA4/axons crossed the
lesion site (a, c) and extended caudally (*p0.001), unlike wild-type (EphA4/) axons that did not cross the lesion site (b, c).
A montage of confocal images of EphA4/spinal cord (ai) showed that the regenerating axons passed through the lesion site
(indicated by dotted line and by H&E-stained section in aii and extended caudally in a straight line with some “waviness” seen
immediately after lesion in aiii, iv, and v). In both panels, rostral is to the right and caudal to the left, and the lesion site is indicated
by dotted lines. Enlarged areas are indicated by boxed areas and arrows. ii in both cases shows an adjacent H&E-stained section
demonstrating the lesion site. Scale bars: i, 250
m; ii, 200
m; iii—v, 50
m. Asterisk in ai indicates the midline.
Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice J. Neurosci., November 10, 2004 24(45):10064 –10073 • 10067
(Fig. 3c), indicating that deletion of the
EphA4 gene results in developmental
guidance defects of the rubrospinal
pathway, as has previously been shown
for the corticospinal tract (Dottori et al.,
1998; Coonan et al., 2001) and the ante-
rior commissure (Dottori et al., 1998).
Functional recovery of EphA4/mice
The axonal regeneration observed in
EphA4/mice also had a functional
correlate. Mice were behaviorally assessed,
first by measuring their stride length
(Bregman et al., 1995) before and from 24
hr to 4 weeks after spinal hemisection. At
24 hr both EphA4/and wild-type mice
showed minimal function. EphA4/
mice regained 100% of their baseline
stride length within 3 weeks, whereas
wild-type mice showed only 70% recovery
(Fig. 3d) and did not improve thereafter.
At up to 3 weeks, mice of both genotypes
were unable to move their ankles or toes
and displayed toe dragging. By 1 month
EphA4/but not wild-type mice
showed ankle and toe movement and be-
gan to bear weight on the plantar surface
of the affected hindpaw.
Given that wild-type mice also showed
improvement in stride length, two other
functional measures were used that did
not improve significantly in wild-type
mice and which required more sophisti-
cated sensory and motor skills; ability to
walk or climb on a grid and hindpaw grip
strength. One month after hemisection, the ipsilateral hindpaw
grip strength (Fig. 3e), and ability to walk or climb on a grid (Fig.
3f) were dramatically improved in EphA4/mice compared
with wild-type. These functions continued to improve up to 3
months after lesion. Nonlesioned EphA4/and wild-type
mice both achieved maximal scores in these tests.
Lack of astrocytic gliosis in EphA4/mice
A striking feature of the hemisected EphA4/spinal cord was
the virtual absence of astrocytic gliosis, as assessed by GFAP ex-
pression, compared with the wild-type (Fig. 4a,b,d,e). At day 7,
the vast majority (90.4%) of the GFAP-positive astrocytes at the
wild-type lesion site were hypertrophic and stained very strongly
for GFAP, whereas only 7.4% of EphA4/astrocytes were hy-
pertrophic (Fig. 4g). Overall, the total number of GFAP-positive
cells was fewer at the EphA4/hemisection over the first 7 d
after lesion, and this was strikingly the case proximal to the lesion
site (Fig. 4h). In nonlesioned cases there was no difference in
astrocyte numbers between EphA4/and wild-type mice
(wild-type 836.8 108.3/mm
compared with EphA4/
825.6 98.1/mm
). The lack of glial response resulted in a
marked reduction in the size of the glial scar of EphA4/mice
at 6 weeks after lesion as assessed by immunostaining for a com-
ponent of the glial scar, CSPG (Fig. 4c,f). Analysis of macropha-
ge–microglia activation at the lesion site 1 week and 6 weeks after
lesion, by use of an antibody to the microglial marker CD11b,
showed no apparent differences in the response of these cells to
the injury in mice of either genotype (supplemental material,
available at
Because EphA4 expression appeared to regulate both the level
of regeneration and gliosis after lesioning, we next examined
whether EphA4 expression was upregulated after spinal hemisec-
tion. Immunostaining and Western analysis (Fig. 5a,d) revealed
that EphA4 expression occurred at very low levels, undetectable
by immunostaining in nonlesioned animals except on some mo-
tor neurons (supplemental material, available at www.jneuro- However, expression and phosphorylation were upregu-
lated after spinal lesion (Fig. 5d), and almost exclusively on
GFAP-expressing astrocytes at the lesion site (Fig. 5a– c). Low
levels of EphA4 were found on anterogradely labeled axons prox-
imal to the lesion site (supplemental material, available at www. A ligand for EphA4, EphrinB3, was also expressed
on regenerating axons, as well as on some astrocytes (supplemen-
tal material, available at
EphA4 expression on astrocytes inhibits neurite outgrowth
We then investigated whether EphA4 expression on astrocytes
inhibits neurite outgrowth of cortical neurons in vitro. E16 cor-
tical neurons were plated onto monolayers of either wild-type or
EphA4/astrocytes, and the length of the longest neurite was
measured 22 hr later. This revealed a twofold to threefold increase
in outgrowth on EphA4/astrocytes compared with wild-type
astrocytes (Fig. 5e– g). This effect appeared to be directly attrib-
utable to expression of EphA4 on the astrocytes, because similar
results were obtained when neurons were grown on 293T cells
transfected with EphA4 (neurite length on nontransfected 293T
Figure 3. EphA4 (/) mice show multiple tract regeneration and improved function. Identification of regenerating neuro-
nal populations was determined by retrograde tracing using Fast Blue (a– c), and each neuron was plotted using an MD3 micro-
scope digitizer and MD-plot software. Unlike lesioned wild-type (WT) mice ( b), multiple axonal tracts regenerated in the lesioned
EphA4/(KO)mice (a, b), with a patternsimilar tothat ofunlesioned controls( c).Regenerated neuronsincluded corticospinal
neurons in layer 5 of the cortex (ai, b), rubrospinal neurons in the red nucleus (RN) (aii, b), as well as neurons in the hypothalamus
(Hyp), the vestibular (VN) and reticular nuclei, and the periaqueductal gray (PAG) matter. Scale bars: (in a) 200
m. Functional
analysis of lesioned mice showed that EphA4/mice recovered substantial function within 1 month (*p0.005; n7WT
and 9 KOmice). One day after lesion, stride length ( d),hindpaw grasping ( e), and the ability towalk on a horizontal orangled (75
grid ( f) were minimal. Stride length was regained in KO mice within 3 weeks, whereas wild-type mice reached a plateau at 70%
recovery. Grasping and grid-walking were significantly (*p0.001; n5 WT and 7 KO mice) improved in KO compared with WT
by 1 month, continuing to improve up to 3 months.
10068 J. Neurosci., November 10, 2004 24(45):10064 –10073 Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice
cells was 80.4 3.3
m compared with 30.2 1.9
m on EphA4-
transfected cells). The increased neurite outgrowth of
EphA4/neurons compared with wild-type neurons, on both
wild-type and EphA4/astrocytes (Fig. 5g), suggests that
EphA4 expressed on the neurons may also inhibit neurite out-
growth, as has been previously suggested (Wahl et al., 2000; Kul-
lander et al., 2001b), and may contribute to the regeneration
observed in EphA4/mice. Inhibition of neurite outgrowth on
astrocytes was potently blocked in a dose-dependent manner by
the addition of monomeric EphrinA5-Fc, which strongly binds to
EphA4 in the astrocyte monolayer; however, it had no effect on
neurons grown on laminin-coated glass slides (Fig. 5h). Con-
versely, addition of complexed EphrinA5-Fc inhibited neurite
outgrowth on glass slides, as previously described (Wahl et al.,
2000; Kullander et al., 2001b) and further inhibited outgrowth on
astrocytes (Fig. 5h). This indicates that blocking of EphA4 on
astrocytes, but not on neurons, enhances neurite outgrowth,
whereas activation of EphA4 on both neurons and astrocytes
inhibits neurite outgrowth. Both results point directly to the ac-
tivation of EphA4 by a ligand as being the mechanism for neurite
inhibition. In vivo, a possible activator of the neurite responses to
EphA4 expression on the astrocytes was EphrinB3, which has been
shown to transduce signals (Palmer et al., 2002) and which was ex-
pressed by regenerating axons in the spinal cord (supplemental ma-
terial, available at
Rho activation and proliferation is
decreased in EphA4/astrocytes
Given that previous studies have demon-
strated that gliosis is mediated by inflam-
matory cytokines, including IFN
and LIF
(Yong et al., 1991; Balasingam et al., 1994;
Sugiura et al., 2000), we then investigated
whether these cytokines play a role in the
upregulation of EphA4 on astrocytes.
and LIF upregulated EphA4 expres-
sion by 56 and 69%, respectively, whereas
interleukin-1 (IL-1) and tumor necrosis
) had no effect (Fig. 6a).
To directly address the question of
whether EphA4 expression is accompa-
nied by downstream activation that could
lead to astrocytic responses, we examined
whether EphA4 is phosphorylated. Both
and LIF upregulated EphA4 phos-
phorylation twofold, in a similar manner
to the addition of a soluble multimeric
EphA4 ligand, EphrinA5-Fc (Fig. 6a). In
addition, this led to a marked increase in
activation of the small GTPase, Rho, a ma-
jor regulator of cytoskeletal changes (Hall,
1998), downstream of Eph receptor sig-
naling (Wahl et al., 2000; Shamah et al.,
2001). Increased Rho activation occurred
both in wild-type spinal cord tissue re-
moved from the lesion site (Fig. 6b) and in
cultured astrocytes (Fig. 6c); no such re-
sponse was observed using cells or tissue
removed from lesioned EphA4/ani-
mals. Activation of Rho in astrocytes, as
well as in neurons and oligodendrocytes,
has also recently been reported in spinal
cord after injury (Dubreuil et al., 2003).
EphA4/astrocyte proliferation and scratch wound repair
are decreased in response to cytokines
The failure of cytokine-induced Rho activation in EphA4/
astrocytes correlated with a failure of cytokine-induced astrocyte
proliferation (Fig. 6d). Although basal levels of astrocyte prolif-
eration were not significantly different in wild-type or
EphA4/astrocytes, wild-type but not EphA4/astrocytes
proliferated in response to LIF and IFN
(Fig. 6d), indicating that
EphA4 expression is a required cofactor for LIF- and IFN
induced astrocyte proliferation. Furthermore, unlike wild-type
astrocytes, Epha4/astrocytes failed to repair a scratch wound
by 72 hr, even in the presence of LIF or IFN
(Fig. 7A–G). Because
are cytokines involved in inducing astrocytic gliosis
in vivo, this provides a mechanism for the failure of overt gliosis
after spinal cord injury in the EphA4/mice.
These studies indicate that EphA4 has the potential to inhibit
axonal regeneration by three different mechanisms. The first of
these, as demonstrated by in vitro assays, is the direct inhibition of
neurite outgrowth mediated by EphA4 on the astrocytes binding
to a receptor–ligand on the axon. Such an action of EphA4 may
provide a mechanism for the inhibition of neurite outgrowth on
astrocytes observed in the presence of IFN
(Fok-Seang et al.,
1998), which we have shown upregulates EphA4 expression.
Figure 4. Astrocyticgliosis and the glial scarare greatlydiminished inEphA4/mice afterinjury. Immunostainingfor GFAP
expression at the lesion site 4 d after spinal cord lesion showed a florid astrocytic gliosis in wild-type mice ( a) that was virtually
absent in EphA4/mice ( d). Under higher magnification, the vast majority of astrocytes in wild-type mice were revealed to be
hypertrophic (white arrows) (b, g), unlike EphA4/astrocytes (black arrows) (e, g). The total number of astrocytes increased
with time after lesion, with greater numbers in EphA4/spinal cords ( h). Immunostaining for chondroitin sulfate proteogly-
can, a component of the glial scar, 6 weeks after lesion, revealed that the scar was diminished in the EphA4/mice ( f)
compared with the wild-type animals ( c). Scale bars: a,d, 200
m; b,e,50
m; c,f, 200
m. *p0.0001 in gand h.
Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice J. Neurosci., November 10, 2004 24(45):10064 –10073 • 10069
These results suggest that EphA4 is yet an-
other directly inhibitory molecule pro-
duced during astrocytic gliosis, in addi-
tion to other inhibitory components, such
as extracellular matrix and myelin-derived
molecules. Although Eph molecules are
generally described as receptors and
Ephrins as ligands, both are membrane-
bound and signaling can occur in either
direction (for review, see Kullander and
Klein, 2002). A likely candidate for trans-
ducing the inhibitory signal is Ephrin-B3,
which was expressed on the axons.
Ephrins have previously been shown to
mediate reverse signaling in cortical neu-
rons in response to Eph stimulation
(Palmer et al., 2002).
The second mechanism may be by ac-
tivation of EphA4 on the regenerating ax-
ons, similar to on E16 cortical neurons.
However, we found EphA4 to be highly
expressed only on astrocytes and motor
neurons, and to be present at low levels on
descending axons in lesioned adult spinal
cord, suggesting that this mechanism may
not play as great a role as the first in axon
The third, and totally unexpected,
mechanism by which EphA4 exerts an in-
hibitory effect involves its vital role in ac-
tivating astrocytes, leading to gliosis and
the formation of a glial scar. Such activa-
tion appears to be dependent on respon-
siveness to cytokine stimulation and may
be dependent on Rho activation. This
cytokine-induced response may be attrib-
utable to the upregulation of EphA4 re-
ceptor expression on the astrocytes, allow-
ing enhanced ligand binding and receptor
activation. It is also possible that the
cytokine-induced astrocyte proliferation
and hypertrophy may be caused by transactivation of EphA4, as
has been shown for FGF2- and PDGF-induced phosphorylation
of EphrinB molecules (Bruckner et al., 1997; Chong et al., 2000),
leading to Rho activation and cytoskeletal rearrangement. The
difference in glial activation seems to be astrocyte specific as there
was no apparent difference in macrophage–microglial activation.
Ephs and Ephrins have been reported to play a role in interactions
between astrocytes and meningeal fibroblasts, excluding fibro-
blasts from the glial scar (Bundesen et al., 2003). However,
whether EphA4 plays any role in regulation of fibroblast reaction
at the lesion site remains to be determined.
The overall functional improvement in the EphA4/mice
is likely to be caused by a lack of astrocytic gliosis and regenera-
tion of ipsilateral axons. The recovery of grip strength and the
ability to successfully walk on a grid reflect functional reconnec-
tion of both descending motor tracts and ascending sensory
tracts. However, although the recovery in stride length is a useful
assay, given that wild-type animals also showed significant im-
provement in the absence of regeneration of ipsilateral descend-
ing axons, as previously described (Bregman et al., 1995), it is
possible that only the enhanced recovery displayed by the
EphA4/mice can be attributed to regeneration and that the
basal improvement is attributable to other causes. The significant
improvement in mice of both genotypes probably reflects the
hemisection model used, with the intact right side of the spinal
cord allowing movement of the right side of the body, which in
turn contributed to movement of the left side, particularly
through the hip joint. The stride length was primarily a measure
of hip movement, and there was no ankle or toe movement at the
early time points in mice of either genotype. Ankle and toe move-
ment did, however, improve in the EphA4/mice by 1 month,
by which time they were beginning to bear weight on their plantar
surface, a phenomenon that was not observed in the wild-type
mice. Another potential cause of stride length recovery is activa-
tion of spinal reflexes, such as the central pattern generator
(Duysens and Van de Crommert, 1998). However, more sensitive
methods, such as grid walking and grasp strength, that did not
produce marked improvement in wild-type mice and that did not
rely on spinal cord reflexes were also assessed. Although grasp
strength showed some modest recovery in the EphA4/mice
at 1 month after injury, with partial grasp and slight movement of
toes and paw, substantial regeneration would be required for
their ability to maintain a strong grasp on a gently moving rod.
The EphA4/mice did not approach this score until 2 months
Figure 5. Expression of EphA4 on astrocytes inhibits neurite outgrowth. After spinal hemisection, EphA4 ( a) and GFAP ( b) are
coexpressedas assessed by immunofluorescence onreactive astrocytes atthe lesion site(c; amerged image ofaand b). EphA4was
also expressed on some neurons (a– c, arrow). Western blot analysis ( d) showed upregulation and phosphorylation of EphA4
(p-EphA4) at the lesion site (les) in comparison with unlesioned control (con) mice; * shows a nonspecific band present in all lanes.
-Actin was used as a loading control and EphA4/spinal cord as an EphA4 expression control. The EphA4 expression on
astrocytes was inhibitory to cortical neuronal neurite outgrowth, because
III-tubulin-positive cortical neurons on EphA4/
astrocytes (e, g) had significantly longer neurites than on EphA4-expressing (EphA4/) astrocytes ( f, g) after 22 hr (*p
0.0001). EphA4/neurite outgrowth was also enhanced on EphA4/and EphA4/astrocytes, compared with that of
wild-type neurons ( g;**p0.0001). h, The inhibition of neurite outgrowth by EphA4 on astrocytes could be blocked in a
dose-dependent manner by addition of monomeric (mono) EphrinA5-Fc, but this had no effect on neurites grown on laminin.
Multimerized (multi) EphrinA5-Fc inhibited neurite outgrowth both on astrocytes and on laminin (*p0.0001). Scale bars, 50
10070 J. Neurosci., November 10, 2004 24(45):10064 –10073 Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice
after injury and further improved at 3 months, at a time when
regenerating axons had passed the lesion site. The improvement
in grid walking also showed a similar time course. Neither of
these measures significantly improved in wild-type mice, sug-
gesting that a functional level of reconnection of both descending
and ascending tracts had occurred in the EphA4/mice.
Although the numbers of labeled axons that were counted
crossing the lesion site were relatively low, this was an underesti-
mate of the actual number of axons crossing the lesion site. The
labeling index of the axons was estimated to be 25% or less, so
that less than one in four axons crossing the lesion site was labeled
with tracer. Axons were also counted in a 250
m wide box over
the white matter, bordering the gray matter, which covered an
average of 75% of the width of the white matter. Furthermore,
axon counts were performed on only 40% of spinal cord sec-
tions; only sections that were intact for the entire length, from the
site of tracer injection to at least 5 mm below the lesion site were
Figure 6. Inflammatory cytokines upregulate EphA4 and Rho activation and astrocyte pro-
liferation.a, Expression of EphA4 was upregulatedin culturedastrocytes after 72hr by IFN
LIF but not TNF
or Il-1, compared with untreated controls (con). These cytokines also induced
EphA4phosphorylation (p-EphA4), similar to EphrinA5-Fc(A5). b, EphA4phosphorylation leads
to activation of Rho (RhoGTP), a cytoskeletal regulator. Rho was activated at the lesion site in
wild-type but not EphA4/lesioned spinal cords (L1, L2), whereas in culture (c), IFN
which is an inducer of astrocytic gliosis, activated Rho in wild-type but not EphA4/astro-
cytes. d, An in vitro astrocyte proliferation assay showed that under basal conditions (con), both
wild-type(WT) and EphA4/(KO) astrocytes proliferated similarlyover 72hr. WT astrocytes
showedincreased proliferation in response toLIF andIFN
,whereas the EphA4/astrocyte
response to these factors was markedly decreased and only significant for LIF at 72 hr. Results
are representative of n3 separate experiments; *p0.001; **p0.005; ***p0.05.
Figure 7. EphA4/astrocytes fail to repair a scratch wound in vitro. Scratch wounds
were performed on wild-type (A, C, E) and EphA4/(B, D, F ) astrocyte monolayers. After 72
hr under basal conditions (A, B) or in the presence of IFN
(C, D) or LIF (E, F ), cells were immu-
nostained for
-actin and DAPI. The number of cells migrating into the wound area was deter-
mined by counting the number of DAPI-stained nuclei ( G)(*p0.001 compared with basal
conditions, representative of n3 separate experiments). Scale bar: (in F) 250
Goldshmit et al. Axonal Regeneration in EphA4-Deficient Mice J. Neurosci., November 10, 2004 24(45):10064 –10073 • 10071
used. Therefore, the actual number of regenerating axons cross-
ing the lesion site was likely to be in the thousands; a number
sufficient for functional improvement.
It is unlikely that unlesioned ipsilateral projections contrib-
uted to the functional recovery within the observed time frame.
The double cutting procedure of the surgery ensured that in most
mice all ipsilateral projections were severed. In a small group
(10%) of both wild-type and EphA4/mice, a small number
of the most lateral projections were spared. These were easily
distinguished from the regenerating fibers as they followed an
uninterrupted, unbranched course, without any apparent collat-
eral sprouting and they were consequently excluded from counts
of regenerating fibers. The presence of these small numbers of
fibers did not contribute to improved function in wild-type mice.
However, given that a small number of CST axons cross the
midline in EphA4/mice (Dottori et al., 1998; Coonan et al.,
2001), and our retrograde tracing experiments showed bilateral
projections of both the CST and the rubrospinal tract in unle-
sioned EphA4/mice, it is possible that during development a
small number of contralateral projections crossed the midline at
a level below the lesion site. After lesion, if only projections from
the contralateral side were labeled, we would have seen an asym-
metric distribution of labeled neurons in the brain, but as we
observed bilateral labeling it indicates that the ipsilateral projec-
tions also regenerated. Whether a small number of contralateral
projections could contribute to functional recovery is unclear,
but as total hemiparalysis was demonstrated for at least the first
few days after the lesion, it is unlikely that these fibers played a
large role in functional recovery. However, collateral sprouting
does appear to play a role in functional recovery in other spinal
cord injury models (Galea and Darian-Smith, 1997; Fouad et al.,
2001; Bareyre et al., 2004). We did not see any evidence of sprout-
ing from the contralateral side above the site of the lesion. The
injection site for the anterograde tracer was at the level of C2–C3,
and collaterals from the intact side would have had to have com-
menced at or above this level to be labeled and tracked down past
the lesion. Axons that cross the midline in EphA4/animals do
not appear to continue down the spinal cord. Rather, they termi-
nate at the level at which they cross (Coonan et al., 2001). We also
did not observe any anterogradely labeled axons cross the midline
to the contralateral side in lesioned or unlesioned animals.
The observed axonal regeneration and functional recovery
also highlight the potential value of modulating the astrocyte
response to injury. Whereas total ablation of astrocytes enhances
tissue damage after neural injury (Bush et al., 1999; Faulkner et
al., 2004), in this study astrocytic gliosis was diminished but not
totally abolished. This appeared to still allow the positive aspects
of gliosis in aiding wound repair, although decreasing the nega-
tive effects, such as the physical barrier of hypertrophic astrocytes
and the formation of the glial scar. Similar enhancement of re-
generation has recently been described in an entorhinal cortex
lesion model in GFAP and vimentin double null mice, which also
produced a very mild gliosis (Wilhelmsson et al., 2004). The de-
creased CSPG expression at the lesion site in the EphA4/mice
is also likely to have allowed enhanced regeneration, as was ob-
served when CSPG glycosaminoglycan side chains were removed
from the lesion site by enzymatic treatment (Grimpe and Silver,
The effects of EphA4 on neuronal regeneration observed in
this study may explain the relative failure of blocking or deleting
other putative inhibitory molecules, such as Nogo (Kim et al.,
2003; Simonen et al., 2003; Zheng et al., 2003), and demonstrates
the presence of another major inhibitory influence in addition to
myelin and other inhibitory components. The finding that
EphA4 mediates glial activation and is in turn regulated by in-
flammatory cytokines such as IFN
and LIF, provides an exciting
possibility for overcoming the inhibitory effects of EphA4 and
promoting axonal regrowth. Not only is it possible to block Eph
activation using monomeric forms of cognate ligands (Davis et
al., 1994), such as EphrinA5 (Fig. 5h), but it may also be possible
to promote neuronal regeneration by blocking the expression of
EphA4 on astrocytes at the injury site by blocking the action of
specific cytokines. Given that preliminary results indicate that
EphA4 expression is induced on astrocytes in primates after spi-
nal cord damage (Y. Goldshmit and M. P. Galea, unpublished
observations), there is also a real possibility of effectively promot-
ing significant axonal regeneration after spinal cord injury in
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    • "Ephrins are attached to the cell membrane either via a glycosylphosphatidylinositol (GPI) anchor linkage (A class) or a transmembrane sequence (B class). Since initial identification as important mediators during development of the central nerve system [16], EPH/Ephrin signaling was shown to be involved in many different cell-cell interactions and cellular processes171819, with EPHA4 as an essential regulator in the correct guidance of spinal cord axons during development[16, 20, 21]. Deregulation of EPH/Ephrin signaling has been demonstrated in a variety of diseases, including diabetes, neurodegenerative disorders and cancer [19, 22]. "
    [Show abstract] [Hide abstract] ABSTRACT: EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.
    Full-text · Article · Sep 2015
    • "Due to the activation of growth impairing pathways by inhibitory substrates and a markedly reduced intrinsic growth capacity of adult lesioned neurons, axonal regeneration, sprouting and functional recovery are severely hampered after spinal cord injury (SCI). Several inhibitory molecules are upregulated after SCI, such as myelin-associated glycoprotein (Mukhopadhyay et al. 1994), Nogo A, oligodendrocyte myelin glycoprotein (McKerracher et al. 1994; Schwab 1998), chondroitin sulfate proteoglycans (Rolls et al. 2009; Yiu and He 2006), semaphorins, and ephrins (Benson et al. 2005; Goldshmit et al. 2004 ). Although these molecules bind to different membranebound receptors, their intracellular signaling converges on Rho kinase (ROCK), which regulates axon cytoskeleton dynamics and axonal outgrowth (Forgione and Fehlings 2013; Sandvig et al. 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback and locomotor deficits. In this study we used AAV-mediated overexpression of BAG1 and ROCK2-shRNA in the red nucleus in order to trace (by co-expression of EGFP) and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein overexpression vs. AAV.shRNA-mediated downregulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Mar 2015
    • "Alternatively, SD-dependent increase in EfnA3 and decrease in EphA4 expression in the cerebral cortex may suggest that sleep loss is not only detrimental to brain recovery after mTBI and could rather produce some positive effects. In fact, the lack of EphA4 was shown to be positively associated with neuronal reconstruction after spinal cord injury (Goldshmit et al., 2004), likely because EphA4 can mediate spine retraction (Murai and Pasquale, 2011). Importantly, EphA4 and EfnA3 have both been implicated in synaptic plasticity and neuron-glia interactions (Murai and Pasquale, 2011), emphasizing the relevance of changes in these elements after TBI. "
    [Show abstract] [Hide abstract] ABSTRACT: Traumatic brain injury (TBI), including mild TBI (mTBI), is importantly associated with vigilance and sleep complaints. Because sleep is required for learning, plasticity and recovery, we here evaluated the bidirectional relationship between mTBI and sleep with two specific objectives: (1) Test that mTBI rapidly impairs sleep–wake architecture and the dynamics of the electrophysiological marker of sleep homeostasis (i.e., non-rapid eye movement sleep delta (1–4 Hz) activity); (2) evaluate the impact of sleep loss following mTBI on the expression of plasticity markers that have been linked to sleep homeostasis and on genome-wide gene expression. A closed-head injury model was used to perform a 48 h electrocorticographic (ECoG) recording in mice submitted to mTBI or Sham surgery. mTBI was found to immediately decrease the capacity to sustain long bouts of wakefulness as well as the amplitude of the time course of ECoG delta activity during wakefulness. Significant changes in ECoG spectral activity during wakefulness, non-rapid eye movement and rapid eye movement sleep were observed mainly on the second recorded day. A second experiment was performed to measure gene expression in the cerebral cortex and hippocampus after a mTBI followed either by two consecutive days of 6 h sleep deprivation (SD) or of undisturbed behavior (quantitative PCR and next-generation sequencing). mTBI modified the expression of genes involved in immunity, inflammation and glial function (e.g., chemokines, glial markers) and SD changed that of genes linked to circadian rhythms, synaptic activity/neuronal plasticity, neuroprotection and cell death and survival. SD appeared to affect gene expression in the cerebral cortex more importantly after mTBI than Sham surgery including that of the astrocytic marker Gfap, which was proposed as a marker of clinical outcome after TBI. Interestingly, SD impacted the hippocampal expression of the plasticity elements Arc and EfnA3 only after mTBI. Overall, our findings reveal alterations in spectral signature across all vigilance states in the first days after mTBI, and show that sleep loss post-mTBI reprograms the transcriptome in a brain area-specific manner and in a way that could be deleterious to brain recovery.
    Full-text · Article · Jan 2015
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