The ion channel TRPM4 in murine experimental autoimmune encephalomyelitis and in a model of glutamate-induced neuronal degeneration

Abstract

Transient receptor potential melastatin member 4 (TRPM4), a Ca²⁺-activated nonselective cation channel, has been found to mediate cell membrane depolarization in immune response, insulin secretion, cardiovascular diseases, and cancer. In murine experimental autoimmune encephalomyelitis (EAE), TRPM4 deletion and administration of glibenclamide were found to ameliorate clinical symptoms and attenuate disease progression. However, the exact role of TRPM4 in EAE, as well as the molecular mechanisms underlining TRPM4 contribution in EAE, remain largely unclear. In the present study, EAE was induced in WT C57BL/6 N mice using myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) and TRPM4 protein and mRNA expression were examined in spinal cord membrane extracts. Our results showed that TRPM4 protein and mRNA are upregulated in EAE, and that their upregulation correlated with disease progression. Moreover, newly-developed TRPM4 inhibitors, named compound 5 and compound 6, were shown to exert a better neuroprotection compared to currently used TRPM4 inhibitors in an in vitro model of glutamate-induced neurodegeneration. These results support the hypothesis that TRPM4 is crucial from early stages of EAE, and suggest that these more potent TRPM4 inhibitors could be used as novel protective therapeutic tools in glutamate-induced neurodegeneration.

Keywords: Experimental autoimmune encephalomyelitis; Glutamate-induced neurodegeneration; HT22; Inhibitors; Multiple sclerosis; TRPM4.

Fig. 1

TRPM4 expression analysis in spinal cords from EAE mice. Female C57BL/6 N WT mice were immunized with MOG_35–55 peptide or with CFA only. Clinical score (a) and body weight (b) were measured for 28 days after immunization. At day 28 post immunization, spinal cords were extracted and membrane proteins were isolated. c Samples from HEK 293 cells transiently transfected with mouse TRPM4 WT plasmid or empty vector (pcDNA4TO) and a spinal cord sample from a healthy WT C57BL/6 N were analyzed for TRPM4 expression, using alpha actin as loading control. A TRPM4 band can be detected at 134 kDa. d TRPM4 membrane expression was analyzed with Western Blot, and alpha subunit of Na⁺/K⁺ ATPase is used as a loading control and for TRPM4 normalization (e) qPCR analysis on Trpm4 gene expression in spinal cords from EAE mice and healthy controls. Data are represented as relative mRNA expression and the expression of Gapdh is used as reference. f Data have been analyzed using unpaired Student’s t-test and are represented as mean ± s.e.m. (*** P < 0.001)

Fig. 2

TRPM4 expression in spinal cords from EAE mice correlates with disease stages. Female C57BL/6 N WT mice were immunized with MOG_35–55 peptide or with CFA only and sacrificed at different days post immunization reflecting different EAE stages. Clinical score and body weight are represented in panel a and b respectively. c and e TRPM4 protein expression at different EAE stages is shown as a relative intensity of protein band and alpha subunit of Na⁺/K⁺ ATPase is used as a loading control and for TRPM4 normalization. d qPCR analysis on Trpm4 gene expression in spinal cords from EAE mice and healthy control at different EAE stages. Data are represented as relative mRNA expression and the expression of Gapdh is used as reference. Data have been analyzed using one-way ANOVA with Sidak’s correction for multiple comparison test and are represented as mean ± s.e.m. (** P < 0.01; *** P < 0.001). Correlation between clinical score and TRPM4 mRNA (f) or TRPM4 protein expression (g) is calculated using Spearman’s correlation analysis. Spearman correlation coefficients (r) and their corresponding p values are shown

Fig. 3

Incubation of HT22 cells with 1 mM glutamate does not alter TRPM4 expression. HT22 cells were incubated with 1 mM glutamate and harvested at different time points after glutamate addition. TRPM4 total (a) as well as surface (b) expression were evaluated, using alpha actin and alpha subunit of Na⁺/K⁺ ATPase as loading controls and for TRPM4 normalization. c and d Total and surface TRPM4 protein expression are shown as a relative intensity of protein band. No significant difference is reported (data are of three independent experiments)

Fig. 4

Newly-developed TRPM4 inhibitors better attenuate glutamate-induced neurodegeneration compared to commonly used compounds. HT22 cells were incubated with 5 mM glutamate after pre-incubation with 5 μM and 10 μM of commonly used TRPM4 inhibitors as well as newly-developed ones. The bar graphs showed the results of cell viability (a) and LDH-release assays (b), where glutamate-induced cell death and membrane disruption were prevented dose-dependently, with newly-developed compounds exerting a more beneficial effect. Data have been analyzed using one-way ANOVA with Sidak’s correction for multiple comparison test and are represented as mean ± s.e.m. (** P < 0.01; *** P < 0.001; n = 10 for each condition)