3.2. Voltage-Gated Ca
2+
Channels in Oligodendroglial Cells
Ca
2+ signalling has been shown to regulate many oligodendroglial cell functions, including proliferation, migration, process extension, differentiation, and myelination
[86][96][110][136][137][138][139][86,96,110,136,137,138,139]. Intracellular Ca
2+ can increase in NG2
+ cells throughout several mechanisms, such as direct influx through plasma membrane voltage-gated Ca
2+ channels (Cav) or ligand-gated channels
[140][141][140,141], as mentioned below, or by the release from internal Ca
2+ stores
[142][143][142,143] as well as Ca
2+-permeable acid-sensing ion channel opening
[144].
Cav immunoreactivity was found in CNS white matter, located in oligodendroglial soma, projections and paranodal wraps
[145] (see also
Table 2). Electrophysiological recordings demonstrated the functional expression of low-voltage and high-voltage activated Ca
2+ currents in OPCs from
corpus callosum and mouse cortex
[81][83][96][97][81,83,96,97], with pharmacological and voltage-dependent properties typical of T-type and L-type Cav, respectively
[97]. Indeed, several studies have reported that Ca
2+ currents through Cav appear to diminish with maturation of OLs from progenitors to mature cells in culture
[83][104][83,104].
These electrophysiological results have been confirmed by RNA-sequencing transcriptome database that revealed high expression of L-type Ca
2+ channel subunits in OPCs and subsequent downregulation in newly formed and myelinating OLs
[98], indicating that Cav plays a role during the first steps of OPC maturation. The presence of Cav was also confirmed by mRNA analysis for L-type (Cav1.2 and Cav1.3), T-type (Cav3.1 and Cav3.2), P/Q-type (Cav2.1), and N-type (Cav2.2) α1 subunits in NG2
+ cells
[110]. In particular, Cav1.2 represents the primary pore-forming subunit in OPCs, as siRNA knockdown of Cav1.2 in NG2
+ cells removes ∼75% of the Ca
2+ elevation following depolarization
[96].
Functional Cav are reported to be necessary for glial cell migration in vivo during olfactory glomeruli formation in the developing antennal lobe of sphinx moth
Manduca sexta [105]. In addition, in deafferented antennal lobes in which glial cells fail to migrate
[106], glial Cav currents are absent, indicating that Cav in glial cells are required to induce or maintain the migration of antennal lobe glial cells into the developing neuropil of the moth
[105].
In addition, data indicate that L-type Cav activation, probably mediated by PDGF, contributes to spontaneous Ca
2+ oscillations in the OPC soma, leading to accelerated migration and process formation
[104]. Furthermore, an increase in amplitude and frequency of Ca
2+ transients is one of the mechanisms underlying AMPA-induced stimulation of OPC migration
[107], as described below. Furthermore, Ca
2+ transients may affect the recycling of cell-adhesion receptors and induce the rearrangement of cytoskeletal components, which are essential for cell movement
[108].
When OPCs are grown in high extracellular K
+, used as a depolarizing stimulus to activate Cav, they are prompted towards maturation, as demonstrated by a more complex morphology and a significant increase in the expression of mature markers
[109]. At the same time, blocking the expression of the Cav α1.2 subunit, that conducts L-type Ca
2+ currents, significantly prevents OPC culture maturation
[96]. Accordingly, Cav1.2 deficient OPCs present inhibited proliferation and disruption of proliferative response to PDGF, the best known and most active mitogen for OPCs
[96]. Interestingly, it was shown that store-operated Ca
2+ entry, as well as Ca
2+ release from intracellular stores, are essential mechanisms for PDGF-mediated mitotic action in OPCs
[86]. A widespread hypothesis is that Ca
2+ entry by L-type channels modulates OPC division and cell maturation through independent intracellular pathways. Cav seem to be essential for cell cycle progression of in mitotic OPCs whereas, in post-mitotic pre-OLs, the same channels are playing an important role in cell maturation. In support to this hypothesis, it was demonstrated that a loss of Cav1.2 in oligodendroglial cells affects axonal contact in co-cultured cortical neurons and consequently inhibits the initial steps of myelination
[96]. Moreover, deletion of Cav1.2 in OPCs reduces OL maturation and myelination in the postnatal mouse brain and impairs remyelination in a CPZ model
[100][101][100,101].
Importantly, it is likely that factors involved in physiological myelination also participate in remyelination of the injured CNS. In this regard, a significant increase in the activity of OPC L-type Cav was found in demyelinated
corpus callosum of CPZ-treated mice, suggesting that these channels may play a key role in the induction and/or survival of newly generated OPCs after an insult
[102]. Cav expression and functions in oligodendroglial cells are summarized in
Table 2.
Cav Channels in Demyelinating Diseases
Several evidences support the notion that aberrant Cav-mediated currents contribute to the pathophysiology of MS or EAE. Increased Ca
2+ 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 Ca
2+ 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 Ca
2+ 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 Ca
2+ 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 Ca
2+ overload activates the Ca
2+-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 Ca
2+ homeostasis and prevention of intracellular Ca
2+ 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 Ca
2+ overload in neurons and immune cells. Nevertheless, there are no trials for clinical translation of Ca
2+ channel blockers so far
[151]. For a summary on the effects of Cav-targeting compounds in demyelination see
Table 1.
3.3. Voltage-Gated K
+
Channels in Oligodendroglial Cells
The OPC resting membrane potential (V
rest) is near the calculated equilibrium potential for K
+ (EK), i.e., −80 mV, suggesting that K
+ channels account for the majority of the resting membrane conductance. The predominant K
+ channel subtypes open at rest (‘leak’ channels) are the inward-rectifier Kir4.1 and two-pore (K2P) K
+ channels
[110]. The RNA-Seq transcriptome database shows that Kir4.1 mRNA is expressed at high levels in NG2
+ cells
[98] (see
Table 2). Kir4.1 mediates inward currents observed in NG2
+ whole cell recordings upon membrane potential hyperpolarization lower than –100 mV. These currents are blocked by low (200 µM) concentrations of extracellular Ba
2+, an inhibitor of Kir channels
[110][114][115][110,114,115]. Kir4.1 facilitates clearance of extracellular K
+ released during axonal firing, thus maintaining resting membrane potential and AP propagation
[46]. Deleting Kir4.1 in mice causes impaired OL maturation and myelination during development, leading to neuronal degeneration
[115] and selective deletion of Kir4.1 from OPCs or mature OLs also results in profound functional impairment and axonal degeneration
[114][115][114,115].
Outward rectifying voltage-gated K
+ channels (Kvs) are also prominent in OPCs, where they are known to regulate cell proliferation and differentiation, and are subsequently downregulated during differentiation
[87][123][87,123] (see
Table 2). Upon depolarization, NG2
+ cells display a non-linear current-to-voltage profile that is shaped by the activation of A-type (I
A) and delayed-rectifier (I
K) K
+ channels. These currents have been extensively characterized in OPCs recorded from cell cultures or in brain slice preparations
[79][80][81][82][117][118][79,80,81,82,117,118]. When challenged by a depolarizing voltage step, 4-aminopyridine (4-AP)-sensitive I
A contribute to the initial ‘peak’ outward current during the depolarizing phase, due to their rapid activation and inactivation kinetics. Moreover, TEA-sensitive I
K, which activate more slowly and do not inactivate, contributes to the sustained ‘steady-state’ current of the depolarizing stimulus
[79][80][81][82][117][118][79,80,81,82,117,118]. The relative proportion of the two current components varies by the region of origin. When compared to cortical NG2
+ cells, white matter OPCs in P5-P10 mice present higher I
K current densities, while I
A density is comparable, resulting in a higher I
K/I
A ratio
[124]. During maturation, outward K
+ conductances, in particular I
K, in OPCs undergo a strong downregulation up to almost completely disappearance in mature OLs
[80][87][80,87]. In parallel to I
K downregulation, there is a gradual increase in the expression of Kir, that represents the main conductance observed in mature OLs
[111], as demonstrated by the Ba
2+-sensitivity of overall OPC conductance increases during maturation
[152].
RT-PCR and immunocytochemical localization in cultured NG2
+ cells have shown robust expression of Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6 and Kv7.2 mRNA and protein, with Kv1.5 and Kv1.6 showing highest levels among Shaker-type delayed rectifiers
[119][120][119,120]. In addition, mRNA for several non-Shaker delayed rectifier channels, Kv7.2 and Kv2.1, are also abundantly expressed
[110]. Kv1.4, the only Shaker-type channel to display A-type properties, presents low expression in NG2
+ cells database
[110]. Other A-type channel subunits that are greatly expressed in OPCs are Kv4.2, Kv4.3, and Kv3.3. Kv1.3 is upregulated during the G1 phase of cell cycle, and blockade of this channel with specific toxins prevents G1/S transition
[120]. Conversely, overexpression of Kv1.3 or Kv1.4 promotes OPC proliferation in the absence of mitogens, while overexpression of Kv1.6 inhibits proliferation in the presence of mitogens
[123]. On the other hand, neither knockdown nor overexpression of Kv1.5 affect OPC proliferation
[119][123][119,123]. Of note, and differently from cell proliferation, differentiation of cultured NG2
+ cells into OLs is not significantly affected by overexpression of Kv subunits but is impaired by the Kv blocker TEA
[87][109][87,109], demonstrating that oligodendroglial cell proliferation and differentiation might be differently regulated
[123].
The large conductance Ca
2+-activated (BK) channel KCa1.1 is highly expressed in NG2
+ cells
[110]. This confirms previous findings that BK channels, which are both voltage- and Ca
2+-dependent, are expressed in cultured NG2
+ cells
[153]. Other K
+ channels may also be important in maintaining oligodendroglial cell functions and integrity, including Kir2.1, Kir7.1 and TASK1 channels
[111][112][113][111,112,113]. For a summary on Kv and Kir expression in oligodendroglial cells, see
Table 2.