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

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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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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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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