Investigation of MicroRNA-134 as a Target against Seizures and SUDEP in a Mouse Model of Dravet Syndrome

Abstract

Dravet syndrome (DS) is a catastrophic form of pediatric epilepsy mainly caused by noninherited mutations in the SCN1A gene. DS patients suffer severe and life-threatening focal and generalized seizures which are often refractory to available anti-seizure medication. Antisense oligonucleotides (ASOs) based approaches may offer treatment opportunities in DS. MicroRNAs are short noncoding RNAs that play a key role in brain structure and function by post-transcriptionally regulating gene expression, including ion channels. Inhibiting miRNA-134 (miR-134) using an antimiR ASO (Ant-134) has been shown to reduce evoked seizures in juvenile and adult mice and reduce epilepsy development in models of focal epilepsy. The present study investigated the levels of miR-134 and whether Ant-134 could protect against hyperthermia-induced seizures, spontaneous seizures and mortality (SUDEP) in F1.Scn1a(+/-)^tm1kea mice. At P17, animals were intracerebroventricular injected with 0.1-1 nmol of Ant-134 and subject to a hyperthermia challenge at postnatal day (P)18. A second cohort of P21 F1.Scn1a(+/-)^tm1kea mice received Ant-134 and were followed by video and EEG monitoring until P28 to track the incidence of spontaneous seizures and SUDEP. Hippocampal and cortical levels of miR-134 were similar between wild-type (WT) and F1.Scn1a(+/-)^tm1kea mice. Moreover, Ant-134 had no effect on hyperthermia-induced seizures, spontaneous seizures and SUDEP incidence were unchanged in Ant-134-treated DS mice. These findings suggest that targeting miR-134 does not have therapeutic applications in DS.

Keywords: Dravet syndrome; SUDEP; miR-134; oligonucleotides; seizure.

Graphical abstract

Figure 1.

Ant-134 dose response curve on hyperthermia-induced seizures in F1.Scn1a(+/−)^tm1kea mice. A, At P17, mice were weaned and intracerebroventricularly injected with 0.1, 0.5, or 1 nmol dose of Ant-134 or scr. At P18, hyperthermia challenge was induced by placing the animals in a thermostat-controlled heating chamber. Graphs show the temperature threshold for mice to develop a seizure (B), median duration of seizures (C), and the respective seizure severity according to a modified Racine scale (D). E, Transcript levels of miR-134 in the ipsilateral hippocampus 24 h after scr or Ant-134 injections. F–I, Expression of validated targets of miR-134 (Limk1, Creb1, and Dcx) and Scn1a levels in scr or Ant-134-treated mice. B–D, Kruskal–Wallis test, median IQR. E–I, One-way ANOVA, mean (SD). **p < 0.01, ***p < 0.001, ****p < 0.0001. One outlier from Ant-134 F1.Scn1a(+/−)^tm1kea-treated mice group was removed according to Grubbs’ test (α = 0.01) in Dcx transcript levels. Representative images of intracerebroventricular injections with methylene blue in P21 mice can be found in Extended Data Figure 1-1. Relative expression of miR-134, its validated targets (Limk1, Creb1, and Dcx) and Scn1a levels in cortical tissue can be found in Extended Data Figure 1-2.

Figure 2.

Ant-134 0.1 nmol does not prevent SRS and SUDEP occurrence in F1.Scn1a(+/−)^tm1kea mice. A, Schematic showing the experimental design used to investigate the occurrence of SRS, SUDEP and miR-134 levels in F1.Scn1a(+/−)^tm1kea mice during the worsening stage of DS. B, Representation of the frequency of SRS experienced by scr and Ant-134 F1.Scn1a(+/−)^tm1kea-treated mice according to a color scale ranging from 0 to 10 seizures or more. C, Quantitative analyses of SRS between P21 and P28. D, E, Seizure duration and severity in Ant-134-treated mice compared with scr. F, SUDEP rates between scr and Ant-134 F1.Scn1a(+/−)^tm1kea-treated mice. G, Taqman results confirming Ant-134 0.1 nmol produced a knock-down in miR-134 levels when compared with scr mice. H, I, EEG representative trace of SRS in scr and Ant-134 F1.Scn1a(+/−)^tm1kea-treated mice. (n = 6/group). C, D, Student’s t test, mean (SD). E, G, Mann–Whitney test, median IQR. F, log-rank test. **p < 0.01.