Involvement of c-jun N-terminal kinase in neurite extension of cultured DRG neurons *Nguyen Huu Tu, Tayo Katano, Shinji Matsumura, Vuong Minh Pham and Seiji Ito Department of Medical Chemistry, Kansai Medical University, Hirakata Osaka, 573-1010, Japan 291.15 Abstract 1. Peripheral nerve regeneration after sciatic nerve axotomy 4. Co-relation between plantar nerve reinnervation and functional recovery 7. Prolongation of the nerve injury marker ATF3 in DRG by JNK inhibitor Weeks after axotomy ATF3/ Nissl (%) * ** 20 40 60 80 100 naive 3d 1 2 3 4 5 6 7 8 Control JNK inh. B Control 3d JNK inh. 3d ATF3 Nissl A Peripheral neurons are able to regenerate after axonal injuries by multiple mechanisms. To understand these mechanisms sheds lights on better nerve healing after injuries both peripherally and centrally. Among them, mitogen-activated protein kinases (MAPKs) have proved effectiveness to nerve regeneration at an early phase after injuries, but the effect of MAPKs at a late phase has not been studied. To answer this question, we have created a transection-regeneration model (see Methods; Unezaki et al. J. Neruosci. Methods 178:308-315, 2009) and examined the effect of MAPK inhibitors on nerve regeneration and functional recovery over 8 weeks. We found out that a JNK inhibitor but not an ERK or p38 inhibitor delayed functional recovery compared to the control for 8 weeks after axotomy. The delay in functional recovery was related to a significantly higher expression of ATF3, a nerve injury signal. We also re-confirmed the effect of JNK inhibitor on neurite outhgrowth by neurotrophic factors including NGF, GDNF and BDNF in vitro: Neurotrophic factors enhanced nerve regeneration, but JNK inhibitor suppressed this effect of neurotrophins on cultured DRG cells. Collectively, we found, for the first time, that JNK is involed in nerve regeneration at the late phase after axotomy and that neurotrophic factors may accelerate peripheral nerve regeneration through the JNK pathway. Motor Neuron Survival Motor Neuron Regeneration Sensory Neuron Regeneration Sensory Neuron Survival (A) Evaluation of nerve regeneration by F-ruby. F-ruby was put close to the proximal (P) and distal (D) stumps of transected sciatic nerve to study peripheral nerve survival and regeneration after axotomy. (B) Quantitation of motor and sensory neurons survival and regeneration at indicated time points. P D silicone tube F- ruby, a neuro tracer A B Number of neurons Weeks after axotomy 500 1000 1500 2000 2500 1 2 3 4 8 * ** NS # 2 w naive A 4 w 8 w Weeks after axotomy Nerve reinnervation (%) B 20 40 60 80 100 120 1 2 3 4 5 6 7 8 C Withdrawal threshold (g) 1 2 3 4 5 6 7 8 Weeks after axotomy 2. Sensory neuron reinnervation to skin after sciatic nerve axotomy naive 6w after axotomy YFP e d Sensory fibers Plantar Nerves A B C D (A) Proper plantar digital nerves in naive and 2, 4 and 8 weeks after axotomy in thy1-YFP mice. The number of nerves in naive mice is 10 and set for 100%. Arrows indicate regenerated nerves. (B) Time course of reinnervation of proper plantar digital nerve in the plantar after axotomy. (C) Time course of functional recovery in thy1-YFP mice. Withdrawal responses started to resume 5 weeks after axotomy. Withdrawal thresholds reached the level similar to that before axotomy at 8 weeks after operation. Reinnervation to the plantar and fingers is well correlated with behavioral response to mechanical stimulation. (A) Immunostaining of ATF3 in L5 DRG. ATF3 (red) was doubly stained with Nissl (green), a neuronal marker. (B) Quantitation of ATF3 in L5 DRG. The expression of ATF3 was significantly higher in JNK inhibitor-treated group than control group over 8 weeks after axotomy. Asterisks denote statistically significant difference between JNK inhibitor-treated and control groups. n = 3 by Student t-test. * p < 0.05, ** p < 0.01. Scale bar 100 μm Introduction JNK, a member of MAPKs, is activated at the injured site of peripheral nerves, resulting in changing regeneration signals in cell body. Whether inhibiton of JNK locally right after injury affects the late functional recovery or not has not been elucidated. To clarify this issue, we have established transection-regeneration model in mice and used DRG culture system and showed that a JNK inhibitor, but not other MAPKs kinese inhibitors, delayed functional recovery to 8 weeks after axotomy. Anti-NGF, anti-GDNF, anti-BDNF decreased functional recovery in vivo. And NGF, GDNF and BDNF significantly increased neurite outgrowth in vitro. However, JNK inhibitor suppressed the effect of neurotrophic factors on neurite outgrowth. These results indicated that JNK has regenerative effect the nerve at late phase and there is a relation between JNK and neurotrophics pathways to accelerating peripheral nerve regeneration. Sensory fiber length (μm / 100μm2 skin) naive 6 weeks 0.1 0.2 0.3 0.4 E 8. Neurotrophic factors accelerate nerve regeneration though JNK pathway (A) Effect of NGF, GDNF, and BDNF on neurite outgrowth in cultured DRG neurons. DRG neurons were cultured for 24h with or without 10 ng/mL of NGF, GDNF or BDNF. If the length of neurites was longer than 2 cell-diameters, the neurons were defined as neuron with neurites. (B) Effect of the JNK inhibitor on neurite outgrowth by NGF, GDNF or BDNF. DRG neurons were incubated for 24 h with 10 ng/mL of NGF, GDNF or BDNF in the presence of 10 μM SP600125; and neurons bearing neurites were counted as described above. The data (the mean ± SD, n = 3 - 4) are expressed as the percentage of total DRG neurons (400 to 600 per each group). ** p < 0.01 vs. the control, by two-way ANOVA test. Scale bar 100 μm. JNK inh Vehicle NGF with neurites (%) Neurons 10 20 30 40 50 10ng ** NeuN/ PGP 9.5 None A B GDNF BDNF JNK.inh (μM) NGF (ng) JNK inh. (μM) GDNF (ng) BDNF (ng) 5. Delay of functional recovery by JNK inhibitor Effect of JNK inhibitor SP 600125 (10 μM), ERK inhibitor (10 μM) or p38 inhibitor (1 μM) on functional recovery from sciatic nerve axotomy by measuring paw-withdrawal thresholds to von Frey filament hairs before and every week after axotomy. JNK inhibitor significantly delayed withdrawal response to mechanical stimuli by 1 week as compared to the control (PBS (-) group), but neither p38 nor ERK inhibitor delayed it, suggesting that JNK is involved in functional recovery from peripheral nerve injury. Data show the mean ± SD (n =6-9). * p < 0.05 vs PBS (-) group. * Withdrawal threshold (g) Weeks after axotomy 1 2 3 4 5 6 7 8 p38 inh ERK inh PBS (-) JNK inh (A) Plantar nerves branches of the naive mice under fluorescent microscope and the area of skin for sensory fiber study (boxed area). (B) Sensory fibers are YFP (+) (green) in skin of naive mice. (C) Plantar Digital Nerves regeneration to the plantar 6 weeks after sciatic nerve cut and the area of investigated skin (boxed). (D) Sensory fiber regeneration in (C) in epidermis (e) and dermis (d) 6 weeks after axotomy. (E) Quantitation of sensory fiber regeneration length (μm/ 100μm2) in naive and 6 weeks after sciatic nerve cut. n = 3, data shows means ± SD. Scale bar 50 μm. Methods: sciatic nerve transection-regeneration model (A) Proximal (P) and distal (D) transected stumps were sutured to the inner wall of a silicone tube. Drugs were continuously delivered into the silicone tube from an osmotic pump for 4 weeks. (B) Sciatic nerve regenerated though the silicone tube (left). Fluorescent positive nerve branches regenerated though a silicone tube, branched and re-innervated to target organs 4 weeks after axotomy (right). A 5-mm gap osmotic pump catheter silicone tube P D Deep tibial N. Medial Sural Cutaneous N. Common peroneal N. Sciatic N. 4 weeks after sciatic nerve axotomy B 3. Motor neuron reinnervation after sciatic nerve axotomy naive A Merged AchR B Reinervation endplates (%) 20 40 60 80 100 naive 6 weeks C 6. Up-regulation of p-JNK in sciatic nerve after axotomy 6w after axotomy YFP NF-200 6h axotomy Naive p-JNK A Merge Intensity of p - JNK 100 200 naive 6h axotomy ** B Neuromuscular junction in thy1-YFP mice Innervation (A) and reinnervation (B) of motor neurons (green) to the endplates (red) on Gastrocnemius muscle 6 weeks after axotomy. (A) Intact motor neuron innervated the muscle and formed the neuromuscular junction at endplates on the muscle of naive mice. Acetylcholine receptors on the endplates were stained with α-bungarotoxin conjugated with Alexa 555 (red) (B) Motor neuron reinnervated to the muscle but partially formed the neuromuscular junction in the ipsilateral muscle 6 weeks after axotomy.(C) Quantitation of % of enplates re-innervated by motor neurons 6 weeks after axotomy and naive. n = 3, means ± SD. . Conclusions JNK inhibitor delayed functional recovery at late phase after injuries, and the acceleration on peripheral nerve regeneration of neurotrophic factors is mediated by the JNK pathway. (A) p-JNK were not seen in naive mice, but up-regulated significantly 6h after axotomy at the proximal site of the sciatic nerve cut. (B) pJNK signal quantitation at the proximal site of the sciatic nerve cut in naive and 6h after axotomy. n = 3, means ± SD, ** p < 0.05, two-tailed Student’s t-test. Scale bar 20 μm