Oligodendrocytes, BK channels and remyelination

Demyelination in multiple sclerosis

Demyelination is the erosion of myelin sheaths, which exposes nerve fibres leading to failure of impulse conduction. It can derive directly from traumatic or ischaemic injury³⁰. Alternatively it originates from attack of myelin related proteins in autoimmune disease³¹. Loss of myelin does not necessarily lead to neuronal death, but overburdens axons by decreasing efficiency of energy homeostasis, making it harder for neurons to meet metabolic demands. Without myelin for saltatory conduction, energy needed to relay impulses increases. This eventually leads to increased functional impairment and susceptibility to further neurodegeneration.

The “sclerosis” of MS is the fibrotic lesion that forms in the brain or spinal cord from gliosis of astrocytes and microglia, often located near vasculature. The BBB appears “leaky” as shown by gadolinium-enhanced magnetic resonance imaging (MRI) from infiltration of blood-borne macrophages, T lymphocytes and B cells, which contribute to demyelination³¹ (Figure 2). After two temporally and spatially distinct acute inflammatory episodes, MS can diagnosed and is classified as relapsing–remitting or primary progressive MS depending on the disease course⁴. As lesions become chronic, factors determining whether inflammation resolves and remyelination occurs are not fully understood. However, demyelination may share pathways with ischaemia and viral infection⁴. Persisting inflammation and remyelination failure and nerve loss contribute to progressive MS¹¹. Without tissue repair, permanent loss of function often ensues.

Figure 2. Demyelination may derive from antibody attack.

Opsonisation by non-specific IgG activates the cytotoxic complement system and ADCC. The emerging importance of B cells is highlighted by recent findings⁴⁹. Additional roles include possibly secreting anti-myelin antibodies and acting as APCs to increase T cell activation 50, labelled 1. Cytotoxic CD8⁺ T cells react against self-antigens expressed by oligodendrocytes. Resident microglia or peripheral macrophages phagocytose myelin residues and debris. Reactive astrocytes, activated microglia and Th cells activated by APCs drive inflammation by secreting pro-inflammatory cytokines (TNFα, IFNγ, interleukins) and neurotoxicity by releasing free radicals (ROS, RNS)³¹. Subsequently to myelin loss, axons degenerate. Figure created with BioRender.

Episodes may resolve incompletely and RRMS invariably involves neurological decline. Motor symptoms generally affect all patients eventually during disease course, but can involve sensory system particularly sight, pyramidal tracts, psychological aspects, brainstem and autonomic functions³². Spinal cord lesions typically cause most of the lower limb disability and are both the white and grey matter³³, which contribute to the atrophy observed. This is observed early in MS brain and spinal cord when measured by atrophy using MRI, as an indicator of neurodegeneration³⁴. Associated neuroaxonal damage, measured as serum and notably intrathecal neurofilament, correlates with disability severity³⁵. Most patients eventually proceed to SPMS, notably those with significant early disease activity⁴. SPMS develops when compensation pathways becomes exhausted and is notably associated with neurodegenerative state with progressive atrophy, enlarging lesions, chronic inflammation and remyelination failure.

Preserving myelin

Oligodendrocytes are limited in their ability to respond to damage and at least in part depend on replacement by their precursors, OPCs³⁶. In the adult CNS, NG2⁺ cells, which include OPCs and neural progenitors, constitute nearly 9% of white matter. Their migration into sites of injury is crucial for remyelination, whereby myelin regenerates spontaneously around demyelinated axons³⁷. Preserving myelin is important because neuroaxonal regeneration is limited. Macrophages have a strong influence, and microglia promote this by clearing myelin debris³⁸. Underlying demyelination and inflammation must resolve before new myelin forms. Remyelination may protect axons from inflammation-mediated neurotoxicity³⁹ and is observed in both acute and chronic lesions, even concomitant to demyelination, and in early MS³¹.

Successful remyelination depends on sufficient OPC pools, their migration and survival, until differentiated into myelinating oligodendrocytes; but this does not guarantee it. In MS, OPCs differentiation may arrest before myelin synthesis completes³⁰. Axonal density is higher in remyelinated than chronic demyelinated plaques. However, demyelination may re-occur more frequently in new myelin because newly differentiated oligodendrocytes may produce thinner and shorter sheaths, possibly from external ischaemic factors of the neuroinflammatory environment impairing proper myelination⁴⁰. Additionally, lesion remyelination occurs 20% more often in acute than chronic lesions, so remyelination may inversely correlate with disease progression or age⁴¹. Therefore, preserving myelin might provide a better neuroprotective strategy than remyelination.

Excessive extracellular glutamate

Excitotoxic stress is caused by excessive or prolonged activation of glutamatergic receptors causing Ca²⁺ overload. This sustains pro-apoptotic pathways involving enzymes and transcription factors like MAPK and NF-κB, which degrade membranes, proteins and intracellular organelles. Increased glutamatergic signalling can be triggered by the energy deficiency from the cellular damage in lesions, mitochondrial dysfunction and oxidative stress^61,62. The last involves highly reactive and damaging free radicals: ROS and RNS. These cause mitochondrial membrane damage by lipid peroxidation, which exacerbates cellular burden and glutamatergic signalling⁶³. At high levels glutamate is thought to induce oxidative stress by means of blockade of the glutamate/cystine antiporter (XC–Cys/Glu) that prevents uptake of cystine and synthesis of the anti-oxidant glutathione, in a form of cell death termed ferroptosis or oxytosis⁶⁴.

Damage to neurons causes axon swelling, where ion channels including voltage-gated sodium channels are upregulated to attempt compensation for impaired conduction^65,66. Excitotoxic damage to myelin may cause this upregulation without necessarily causing overt demyelination⁶⁶. Axon swelling impairs network connectivity in MS, where sustained glutamatergic activation associates significantly with increased neurological disability⁶⁷.

Glutamate is upregulated in MS CSF (p<0.001) and carrying the polymorphism rs794185 that further increases this associates with neurodegeneration^67,68. The major source of glutamate production is difficult to discern, but evidence suggests neuroinflammation is important. Pro-inflammatory cytokines TNFα and IL-1β cause neurotoxicity by downregulating astrocytic glutamate transporter and glutaminase which accumulates glutamate in the extracellular space^61,69,70. IL-1β but not TNFα are established as significantly upregulated in MS CSF^70,71. Immune activation upregulated the cysteine glutamate exchanger on macrophages and microglia and in MS patients⁷². To synthesise important antioxidant glutathione this exchanger releases glutamate extracellularly.

Table 2 describes drugs targeting excitotoxicity in MS, highlighting the still unmet clinical need. These therapies are inadequate clinically because antagonists of glutamatergic pathways can downregulate excitatory CNS conduction, which importantly can cause serious adverse events. Selectivity could be improved by targeting receptor subunits specific to glial cells and that are more permeable to pathological Ca²⁺ accumulation, like NR1 and NR3 NMDAR subunits⁷³. Sodium channel blockers provide an alternative means to control excitotoxicity and some benefit has been noted in the more recent clinical trials, but they are poorly tolerated leading to non-compliance^74,75.

Oligodendrocytes are deficient in their response to excitotoxic stress

Oxidative damage to proteins and lipids is substantially increased in acute demyelinating lesions compared to healthy white matter. Hypertrophic astrocytes and foamy macrophages are able to limit this damage by upregulating antioxidant superoxide dismutase, but not other components of lesion tissue including neurons and oligodendrocytes⁷⁶. Oligodendrocytes have a particularly inefficient antioxidant protection. These have a reduced ability to synthesise glutathione⁷⁷ and their death positively correlates with concentration of the highly reactive lipid peroxidation product 4-HNE⁷⁸. Oligodendrocytes are also the main cells that store iron in a balance that is susceptible to conversion to its oxidative divalent form⁷⁹. Their susceptibility to excess glutamate activation specifically is supported by in vitro studies. Only upon inhibition of glutamatergic receptors in oligodendrocytes-only cultures were the apoptotic indicators DNA fragmentation and caspase-3 abolished^70,80.

Experimental autoimmune encephalomyelitis (EAE) is an established MS model induced by adoptive transfer of anti-myelin protein T cells. In EAE mice, 60% more of the oligodendrocytes population was preserved with the AMPA/kainate receptor inhibitor NBQX compared with administering phosphate buffered saline (PBS) only, which also improved neurologic impairment score (p <0.01)⁸¹. AMPAR-mediated Ca²⁺ influx activates a sustained phosphorylation of ERK1/2 to activate proapoptotic pathways in oligodendrocytes and mitochondrial impairment in a manner similar to ischaemia⁶². Ca²⁺-permeable AMPARs are upregulated only at MS lesions, but not in regions of healthy tissue⁸⁹, so Ca²⁺ permeability might indicate upregulation of excitotoxic responses with demyelination. Considering the complex pathological microenvironment of lesions, glutamatergic receptor inhibition alone might not prevent cytotoxicity locally in MS. Pro-inflammatory damage spreads centrifugally from the lesion centre⁴, so inhibition might instead prevent spread of excitotoxins.

AMPAR/kainate receptors are mainly expressed on oligodendrocytes soma, while myelin mainly expresses NMDARs⁹⁰. Excitotoxic stress to myelin can cause decompaction of myelin sheath⁹¹, which can impair neuronal metabolism before overt demyelination. Since damaged or degraded myelin sheaths increase neuronal metabolic burden and expose axons to inflammation related toxins, this suggests therapeutically protecting myelin from excitotoxic stress may be neuroprotective in MS. A characteristic feature of MS is a dying back oligodendrogliopathy which, in a similar way to complement activation by direct antibody attack⁴, might also be caused by activation of catalases and mitochondrial redox damage at myelin processes which retrogradely affects oligodendrocytes.

NMDARs induce weaker Ca²⁺ currents compared with AMPARs but sustain these for longer⁵³. The small cytosolic compartment of myelin may quickly accumulate Ca²⁺ concentrations sufficiently high to be toxic. All compartments needed for NMDARs to be functional have been detected with immunoblotting: NR1, NR2 and NR3⁹⁰. These require activation by both glutamate and its co-agonist glycine. Release of only glutamate from myelinated axolemmas has been established⁵⁸. The Mg²⁺ block characteristic of NMDARs can be released by a slight depolarisation⁵³, which may justify the expression of AMPARs on myelin at lower concentrations. Especially because AMPARs inhibitors only partially abolished the Ca²⁺ current through myelin, but completely at oligodendrocytes soma, while non-selective ionotropic receptor inhibitor completely abolished at both locations⁹⁰. This suggests a mediating effect by AMPAR.

However, no significant decrease of NMDAR mediated Ca²⁺ into oligodendrocytes when their inhibitors, NBQX or D-AP5 respectively, were added after ischaemia⁹¹. The authors proposed excitotoxicity does not derive directly from glutamatergic Ca²⁺ influx, but from the resulting K⁺ and H⁺ increase because the NMDA evoked current correlated with K⁺ increase. The resulting decrease in pH (from K⁺ and from the hypoxic cell) might activate H⁺-gated TRP channels which then caused about 70% of the Ca²⁺ rise⁹¹. TRP block reduced myelin decompaction, so it is possible these channels are more responsible for the ischaemic excitotoxicity to oligodendrocytes than direct ionotropic receptor activation. Alternatively, the majority of Ca²⁺ may derive from a secondary source, such as from subsequently activated voltage gated calcium channels (VGCCs) or the reversal of the Na⁺/Ca²⁺ exchanger which can occur in conditions of excessive depolarisation⁸⁹.

Dying oligodendrocytes release high levels of Fe²⁺ which directly contributes to oxidative injury to neurons⁷⁹. This accumulates at acute demyelinating lesions, phagocytosed and released through oxidative burst. Ferrous iron, Fe²⁺, is a mediator of the Fenton reaction that synthesises hydroxyl and H₂O₂ radicals⁷⁹. Excitotoxic stress will damage oligodendrocytes, which will in turn release more oxidative stress, although contribution of oligodendrocytes excitotoxicity is still unclear because complex to quantify.

BK channels

Large conductance calcium-activated, voltage gated potassium channels (BK channels) are the most diverse within the family of transmembrane protein channels, which also includes small and intermediate K⁺ conductance (SK and IK) channels⁹². These are activated by thresholds of voltage or Ca²⁺ transients and accordingly control membrane potential by mediating efflux of the required amount of hyper-polarising K⁺⁹³. They can also be activated by other metal ions such as Mg²⁺, but also by pH, arachidonic acid and nitric oxide. Encoded by the KCNMA1 (or SLO) gene, BK channels constitute a heterodimer of pore-forming α-subunits and a monomer comprising a voltage-sensing and a calcium-sensing module⁹⁴. Ubiquitous, BK channels are overexpressed in regions of high Ca²⁺ concentrations⁹⁵. By mediating K⁺ transients out of cells, BK channels can also regulate K⁺ homeostasis, cell volume, and therefore have various functions including neuronal excitability, smooth muscle relaxation, blood pressure control and electrical tuning of cochlear hair cells⁹⁶.

The highly dynamic physiological properties of BK channels are partly due to the numerous α-subunit splice variants, which makes their translated protein structure highly versatile physiologically. For example, a cysteine-rich 59-amino-acid insert between RCK domains called STREX variant can be added to the C-terminus⁹⁷, resulting in increased sensitivity to activation, inducing higher neuronal firing frequencies. Additionally, BK channels assemble auxiliary subunits, such as β subunits (β1–4)⁹⁸. These can modify activity, including modifying sensitivity to its activators, voltage or Ca²⁺, or by activating protein kinases⁹⁹. Furthermore, the association with γ subunits, which are leucine rich repeat containing proteins, can increase stimulability of the BK channel by decreasing the negative voltage difference threshold¹⁰⁰. Ultimately, this increases the range of pharmacological applications of these channels.

BK channels regulate neuronal excitability

In the CNS, BK channels are abundantly expressed on axons, dendrites, soma and synaptic terminals in widespread CNS regions. Here, these can control the fast phase of after-hyperpolarisation. Additionally, these can control AP output by changing the magnitude and duration of incoming Ca²⁺ spikes at dendrites¹⁰¹. This will determine AP duration and firing frequency¹⁰². BK channels can mediate their activities and their responses specifically for their cellular location and type of neuronal cell by co-localising with functionally distinct VGCCs¹⁰². BK channels have been shown to co-localise with L-/, P/Q-, or N-/ types of VGCCs^103,104. Depending on the frequency of basal firing, the BK channels at that neuronal cell will typically provide the opposite effect to modulate and re-set the phase, ultimately to flatten the frequency-current curve and control neuronal excitability. This would occur in a manner similar to hyperpolarisation activated by cyclic nucleotide gated channels, that set the “pacemaker” firing frequency in the brain¹⁰⁵. Overall, studies of BK channels indicate these tune the neuronal signal by amplifying it if weak or reducing it if too strong, rather than stringently enhance inhibition or excitation^106–108.

BK channels also have an important role in directly mediating neurotransmitter release, this is supported by their co-localisation to VGCCs with those of the P/Q-type being most frequently observed. This co-localisation occurs predominantly at dendrites where it regulates dendritic spike generation relative to neurotransmitter release¹⁰⁹. This is consistent with localisation of the BK α subunits at presynaptic terminals in functionally important axon tracts¹¹⁰. At these locations, BK channels limited the Ca²⁺ mediated neurotransmitter release by decreasing presynaptic APs duration¹¹⁰. Indeed, release of neurotransmitter from vesicles is triggered by Ca²⁺ elevated locally through VGCCs, once the propagated AP reaches the terminal¹¹¹. Typically, BK channels would reduce neurotransmitter release, because these are able to reduce the amplitude of the presynaptic AP. An important demonstration of this is the effect on neurotransmitter release by CA3 hippocampal neurons and associated APs upon addition of BK channel blockers. The resulting spontaneous EPSCs increased in amplitude and frequency¹¹⁰. This inhibition ultimately reduces release of glutamate, but does not occur for inhibitory neurotransmitter GABA¹¹². Therefore, BK channels are key to avert overexcitation of the post synaptic neuron.

Mediators of excitotoxic stress

Physiologically, BK channels can prevent too much neurotransmitter from causing excessive depolarisation and Ca²⁺ accumulation post-synaptically. In mice where acute focal cerebral ischemia was induced by middle cerebral artery occlusion, the neurological symptoms were significantly higher with knockout of the BK α subunit compared to wild type¹¹³. This may imply glutamate-induced oxidative stress, and consequences for acute and chronic neurodegeneration. This negative feedback by BK channels might only occur if propagated APs are high enough to induce levels of intracellular Ca²⁺ and neurotransmitter similar to those observed in pathological conditions. For example, only upon addition of 4-AP, a non-specific inhibitor of voltage gated K⁺ channels, were BK channels activated to decrease AP amplitude post-synaptically and decrease neurotransmitter release¹¹⁴. No amplified repolarisation or reduced neurotransmitter release by BK channels was observed without 4-AP. This is specific to excitatory neurotransmitter release, because a concentration dependent reduction in ischaemia mediated by NMDAR correlated with increased opening of BK channels by the activator NS1619¹¹⁵. By creating a negative feedback control to disproportionate neurotransmitter release, BK channels may be an emergency break to prevent hyperexcitability and subsequent toxicity.

Activating BK channels to protect oligodendrocytes

Much of the available evidence relates to neurons, but if there is a functional link between the role of BK channels and oligodendrocytes in mediating this excitotoxic stress, targeting this could possibly provide an avenue for disease modifying therapy in MS.

Although big conductance, calcium-activated potassium (BKCa) channels, notably KCNMB4 isoforms are neuronally expressed¹¹⁶, it is evident that KCNMB4 is also present and differentially expressed by oligodendrocytes^117,118. OPCs were associated with high expression of KCNMA1 and KCNMB2 (Figure 3A. 3B), at a time when they express many ion channels perhaps as part of the pre-myelination glial-neuronal synapse¹¹⁹. However, it is evident that oligodendrocyte maturation and myelination was associated with their relative loss and the upregulation of the KCNMB4 BK isoform (Figure 3A, 3B). In addition transcriptomic expression of KCNMA1 and KCNMB4 in NG2+ cells has been found¹²⁰.

Figure 3. Oligodendrocyte expression of BKCa channels in humans and mice.

The expression of: BK channels; platelet-derived growth factor receptor alpha (PDGFRA) and chondroitin sulphate proteoglycan four (CSPG4/NG2) as markers for oligodendrocyte precursor cells (OPC) and committed oligodendrocyte precursors ((COP); myelin oligodendrocyte glycoprotein (MOG) and proteolipid protein one (PLP1) as markers of mature oliogdendrocytes (OL); human glutamatergic neurons (Neuro2 GAD2 0.02, SLC17A7 2.11 (Jäkel et al. 2019); and aquaporin 4 (AQP4) and glial fibrillary acidic protein (GFAP) as markers for astrocytes channels was extracted from public data bases (A) Expression of BK channels in human cells in human white matter tissues extracted from the oligointernode (https://ki.se/en/mbb/oligointernode¹¹⁷. (B) Expression of human and mouse BK channels from cortical brain tissue using 10X single cell RNAseq from the Allen Brain Atlas (www.portal.brain-map.org) (C) BK expression in OPC and mature oligodendrocytes from RNAseq data from the Oligointernode portal¹²⁶ and the Brain RNA-Seq portal (www.brainrnaseq.org¹²⁷). Data is expressed as fragments per kilobase of transcript per million mapped reads (FPKM). * = data values reduced 10 times ** = data values reduced 100 times.

Human KCNMB4 expression increases as OPCs mature into oligodendrocytes and was increased in myelinating oligodendrocytes (Figure 3A). This is perhaps consistent with elevated KCNMB4 expression in chronic inactive multiple sclerosis lesions¹¹⁷. In contrast mouse OPC and oligodendrocytes do not seem to express much Kcnmb2 (Figure 3B, 3C). However, as occurs in humans, Kcnma1 is most marked in the OPC and is down-regulated as oligodendrocytes mature and myelinate (Figure 3C). Likewise, Kcnmb4 can sometimes be found at higher levels in OPCs, but persists in mature oligodendrocytes to be the dominant BK channel isoform (Figure 3B, 3C). Kcnmb4 is expressed on the cell membrane and is also expressed in mitochondria¹²¹. Loss of Kcnma1 message during development is consistent with protein expression and functional calcium-induced signalling activity¹¹⁸ and may play a role in oligodendrocyte differentiation.

Additionally, electrophysiological recordings of increased oligodendrocytes depolarisation corresponded to the increased intracellular fluorescence from labelled Ca²⁺ upon glutamate-induced stimulation; which occurred only when the BK channel blocker iberiotoxin was added¹¹⁸. This suggests a role of BK channels to regulate Ca²⁺ influx to protect oligodendrocytes from excitotoxic stress. Other evidence indirectly supports this. As such the fundamental subunits of the NMDARs, NR1, NR2 and NR3 co-localise with myelin protein from primary optic nerve oligodendrocytes upon immunohistochemical staining⁹⁰. Blocking NMDARs substantially blocked myelin damage upon chemically induced ischaemia in vitro⁹⁰. This was the first evidence of axo-myelinic signalling, indicating that glutamate released from the axon can cause Ca²⁺ to enter oligodendrocytes through the myelin sheath. Importantly, it has been found that mature oligodendrocytes express NMDARs, and that small quantities of excitatory neurotransmitters diffusing between axon and myelin could form sufficiently high concentrations to give rise to large Ca²⁺ transients within mature oligodendrocytes¹²². In health, oligodendrocytes already communicate with axons through NMDAR for trophic support⁵⁹ and BK channels form complexes with this receptor¹²³. Therefore, when activated, BK channels could protect oligodendrocytes from axon-induced excitotoxicity by increasing hyperpolarisation. Prolonging APs may increase the duration of the desensitised state of ionotropic channels and VGCCs to limit Ca²⁺ influx. In demyelinating pathology, the excessive excitotoxicity could inhibit the endogenous protection by BK channels to oligodendrocytes. The addition of an activator could re-open these, re-establishing protective effects. A counter argument is that high extracellular potassium is primarily responsible by increasing length of neuronal depolarised state. Damaged oligodendrocytes may have a dysfunctional inward rectifier potassium channel, so K⁺ clearance is faulty¹²⁴. Large levels of excitatory stimulation of myelin may result because when neurons are demyelinated or damaged they upregulate sodium channels, and subunits which maintain the depolarised state⁶⁵. In this scenario, BK channel activators might be counter-productive by increasing extracellular K⁺, but possibly only if K⁺ clearance is faulty.

BK channel activators could be used therapeutically to preserve function in demyelinating diseases, particularly MS. As described above, currently the standard treatment for MS targets inflammation, but curbing the pathological attack by the immune system does not protect from demyelination or excitotoxicity. Therefore, it does not prevent neurodegeneration or restore functionality lost¹¹. In MS, BK channels are expressed in both myelin and the axons it covers. Crucially, in chronically injured white matter, their activation upon Ca²⁺ influx was observed only upon axon exposure subsequent to chronic spinal cord injury¹²⁵. Addition of the BK channel activator isopimaric acid preserved myelination after spinal cord injury in rats⁵⁰, where functionality correlated with preserved myelinated tracts. This suggests that a BK channel activator could target demyelination to preserve functionality in MS.

Only a few BK channel activators have been studied in the clinic, BMS-204352 (Maxipost) was developed for stroke while andolast is reported to be in phase III for asthma^128,129. Unoprostone isopropyl is an atypical prostanoid used topically in the treatment of glaucoma¹³⁰. VSN16R was recently trialled in people with MS for muscle spasticity^116,131,132. This trial focussed on spasticity endpoints up to a week after administration of the drug and no remyelination parameters were studied¹³².

Underlying data

No data are associated with this article.