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. 2019 Feb 1;142(2):362-375.
doi: 10.1093/brain/awy324.

Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

Rosie K A Bunton-Stasyshyn  1 , Jacy L Wagnon  1 , Eric R Wengert  2   3 , Bryan S Barker  2   3 , Alexa Faulkner  4 , Pravin K Wagley  5 , Kritika Bhatia  6 , Julie M Jones  1 , Marissa R Maniaci  1 , Jack M Parent  4   6 , Howard P Goodkin  3   5 , Manoj K Patel  2   3 , Miriam H Meisler  1   6
Affiliations

Prominent role of forebrain excitatory neurons in SCN8A encephalopathy

Rosie K A Bunton-Stasyshyn et al. Brain. .

Abstract

De novo mutations of the sodium channel gene SCN8A result in an epileptic encephalopathy with refractory seizures, developmental delay, and elevated risk of sudden death. p.Arg1872Trp is a recurrent de novo SCN8A mutation reported in 14 unrelated individuals with epileptic encephalopathy that included seizure onset in the prenatal or infantile period and severe verbal and ambulatory comorbidities. The major biophysical effect of the mutation was previously shown to be impaired channel inactivation accompanied by increased current density. We have generated a conditional mouse mutation in which expression of this severe gain-of-function mutation is dependent upon Cre recombinase. Global activation of p.Arg1872Trp by EIIa-Cre resulted in convulsive seizures and lethality at 2 weeks of age. Neural activation of the p.Arg1872Trp mutation by Nestin-Cre also resulted in early onset seizures and death. Restriction of p.Arg1872Trp expression to excitatory neurons using Emx1-Cre recapitulated seizures and juvenile lethality between 1 and 2 months of age. In contrast, activation of p.Arg1872Trp in inhibitory neurons by Gad2-Cre or Dlx5/6-Cre did not induce seizures or overt neurological dysfunction. The sodium channel modulator GS967/Prax330 prolonged survival of mice with global expression of R1872W and also modulated the activity of the mutant channel in transfected cells. Activation of the p.Arg1872Trp mutation in adult mice was sufficient to generate seizures and death, indicating that successful therapy will require lifelong treatment. These findings provide insight into the pathogenic mechanism of this gain-of-function mutation of SCN8A and identify excitatory neurons as critical targets for therapeutic intervention.

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Figures

Figure 1
Figure 1
Structure and basal expression of the conditional R1872W allele of Scn8a. (A) The conditional allele contains two copies of exon 26, the final exon of Scn8a. Both exons 26a and 26b were derived from a targeting vector with eight silent substitutions in exon 26 that were introduced to prevent digestion by the TALEN endonucleases used for targeting of the Scn8a locus (Jones and Meisler, 2014; see Supplementary Fig. 1 for details). The upstream exon 26a contains wild-type arginine codon 1872 (R) and the 3′ UTR from the bGH gene, and is flanked by loxP sites. The downstream exon 26b contains the same eight silent substitutions as well as the mutant tryptophan codon 1872 (W) and terminates in the endogenous 3′ UTR. Exposure to Cre recombinase will delete exon 26a and lead to expression of exon 26b encoding the mutant sodium channel. (B) Southern blot of genomic DNA from eight potential founder mice. Correct targeting of the endogenous Scn8a locus in DNA from founder 2176 (asterisk) is demonstrated by the 5.4 kb KpnI restriction fragment hybridizing with the 5′ probe 1 as well as the 3.5 kb HincII hybridizing with 3′ probe 2 (Supplementary Fig. 1B). The full gel is shown in Supplementary Fig. 1B. Related data in Supplementary Fig. 1C show the sequenced regions of long-range junction PCR products that confirmed the structure of the conditional allele. (C) RT-PCR products were amplified with forward primer in exon 25 and reverse primer either in the bGH 3′ UTR or the endogenous 3′ UTR. The wild-type allele expresses the endogenous 3′ UTR and the conditional allele expresses only the bGH 3′ UTR in the absence of Cre recombinase. (D) Sequences of the RT-PCR products amplified using primer pair 6 (forward primer in exon 25, reverse primer in exon 26) demonstrate expression of exon 26 from the wild-type allele and exon 26a from the conditional allele. Boxed nucleotides indicate the location of markers of the conditional allele: three of the synonymous nucleotide substitutions and tryptophan codon 1872. (E) Quantitative RT-PCR of whole brain RNA with the TaqManTM assay for Scn8a and comparison with the control transcript of the Tata-binding protein (Tbp) demonstrates comparable level of transcript from the wild-type allele and the conditional allele. Three animals of each genotype were assayed.
Figure 2
Figure 2
Ubiquitous activation of the conditional Scn8a allele by EIIA-Cre. (A) Genotyping of genomic DNA samples from brain and tail of heterozygous Scn8acond/+,EIIa-Cre mice expressing one wild-type and one conditional allele demonstrates Cre-mediated deletion of exon 26a. Genomic DNA was amplified with primer pair 2 located in intron 25 (Supplementary Table 1 and Supplementary Fig. 2) and the PCR product was digested with KpnI. (B) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. There is expression of exon 26b from the conditional allele, due to the excision of exon 26a by Cre recombinase. In addition, exon 26 is expressed from the wild-type allele in the heterozygous mice. B = Bsrb1. (C) Sanger sequencing of the RT-PCR products demonstrates the heterozygous expression of wild-type exon 26 and exon 26b. Heterozygous synonymous mutations are boxed and the heterozygous R1872W mutation is marked with an arrow. The location of the sequenced nucleotides within the amplified exon is indicated by dotted lines.
Figure 3
Figure 3
Phenotypic effects of activation of the conditional allele by ubiquitously expressed EIIa-Cre. (A) Scn8acond/+ heterozygotes expressing EIIa-Cre exhibited sudden lethality at the end of the second week of life, with median survival of 15 days. Scn8acond/+ heterozygotes that did not inherit EIIa-Cre exhibited normal life span. (B) EEG recording of a fatal seizure in an Scn8acond/+,EIIa-Cre mouse on P15. The recording was obtained from bipolar insulated stainless-steel electrodes in the left parietal cortex (top trace) and right parietal cortex (second trace). The EMG artefact in the third trace reflects the period of running evolving to tonic extension. Death occurred 3 min 20 s after the onset of running. The bottom trace was obtained from an electrode placed subcutaneously in the right thorax and demonstrates sudden slowing of the heart rate from 756 bpm prior to the running fit to 156 bpm immediately afterwards.
Figure 4
Figure 4
Hyperactivity of cortical neurons expressing the Scn8aR1872W mutant sodium channel. (A and B) Representative slice recordings from control (14 cells from four mice) and Scn8acond/+,EIIa-Cre CA1 neurons (16 cells from five mice). (C) Action potential firing frequency at 400 pA current injection. (D and E) Representative slice recordings from control (12 cells from four mice) and Scn8acond/+,EIIa-Cre layer V cortical neurons (16 cells from five mice). (F) Action potential firing frequency at 400 pA current injection. Data shown represent means ± standard error of the mean (SEM) *P < 0.05, ***P < 0.001.
Figure 5
Figure 5
Effects of expression of Scn8aR1872W in forebrain neurons. (A) Survival of conditional mice expressing cell-specific Cre constructs. Nestin-Cre is broadly expressed in neural cells; median survival 21 days. Emx1-Cre is expressed in excitatory neurons of the forebrain; median survival 46 days. Gad2-Cre is expressed in inhibitory neurons. (B) Brain transcripts encoding arginine 1872 (173 bp RT-PCR fragment) and tryptophan 1872 (273 bp RT-PCR fragment) in mice expressing various Cre recombinases.
Figure 6
Figure 6
Activation of Scn8aR1872W in adult mice leads to seizures and lethality. Scn8acond / +,CAG-Cre-ER mice (n = 6) were treated at 8 weeks of age with five daily injections of tamoxifen. Seizures were monitored visually for 8 h per day. (A) The first seizures were observed between 2 and 4 weeks after tamoxifen injection. (B) Death occurred between 1 and 3 months after seizure onset. No seizures or deaths were observed in Scn8acond/+ littermates lacking Cre (n = 3). (C) RT-PCR of brain RNA using primer pair 6 in exons 25 and 26. Brain transcripts encoding tryptophan 1872 (273 bp RT-PCR fragment) are present only in tamoxifen-treated Scn8acond/+,CAG-Cre-ER mice and not in littermate controls lacking CAG-Cre-ER.
Figure 7
Figure 7
GS967/Prax330 extends the survival of Scn8acond/+,EIIa-Cre mice and reduces activity of the mutant channel in transfected cells. (A) GS967/Prax330 was added to the mouse chow at 8 mg/kg beginning on P1. Untreated mice received normal chow. Mean survival was increased from 15 days for untreated mice (n = 55) to 21 days for treated mice (n = 14). Kaplan-Meier log-rank (Mantel-Cox) test, χ2(1) = 32, P < 0.0001. (B–E) ND7/23 cells were transfected with Scn8aR1872W cDNA and incubated in the presence or absence of 1 µM GS967/Prax330. (B) Averaged current-voltage (I-V) relation for cells expressing mutant Nav1.6 ± GS967/Prax330. Peak currents were normalized to cell capacitance. (C) Voltage dependence of steady-state inactivation. Smooth lines correspond to the least squares fit when average data were fit to a single Boltzmann equation. (D) Representative normalized current traces recorded during a 100 ms depolarizing pulse from −120 mV to 0 mV illustrating the reduction of persistent sodium current for p.Arg1872Trp in the presence of GS967/Prax330. (E) Quantification of the ratio of peak to persistent sodium current. Data are means ± SEM statistical significance (*P < 0.05).
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