Altered Expression of Ion Channels in White Matter Lesions of Progressive Multiple Sclerosis: What Do We Know About Their Function?

Despite significant advances in our understanding of the pathophysiology of multiple sclerosis (MS), knowledge about contribution of individual ion channels to axonal impairment and remyelination failure in progressive MS remains incomplete. Ion channel families play a fundamental role in maintaining white matter (WM) integrity and in regulating WM activities in axons, interstitial neurons, glia, and vascular cells. Recently, transcriptomic studies have considerably increased insight into the gene expression changes that occur in diverse WM lesions and the gene expression fingerprint of specific WM cells associated with secondary progressive MS. Here, we review the ion channel genes encoding K+, Ca2+, Na+, and Cl− channels; ryanodine receptors; TRP channels; and others that are significantly and uniquely dysregulated in active, chronic active, inactive, remyelinating WM lesions, and normal-appearing WM of secondary progressive MS brain, based on recently published bulk and single-nuclei RNA-sequencing datasets. We discuss the current state of knowledge about the corresponding ion channels and their implication in the MS brain or in experimental models of MS. This comprehensive review suggests that the intense upregulation of voltage-gated Na+ channel genes in WM lesions with ongoing tissue damage may reflect the imbalance of Na+ homeostasis that is observed in progressive MS brain, while the upregulation of a large number of voltage-gated K+ channel genes may be linked to a protective response to limit neuronal excitability. In addition, the altered chloride homeostasis, revealed by the significant downregulation of voltage-gated Cl− channels in MS lesions, may contribute to an altered inhibitory neurotransmission and increased excitability.

Despite significant advances in our understanding of the pathophysiology of multiple sclerosis (MS), knowledge about contribution of individual ion channels to axonal impairment and remyelination failure in progressive MS remains incomplete. Ion channel families play a fundamental role in maintaining white matter (WM) integrity and in regulating WM activities in axons, interstitial neurons, glia, and vascular cells. Recently, transcriptomic studies have considerably increased insight into the gene expression changes that occur in diverse WM lesions and the gene expression fingerprint of specific WM cells associated with secondary progressive MS. Here, we review the ion channel genes encoding K + , Ca 2+ , Na + , and Cl − channels; ryanodine receptors; TRP channels; and others that are significantly and uniquely dysregulated in active, chronic active, inactive, remyelinating WM lesions, and normal-appearing WM of secondary progressive MS brain, based on recently published bulk and single-nuclei RNA-sequencing datasets. We discuss the current state of knowledge about the corresponding ion channels and their implication in the MS brain or in experimental models of MS. This comprehensive review suggests that the intense upregulation of voltage-gated Na + channel genes in WM lesions with ongoing tissue damage may reflect the imbalance of Na + homeostasis that is observed in progressive MS brain, while the upregulation of a large number of voltage-gated K + channel genes may be linked to a protective response to limit neuronal excitability. In addition, the altered chloride homeostasis, revealed by the significant downregulation of voltage-gated Cl − channels in MS lesions, may contribute to an altered inhibitory neurotransmission and increased excitability.
Keywords: multiple sclerosis, progressive, white matter, lesions, ion channels, transcriptome INTRODUCTION Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) affecting more than 2 million people worldwide. MS lesions in CNS white matter (WM) are multiple focal areas of myelin loss accompanied by inflammation, gliosis, phagocytic activity, and axonal damage (Compston and Coles, 2008;Kuhlmann et al., 2017;Filippi et al., 2018;Rommer et al., 2019). Available MS therapies have little benefit for secondary-progressive MS (SPMS) patients, who develop progressive disability after a disease course characterized by inflammatory attacks. Therefore, promoting neuroprotection and remyelination are important therapeutic goals to prevent irreversible neurological deficits and permanent disability.
Ion channels play a fundamental role in maintaining WM integrity and regulating function of axons, interstitial neurons (Sedmak and Judas, 2021), glia, and vascular cells. Dysregulation of ionic homeostasis in the WM during demyelination is decisive for axonal damage and cell death and may interfere with tissue repair processes (Boscia et al., 2020). Furthermore, MS may involve an acquired channelopathy (Waxman, 2001;Schattling et al., 2014). Hence, selectively targeting ion channels in WM represents an attractive strategy to overcome axonal and glial impairment and prevent disease progression.
Recently, transcriptomic studies have considerably increased our insight into gene expression changes occurring in the MS brain (Elkjaer et al., 2019;Jakel et al., 2019;Schirmer et al., 2019). Aiming at identifying the ion channel genes governing WM dysfunction in SPMS brain, we analyzed the recent bulk RNAsequencing (RNA-seq) datasets by using the MS-Atlas (Elkjaer et al., 2019;Frisch et al., 2020). We put a special emphasis on the distribution of shared and unique genes encoding ion channels in chronic active (CA), active (AL), inactive (IL), and remyelinating (RL) lesions, and normal-appearing white matter (NAWM) compared to control WM (Figures 1A,B, Table 1). We identified uniquely expressed ion channel genes: 34 genes in CA, 9 in IL, 1 in AL, as well as 2 genes in all lesions and NAWM (Figures 1,  2, Table 1). The CA lesions displayed the highest number of upregulated ion channels genes while downregulated ion channels genes were more consistently found in ILs ( Figure 1C). Next, we explored recent single-nuclei RNA-seq (snRNA-seq) datasets to identify the expression of dysregulated ion channel genes in cell clusters in the WM of control and SPMS brain (Jakel et al., 2019; Tables 1, 2, Figure 3). FIGURE 1 | The transcriptional landscape of ion channels in different types of white matter brain lesions from patients with secondary progressive multiple sclerosis. (A) The percentage of significantly differentially expressed genes coding for ion channels among all dysregulated genes and within each lesion type [chronic active (CA), active (AL), inactive (IL), and remyelinating (RL)] and normal-appearing white matter (NAWM) compared to control white matter are indicated. (B) The Venn diagram shows the number of lesion-specific differentially expressed genes coding for ion channels and the number of overlapping genes among the lesion types. (C) The number of significantly differentially upregulated (red) and downregulated (blue) genes in each type of while matter lesion and NAWM compared to control white matter are indicated.
rat cultured microglia, and amoeboid microglia within corpus callosum during development, but barely in resting microglia by P21 (Fordyce et al., 2005;Li F. et al., 2008;Wu et al., 2009). In microglia, K v 1.1 and K v 1.2 expression was linked to cell activation (Eder, 1998), and their upregulation induced by lipopolysaccharide (LPS), ATP, or hypoxia is involved in the release of pro-inflammatory cytokines and intracellular production of reactive oxygen species (ROS) and nitric oxide (NO) (Li F. et al., 2008;Wu et al., 2009).

Expression and Function in MS
Bulk RNA-seq found upregulation of K v 1.1, K v 1.2, and Kv1.4 transcripts in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq detected significant K v 1.2 expression in neuronal clusters, slight increase of K v 1.4 transcripts in neuronal but not glial clusters, and no K v 1.1 transcript (Tables 1, 2; Jakel et al., 2019).
The CA lesion is characterized by ongoing tissue damage and, functionally, K v 1.2 upregulation in CA lesions may be a hallmark of axonal damage. While recent data found that KCNA1 gene is downregulated during demyelination in the cuprizone model (Martin et al., 2018), in animal models of MS, K v 1.2 (and also K v 1.1) ectopically redistributes to nodes and internodes of WM axons (McDonald and Sears, 1969;Wang et al., 1995;Sinha et al., 2006;Jukkola et al., 2012;Zoupi et al., 2013;Kastriti et al., 2015), while in human MS, the dislocation of K v 1.2 channels is associated with paranodal pathology, particularly in NAWM regions, and contributes to axonal dysfunction (Howell et al., 2010;Gallego-Delgado et al., 2020). The upregulated and redistributed K v 1.2 and K v 1.1 channels may hyperpolarize the axonal resting membrane potential (V rest ), elevate the amount of depolarization necessary for AP initiation, and impair AP conduction (Wang et al., 1995;Sinha et al., 2006;Jukkola et al., 2012). Pharmacological inhibition of K v 1.1 and K v 1.2 channels, e.g., with 4-aminopyridine, enhances axonal conduction and improves MS symptoms (Lugaresi, 2015).
It is difficult to speculate regarding K v 1.4 function in MS because data are not consistent. In animal models of MS and spinal cord injury (SCI), this developmentally restricted subunit re-appears/increases in OPCs, OLs, and astrocytic processes around lesion sites (Herrero-Herranz et al., 2007;Jukkola et al., 2012), but not in WM axons or microglia (Edwards et al., 2002;Jukkola et al., 2012). Mice lacking K v 1.4 exhibit reduced myelin loss in the spinal cord WM during EAE but no change of demyelination/remyelination in the corpus callosum in the cuprizone model (Gonzalez-Alvarado et al., 2020). However, it is unclear whether function of K v 1.4 subunits is relevant for glial cells in human MS because snRNA-seq barely detected K v 1.4 transcripts in glia clusters ( Table 2).    (Elkjaer et al., 2019) and data are collected from the database available at www.msatlas.dk (Frisch et al., 2020). b Cell type-specific clusters with significant expression of ion channels genes in human brain WM. The information is based on the snRNAseq from the WM of individuals with SPMS and non-neurological control subjects (Jakel et al., 2019), and data are collected from the database available at https://ki.se/mbb/oligointernodeen/ where the encoded subunits were listed according to the IUPHAR nomenclature. Fold changes of up-regulated genes are shown in bold. channels influence AP duration during high-frequency firing and regulate neuronal excitability (Guan et al., 2007). K v 2.1 mutations are associated with neonatal encephalopathy epilepsies and neurodevelopmental delays (Torkamani et al., 2014;Thiffault et al., 2015;de Kovel et al., 2017).

Expression and Function in MS
Bulk RNA-seq revealed upregulation of K v 2.1 and K v 2.2 transcripts in CA lesions of SPMS brain (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq found K v 2.1 and K v 2.2 in neuronal clusters (Tables 1, 2; Jakel et al., 2019). During EAE, K v 2.1 protein expression was downregulated in spinal cord motor neurons (Jukkola and Gu, 2015). Remarkably, K v 2.1 channels exist as freely dispersed conducting channels, or form electrically silent somatodendritic clusters (Schulien et al., 2020). Upregulated clustered K v 2.1 channels promote functional coupling of L-type Ca 2+ channels in plasma membrane to ryanodine receptors (RyRs) of the endoplasmic reticulum (ER) (Deutsch et al., 2012;Kirmiz et al., 2018;Vierra et al., 2019) and may modulate intracellular Ca 2+ level contributing to cell damage, while dispersal of K v 2.1-clusters blocks apoptogenic K + currents and provides neuroprotection (Sesti et al., 2014;Justice et al., 2017). Hence, to elucidate the functional role of K v 2 upregulation in MS (Table 1), it will be important to determine whether it reflects an increase in clustered or dispersed K v 2 channels.
The KCNC3 gene encodes for the K v 3.3 subunit, which, together with K v 3.1, K v 3.2, and K v 3.4, belongs to the K v 3 channel subfamily (Shaw). The K v 3.3 and K v 3.4 mediate transient Atype K + currents, while K v 3.1 and K v 3.2 mediate sustained K + currents.

Neurons
K v 3 channels localize to axonal and somatodendritic domains, and play a critical role in regulating AP firing at high frequency (Rasmussen and Trimmer, 2019). KCNC3 mutations result in spinocerebellar ataxia type-13 and cerebellar neurodegeneration (Rasmussen and Trimmer, 2019).

Expression and Function in MS
Bulk RNA-seq showed significant K v 3.3 upregulation in CA lesions (Figure 2, Table 1), while snRNA-seq revealed its predominant distribution in neuronal clusters (  (Jukkola et al., 2017), and the deletion of K v 3.1, which forms hetero-tetramers with K v 3.3, reduced EAE severity in mice (Jukkola et al., 2017). The Venn diagram represents the number of overlapping and lesion-specific differentially expressed genes coding for ion channels in chronic active (CA), active (AL), inactive (IL), and remyelinating (RL) lesions and in normal-appearing white matter (NAWM) compared to control white matter. Right panel: The heatmap shows two genes, coding for ion channels KCNH8 and TRPV6 that are significantly altered in all lesion types compared to control white matter. Scale bar indicates fold changes. (B) The Venn diagram, the heatmap, and the scale bar show the single ion channel gene, KCNK10, which is uniquely downregulated in active lesion (AL). (C) The Venn diagram, the heatmap, and the scale bar show the eight genes coding for ion channels that are uniquely significantly differentially dysregulated in inactive lesion (IL). (D) The Venn diagram, the heatmap, and the scale bar show the 33 genes coding for ion channels that are significantly and differentially dysregulated compared to control white matter in chronic active lesion (CA). The red box in Venn diagrams marks the genes that are specifically dysregulated in the corresponding type of lesion.

Kv4.2 (KCND2)
The KCND2 gene encodes for the K v 4.2 subunit that (together with K v 4.1 and K v 4.3) is a member of the K v 4 channel subfamily (Shal) and is highly expressed in the brain (Alfaro- Ruiz et al., 2019). K v 4 channels activate at subthreshold potentials and then inactivate and recover rapidly. They mediate transient A-type K + current (Bahring et al., 2001;Birnbaum et al., 2004).

Neurons
K v 4.2 subunits are highly expressed in soma and dendrites of hippocampal neurons and interneurons. They regulate the threshold for AP initiation and repolarization, frequencydependent AP broadening, and AP back-propagation (Nerbonne et al., 2008). K v 4.2 mutations are associated with infant-onset epilepsy and autism.

Expression and Function in MS
Bulk RNA-seq found significant K v 4.2 upregulation in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq reported significant expression of K v 4.2 transcripts in neuronal, OPCs, and committed OPCs (COP) clusters ( Table 2; Jakel et al., 2019). K v 4.2 subunit may contribute to oligodendrocyte dysfunction in SPMS brain because dysregulated KCND2 transcripts are associated with oligodendrocyte dysfunction in mental illnesses (Vasistha et al., 2019).
K v 7.2, K v 7.3, and K v 7.5 (KCNQ2, KCNQ3, and KCNQ5) The KCNQ genes encode for K v 7.1-K v 7.5 (KCNQ1-KCNQ5) family members that underlie a voltage-gated non-inactivating outward K + current, known as M current (I M ).

Neurons
The K v 7.2/3 or K v 7.3/5 hetero-tetramers represent the dominant subunit composition in neurons (Wang et al., 1998;Cooper et al., 2000;Kharkovets et al., 2000), while K v 7.4/K v 7.5 is dominant in vascular smooth muscles (Brueggemann et al., 2014). The K v 7.2and K v 7.3 subunits co-cluster with Na v channels at AIS and nodes of Ranvier in rodent somatosensory cortex and spinal cord WM and gray matter (GM) (Pan et al., 2006;Cooper, 2011;Battefeld et al., 2014). K v 7.5 localizes to soma and dendrites of cortical and hippocampal neurons and contributes to afterhyperpolarization currents (Tzingounis et al., 2010). The K v 7 channels stabilize V rest , influence neuronal subthreshold excitability, and regulate spike generation (Jentsch, 2000;Miceli et al., 2008). By reducing the steady-state inactivation of nodal Na v channels, the K v 7 channels increase the availability of transient Na v currents at nodes of Ranvier, thereby accelerating the AP upstroke and elevating short-term axonal excitability (Hamada and Kole, 2015). In the perisomatic region, K v 7 channels counteract the persistent Na v current and restrain repetitive firing (Pan et al., 2006;Cooper, 2011). Variants of KCNQ2/KCNQ3 or KCNQ4 genes cause developmental/epileptic disorders and hearing loss (Soldovieri et al., 2011;Miceli et al., 2013).

Expression and Function in MS
Bulk RNA-seq found upregulation of KCNQ2-3-5 transcripts in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq reported KCNQ2-3-5 expression in neuronal clusters and KCNQ3 expression in immune oligodendroglia (ImOLG) and microglia/macrophages clusters (Tables 1, 2; Jakel et al., 2019). K v 7.3 upregulation may reflect increased necessity of the channels along the axons because K v 7.3 subunit extensively redistributes to internodes of acutely and chronically demyelinated GM axons in the cuprizone model (Hamada and Kole, 2015). It is tempting to speculate that K v 7 upregulation may be beneficial during MS. First, K v 7 channels may increase the availability of transient Na v current via membrane hyperpolarization supporting AP conduction in demyelinated axons (Battefeld et al., 2014). Second, K v 7 channels may mitigate inflammation-induced neuronal excitability because, following LPS exposure, the I M inhibition underlies hyperexcitability of hippocampal neurons that is reversed by a nonselective K v 7-opener retigabine (Tzour et al., 2017). Although retigabine also exerts neuroprotective effects in several neurodegenerative conditions (Boscia et al., 2006;Nodera et al., 2011;Wainger et al., 2014;Bierbower et al., 2015;Li et al., 2019;Vigil et al., 2020;Wu et al., 2020), a clinical trial with retigabine analog flupirtine failed to demonstrate neuroprotective effects during MS (Dorr et al., 2018). Furthermore, blockade of K v 7 channels with XE-991 inhibited migration of LPS-treated pro-inflammatory microglia in vitro (Vay et al., 2020), suggesting that these channels may promote the pro-inflammatory role of microglia also during MS. Hence, neuronal and glial K v 7 channels may have diverse functions during MS.    K v 8.1 and K v 9.2 (KCNV1 and KCNS2) Neurons KCNV1 and KCNS2 genes encode for electrically silent (K v S) K v 8.1-and K v 9.2 subunits that assemble into hetero-tetrameric channels with K v 2 subunits (Bocksteins, 2016). A number of channelopathies is ascribed to K v S subunits (Salinas et al., 1997a;Liu et al., 2016;Allen et al., 2020), pointing to their important physiological role.

Expression and Function in MS
Bulk RNA-seq showed upregulation of KCNV1 and KCNS2 genes in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq detected KCNV1 and KCNS2 in neuronal clusters ( Table 2; Jakel et al., 2019). Co-assembly between K v 8.1 and K v 2.1 reduces K v 2.1 current density (Hugnot et al., 1996;Castellano et al., 1997): the high stoichiometry of the K v 8.1 subunit suppresses surface expression and favors retention of heteromeric channels in the ER (Salinas et al., 1997b). Neurons with reduced K v 2.1-mediated currents demonstrate broadened APs (Du et al., 2000) underlying hyper-synchronized highfrequency firing observed during epilepsy. Hence, upregulated K v S subunits in CA lesions may influence the localization of clustered K v 2 subunits in SPMS brain and affect AP firing and/or propagation.

Neurons
All EAG channels are expressed in the CNS neurons (Ludwig et al., 2000;Papa et al., 2003;Zou et al., 2003), but only ergmediated currents have been verified using suitable blockers (Bauer and Schwarz, 2018).

Expression and Function in MS
Bulk RNA-seq detected increased KCNH5(eag2) and KCNH7(erg3) transcripts in CA lesions and downregulation of KCNH8(elk1) transcript in all lesions and NAWM (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq found significant expression of KCNH5 and KCNH7 transcripts in neuronal clusters and KCNH8 in mature oligodendrocyte clusters (Tables 1, 2; Jakel et al., 2019). The functional role of eag2, erg3, and elk1 during MS may be related to altered neuronal excitability. Indeed, human eag1 and eag2 gain-of-function mutations underlie severe neurological disorders associated with epileptic seizures (Allen et al., 2020). The erg channels that are active at subthreshold potentials stabilize the V rest and dampen excitability (Fano et al., 2012). Erg3 knockdown in mice increases intrinsic neuronal excitability and enhances seizure susceptibility, while treatment with erg activator reduces epileptogenesis (Xiao et al., 2018). Erg3 expression is decreased in the brain of epilepsy patients. Remarkably, association of KCNH7(erg) intronic polymorphisms with MS pathogenesis was speculated although never substantiated (Martinez et al., 2008;Couturier et al., 2009).
Only TREK-1 transcripts were detected in microglia (Hammond et al., 2019). In astrocytes, TREK channels contribute to passive conductance and glutamate release (Zhou et al., 2009;Woo et al., 2012). TREK-1 and TREK-2 may be activated by a wide range of physiological and pathological stimuli reminiscent of inflammatory environment including membrane stretch, heat, intracellular acidosis, and cellular lipids (Ehling et al., 2015).

Expression and Function in MS
Bulk RNA-seq found upregulated TREK-1 transcripts in CA lesions, but a divergent modulation was observed for TREK-2 mRNAs in ALs (Figure 2, Tables 1, 2; Elkjaer et al., 2019;Frisch et al., 2020). KCNK2 and KCNK10 transcripts were detected in neuronal and oligodendrocyte clusters, but scarcely observed in astrocytes ( Table 2; Jakel et al., 2019). TREK-1 upregulation in CA lesions most likely reflects a protective response because TREK-1 plays a neuroprotective role during neurological diseases, including MS (Djillani et al., 2019). TREK-1 reduces neuronal excitability by hyperpolarizing the membrane potential (Honore, 2007) and is required for rapid AP repolarization at the node of Ranvier in mammalian afferent myelinated nerves, while TREK-1 loss-of-function retards nerve conduction and impairs sensory responses in animals (Kanda et al., 2019). Treatment of mice with TREK-1 activators, riluzole (Gilgun-Sherki et al., 2003), or alpha-linolenic acid attenuates EAE course (Blondeau et al., 2007), while these effects are reduced in TREK-1 −/− mice (Bittner et al., 2014). TREK-1 function is also important for non-neuronal cells because aggravated EAE course in TREK-1 −/− mice is associated with increased numbers of infiltrating T cells and higher endothelial expression of ICAM1 and VCAM1 (Bittner et al., 2013), and TREK-1 is reduced in the microvascular endothelium in inflammatory MS brain lesions (Bittner et al., 2013). TREK-2 downregulation in AL, a lesion type characterized by myelin breakdown and infiltration by inflammatory cells (Elkjaer et al., 2019;Frisch et al., 2020), may contribute to reduced glutamate and K + buffering and neuronal over-excitation because TREK-2 helps maintain the membrane potential and low extracellular glutamate and K + level during ischemia (Gnatenco et al., 2002;Rivera-Pagan et al., 2015).
Na + -and Ca 2+ -Activated K + Channels K Na 1.1 (KCNT1) Neurons The KCNT1 and KCNT2 genes encode for Slack and Slick K + channels that are activated by Na + influx (Bhattacharjee and Kaczmarek, 2005). They localize to soma and axons of neurons (Bhattacharjee et al., 2002;Brown et al., 2008;Rizzi et al., 2016) and are involved in the generation of slow afterhyperpolarization, regulation of firing patterns, and setting and stabilizing the V rest (Franceschetti et al., 2003). Alterations in KCNT1 and KCNT2 genes are linked to early-onset epileptic encephalopathies and Fragile-X-syndrome (Kim and Kaczmarek, 2014).
The KCNN3 gene encodes for the SK3 subunit of smallconductance Ca 2+ -activated K + channels (SK channels). They mediate Ca 2+ gated K + current and thus couple the increase in intracellular Ca 2+ concentration to hyperpolarization of the membrane potential.

Neurons
SK3 channels are found on dendrites and AIS (Abiraman et al., 2018). They play a role in AP propagation and regulation of neuronal excitability (Stocker, 2004). They protect against excitotoxicity by maintaining Ca 2+ homeostasis after NMDA receptor activation (Dolga et al., 2011).

Inward Rectifier K + Channels (K ir )
KCNJ gene family encodes K ir channels and comprises 16 subunits of K ir 1-K ir 7 subfamilies categorized into four groups: (1) classical (K ir 2.x); (2) G-protein-gated (K ir 3.x); (3) ATPsensitive (K ir 6.x); and (4) K + -transport channels (K ir 1.x, K ir 4.x, K ir 5.x, K ir 7.x) (Hibino et al., 2010). At a comparable driving force, K ir channels allow greater influx than efflux of K +ions. Their high open probability at negative transmembrane voltages makes them well-suited to set the V rest and to control cell excitability.

Neurons
K ir 3.1/K ir 3.2 hetero-tetramers are found in the somatodendritic compartment of neurons. Activation of GIRK channels is mediated by G-protein-coupled receptors including muscarinic, metabotropic glutamate, somatostatin, dopamine, endorphins, endocannabinoids, etc. GIRK channels are important for K + homeostasis and maintenance of V rest near the K + equilibrium potential. GIRK current hyperpolarizes neuronal membrane reducing spontaneous AP firing and inhibiting neurotransmitter release (Luscher and Slesinger, 2010). GIRK signaling contributes to learning/memory, reward, pain, anxiety, schizophrenia, addiction, and other processes (Mayfield et al., 2015). K ir 3.2 mutations in mice lead to a loss of K + selectivity and increased Na + permeability of the channel, resulting in the weaver phenotype (Liao et al., 1996;Surmeier et al., 1996).

Expression and Function in MS
RNA-seq revealed KCNJ6 upregulation in the CA lesions (Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq predominantly found KCNJ6 transcripts in neuronal clusters (Jakel et al., 2019; Table 1). The functional role of K ir 3.2 channels in MS may be related to membrane hyperpolarization and compensation of excessive neuronal excitability driving neurodegeneration.

Neurons
In cultures, K ir 5.1 immunoreactivity was detected in somatodendritic compartments where PSD-95 immunoreactivity was also localized. The K ir 5.1/PSD-95 complex may exist at dendritic spines in vivo and play a role in synaptic transmission (Tanemoto et al., 2002).

Expression and Function in MS
Bulk RNA-seq revealed K ir 5.1 upregulation in CA lesions ( Table 1; Elkjaer et al., 2019;Frisch et al., 2020). SnRNA-seq detected K ir 5.1 in OPCs clusters and scarcely in astrocytes. The KCNJ16 gene is upregulated during demyelination and acute remyelination in mouse cuprizone model (Martin et al., 2018). Upregulation of K ir 5.1 may reflect the role of the oligodendroglial K ir 4.1/K ir 5.1 channels in K + clearance during MS and may represent a mechanism to compensate K ir 4.1 reduction in MS brain (Schirmer et al., 2014). Alternatively, K ir 5.1 upregulation may underlie reduced K ir 4.1 function in MS because presence of K ir 5.1 subunit confers loss of functional activity to K ir 4.1/K ir 5.1 channels under oxidative stress (Jin et al., 2012).
Voltage-Gated Na + Channels (Na v ) In the mammalian brain, Na v are composed of α-subunit (260 kDa) and one or several β-subunits (β1-β4, of 33-36 kDa) (Goldin et al., 2000). The α-subunit forms the channel pore and acts as a voltage sensor; β-subunits play a modulatory role and influence voltage dependence, gating kinetics, and surface expression of the channel (Goldin et al., 2000;Yu and Catterall, 2003;Namadurai et al., 2015). The nine Na V 1.1-Na V 1.9 α-subunits are encoded by the corresponding genes SCN1A-SCN5A and SCN8A-SCN11A. In addition, Na X isoform was described,which is encoded by the SCN6/7A gene.

Na V 1.1 (SCN1A) Neurons
Na V 1.1 channels localize to the somatodendritic compartment of principal neurons and AIS of GABAergic interneurons, spinal cord motor neurons, and retinal neurons (Ogiwara et al., 2007;Duflocq et al., 2008;Dumenieu et al., 2017). Na V 1.1 channels are also present at the nodes of Ranvier of the cerebellar WM, fimbria, corpus callosum, and spinal cord WM (Ogiwara et al., 2007;Duflocq et al., 2008;O'Malley et al., 2009). They play a role during saltatory conduction along myelinated axons and are essential for maintaining the sustained firing of GABAergic interneurons and Purkinje cells, thus controlling the excitability of neuronal networks (Duflocq et al., 2008;Dumenieu et al., 2017). Mutations in Na V 1.1 channels result in various types of epilepsy and reduced volume of brain GM and WM (Lee et al., 2017;Scheffer and Nabbout, 2019).

Glia
Human astrocytes show negligible immunolabelling for Na V 1.1 and no upregulation in the WM of MS patients (Black et al., 2010). Transcriptome analysis revealed low level of SCN1A in mouse cortical and hippocampal astrocytes (Batiuk et al., 2020). RNA-seq detected SCN1A in oligodendrocytes and OPCs throughout the CNS (Larson et al., 2016;Marques et al., 2016;Falcao et al., 2018). The functional role of Na V 1.1 channels in astrocytes and oligodendroglia remains unknown. Transcriptome studies have not detected SCN1A in microglia prepared from brain homogenates (Hammond et al., 2019), but Na V 1.1 protein was found in microglia derived from neonatal rat mixed glial cultures (Black et al., 2009). Na V 1.1 channels may be involved in regulation of phagocytosis and/or release of IL-1α, IL-β, and TNF-α from microglia (Black et al., 2009). The Na V 1.1 mRNA was detected in astrocytoma, oligodendroglioma, and glioblastoma samples from patients where these channels may contribute to the pathophysiology of brain tumors (Schrey et al., 2002).

Expression and Function in MS
Bulk RNA-seq detected SCN1A upregulation in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq revealed significant expression of Na V 1.1 transcripts in neuronal, committed OPC, and OPC clusters (Tables 1, 2; Jakel et al., 2019). Experimental models do not provide clues regarding the functional role of Na V 1.1 channels in MS: Na V 1.1 expression was increased or unaltered in the optic nerve during EAE (Craner et al., 2003;O'Malley et al., 2009), while in the spinal cord, these channels clustered at the nodes of Ranvier and localized along the demyelinated regions (O'Malley et al., 2009). SCN1A upregulation in human MS may reflect the necessity of the channel for redistribution along the demyelinated axons and support of AP propagation.
The Na V 1.2 channels localize to the AIS, immature nodes of Ranvier, and in non-myelinated axons during early development.
As nervous system matures, Na V 1.2 channels are replaced by Na V 1.6 channels (Boiko et al., 2001;Osorio et al., 2005;Dumenieu et al., 2017), although in some neurons, they remain into adulthood. Na v 1.2 channels of the AIS control backpropagation of APs into the somatodendritic compartment, while Na V 1.6 channels are being placed at distal parts of the AIS control initiation and propagation of AP into the axon (Boiko et al., 2003;Hu et al., 2009). Na V 1.2 channels are also diffusely distributed along non-myelinated axons in the adult CNS where they may support slow spike propagation (Arroyo et al., 2002;Dumenieu et al., 2017).

Glia
Na V 1.2 protein was found in rat astrocytes isolated from the spinal cord and optic nerve (Black et al., 1995), but only limited Na V 1.2 expression was observed in human astrocytes in control and MS tissue (Black et al., 2010). The RNA-seq detected SCN2A expression in oligodendrocytes and OPCs (Larson et al., 2016;Marques et al., 2016). Knockdown of Na V 1.2 in preoligodendrocytes of the auditory brainstem resulted in reduced number and length of cellular processes and decreased MBP level, indicating that Na V 1.2 channels are important for structural maturation of myelinating cells and myelination (Berret et al., 2017). Microglia expresses no/little functional Na V 1.2 channels (Black et al., 2009;Pappalardo et al., 2016;Hammond et al., 2019).

Expression and Function in MS
Bulk RNA-seq detected upregulation of SCN2A gene in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq showed abundant SCN2A expression in neuronal clusters (Tables 1, 2; Jakel et al., 2019). The upregulation may reflect re-expression of Na V 1.2 protein, in line with previous reports showing diffuse distribution of Na V 1.2 channels along the demyelinated axons in human MS lesions within optic nerve and spinal cord (Craner et al., 2004b). Axonal Na V 1.2 channels may contribute to preservation of AP propagation and re-establishment of myelin sheathes (Coman et al., 2006), as it occurs during development. On the other hand, Na V 1.2 channels may promote axonal damage by increasing the intracellular Na + concentration that triggers reversal of Na + /Ca 2+ exchanger (NCX) and Ca 2+ overload in the axoplasm (Friese et al., 2014;Schattling et al., 2016). In line with this, human gain-of-function mutation in the mouse SCN2A gene triggers axonal damage, neurodegeneration, disability, and lethality in the mouse model of MS (Schattling et al., 2016). Expression of "developmental" Na V 1.2 channels in axons was also found in animal models of MS, i.e., in adult Shiverer mice that lack myelin (Westenbroek et al., 1992;Boiko et al., 2001), in transgenic mice overexpressing proteolipid protein that initially have normal myelination but then lose myelin (Rasband et al., 2003), and in the demyelinated optic nerve and spinal cord during EAE (Craner et al., 2003(Craner et al., , 2004aHerrero-Herranz et al., 2008). However, other data showed that in chronic spinal cord MS lesions, Na V 1.2 channels localize on astrocytic processes surrounding the axons rather than on axons themselves (Black et al., 2007), and Na V 1.2 expression/distribution was unchanged in the spinal cord of myelin-deficient rats (Arroyo et al., 2002).

Na V 1.3 (SCN3A) Neurons
Na V 1.3 channels are highly expressed in rodent and human CNS throughout the embryonic development (Black and Waxman, 2013). Some studies reported that their expression decreases during the first weeks after birth, while others found Na V 1.3 immunoreactivity in GM and/or WM of adult rat and human brain (Whitaker et al., 2001;Lindia and Abbadie, 2003;Thimmapaya et al., 2005;Cheah et al., 2013). Na V 1.3 channels mainly localize to the somatodendritic compartment of neurons but were also detected along the axons including myelinated fibers where they may contribute to initiation and propagation of APs (Whitaker et al., 2001;Lindia and Abbadie, 2003;Cheah et al., 2013;Wang et al., 2017). In the developing brain, Na V 1.3 channels regulate proliferation and migration of cortical progenitors that do not fire APs (Smith et al., 2018).

Glia
The mRNA and Na V 1.3 protein were detected in astrocytes (Black et al., 1995). RNA-seq demonstrated SCN3A expression in oligodendroglial cells and suggested higher expression in OPCs vs. mature oligodendrocytes (Larson et al., 2016;Marques et al., 2016). Na V 1.3 expression in microglia was negligible or absent (Black et al., 2009;Hammond et al., 2019). Heterogeneous expression (from weak to strong) of Na V 1.3 mRNA occurred in human astrocytoma, oligodendroglial tumors, and glioblastoma (Schrey et al., 2002). Functions of Na V 1.3 channels in glia remain unknown.

Expression and Function in MS
Bulk mRNA-seq reported upregulation of SCN3A gene in the CA lesions (Elkjaer et al., 2019;Frisch et al., 2020). The snRNAseq found significant SCN3A expression in neuronal and OPCs clusters (Jakel et al., 2019; Tables 1, 2). SCN3A upregulation during MS may reflect augmented expression of Na V 1.3 protein in axons that is necessary for supporting/re-establishment of AP propagation in injured WM, because increased Na V 1.3 levels are known to be associated with higher neuronal firing. For instance, mRNA and Na V 1.3 protein were upregulated in spontaneously epileptic rats (Guo et al., 2008), and expression in hippocampal neurons of a novel coding variant SCN3A-K354Q resulted in enhanced Na v 1.3 currents, spontaneous firing, and paroxysmal depolarizing shift-like depolarizations of the membrane potential (Estacion et al., 2010). Na V 1.6 (SCN8A) Neurons Na V 1.6 channels cluster at high-density at the AIS and nodes of Ranvier of GM and WM axons, but can be also located on the soma, dendrites, and synapses although at a lower density Dumenieu et al., 2017;Johnson et al., 2017;Eshed-Eisenbach and Peles, 2020). The expression level of Na V 1.6 channels is low during development, but significantly increases as the nervous system matures (Boiko et al., 2001;Osorio et al., 2005;Dumenieu et al., 2017). In the adult CNS, Na V 1.6 channels are the major Na + channels responsible for initiation and propagation of APs (Boiko et al., 2003;Hu et al., 2009). Loss of Na v 1.6 activity results in decreased neuronal excitability, while gain-of-function mutations potentiate excitability (O'Brien and Meisler, 2013). SCN8A mutations in mice result in ataxia, tremor, and dystonia; in humans, SCN8A haploinsufficiency is associated with intellectual disability, while hyperactivity can contribute to pathogenesis of epileptic encephalopathy (O'Brien and Meisler, 2013;Meisler, 2019).

Glia
RNA-seq detected SCN8A transcripts in mouse oligodendrocyte lineage (Marques et al., 2016), but they were negligible in microglia (Hammond et al., 2019). Immunoreactivity for Na V 1.6 was observed in cultured spinal cord astrocytes and in brain microglia in vitro and in situ (Reese and Caldwell, 1999;Black et al., 2009;Black and Waxman, 2012;Hossain et al., 2013), but their functional role is unknown.

Expression and Function in MS
Bulk mRNA-seq found upregulation of SCN8A gene in CA lesions (Elkjaer et al., 2019;Frisch et al., 2020), while snRNAseq did not detect SCN8A transcripts (Jakel et al., 2019) (Tables 1, 2). Upregulation of SCN8A may reflect increased diffuse distribution of the channels along the demyelinated axons; it may be important for remyelination but may also contribute to axonal damage. Re-distribution of Na V 1.6 channels, in parallel to their loss from the nodes of Ranvier, was reported previously in chronic, active, and inactive MS plaques within cerebral hemisphere, cerebellum, and spinal cord WM tissue from MS patients (Craner et al., 2004b;Black et al., 2007;Howell et al., 2010;Bouafia et al., 2014), as well as in several CNS regions affected by demyelination in animal models, including optic nerve and spinal cord WM (Craner et al., 2003(Craner et al., , 2004aHassen et al., 2008;Howell et al., 2010). Expression of Na V 1.6 channels is disrupted at the nodes of Ranvier of WM axons in Shiverer mice that lack compact myelin (Boiko et al., 2001(Boiko et al., , 2003, and in transgenic mice overexpressing proteolipid protein that initially have normal myelination but then lose myelin (Rasband et al., 2003). During EAE in animals, Na V 1.6 co-localizes with NCX and may contribute to persistent Na + influx, increased Na + level in the axoplasm, reversal of NCX, and intra-axonal Ca 2+ overload leading to axonal damage (Craner et al., 2004a). Interestingly, robust increase in Na V 1.6 expression was detected also in microglia/macrophages and was associated with microglia activation and phagocytosis in human MS brain and in the EAE model (Craner et al., 2005). SCN8A deletion resulted in reduced inflammation and improved axonal health during EAE (Alrashdi et al., 2019). Hence, microglial Na V 1.6 may contribute to the pathophysiology of MS as well, yet, snRNA-seq did not detect SCN8A in WM glia clusters (Tables 1, 2).

Na V 1.9 (SCN11A) Neurons
Although Na V 1.9 channels are mainly expressed in sensory ganglia neurons (Wang et al., 2017), Na V 1.9 mRNA and/or protein were detected in soma and/or proximal processes of neurons in the olfactory bulb, hippocampus, cerebellar cortex, supraoptic nucleus, and spinal cord of rodents and humans (Jeong et al., 2000;Blum et al., 2002;Subramanian et al., 2012;Wetzel et al., 2013;Black et al., 2014;Kurowski et al., 2015). Information regarding axonal labeling for Na V 1.9 is lacking. Na V 1.9 channels regulate excitation in hippocampal neurons in concert with BDNF and TrkB, control activity-dependent axonal elongation in spinal cord motoneurons, and mediate sustained depolarizing current upon activation of M1 muscarinic receptors in cortical neurons (Blum et al., 2002;Subramanian et al., 2012;Kurowski et al., 2015). It is uncertain whether, similar to their role in the PNS (Cummins et al., 1999;Wang et al., 2017), Na V 1.9 channels contribute to the regulation of V rest and AP threshold in the CNS neurons.

L-Type VGCCs
The α1-subunit of L-type VGCCs is encoded by CACNA1S (Ca V 1.1), CACNA1C (Ca V 1.2), CACNA1D (Ca V 1.3), or CACNA1F (Ca V 1.4) genes. High sensitivity to dihydropyridine modulators distinguishes L-type Ca 2+ channels from other types of VGCCs. In the CNS, mainly Ca V 1.2 and Ca V 1.3 subunits are expressed (Lipscombe et al., 2004;Zamponi et al., 2015), but Ca V 1.1 subunit was detected in human and rat basal ganglia where it is co-expressed with RyRs in GABAergic neurons (Takahashi et al., 2003).
Ca V 1.2 channels open upon membrane depolarization beyond −30 mV, and mediate direct Ca 2+ entry from the extracellular space into the cytoplasm. In addition, they may act as voltage sensors, transducing membrane depolarization to the RyRs activation and subsequent Ca 2+ release from the ER via the mechanism of Ca 2+− induced Ca 2+ release (CICR) (Ouardouz et al., 2003;Micu et al., 2016;Vierra et al., 2019). Clustering and functional coupling of plasmalemmal Ca V 1.2 channels to RyRs of the ER is mediated by the K V 2.1 channels (Vierra et al., 2019).
Functional expression of Ca V 1.2 channels in microglia is still debated (Hopp, 2021). Sequencing data showed no/low CACNA1C expression in microglia (Hammond et al., 2019), and no Ca V 1.2 was found in cultured microglia even upon stimulation with TNF-α/IFN-γ (Schampel et al., 2017). However, increased immunolabelling for α1C-subunit of L-type VGCCs was observed in reactive microglia during excitotoxicity in rat hippocampus (Espinosa-Parrilla et al., 2015).

Expression and Function in MS
Bulk RNA-seq detected increased CACNA1C expression in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq showed significant CACNA1C expression in neuronal and pericyte clusters (Jakel et al., 2019; Tables 1, 2), while low expression in OPCs and astrocyte clusters. In mouse models of MS, application of L-type VGCCs blockers reduces brain and spinal cord WM damage, decreases mitochondrial pathology in nerve fibers, attenuates axonal loss, increases oligodendrocyte survival, and promotes remyelination (Brand-Schieber and Werner, 2004;Schampel et al., 2017;Ingwersen et al., 2018;Zamora et al., 2020). These findings suggest that Ca V 1.2 channels contribute to damage during MS. However, expression and activity of Ca V 1.2 channel increased in OPCs within the demyelinated lesions in the mouse corpus callosum after cuprizone treatment (Paez et al., 2012), and deletion of Ca V 1.2 specifically in OPCs resulted in reduced myelination and lower MBP and MOG expression (Santiago Gonzalez et al., 2017). Hence, activity of L-type channels in oligodendroglial lineage is crucial for remyelination in this MS model, but it is unclear whether oligodendroglial Ca V 1.2 channels also play a role during MS in humans. Upregulation of Ca V 1.2 channels in pericytes may reflect altered microcirculation in MS lesions, in analogy to the role of L-type VGCCs in pericytes outside the brain (Hashitani and Mitsui, 2019).

Ca V 1.3 (CACNA1D) Neurons
Ca V 1.3 channels localize primarily in neuronal cell bodies and dendrites in GM (Hell et al., 1993;Zhang et al., 2005) but were also found in the developing rat optic nerve, corpus callosum (Huang et al., 2012), and axons in spinal dorsal columns of adult rats where they form clusters with RyR2s (Ouardouz et al., 2003). Ca V 1.3 channels activate at the membrane potential of −55 mV (Lipscombe et al., 2004) and are important players in generating the pacemaking activity and spontaneous firing (Zuccotti et al., 2011). Ca V 1.3 channels control Ca 2+ -dependent post-burst after-hyperpolarization in CA1 pyramidal neurons, and their activity may trigger Ca 2+ -dependent intracellular signaling pathways (Gamelli et al., 2011;Striessnig et al., 2014). Ca V 1.3 channels may contribute to the mechanisms of memory because their increased expression correlates with memory loss during aging while their inhibition improves age-related memory deficits (Veng et al., 2003). Deletion of Ca V 1.3 channels results in increased firing rates of amygdala neurons (probably caused by a reduced slow component of post-burst after-hyperpolarization) and underlies altered fear consolidation in Ca V 1.3 knockout mice (McKinney et al., 2009). Ca V 1.3 channels are important for formation of cellular architecture: their various splice variants regulate morphology of dendritic spines while their deletion results in reduced morphology of axonal arbors (Hirtz et al., 2012;Stanika et al., 2016).

Glia
Ca V 1.3 mRNA and/or protein were detected in cultured or freshly isolated rat brain astrocytes; Ca V 1.3 channels may mediate intracellular Ca 2+ increase directly and via Ca 2+mediated activation of RyRs (Latour et al., 2003;Yan et al., 2013;Du et al., 2014;Enders et al., 2020). Ca V 1.3 expression increases in reactive astrocytes after status epilepticus in mice, suggesting that role in initiation, maintenance, or spread of seizures (Xu J. H. et al., 2007). Yet, other studies have not found Ca V 1.3 channels in astrocytes (D'Ascenzo et al., 2004).
Ca V 1.3 channels are expressed in cortical and hippocampal OPCs where they, in concert with other Ca 2+ channels, may mediate Ca 2+ entry from the extracellular space and/or trigger CICR from the ER (Haberlandt et al., 2011;Cheli et al., 2015). Knockdown of Ca V 1.3 reduces Ca 2+ influx but does not affect expression level of myelin proteins, proliferation, or morphological differentiation of OPCs (Cheli et al., 2015). In the adult rat spinal cord WM, Ca V 1.3 channels are expressed by APC-positive oligodendrocytes, may mediate oligodendrocyteaxon signaling, and/or contribute to Ca 2+ -dependent injury following trauma (Sukiasyan et al., 2009). Static magnetic stimulation may alter Ca V 1.3 gene expression level in human cultured OPCs (Prasad et al., 2017), suggesting that external manipulations may be a useful approach to modulate L-type VGCCs in oligodendroglial cells during diseases.

Expression and Function in MS
Bulk RNA-seq showed CACNA1D upregulation in CA lesions (Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq detected significant expression of CACNA1D in neuronal clusters (Jakel et al., 2019 ; Tables 1, 2). Administration of L-type VGCCs blockers resulted in multiple beneficial effects in animal MS models (see above), suggesting that Ca V 1.3 channels, perhaps in concert with Ca V 1.2 channels, contribute to tissue damage during MS.

Neurons
Ca V 2.1 channels localize on axonal synaptic terminals and play a fundamental role in neurotransmitter release: their direct interaction with the SNARE proteins and synaptotagmin is required for positioning the docked synaptic vesicles near the Ca 2+ channels for fast vesicular exocytosis (Rettig et al., 1996;Zamponi et al., 2015;Mochida, 2019). Ca V 2.1 channels are also present at somatodendritic compartments of neurons (Catterall, 2000;Zamponi et al., 2015;Mochida, 2019) where they colocalize with BK and SK channels and provide Ca 2+ for activation of these channels (Berkefeld et al., 2006;Indriati et al., 2013;Irie and Trussell, 2017). Ca 2+ enters through the Ca V 2.1 channels and triggers further Ca 2+ release from the intracellular stores upon activation of RyRs on the ER (Berkefeld et al., 2006;Indriati et al., 2013;Irie and Trussell, 2017). These mechanisms control neuronal firing even in the millisecond time scale (Irie and Trussell, 2017). Somatodendritic Ca V 2.1 channels regulate gene expression, local Ca 2+ signaling, and cell survival (Pietrobon, 2010).
Ca V 2.1 channels are also present in the WM, i.e., corpus callosum and developing optic nerve (Alix et al., 2008;Nagy et al., 2017). In the optic nerve, Ca V 2.1 channels are transiently clustered in the axolemma at the sites where the underlying vesicular and tubular elements are fusing with the axonal membrane (Alix et al., 2008). Some of these sites later become nodes of Ranvier, and mutations of the α1A-subunit results in malformation of the nodes of Ranvier (Alix et al., 2008). In the corpus callosum, Ca V 2.1 channels mediate fast release of glutamatergic vesicles at axon-OPC synapses, and blockade of these channels in slices reduces release at axon-glia synapses by 88% (Nagy et al., 2017).
Ca V 2.1 channels may play a role in nociception because inflammatory and neuropathic pain is altered in mice with deletion of Ca V 2.1 channels (Pietrobon, 2010). Mutations in the CACNA1A gene underlie familial hemiplegic migraine type 1, spinocerebellar ataxia type 6, and episodic ataxia type 2, and may be associated with increased risk of epilepsy (Pietrobon, 2010;Rajakulendran et al., 2012;Izquierdo-Serra et al., 2020).

Glia
RT-PCR detected α1A-subunit in mouse cortical astrocytes in culture, but Ca V 2.1 channels did not mediate Ca 2+ entry into astrocytes (Cheli et al., 2016b). Exposure of mouse primary astrocytes to β-Amyloid did not affect Ca V 2.1 transcript level (Daschil et al., 2014). However, increased expression of Ca V 2.1 channels was observed in reactive astrocytes after status epilepticus in mice, suggesting their role in initiation, maintenance, or spread of seizures (Xu J. H. et al., 2007). Ca V 2.1 channels are expressed in hippocampal OPCs, and in premyelinating oligodendrocytes of the brainstem (Haberlandt et al., 2011;Barron and Kim, 2019). In brainstem oligodendrocytes, opening of Ca V 2.1 channels is triggered upon depolarization mediated by glutamate (via AMPA receptors) or high K + , as well as upon electrical stimulation of axons (Barron and Kim, 2019), suggesting that Ca V 2.1 channels mediate Ca 2+ influx into the oligodendroglial cells upon neuronal activity in vivo. In this way, neuronal activity may trigger and/or modulate Ca 2+ -dependent signaling in oligodendroglial cells. RNA-seq detected CACNA1A gene in microglia (Hammond et al., 2019). Ca V 2.1 channels may contribute to glioblastoma progression because their inhibition reduced proliferation of glioblastoma cells, although to a lesser extent than blockade of N-type channels (Nicoletti et al., 2017).

Expression and Function in MS
Bulk RNA-seq found CACNA1A upregulation in CA lesions (Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq revealed significant expression of CACNA1A transcripts in neuronal and OPCs clusters (Jakel et al., 2019 ; Tables 1, 2). CACNA1A upregulation in MS may reflect the necessity to build new nodes of Ranvier on demyelinated axons within the CA lesions. In oligodendroglial cells, Ca 2+ entry through Ca V 2.1 channels may be required for activation of intracellular signaling pathways necessary for differentiation of OPCs and pre-myelinating oligodendrocytes.

Ca V 2.3 (CACNA1E), R-Type VGCCs Neurons
Ca V 2.3 channels are localized to the dendritic spines and pre-synaptically (Parajuli et al., 2012). Ca V 2.3-mediated Ca 2+ currents activate upon strong membrane depolarization and are distinguished by sensitivity to low NiCl 2 concentrations and SNX-482 toxin. Presynaptic R-type channels play a role in neurotransmitter release (Wu et al., 1999;Gasparini et al., 2001) and synaptic plasticity (Dietrich et al., 2003;Yasuda et al., 2003;Takahashi and Magee, 2009), but their efficiency in triggering neurotransmitter release may be lower compared to P/Q-or N-type VGCCs if they are placed distantly from vesicle release sites (Wu et al., 1999). Dendritic R-type channels are coupled to SK channels and provide Ca 2+ influx for their activation during excitatory postsynaptic potentials and back-propagating APs (Bloodgood and Sabatini, 2008;Jones and Stuart, 2013). The capacity of dendritic SK channels to promote generation of dendritic Ca 2+ spikes also depends on Ca V 2.3 activation (Bock et al., 2019). Besides, Ca 2+ influx via Ca V 2.3 channels may be necessary for activation of K v 4.2 channels (Wang et al., 2014). The Ca V 2.3 channels also form complexes with BK channels, and this functional interaction modulates AP properties and shortterm plasticity in hippocampal neurons (Gutzmann et al., 2019). Studies in KO mice revealed that Ca v 2.3 channels are involved in the mechanisms of sleep modulation, fear response, pain, and seizures (Saegusa et al., 2000;Lee et al., 2002;Weiergraber et al., 2007;Siwek et al., 2014;Zamponi et al., 2015;Wormuth et al., 2016). Deletion of Ca V 2.3 channels in mice resulted in larger infarct size after middle cerebral artery occlusion in vivo and larger Ca 2+ entry into the cells upon oxygen-glucose deprivation in slices, suggesting that Ca V 2.3 channels are protective during ischemic tissue damage (Toriyama et al., 2002).

Expression and Function in MS
Bulk RNA-seq showed CACNA1E upregulation in CA lesions (Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq found significant expression of CACNA1E transcripts in neuronal clusters (Jakel et al., 2019 ; Tables 1, 2). The functional role of Ca V 2.3 channels in MS is unknown.
Cav3.1, Ca3.2, and Ca3.3 (CACNA1G, CACNA1H, and CACNA1I) Neurons Ca v 3 isoforms display distinct distribution patterns with prominent somatodendritic expression in thalamic and hippocampal neurons (McKay et al., 2006). Ca 2+ imaging and pharmacological experiments showed that Ca v 3.2 and Ca v 3.3 subtypes located in the AIS influence the generation and the timing of APs (Bender and Trussell, 2009;Kole and Stuart, 2012). In rodent WM, Ca v 3 transcripts were detected at low level (Aguado et al., 2016), and information on cellular distribution is lacking.

Glia
Some studies detected Ca v 3.1 transcripts and proteins in rat cortical astrocytic cultures (Latour et al., 2003), while others found only scarce Ca v 3.1 expression in cultured astrocytes (Cheli et al., 2016b;Kim et al., 2018). Divergent findings showed that Ca v 3.2 immunoreactivity was absent (Chen et al., 2015) or present (Li et al., 2017) in rat spinal cord astrocytes. Ca v 3.1 and Ca v 3.2 transcripts were detected in clonal oligodendroglial CG4 cell line (Rui et al., 2020) and in OPCs isolated from mouse cortex (Zhang et al., 2014) or hippocampal slices (Haberlandt et al., 2011). In microglia, RNA-seq did not detect the Ca v 3 isoforms (Hammond et al., 2019).

Expression and Function in MS
Bulk RNA-seq revealed upregulation of Ca v 3.2 and Ca v 3.3 genes in CA lesions and upregulation of Ca v 3.1 in ILs (Elkjaer et al., 2019;Frisch et al., 2020; Table 1). The snRNA-seq detected Ca v 3.1and Ca v 3.3 transcripts in neuronal clusters, while it did not detect the Ca v 3.2 (Jakel et al., 2019; Tables 1, 2). Genome-wide sequencing identified significant association of a Ca v 3.2 mutation (CACNA1Hp.R1871Q) with patients suffering relapsing-remitting MS (Sadovnick et al., 2017). Ca v 3 upregulation in MS lesions may be triggered by inflammatory mediators and may contribute to axonal dysfunction. Indeed, prostanoids and hydrogen sulfide modulate Ca v 3.2 expression and function, and increased Ca v 3.2 channel activity and axonal accumulation is associated with inflammation and pain (Sadovnick et al., 2017;Chen et al., 2018). T-type currents contribute to Ca 2+ -mediated injury of spinal cord WM axons triggered by anoxia (Imaizumi et al., 1999) and to peripheral nerve injury (Watanabe et al., 2015). L/T-type VGCC blocker lomerizine prevents retinal ganglion cell death after diffuse axonal injury (Karim et al., 2006).
Animal studies suggest that Ca v 3.1 upregulation in IL, a lesion type with complete demyelination and substantial axonal loss, may play a detrimental role. Specifically, the Ca v 3.1deficient mice are markedly resistant to EAE induction, and this effect may be mediated by lower production of granulocytemacrophage colony-stimulating factor (a cytokine implicated in EAE susceptibility) by CNS-infiltrating Th1 and Th17 cells (Wang et al., 2016). The Ca v 3.1 subunit is a functionally predominant T-type channel in CD4 + T cells (Trebak and Kinet, 2019). The Ca v 3.1-mediated Ca 2+ increase is critical for calcineurin-NFAT activation driving transcription of cytokines in T cells, and T cells from Ca v 3.1-deficient mice show decreased IL-17A, IL-17F, and IL-21 production. The development of isoformspecific modulators should help in establishing the differential role of Ca v 3 subtypes in MS lesions.

Expression and Function in MS
Bulk RNA-seq found upregulation of RyR2 transcripts in CA lesions and downregulation of RyR3 in ILs (Table 1; Elkjaer et al., 2019;Frisch et al., 2020). The snRNA-seq revealed significant expression of RyR2 in neuronal clusters and of RyR3 in the astrocyte1 cluster (Table 1; Jakel et al., 2019). RyR subunits probably play a differential role in perturbed intracellular Ca 2+ homeostasis in WM cells of SPMS brain. RyR2 in CA lesions may contribute to axonal dysfunction because intraaxonal Ca 2+ overload mediated by RyRs and IP3Rs activates the mitochondrial permeability transition pore and contributes to axonal dieback and degeneration following WM ischemic injury (Ouardouz et al., 2003;Stirling and Stys, 2010;Kesherwani and Agrawal, 2012) and SCI (Stirling et al., 2014;Liao et al., 2016). The RyRs inhibitor ryanodine significantly attenuates mitochondrial dysfunction (Villegas et al., 2014), axonal dieback, and secondary axonal degeneration in injured WM (Thorell et al., 2002;Stirling et al., 2014;Orem et al., 2017). In line, mice with RyR2 gain-of-function mutation exhibit more axonal damage than wild-type controls following SCI (Stirling et al., 2014), while RyR2 knockdown attenuates mitochondrial dysfunction and ER stress and improves functional recovery (Liao et al., 2016).
Functional RyR3s may contribute to astrocyte migration in response to injury, which is important for tissue remodeling and wound healing. In fact, RyR3s control astrocyte motility because astrocytes from RyR3 KO mice display reduced migratory activity (Matyash et al., 2002). Conversely, RyR3 downregulation in ILs may influence the formation of dense astrocytic scar imposing a major barrier to axonal and myelin regeneration. RyR3s also contribute to intracellular Ca 2+ transients during OPCs differentiation, while RyR3 inhibition prevents OPCs development . Interaction between RyRs and NCX in oligodendrocyte processes may represent an amplification mechanism to generate Ca 2+ transients required for oligodendrocyte differentiation in vitro (Casamassa et al., 2016;Hammann et al., 2018;de Rosa et al., 2019;Boscia et al., 2020). However, it remains unclear whether these mechanisms play a role in human MS. The development of selective modulators will help to establish function of RyRs in MS.

TRP Channels
Transient receptor potential (TRP) channels are tetrameric nonselective cation channels which encompass 30 different types (Nilius and Owsianik, 2011). Upon TRP channel activation, the membrane potential depolarizes, leading to activation or inactivation of voltage-gated ion channels and regulation of Ca 2+ signaling (Gees et al., 2010). Various intracellular or extracellular stimuli, including chemical and osmotic stress, can trigger activation of TRP channels (Clapham, 2003). TRP channels are involved in pain, regulation of neurotransmitter release, and immune functions. Vanilloid TRP channels (TRPV), melastatin TRP channels (TRPM), and polycystin TRP channels (TRPP) have been detected in WM lesions of patients with progressive MS.

Expression and Function in MS
Bulk RNA-seq showed significant TRPV1 downregulation in CA lesions (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq barely detected TRPV1 ( Table 2; Jäkel and Williams, 2020). The downregulated TRPV1 in CA lesions may influence neural plasticity and glia response both in the hypocellular inactive demyelinated core and in the hypercellular rim filled with activated glia. However, it is unclear whether dysfunctional TRPV1 has pro-and anti-inflammatory roles, and whether it favors or prevents CA lesion expansion and progression, because experimental findings are inconsistent. In rodents, administration of TRPV1 agonists reduced EAE severity (Tsuji et al., 2010), while the TRPV1 antagonist capsazepine, although ineffective for EAE severity (Paltser et al., 2013), reversed the beneficial effects of the endocannabinoid uptake inhibitor (Cabranes et al., 2005). Beneficial effects of TRPV1 may be mediated by its ability to promote micro-vesicle release from microglia, which enhances glutamatergic transmission in neurons (Marrone et al., 2017). However, on the other hand, TRPV1 stimulation induces the pro-inflammatory phenotype of microglia while downregulation promotes the anti-inflammatory phenotype (Hassan et al., 2014;Marrone et al., 2017). TRPV1 also regulates microglia migration, cytokine production, ROS generation, phagocytosis, and death (Kim et al., 2006;Schilling and Eder, 2009;Miyake et al., 2015). Furthermore, TRPV1 mediates migration and chemotaxis of astrocytes, their activation during stress and injury (Ho et al., 2014), and inflammasome activation. The picture becomes even more complex because TRPV1-KO mice show higher lethality during EAE peak but better recovery in the chronic stage (Musumeci et al., 2011). In addition, genetic deletion of TRPV1 in mice resulted in significant protection in the MOG-EAE model, and less severe breakdown of BBB (Paltser et al., 2013). Interestingly, patients with severe MS progression show over-representation of singlenucleotide polymorphisms (SNPs) in the TRPV1 gene (Paltser et al., 2013) that can affect the expression and activity of the channel and cortical excitability, and modulate pain Mori et al., 2012;Stampanoni Bassi et al., 2019).

TRPV6
TRPV6 channels are distinguished by high Ca 2+ selectivity (van de Graaf et al., 2006) and constitutive activity at low intracellular Ca 2+ levels and V rest (Vennekens et al., 2000). TRPV6 channels can form homo-or hetero-tetramers. TRPV5-6 are mainly expressed in epithelial and bone cells .

Neurons and Glia
In the mouse brain, TRPV6 channels are expressed in neurons, while transcripts were detected in astrocytes by RNA-seq (Riccio et al., 2002;Nijenhuis et al., 2003;Batiuk et al., 2020).

Expression and Function in MS
Bulk RNA-seq found TRPV6 downregulation in all MS lesion types and in NAWM (Figure 2, Table 1; Elkjaer et al., 2019;Frisch et al., 2020), but snRNA-seq failed to detect TRPV6 transcripts ( Table 2; Jakel et al., 2019). Little is known about the functional role of TRPV6 in brain cells. However, TRPV6 deletion in trophoblasts correlates with altered extracellular matrix (ECM) formation in the labyrinth during pregnancy (Winter et al., 2020). Hence, it will be important to investigate whether TRPV6 downregulation contributes to ECM alterations observed in SPMS lesions and believed to be a key remyelinationinhibiting factor.

Expression and Function in MS
Bulk RNA-seq showed increased TRPM2 expression in the ILs ( Table 1; Elkjaer et al., 2019;Frisch et al., 2020). SnRNA-seq found TRPM2 in neuronal, microglia, and ImOLG clusters. The functional role of TRPM2 channels in ILs, lesions that display reduced microglia density, axonal loss, and upregulation of stress response genes (Elkjaer et al., 2019;Frisch et al., 2020), may be related to neuronal and microglia damage. Indeed, TRPM2 channel is upregulated by diverse pathological stimuli (Malko et al., 2019) and is an important element during oxidative stress, mitochondrial dysfunction (Freestone et al., 2009), and neurodegenerative disorders (Chung et al., 2011). Constitutive TRPM2 activation is triggered by ROS and leads to pathological Ca 2+ signaling and cell death (Eisfeld and Luckhoff, 2007;Naziroglu and Luckhoff, 2008). Knockout of TRPM2 gene in mice, or blocking the channels with miconazole, improves pathological outcome in EAE and attenuates painful behavior (Melzer et al., 2012;So et al., 2015;Tsutsui et al., 2018). TRPM2-KO mice show reduction of CXCL2 chemokine production by CNS-infiltrating macrophages and suppressed neutrophil infiltration of the brain tissue (Tsutsui et al., 2018). These findings suggest that TRPM2 may represent a promising target in SPMS.

Expression and Function in MS
Bulk RNA-seq detected significant downregulation of PKD2 or PKD2L2 in ILs and CA lesions, respectively (Elkjaer et al., 2019;Frisch et al., 2020) (Figure 2, Table 1). SnRNA-seq detected PKD2 transcripts in neuronal and glia clusters but did not detect PKD2L2 ( Table 2). It is unclear whether TRPP downregulation in MS lesions is beneficial or detrimental. On one hand, it may be detrimental because TRPP1 and TRPP3 channels are important for maintaining Ca 2+ homeostasis and contribute to cell proliferation (Xiao and Quarles, 2010;, while TRPP1 knockdown results in increased susceptibility to stress-induced cell death in kidney epithelial cells (Brill et al., 2020). On the other hand, overexpression of TRPP contributes to apoptosis (Xiao and Quarles, 2010;, and TRPP1 is upregulated as a direct consequence of ER and oxidative stress during pathological conditions.

Chloride Channels
ClC channels mediate voltage-dependent transmembrane transport of Cl − . They are expressed in plasmalemma and intracellular membranes forming transmembrane dimers (Weinreich and Jentsch, 2001). ClC proteins can function as Cl − channels or as Cl − /H + exchangers. ClCs regulate V rest in skeletal muscle, trans-epithelial Cl − reabsorption in kidneys, and intracellular pH and Cl − concentration through coupled Cl − /H + exchange in several cell types including brain cells.

Neurons
CIC-2 localizes on inhibitory interneurons and regulates GABA A receptor-mediated synaptic inputs from basket cells (Foldy et al., 2010). Cl − extrusion by ClC-2 following hyperpolarization ensures the maintenance of low intracellular Cl − concentration following synaptic inhibition (Foldy et al., 2010). The link of ClC-2 mutations with generalized epilepsies in humans suggests an important role of ClC-2 in regulating neuronal excitability (Kleefuss-Lie et al., 2009).

Expression and Function in MS
Bulk RNA-seq showed significant CLCN2 downregulation in CA lesions (Elkjaer et al., 2019;Frisch et al., 2020; Figure 2, Table 1). SnRNA-seq detected CLCN2 transcripts in oligodendrocyte clusters, while they were only faintly observed or absent in other clusters ( Table 2). Several findings suggest that CLCN2 downregulation in MS may reflect altered WM integrity and/or contribute to the mechanisms of myelin destruction: first, ClCN2 −/− mice exhibit abnormal WM morphology (Blanz et al., 2007); second, loss-of-function CLCN2 mutations lead to leukodystrophy; third, loss of cell adhesion molecule GlialCAM, which binds to ClC-2 in glia, is associated with leukodystrophy (Jeworutzki et al., 2012;Hoegg-Beiler et al., 2014). Of note, though, is a recent report showing that leukodystrophy fully develops only when ClC-2 is disrupted in both astrocytes and oligodendrocytes (Goppner et al., 2020). It remains to be investigated whether CLC-2 loss in glia contributes to the failure of myelin repair in human CA lesions.

Connexins
Connexins (Cxs) are transmembrane proteins with channel and non-channel functions. Channel functions include the formation of gap junctions (GJs) and hemichannels (HCs) (Saez et al., 2003;Wang et al., 2013;Gajardo-Gomez et al., 2016), while nonchannel functions involve adhesion properties and intracellular signaling (Zhou and Jiang, 2014;Leithe et al., 2018). More than 20 Cxs genes have been described in humans, and 11 of them are expressed in the brain (Willecke et al., 2002;Theis et al., 2005). Cxs are essential players in ionic homeostasis, intercellular Ca 2+ signaling and Ca 2+ waves propagation, gliotransmission, synaptic transmission and plasticity, brain metabolism, brainblood barrier development and integrity, and myelination (Takeuchi and Suzumura, 2014). In the WM, GJs are essential for K + buffering in response to neuronal activity, they facilitate transport of nutrients and ions from oligodendrocyte soma to myelin layers and from astrocytes to oligodendrocytes (Bradl and Lassmann, 2010). In the WM, HCs are involved in metabolic coupling and energy supply to neurons, and provide a major pathway for glucose entry into OPCs and oligodendrocytes (Niu et al., 2016).

Cx37 (GJA4)
Cx37, encoded by GJA4 gene, predominantly builds heterotypic GJs with Cx40 and Cx43 in vascular cells and plays an essential role in vasomotor activity, endothelial permeability, and maintenance of body fluid balance (Falcao et al., 2018;.

Expression and Function in MS
Bulk RNA-seq revealed significant GJA4 upregulation in ILs (Elkjaer et al., 2019;Frisch et al., 2020), while snRNA-seq showed high GJA4 expression in pericyte cluster (Jakel et al., 2019; Tables 1, 2). In chronically demyelinated axons, as those within ILs, hypoxia due to imbalance between increased energy demand and reduced ATP production because of mitochondrial dysfunction may drive angiogenesis. However, while providing trophic factors for tissue remodeling, angiogenesis may contribute to hypoperfusion and neurovascular uncoupling (Girolamo et al., 2014). Interestingly, Cx37 knockdown with siRNA in human umbilical vein endothelial cells diminishes capillary branching (Gartner et al., 2012), but Cx37 −/− mice develop a more extensive vasculature under ischemic conditions and show enhanced recovery after hind limb ischemia (Fang et al., 2011). In the future, it will be important to investigate whether Cx37 protein contributes to aberrant cerebrovascular and angiogenic responses in human ILs during MS.

Pannexins
The Pannexin (Px) family consists of three members, encoded by Panx1, Panx2, and Panx3 genes. Pannexins do not form GJ in vivo but operate as plasma membrane channels (pannexons) and participate in paracrine and autocrine signaling in brain GM and WM (Sosinsky et al., 2011;Sahu et al., 2014;Dahl, 2015).

Expression and Function in MS
Bulk RNA-seq showed significant PANX1 upregulation in ILs ( Table 1; Elkjaer et al., 2019;Frisch et al., 2020), but snRNAseq did not detect PANX1 transcripts (Jakel et al., 2019). ILs are lesions with little/no inflammatory activity but with sharply demarcated hypocellular areas of demyelination and axonal degeneration. Px1 activation is known to enable ATP release, and ATP is a "find me" signal promoting chemotaxis of microglia/macrophages to the injury site for fast clearance of dead cells and a molecule important for myelination (Chekeni et al., 2010;Gajardo-Gomez et al., 2016). Hence, PANX1 upregulation in ILs may be a compensatory mechanism that stimulates glial activity. On the other hand, upregulated Px1 mRNA expression in cerebellum and spinal cord in chronic EAE contributes to WM damage (Lutz et al., 2013). Uncontrolled opening of P2X 7 R-Px1 complex in response to demyelination triggers excessive glutamate and ATP release, altered Ca 2+ dynamics, excitotoxicity, damage of axons, and myelin (Orellana et al., 2011;Crespo Yanguas et al., 2017). Knockout or blockade of Px1 with probenecid in rodents restrains EAE symptoms and results in reduced inflammation and decreased oligodendrocyte damage (Hainz et al., 2017), suggesting that Px1 activity supports damage during MS. More studies are required to establish how Px1 should be modulated in order to halt neurodegeneration during MS.

Expression and Function in MS
Bulk RNA-seq found downregulation of the auxiliary subunit gamma (CASPERG) and epsilon (CATSPERE) in CA lesions and ILs, respectively ( Table 1; Elkjaer et al., 2019;Frisch et al., 2020). SnRNA-seq did not find CATSPERE transcripts and barely detected CATSPERG in neuronal and glia clusters. It is difficult to speculate on the role of CatSper channels in MS lesions because characterization of these subunits is limited to sperm cells, and no data on CatSper protein expression or function in the brain are available.

CONCLUSIONS
Understanding how distinct ion channels regulate CNS ionic homeostasis in WM neurons, axons, glia, and vascular cells under chronic demyelinating conditions is of critical importance for the development of novel therapeutic strategies to prevent neurodegeneration and disability progression and improve functional recovery and repair in MS. Recent Bulk RNA-seq (Elkjaer et al., 2019;Frisch et al., 2020) revealed a considerable number of ion channel genes that are altered in different types of WM lesions of the SPMS brain, particularly in WM CA lesions, a type of lesion that develops in MS patients despite disease-modifying therapy and predicts a more aggressive disease course (Absinta et al., 2019;Elliott et al., 2019). SnRNA-seq found that transcripts for dysregulated ion channels belong to the clusters of neurons, astrocytes, oligodendrocyte lineage, microglia/macrophages, and pericytes (Jakel et al., 2019). The dysregulation of ion channel genes in MS may be detrimental or beneficial for functions of neurons, including interstitial neurons. Intense upregulation of genes encoding voltage-gated Na + channels in CA lesions may reflect the imbalance of Na + homeostasis observed in SPMS brain (Inglese et al., 2010). Conversely, the upregulation of a large number of voltagegated K + channel genes may be linked to a protective response to limit neuronal excitability. The altered Cl − homeostasis, revealed by the significant downregulation of voltage-gated Cl − channels in MS lesions, may contribute to an altered inhibitory neurotransmission and increased excitability. Depending on the type of alterations, dysregulated ion channels in MS may favor AP propagation and dampen neuronal hyperexcitability or, on the contrary, may contribute to axonal dysfunction and cell death. Altered expression and/or function of ion channels may also influence key properties of glia including proliferation, migration, spatial buffering, cytokine release, cell metabolism, myelin repair, angiogenesis, BBB permeability, and several other important functions.
We described the importance of uniquely dysregulated genes well-known to play a role in WM dysfunction in the MS brain (KCNA1, KCNA2, SCN2A, and SCN8A), or in experimental models of MS (KCNC3, KCNQ3, KCNK2, CACNA1C, CACNA1G, TRPV1, TRPM2, and PANX1). Furthermore, we highlighted the importance of ion channel genes that are uniquely dysregulated in SPMS lesions but have never been previously explored in MS brain. Those genes are expressed in OPC (KCND2, SCN1A, SCN3A, and CACNA1A), ImOLG (KCNQ3), mature oligodendrocyte (KCNH8), microglia (KCNQ3), astrocyte (KCNN3 and RYR3), and pericyte (GJA4 and CACNA1C) clusters of healthy and SPMS brain. It remains to be investigated whether and how the ionic imbalance in different glial cells, particularly oligodendroglia, contributes to impaired recovery and failure of myelin repair.
Several genes, including KCNA1, SCNA8, SCN11A, CACNA1H, PKD2L2 TRPV6, PANX1, and CATSPERE transcripts, were detected in bulk transcriptome (Elkjaer et al., 2019;Frisch et al., 2020), but were not found by snRNA-seq (Jakel et al., 2019). This discrepancy may be explained by several observations: (1) the transcriptional profiling may vary when lesions analyzed by different studies come from different WM regions (Jäkel and Williams, 2020); (2) snRNA-seq analysis lacks information on gene expression in WM axons that may also contain ion channel transcripts; and (3) snRNA-seq only includes RNA transcripts from the nucleus and may therefore lack RNA transcripts from cytoplasm.
Future experiments on dysregulated ion channels predicted by transcriptomic analysis are expected to provide a better understanding of the molecular mechanism of MS progression and may pave the way for the identification of new therapeutic targets to limit lesion expansion, reduce neurological impairment, and stimulate functional recovery.