**4. Ion channel modulation**

Among the molecules that make up neurons, ion channels are especially important, because they provide them their signaling abilities. In multiple sclerosis, there were described several types of ion channels dysfunctioning:


In MS, excessive accumulation of Ca2+ ions is known to contribute to axonal degeneration in the central nervous system (CNS) through the activation of acid-sensing ion channel type 1a (ASIC1). ASIC1 is considered a mediator of neuronal injury in stroke and CNS inflammation due to its ability to modulate Na+ and Ca2+ flux. So, it could be possible to attenuate axonal loss by disrupting the ASIC1a gene or by using a nonspecific blocker of these channels, such as amiloride (a diuretic with a proven safety record) [82]. Recently, a single-arm, longitudinal trial of amiloride showed an important reduction of brain atrophy in the primary progressive form of MS. The aim of Amiloride Clinical Trial in Optic Neuritis (ACTION), an ongoing phase II clinical trial, is to demonstrate the neuroprotective effect of amiloride in acute optic neuritis (a common manifestation of MS) using a multimodal approach that combines structural and functional outcomes with clinical measures [83].

Ginseng has been used in traditional medicine for over 2000 years due to its antianxiety, antidepressant, and cognition-enhancing properties. Moreover, its effects on the brain are related to glutamatergic and monoaminergic transmission, estrogen signaling, nitric oxide production, neuronal survival, apoptosis, neural stem cells, and neuroregeneration. The efficacy of ginsenoside Rd has been studied in mice with EAE. The results were promising because the ginsenoside reduced the permeability of the blood–brain barrier, regulated the

Based on the observational studies that showed that low levels of vitamin D represent a risk factor for the development of MS [66, 67], treatment with vitamin D has become increasingly attractive and has been tested in both experimental and clinical trials. Vitamin D appeared to modulate upon immune responses and inflammation, but clinical studies have not yet shown

In addition to pharmaceutical compounds, clinical and basic research studies have also highlighted that voluntary exercise can promote both neuroprotection and neuroregeneration [70, 71]. An experiment conducted in mice with EAE showed that the exercising mice (on a running wheel) presented a less severe neurological disease score, later disease onset and a significant reduction of inflammatory cell infiltration and demyelination in the ventral white matter tracts of the lumbar spinal cord [71]. Studies of patients with MS also support these observations, physical excesses determining not only improvement of muscle function and

Among the molecules that make up neurons, ion channels are especially important, because they provide them their signaling abilities. In multiple sclerosis, there were described several

**•** Ectopic distribution of calcium channels, up-regulated within the axon membrane, during the demyelinating process. Increased intracellular calcium levels activate calcium-depend‐ ent proteases (calpains) that can degrade axonal proteins, contributing to the axonal injury. Blocking the calcium channels can protect myelinated axons from axotomy-induced and

**•** Transcriptional channelopathy that described in cerebellar Purkinje neurons. Studies showed that Nav1.8 gene (normally inactivated in the cerebellum) is aberrantly activated in Purkinje neurons, producing the Nav1.8 protein, possibly responsible for cerebellar

**•** Ion channel dysfunctioning during remyelination—redistribution and clustering of ion

In MS, excessive accumulation of Ca2+ ions is known to contribute to axonal degeneration in the central nervous system (CNS) through the activation of acid-sensing ion channel type 1a

secretion of INF-gamma and IL-4 and decreased disease severity [65].

walking endurance, but also of cognitive abilities [72–75].

anoxia-induced degeneration (see **Figure 2**) [76].

a clear benefit [68, 69].

292 Trending Topics in Multiple Sclerosis

**4. Ion channel modulation**

types of ion channels dysfunctioning:

deficits [77].

channels [78–81].

**Figure 2.** Mechanisms of demyelination-related neurodegeneration. Demyelination can result progressively in ionic disequilibria, energy crisis, conduction block, and eventually neurodegeneration. (A) a normal node of Ranvier with juxtaparanodal, paranodal, and nodal regions intact, depicting Na+ , K+ , and Ca2+ ions flowing through their respective channels with mitochondria supplying the ATP for energy-dependent Na+ K+ ATPases that re-establish the ion gradi‐ ents depleted by ion flux through channels. Numerous different ion channels are present in the axon but only a small subset is depicted here; (B) partial demyelination results in dispersal of nodal ion channels, energy insufficiency, and disequilibria of ion gradients; (C) complete demyelination can result in conduction block and axonal degeneration due to the accumulation of intracellular Ca2+ that results from energy crisis and disruption of ionic balances. Abbreviations: Kv1—potassium channel type 1; Nav1.6 and Nav1.2—sodium channel types 1.6 and 1.2; Na+ Ca+ Exchanger—Na-Ca exchange pump; Na+ K+ ATPase—ATP (energy)-dependent Na-K exchange pump; CASPR1—contactin-associated pro‐ tein 1 (interaction molecule between myelinating cell with axon); NF155—neurofascin 155 (predominant interaction molecule between myelin and axon at paranodal axo-glial junction) http://www.mdpi.com/1422-0067/16/9/21215.

4-Aminopyridine (Fampridine) is a potassium channel blocker that improves axonal conduc‐ tivity in demyelinated lesions by targeting the potassium channel subtypes Kv1.1, Kv1.2, and Kv1.4 and thus correcting the leakage of potassium ions. Even if it has no impact upon disease incidence and severity, it has been already approved for improvement of fatigue, walking speed, and strength in MS patients [84].

Other potential agents that can target ion channels are lamotrigine, phenytoin, flecainide, topiramate, carbamazepine, and glibenclamide, but even if some of them have some positive results in animal studies, there is lack of clinical data regarding their efficacy in MS [85].

### **5. Remyelinating strategies in MS**

For successful remyelination to take place, OPCs must undergo several necessary and sequential steps. This very intricate process can fail if not regulated effectively. In the first step —the activation phase—OPCs must proliferate, which involves the expression of several genes and transcription factors by either activated microglia or astrocytes within the lesion [86, 87]. Mediators such as the proteins Cdk2 and p27Kip-1, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and other factors have been demonstrated to have a prolifera‐ tive effect in tissue cultures. In the second step—the migration or recruitment phase—OPCs are guided to migrate to the site of demyelination by chemotactic factors such as semaphorin receptors, neuropilins, and plexins. Semaphorin 3A impairs OPC migration to the lesion site, whereas semaphorin 3F promotes OPC migration and remyelination [88]. PDGFα is the archetypal chemotactic factor for OPCs, although it is difficult to separate its chemotactic effects from its effect on OPC proliferation. In the third step, OPCs must differentiate into remyelinating oligodendrocytes in a process driven by transcription factors such as Nkx2.2 and Olig2 [89].

Many changes in both the cytoarchitecture and microenvironment of the MS brain could prevent remyelination by endogenous OPCs. Extracellular matrix components, including fibronectin, hyaluronic acid (HA), and chondroitin sulfate proteoglycans (CSPGs), can block the differentiation of OPCs and premyelinating oligodendrocytes [90]. Components of damaged myelin, such as myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and NOGO-A, which signal through the Nogo 1 receptor and its coreceptors p75, TROY and LINGO-1 (leucine-rich repeat- and Ig domain-containing Nogo receptor-interacting protein 1) inhibit both axonal regeneration and oligodendrocyte differ‐ entiation and remyelination [91, 92]. The differentiation phase can also be influenced by intrinsic signaling pathways (Notch signaling, Wnt signaling, and Retinoid X receptor (RXR) signaling) and extrinsic competitors (LINGO-1, semaphorin 3A, sonic hedgehog (Shh), fibroblast growth factor, insulin-like growth factor 1 (IGF-1), BDNF, chemokine CXCL 12, and bone morphogenic proteins (BMPs). The Notch signaling pathway is an important regulator of the balance between OPC proliferation and differentiation in the developing CNS as well as PNS. Notch 1 is a surface receptor expressed by both developing and mature oligodendro‐ cytes. The ligand engaged with the Notch receptor determines whether the canonical or noncanonical signaling pathway is activated. The canonical Notch 1 signaling pathway, which is mediated through Jagged 1, prevents OPC differentiation, whereas the non-canonical signal‐ ing pathway mediated through contactin promotes differentiation [93]. The canonical Wnt-βcatenin signaling pathways negatively regulate the production and differentiation of oligodendrocytes during both developmental myelination and remyelination. Some data suggest that the inhibition of Wnt via Axin2 promotes oligodendrocyte differentiation and remyelination [94].

Other potential agents that can target ion channels are lamotrigine, phenytoin, flecainide, topiramate, carbamazepine, and glibenclamide, but even if some of them have some positive results in animal studies, there is lack of clinical data regarding their efficacy in MS [85].

For successful remyelination to take place, OPCs must undergo several necessary and sequential steps. This very intricate process can fail if not regulated effectively. In the first step —the activation phase—OPCs must proliferate, which involves the expression of several genes and transcription factors by either activated microglia or astrocytes within the lesion [86, 87]. Mediators such as the proteins Cdk2 and p27Kip-1, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and other factors have been demonstrated to have a prolifera‐ tive effect in tissue cultures. In the second step—the migration or recruitment phase—OPCs are guided to migrate to the site of demyelination by chemotactic factors such as semaphorin receptors, neuropilins, and plexins. Semaphorin 3A impairs OPC migration to the lesion site, whereas semaphorin 3F promotes OPC migration and remyelination [88]. PDGFα is the archetypal chemotactic factor for OPCs, although it is difficult to separate its chemotactic effects from its effect on OPC proliferation. In the third step, OPCs must differentiate into remyelinating oligodendrocytes in a process driven by transcription factors such as Nkx2.2

Many changes in both the cytoarchitecture and microenvironment of the MS brain could prevent remyelination by endogenous OPCs. Extracellular matrix components, including fibronectin, hyaluronic acid (HA), and chondroitin sulfate proteoglycans (CSPGs), can block the differentiation of OPCs and premyelinating oligodendrocytes [90]. Components of damaged myelin, such as myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and NOGO-A, which signal through the Nogo 1 receptor and its coreceptors p75, TROY and LINGO-1 (leucine-rich repeat- and Ig domain-containing Nogo receptor-interacting protein 1) inhibit both axonal regeneration and oligodendrocyte differ‐ entiation and remyelination [91, 92]. The differentiation phase can also be influenced by intrinsic signaling pathways (Notch signaling, Wnt signaling, and Retinoid X receptor (RXR) signaling) and extrinsic competitors (LINGO-1, semaphorin 3A, sonic hedgehog (Shh), fibroblast growth factor, insulin-like growth factor 1 (IGF-1), BDNF, chemokine CXCL 12, and bone morphogenic proteins (BMPs). The Notch signaling pathway is an important regulator of the balance between OPC proliferation and differentiation in the developing CNS as well as PNS. Notch 1 is a surface receptor expressed by both developing and mature oligodendro‐ cytes. The ligand engaged with the Notch receptor determines whether the canonical or noncanonical signaling pathway is activated. The canonical Notch 1 signaling pathway, which is mediated through Jagged 1, prevents OPC differentiation, whereas the non-canonical signal‐ ing pathway mediated through contactin promotes differentiation [93]. The canonical Wnt-βcatenin signaling pathways negatively regulate the production and differentiation of oligodendrocytes during both developmental myelination and remyelination. Some data

**5. Remyelinating strategies in MS**

294 Trending Topics in Multiple Sclerosis

and Olig2 [89].

Remyelination is not regulated by a single molecule or mediator but through a combination of signaling pathways that act on OPCs and oligodendrocytes as well as on other cellular players such as microglia, astrocytes, and even blood vessels. The discovery of new molecular players and of pharmacological strategies to act on them is currently a priority of the field so that new therapeutic agents that can change the natural history of MS can be developed.

Currently, from all potential remyelinating strategies for MS that stimulate OPC differentiation and enhance remyelination that include all the pathways and the signaling molecules descri‐ bed above [95–100], only anti-LINGO-1 antibodies have been tested in clinical trials. A phase II trial is ongoing and will provide additional information about safety, tolerability, and efficacy (NCT01864148).

The transplantation of exogenous OPCs into the CNS appears to be an attractive solution for MS, but unanswered questions render this procedure unfeasible in MS; these open questions include how to overcome the limited migration potential of transplanted OPCs, how to control the proliferation and differentiation process, and how to avoid immunosuppression treatment [101].
