Normalization of network activity in an epilepsy model with a constitutively active <i>GABBR2</i> variant

The neurotransmitter GABA activates G protein-coupled GABAB receptors (GBRs) that mediate neuronal inhibition in the brain. These receptors function as obligate heterodimers, consisting of the GB1 and GB2 subunits, with GB1 binding GABA and GB2 interacting with the G protein. The monoallelic variants p.A567T, p.S695I, and p.I705N in the GABBR2 gene, which encodes the GB2 subunit, have been associated with epileptic encephalopathy and Rett-like disorders. The clinical phenotypes overlap with those seen in individuals with monoallelic loss-of-function variants in GABBR1, the gene encoding the GB1 subunit. To investigate the effects of these GABBR2 variants on GBR function, we expressed the variants in heterologous cells and evaluated their pharmacological profiles using a luciferase reporter assay. Furthermore, we introduced the epileptic encephalopathy-associated p.I705N variant into the mouse Gabbr2 gene to examine its impact on neuronal and network activity. These mice were analyzed using proteomic approaches, in combination with in vitro and in vivo electrophysiological techniques. Finally, we evaluated whether the observed network alterations could be reversed pharmacologically. In heterologous cells, all variants displayed strong constitutive activity, reaching 50-100% of the maximal GABA-induced activity of wild-type receptors. This gain-of-function effect was evident regardless of whether the variants were expressed as individual subunits or as heterodimeric receptors. EEG recordings from Gabbr2I704N/+ mice revealed abnormal high-amplitude synchronization in the δ frequency band, without overt seizures. Electrophysiological recordings from brain slices confirmed an increase in constitutive activity in both pre- and postsynaptic GBRs, but also revealed a significant reduction in receptor responsiveness to agonists. Proteomic analysis of brain tissue further revealed a downregulation of both GB1 and GB2 subunits, along with several G protein signaling components. This downregulation likely serves as an adaptive response to the heightened constitutive activity, reducing not only the activity of the variant receptors but also the signaling of wild-type receptors. In vivo recordings from the auditory cortex of awake Gabbr2I704N/+ mice revealed reduced spontaneous neuronal activity and a slower decline in neuronal activity following auditory stimuli. Treatment with a positive allosteric modulator of GBRs normalized spontaneous network activity and the termination of neuronal activity after sensory stimulation in these mice. In conclusion, our findings indicate that the clinical phenotypes associated with constitutively active GABBR2 variants are driven by an adaptive downregulation of GBRs and their key signaling components. Therefore, in monoallelic individuals, positive allosteric modulators that enhance wild-type receptor activity may provide a promising therapeutic strategy.