Energy coupling of axon-Schwann cells by Nax The 44th NIPS International Symposium, The 5th Asian Pain Symposium December 18-20,2013 ◆ Okazaki Conference Center Energy coupling of axon-Schwann cells by Nax and endothelin in nerve regeneration Nguyen H.Tu1, Tayo Katano1, Takeshi H Hiyama2,3, Masaharu Noda2,3 and Seiji Ito1 1Department of Medical Chemistry, Kansai Medical University, Hirakata Osaka, 573-1010, Japan 2Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki 444-8787, Japan 3School of Life Science, The Graduate University for Advanced Studies, Okazaki 444-8787, Japan　 Abstract Fig.4 Effects of Nax and lactate on the functional recovery from peripheral nerve transection. Wild-type Saline CIN Nax-/- Lactate Lactate+CIN Nax, a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of adult peripheral nervous system, but the pathophysiological role remains unclear. After a nerve transection, Schwann cells demyelinate, proliferate, and dedifferentiate in the injury site, and supply multiple factors for promoting axon regeneration and remyelination. In this study, we examined the role of Nax in peripheral nerve regeneration after the sciatic nerve transection in wild-type and Nax-knockout (Nax-/-) mice by using our tubing and osmotic pump model. The Nax mRNA level of proximal end of the sciatic nerve at the transection was down-regulated 1 week after the operation and recovered for 4 weeks in a time-dependent manner. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Nax-/- mice. Histological analyses showed a decrease in the number of regenerated myelinated axons in Nax-/- sciatic nerves. The delay in the recovery in Nax-/- mice was improved by lactate and inhibited by a monocarboxylate transporter inhibitor. In vitro experiments using cultured Schwann cells showed that lactate release was enhanced by endothelin (ET)-1 and blocked by an ET receptor type B antagonist. The amount of lactate release by ET-1 was lower in Nax-/- mice than that from wild-type mice. The present study shows, for the first time, that Nax is functionally coupled to ET for lactate release from non-myelinating Schwann cells via ET receptor type B and involved in the regeneration process of the injured nerve. The functional recovery from sciatic nerve transection in wild-type and Nax-/- mice administered with saline, 2 mM lactate or/and 5 mM CIN. The recovery was assessed every week after the operation by paw-withdrawal thresholds to von Frey filament stimuli applied to the plantar. The mechanical force of 3.63 g was used as a cut-off value and indicated by a dotted line. Data show the mean ± SD (n=5-6). *P < 0.05 vs wild-saline group, #P < 0.05 vs Nax-/--saline group, and +P < 0.05 vs Nax-/--lactate group by Mann-Whitney's U test. Withdrawal thresholds (g) Weeks after operation 4 3 2 1 Pre 5 6 7 8 * + # Fig.5 Sensory neurons regenerated after sciatic nerve transection in Wild-type mice Silicone tube F-Ruby 10% for 30 minutes Pain 87:149-158, 2000 B F-ruby Naïve 2 w 4 w 8 w A Introduction Nax is localized at astrocytes and ependymal cells in the circumventricular organs (CVOs) in the central nervous system, and the dorsal root ganglion (DRG) neurons and non-myelinating Schwann cells in the peripheral nervous system. In the subfornical organ (SFO) in the CVOs, Nax functions as a sodium-level sensor of the body fluid. The threshold level in vitro was revealed to be ~150 mM for the extracellular [Na+] by using SFO cells and DRG neurons. In the SFO, Na+ influx via Nax leads to an increase of glucose uptake and activation of anaerobic glucose metabolism of Nax-positive glial cells. Subsequent enhancement of lactate release from the cells stimulates activity of GABAergic neurons in the SFO, which probably control salt intake. In contrast to the CNS, a function of Nax in the peripheral nervous system, especially in non-myelinating Schwann cells, remains to be elucidated. In this study, we examined the peripheral nerve regeneration after the sciatic nerve transection in wild-type and Nax-/- mice by using our tubing and osmotic pump model (Fig. 1; Unezaki et al. J. Neurosci. Methods, 178:308-315, 2009). Nerves Regenerative Evaluation Model: F-ruby (Dextran, Tetramethylrhodamine, 10,000 MW, Lysine Flexible, Molecular Probes) 10%, 5 μL were applied for 30 minutes on distal site of the silicone tube to label regenerative neurons. Sensory neurons in L5 DRG of Naïve, 2 weeks, 4 weeks and 8 weeks after sciatic nerve transection, labeled by F-ruby. Fig.6 Effects of Nax and lactate on the functional recovery from peripheral nerve transection. (A) Paw withdrawal threshold for the selective current stimuli applied to the plantar. The pulses at 2000, 250, and 5 Hz stimulate Ab-, Ad-, and C-fibers, respectively. Data show the ratio of the threshold value of operated side to that of contralateral side, by the mean ± SD. (n= 5- 6). (B) The cutaneous nerves in the contralateral and ipsilateral sides were immunostained with anti-NF-200 antibody 13-14 weeks after the operation. The area of NF200-positive fiber in the dermis was quantified. Data show the mean ± SD (n=2 for contralateral, n=4 for ipsilateral). A B 0.5 1 1.5 2 2000 250 5 Relative withdrawal thresholds Wild-Saline * Current stimulus (Hz) Nax-/--Saline Nax-/--Lactate 0.5 1 1.5 2 NF200-IR (+) area / 100mm2 * Wild-Contra Nax-/--Contra Wild-Saline Nax-/--Saline Nax-/--Lactate Methods 1. Nerve regeneration model The model was made as described previously by Unezaki et al.(2009). Figure Diagram of sciatic nerve transection-regeneration model. Transected proximal (p) and distal (d) nerve stumps were sutured to a silicone tube. A drug was continuously delivered into the silicone tube from an osmotic pump for 4 weeks. 5mm gap osmotic pump catheter silicone tube P D 2. Schwann cell culture Murine primary Schwann cells were isolated from sciatic nerves of 3–5-day-old mice, cultured and purified with polyclonal rabbit anti-nerve growth factor receptor p75 using a modified method based on that described previously (Jin et al., 2008). Purity of Schwann cells was over 95% as evaluated by the mean percentage of S100-positive cells with respect to the total number of cells counted. Fig.7 Morphometric analyses of regenerated sciatic nerves 8 weeks after the operation. (A) Quantification of total (TA), myelinated (MA), and unmyelinated (UMA) axon numbers in regenerated nerves. Data show the mean ± SD (n = 5). (B) The distribution of Remak bundles in regenerated nerves, which were categorized into five groups based upon the number of ensheathed axons. (C and D) Histograms showing the distribution of myelinated (C) and unmyelinated (D) axon diameters in regenerated nerves. Data show axon numbers by the mean ± SD (n = 5). A B D 1 2-5 6-9 10-17 >=18 Axons in Remak bundle 20 40 60 % of total bundles 2000 4000 6000 TA MA UMA Total axon numbers * Myelinated axons 200 400 600 ~0.5 ~1 ~1.5 ~2 >8 Axon numbers ~2.5 ~3 ~3.5 ~4 ~4.5 ~5 ~5.5 ~6 ~6.5 ~7 ~7.5 ~8 ~0.1 ~0.2 ~0.3 ~0.4 >1.5 Axon diameter (mm) ~0.6 ~0.7 ~0.8 ~0.9 ~1.0 ~1.1 ~1.2 ~1.3 ~1.4 Unmyelinated axons C Wild Nax-/- Fig.1 Nax expression in non-myelinating Schwann cells in peripheral nerves. C B A Peripherin / Nax / YFP E D G H I F K J Nax+/+ S100 / Nax / YFP Nax-/- Nax / GFAP GFAP Nax Nax / YFP / DIC Wild Transverse sections of the normal sciatic nerve (A–I) and plantar (J and K). (A-C) Expression of peripherin or S100 (red) and Nax (blue) with YFP-labeled axons (green) in in Nax+/+ (A and B) and Nax-/- (C) nerves. The arrows indicate the S100 expression in myelinating Schwann cells. (D–I) Expression of Nax (red in D, F, G and I) and glial fibrillary acidic protein (GFAP; green in E, F, H and I) in wild-type (D–F) and Nax-/- (G–I) nerves. The arrows in “D–F” indicate the coexpression of Nax and GFAP. (J and K) Expression of Nax (red) with YFP-labeled axons (green) in Nax+/+ (J) and Nax-/- (K) plantar footpads. The images are shown overlaid with the differential interference contrast image. The arrows indicate the Nax expression in epidermis. Scale bars, 20 (B and C), 50 (D–I), and 100 (A, J, and K) mm. Fig.8 Effects of ET on Schwann cells. (A) RT-PCR of the expression of Nax, MTC1, MTC4, and ETBR in 9-day cultured Schwann cells without and with the treatment of 10 mM forskolin (FK) for 24 and 48 h. Elongation factor 1a (EF) was amplified as a control. Elongation factor 1α (EF) was amplified as a control. (B–D) Immunostaining for anti-Nax (red) with nuclear counterstaining (blue) of wild-type (B and C) and Nax-/- (D) cells on day 5 in culture with forskolin for 48 h. The arrows indicate Nax-expressed cells. Scale bars, 20 (B and D) and 10 (C) µm. (E) Lactate release. Cells were incubated without or with 160 mM Na+, 100 nM ET-1, ET-1 plus 100 mM BQ123 (ET-1+BQ123) or 100 mM BQ788 (ET-1+BQ788) for 2 h. The fold change from no treatment (None) was calculated, and the data show the mean ± SD (n=3-5 preparations). (F) Intracellular Na+ concentration. To evaluate the Na+ influx in Schwann cells, SBFI, a fluorescent sodium indicator, was loaded into cells, and the change of fluorescence ratio (F340 / 380) was recorded over 25 min. The increasing ratio (F340 / F380) by the treatment was calculated. Data show the mean ± SD (n=267-475). A FK (h) 24 48 Wild Nax-/- Nax MCT1 EF MCT4 ETBR C B D Wild Nax-/- Fig.2 No obvious abnormality was detected in the sciatic nerve of Nax-/- mice. (A–D) Electron micrographs of the transverse section of sciatic nerve of wild-type (A and B) and Nax-/- (C and D) mice. Scale bars, 10 (A and C) and 2 (B and D) mm. (E) Morphometric analysis of the wild-type and Nax-/- sciatic nerves. Data show the mean ± SD (n =3). Wild Nax-/- A B C D E E F 1 2 3 4 None Na+160mM ET-1 ET-1+BQ123 Lactate release (fold change) Wild Nax-/- * # ET-1+BQ788 0.9 1.0 1.1 1.2 Na+ uptake (relative fluorescent ratio) * Na+170mM ET-1 Fig.3 Expression of Nax in sciatic nerve at the transection site after operation. D Normalized Fold Expression Time (weeks) Proximal Distal 0.5 1 1.5 2 3 4 Nax / YFP A B C Contra Proximal Distal Conclusion Nax, a sodium concentration-sensitive sodium channel, is expressed in non-myelinating Schwann cells of adult peripheral nervous system. We found that functional recovery of the hind paw responses from the sciatic nerve transection was delayed in Nax-/- mice. The present study demonstrates that Nax is functionally coupled to endothelin for lactate release via endothelin receptor type B and involved in peripheral nerve regeneration. (A–C) Expression of Nax (red) with YFP-labeled axons (green) in the transverse section of contralateral nerve (A) or proximal (B) and distal (C) nerve end 2 mm from the transection site at 2 weeks after the operation. Scale bars, 100 mm. (D) Expression level of Nax mRNA in proximal and distal ends at the transection site. The Ct value of proximal end before the operation is taken as 1 and other Ct values are expressed as fold expression. Data are expressed as the mean ± SEM (n = 3 for before; n = 4 for after operation). Kansai Medical University Department of Medical Chemistry