The main clinical symptoms of spasticity, hyperreflexia and spasms, develop after spinal cord injury (SCI) due in part to the hyperexcitability of motoneurons (Hultborn, 2003; Gorassini, 2004; Nielsen et al., 2007; D'Amico et al., 2014; Thomas et al., 2014). Understanding the cellular pathophysiological processes underlying this hyperexcitability might offer new therapeutic perspectives for spasticity.

SCI enhances the intrinsic excitability of motoneurons by upregulating their persistent sodium (I_NaP) and calcium (I_CaP) currents, leading to muscle spasms and hyperreflexia in both humans and adult rats (Li and Bennett, 2003; Li et al., 2004; Harvey et al., 2005; Harvey et al., 2006b; Harvey et al., 2006a; ElBasiouny et al., 2010; Theiss et al., 2011; Brocard et al., 2016). In addition, a concomitant synaptic disinhibition of motoneurons due to a decrease of the main chloride extruder KCC2 also takes place after SCI in both humans and adult rodents (Boulenguez et al., 2010; Mòdol et al., 2014; Klomjai et al., 2019). In vitro experiments from neonatal rats show that this form of disinhibition stems primarily from an impaired Cl⁻ extrusion, typically identified by a depolarizing shift of the reversal potential of inhibitory postsynaptic potentials (E_IPSP) (Boulenguez et al., 2010), which may facilitate the recruitment of persistent inward currents after SCI (Venugopal et al., 2011). Thus, up-regulation of persistent inward currents concomitant with down-regulation of KCC2 may have a synergistic effect causing spasticity.

Remarkable advances have been made in the molecular mechanisms involved in alterations of persistent inward currents after SCI. SCI-induced constitutive 5-HT_2B/C receptor activity leads to an increase in I_CaP (Murray et al., 2010), while calpain-mediated proteolysis of Nav1.6 channels up-regulates I_NaP (Brocard et al., 2016). Although the mechanisms involved in alterations of KCC2 after SCI remain elusive, it is worth mentioning that calpain-mediated cleavage of KCC2 depolarizes the E_IPSP in some pathophysiological conditions (Puskarjov et al., 2012; Zhou et al., 2012; Wan et al., 2018). Our study investigates whether SCI-induced activation of calpains is upstream of the up- and down-regulation of I_NaP and KCC2 in motoneurons after SCI. If so, we aim at demonstrating whether a cooperation between calpain-mediated alterations of I_NaP and E_IPSP is a necessary element driving spasticity. For this purpose, we characterized signs of spasticity from the neonatal rat SCI model to obtain easier in vitro correlates of the hyperexcitability of lumbar motoneurons controlling hindlimb muscles.

Numerous studies on adult animal models delineate the hyperexcitable state of motoneurons as one of the causes of spasticity, presumably because of an increase of I_NaP and a decrease of KCC2 expression. The present study performed in neonatal rats identifies the activation of calpains as the upstream mechanism of the hyperexcitability of motoneurons after SCI. Calpain-mediated cleavage of Nav channels and KCC2 up- and down-regulates I_NaP and inhibition, respectively, in lumbar motoneurons. The reduction of calpain activity normalizes I_NaP and the strength of inhibitory transmission, and thereby alleviates spasticity. Altogether, these findings uncover a new cellular mechanism contributing to spasticity and provide novel therapeutic targets. Rather than targeting Nav channels or KCC2 independently, inhibition of calpains appears as a promising therapy by targeting two pathways that are crucial for the development of spasticity, ‘killing two birds with one stone’.

The main spasticity symptoms develop within one week following SCI in neonatal rats, and correlate with an early appearance of hyperexcitability in the spinal cord. The emergence of spasticity is surprisingly faster than that of adult animals, which appears several weeks after the injury. A more active calpain-mediated proteolysis during early development may explain this age-dependent discrepancy. Indeed, calpain proteolysis is necessary for the pruning of axons during development (Yang et al., 2013) and the natural calpain inhibitory peptide, calpastatin, increases slowly in the CNS during the first weeks of life (Li et al., 2009). The lower presence of this inhibitor in newborn rats may explain why hyperexcitability after a neonatal SCI developed faster. Also at birth, rat motoneurons overexpress NMDA receptors (Vinay et al., 2000) which have been demonstrated to be efficient in activating calpains in pathological conditions (Zhou et al., 2012). In sum, the low expression of calpastatin combined with a high expression of NMDA receptors may contribute to a fast increase of motoneuronal excitability after a SCI in newborn rats.

This hyperexcitability emerges in vitro in the form of high-frequency spontaneous bursting activity and enhanced, prolonged sensory-evoked LLR. We provided evidence that both are dependent on I_NaP and KCC2 dysfunction. I_NaP plays an important role in the operation of the spinal locomotor network (Tazerart et al., 2007; Tazerart et al., 2008; Brocard et al., 2010; Bouhadfane et al., 2013; Brocard et al., 2013) and its increase after SCI promotes prolonged plateau potential firing in motoneurons and hyperreflexia, both in animal models and humans (Li and Bennett, 2003; Li et al., 2004; Kitzman, 2009; Theiss et al., 2011). Supersensitivity of motoneurons to residual serotonin after SCI leads to the facilitation of I_NaP (Harvey et al., 2006a; Harvey et al., 2006b). Here, we demonstrate that calpain-dependent cleavage of Nav channels also upregulates I_NaP in lumbar motoneurons. If serotonin recruits calpains after SCI, similarly to what observed in Aplysia motoneurons (Bougie et al., 2012), remains to be tested.

The exact mechanisms involved in the facilitation of I_NaP by calpain are not fully understood. As previously observed after a traumatic brain injury (von Reyn et al., 2009), proteolyzed Nav channels remain in the plasma membrane and thus might interfere with biophysical properties of full-length voltage-gated channels (Michailidis et al., 2014). On the other hand, some determinants governing the inactivation of Nav channels are sensitive to proteases, which can increase I_NaP (Armstrong et al., 1973; Gonoi and Hille, 1987; Clarkson, 1990). Whatever the mechanisms, a clear relationship is given by our previous study in which calpain-mediated cleavage of Nav1.6 channels in HEK-293 cells up-regulates I_NaP (Brocard et al., 2016). Because Nav1.6 channels are highly expressed in spinal motoneurons (Alessandri-Haber et al., 2002; Duflocq et al., 2008; Brocard et al., 2016), their cleavage likely contributes to increase I_NaP after SCI. This is consistent with our result that pharmacological inhibition of Nav1.6 channels significantly reduced hyperexcitability after SCI. Further support comes from mutation of Nav1.6 channels that leads to severe myoclonic spasms with a fivefold increase of I_NaP (Veeramah et al., 2012). It is noteworthy that the rescue of I_NaP by MDL28170 is only partial. Although an incomplete block of calpains by MDL28170 cannot be excluded, calpain-independent mechanism(s) may contribute to increase I_NaP. Since the activation of 5HT₂ receptors facilitates I_NaP (Harvey et al., 2006b), the constitutive activation of 5HT₂ receptors after SCI (Murray et al., 2010) might be one of them. Also, the amount of full-length Nav channels (~250 kDa) at the plasma membrane never changed after either the post-SCI activation of calpains (Brocard et al., 2016) or their inhibition by a MDL28170 treatment (see Figure 3F). As a compensation mechanism depending on the number of proteolytic fragments, it is possible that the translocation of fragments into the nucleus maintains constant the expression level of the full-length channel at the membrane by regulating the Nav channel transcription (Onwuli et al., 2017).

Synaptic disinhibition of motoneurons after SCI also contributes to spasticity and results from a decreased expression of KCC2 (Boulenguez et al., 2010). Although a down-regulation of KCC2 has been described after SCI (Boulenguez et al., 2010; Côté et al., 2014; Mòdol et al., 2014; Chang et al., 2018; Chen et al., 2018), the causal molecular mechanisms remain unknown. Here, we provide evidence that activation of calpains is one of the molecular basis for disinhibition of motoneurons after SCI. A direct relationship between calpain and KCC2 is given by our studies on spinal cord homogenates, in which exogenous application of active calpains reduces KCC2 in its oligo and monomeric forms. The sensitivity of KCC2 to calpain is consistent with the presence of two predicted sites (PEST motifs) for cleavage by calpain within the C-terminal domain (Mercado et al., 2006; Acton et al., 2012). Since the ability of KCC2 to extrude Cl^- requires the C-terminal domain (Mercado et al., 2006; Acton et al., 2012) and that the KCC2 antibody targets the C-tail, its cleavage by calpains after SCI likely accounts for both the immunostaining decrease of KCC2 at the plasma membrane and the loss of chloride extrusion resulting in the depolarizing shift of E_IPSP in motoneurons (Boulenguez et al., 2010). The tight relationship between the calpain-mediated proteolysis of KCC2 and the altered Cl^- homeostasis is supported by our observation that an acute inhibition of calpains by MDL28170 rapidly restores both E_IPSP and KCC2. The rapid rescue of KCC2 by the acute application of MDL28170 is in agreement with the fast pharmacodynamic of MDL28170 to cross the brain blood barrier (Markgraf et al., 1998) and the extremely high rate (minutes) in KCC2 turnover at the plasma membrane (Lee et al., 2007). Regarding the rescue of KCC2 by MDL28170, oligomers appear more sensitive to calpains than monomers after SCI. As phosphorylation regulates the vulnerability of substrates to calpains (Sprague et al., 2008), the post-SCI dephosphorylation of KCC2 at the serine 960 (Mòdol et al., 2014), in the vicinity of PEST motifs, might contribute to sensitize more oligomers to calpains.

Our results suggest that KCC2 and I_NaP appear to cooperate in promoting spinal hyperexcitability after SCI. In line with this, a modeling investigation demonstrated that the depolarizing shift of E_IPSP after SCI facilitates the recruitment of persistent inward currents in motoneurons (Venugopal et al., 2011). The depolarizing shift of E_IPSP after a decrease in KCC2 may enable the unusual long-lasting depolarization reported in motoneurons after SCI (Li et al., 2004), which is required to recruit I_NaP. In turn, I_NaP will promote plateau potentials resulting in self-sustaining spiking in motoneurons (Li and Bennett, 2003; Bouhadfane et al., 2013) that drives spasticity (Bennett et al., 2001; Gorassini, 2004; Li et al., 2004). Furthermore, termination of plateau potentials by hyperpolarizing the motoneuron (Hounsgaard et al., 1984) will be much more difficult in a context of disinhibition; this may explain the fasciculation-like contractions commonly recorded in spastic subjects (Gorassini, 2004; Winslow et al., 2009; Zijdewind and Thomas, 2012). Therefore, I_NaP-blockers such as riluzole, or KCC2 enhancers likely alleviate spasticity (Brocard et al., 2016; Liabeuf et al., 2017) by decoupling the tandem response driven by enhanced I_NaP and decreased KCC2. The riluzole is currently approved for humans affected by Amyotrophic Lateral Sclerosis (ALS). Our data provide strong preclinical evidence for translation to chronic SCI subjects, a process that will likely be facilitated by clinical trials that are currently in progress to test the neuroprotective effects of riluzole in the acute phase of SCI (Grossman et al., 2014).

We identify calpain as the upstream mechanism responsible for the hyperexcitability of motoneurons after SCI. Calpains exist in the CNS mainly as two major isoforms, µ-calpain (calpain-I) and m-calpain (calpain-II) that differ on the range of [Ca²⁺] required for their activation (µM and mM, respectively). As previously observed in adult rodents (Springer et al., 1997; Du et al., 1999; Schumacher et al., 1999; Yu et al., 2013), we found that the expression of calpain-I increases after SCI in neonatal rats. However, the protease protein expression does not necessarily correspond to enzyme’s catalytic activity. Furthermore, calpain-I and -II may have opposite functions. After a traumatic brain injury (TBI), calpain-I and -II appear neuroprotective and neurodegenerative, respectively (Baudry and Bi, 2016). Therefore, the respective contribution of calpain-I and -II in the hyperexcitability of motoneurons after SCI remains to be clarified.

Calpain-mediated changes in motoneurons alone are unlikely to account for all the post-injury spinal hyperexcitability. Premotor excitatory interneurons, including locomotor network-related interneurons, may play a critical role in initiating muscle spasm activity (Husch et al., 2012; Bellardita et al., 2017; Lin et al., 2019). In line with this, we found that most sensory-evoked LLRs are composed of a fictive locomotor episode, suggesting enhanced sensory recruitment of the locomotor central pattern generator (CPG). This leads us to believe that part of muscle spasm, and especially clonus (involuntary rhythmic contractions), might be a manifestation of a hyperexcitable locomotor CPG. Enhanced sensory-evoked LLR has been previously described in the sacrocaudal spinal cord from adult SCI rodents but differs in some aspects from our results with neonatal lumbar motoneurons. Indeed, I_CaP contributes to sacrocaudal hyperreflexia by promoting plateaus in motoneurons (Li and Bennett, 2003; Li et al., 2004) while disinhibition appears negligible (Bellardita et al., 2017). The I_CaP appears to have a negligible role in lumbar hyperreflexia from neonatal SCI rats, in line with previous work showing a full maturation of L-type Ca²⁺ channels at second/third post-natal weeks (Jiang et al., 1999). However, we also demonstrate that some of these channels are already expressed in the first post-natal week, as their pharmacological activation by Bay-K triggered LLRs. Thus, it is likely that the contribution of L-type Ca²⁺ channels to the LLR increases with age. Alternatively, temperature and ionic composition of the extracellular medium may account for the relative contribution of I_NaP and I_CaP in generating LLRs. In vitro studies in adults were performed at room temperature with high extracellular [Ca²⁺] (≥2.5 mM) (Li and Bennett, 2003; Li et al., 2004; Bellardita et al., 2017), far from physiological conditions [body temperature 37°C; 1.2 mM of Ca²⁺ in the CSF (Nicholson et al., 1977; Jones and Keep, 1988; Brocard et al., 2013). As a consequence, Ca²⁺ currents are potentiated (Carlin et al., 2000; Carlin et al., 2009), I_NaP is reduced (Tazerart et al., 2008) and thermosensitive I_NaP-dependent plateaus in motoneurons are dampened down (Bouhadfane et al., 2013). Also, the disinhibition seen in the SCI lumbar enlargement, linked to the decrease of KCC2 (Boulenguez et al., 2010), seems unimportant in the sacrocaudal spinal cord (Bellardita et al., 2017). Diversity of KCC2 expression between motoneurons innervating distinct muscles (Ueno et al., 2002) or variation in the sacral inhibitory circuitry (Jankowska et al., 1978) may account for this discrepancy. Spinal cord level-specific differences in neuronal responses to SCI are probable with respect to the special anatomy and function of the sacrocaudal spinal cord (Ritz et al., 1992; Ritz et al., 2001).

To conclude, our study sheds light on the etiology of spasticity and opens novel perspectives to develop therapies by targeting calpains. The discovery of calpain as a new upstream mechanism leading to spasticity, is of special importance given the lack of translational results obtained from previously tested therapeutic approaches. Since spinal maladaptive mechanisms triggered by calpain start taking place within the first hours after injury in our SCI model, it is conceivable that early therapies against calpain might show a higher effectiveness. Because altered chloride homeostasis and proteolysis of Nav channels have been implicated in other neurological disorders such as traumatic brain injury for which calpains are recruited (Kahle et al., 2008; von Reyn et al., 2009; Wang et al., 2018), the involvement of calpains might be broadened to other pathologies leading to an excitatory/inhibitory imbalance.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures and figure supplements.

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Author details

1. Vanessa Plantier

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Performed and analyzed most of in vitro and in vivo experiments

   Contributed equally with

   Irene Sanchez-Brualla

   Competing interests

   No competing interests declared

2. Irene Sanchez-Brualla

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Performed and analyzed most of in vitro and in vivo experiments

   Contributed equally with

   Vanessa Plantier

   Competing interests

   No competing interests declared

3. Nejada Dingu

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Performed and analyzed some in vitro experiments

   Competing interests

   No competing interests declared

4. Cécile Brocard

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Performed and analyzed most of biochemistry and immunohistochemistry experiments

   Competing interests

   No competing interests declared

5. Sylvie Liabeuf

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Formal analysis, Methodology, Performed some biochemistry experiments

   Competing interests

   No competing interests declared

6. Florian Gackière

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Data curation, Formal analysis, Methodology, Performed and analyzed most of intracellular recordings

   Competing interests

   No competing interests declared

7. Frédéric Brocard

   Institut de Neurosciences de la Timone (UMR7289), Aix-Marseille Université and CNRS, Marseille, France

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration, Designed, supervised the whole project, performed and analyzed some in vitro experiments and wrote the manuscript

   For correspondence

   frederic.brocard@univ-amu.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9444-9586

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