Acid sensing ion channel 2: A new potential player in the pathophysiology of multiple sclerosis

Abstract

Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1^-/- and Asic2^-/- mice and observed a significant reduction of clinical score in Asic1^-/- mice and a significant reduction in the clinical score in Asic2^-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1^-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2^-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1^-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.

Keywords: ASIC1; ASIC2; experimental autoimmune encephalomyelitis; mechanosensation; mouse models.

Figure 1

(a) Clinical score of mice after immunization with MOG _35‐55 peptide. Values are means + SEM and data are cumulative of two independent experiments with similar results with a total of 14 wild‐type (WT), 10 Asic1^−/− and 14 Asic2^−/− mice. * p = 0.019 (strain factor); p < 0.001 (time factor); p < 0.001 (strain × time factor) vs. WT mice; # p = 0.013 for day 20; p = 0.004 for day 21; p = 0.013 for day 22; p = 0.032 for day 23 of WT mice vs. the respective values in Asic2^−/− mice (Two‐way ANOVA Repeated Measures + Bonferroni post hoc test). (b) Comparison between the observed difference between the mean clinical scores of Asic2^−/− and wild‐type (WT) and the pattern of difference simulated under the null hypothesis of no difference. (c) Comparison between the observed difference between the mean clinical scores of Asic1^−/− and wild‐type (WT) and the pattern of difference simulated under the null hypothesis of no difference. In (b) and (c) data points lying outside the lower confidence band can be interpreted as evidence of a departure from the null hypothesis of no difference

Figure 2

Histopathological analysis of spinal cord in EAE mice. (a) Representative spinal cord sections from wild‐type (WT), Asic1^−/− , and Asic2^−/− mice at 45 days after immunization. Sections of the spinal cord anterior horn were stained with hematoxylin and eosin (E&H) to assess inflammation and immunostained for MCHII, CD4⁺, CD8⁺ infiltrating cells. (b) Graphs show the quantification of the extent of inflammation and infiltrating MCHII, CD4⁺ and CD8⁺ cells in the anterior horn of the spinal cord. Values are means + SEM (n = 3 animals for each group). *p = 0.014 for CD4⁺ cells/mm² vs. WT mice; ** p = 0.008 for CD4⁺ cells/mm² vs. WT mice; p < 0.001 for % white matter with CD4⁺ immunoreactivity vs. WT mice; p < 0.001 for CD8⁺ cells/mm² vs. WT mice; p < 0.001 for % white matter with CD8⁺ immunoreactivity vs. WT mice; p = 0.091 for MHCII cells/mm² vs. WT mice; p < 0.002 for % white matter with MHCII immunoreactivity vs. WT mice (One‐way ANOVA + Fisher's LSD). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Mechanical allodynia assessment in mice. Mechanical pain thresholds were assessed in wild‐type (WT; n = 20), Asic1^−/− (n = 12) and Asic2^−/− (n = 21) mice under basal physiological conditions (before immunization), and in pathological conditions at 28 days and at 40 days after immunization with MOG _35‐55 peptide. Values are means ± SEM and are cumulative of two independent experiments. * p < 0.001 vs. WT and Asic1^−/− mice under basal conditions (before immunization), and # p = 0.007 WT mice at 28 days vs. WT mice before immunization; p = 0.025 Asic1^−/− mice at 28 days vs. Asic1^−/− mice before immunization; p < 0.001 Asic2^−/− mice at 28 days vs. Asic2^−/− mice before immunization; p = 0.016 WT mice at 40 days vs. WT mice before immunization; p < 0.001 Asic2^−/− mice at 40 days vs. Asic2^−/− mice before immunization (One‐way ANOVA + Fisher's LSD)

Figure 4

ASIC2 mRNA levels in the brain of 32 control cases and 62 multiple sclerosis (MS) cases. Single values are shown along with means ± SEM expressed as % to cumulate data coming from different quantitative PCR runs. * p = 0.0457 (Student')s t test)