Sleep-related hypermotor epilepsy (SHE), also known as nocturnal frontal lobe epilepsy, is a type of focal epilepsy. Seizures in SHE are phenotypically comparable to those in frontal lobe epilepsy and occur predominantly during the nonrapid eye movement (NREM) phase of sleep (Tinuper et al., 2016). The reasons why seizures in SHE primarily arise during NREM sleep remain unclear (Becchetti et al., 2020). Genetic mutations are common in patients with SHE and are believed to be associated with its pathogenesis (Becchetti et al., 2015; Licchetta et al., 2020). Up to 15% of patients have variants in the nicotinic acetylcholine receptor (nAChR) genes CHRNA4, CHRNA2, and CHRNB2 (Becchetti et al., 2015; Licchetta et al., 2020). A small number of patients have variants in DEPDC5, NPRL2, and NPRL3, which control the activity of mammalian target of rapamycin complex 1, as well as variants in KCNT1, which encodes a sodium-activated potassium channel subunit (Heron et al., 2012; Ishida et al., 2013; Korenke et al., 2016; Ricos et al., 2016). In our previous study, we detected novel rare variants in GABRG2, the gene encoding the γ2 subunit of the γ-aminobutyric acid type A receptor (GABAAR), in patients with SHE. Further in vitro investigations demonstrated that these GABRG2 variants (NM_198904.2: c.269C > T, p.T90M; NM_198904.2: c.950C > A, p.T317N; and NM_198903: c.649C > T, p.Q217X) hamper surface expression, cause the endoplasmic reticulum (ER) retention, and impair the channel-gating functions of GABAAR in neurons (Jiang et al., 2022). However, it remains to be elucidated how GABRG2 variants cause seizures to occur in NREM sleep in vivo.

Functional abnormalities in the thalamocortical loop contribute to the propagation of epileptic discharges, particularly during sleep (Beenhakker and Huguenard, 2009; Grigg-Damberger and Foldvary-Schaefer, 2021; Halász, 2012; Luthi, 2014; Peyrache et al., 2011). Animal models harboring nAChR gene variants, which are the major molecular causes of SHE discovered so far (Licchetta et al., 2020), have demonstrated that functional abnormalities in the motor thalamic nuclei (MoTN) and secondary motor cortex (M2) may contribute to the pathogenesis of SHE seizures (Fukuyama et al., 2020a; Fukuyama et al., 2020b). Notably, the functional connection between the MoTN and M2 is mainly controlled by synaptic GABAergic inhibition, which is predominantly mediated by GABAARs (Anaclet et al., 2014; McCormick and Bal, 1997). Based on our previous findings, we hypothesized that impaired GABAAR function caused by GABRG2 variants (such as T317N) might lead to dysfunction in the MoTN-M2 pathway, resulting in seizures during NREM sleep in patients with SHE.

In the present study, to verify this hypothesis and explore how variants in GABRG2 cause SHE in vivo, we designed and generated a knock-in (KI) mouse expressing the mouse Gabrg2 T316N variant (Gabrg2T316N/+) which corresponds to the human variant T317N in GABRG2, using CRISPR/Cas9 technology. We chose to model the T317N variant because it arises de novo, and patients carrying this variant exhibit more severe clinical phenotypes (Jiang et al., 2022). We investigated the neuroanatomical characteristics of this mouse model. Continuous video electroencephalogram (EEG) monitoring and in vivo multichannel electrophysiological recordings were performed to explore alterations in the sleep-wake cycle, seizure patterns, and local field potentials (LFPs) of the MoTN-M2 pathway. The molecular pathology associated with the expression of GABAAR subunits was further examined in this KI mouse model. Our study established a mouse model carrying variants of GABRG2 to explore the pathogenesis of SHE and provided evidence that hypersynchronization between MoTN and M2 contributes to seizure attacks during NREM in SHE.