Cacna1g is a genetic modifier of epilepsy caused by mutation of voltage-gated sodium channel Scn2a

Summary

More than 1200 mutations in neuronal voltage-gated sodium channel genes have been identified in patients with several epilepsy syndromes. A common feature of genetic epilepsies is variable expressivity among individuals with the same mutation. The Scn2a^Q54 transgenic mouse model has a mutation in Scn2a that results in spontaneous epilepsy. Scn2a^Q54 phenotype severity varies depending on the genetic strain background, making it a useful model for identifying and characterizing epilepsy modifier genes. Scn2a^Q54 mice on the [C57BL/6JxSJL/J]F1 background exhibit earlier seizure onset, elevated spontaneous seizure frequency, and decreased survival compared to Scn2a^Q54 mice congenic on the C57BL/6J strain. Genetic mapping and RNA-Seq analysis identified Cacna1g as a candidate modifier gene at the Moe1 locus, which influences Scn2a^Q54 phenotype severity. In this study, we evaluated the modifier potential of Cacna1g, encoding the Cav3.1 voltage-gated calcium channel, by testing whether transgenic alteration of Cacna1g expression modifies severity of the Scn2a^Q54 seizure phenotype. Scn2a^Q54 mice exhibited increased spontaneous seizure frequency with elevated Cacna1g expression and decreased seizure frequency with decreased Cacna1g expression. These results provide support for Cacna1g as an epilepsy modifier gene and suggest that modulation of Cav3.1 may be an effective therapeutic strategy.

Introduction

Mutations in the voltage-gated sodium channel (VGSC) genes SCN1A, SCN2A, SCN3A, and SCN8A are responsible for several human epilepsies. More than 1200 mutations in SCN1A, SCN2A, and SCN8A have been identified in patients with various epilepsy syndromes, including Genetic Epilepsy with Febrile Seizures Plus, Benign Familial Neonatal-Infantile Seizures, and early infantile epileptic encephalopathies. Variable expressivity is a common feature of epilepsies caused by VGSC mutations.

Mouse epilepsy models provide a tractable system to map and characterize modifier genes. A common feature of these models is strain-dependent phenotype severity, suggesting the contribution of genetic modifiers. Scn2a^Q54 (Q54) transgenic mice have a gain-of-function mutation that results in spontaneous focal motor seizures and strain-dependent epilepsy severity.^{1; 2} On the C57BL/6J (B6) strain, the Q54 mutation (B6.Q54) results in mild, adult-onset epilepsy. When B6.Q54 is crossed to SJL/J (SJL), the resulting F1.Q54 mice experience juvenile-onset spontaneous seizures and reduced lifespan. We previously mapped modifier loci responsible for the strain difference in seizure frequency exhibited by Scn2a^Q54 mice: Moe1 (modifier of epilepsy 1) and Moe2 on chromosome 11 and 19, respectively.² Kcnv2 was identified as a modifier gene at Moe2.³ Recent work indicated multiple genes likely underlie the Moe1 peak, including Cacna1g and Hlf.⁴ Genetic abalation of Hlf, encoding a PAR bZIP transcription factor, exacerbated epilepsy in the Scn2a^Q54 and Scn1a^+/− Dravet mouse models.⁵

Cacna1g resides within the Moe1 locus and RNA-seq showed strain-dependent differences in Cacna1g transcript expression and alternative splicing.⁴ Cacna1g encodes the pore-forming α1G/Cav3.1 subunit, a low-voltage activated calcium channel of the T-type calcium channel family. Patient and animal model data have demonstrated a link between T-type currents and absence seizures. Ethosuximide (ETX), a T-type channel blocker, is widely used for treatment of absence epilepsy in patients. In mice, targeted deletion of Cacna1g diminishes GABA_B-receptor agonist-induced spike-wave discharges (SWDs), while overexpression of Cacna1g is associated with absence seizures and abnormal thalamocortical synchronization.^6–8 A role for Cacna1g in non-absence epilepsy has been remained elusive.

This study evaluated the modifier effect of Cacna1g on the Scn2a^Q54 mouse epilepsy model. We tested the effect of transgenic upregulation or heterozygous deletion of Cacna1g on spontaneous seizure frequency of Scn2a^Q54 mice.

Methods

Mice

A B6-derived BAC clone containing Cacna1g (RP23-65I14) was obtained from BACPAC Resources (http://bacpac.chori.org/home.htm). Pronuclear injections into fertilized SJL or B6 oocytes were performed by the Vanderbilt University Transgenic Mouse/ESC Shared Resource and the University of Michigan Transgenic Animal Model Core, respectively. Injections resulted in two Cacna1g BAC transgenic founders: (1) SJL.Cacna1gLOW (SJL.1GL), maintained by continued backcrossing of hemizygous transgenic mice to SJL/J (Jackson Labs); and (2) B6.Cacna1gHIGH (B6.1GH), maintained by continued backcrossing of hemizygous transgenic mice to C57BL/6J (Jackson Labs). B6(129S4)-Cacna1g^tm1Stl/J mice were obtained (Jackson Labs #021932), bred to C57BL/6-Tg(Zp3-cre)93Knw/J (Jackson Labs #003651), and maintained by continued backcrossing of heterozygous Cacna1g^KO/+ (B6.1G^KO/+) offspring to B6.Scn2a^Q54 transgenic mice [Tg(Eno2-Scn2a1*)Q54Mm] congenic on B6 (B6.Q54) were maintained by continued backcrossing of hemizygous B6.Q54 males to B6 females.¹

Transgenic SJL.1GL (SJL/J background) and B6.1GH (C57BL/6J background) females were crossed with B6.Q54 males to generate double transgenic (F1.Q54;1GL or B6.Q54;1GH) mice and single transgenic littermate controls. B6.1G^KO/+ females were crossed to B6.Q54 mice, and B6.Q54;1G^KO/+ male offspring were subsequently crossed to SJL/J females to generate F1.Q54;1G^KO/+ mice and single transgenic littermate controls.

Mice were group-housed with access to food and water ad libitum. All studies were approved by the Vanderbilt University and Northwestern University Animal Care and Use Committees in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The principles outlined in the ARRIVE guideline and Basel declaration (including the 3R concept) were considered when planning the experiments.

Genotyping

Mice were genotyped by PCR of DNA isolated from postnatal day 14 (P14) tail biopsies. SJL.1GL and B6.1GH transgenic mice were identified using 1G-SP6 and 1G-T7 primers that amplify 200 bp and 220 bp products, respectively. The Q54 transgene and 1G^KO/+ allele were genotyped as described.^{1; 9} Primer sequences used for genotyping, BAC integrity, copy number, and expression analysis will be provided upon request.

BAC Integrity and Copy Number

BAC integrity was evaluated by PCR amplification of genomic DNA. SP6 and T7 promoters flanked each end of the BAC insert, while microsatellite markers were located approximately every 32 Kb, including two within Cacna1g. B6.1GH mice were backcrossed twice to SJL to obtain informative mice for integrity analysis of the B6-derived BAC.

Transgene copy number was assessed by quantitative PCR (qPCR) using Cacna1g and reference Scn5a Taqman assays previously described.^{8; 10} At least n>4 independent DNA samples from hemizygous transgenics and wildtype controls were used to estimate copy number. Standard curves were generated by spiking SJL or B6 genomic DNA with BAC plasmid DNA equivalent to 0, 5, 10 and 20 copies per diploid genome.

Transcript Analysis and Protein Analysis

RNA was extracted from whole brains of six week old mice using Trizol (Life Technologies). The six week time point was selected to match our previous RNA-seq transcriptome analysis that identified Cacna1g as a candidate modifier.⁴ Total RNA was reverse transcribed using SuperScript III (Life Technologies). Quantitative digital droplet PCR (ddPCR) was performed using ddPCR Supermix for Probes (No dUTP) (Bio-Rad) and TaqMan Gene Expression Assays (Life Technologies) for Cacna1g (FAM-Mm00486571_m1) and TATA binding protein (Tbp) (VIC-Mm00446971_m1). Reactions were partitioned into 20,000 droplets (1 nl each) in a QX200 droplet generator (Bio-Rad). Thermocycling conditions were 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute (ramp rate of 2°C/sec), and a final inactivation step of 98°C for 10 minutes. Following amplification, droplets were analyzed with a QX200 droplet reader and QuantaSoft v1.6.6.0320 software (Bio-Rad). All assays lacked detectable signal in no-RT and no-template controls. Relative transcript levels were expressed as a ratio of Cacna1g/Tbp concentration and compared between groups by Student’s T-test.

Membrane protein fractions (50 μg) were prepared from whole brains of six week old mice and separated on 4–20% gradient SDS-PAGE gels (Biorad) and transferred to nitrocellulose. Membranes were assayed using mouse monoclonal Cav3.1 (Neuromab, N178A/9; 1:500) and β-tubulin (Sigma, TUB 2.1; 1:2,000) primary antibodies, and peroxidase-conjugated goat anti-mouse (1:50,000; Jackson ImmunoResearch) secondary antibodies. Signals were detected with SuperSignal West Femto (Pierce) and densitometry was performed using ImageJ. Cav3.1 expression was normalized to the corresponding β-tubulin band. Expression differences were compared between groups by Student’s T-test.

Phenotyping

Transgenic mice were phenotyped as previously described.⁴ Briefly, at three and/or six weeks of age, littermates were placed in a clean cage and underwent 30 minute video-taped observations between 1:00 and 5:00pm. Prior extensive video-electroencephalogram monitoring of Q54 mice demonstrated a strong correlation between behavioral and electroencephalographic seizures (κ = 0.988), validating visual assessment of seizures.^{1; 11} Behavioral seizures were assessed offline by an observer blinded to genotype, counting the number of focal motor seizures (FMS) with forelimb clonus and repetitive movements lasting one to five seconds. For evaluation of transgenic lines SJL.1GL and B6.1GH, seizure counts from three and six weeks were combined to obtain a 60-minute seizure frequency for each animal. For F1.Q54;1G^KO/+ evaluation, seizure counts from the three week time-point were used for analysis. Other seizure types, including generalized tonic-clonic seizures (GTCS), were rarely observed at these ages. Seizure frequencies were compared between genotypes using Student’s T-test (parametric) or Mann-Whitney rank-sum test (non-parametric).

Results and Discussion

We previously mapped Moe1, a dominant modifier locus on chromosome 11 that influenced Scn2a^Q54 seizure frequency. Based on fine-mapping and RNA-Seq analysis, we nominated Cacna1g as a candidate modifier gene at Moe1.⁴ To evaluate the modifier effect of Cacna1g on the Scn2a^Q54 seizure phenotype, we generated two independent Cacna1g BAC transgenic mouse lines: SJL.Cacna1gLOW (SJL.1GL) and B6.Cacna1gHIGH (B6.1GH), with low and high transgene expression, respectively. During routine breeding of SJL.1GL and B6.1GH lines, X-linked inheritance patterns became apparent. Due to the potential for X-inactivation of the BAC transgenes as a confounding factor, we focused our efforts on studying males.

We confirmed integrity of the 1GL and 1GH BAC constructs using vector primers and microsatellite markers spanning the BAC. Copy number analysis estimated 5–8 transgene copy integrations for both SJL.1GL and B6.1GH lines. A modest increase in Cacna1g transcript expression was observed in hemizygous SJL.1GL compared to wildtype littermates (p<0.005) (Figure 1A). Although there was not a detectable increase in Cav3.1 protein (data not shown), this is likely due to lower sensitivity of western blotting relative to digital droplet PCR (ddPCR). Hemizygous B6.1GH had an approximately 3-fold increase in Cacna1g transcript expression (p<0.0001) (Figure 1B) and Cav3.1 protein expression (p<0.001) (Figure 1C) relative to wildtype littermates.

Figure 1. Characterization of Cacna1g transgenic mice.

(A,B) To assess expression at the level of mRNA, a quantitative digital droplet PCR (ddPCR) Taqman assay was performed on hemizygous transgenic (SJL.1GL or B6.1GH) male offspring and controls (SJL.WT or B6.WT). The expression of Cacna1g in six week old whole-brain was normalized to Tbp expression. P-value was determined by unpaired Student’s T-test. Error bars represent upper and lower limits derived from SEM. (A) Normalized Cacna1g expression was observed to be: SJL.WT = 1.0 (n=7) and hemizygous SJL.1GL = 1.27 (n=4). p<0.005. (B) Normalized Cacna1g expression was observed to be: wildtype control males = 1.0 (n=6) and hemizygous B6.1GH males = 2.66 (n=6). p<0.0001. (C & D) Whole-brain membrane fractions prepared from six week old B6.1GH (C) or B6.1G^KO/+ and B6.1G^KO/KO (D) mice were assayed using mouse monoclonal Cav3.1 (Neuromab, N178A/9) (upper panels) and β-tubulin antibodies (Sigma, TUB 2.1) (lower panels). Expression of Cav3.1 was determined by densitometry using ImageJ. (C) Cav3.1 expression was significantly elevated in B6.1GH hemizygous males (2.69; n=5) relative to wildtype controls (1; n=4) (p < 0.001; Student’s T-test). (D) Cav3.1 expression was decreased by approximately half in 1G^KO/+ heterozygotes and absent in 1G^KO/KO homozygotes.

To determine if Cacna1g could modify the Scn2a^Q54 phenotype, we evaluated the effect of elevated Cacna1g expression on the Scn2a^Q54 seizure frequency. Double transgenic F1.Q54;1GL males had significantly more FMS than F1.Q54 controls (p<0.004) (Figure 2A). Similarly, double transgenic B6.Q54;1GH males exhibited significantly more FMS than B6.Q54 controls (p<0.0001) (Figure 2B). Single transgenic F1.1GL (n=17) and B6.1GH (n=10) controls did not exhibit any observable FMS. This is consistent with previous reports that Cacna1g BAC transgenic mice exhibited frequent behavioral arrest, but lacked evident motor seizures.⁸ Our results demonstrate that elevated levels of Cav3.1 can worsen seizures, consistent with our prior observation that mice with the susceptible Moe1 allele have higher Cacna1g expression.⁴

Figure 2. Effect of genetic modulation of Cacna1g on Scn2a^Q54 seizure frequency.

(A–C) Average number of focal motor seizures (FMS) for each genotype is shown. Error bars represent SEM. (A) Average seizure frequencies in 60 minutes were compared between F1.Q54 (22.3 ± 1.76) and F1.Q54;1GL (29.6 ± 1.68) using Student’s T-test (p<0.004). (B) Average seizure frequencies in 60 minutes were compared between B6.Q54 (0.45 ± 0.15) and B6.Q54;1GH (6.68 ± 1.37) using Mann-Whitney rank-sum test (p<0.0001). (C) Average seizure frequencies in 30 minutes were compared between F1.Q54 (11.47 ± 1.9) and F1.Q54;1G^KO/+ (6.93 ± 1.0) using Student’s T-test (p<0.05).

To determine if decreased Cav3.1 activity could modify the Scn2a^Q54 phenotype, we evaluated the effect of heterozygous deletion of Cacna1g. Global heterozygous null Cacna1g^KO/+ mice congenic on B6 were generated by crossing a previously reported floxed Cacna1g allele with mice expressing Cre recombinase driven by the Zp3 promoter.⁹ We validated our Cacna1g^KO/+ strain by Western blot. We observed reduced Cav3.1 expression in heterozygous null Cacna1g^KO/+ mice and loss of Cav3.1 expression in homozygous null Cacna1g^KO/KO mice (Figure 1D). Heterozygous deletion of Cacna1g resulted in decreased FMS frequency in F1.Q54;1G^KO/+ mice compared to littermate F1.Q54 mice (p<0.05) (Figure 2C). This is consistent with our prior observation that mice with the protective Moe1 allele have lower Cacna1g expression.⁴

Our genetic results support Cacna1g as a genetic modifier of epilepsy. Recent evidence from other studies suggests T-type calcium channels can contribute to additional epilepsies beyond absence. Two novel T-type channel blockers, TTA-A2 and Z944, reduced tonic seizure frequency in the maximal electroshock (MES) model or delayed kindling, respectively.^{12; 13} This pharmacological inhibition of rodent T-type channels effectively reduced seizures, mimicking the phenotype of homozygous null Cacna1g^−/− mice, which are resistant to induction of tonic seizures by MES and display reduced epileptogenicity in response to kainate.^{12; 14} Similarly, the results of our study showed Cacna1g-mediated modulation of focal motor seizures in a genetic model. Future studies will evaluate the effect of T-type channel blockers on the Scn2a^Q54 epilepsy model.

Taken together, a growing body of genetic and pharmacological evidence supports a contribution of T-type calcium channels, including Cacna1g, to non-absence epilepsy, suggesting that it may be a therapeutic target with broader applicability.