European Journal of Neuroscience, pp. 1–11, 2014

doi:10.1111/ejn.12677

The Ephrin receptor EphA4 restricts axonal sprouting and enhances branching in the injured mouse optic nerve
mie Jordi,1,2 Martin E. Schwab1,2 and Vincent Pernet1,2 Sandrine Joly,1,2 Noe 1 2

Brain Research Institute, University of Zurich, Winterthurerstrasse 190, Zurich, CH-8057, Switzerland Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland

Keywords: axonal guidance, axonal regeneration, retinal ganglion cells, tissue clearing, trauma

Abstract The lack of axonal regeneration in the adult central nervous system is in part attributable to the presence of inhibitory molecules present in the environment of injured axons such as the myelin-associated proteins Nogo-A and MAG and the repulsive guidance molecules Ephrins, Netrins and Semaphorins. In the present study, we hypothesized that EphA4 and one of its potential binding partners EphrinA3 may participate in the inhibition of adult axon regeneration in the model of adult mouse optic nerve injury. Axonal regeneration was analysed in three dimensions after tissue clearing of EphA4 knockout (KO), EphrinA3 KO and wild-type €ller glia endfeet in the retina and in astrocytes in (WT) optic nerves. By immunohistochemistry, EphA4 was highly expressed in Mu the retina and the optic nerve, while EphrinA3 was present in retinal ganglion cells and oligodendrocytes. Optic nerve crush did not cause expression changes. Significantly more axons grew in the crushed optic nerve of EphA4 KO mice than in WT or EphrinA3 KO animals. Single axon analysis revealed that EphA4 KO axons were less prone to form aberrant branching than axons in the other mouse groups. The expression of growth-associated proteins Sprr1a and Gap-43 did not vary between EphA4 KO and WT retinae. However, glial fibrillary acidic protein-expressing astrocytes were withdrawn from the perilesional area in EphA4 KO, suggesting that gliosis down-regulation may locally contribute to improve axonal growth at the injury site. In summary, our threedimensional analysis of injured mouse optic nerves reveals beneficial effects of EphA4 ablation on the intensity and the pattern of optic nerve axon regeneration.

Introduction The lack of axonal regeneration in the central nervous system (CNS) leads to irreversible neurological deficits after traumatic injuries. To elucidate the molecular mechanisms controlling axonal regeneration, the experimental optic nerve crush paradigm has been intensively used in adult mice. With this model, important intrinsic neuronal growth modulators have been unveiled such as those involved in the PTEN/mTOR or the Stat3 signaling pathways (Leaver et al., 2006; Park et al., 2008; Smith et al., 2009; Leibinger et al., 2012; Pernet et al., 2013a,b). The inhibitory environment composed of myelin-associated proteins such as Nogo-A and MAG (Schwab, 2010; Pernet & Schwab, 2012) and some guidance molecules of the Netrin, Ephrin and Semaphorin families repress CNS axon growth by destabilizing the neuronal cytoskeleton in a RhoA/ ROCK signaling-dependent manner (Goldberg et al., 2004; Goldshmit et al., 2004; Low et al., 2008; Duffy et al., 2009). However, the role of many members of guidance molecule families in adult CNS axonal regeneration remains unknown.

Correspondence: Dr V. Pernet, 1Brain Research Institute, as above. E-mails: [email protected] and [email protected] S.J. and N.J. contributed equally to this work. Received 26 March 2014, revised 10 June 2014, accepted 16 June 2014

Axonal guidance defects have been proposed to limit the range of growth in the visual pathway after retinal ganglion cell (RGC) growth program stimulation (Fischer et al., 2004b; Kurimoto et al., 2010; Luo et al., 2013; Pernet et al., 2013b). The moderate axonal regeneration obtained in the crushed optic nerve by transfecting RGCs with Stat3 was associated with a high proportion of axonal U-turns at the front of growth, suggesting that inhibitory molecules in the nerve may repel growth cones toward the retina. The blockade of ROCK resulted in straighter axonal trajectories and potentiated Stat3-mediated axonal regeneration in the optic nerve (Pernet et al., 2013b). In another study, axonal regeneration induced by mTOR and Stat3 co-activation in RGCs was possible only up to the optic chiasm from where axons failed to extend in the correct direction, i.e. in the contralateral optic tract (Luo et al., 2013). A better understanding of adult axon guidance mechanisms therefore appears essential to improve axonal regeneration in the injured visual system. During development, Ephrins and their Eph receptors play an important role in the establishment of the retinotopic projection map (Scicolone et al., 2009; Feldheim & O’Leary, 2010). In the injured adult visual system, Ephrins/Eph may influence axonal regeneration as well (Liu et al., 2006; Du et al., 2007; Duffy et al., 2012; Fu & Sretavan, 2012). The ablation of the oligodendrocyte protein EphrinB3 significantly enhanced axonal regeneration in the injured optic nerve and corticospinal tract (Duffy et al., 2012). In the spinal cord,

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

2 S. Joly et al. these effects may be mediated by the neuronal expression of EphA4 that can bind diverse EphrinB and EphrinA ligands (Scicolone et al., 2009). Indeed, EphA4 knockout (KO) mice showed improved axonal regeneration after spinal cord dorsal hemisection (Goldshmit et al., 2004, 2011). In contrast, the role of EphA4 has never been addressed in the classical model of optic nerve injury. In the present study, we analysed the possible inhibitory effects of EphA4 and EphrinA3, a new potential axon growth inhibitor (Kempf et al., 2013), on optic nerve axon regeneration.

Materials and methods Animals Two-/4-month-old-male C57BL/6 mice were used for optic nerve crush injuries and for tissue analysis. All animal experiments were performed with the acceptance and in accordance with the guidelines of the Veterinary Office of the Canton of Zurich, the Council Directive 2010/63EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. EphA4 KO mice were provided by Prof. R. Klein (Max Planck Institute for Neurobiology, Martinsried, Germany) (Dottori et al., 1998; Kullander et al., 2001). EphrinA3 KO mice were a gift of Prof. E. Pasquale (La Jolla, San Diego, CA, USA) (Carmona et al., 2009). Three-dimensional (3D) analysis of axonal regeneration in the cleared optic nerve The optic nerve micro-crush injury was carried out at ~0.25 mm from the eyeball with a 9-0 suture to minimize the size of the injury (Pernet et al., 2012). To relieve pain, animals received analgesic (buprenorphin, 0.01–0.05 mg/kg body weight) subcutaneously during the optic nerve crush surgery. To anterogradely trace injured axons, cholera toxin b subunit conjugated to alexa-594 [CTb-594, 0.5% in phosphate-buffered saline (PBS), 1.5–2 lL; Molecular Probes, Zug, Switzerland] was injected in the vitreous of mice the day preceding intracardial perfusion with paraformaldehyde (PFA, 4%), on day 14 after the lesion (Pernet et al., 2012, 2013a,b). For 3D analysis of CTb-594-labeled axons, fixed optic nerves were cleared according to the adapted protocol of Dodt et al. (2007) and as recently described (Pernet et al., 2013b). In brief, after dehydration in ethanol and in hexane, optic nerves were immersed in the clearing solution composed of benzylalcohol and benzylbenzoate (1 : 2) (Sigma-Aldrich, Buchs, Switzerland). Resulting transparent optic nerves containing fluorescent axons were imaged using a confocal Leica SP5 inverted microscope (Leica Microsystems, Mannheim, Germany) equipped with a 63 9 glycerine immersion objective [numerical aperture (NA) 1.3]. 3D reconstructions of CTb-594-containing axons in the optic nerve were allowed after image stack stitching with the XuvTools software (Emmenlauer et al., 2009) and macro-stack exportation to the Imaris Software (Bitplane, Zurich, Switzerland) for trajectory analysis. The total number of sprouting axons was counted throughout the whole thickness of the optic nerve at 100, 200, 300, 400, 500, 800 and 1000 lm past the injury site. Using the Filament Tracer’s advanced manual tracing mode (AutoDepth), single axon branching and trajectories were examined and calculated for the 20 longest axons. Branched axons were defined as axons presenting one or more collateral extensions in the last 200 lm of their course. Snapshots of CTb-594-filled axon projections were captured in the orthogonal mode. For statistical analysis, a one-

way analysis of variance (ANOVA) followed by a Tukey post hoc test was applied. Animals presenting ischemia or retinal hemorrhages were excluded from the analysis. Neuronal survival Retinal ganglion cell survival was estimated 2 weeks after intraorbital optic nerve crush according to a standard protocol (Pernet et al., 2005, 2012, 2013a,b). After fixation in 4% paraformaldehyde, the retinae were rapidly flat-mounted and RGCs were labelled by immunofluorescence using a rabbit anti-b3Tubulin antibody (1 : 200, #ab18207; Abcam, Cambridge, UK) diluted in PBS containing 0.3% Triton X-100, 5% normal serum and 0.05% sodium azide. The anti-b3Tubulin antibody was raised against a synthetic peptide consisting of the C terminus of human neuronspecific b3Tubulin, following the manufacturer’s description. Western blot analysis detected a single band in mouse brain lysates. After washing, the retinae were incubated with corresponding secondary antibodies at 4 °C. To estimate the density of surviving neurons, RGCs stained for b3Tubulin were imaged in the four quadrants of the retina using a Leica SPE-II confocal microscope at 40 9 (NA 1.25), with a step size of 0.5 lm and a resolution of 1024 9 1024 pixels (0.27 lm per pixel). The number of RGC bodies was counted in regions of 62 500 lm2 at 1 mm and 1.5 mm from the optic disk in each quadrant. Immunohistochemistry on retinal and optic nerve sections Adult mice were injected with an overdose of anesthetic and intracardially perfused with PBS (0.1 M) and 4% PFA. Eyecups were quickly dissected by removing the cornea and the lens, and postfixed in 4% PFA overnight at 4 °C. The day after, eyecups were cryoprotected in 30% sucrose overnight and embedded in OCT compound (Tissue-TEK, Sakura) with a liquid nitrogen-cooled bath of 2-methylbutane. Optic nerves and retinal sections were cut (14 lm) with a cryostat microtome and collected on Superfrost Plus glass slides (Menzel-Glaser, Braunschweig, Germany). For immunohistochemisty, tissue sections were incubated in a solution containing 5% bovine serum albumin (BSA) or normal serum, 0.3% Triton X-100 in PBS for 1 h at room temperature to avoid unspecific staining. Then, primary antibodies were added in 5% BSA or normal serum, 0.3% Triton X-100 in PBS overnight at 4 °C. After PBS washes, tissue sections were incubated with the appropriate secondary antibody for 1 h at room temperature, and mounted with MOWIOL anti-fading medium [10% Mowiol 4-88 (w/v) (Calbiochem, Cambridge, UK), in 100 mM Tris, pH 8.5, 25% glycerol (w/v) and 0.1% 1,4-diazabicyclo[2.2.2]octane (DABCO)]. Primary antibodies were: rabbit anti-EphA4 [F88 antiserum kindly provided by the laboratory of Prof. A. Turnley, University of Melbourne, Australia (Goldshmit et al., 2011)], mouse anti-glutamine synthetase (GS, 1 : 500, #MAB302; Chemicon, Zug, Switzerland), rabbit antiEphrinA3 (1 : 100, #36-7500; Invitrogen, Zug, Switzerland), mouse anti-b3Tubulin (1 : 1000, #G712A; Promega, Madison, WI, USA), rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 500, #Z0334; Dako, Carpinteria, CA, USA), mouse anti-adenomatous polyposis coli (APC, 1 : 100, #OP80; Calbiochem). The F88 antiserum was raised by immunizing rabbits against a peptide corresponding to amino acids 938–953 in the intracellular sterile alpha motif (SAM) domain of EphA4 and its specificity was previously validated (Goldshmit et al., 2004). The mouse anti-GS antibody was generated using GS purified from sheep brains and allowed to detect a single band by Western blotting. This antibody specifically labels M€ uller glial cells in the rat

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

EphA4 inactivation improves optic nerve regeneration 3 (Riepe & Norenburg, 1977) and mouse retina (Bringmann et al., 2013). The EphrinA3 antibody was obtained by immunizing rabbits with a synthetic peptide derived from the C terminus of the mouse EphrinA3 protein, and its reactivity was confirmed by Western blotting with mouse EphrinA3 recombinant protein, according to the description of the manufacturer. The b3Tubulin monoclonal antibody (clone 5G8) is a protein G-purified IgG1 raised in mice against a peptide (EAQGPK) corresponding to the C terminus of b3Tubulin. It only recognizes neurons and yields a single band on Western blots. The GFAP polyclonal antibody (Dako) is the purified immunoglobulin fraction of the rabbit serum raised against GFAP isolated from bovine spinal cord. The mouse anti-adenomatous polyposis coli antibody is an IgG2b that was produced by immunization against a recombinant peptide corresponding to amino acids 1–226 of APC. APC is a common marker for mature oligodendrocytes (Bhat et al., 1996; McTigue et al., 2001). Immunofluorescence pictures were taken with a Leica SPE-II confocal microscope equipped with a 40 9 oil immersion objective (NA 1.25). Western blotting Lysates were prepared by homogenizing retinae in lysis buffer (20 mM Tris–HCl, 0.5% CHAPS, pH 8) containing protease inhibitors (Complete mini; Roche Diagnostics, Indianapolis, IN, USA) for 60 min on ice. Proteins in the supernatant were collected in Eppendorf tubes, and centrifuged for 15 min at 15 000 g at 4 °C. Retinal proteins (20 lg per well) were resolved by electrophoresis on a 4– 12% gradient polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were pre-incubated in a blocking solution of 5% BSA dissolved in TBST (Tris-base 0.1 M, 0.2% Tween 20, pH 7.4) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C and after washing, with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody (1 : 10 000–1 : 25 000; Pierce Biotechnology, Rockford, IL, USA). Primary antibodies were mouse anti-EphA4 (1 : 200, #37-1600, clone 4C8H5; Invitrogen), rabbit anti-EphrinA3, (1 : 100, #36-7500; Invitrogen) and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 20 000, #ab8245, clone 6C5; abcam). The EphA4 mouse antibody is a protein A-purified IgG1j that was raised using a synthetic peptide derived from the C terminus of the human EphA4 receptor protein as immunogen. It recognizes a single band at ~110–120 kDa on Western blots. The anti-GAPDH antibody is a protein A-purified IgG1 obtained after immunization against the rabbit muscle GAPDH protein and detects a single band at ~36 kDa by western blotting. Proteins were detected by adding SuperSignal West Pico Chemiluminescent Substrate (Pierce) and after exposure of the blot in a Stella detector. Semi-quantitative real time RT-PCR (qRT-PCR) Transcript levels were determined by qRT-PCR measurements in retinal lysates as detailed previously (Pernet et al., 2012, 2013a,b). Relative quantification was calculated using the comparative threshold cycle (DDCT) method. cDNA levels were normalized to Gapdh (reference gene) and a control sample (calibrator set to 1) was used to calculate the relative values. For each gene, the PCR amplification efficiency was established from the slope of the calibration curve according to the equation: E = 10(
1/slope) (Pfaffl, 2004). Each reaction was performed in triplicate and at least three mice per condition were analysed. Primer sequences used were for Gap43: forward: 50 -TGCTGTCACTGATGCTGCT-30 , reverse: 50 -GGCTTCGT CTACAGCGTCTT-30 ; for Sprr1a: forward: 50 -GAACCTGCTCTT

CTCTGAGT-30 , reverse: 50 -AGCTGAGGAGGTACAGTG-30 ; for Cntf: forward: 50 -CTCTGTAGCCGCTCTATCTG-30 , reverse: 50 -GG TACACCATCCACTGAGTC-30 ; for Lif: forward: 50 -AATGCCACCTGTGCCATACG-30 , reverse: 50 -CAACTTGGTCTTCTCTGTCCCG30 ; for Bdnf: forward: 50 -CAAAGCCACAATGTTCCACCAG-30 , reverse: 50 -GATGTCGTCGTCAGACCTCTCG-30 ; for Fgf2: forward: 50 -TGTGTCTATCAAGGGAGTGTGTGC-30 , reverse: 50 -ACCAA CTGGAGTATTTCCGTGACCG-30 ; for Gfap: forward: 50 -CCA CCAAACTGGCTGATGTCTAC-30 , reverse: 50 -TTCTCTCCAAAT CCACACGAGC-30 ; for Vimentin: forward: 50 -TACAGGAAGCTG CTGGAAGG-30 , reverse: 50 -TGGGTGTCAACCAGAGGAA-30 ; for Gapdh: forward: 50 -CAGCAATGCATCCTGCACC-30 , reverse: 50 -TGGACTGTGGTCATGAGCCC-30 . Measurement of GFAP-free regions in injured optic nerves GFAP-expressing astrocytes were labeled by immunohistochemistry (rabbit anti-GFAP, 1 : 500, #Z0334; Dako) on longitudinal sections of optic nerves, 5 days after crush injury. Axons and cell nuclei were stained with an anti-b3Tubulin antibody (1 : 1000, #G712A; Promega) and Dapi (40 ,6-diamidino-2-phenylindole, dilactate, 1 : 40 000, #D3571; Invitrogen) respectively, on the same sections. Fluorescence images were acquired with a Leica SP5 confocal microscope at 409. The region that did not contain clear, homogenous GFAP staining was measured in wild-type (WT) and EphA4 KO nerves with the NIH ImageJ software as previously described (Sengottuvel et al., 2011). The average length of the gap was calculated by dividing the GFAP-negative area by the width of the optic nerve at the injury site. Four to six sections per animal were analysed and four mice per group were used to calculate the distance [mean  standard error of the mean (SEM)]. Data analysis Bar graphs are presented as mean  SEM. Statistical analyses were performed by using a one-way ANOVA followed by the Tukey post-hoc test, or by unpaired t-test as indicated in figure legends (GraphPad Software, Prism 5).

Results EphA4 and EphrinA3 are expressed in the adult mouse retina and optic nerve The expression of EphA4 and one of its ligands, EphrinA3, was examined by immunohistochemistry on 2-month-old mouse retinal cross-sections (Fig. 1). EphA4 was mainly present in GS-containing M€ uller glia processes and their endfeet in the ganglion cell layer and optic fiber layer (Fig. 1A–C, a–c), a distribution consistent with a previous study (Petros et al., 2006). In contrast, EphrinA3 signal was highest in b3Tubulin-labeled RGC bodies, and was detected at a lower intensity in cells of the inner nuclear layer (Fig. 1D–F, d–f). By Western blot analysis, injury of RGC axons in the optic nerve did not change the total EphA4 or EphrinA3 levels in retinal lysates after 5 days (Fig. 1G and H; Fig. S1A). In addition, the signal for EphA4 did not appear stronger by immunohistochemistry in retinal cross-section 5 days after optic nerve crush compared with intact retinae (Fig. S1B). Immunohistochemical analysis on intact optic nerve sections revealed that EphA4-expressing cells were stained for GFAP, a specific marker for astrocytes (Fig. 1I), whereas EphrinA3expressing cells contained the oligodendrocyte marker APC (Fig. 1J). Five days after optic nerve crush, a strong signal for

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

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Fig. 1. EphA4 and EphrinA3 expression in retinae and in the optic nerves. (A–C) Using immunohistochemistry on uninjured mouse retina cross-sections, EphA4 was expressed in M€uller glial cells and localized in a highly polarized way in their endfeet labeled with glutamine synthetase (GS) and as shown at higher magnification (a–c). (D–F) In contrast, immunohistochemical staining revealed that EphrinA3 was present in RGCs identified with b3Tubulin used as a specific marker. Other cells in the inner nuclear layer also showed a signal for EphrinA3. (d–f) Higher magnification of the ganglion cell layer. (G, H) Western blot analysis did not allow us to observe expression changes for EphA4 or EphrinA3 between intact and injured retinal lysates (20 lg protein per lane). (I, J) In the intact adult optic nerve, EphA4 was endogenously expressed in GFAP-containing astrocytes while rows of APC-positive oligodendrocytes contained EphrinA3 protein. Images are single confocal microscope scans from anterior regions of intact optic nerves. (K, L) Five days after optic nerve crush, EphA4 and EphrinA3 remained highly expressed in the perilesional area, and in the proximal and distal segments of the optic nerve. Images in A–F, K and L represent merged confocal microscope stacks. White stars indicate the injury site. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; OFL, optic fiber layer. Scale bars: A–F, I–J = 50 lm, K, L = 200 lm.

EphA4 and for EphrinA3 was observed on optic nerve sections, before and after the injury site in the proximal and distal segments (Fig. 1K and L). For EphA4, the immunostaining intensity at the level of the injury site appeared slightly higher (mean  SD, 100  17.2%, n = 7 slices) by densitometric analysis than in the distal segment of the optic nerve (76.6  13.5%, ANOVA, P < 0.05), but was not significantly different when compared with the proximal side of the optic nerve (85.1  8.1%). The expression of EphA4

and EphrinA3 in optic nerve glia suggests that the two proteins may inhibit axonal regeneration after axonal lesion. EphA4 gene deletion improves axonal regenerative sprouting in the injured optic nerve EphA4 is known to mediate repulsive interactions for growing axons (Goldshmit et al., 2004, 2011); the possible implication of EphA4

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

EphA4 inactivation improves optic nerve regeneration 5 and EphrinA3 (Kempf et al., 2013) on axon regrowth was therefore tested by comparing axonal regeneration in the optic nerve of WT, EphrinA3 KO and EphA4 KO mice, 2 weeks after complete crush injury (Fig. 2). To anterogradely trace growing axons, CTb-594 was intravitreously injected on day 13 post-lesion. Two weeks after injury, whole, unsectioned optic nerves were subjected to tissue clearing, a chemical procedure that rendered myelinated nerves transparent and preserved the fluorescent signal of the CTb-594 (Erturk et al., 2012; Luo et al., 2013; Pernet et al., 2013b). Regenerating axons were visualized and analysed in three dimensions (3D) by confocal microscopy imaging and nerve reconstruction

using the Imaris software. Very few axons crossed the lesion site (white stars) in WT and EphrinA3 KO optic nerves (Fig. 2A and B, A0 and B0 ). In contrast, up to three times as many axons as in WT mice were observed extending beyond the injury site in EphA4 KO optic nerves (Fig. 2C and C0 ). The number of axonal fibers was significantly higher in EphA4 KO animals (mean  SEM, 161  12 axons per optic nerve, n = 7 mice) than in the WT (57  4 axons per optic nerve, n = 7 mice) or EphrinA3 KO (60  5 axons per optic nerve, n = 5 mice) groups, at up to 500 lm past the crush site (ANOVA, **P < 0.01, ***P < 0.001; Fig. 2G). On average, the length of the 20 longest axons was not different between the three

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Fig. 2. Axonal regeneration is improved in EphA4 KO but not in EphrinA3 KO optic nerves after injury. (A–C) The growth of injured RGC axons was examined 2 weeks after optic nerve crush (white stars) in the whole, cleared optic nerve. Axons were anterogradely traced by intravitreous injection of CTb-594 the day preceding perfusion. Fixed whole-mounted, cleared optic nerves were imaged by confocal microscopy at high magnification (63 9) and reconstructed in XuvTools. Pictures are snapshots of CTb-594-labelled axons acquired in Imaris and imaged through the whole optic nerve thickness. In A0 –C0 , axonal sprouts beyond the injury site appear more numerous in EphA4 KO than in EphrinA3 KO or WT mice. (G) The quantification revealed significantly more axonal fibers in lesioned EphA4 KO optic nerves than in the other groups up to 500 lm past the crush site (one-way ANOVA, **P < 0.01; ***P < 0.001). (H) The length of the 20 longest axons did not vary between genotypes. (D–F, I) The density of b3Tubulin-labeled RGCs was not different between groups on retinal flat-mounts from injured mice. Quantifications represent mean  SEM. NS, not significant change. Scale bars = 100 lm. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

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Fig. 3. 3D analysis of growing axons in transparent injured optic nerves. (A–C) The directionality and pattern of regenerating CTb-594-labeled axons were analysed in the distal segment of optic nerves of WT, EphrinA3 KO and EphA4 KO animals. (A0 –C0 ) The 20 longest axons of each optic nerve were traced in color in the Imaris software. (A″–C″) Colored axons revealed the presence of short (arrowhead) and long (arrows) axonal branches in WT or EphrinA3 KO mice. In contrast, in EphA4 KO nerves only few axons exhibited collateral sproutings. Red arrowhead indicates an example of a U-turn. (D) Quantitatively, fewer axons had branches in EphA4 KO than in WT or EphrinA3 KO mice (one-way ANOVA, *P < 0.05). (E) The proportion of axonal U-turns did not differ between genotypes. Quantifications represent mean  SEM. Scale bar = 100 lm.

genotypes (Fig. 2H), suggesting that other growth-restricting factors are present in the injured nerve and that the range of enhanced axonal outgrowth in EphA4 KO mice is limited to relatively short distances after the injury site. In addition, the density of surviving RGCs did not vary between WT (746  65 RGCs/mm2, n = 5 mice), EphrinA3 KO (739  29 RGCs/mm2, n = 5 mice) and EphA4 KO (930  149 RGCs/mm2, n = 4 mice) retinal flat-mounts (Fig. 2D–F and I). These data indicate that EphA4 exerts clear

inhibitory effects on adult optic nerve axon growth in the perimeter of the lesion in vivo. EphA4 enhances branching in regenerating axons after injury To gain more insights into the pattern of EphA4 gene deletioninduced axonal regrowth, the degree of axonal branching and the proportion of axonal U-turns were analysed in 3D. To do so, the last

Fig. 5. Gliosis in the axotomized retina and optic nerve. (A, B) To monitor gliosis in injured retinae of WT and EphA4 KO animals, the mRNA levels of Gfap and Vimentin, two specific markers, were assessed by qRT-PCR. In response to optic nerve crush, the gene expression of the two proteins was increased to a similar extent in the two genotypes. Means are from three individual mice  SEM. (C) The distribution of astrocytes around the lesion site was visualized on longitudinal sections of optic nerves by immunohistochemistry for GFAP, 5 days after crush. Axons and cell nuclei were detected with an anti-b3Tubulin antibody and with Dapi, respectively. A relatively small GFAP-free region was observed at the WT lesion sites. In contrast, a larger gap of GFAP staining appeared around EphA4 KO nerve lesions. (D) The average distance of the GFAP-positive astrocytes from the injury was longer in EphA4 KO than in WT nerves. Means are from four individual mice  SEM. For statistical analysis, a Student’s t-test was applied (*P < 0.05). Scale bar = 100 lm. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

EphA4 inactivation improves optic nerve regeneration 7

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Fig. 4. Gene expression analysis of growth-associated proteins and neurotrophic factors. mRNA levels were measured by qRT-PCR for growth-associated proteins (A, B) and neurotrophic factors (C–F) in WT and EphA4 KO retinal lysates without injury and 5 days after optic nerve crush. The optic nerve crush induced a marked up-regulation of each transcript. Only Lif and Bdnf transcripts showed a higher up-regulation in EphA4 KO retinae than in WT controls (t-test, *P < 0.05). NS, not significant change. Means are from three individual mice  SEM. C A

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© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

8 S. Joly et al. 200 lm of the 20 longest axons was analysed in each group after reconstruction of distal segments of the optic nerves with the Imaris software, 2 weeks after injury (Fig. 3A0 , B0 and C0 ). In agreement with our previous study (Pernet et al., 2013b), 34  7% of the axons regenerating past the lesion site had side branches in WT mice (n = 7 mice), and the branches were relatively short. A very similar situation was found in EphrinA3 KO mice (25  3% branched axons, n = 5 mice). Strikingly, collateral branches were significantly less frequent in EphA4 KO optic nerves (14  2%, n = 7 mice) than in WT or EphrinA3 KO mice (Fig. 3D). In the perilesional part of WT nerves, up to 0.5 mm from the lesion, about 25% of all regenerating axons showed U-turns, a proportion consistent with previous observations (Pernet et al., 2013b). This number was unchanged in the absence of EphA4 (24  4%) or EphrinA3 (22  1%) compared with WT littermates (22  4%) (Fig. 3E). These results suggest that the occurrence of axonal branching is lowered when EphA4 is removed, but the repulsive events leading to U-turns are not EphA4-dependent.

GFAP antibody on injured optic nerve sections (Fig. 5C). On the same sections, axons were labeled with a b3Tubulin antibody and cell nuclei were stained with Dapi. In the retina, qRT-PCR measurements showed similar up-regulations of Gfap and vimentin mRNAs in WT and in EphA4 KO mice after nerve crush (Fig. 5A and B). The time course of GFAP expression in injured WT optic nerves revealed that a large astrocyte-free region appeared around the injury site as early as 1 day after lesion (Fig. S2A and B). Then, GFAPexpressing astrocytes progressively invaded the perilesional zone between day 3 (Fig. S2C and D) and day 5 (Fig. S2E and F), until day 14 when most of the gap was filled (Fig. S2G and H). On longitudinal optic nerve sections of EphA4 KO mice, however, the GFAP-negative region was significantly larger than in WT mice (Fig. 5C and D), possibly as a result of altered astrocyte migration or activation (Fig. 5D). The attenuation of the astrocyte reaction at the lesion site may therefore positively influence the regeneration of EphA4 KO axons.

Discussion LIF and BDNF but not growth program-associated proteins are mildly enhanced by EphA4 ablation We then sought to determine if the growth program of injured RGCs was enhanced by Epha4 gene deletion. By qRT-PCR, we measured the expression of growth-associated protein 43 (Gap-43) and small proline-rich repeat protein 1a (Sprr1a) genes that were previously found to correlate with the effective axonal regeneration in the optic nerve (Pernet et al., 2012, 2013a,b). Five days after injury, the mRNA levels of Gap-43 and Sprr1a were increased in these retinae compared with intact mice, but to a similar extent in EphA4 and WT retinal samples (Fig. 4A and B). The transcript levels of several neurotrophic factors known to occur in the lesioned retina, such as ciliary neurotrophic factor (Cntf) (Leaver et al., 2006; Pernet et al., 2013a), leukemia inhibitory factor (Lif) (Leibinger et al., 2009), brain-derived neurotrophic factor (Bdnf) (Mansour-Robaey et al., 1994; Sawai et al., 1996; Pernet & Di Polo, 2006) and fibroblast growth factor 2 (Fgf2) (Sapieha et al., 2003), were also measured (Fig. 4C–F). All these factors were increased by 3.5–8-fold in WT injured retinae. In EphA4 KO, the levels of Lif and Bdnf expression were, respectively, 40 and 60% as high in EphA4 KO as in WT retinae in the intact as well as in the injured condition (Fig. 4D and E). Thus, the improvement of axonal outgrowth in EphA4 KO optic nerves may be due to a local dis-inhibition of the growth cones in the lesion environment rather than an enhanced growth factor stimulation (Sengottuvel et al., 2011), or to indirect effects involving astrocytic gliosis at the injury site in the absence of EphA4 (Fig. 1K). Optic nerve gliosis is attenuated in EphA4 KO mice after injury Glial scars can act as mechanical as well as biochemical barriers for regenerating axons (Kawano et al., 2012). We hypothesized that the higher number of regenerating axons observed in the EphA4 KO optic nerves could be due to changes in gliosis in the optic nerve or the retina. Reactive astrocytes have previously been shown to secrete chondroitin sulfate proteoglycans (CSPGs) that inhibit axonal extension through the lesion area (Selles-Navarro et al., 2001; Shen et al., 2009). To follow gliosis in the retina and the activation of astrocytes in the vicinity of severed axons in the optic nerve, we evaluated the gene expression of two gliosis markers, glial fibrillary acidic protein (Gfap) and Vimentin in retinal lysates (Fig. 5A and B), and we stained astrocytes by immunohistochemistry using a

The EphA4 protein could be detected in M€ uller glia in the retina and in astrocytes in the retina and the optic nerve but not in RGCs themselves with or without optic nerve crush. Axonal outgrowth was increased across the optic nerve injury site of EphA4 KO mice but was not different from WT mice after EphrinA3 deletion. Highresolution 3D imaging allowed us to observe fewer collateral extensions by the sprouting EphA4 KO axons compared with WT or EphrinA3 KO animals, where about one-third of the regenerating axons had short side branches. The elevation of growing fiber numbers and the reduction of branches suggest a contribution of EphA4 to axonal growth and patterning in the injured adult optic nerve in vivo. Deletion of the EphA4 gene enabled significantly more axons to grow past the injury site in the KO optic nerves. In the absence of EphA4, the number of sprouting axons was enhanced 3–5-fold at 100–500 lm past the injury site compared with WT animals, a relatively important effect when compared with the blockade of other extrinsic growth inhibitors, molecular mechanisms of which depend on the intracellular activation of the RhoA/ROCK pathways (Schwab, 2010). For example, in the rat optic nerve, the blockade of RhoA or NgR1 elicited a 2–3-fold increase in the number of growing axons at 100–500 lm after the injury site, 2 weeks post-lesion (Lehmann et al., 1999; Fischer et al., 2004a). The axonal growth improvement obtained after EphrinB3 gene ablation (Duffy et al., 2012) was very similar to the intensity of growth that we observed in EphA4 KO mice. EphrinB3 can bind to and interact with EphA4 (also in reverse signaling direction) and therefore could be a component of the pathway leading to the growth effects observed here (Zimmer et al., 2011; Paixao et al., 2013). EphA4 neutralization could be considered to potentiate axonal regeneration induced by the intracellular activation of Stat3 or mTOR. Indeed, alone, the inactivation of extrinsic inhibitory mechanisms promotes more modest sprouting of optic nerve fibers than the manipulation of intrinsic growth mechanisms, but may be essential to drive complete axonal regeneration to the relevant targets in combinatorial treatments (see below). Further experiments will be necessary to evaluate the potential of EphA4 blockade on the potentiation of growth-promoting intrinsic molecules and/or the blockade of other extrinsic growth inhibitors. Optic nerve axons do not form branches in the normal adult nerve. In crush lesioned WT nerves about one-third of the regenerating axons showed – usually short – side branches. When regenera-

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

EphA4 inactivation improves optic nerve regeneration 9 tion was enhanced by Stat3 overexpression in RGCs and simultaneously blocking the Rho-kinase ROCK, side branch formation occurred in > 40% of the growing axons (Pernet et al., 2013b). In contrast, branch formation was reduced to 14% in the EphA4 KO mice. Ultimately, the hope is to promote long-distance growth of unbranched axons to repair the retinal connection to the targets in the diencephalon and superior colliculus, and manipulation of EphA4 may help to achieve this important goal. Whether EphA4 can influence axonal branching by reverse signaling is not known, however (Knoll & Drescher, 2002). It is also not clear if EphA4 has direct chemorepulsive properties for the growing RGC axons; in stripe assays with embryonic retinae EphA4 failed to inhibit RGC axons (Petros et al., 2006). EphrinA3, which is expressed by adult mouse RGCs, is unlikely as an interaction signaling partner, as our results did not show enhanced regenerative growth in the EphrinA3 KOs and in contrast to the EphA4 KOs. Future experiments will be required to determine if Ephrins can elicit reverse signaling in adult RGCs in response to EphA4 stimulation presented by astrocytes in the injured optic nerve. In our study, the improved axonal regeneration observed in EphA4 KO mice is unlikely to come from the absence of EphA4 on the surface of RGC axons. By immunohistochemistry, we could only detect EphA4 in M€uller glia and in optic nerve astrocytes but not in RGCs, with or without axotomy. This cellular pattern of distribution of EphA4 is consistent with previous studies that reported that EphA4 expression was restricted to astrocytes in the optic nerve head and in the retinal fiber layer (Petros et al., 2006; Du et al., 2007) while EphrinA3 was found in RGCs and in lateral geniculate nucleus neurons (Pfeiffenberger et al., 2005). Mice deficient for EphA4 show normal decussation of optic fibers at the chiasm (Petros et al., 2006), suggesting that EphA4 expression and function differ in the visual system and, for example, the spinal cord (Dottori et al., 1998; Kullander et al., 2001). It is not clear why, despite its strong endogenous expression in the retina and in the optic nerve, EphrinA3 deletion did not affect regeneration in the injured optic nerve. EphrinA3 can inhibit cortical neurite outgrowth in culture (Kempf et al., 2013). It is possible that the removal of EphrinA3 alone is not sufficient to enhance the growth capacity of injured RGCs due to compensatory inhibition mediated by other EphrinAs. Consistent with this idea, the pattern of RGC axon projections in the dorsolateral geniculate nucleus was not shown to be affected in EphrinA2, EphrinA3 and EphrinA5 single KO mice compared with WT animals but presented striking aberrations in triple mutants deprived of the three EphrinA2/A3/A5 (Pfeiffenberger et al., 2005). Moreover, the normal expression of EphrinA3 in RGCs may neutralize the inhibitory action of EphrinA3 presented by oligodendrocytes to cut axons via cis interaction between EphrinA3 and EphA receptors at the RGC membrane, as previously suggested for EphrinA2 and EphrinA5 in immature RGCs (Hornberger et al., 1999; Carvalho et al., 2006). To test this hypothesis, selective manipulations of EphrinA3 expression in RGCs or in oligodendrocytes could be carried out in conditional mutants or by using adenoassociated virus vectors. The density of GFAP-positive astrocytes was clearly lower at and around the injury site in EphA4 KO optic nerves 5 days after injury. Reduced gliosis may facilitate sprouting and short-distance growth of axons, a mechanism that could explain the higher numbers of regenerating fibers seen in the present study. A similar observation was made in the spinal cord after corticospinal tract lesions; the weaker gliosis of astrocytes in the injury area was associated with the lower expression of growth inhibitory CSPGs

and enhanced axonal growth (Goldshmit et al., 2004; Kempf et al., 2013). However, the expression pattern of CSPGs was not correlated with that of GFAP at the level of the lesion site in the optic nerve (Selles-Navarro et al., 2001). In addition, the attenuation of the astrocyte reaction in EphA4 KO mice after spinal cord injury has been challenged by later studies (Herrmann et al., 2010; Dixon et al., 2012). In fact, this apparent discrepancy between the last two studies and our results may stem from differences in tissue composition between the spinal cord and the optic nerve and from the type of injuries applied (crush in the optic nerve vs. hemi-section in the spinal cord). After spinal cord injury, the up-regulation of GFAP was used as an indicator to assess astrogliosis. In similar experimental conditions (e.g. mouse genetic background, dorsal hemi-lesion, GFAP antibodies), a lower intensity of GFAP immunostaining was reported in EphA4 KO mice (Goldshmit et al., 2004) but was later contested (Herrmann et al., 2010; Dixon et al., 2012). In our study, possible variations in GFAP expression were not addressed in the optic nerve where astrocytes constitutively express GFAP. However, a similar up-regulation of Gfap mRNA, probably from M€ uller glia, was observed by qRT-PCR in EphA4 KO as in WT retinae, before and after injury. In the optic nerve of EphA4 KO mice, the enlarged GFAP-free region around the injury site could be due to the slower migration of astrocytes to the injury area than in WT mice. In vitro, the migration of EphA4 KO astrocytes was shown to be altered in a scratch wound assay (Goldshmit et al., 2004), perhaps as a result of disturbances in cytoskeleton stability (Sengottuvel et al., 2011). Therefore, the decrease in GFAP-labelled astrocytes that we found in the lesion area of EphA4 KO optic nerves may result from a migration defect and may not be considered as a sign of general gliosis alteration. It will be important in future studies to clarify the role of EphA4 in astrocyte motility in CNS regions such as the brain and the spinal cord; in this regard, heterogeneous reactions between astrocyte subsets have been described and could differ in the optic nerve compared with the spinal cord (Bardehle et al., 2013). Additional effects in EphA4 KO mice could include complex changes in blood vessel remodeling (Goldshmit et al., 2006) and inflammatory responses (Munro et al., 2012). It remains to be studied if such local reactions at the lesion site or the relief of an EphA4-mediated direct negative signal on the growing axons is responsible for the enhanced regenerative sprouting observed in the present study. In summary, our results show that the absence of EphA4 enhances the growth of lesioned axons in the optic nerve of adult mice without increasing unwanted axonal branching. These results could be used to ameliorate long-distance axonal regeneration in combination with other growth-stimulatory treatments in the injured visual system and in other parts of the CNS.

Supporting Information Additional supporting information can be found in the online version of this article: Fig. S1. EphA4 expression in the intact and axotomized retina. Fig. S2. Time course of GFAP immunostaining changes in the injured optic nerve.

Acknowledgements This work was supported by Swiss National Science Foundation (SNF) grant SNF 31003A-149315–1, the SNF National Center of Competence in Research ‘Neural Plasticity and Repair’ (to MES) and the Velux Stiftung

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11

10 S. Joly et al. (project #817) (to VP). We thank Franziska Christ for her technical help, Professor O. Raineteau for sharing with us his SPE-II confocal microscope, Professor A. Turnley for providing us with EphA4 antibody, and Professors R. Klein and E. Pasquale for giving KO mouse strains. For 3D analysis, imaging was performed with equipment maintained by the Center for Microscopy and Image Analysis, University of Zurich (ZMB). The authors declare no competing interests.

Abbreviations 3D, three-dimensional/three dimensions; ANOVA, analysis of variance; APC, adenomatous polyposis coli; CNS, central nervous system; CSPG, chondroitin sulfate proteoglycan; CTb, cholera toxin b subunit; GAP-43, growth-associated protein 43; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCL, ganglion cell layer; GFAP, glial-fibrillary acidic protein; GFP, green fluorescent protein; GS, glutamine synthetase; KO, knockout; MAG, myelinassociated glycoprotein; mTOR, mammalian target of rapamycin; NA, numerical aperture; OCT, optimal cutting temperature; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PIAS3, protein inhibitor of activated Stat3; PTEN, phosphatase and tensin homolog; qRT-PCR, quantitative realtime PCR; RGCs, retinal ganglion cells; ROCK, rho-kinase; RPL19, ribosomal protein L19; SOCS3, suppressor of cytokine signaling 3; SPRR1A, small proline-rich protein 1A; STAT3, signal transducer and activator of transcription 3; WT, wild-type.

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© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 1–11