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. 2023 Jul 7;16(7):972.
doi: 10.3390/ph16070972.

Neuroprotection in an Experimental Model of Multiple Sclerosis via Opening of Big Conductance, Calcium-Activated Potassium Channels

Gareth Pryce  1 , Sofia Sisay  1 , Gavin Giovannoni  1 , David L Selwood  2 , David Baker  1
Affiliations

Neuroprotection in an Experimental Model of Multiple Sclerosis via Opening of Big Conductance, Calcium-Activated Potassium Channels

Gareth Pryce et al. Pharmaceuticals (Basel). .

Abstract

Big conductance calcium-activated (BK) channel openers can inhibit pathologically driven neural hyperactivity to control symptoms via hyperpolarizing signals to limit neural excitability. We hypothesized that BK channel openers would be neuroprotective during neuroinflammatory, autoimmune disease. The neurodegenerative disease was induced in a mouse experimental autoimmune encephalomyelitis model with translational value to detect neuroprotection in multiple sclerosis. Following the treatment with the BK channel openers, BMS-204253 and VSN16R, neuroprotection was assessed using subjective and objective clinical outcomes and by quantitating spinal nerve content. Treatment with BMS-204253 and VSN16R did not inhibit the development of relapsing autoimmunity, consistent with minimal channel expression via immune cells, nor did it change leukocyte levels in rodents or humans. However, it inhibited the accumulation of nerve loss and disability as a consequence of autoimmunity. Therefore, in addition to symptom control, BK channel openers have the potential to save nerves from excitotoxic damage and could be useful as either stand-alone neuroprotective agents or as add-ons to current disease-modifying treatments that block relapsing MS but do not have any direct neuroprotective activity.

Keywords: BK channel; autoimmunity; experimental autoimmune encephalomyelitis; multiple sclerosis; neuroprotection.

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Conflict of interest statement

D.B., G.P. and D.L.S. have filed patents on VSN16, and related compounds, in relation to symptomatic and neuroprotective activities in MS and other diseases. Although not considered relevant, G.G. and D.B. have received funds for meetings presentations and as consultants for most companies within the MS-related disease modifying drugs, including Abbvie, Bayer, Biogen, Johnson & Johnson, Merck, Novartis, Roche, Sanofi-Genzyme, Siphon, Teva.

Figures

Figure 1
Figure 1
Lymphocytes exhibit limited BK channel expression. Tissue expression of BK channel subunits was extracted from public databases under Creative Commons licenses (A) Messenger RNA expression of mouse Kcnma1 (1424848_at), Kcnmb2 (1431844_at) and Kcnmb4 (144941_at) data were extracted from the mouse Gene Atlas MOE430, gcrma following RNAseq using Affymetrix MOE430_2 microarrays (www.biogps.org (accessed on 6 July 2023) [28,29,30]). The results represent mean ± SD expression in arbitrary units (a.u.). (B,C) Data were extracted from the Human Protein atlas [32,33] KCNMA1 protein expression was assessed using immunoperoxidase staining of sections in (B) human lymph node (https://www.proteinatlas.org/ENSG00000156113-KCNMA1/tissue/lymph+node#img (accessed on 6 July 2023)) and (C) human cerebellum (https://www.proteinatlas.org/ENSG00000156113-KCNMA1/tissue/cerebellum#img (accessed on 6 July 2023)). Follicular and marginal zone (MZ) B-cells and paracortical (PC) T-cells exhibited no or low-level expression of KCNMA1. Expression was detected in lymph node blood vessels consistent with vascular KCNMA1 and KCNMB1 expression and was evident in the grey matter within the cerebellum. (D) Single-cell RNAseq expression data of BK channel subunits within human tonsils were extracted from public databases and assessed using Illumina NextSeq 50010X. The results represent Uniform manifold approximation and projection (UMAP) plots from data based on results from up to 32,000 cells from 7 donors, showing the distribution in immune subsets and expression of CD3D (T cell marker), CD20/MSA41 (B cell marker), and KCNMA1, KCNMB1, and KCNMB4. (www/tonsilimmune.org (accessed on 6 July 2023) [34]). (E) Single-cell RNAseq expression data of BK channel subunits within human brain was extracted from public databases [35] and assessed using 10X Genomics Chromium single cell 3′ chips. Figures show UMAP representations of KCNMA1, KNCMB2, and KCNMB4 in different cell lineages.
Figure 2
Figure 2
Inhibition of VSN16R potassium channel opening using BK channel antagonists. Automated whole-cell analysis of individual human Ea.hy926 cells demonstrating the influence of 15 μM (A) BMS-204352 or (B) VSN16R alone or with co-administration with 2 μM paxilline or (C) 100 nM martentoxin.
Figure 3
Figure 3
VSN16R is not immunosuppressive during the initial attack of EAE. Adult ABH mice were injected with spinal cord homogenate in Freund’s adjuvant on days 0 and 7. Animals were treated daily with either vehicle (0.1 mL water. Hexagon n = 8) or 40 mg/kg p.o. VSN16R in water (circle n = 10) from day 10 onwards. Clinical signs were grade 0 = normal, 1 = limb tail, 2 = impaired righting reflex, 3 = hind limb paresis, 4 = hind limb paralysis, and 5 = moribund. The results represent the mean ± SEM daily score of all animals developing neurological disease.
Figure 4
Figure 4
VSN16R is not immunosuppressive in the relapse phase of EAE but is neuroprotective. Adult ABH mice were injected with spinal cord homogenate in Freund’s adjuvant on days 0 and 7 and on day 28 to induce a relapse. Following accelerating-rotarod analysis on day 27, animals were randomized to either 0.1 mL water (circle), 50 mg/kg p.o. VSN16R (triangle), 100 mg/kg p.o. VSN16R in water (inverse triangle), or 20 mg/kg i.p. BMS-204352 (diamond) in DMSO: cremophor: PBS (1:1:18. N = 15/group). Rotarod activity was retested on day 48 post-inoculation. The results represent (A) The mean ± SEM daily score of animals developing neurological disease graded: 0 = normal, 1 = limb tail, 2 = impaired righting reflex, 3 = hind limb paresis, 4 = hind limb paralysis, 5 = moribund (B) The mean ± SEM percentage loss of motor co-ordination due to relapse, based on time to fall from an accelerating rotarod, compared to baseline on day 28. (C) The mean ± SEM total neurofilament level within the whole spinal was assessed using a neurofilament-specific ELISA. (D) The mean ± SEM total, hypo-phosphorylated neurofilament levels within the whole spinal were assessed using an SMI32-specific ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to vehicle control.
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