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Cleavage of Na+ channels by calpain increases persistent Na+ current and promotes spasticity after spinal cord injury

Nature Medicine volume 22, pages 404–411 (2016)Cite this article

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Abstract

Upregulation of the persistent sodium current (INaP) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate INaP and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of INaP in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of INaP. The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, INaP in motoneurons and spasticity in rats with SCI. Similarly, the blockade of INaP by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of INaP and spasticity.

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Figure 1: Upregulation of motoneuron AIS Nav α-subunits after SCI.
Figure 2: Cleavage of Nav1.6 channels by calpain.
Figure 3: Calpain inhibition prevents the cleavage of Nav channels and the INaP increase after SCI.
Figure 4: Riluzole decreases spasms in rats with SCI.
Figure 5: Calpain inhibition restores Nav expression and decreases spasms in rats with SCI.
Figure 6: Calpain inhibition has a long-lasting effect on the restoration of Nav expression and the reduction of spasms after SCI.

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References

  1. Biering-Sørensen, F., Nielsen, J.B. & Klinge, K. Spasticity-assessment: a review. Spinal Cord 44, 708–722 (2006).

    PubMed  Google Scholar 

  2. Boulenguez, P. & Vinay, L. Strategies to restore motor functions after spinal cord injury. Curr. Opin. Neurobiol. 19, 587–600 (2009).

    CAS  PubMed  Google Scholar 

  3. Boulenguez, P. et al. Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury. Nat. Med. 16, 302–307 (2010).

    CAS  PubMed  Google Scholar 

  4. Harvey, P.J., Li, X., Li, Y. & Bennett, D.J. 5-HT2 receptor activation facilitates a persistent sodium current and repetitive firing in spinal motoneurons of rats with and without chronic spinal cord injury. J. Neurophysiol. 96, 1158–1170 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Li, X., Murray, K., Harvey, P.J., Ballou, E.W. & Bennett, D.J. Serotonin facilitates a persistent calcium current in motoneurons of rats with and without chronic spinal cord injury. J. Neurophysiol. 97, 1236–1246 (2007).

    CAS  PubMed  Google Scholar 

  6. Heckman, C.J., Gorassini, M.A. & Bennett, D.J. Persistent inward currents in motoneuron dendrites: implications for motor output. Muscle Nerve 31, 135–156 (2005).

    CAS  Google Scholar 

  7. Hounsgaard, J., Hultborn, H., Jespersen, B. & Kiehn, O. Bistability of α-motoneurones in the decerebrate cat and in the acute spinal cat after intravenous 5-hydroxytryptophan. J. Physiol. (Lond.) 405, 345–367 (1988).

    PubMed Central  Google Scholar 

  8. Li, Y., Gorassini, M.A. & Bennett, D.J. Role of persistent sodium and calcium currents in motoneuron firing and spasticity in chronic spinal rats. J. Neurophysiol. 91, 767–783 (2004).

    CAS  PubMed  Google Scholar 

  9. Gorassini, M.A., Knash, M.E., Harvey, P.J., Bennett, D.J. & Yang, J.F. Role of motoneurons in the generation of muscle spasms after spinal cord injury. Brain 127, 2247–2258 (2004).

    PubMed  Google Scholar 

  10. Murray, K.C. et al. Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors. Nat. Med. 16, 694–700 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Tazerart, S., Vinay, L. & Brocard, F. The persistent sodium current generates pacemaker activities in the central pattern generator for locomotion and regulates the locomotor rhythm. J. Neurosci. 28, 8577–8589 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Brocard, F. et al. Activity-dependent changes in extracellular Ca2+ and K+ reveal pacemakers in the spinal locomotor-related network. Neuron 77, 1047–1054 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Brocard, F., Tazerart, S. & Vinay, L. Do pacemakers drive the central pattern generator for locomotion in mammals? Neuroscientist 16, 139–155 (2010).

    CAS  PubMed  Google Scholar 

  14. Tazerart, S., Viemari, J.C., Darbon, P., Vinay, L. & Brocard, F. Contribution of persistent sodium current to locomotor pattern generation in neonatal rats. J. Neurophysiol. 98, 613–628 (2007).

    CAS  PubMed  Google Scholar 

  15. Bouhadfane, M., Tazerart, S., Moqrich, A., Vinay, L. & Brocard, F. Sodium-mediated plateau potentials in lumbar motoneurons of neonatal rats. J. Neurosci. 33, 15626–15641 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Fukuoka, T., Kobayashi, K. & Noguchi, K. Laminae-specific distribution of alpha-subunits of voltage-gated sodium channels in the adult rat spinal cord. Neuroscience 169, 994–1006 (2010).

    CAS  PubMed  Google Scholar 

  17. Duflocq, A., Le Bras, B., Bullier, E., Couraud, F. & Davenne, M. Nav1.1 is predominantly expressed in nodes of Ranvier and axon initial segments. Mol. Cell. Neurosci. 39, 180–192 (2008).

    CAS  PubMed  Google Scholar 

  18. Croall, D.E. & Ersfeld, K. The calpains: modular designs and functional diversity. Genome Biol. 8, 218 (2007).

    PubMed  PubMed Central  Google Scholar 

  19. von Reyn, C.R. et al. Calpain mediates proteolysis of the voltage-gated sodium channel α-subunit. J. Neurosci. 29, 10350–10356 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kunz, S. et al. The calpain inhibitor MDL 28170 prevents inflammation-induced neurofilament light chain breakdown in the spinal cord and reduces thermal hyperalgesia. Pain 110, 409–418 (2004).

    CAS  PubMed  Google Scholar 

  21. Arataki, S. et al. Calpain inhibitors prevent neuronal cell death and ameliorate motor disturbances after compression-induced spinal cord injury in rats. J. Neurotrauma 22, 398–406 (2005).

    PubMed  Google Scholar 

  22. Markgraf, C.G. et al. Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats. Stroke 29, 152–158 (1998).

    CAS  PubMed  Google Scholar 

  23. Harvey, P.J., Li, X., Li, Y. & Bennett, D.J. Endogenous monoamine receptor activation is essential for enabling persistent sodium currents and repetitive firing in rat spinal motoneurons. J. Neurophysiol. 96, 1171–1186 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Harvey, P.J., Li, Y., Li, X. & Bennett, D.J. Persistent sodium currents and repetitive firing in motoneurons of the sacrocaudal spinal cord of adult rats. J. Neurophysiol. 96, 1141–1157 (2006).

    CAS  PubMed  Google Scholar 

  25. Li, Y. & Bennett, D.J. Persistent sodium and calcium currents cause plateau potentials in motoneurons of chronic spinal rats. J. Neurophysiol. 90, 857–869 (2003).

    CAS  PubMed  Google Scholar 

  26. Urbani, A. & Belluzzi, O. Riluzole inhibits the persistent sodium current in mammalian CNS neurons. Eur. J. Neurosci. 12, 3567–3574 (2000).

    CAS  PubMed  Google Scholar 

  27. Svenningsen, P. et al. Physiological regulation of epithelial sodium channel by proteolysis. Curr. Opin. Nephrol. Hypertens. 20, 529–533 (2011).

    CAS  PubMed  Google Scholar 

  28. Vallet, V., Chraibi, A., Gaeggeler, H.P., Horisberger, J.D. & Rossier, B.C. An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature 389, 607–610 (1997).

    CAS  PubMed  Google Scholar 

  29. Armstrong, C.M., Bezanilla, F. & Rojas, E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J. Gen. Physiol. 62, 375–391 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Undrovinas, A., Maltsev, V.A. & Sabbah, H.N. Calpain inhibition reduces amplitude and accelerates decay of the late sodium current in ventricular myocytes from dogs with chronic heart failure. PLoS One 8, e54436 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Murray, K.C., Stephens, M.J., Ballou, E.W., Heckman, C.J. & Bennett, D.J. Motoneuron excitability and muscle spasms are regulated by 5-HT2B and 5-HT2C receptor activity. J. Neurophysiol. 105, 731–748 (2011).

    PubMed  Google Scholar 

  32. Palecek, J., Lips, M.B. & Keller, B.U. Calcium dynamics and buffering in motoneurones of the mouse spinal cord. J. Physiol. (Lond.) 520, 485–502 (1999).

    CAS  Google Scholar 

  33. Kuo, J.J., Lee, R.H., Zhang, L. & Heckman, C.J. Essential role of the persistent sodium current in spike initiation during slowly rising inputs in mouse spinal neurones. J. Physiol. (Lond.) 574, 819–834 (2006).

    CAS  Google Scholar 

  34. Schoch, K.M. et al. Brain injury-induced proteolysis is reduced in a novel calpastatin-overexpressing transgenic mouse. J. Neurochem. 125, 909–920 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Hunanyan, A.S. et al. Alterations of action potentials and the localization of Nav1.6 sodium channels in spared axons after hemisection injury of the spinal cord in adult rats. J. Neurophysiol. 105, 1033–1044 (2011).

    PubMed  Google Scholar 

  36. Hains, B.C. & Waxman, S.G. Sodium channel expression and the molecular pathophysiology of pain after SCI. Prog. Brain Res. 161, 195–203 (2007).

    CAS  PubMed  Google Scholar 

  37. Craner, M.J. et al. Sodium channels contribute to microglia/macrophage activation and function in EAE and MS. Glia 49, 220–229 (2005).

    PubMed  Google Scholar 

  38. Zhou, H.Y. et al. N-methyl-D-aspartate receptor- and calpain-mediated proteolytic cleavage of K+-Cl− cotransporter-2 impairs spinal chloride homeostasis in neuropathic pain. J. Biol. Chem. 287, 33853–33864 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Banik, N.L., Matzelle, D.C., Gantt-Wilford, G., Osborne, A. & Hogan, E.L. Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury. Brain Res. 752, 301–306 (1997).

    CAS  PubMed  Google Scholar 

  40. Springer, J.E., Azbill, R.D., Kennedy, S.E., George, J. & Geddes, J.W. Rapid calpain I activation and cytoskeletal protein degradation following traumatic spinal cord injury: attenuation with riluzole pretreatment. J. Neurochem. 69, 1592–1600 (1997).

    CAS  PubMed  Google Scholar 

  41. Schumacher, P.A., Eubanks, J.H. & Fehlings, M.G. Increased calpain I–mediated proteolysis, and preferential loss of dephosphorylated NF200, following traumatic spinal cord injury. Neuroscience 91, 733–744 (1999).

    CAS  PubMed  Google Scholar 

  42. Ray, S.K. et al. Calpain activity and translational expression increased in spinal cord injury. Brain Res. 816, 375–380 (1999).

    CAS  PubMed  Google Scholar 

  43. Li, Z., Hogan, E.L. & Banik, N.L. Role of calpain in spinal cord injury: increased calpain immunoreactivity in rat spinal cord after impact trauma. Neurochem. Res. 21, 441–448 (1996).

    CAS  PubMed  Google Scholar 

  44. Yu, C.G. et al. Calpain 1 knockdown improves tissue sparing and functional outcomes after spinal cord injury in rats. J. Neurotrauma 30, 427–433 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. Du, S. et al. Calcium influx and activation of calpain I mediate acute reactive gliosis in injured spinal cord. Exp. Neurol. 157, 96–105 (1999).

    CAS  PubMed  Google Scholar 

  46. Shields, D.C., Schaecher, K.E., Hogan, E.L. & Banik, N.L. Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion. J. Neurosci. Res. 61, 146–150 (2000).

    CAS  PubMed  Google Scholar 

  47. Wienecke, J., Westerdahl, A.C., Hultborn, H., Kiehn, O. & Ryge, J. Global gene expression analysis of rodent motor neurons following spinal cord injury associates molecular mechanisms with development of postinjury spasticity. J. Neurophysiol. 103, 761–778 (2010).

    CAS  PubMed  Google Scholar 

  48. Cifra, A., Mazzone, G.L. & Nistri, A. Riluzole: what it does to spinal and brainstem neurons and how it does it. Neuroscientist 19, 137–144 (2013).

    PubMed  Google Scholar 

  49. Theiss, R.D., Hornby, T.G., Rymer, W.Z. & Schmit, B.D. Riluzole decreases flexion withdrawal reflex but not voluntary ankle torque in human chronic spinal cord injury. J. Neurophysiol. 105, 2781–2790 (2011).

    CAS  PubMed  Google Scholar 

  50. Zijdewind, I. & Thomas, C.K. Firing patterns of spontaneously active motor units in spinal cord–injured subjects. J. Physiol. (Lond.) 590, 1683–1697 (2012).

    CAS  Google Scholar 

  51. Kitzman, P.H. Effectiveness of riluzole in suppressing spasticity in the spinal cord injured rat. Neurosci. Lett. 455, 150–153 (2009).

    CAS  PubMed  Google Scholar 

  52. Lampert, A., Hains, B.C. & Waxman, S.G. Upregulation of persistent and ramp sodium current in dorsal horn neurons after spinal cord injury. Exp. Brain Res. 174, 660–666 (2006).

    CAS  PubMed  Google Scholar 

  53. Hama, A. & Sagen, J. Antinociceptive effect of riluzole in rats with neuropathic spinal cord injury pain. J. Neurotrauma 28, 127–134 (2011).

    PubMed  Google Scholar 

  54. Grossman, R.G. et al. A prospective, multicenter, phase I matched-comparison group trial of safety, pharmacokinetics, and preliminary efficacy of riluzole in patients with traumatic spinal cord injury. J. Neurotrauma 31, 239–255 (2014).

    PubMed  PubMed Central  Google Scholar 

  55. Ray, S.K. & Banik, N.L. Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeutic potential of calpain inhibitors for prevention of neurodegeneration. Curr. Drug Targets CNS Neurol. Disord. 2, 173–189 (2003).

    CAS  PubMed  Google Scholar 

  56. Yu, C.G., Joshi, A. & Geddes, J.W. Intraspinal MDL28170 microinjection improves functional and pathological outcome following spinal cord injury. J. Neurotrauma 25, 833–840 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Wang, K.K. Developing selective inhibitors of calpain. Trends Pharmacol. Sci. 11, 139–142 (1990).

    CAS  PubMed  Google Scholar 

  58. Mehdi, S. Cell-penetrating inhibitors of calpain. Trends Biochem. Sci. 16, 150–153 (1991).

    CAS  PubMed  Google Scholar 

  59. Goll, D.E., Thompson, V.F., Li, H., Wei, W. & Cong, J. The calpain system. Physiol. Rev. 83, 731–801 (2003).

    CAS  PubMed  Google Scholar 

  60. Wu, Y. et al. Delayed post-injury administration of riluzole is neuroprotective in a preclinical rodent model of cervical spinal cord injury. J. Neurotrauma 30, 441–452 (2013).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We dedicate this article to the memory of our dear colleague and friend Laurent Vinay, who passed away on 26 March 2015. Vinay was absolutely dedicated to science and, specifically, to the field of spinal cord research. Everyone who interacted with him was marked by his kindness, humanity and devotion. Vinay will leave a great void behind. This study was supported by the International Spinal Research Trust (to L.V.; STR110), the Fondation pour la Recherche Médicale (to L.V. and F.B.; DEQ20130326540) and the French Institut pour la Recherche sur la Moelle épinière et l'Encéphale (to F.B.). P.B. is supported by the French National Institute of Health and Medical Research (INSERM). We thank the company NSrepair for their help in surgery and postoperative care. We thank A.J. Powell and M. Bird for providing us with the HEK293 cell line stably expressing Nav1.6 (GlaxoSmithKline, Stevenage, UK).

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  1. Cécile Brocard, Vanessa Plantier and Pascale Boulenguez: These authors contributed equally to this work.

Authors and Affiliations

  1. Team P3M, Institut de Neurosciences de la Timone, UMR7289, Aix-Marseille Université and Centre National de la Recherche Scientifique (CNRS), Marseille, France

    Cécile Brocard, Vanessa Plantier, Pascale Boulenguez, Sylvie Liabeuf, Mouloud Bouhadfane, Annelise Viallat-Lieutaud, Laurent Vinay & Frédéric Brocard

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  1. Cécile Brocard
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Contributions

C.B. designed and performed the immunohistochemistry, western blot and surgery. V.P. performed in vivo experiments, contributed to in vitro experiments and to surgery. P.B. designed and performed in vivo experiments. S.L. performed cell culture and participated in immunohistochemistry and western blot. M.B. participated in some in vitro experiments. A.V.-L. contributed to surgery and postoperative care. L.V. provided valuable expertise in the field of SCI research. F.B. designed and supervised the whole project and contributed to the in vitro experiments. C.B., V.P., P.B., L.V. and F.B. analyzed data. L.V. and F.B. wrote the manuscript.

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Correspondence to Frédéric Brocard.

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Brocard, C., Plantier, V., Boulenguez, P. et al. Cleavage of Na+ channels by calpain increases persistent Na+ current and promotes spasticity after spinal cord injury. Nat Med 22, 404–411 (2016). https://doi.org/10.1038/nm.4061

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