Altered GABAA receptor expression in brainstem nuclei and SUDEP in Gabrg2+/Q390X mice associated with epileptic encephalopathy

Abstract

Sudden unexpected death in epilepsy (SUDEP) is the leading cause for death in individuals with epilepsy. The frequency of SUDEP correlates with the severity of epilepsies and lack of response to antiepileptic drug treatment, but the underlying mechanisms of SUDEP have not been elucidated fully. GABRG2(Q390X) is a mutation associated with the epileptic encephalopathy Dravet syndrome (DS) and with genetic epilepsy with febrile seizures plus (GEFS+) in patients. The Gabrg2+/Q390X knockin (KI) mouse phenocopies the major features of DS and GEFS+ and has SUDEP throughout life. The Gabrg2+/− knockout (KO) mouse is associated with infrequent absence seizures and represents a model of mild absence epilepsy syndrome without increased mortality. To explore the basis for SUDEP in DS and GEFS+, we compared mutant γ2 subunit and wild-type α1 and β2/3 subunit expression in mice in brainstem nuclei associated with respiratory function including the solitary tract, pre-Botzinger complex and Kolliker-Fuse nuclei. We found that synaptic GABAA receptors were reduced while intracellular nonfunctional γ2(Q390X) subunits were increased in the heterozygous DS and GEFS+ KI mice, but not in the heterozygous absence epilepsy KO mice. Given the critical role of these nuclei in cardiorespiratory function, it is likely the impaired GABAergic transmission and neuronal dysfunction in these brainstem nuclei are involved in the cardiorespiratory collapse in SUDEP. The study provides novel mechanistic insights into cardiorespiratory failure of SUDEP.

Heat induced temperature dysregulation and seizures in Dravet Syndrome/GEFS+ Gabrg2+/Q390X mice

“…Three out of 32 Gabrg2 +/Q390X mice died after seizure induction by temperature elevation, 22 out of 35 Gabrg2 +/Q390X mice died after PTZ induction (Figure 3F), but none of wild-type mice died after seizure induction by either method. The increased mortality of Gabrg2 +/Q390X mice during seizure inductions was consistent with our previous report that the Gabrg2 +/Q390X mice had spontaneous death with evidence favoring cardiorespiratory failure, which is a putative cause of sudden unexpected death in epilepsy (SUDEP) 14;24 . However, to our surprise, none of Gabrg2 +/Q390X mice in DBA/2J background died during seizure induction by temperature elevation and only 1 out 10 mice died during PTZ seizure induction (Figure 3G), suggesting that some unknown mechanisms modify the phenotype of Gabrg2 +/Q390X mice in the two different genetic backgrounds.…”

Beyond Epilepsy and Autism: Disruption of GABRB3 Causes Ocular Hypopigmentation

“…A similar phenomenon has been noticed in the phenotype for another GABA A receptor subunit gene GABRG2 . In Gabrg2 knockout mice, the homozygous knockout was lethal while the heterozygous knockout only had mild absence epilepsy and anxiety but a normal survival (Warner et al, 2016; Xia et al, 2016). The lack of pigmentation in the Gabrb3 −/− mice is likely to be due to the defects in melanin transport and melanosome maturation instead of melanin synthesis because there was no change with tyrosine hydroxylase staining (Figure S3).…”

Overexpressing wild‐type γ2 subunits rescued the seizure phenotype in Gabrg2+/Q390X Dravet syndrome mice

“…To study the cellular expression of GABA A receptor subunits, the brains were fixed in 4% paraformaldehyde for 30 min, and maintained in 30% sucrose before sectioning on a cryostat at 30 µm before staining. The rabbit anti-α1 antibody (1:100; Abcam, #ab33299), the mouse anti-β2/3 (1:200 ; Millipore, #MAB341) antibody, and the nuclei staining reagent To-pro-3 (1:500) were used 20 . The images were acquired under confocal microscopy with objective of 63X and analyzed by ImageJ.…”

“…The rabbit anti-a1 antibody (1:100; Abcam, #ab33299), the mouse anti-b2/3 (1:200; Millipore, #MAB341) antibody, and the nuclei staining reagent Topro-3 (1:500) were used. 20 The images were acquired under confocal microscopy with objective of 63X and analyzed by ImageJ.…”