Following spinal cord injury (SCI), spasticity resulting from hyperexcitability of motor neurons is common. A recent study has shown that calpain-mediated cleavage of sodium channels in motor neurons contributes to this complication of SCI and provides evidence that pharmacological inhibition of a calpain could be a useful therapeutic strategy for such spasticity.
Upregulation of the persistent sodium current (INaP) underlies much of the increased excitability of motor neurons after SCI. To investigate the mechanisms that link SCI with INaP, the authors of the current study performed spinal cord lesion in rodents followed by immunohistochemistry of spinal cord samples. Compared with sham-operated rodents, SCI was associated with upregulation of the voltage-gated sodium channel Nav1.6 in motor neurons.
Next, the authors carried out further analysis of Nav1.6 expression using western blotting on the membrane fractions of rodent motor neurons. A Nav-specific antibody revealed an ~250 kDa band consistent with full-length Nav. Notably, although the intensity of the ~250 kDa band was unchanged by SCI, an ~120 kDa cleavage product was strongly upregulated.
In adult rat spinal cord homogenates, addition of calcium generated a similar ~120 kDa band, suggesting the involvement of a calcium-dependent protease, which the investigators posited could be a calpain. To test this hypothesis, they pretreated the spinal cord homogenates with MDL28170, a calpain inhibitor, and indeed saw no formation of an ~120 kDa band in response to calcium. Together, these findings suggest that calpains mediate the cleavage of Nav1.6 following SCI.
To investigate how this mechanism might contribute to upregulation of INaP, the team performed patch clamp and voltage clamp recordings in neonatal rat motor neurons after SCI. Compared with vehicle treatment, intraperitoneal injections of MDL28170 for 8 days following SCI significantly reduced the formation of the ~120 kDa band on western blots and reduced the amplitude and the density of INaP. Moreover, calpains similarly increased INaP in HEK293 cells expressing Nav1.6.
Finally, in adult rats with SCI, injection of MDL28170 daily for 10 days, beginning 30–60 days after injury, suppressed the cleavage of Nav and significantly reduced muscle spasms relative to vehicle-treated SCI rats. Importantly, the beneficial effects of MDL28170 were maintained at 3 weeks after discontinuation of treatment.
The authors noted some residual upregulation of INaP after calpain inhibition with MDL28170, and they suggest that this could be due to mechanisms involving 5-HT receptors, which are known to be upregulated after SCI and to stimulate INaP.
This study suggests inhibition of calpains, even long after SCI, could have beneficial effects on subsequent spasticity.
Brocard, C. 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)
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Kingwell, K. Clamping down on calpains to treat injury-induced spasticity. Nat Rev Drug Discov 15, 310 (2016). https://doi.org/10.1038/nrd.2016.83