Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves

YEXNR-11837; No. of pages: 11; 4C: Experimental Neurology xxx (2014) xxx–xxx

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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

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Regular Article

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Abhijeet R. Joshi a,b,⁎, Ilja Bobylev a,b, Gang Zhang c, Kazim Sheikh c, Helmar C. Lehmann a,b,⁎

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Article history: Received 30 May 2014 Revised 27 August 2014 Accepted 14 September 2014 Available online xxxx

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Keywords: Regeneration Nerve injury Cytoskeleton Remyelination

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Department of Neurology, University of Cologne, Germany Center for Molecular Medicine Cologne, Cologne, Germany Department of Neurology, University of Texas Health Sciences Centre, Houston, TX, USA

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The small GTPase RhoA and its down-stream effector Rho-kinase (ROCK) are important effector molecules of the neuronal cytoskeleton. Modulation of the RhoA/ROCK pathway has been shown to promote axonal regeneration, however in vitro and animal studies are inconsistent regarding the extent of axonal outgrowth induced by pharmacological inhibition of ROCK. We hypothesized that injury to sensory and motor nerves result in diverse activation levels of RhoA, which may impact the response of those nerve fiber modalities to ROCK inhibition. We therefore examined the effects of Y-27632, a chemical ROCK inhibitor, on the axonal outgrowth of peripheral sensory and motor neurons grown in the presence of growth-inhibiting chondroitin sulfate proteoglycans (CSPGs). In addition we examined the effects of three different doses of Y-27632 on nerve regeneration of motor and sensory nerves in animal models of peripheral nerve crush. In vitro, sensory neurons were less responsive to Y-27632 compared to motor neurons in a non-growth permissive environment. These differences were associated with altered expression and activation of RhoA in sensory and motor axons. In vivo, systemic treatment with high doses of Y-27632 significantly enhanced the regeneration of motor axons over short distances, while the regeneration of sensory fibers remained largely unchanged. Our results support the concept that in a growth non-permissive environment, the regenerative capacity of sensory and motor axons is differentially affected by the RhoA/ROCK pathway, with motor neurons being more responsive compared to sensory. Future treatments, that are aimed to modulate RhoA activity, should consider this functional diversity. © 2014 Published by Elsevier Inc.

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Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves

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Axonal injury is a common feature in a large spectrum of metabolic, inflammatory and traumatic peripheral nerve conditions. In optimal circumstances injured peripheral axons are able to fully regenerate, however, in humans axonal regeneration is often compromised resulting in poor functional outcome and recovery. Factors that limit the regenerative capacity of injured axons in humans are relatively slow axonal regrowth rate, large distances that have to overcome by the regrowing axons and the presence of growth-inhibiting molecules, such as the chondroitin sulfate proteoglycans (CSPGs) that are present in the extracellular matrix (ECM) of peripheral nerves (Chen et al., 2005; Heine et al., 2004; Höke, 2005, 2006; Scheib and Höke, 2013; Zuo et al., 2002). The development of treatments to promote axonal regeneration is therefore of great interest and a growing body of evidence suggests that small GTPases including RhoA and its downstream effector Rho-kinase (ROCK) in axons may represent a promising target. In its GTP-bound activated form, RhoA activates ROCK that results in

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Introduction

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⁎ Corresponding authors at: Department of Neurology, University Hospital of Cologne, Kerpener Straße 62, D-50937 Köln, Germany. Fax: +49 221 478 87309. E-mail address: [email protected] (H.C. Lehmann).

phosphorylation of various target proteins including myosin light chain (MLC), which mediates rearrangements of the neuronal cytoskeleton (Fu et al., 2007). The remodeling of the actin cytoskeleton in axon tips/growth cones is considered essential for successful axon elongation and growth. In contrast to the growth-promoting effects of the GTPases Rac1 and Cdc42, the activation of GTPase RhoA and its main downstream effector ROCK, lead to neurite outgrowth arrest and collapse of growth cones (Filbin, 2003; Fournier et al., 2003; Lehmann et al., 1999). Pharmacological inhibition of ROCK by the chemical compound Y-27632 is known to substantially promote axonal regeneration of sensory dorsal root ganglia (DRG) neurons in vitro (Cheng et al., 2008; Fournier et al., 2003; Gopalakrishnan et al., 2008). Moreover, the local or systemic application of fasudil, another ROCK inhibitor, has been shown to improve axonal regeneration after sciatic nerve injury in rodents (Cheng et al., 2008; Hiraga et al., 2006). Notably, in those studies the beneficial effects of systemic treatment with fasudil were more pronounced on the recovery of measures for motor function. Thus we sought to evaluate the effects of Y-27632, a well-characterized chemical ROCK inhibitor, on peripheral nerve regeneration in vitro and in vivo, thereby focusing on potential divergent effects in motor and sensory axons.

http://dx.doi.org/10.1016/j.expneurol.2014.09.012 0014-4886/© 2014 Published by Elsevier Inc.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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nerve growth factor. Cells were treated either with Y-27632 (10 μM; 98 Calbiochem; Selleckchem) or vehicle for 24 h. 99

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Dissociated sensory and motor neuronal cultures were prepared from E15 rat embryos and 8 day old rat pups (p8) as described (Camu and Henderson, 1992; Lehmann et al., 2007). For motor neuronal culture, spinal cords from rat embryos were dissected out and subsequently digested in trypsin for 30 min at 37 °C. Then the tissue was washed in 10% FBS and triturated in Leibowitz-15 (L-15) medium to obtain the cell suspension. For sensory neuronal culture, the entire spinal column was dissected out from 8 day old rat pups and cut to reveal the spinal cord with attached DRGs. Around 30–40 DRGs were removed from each column with microforceps and digested with trypsin for 30 min at 37 °C or 0.25% collagenase for 3 h at 37 °C. After incubation, tissue was dissociated to obtain the cell suspension, which was subsequently filtered with a 40 μm cell strainer to remove undissociated nerve tissue. Neurons were plated in low density on coverslips coated with collagen or collagen and CSPGs (5 μg/ml; Millipore) and maintained in Neurobasal medium (Gibco, Life Technology) containing 1% FBS (Gibco, Life Technology), 2 M L-glutamine, N2 supplement (Gibco, Life Technology) and 10 ng/ml

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RNA was extracted from the neuronal cultures using RNeasy plus mini kit (Qiagen), followed by the reverse transcription of mRNA to cDNA using QuantiTect reverse transcription kit (Qiagen). Quantitative real time PCR was carried out on Rotor Gene 2000 (Corbett Life Sciences). Primers for specific genes were designed online with Primer3.

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RT-PCR

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Cells were fixed for 30 min in 4% paraformaldehyde 24 h after treatment with Y-27632 and stained with neuron specific antibody against β-III-tubulin (1:5000; Promega) and developed with IgG1-specific Alexa Fluor conjugated secondary antibody (1:200; Molecular Probes). Images were acquired using a fluorescence microscope (Keyence BZ9000) and analyzed with ImageJ. The length of longest neurite of each neuron was measured using ImageJ (n = 60–80 neurons per group).

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Neurite outgrowth assay

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Fig. 1. Effects of Y-27632 on neurite outgrowth of motor neurons and dorsal root ganglia neurons in vitro. Motor (A) and sensory (B) neurons, cultured on collagen and treated with Y27632 (lower figures) have longer neurites compared to controls (upper figures, bar = 20 μm (A), 50 μm (B)). (C, D) Quantification shows that Y-27632 treated neurons have significantly longer neurites than controls. *** b 0.0001, n = 60–80 neurons per group.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Fig. 2. Effects of Y-27632 on motor and sensory axonal outgrowth in the presence of CSPGs. Motor neurons (A) and sensory neurons (B) were cultured on collagen (upper figures), or on CSPGs (middle and lower figures) and treated with Y-27632 (lower figures). (Bar = 20 μm (B), 40 μm (A)). Quantification shows a significant decrease in axonal outgrowth of motor (C) and sensory neurons (D) on CSPGs. Treatment with Y-27632 overcomes the inhibitory effect of CSPGs in motor but not in sensory neurons. * b 0.05, *** b 0.0001, n = 60–80 neurons per group.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Protein analysis was done as per Jianjian Shi et al. (Shi et al., 2013) with some modifications. Briefly cells were solubilized with RIPA buffer (Thermo Scientific) with protease and phosphatase inhibitor cocktail (Thermo Scientific). Cell lysate was centrifuged at 15000 × g for 15 min at 4 °C. Supernatant was resolved on 12% SDS-PAGE gels (Life Technology) and transferred onto nitrocellulose membrane (GE Healthcare). Blots were probed with primary antibodies to RhoA (sc418, Santa Cruz Biotechnology), Myosin Light Chain 2 (MLC2) (#3672, Cell Signaling Technology) and phosphorylated Myosin Light Chain 2 (p-MLC2) (#3671, Cell Signaling Technology). Membranes were washed and blotted with corresponding secondary antibodies conjugated with horseradish peroxidase (NXA931, NA9340V, GE Healthcare). Membranes were then developed with SuperSignal West Dura Chemiluminescent Substrate (#34075, Thermo Scientific). All blots were normalized to actin (ab3280, Abcam).

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RhoA activity assay

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RhoA activity was measured using RhoA activation assay kit (Cytoskeleton Inc.), which detects only GTP bound RhoA in total protein extracted from cell lysates. RhoA activity was measured as per manufacturer's instructions. Briefly, protein was extracted from neuronal culture using lysis buffer. Equal quantity of protein extracts (30 μg) were incubated in the wells coated with RhoA binding domain of Rho effector protein for 30 min at 4 °C. The plate was further incubated with primary antibody against RhoA and secondary horseradish peroxidase-conjugated antibody for 45 min each at room temperature. Absorbance was measured at 490 nm to determine the amount of active RhoA.

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In vivo nerve crush

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All animal procedures were in accordance with the German Laws for Animal Protection and were approved by the local animal care committee and local governmental authorities. Axons in the femoral nerve separate distally in motor branch to the quadriceps muscle and a sensory branch to the skin (Madison et al., 1996). In 12- to 14-week old C57BL/6 mice, motor branch (n = 14) and sensory branch (n = 14) of femoral nerve were crushed in separate animals with a forceps for 20 s resulting in axonal degeneration. Animals were then treated either with vehicle or Y-27632 (10 mg/kg body weight i.p., Selleckchem, Batch # S104910) at the day of surgery. After 10 days of crush, animals were perfused with 4% paraformaldehyde and the femoral nerve branches were isolated for morphometry. For sciatic nerve crush, in 12- to 14-week-old C57BL/6 mice the sciatic nerves (n = 6) were crushed 35 mm above the middle toe for 30 s with fine forceps as described previously (Lehmann et al., 2007). Compared to femoral nerve crush, higher dose was used in sciatic nerve crush to facilitate the regeneration over longer distance. Animals were administered 10 doses of either 20 mg/kg Y-27632 (Calbiochem; Batch # D00021370), 50 mg/kg Y-27632, or the similar volumes of vehicle only on days 3, 5, 6, 7, 8, 11, 12, 13, 14 and 15 intraperitoneally (i.p.). On day 17 after the nerve crush, mice were perfused with 4% paraformaldehyde and the sciatic, tibial nerves, and hindpaws (n = 4 each for control and Y-27632-treated groups) were harvested.

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Histological evaluation

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For analysis of intraepidermal nerve fiber density and reinnervation of neuromuscular junctions in the hind paw, 50 μm transverse sections were prepared and immunostained with antibodies against synaptophysin, calcitonin gene-related peptide (CGRP, Chemicon, CA) and Protein Gene Product 9.5 (PGP9.5, Chemicon), as described previously (Griffin et al., 2010). Teased fibers were prepared from small segments of sciatic nerves distal to the crush as described previously (Stoll et al., 1989) and internodal lengths of 30–50

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Sciatic, tibial and femoral nerves as well as hindpaws were immersion-fixed overnight. For morphology and morphometric studies, nerve segments 3 mm (sciatic nerve, femoral motor, femoral sensory) and 15 mm (tibial nerve) distal to the crush site were embedded in epon and 1-μm cross sections were stained with toluidine blue, as described (Lehmann et al., 2007). All myelinated axons in a single whole cross section of the nerve were counted for quantification at light level (40×) by using stereotactic imaging software. In cross sections of the sciatic nerve the axon histogram, mean axon caliber, and mean g-ratio (axon diameter/fiber diameter) were analyzed using ImageJ software.

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On day 9 and 16 the compound muscle action potential (CMAP) amplitudes were recorded by needle electrode insertion in the hindpaw (sole) and stimulation at the sciatic notch using a PowerLab signal acquisition set-up (AD Instruments) under controlled body temperature, as described (Lehmann et al., 2007).

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The following primers were used: RhoA forward: 5′-CATCCCAGAAAAGT GGACTCC-3′; RhoA reverse: 5′-CCTTGTGTGCTCATCATTCCG-3′; GAPDH forward: 5′-CCTGTTCATCCCTCCACACATC-3′; GAPDH reverse: 5′-CCAG TGATTTTCCAGCCCTAATC- 3′; HPRT forward: 5′-GCAGTACAGCCCCAAA ATGG- 3′; and HPRT reverse: 5′-GGTCCTTTTCACCAGCAAGCT- 3′. The relative gene expression was measured by comparative CT method.

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Fig. 3. Relative mRNA expression of RhoA in motor neurons (B) and DRG neurons (A) cultured on wells coated with either collagen or collagen and CSPGs. * b 0.05, ** b 0.01.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Fig. 4. Relative protein expression of RhoA and activation. Protein expression of RhoA is unchanged on CSPG coated wells in motor and sensory neurons but is decreased significantly in motor neurons (A, C) but not in sensory neurons (B, D) after treatment with Y-27632. The fraction of active RhoA to total RhoA is unchanged on CSPG coated wells and decreased upon treatment with Y-27632 in motor (E) and sensory neurons (F), but to more extent in motor neurons compared to sensory neurons. 1: −CSPG, 2: +CSPG, 3: +CSPG, +Y-27632; * b 0.05, ** b 0.01.

Fig. 5. Relative protein expression of MLC and p-MLC in motor and sensory neurons. Ratio of p-MLC to total MLC is decreased significantly in motor neurons (A, C) after treatment with Y-27632 on CSPG coated wells but not in sensory neurons (B, D). 1: − CSPG, 2: + CSPG, 3: + CSPG, + Y-27632; * b 0.05.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Fig. 6. Effect of Y-27632 on axonal regeneration in motor (A) and sensory (B) branch of femoral nerve after treatment with vehicle (upper figures) and Y-27632 (lower figures). Morphometric analysis shows that motor nerve (C) shows enhanced regeneration compared to the sensory nerve (D) after Y-27632 treatment (Bar = 20 μm). *** b 0.0001, n = 14 mice each group.

internodes (per nerve) were determined in Y-27632 and vehicle treated groups (n = 4 each group).

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Data were statistically analyzed using GraphPadPrism 5.0 (GraphPad Software). All numerical results are presented as means ± SEM. Differences between groups were compared with Student's t test or ANOVA with corrections for multiple comparisons. P b 0.05 was considered statistically significant.

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Y-27632 enhances neurite outgrowth in sensory and motor neuron cultures on a growth permissive substrate

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In order to determine if the inhibition of ROCK by Y-27632 promotes neurite outgrowth in vitro, primary motor and sensory neurons were cultured on a growth promoting substrate (collagen) and exposed to

Y-27632. As reported previously (Cheng et al., 2008; Fournier et al., 2003), exposure of neuronal cell cultures to Y-27632 promoted neurite outgrowth in vitro. We observed a more than 1.5 fold increase in the neurite outgrowth in cell cultures of the two fiber modalities when treated with 10 μM Y-27632 compared to vehicle treated control cultures (Fig. 1).

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Sensory and motor neurons respond differently to Y-27632 in an axon 219 growth inhibitory environment 220 Because the activity state of RhoA in neurons is intensely modulated by growth inhibitory molecules such as myelin associated glycoprotein (MAG) or CSPGs, we next determined the effect of Y-27632 in sensory and motor neuron cultures that were grown on a growth inhibitory substrate. Therefore we cultured primary sensory and motor neurons on wells that were coated either with collagen or with a mixture of collagen and CSPGs. In compliance with previous reports (Snow et al., 1996), we observed that CSPGs decrease the axonal outgrowth compared to control in motor and sensory neurons (Figs. 2A and B). Notably,

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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only motor but not sensory neurons were able to overcome the inhibitory effect of CSPGs after treatment with Y-27632 (Figs. 2C and D).

Total RhoA expression in motor and sensory neurons in an inhibitory environment

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Fig. 7. CMAP amplitudes recorded in the hindpaw on days 9 and 16 after nerve crush were higher in Y-27632-treated (50 mg/kg) (A) as compared to animals treated with low-dose Y-27632 and control (B). (C) Mean CMAP amplitudes (± SEM) at day 10 and day 17 for Y-27632-treated (12.5 mg, ▼), (5 mg, ■) and vehicle-treated (▲) groups. (D) Representative synaptophysin staining shows enhanced reinnervation in hindpaw of Y-27632 treated animals in comparison to control. Bar = 75 μm. (E) Quantification of synaptophysin staining showed significant higher reinnervation in group treated with high dose of Y-27632. * b 0.05, n = 6 mice each group.

We next asked if expression levels of RhoA may account for the different response of motor and sensory neurons to Y-27632. Therefore we investigated the relative expression of RhoA mRNA in motor and sensory neurons in a non-permissive environment. In motor neurons, the relative mRNA expression of RhoA was significantly increased on CSPGs coated wells and decreased significantly on CSPGs coated wells treated with Y-27632 (Fig. 3A). In contrast, sensory neurons showed no significant change in relative mRNA expression of RhoA on CSPGs coated wells (Fig. 3B). On protein level we did not observe an increase of RhoA expression in neurons exposed to CSPGs. However total RhoA expression significantly decreased in Y27632 treated motor neurons (Figs. 4A, C). In contrast, there was no change in expression of RhoA in sensory neurons after Y-27632 treatment (Figs. 4B, D).

Y-27632 decreases RhoA activation in motor neurons

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RhoA ELISA assay was used to measure total RhoA and the GTP bound active form of RhoA in sensory and motor neurons in vitro. RhoA activity was unaffected on CSPGs coated wells but decreased significantly in sensory and motor neurons when Y-27632 was added. RhoA activity was lower in motor neurons compared to sensory neurons after Y-27632 treatment (Figs. 4E, F).

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Y-27632 decreases MLC phosphorylation in motor neurons but not in sensory neurons

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MLC is the downstream effector of RhoA/ROCK pathway. Activation of RhoA/ROCK results in phosphorylation of MLC (p-MLC) which increases actomyosin contractility in neurons, leading to growth cone collapse and regeneration inhibition (Filbin, 2003). Thus we measured ratio of p-MLC to total MLC in neuronal cell cultures that were grown on CSPGs. Phosphorylation of MLC was not significantly altered in sensory neurons but decreased significantly in motor neurons after treatment with ROCK inhibitor Y-27632 (Fig. 5).

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Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Fig. 8. Treatment with Y-27632 does not alter the intraepidermal density of PGP9.5 positive (A, C) and CGRP positive nerve fibers (B, D). Bars = 100 μm, n = 6 each group.

Femoral nerve crush

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To study in vivo the regeneration of sensory and motor nerves separately, we used a femoral nerve crush model as the femoral nerve divides into a pure sensory and a pure motor branch. We investigated the regeneration in the nerves by counting the total number of regenerated axons in control and Y-27632 treated group 10 days after crush of each nerve. We observed that the number of regenerated axons from motor neurons were significantly higher in Y-27632 treated compared to control group (427 vs. 732; Fig. 6A). In accordance with in vitro data, sensory nerves showed no significant regeneration after treatment with Y-27632 (269 vs. 285; Fig. 6B).

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We examined the effects of two different Y-27632 doses (20 mg/kg/ dose (total 5 mg over 10 days) and 50 mg/kg/dose (total 12.5 mg over 10 days)) on axonal regeneration in a mixed nerve after proximal injury. Nerve conduction studies showed that animals that received high dose, but not low dose Y-27632 had higher CMAP amplitudes in comparison to controls (Figs. 7A–C). Likewise, immunostaining for synaptophysin (Figs. 7D–E), which was used as a presynaptic marker of reinnervated neuromuscular junctions in hind paws, showed a comparable staining area in control and low dose Y-27632 treated animals, but a significant larger area of reinnervated neuromuscular junctions in animals that received high dose Y-27632. These findings indicate that muscle (target) reinnervation was enhanced in high dose Y-27632-treated animals. Regeneration of sensory fibers was assessed by quantification of PGP 9.5 and CGRP stained intraepidermal nerve fiber density in the hind paws of Y-27632- and vehicle-treated mice. As shown in Figs. 8A and C, the number of PGP 9.5 stained fibers was not statistically significant between the three groups of animals (Figs. 8B, D). Likewise, we found that numbers of regenerated myelinated axons in sciatic and tibial nerves were also comparable in all three groups (Figs. 9A, B). It

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implicates that Y-27632 treatment mainly affects regeneration of motor fibers but not sensory myelinated, because more than 70% of myelinated nerve fibers in mouse sciatic nerve are sensory afferents (Duchen and Scaravilli, 1977). Morphological analysis further revealed that the systemic treatment with Y-27632 was associated with changes in myelin thickness (g-ratio), and internodal length in Y-27632 treated animals. Mean axon diameter (sciatic nerves: 3.75 ± 0.26 μm and 3.19 ± 0.54 μm in Y-27632-treated and 3.55 ± 0.82 μm in vehicle-treated; tibial nerves: 2.19 ± 0.29 μm and 2.37 ± 0.22 μm in Y-27632-treated; 2.13 ± 0.40 μm in vehicle-treated) and axon caliber distribution (histograms) did not show significant differences between untreated and Y-27632-treated animals (Figs. 10A–C). However, quantification of g-ratio, a parameter that indicates myelin sheath thickness in relation to the axon diameter, revealed a modest but significant increase in myelin sheath thickness at sciatic nerve level in high dose Y-27632-treated animals compared to controls and low-dose treated animals (Fig. 10D). Teased fiber preparations from sciatic nerves showed that mean length of regenerated myelinated internodes was modestly but significantly reduced in low and high dose Y27632-treated groups compared to controls (Figs. 10E, F). These changes in the myelin morphology did not affect nerve conduction properties, since nerve conduction studies showed no differences in distal motor latencies (12.09 ± 0.24 in Y-27632-treated and 12.13 ± 0.48 ms in vehicle-treated animals).

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The RhoA-ROCK signaling axis plays a crucial role in mediating axon growth inhibition of myelin-derived molecules to central nerve fibers. Embryonic and adult peripheral neurons also express RhoA (Bowerman et al., 2009, 2010; Kobayashi et al., 2004) and it has been demonstrated that injury to peripheral nerve fibers increases the expression of total RhoA and its activated GTP-bound form (Cheng et al.,

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injury in the PNS, Schwann cells express CSPGs that can rapidly aggregate to build a growth-inhibiting barrier for regenerating axons (Chen et al., 2005; Heine et al., 2004; Zuo et al., 1998, 2002). Outgrowth inhibition by CSPGs is mediated at least in part by activation of RhoA and local or systemic inhibition of the RhoA/ROCK signaling cascade has previously been shown to enhance axonal regeneration (Chen et al., 2005; Hiraga et al., 2006). Moreover in peripheral neurons, CSPGSs can activate local translation of RhoA in the growth cones and thereby increases the growth inhibitory effect. The inhibition of ROCK in neurons exposed to CSPGs results in a decreased expression of the upstream molecule RhoA in growth cones (Walker et al., 2012). We choose the model of femoral nerve crush to study the in vivo effects of Y-27632 on sensory and motor nerve regeneration separately. In line with the results from our in vitro data we observed that a single injection of high dose Y-27632 resulted in a higher numbers of regenerated axons in the motor branch of the femoral nerve as compared to the sensory branch and vehicle treated controls. In an experimental paradigm of peripheral nerve injury (sciatic nerve crush) the overall axon growth promoting effect was less distinct. We found that Y-27632 modestly enhanced axon regeneration of motor myelinated fibers as assessed by functional and morphological measures of target reinnervation. This effect was seen with the higher dose but not the lower dose of Y-27632. Dose dependent improvement in axonal sprouting has also been reported in spinal cord injury models (Chan et al., 2005). Whether the number of regenerating motor fibers was increased after this treatment is not addressed because of lack of reliable markers for motor and sensory myelinated axons in mixed nerves, but it clearly induced more robust reinnervation of neuromuscular junctions (NMJ) in treated animals compared to controls. A possible explanation for the reduced axonotrophic efficacy of Y27632 after sciatic crush compared to femoral nerve crush is that in the sciatic nerve axons have to regrow over much longer distances and may therefore be exposed to larger accumulations of CSPGs and other growth-inhibiting molecules. Even repeated injections of Y-27632 may therefore be insufficient to completely reverse the axon growth inhibitory effect of CSPGs and possible CSPGs induced local RhoA translation (Walker et al., 2012). Previously, fasudil, a ROCK inhibitor with similar properties as Y27632, has also been demonstrated to enhance axon regeneration in the PNS (Chen et al., 2005; Hiraga et al., 2006). Chen and colleagues infused fasudil in regenerative conduits connecting transected sciatic nerve segments in rats. This local exposure is likely to result in much higher concentrations compared to a systemic treatment in our study, which may explain differences in the extent of observed proregenerative effects. In the study by Hiraga and colleagues, fasudil was administered up to 8 weeks, and promoted axon regeneration as demonstrated by increased CMAP amplitudes and increased number of regenerating myelinated axons after this time period. We choose 17 days post-crush time point in our sciatic crush model for morphological analysis because in our experimental paradigm target reinnervation in the hind paw occurs at this time point and target reinnervation is considered the most meaningful measure of axon regeneration and recovery. Different time points of examination (17 days versus 3–7 weeks) and differences in methodologies may account for the discrepancy between morphological findings of the present study and the report by Hiraga and colleagues. Moreover, fasudil and Y-27632 may have different pharmacological properties that may influence tissue accessibility and target concentration, despite comparable Ki values of fasudil and Y-27632 (Shimokawa, 2002). Our observation that Y-27632 treatment alters myelin thickness and internodal length of regenerating fibers are in line with a previous study reporting that Y-27632 modulates myelin thickness and internodal length of myelinated Schwann cells in Schwann cell and

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2008; Hiraga et al., 2006). Here we provide evidence that peripheral motor and sensory neurons show substantial differences in their response to pharmacological modulation of this pathway. Our in vitro data indicate that in the growth inhibitory environment created by molecules CSPGs, sensory and motor neurons show no alteration in levels of RhoA activation. However, in motor neurons, ROCK inhibition results in a decreased expression and activation of the upstream molecule RhoA and hence lower levels of phosphorylated MLC compared to sensory. Therefore the modulation of the downstream effector ROCK by Y-27632, a chemical ROCK inhibitor, results in diverse effects on the axonal regeneration of the two fiber modalities in the presence of CSPGs. Consequently, in an animal model of peripheral nerve regeneration, sensory neurons were less responsive to systemic treatment with Y-27632 compared to motor neurons. Our results are in line with previous studies demonstrating dynamic changes in transcription and translation of neuronal RhoA in response to growth-inhibitory molecules (Conrad et al., 2005; Erschbamer et al., 2005; Walker et al., 2012) and provide further evidence for a feedback mechanism of ROCK to RhoA expression and activity (Ito et al., 2006; Kobayashi et al., 2002; Olson, 2004; Tang et al., 2012). CSPGs are major components of the glial scar that contribute to incomplete recovery after nerve fiber damage. As a result of nerve

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Fig. 9. Systemic treatment with Y-27632 does not significantly alter the total numbers of regenerated myelinated nerve fibers at the level of the sciatic nerve (A) or tibial nerve (B), n = 6 each group.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Fig. 10. Morphometric analysis of sciatic and tibial nerves. Axon diameter histograms of sciatic (A) and tibial (B) nerves of Y-27632- (dashed line) and vehicle-treated animals (solid line). (C) Representative light micrographs of Y-27632-treated and control animals. (Bar = 10 μm). (D) g-ratio (axon diameter/fiber diameter) is significantly decreased in sciatic nerves of high dose Y-27632-treated animals but not in those of low dose treated animals and controls (mean ± SEM). (E) Teased fiber preparations showing shorter internodes in Y-27632-treated (bottom panel) nerves compared to controls (top panel) (Bar = 50 μm). (F) Quantification of internodal lengths showing significantly shorter internodes in Y-27632-treated groups compared to controls. * b 0.05, n = 6 each group.

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DRG neuron co-cultures (Melendez-Vasquez et al., 2004). Our findings in animal studies are much less striking than this previous cell culture study likely due to differences in effective bioavailability of drug in animal and myelinating culture models. Nevertheless the altered myelin morphology in the mixed sciatic nerve argues against different accessibility and target concentration of Y-27632 as potential explanation for the different responses of motor and sensory axons to this treatment. Overall, our studies indicate that ROCK signaling in axons and Schwann cells can be modulated by systemic administration of a

chemical inhibitor with effects on axon regeneration and remyelination. However ROCK inhibition by Y-27632 has divergent effects on regeneration of sensory and motor neurons in a nongrowth-permissive environment. It is less effective in sensory as compared to motor neurons, because of differences between the two fiber modalities in terms of expression and activation of RhoA. Our study implicates that neuropathic conditions with predominant or exclusive motor fiber damage may represent the most eligible targets for future assessment of non-cell autonomous delivery of inhibitors, such as Y-27632 to enhance axon regeneration.

Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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Abhijeet Joshi was supported by the Deutscher Akademischer Austausch Dienst (DAAD). Drs. Sheikh and Zhang are supported by grants from the National Institutes of Health (NS42888 and NS054962). The technical assistance of Claudia Drapatz is greatly acknowledged.

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Please cite this article as: Joshi, A.R., et al., Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.09.012

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