Collapse of feed-forward inhibition underpins hyperexcitability in GABAA gain-of-function epilepsy

Gain-of-function (GOF) variants in GABAA receptors are increasingly recognised as a cause of severe developmental and epileptic encephalopathies (DEE). However, the mechanisms by which enhanced GABAA receptor activity leads to neuronal network hyperexcitability remains unclear. We engineered a novel mouse model based on the human GOF GABRB3 p.(Glu77Lys) variant (β3E77K), identified in two individuals diagnosed with DEE, to explore the mechanisms underlying GABAA receptor GOF disease. The phenotypes of β3E77K mice included embryonic lethality consistent with a severe early developmental impact of the GOF GABAA receptor variant. These mice display spike-wave-like discharges that were exacerbated by vigabatrin and ameliorated by valproate matching the clinical observations of GOF GABRB3 patients. β3E77K mice also have increased proconvulsant-induced seizure susceptibility and a broad increase in ECoG spectral power amplitude, indicative of cortical hyperexcitability. Additionally, neurological assessments revealed hypoactivity and weakened grip-strength. Ex-vivo electrophysiological recordings demonstrated increased GABAA receptor-mediated current amplitudes at both excitatory and inhibitory synapses in CA1 hippocampus, consistent with the GOF molecular phenotype previously identified in functional studies. In cortical layer 2/3, inhibitory interneurons showed increased synaptic GABAA receptor-mediated current amplitude, while synapses onto pyramidal neurons exhibited reduced inhibitory currents. Enhanced GABAA receptor-mediated synaptic activity among layer 2/3 interneuron populations caused a use-dependent collapse of feed-forward inhibition resulting in increased pyramidal neuron excitability. Computational modelling supported this disinhibition mechanism, showing that enhanced GABAA receptor synaptic strength between interneurons diminishes inhibitory synaptic conductance onto pyramidal cells. Our findings highlight the critical role of interneuron network dysfunction in driving cortical hyperexcitability caused by GOF GABAA receptor variants. They provide a novel pathogenic mechanism in DEE that could have broader implications for disorders involving dysfunction in GABAergic neurons. The β3E77K mouse also provides a unique preclinical model to test therapeutic strategies in GOF GABAA receptor disease.