In healthy conditions, Nav1.2 is expressed on immature, unmyelinated neurons, where it supports impulse transmission. However, this channel subtype is no longer detectable in adult mice myelinated neurons, where the closely related Nav1.6 channel supports saltatory conduction of nerve impulses at nodes of Ranvier [128]. Nav1.2 channels produce rapidly activating and inactivating currents and appear to support action-potential conduction, which occurs before myelination. Nav1.6 channels, on the other hand, produce a persistent current that follows the rapidly activating and inactivating currents, and Nav1.6 is co-expressed together with the Na⁺/Ca²⁺ exchanger (NCX) along degenerating axons in EAE ^[93][129][93,129]. This event leads to sustained depolarization causing an intracellular Ca²⁺ overload that triggers mitochondrial dysfunction and leads to neuronal cell death ^[90][129][90,129]. During demyelination, Nav1.2 and Nav1.6 are found to be overexpressed at demyelinated sites of the axonal membrane in EAE models [90]. Thus, redistribution of Na⁺ channels might represent an initial compensatory process preserving conductance and axonal integrity but, at later stages, accelerates chronic neurodegeneration [46]. Accordingly, in post-mortem tissues from patients with secondary progressive MS, both Nav1.6 and Na⁺-Ca²⁺ exchanger (NCX) colocalize with amyloid precursor protein, a marker of axonal injury [93]. Of note, data demonstrated that NCX3 is strongly upregulated during oligodendrocyte development [130] and its activity is required for the maintenance of Ca²⁺ transients necessary to myelin synthesis in oligodendrocyte cultures [131]. Recent evidence confirmed this hypothesis and deepened knowledge in the field by demonstrating that neuronal activity, presumably in form of local extracellular [K⁺] changes, might locally influence NCX-mediated Ca²⁺ transients in OLs thus triggering local MBP synthesis in the vicinity of an active axon [132].

Furthermore, an abnormal upregulation of mRNA and protein for Nav1.8 was found within cerebellar Purkinje cells in EAE mice and MS humans [91]. This suggests that abnormal electrical signalling within the cerebellum due to a transcriptional channelopathy could contribute to MS [91]. In confirm, the Nav1.8 subtype in EAE mice is found to be aberrantly expressed in Purkinje neurons and Nav1.8-selective blockers can partially reverse EAE-induced neurological deficits, including abnormal Purkinje cell firing [53]. Indeed, the new orally bioavailable Nav1.8-selective blocker PF-01247324 improved motor coordination and cerebellar-like symptoms in transgenic mice expressing Nav1.8 in Purkinje neurons and in wild-type mice with EAE [53].

Wang et al. found that the expression of Nav1.5 was significantly increased in reactive astrocytes within MS lesions, while Nav1.1-Nav1.3 and Nav1.6 exhibited only minor changes [92], thus suggesting a role of Nav in the functional response of reactive glial cells in MS lesions. In accordance, it was found that the Nav blocker phenytoin can attenuate microglial activation, migration and phagocytosis ^[56][133][56,133], as well as the release of IL-1 and TNF-α proinflammatory mediators, in EAE lesions [133]. Several studies have also shown that Nav blockers can reduce axonal injury and improve the clinical score of MS patients ^[56][134][56,134]. Indeed, safinamide, at both high and low doses, significantly protected against neurological deficit as well as microglial activation and increased the pool of protective M2 type microglial cells in EAE mice [54]. In cultured microglia, safinamide suppressed superoxide production via inhibition of NADPH oxidase, an enzyme which is primarily responsible for the generation of reactive oxygen species. As these effects of safinamide were comparable with that of the cognate Na⁺ channel inhibitor flecainide, the mechanism behind safinamide mediated protection was attributed to the inhibition of Navs ^[54][55][54,55].

In confirm, the use of Nav blockers, such as flecainide, phenytoin and, more recently, lamotrigine preserved both axonal integrity and electrical conduction in EAE mice. Importantly, these agents maintained conduction along the CNS and reduced disability scores in treated animals ^[56][57][56,57]. Phenytoin was also tested in a phase II clinical trial on 86 participants affected by acute demyelinating optic neuritis, a common symptom of MS, and has proved to be neuroprotective ^[58][59][58,59]. On the other hand, lamotrigine, also tested in a phase II clinical trial on 120 patients with secondary progressive MS, did not affect the rate of changes in partial cerebral volume over 24 months vs. placebo. However, a relevant side effect was pointed out by the study as a reduction in white matter volume was detected in patients during the first 12 months after lamotrigine administration, an effect that was however lost upon discontinuation of treatment ^[58][60][58,60]. Finally, paroxysmal dysarthria and ataxia in patients with MS can respond to treatment with Na⁺ channel blockers such as carbamazepine [61].

Although encouraging preclinical data, Nav-targeting compounds still miss clinical translation due to rebound effects after discontinuation of the drug. Only phenytoin differs from related compounds as both preclinical and clinical studies unveiled its neuroprotective properties, based on Nav blockade [135]. For a summary on the effects of Nav-targeting compounds in demyelination see Table 1.

Several evidences support the notion that aberrant Cav-mediated currents contribute to the pathophysiology of MS or EAE. Increased Ca²⁺ influx through Cav was assumed to facilitate neurological impairment and histological damage in EAE mice and, by inference, MS [146]. Pregabalin (Lyrica^®) is prescribed to MS patients to treat neuropathic pain by targeting Cav [62] and, in addition, it could provide neuroprotection by inhibiting exaggerated Cav currents during excitotoxicity and neuroinflammation. Indeed, it has been demonstrated that Pregabalin treatment alleviates EAE symptoms in mice possibly by reverting, at neuronal level, intracellular Ca²⁺ overload in EAE lesions [63]. However, in the same paper, the authors pointed to a significant reduction of hippocampal long-term potentiation in pregabalin-treated EAE mice, thus warning of potential side effects on memory and learning processes ^[58][63][58,63]. In accordance with deleterious effects on memory, two recent clinical studies showed that perioperative pregabalin reduced spatial working memory in humans [64] and its misuse led to cognitive impairment [65].

An abnormal redistribution of N-type Ca²⁺ channels was found in acutely injured axons, followed by rearrangement of the axonal membrane after injury [147]. Since Nav are known to redistribute along demyelinated axons [46], a similar mechanism may also exist for Cav. Additionally, the N-type Cav2.2 was detected also on mature OLs [99] and the expression of the pore forming α1B-subunit of Cav2.2 was found in MS and EAE plaques and was overexpressed in active lesions [103].

In mice, it has been shown that, after knockout of Cav1.2, axonal myelination is inhibited and OPC maturation disturbed [100]. However, the L-type Ca²⁺ channel (Cav1.2, Cav1.3, Cav1.4) blockers Bepridil and nitrendipine had comparable beneficial effects in reducing neuroinflammation and axonal pathology on EAE mice [66]. When nimodipine was administered preventively at the time point of disease induction, EAE severity and demyelination decreased [67]. Recently, nimodipine has been reported to have positive effects on Schwann cells, astrocytes and neurons, being associated to increased phosphorylation of either protein kinase B and the cyclic adenosine monophosphate response element-binding protein (CREB) ^[68][148][68,148]. It is known that axonal Ca²⁺ overload activates the Ca²⁺-dependent protease calpain, leading to disruption of the cytoskeleton and to other structural and functional alterations of the axon [149]. Of note, nimodipine also downregulated the expression of calpain as well as the pro-apoptotic protein caspase 3, whereas calbindin expression was upregulated, indicating that modulation of Ca²⁺ homeostasis and prevention of intracellular Ca²⁺ overload might be responsible for the neuroprotective properties of this Cav blocker ^[68][69][68,69].

Interestingly, recent studies have reported that the Cav1.2 channel blockers nimodipine and verapamil exert their neuroprotective effects through anti-inflammatory properties [70], possibly preventing microglial activation [71] and down-regulating TNFα and IL1β expression in the hippocampus ^[72][73][72,73]. However, microglial cells do not express functional Cav1.2 channels [150], thus the anti-inflammatory effects of these drugs are likely mediated by their block on other cell types [72]. Recently, it was found that animals injected with nimodipine during CPZ-induced demyelination displayed a reduced astrocyte and microglia activation and proliferation as well as a faster and more efficient brain remyelination [74]. Cav1.2 channels are not present in mature OLs ^[96][104][96,104], but they are expressed by OPCs where they are essential for maturation ^[100][101][100,101], as mentioned above. This suggests that reducing Cav currents inhibits astrocyte and microglia activation during demyelination. Consequently, pool of proliferating OPC increases as well as the number of myelinating OLs, leading to a beneficial effect for myelin regeneration [74]. However, deletion of the Cav1.2 channels in GFAP⁺ astrocytes did not prevent myelin damage during CPZ treatment.

Hence, it appears that Cav blockers might represent promising targets for demyelinating diseases as they concur to pathological intracellular Ca²⁺ overload in neurons and immune cells. Nevertheless, there are no trials for clinical translation of Ca²⁺ channel blockers so far [151]. For a summary on the effects of Cav-targeting compounds in demyelination see Table 1.

As mentioned above, deleting Kir4.1 channels in mice causes impaired myelination and OL maturation during development, leading to neuronal degeneration [115]. In confirm, serum levels of antibodies against Kir4.1 were enriched in MS patients, suggesting that Kir4.1 could be a target of antibody responses in this pathology [116].

In myelinated axons, Kv1.1 and Kv1.2 are located underneath the myelin sheath in the paranodes or internodal regions [46], but, after demyelinating insults, alterations in K⁺ channel expression and distribution along the axon are reported. Indeed, a reduction of Kv1.2, Kv1.4, and Kv2.1 has been demonstrated in EAE mice, correlating with disease severity [122]. Moreover, Calvo et al. found that, although Kv1.1 and Kv1.2 expression levels decrease, they redistributed form the juxtaparanode into the paranode in a spinal nerve transection (SNT) model of neuropathic pain [154]. In contrast, Kv1.4 and 1.6 expression increases within justaparanodes and paranodes [154]. These findings are consistent with previous studies reporting a Kv mislocalization in EAE animals and in post-mortem human MS lesions [121]. In accordance, administration of a Kv1.1 selective blocker (BgK-F6A) ameliorates disease course in EAE mice [75], and treatment with the I_A blocker 4-AP enhances axonal conduction [76]. As I_A is widely diffused along demyelinated axons and contributes to MS symptoms [155], in 2010 dalfampridine, an extended-release form of 4-AP, has been approved by the FDA to improve walking in MS patients [77]. The mechanism by which Kv blockers could exert neuroprotection is ascribed to the block of excessive K⁺ outflow from axons causing extracellular K⁺ homeostasis outbalance that postpones the K⁺ reversal potential to more positive values. Consequently, a gradual depolarization of neuronal membrane occurs with a consequent inactivation of Nav leading to impaired action potential propagation. Hence, Kv block may help preserving signal conduction. Furthermore, as axonal K⁺ outflow leads to intracellular K⁺ depletion causing water loss and disinhibition of proapoptotic enzymes [156], these compounds might also provide neuroprotection by preserving intracellular K⁺ homeostasis. Of note, a recent paper demonstrated that, although Kv1.4 deficiency decreases OPC proliferation in vitro, it does not influence de- or remyelination in the CPZ model [157]. Moreover, in the same study, Kv1.4 deficiency leads to an ameliorated course of EAE and results in reduced Th1 responses. These data argue for a peripheral effect of Kv1.4 on immune cells, possibly via glial cells. Since the authors did not observe any change in the CPZ model, it is tempting to speculate that the benefits observed in the EAE model are not only due to remyelination, but also to a reduced impact of immune system on the CNS, as discussed previously ^[158][159][158,159]. For a summary on the effects of Kv-targeting compounds in demyelination see Table 1.