Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome

Abstract

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2a^Q54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a^+/- Dravet phenotype. Scn1a^+/- mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a^+/- epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.

Keywords: Epilepsy; Genetics; Mouse model; Seizures; Voltage-gated calcium channels; Voltage-gated ion channels.

Figure 1. Effect of reduced Cacna1g expression on Scn1a^+/− survival and spontaneous seizure rate

(A) Survival over 8 weeks is shown for control 1A^+/− (black), 1G^+/− (blue) mice and 1A^+/−;1G^+/− (red) mice. Reduced expression of Cacna1g resulted in a significant improvement in survival of 1A^+/−;1G^+/− versus 1A^+/− mice. p < 0.0001 (Mantel-Cox Logrank), with n=43–58 per group. (B) Spontaneous seizure rate during a 48 hour window is displayed. Fewer seizures per hour were observed in 1A^+/−;1G^+/− mice (red circles) relative to control 1A^+/− (black circles) littermates. *p < 0.0009 (Mann-Whitney U test). Average seizure frequencies are depicted by the horizontal line and error bars represent standard error of the mean (SEM), with n = 22 per group. (C) Spontaneous seizure rate during a 60 hour window following a hyperthermia-induced priming seizure is displayed. No difference in seizures per hour were observed in 1A^+/−;1G^+/− mice (red squares) relative to control 1A^+/− (black squares) littermates. p > 0.05 for both males and females (Mann-Whitney U test). Average seizure frequencies are depicted by the horizontal line ± SEM, with n = 21 per group.

Figure 2. Cacna1g mRNA levels increase after seizure activity

(A) To assess expression at the level of messenger RNA (mRNA), a quantitative droplet digital PCR (ddPCR) Taqman assay was performed on whole brain samples collected from P19 1A^+/− mice 24 hours after either a priming event (hyperthermia-induced GTCS) or a sham treatment. The expression of Cacna1g was normalized to Tbp expression. p-value was determined by unpaired Student’s t-test with Welch’s correction. Error bars represent upper and lower limits derived from standard error of the mean. Mean Cacna1g expression was observed to be the following: Sham = 3.13 (n = 8) and Primed = 3.56 (n = 8); *p < 0.05. (B) Differences in hippocampal Cacna1g expression between 1A^+/− mice with and without observed seizure activity prior to sample collection. RNA-seq normalized count values are shown. *FDR adjusted p-value = 2.57e⁻²⁸ (Benjamini-Hochberg).