Interneuron Dysfunction in a New Mouse Model of SCN1A GEFS

Abstract

Advances in genome sequencing have identified over 1300 mutations in the SCN1A sodium channel gene that result in genetic epilepsies. However, it still remains unclear how most individual mutations within SCN1A result in seizures. A previous study has shown that the K1270T (KT) mutation, linked to genetic epilepsy with febrile seizure plus (GEFS+) in humans, causes heat-induced seizure activity associated with a temperature-dependent decrease in GABAergic neuron excitability in a Drosophila knock-in model. To examine the behavioral and cellular effects of this mutation in mammals, we introduced the equivalent KT mutation into the mouse (Mus musculus) Scn1a (Scn1a^KT) gene using CRISPR/Cas9 and generated mutant lines in two widely used genetic backgrounds: C57BL/6NJ and 129X1/SvJ. In both backgrounds, mice homozygous for the KT mutation had spontaneous seizures and died by postnatal day (P)23. There was no difference in mortality of heterozygous KT mice compared with wild-type littermates up to six months old. Heterozygous mutants exhibited heat-induced seizures at ∼42°C, a temperature that did not induce seizures in wild-type littermates. In acute hippocampal slices at permissive temperatures, current-clamp recordings revealed a significantly depolarized shift in action potential threshold and reduced action potential amplitude in parvalbumin (PV)-expressing inhibitory CA1 interneurons in Scn1a^KT/+ mice. There was no change in the firing properties of excitatory CA1 pyramidal neurons. These results suggest that a constitutive decrease in inhibitory interneuron excitability contributes to the seizure phenotype in the mouse model.

Keywords: CRISPR/Cas9; GEFS+; SCN1A; epilepsy; parvalbumin interneurons; seizures.

Figure 1.

Generation of Scn1a^KT/+ mouse using CRISPR/Cas9. A, The GEFS+ causing K1270T mutation (asterisk) is located in S2 transmembrane segment of Domain III of the α subunit of Nav1.1 ion channel encoded by the human SCN1A gene. B, Location of the guide RNA relative to the Cas9 cut site and the locus of the K1259T mutation in the mouse Scn1a gene and the Nav1.1 protein sequence. Repair template sequence with the base pair changes introducing the K-T mutation and the EcoRV cut site. All edited nucleotides are shown in lower case letters and the HDR region is represented by underlined letters. Two additional silent mutations (asterisks) were added to prevent re-cutting by Cas9 following HDR. C, Outline of the steps followed to generate Scn1a^KT mouse colonies in B6NJ and 129X1 genetic backgrounds from a single founder G₀ male. D, DNA sequence comparison between a wild-type (Scn1a^+/+) and a heterozygous (Scn1a^KT/+) mouse showing missense K-T mutation and another silent mutation that results in an EcoRV cut site. DNA chromatograms of Scn1a^+/+and Scn1a^KT/+ mice showing no off-target effects is shown in Extended Data Figure 1-1. E, A representative agarose gel shows PCR amplified DNA bands digested with EcoRV which distinguishes between mice homozygous for the mutant allele, Scn1a^KT/KT (223 and 165 bp), Scn1a^+/+ wild-type mice homozygous for the wild-type allele (388 bp), and heterozygous Scn1a^KT/+ mice carrying one copy each of wild-type (388 bp) and mutant (223 and 165 bp) alleles.  Figure Contributions: Grant R. MacGregor and Jonathan C. Neumann designed the CRISPR/Cas9 strategy to generate transgenic mouse. Soleil S. Schutte, Antara Das performed DNA sequencing and genotyping. Grant MacGregor, Olga Safrina, and Daniel R. Benavides performed off-target screening, Antara Das and Olga Safrina, analyzed the off-target data.

Figure 2.

Homozygous (Scn1a^KT/KT) mice have a shortened lifespan in both genetic backgrounds. A, B, Survivorship plots of wild-type (Scn1a^+/+) and mutant mice over a period of one month in 129X1 and B6NJ strains, respectively. Homozygous (Scn1a^KT/KT) mice in both backgrounds displayed reduced mean lifespan (129X1 = 19.9 d; B6NJ = 20.3 d) compared with heterozygous (Scn1a^KT/+) and wild-type (Scn1a^+/+) littermates assayed in parallel. C, D, Body weight of mutant mice (Scn1a^KT/+ and Scn1a^KT/KT) was not different from wild-type (Scn1a^+/+) littermates before the age of P20 (Mann–Whitney test, p < 0.05). Data are represented as mean ± SEM. Individual data points of mouse survival and body weights observed across time is listed in detail in Extended Data Table 2-1. Figure Contributions: Antara Das performed the experiment and analyzed the results.

Figure 3.

Heterozygous mice (Scn1a^KT/+) exhibit heat-induced seizures and spontaneous seizures. A, Custom-built heating chamber and schematic of the heating protocol used for inducing seizures. B, C, Change in mean body temperature over time in wild-type (Scn1a^+/+) and heterozygous mice (Scn1a^KT/+) in 129X1 and B6NJ strains, respectively. D, Percentage of mice exhibiting heat-induced seizures in both strains. E, Seizure threshold temperature and (F) latency to heat-induced seizures in wild-type (Scn1a^+/+) and heterozygous (Scn1a^KT/+) mice in both strains. G, Maximum Racine scores of heat-induced seizures exhibited by heterozygous (Scn1a^KT/+) mice in both genetic strains are shown. Each dot represents maximum Racine score in a single mouse. Data shown in panels B–F are mean ± SEM. Asterisks (*) indicate significant differences at p < 0.05. n.s., not significant. Number of animals are shown in parentheses. Representative electrographic (EEG) traces from a wild-type (Scn1a^+/+) and a heterozygous (Scn1a^KT/+) mouse on 129X1 strain is shown in Extended Data Figure 3-1. Figure Contributions: Antara Das and Lisha Zeng performed the heat-induced seizures experiment, Antara Das and An T. Pham performed the EEG experiments, Antara Das analyzed the results.

Figure 4.

Reduced excitability of PV interneurons in Scn1a^KT/+mice. A, left, Representative wild-type and heterozygous mouse brain sections depicting td-Tomato labeled PV interneurons (red) and DAPI (blue). so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar: 50 μm. Co-immunolabeling of td-Tomato positive PV interneurons with anti-PV antibody is shown in Extended Data Figure 4-1. A, right, Mean cell density of td-Tomato-labeled PV interneurons is not different between wild-type and heterozygous mice (n = 3 mice per genotype, two tailed Student’s t test). B, Representative traces of PV interneurons from wild-type (Scn1a^+/+) and heterozygous (Scn1a^KT/+) littermates at different current injection steps. C, Input-output curves showing AP firing frequency in PV interneurons against current injection steps between 0 and 900 pA, data points for 50-pA step increment is shown here. No difference in PV firing frequency between Scn1a^KT/+(gray curve, open circles) and wild-type Scn1a^+/+mice (black curve, triangles) was seen (two-way ANOVA with repeated measures, p > 0.05). D, Phase plots of the first derivative (dv/dt) was plotted against membrane potential (Vm) for the representative AP traces shown in panel E. Arrow indicates the AP threshold. E, Expanded representative traces of first AP fired from a PV interneuron recorded from a Scn1a^+/+and Scn1a^KT/+mouse. Circles represent the AP threshold determined from phase plots in panel D and the peak of action potentials. Arrows represent AP amplitude. F, AP threshold is more depolarized in Scn1a^KT/+ mice compared with wild-type Scn1a^+/+ mice. AP amplitude is also reduced in Scn1a^KT/+ mice compared with Scn1a^+/+ littermates. G, AP half-width is increased in Scn1a^KT/+ mice. F–H, All statistical comparisons done by Mann–Whitney test. Data shown are average of 14 and 9 cells from Scn1a^+/+ and Scn1a^KT/+ littermates, respectively, recorded from at least three different mice. Data are represented as mean ± SEM. Asterisks indicate data means are significantly different. * represents p < 0.05, and ** represents p < 0.01. Figure Contributions: Antara Das did the PV cell counts, Bingyao Zhu performed the electrophysiological recordings, Bingyao Zhu and Antara Das analyzed the results.

Figure 5.

Firing property of CA1 excitatory neurons in Scn1a^KT/+ mice remains unaltered. A, Representative traces of action potential firing from CA1 excitatory cells in wild-type (Scn1a^+/+) and heterozygous (Scn1a^KT/+) mice in response to increasing current injection steps. B, No difference in firing frequency at a series of current injection steps was found between heterozygous Scn1a^KT/+ mice and Scn1a^+/+ littermates (two-way ANOVA, p > 0.05). C, Phase plots (dv/dt vs membrane potential) of the representative AP traces shown in panel D. Arrow indicates the AP threshold. D, Expanded single AP traces from Scn1a^+/+ and Scn1a^KT/+ mice. Circles represent the inflection point (AP threshold) and the peak of action potentials, arrow indicate AP amplitude. E–G, No change observed in AP threshold, AP amplitude, and AP half-width between heterozygous Scn1a^KT/+ and Scn1a^+/+ mice (two-tailed unpaired Student’s t test, p > 0.05). Data shown is average of 12 and 17 cells from Scn1a^+/+ and Scn1a^KT/+ littermates, respectively, recorded from at least eight different mice. Data represented as mean ± SEM. Figure Contributions: Antara Das and Yunyao Xie performed the experiments, Antara Das analyzed the results.