Inhibition of MEK-ERK signaling reduces seizures in two mouse models of tuberous sclerosis complex

Abstract

Tuberous sclerosis complex (TSC) is a monogenic disorder characterized by hyperactivation of the mTOR signaling pathway and developmental brain malformations leading to intractable epilepsy. Although treatment with the recently approved mTOR inhibitor, everolimus, results in clinically relevant seizure suppression in up to 40% of TSC patients, seizures remain uncontrolled in a large number of cases, underscoring the need to identify novel treatment targets. The MEK-ERK signaling pathway has been found to be aberrantly activated in TSC and inhibition of MEK-ERK activity independently of mTOR rescued neuronal dendrite overgrowth in mice modeling TSC neuropathology. Here, we evaluated the efficacy of MEK-ERK inhibition on seizures in two mouse models of TSC. We found that treatment with the MEK inhibitor PD0325901 (mirdametinib) significantly reduced seizure activity in both TSC mouse models. These findings support inhibiting MEK-ERK activity as a potential alternative strategy to treat seizures in TSC.

Keywords: Epilepsy; MAPK; MEK inhibitor; MEK-ERK signaling; Seizures; Tuberous sclerosis complex.

Figure 1:. PD0325901 treatment decreases seizures in Tsc1^hGFAP cKO mice.

(A). Schematic of the experimental paradigm. Tsc1^hGFAP cKO mice were treated with vehicle, 1.5 mg/kg PD0325901, or 6 mg/kg PD0325901 once daily from P21 to P54–55 (treatment days D1 to D34–35). Mice were monitored with continuous video-EEG recording starting at P35 (D15). Brain tissue was collected at the end of the experiment on P54–55 (D34–35) for western blot analysis. (B) Schematic of the EEG montage. (C) Representative EEG trace showing electrographic seizure activity in a Tsc1^hGFAP cKO mouse. (D) Quantification of seizure frequency (average daily seizures) in vehicle- and PD0325901-treated Tsc1^hGFAP cKO mice. *p<0.05 by Kruskal-Wallis test with Dunn’s post-hoc test, n=12 (vehicle), 12 (1.5 mg/kg PD0325901), and 15 (6 mg/kg PD0325901) mice. (E) Animal bodyweight over the course of treatment. Top graph shows the weights of female mice and bottom graph shows the weights of male mice. *p<0.05 by mixed-effects analysis with Tukey’s post-hoc test, n=8 female/4 male (vehicle), 6 female/6 male (1.5 mg/kg PD0325901), and 8 female/7 male (6 mg/kg PD0325901) mice. (F) Representative western blots showing p-ERK1/2, ERK1/2, and GAPDH levels in whole brain lysates from vehicle- and PD0325901-treated Tsc1^hGFAPCKO mice. (G) Western blot quantification of p-ERK1/2/ERK1/2 levels (normalized to GAPDH). *p<0.05, ***p<0.001 by one-way ANOVA with Tukey’s post-hoc test, n=7 (vehicle), 12 (1.5 mg/kg PD0325901), and 14 (6 mg/kg PD0325901) mice. (H) Scatterplot of seizures/24 hours vs. p-ERK1/2/ERK1/2 levels. r=0.3820, p=0.0283 by Spearman correlation, n=7 (vehicle), 12 (1.5 mg/kg PD0325901), and 14 (6 mg/kg PD0325901) mice. Error bars are ±SEM.

Figure 2:. PD0325901 treatment decreases seizures in Rheb^CA mice.

(A) Schematic of the experimental paradigm. Rheb^CA mice were generated by in utero electroporation (IUE) at embryonic day (E) 15.5. Mice at 2–4 months of age were monitored with continuous video-EEG recording for 5 days to establish baseline activity. Mice were then treated with either vehicle or 5 mg/kg PD0325901 once daily for 10 days while undergoing video-EEG recording. B) Schematic of the EEG montage. (C) Representative EEG trace showing electrographic seizure activity in a Rheb^CA mouse. (D) Quantification of seizure frequency (average daily seizures) in vehicle- and PD0325901-treated Rheb^CA mice. The graphs show pairwise comparisons between baseline (days D1–5) and treatment (D11–15) within individual animals. Group means are indicated by the bars. *p=0.0059 by Wilcoxon matched-pairs signed-rank test, n=11 (vehicle) and 11 (PD0325901) mice. E) Representative western blots showing p-ERK1/2, ERK1/2, and β-tubulin levels within microdissected cortices containing the electroporated region from vehicle- and PD0325901-treated Rheb^CA mice. (F) Western blot quantification of p-ERK1/2/ERK1/2 levels (normalized to β-tubulin). *p=0.0440 by unpaired t-test, n=5 (vehicle) and 3 (PD0325901) mice. Error bars are ±SEM.