Gain-of-function mutation in Gnao1: a murine model of epileptiform encephalopathy (EIEE17)?

Abstract

G protein-coupled receptors strongly modulate neuronal excitability but there has been little evidence for G protein mechanisms in genetic epilepsies. Recently, four patients with epileptic encephalopathy (EIEE17) were found to have mutations in GNAO1, the most abundant G protein in brain, but the mechanism of this effect is not known. The GNAO1 gene product, Gαo, negatively regulates neurotransmitter release. Here, we report a dominant murine model of Gnao1-related seizures and sudden death. We introduced a genomic gain-of-function knock-in mutation (Gnao1 (+/G184S)) that prevents Go turnoff by Regulators of G protein signaling proteins. This results in rare seizures, strain-dependent death between 15 and 40 weeks of age, and a markedly increased frequency of interictal epileptiform discharges. Mutants on a C57BL/6J background also have faster sensitization to pentylenetetrazol (PTZ) kindling. Both premature lethality and PTZ kindling effects are suppressed in the 129SvJ mouse strain. We have mapped a 129S-derived modifier locus on Chromosome 17 (within the region 41-70 MB) as a Modifer of G protein Seizures (Mogs1). Our mouse model suggests a novel gain-of-function mechanism for the newly defined subset of epileptic encephalopathy (EIEE17). Furthermore, it reveals a new epilepsy susceptibility modifier Mogs1 with implications for the complex genetics of human epilepsy as well as sudden death in epilepsy.

Fig. 1

Gnao1 ^+/G184S mice exhibit strain-dependent lethality, seizures, and altered EEGs. a Gnao1 ^+/G184S mutant mice on a C57BL/6J background (N7 B6) die prematurely (Gehan–Breslow–Wilcoxon df(1) = 12.8, p < 0.001). b Early lethality is independent of gender. c Video monitoring revealed a seizure just prior to death (video in supplement). d, e EEG monitoring of Gnao1 mutant and wt mice demonstrate a greater than tenfold increase in interictal epileptiform discharges (IEDs) in B6 Gnao1 ^+/G184S mutant females compared to littermate wt controls (unpaired t test t = 7.37 df = 2.2, p < 0.02). Vertical bars demark the passage of 1 s. Data represent mean ± SEM. *p < 0.02

Fig. 2

Gnao1 ^+/G184S mice sensitize faster to repeated low-dose PTZ. The time to undergo sensitization to PTZ kindling (see Methods for definition) was recorded. a B6 female Gnao1 ^+/G184S sensitize more rapidly than littermate controls (Gehan–Breslow–Wilcoxon df(1) = 5.74, p < 0.05). b Similarly Gnao ^+/G184S males also sensitize more rapidly than littermate controls (Gehan–Breslow–Wilcoxon df(1) = 6.88, p < 0.01). c B6 heterozygous knockout Gnao1 ^+/− mutant females do not sensitize more rapidly than littermate controls (p > 0.05)

Fig. 3

Adult lethality and kindling sensitivity are suppressed on a 129 background. a While B6 (N7) Gnao1^+/G184S mutant mice die prematurely, mutants on a 129SvImJ strain background do not. b Similarly, the enhanced kindling sensitivity of Gnao1^+/G184S mutants is not seen on a 129 strain background. c The effect of the 129 background is dominant as 129/B6 F1 mice carrying the Gnao1 mutation do not show enhanced adult lethality. d Similarly, the increased kindling sensitivity of Gnao1^+/G184S mutants on the B6 background is partially ameliorated on the F1 background

Fig. 4

Mapping a modifier of the strain-dependent lethality on Chromosome 17. A cohort of 18 Gnao1 ^+/G184S mutant mice derived from a single N6 male founder was monitored for longevity. Whole genome SNP analysis was done comparing short-lived (black, dead by 25 weeks) versus long-lived (red, alive at 25 weeks). Regions of retained 129S alleles were assessed. a Plots show the percentage of the mutant mice carrying 129 alleles at a particular location in the genome (see b and Fig. S3 for specific marker locations). All mutant mice had 129 alleles linked to the Gnao1 locus on Chr8. b Only one region, Chr 17, showed preferential retention of 129 alleles in the long-lived population. c Confirmation of the association of retained 129 alleles on Chr17 with longer survival was undertaken in an independent cohort of 44 mice. Those mice retaining 129 alleles on Chr 17 at both 47 and 62 Mb (but not at 41 and 70 Mb) showed increased survival (c Gehan–Breslow–Wilcoxon df(1) = 4.9, p < 0.05)

Fig. 5

Chromosome 17 modifier region also affects sensitization to kindling of B6 Gnao1 ^+/G184S mutants but not that of WT mice. a Female Gnao1 mutant mice homozygous for B6 alleles at Chr17 (47–62 MB) sensitize to kindling faster than those heterozygous for 129 alleles at those loci. So the Chr17 region that protects against early lethality also delays sensitization to PTZ kindling (Gehan–Breslow–Wilcoxon df(1) = 7.9, p < 0.01). b The modifier is dependent on G_o mutation status. b In a sample of similar size to that in 5A, the Chr 17 modifier region did not significantly alter sensitization to PTZ kindling in B6 Gnao1 wt mice