Rapamycin rescues neuronal hyperexcitability and seizures driven by megalencephaly-causing AKT3 activation

Abstract Background AKT serine/threonine kinase 3 is highly expressed in the brain, and activating variants cause megalencephaly and cortical malformations frequently associated with intractable epilepsy. However, the underlying disease mechanisms and therapeutic strategies remain poorly understood. Although the AKT inhibitor capivasertib may provide targeted treatment, its long-term use from infancy is limited by adverse effects. Identification of safer downstream therapeutic targets is therefore required. A heterozygous AKT3 variant (NM_005465.7:c.233 A > G, p.Q78R) was identified in a patient with megalencephaly and cortical malformations. Patient-derived induced pluripotent stem cells and isogenic control lines were established using CRISPR/Cas9. These cells were differentiated into glutamatergic neurons. A genome-edited mouse model carrying the same variant was established. In vitro and in vivo models were used to investigate disease mechanisms and therapeutic responses. Results Patient-derived neurons exhibited somatic enlargement, increased mechanistic target of rapamycin complex 1 (mTORC1) activity, enhanced spontaneous firing, and increased synchrony. Transcriptomic analysis showed comparable neuronal differentiation and maturation between patient-derived and control neurons, while revealing distinct gene expression profiles, including dysregulation of protocadherin family genes implicated in synaptogenesis and neuronal connectivity. Genome-edited mice demonstrated reduced survival, megalencephaly, neuronal hypertrophy, and decreased latency to chemically-induced seizures. The mTOR inhibitor rapamycin reduced neuronal hyperexcitability in vitro and increased seizure threshold in vivo. Conclusion Complementary human cellular and mouse models recapitulating epilepsy-related phenotypes driven by activating AKT3 variants were established. These phenotypes were demonstrated to be amenable to rescue through pharmacological inhibition. Importantly, the consistent therapeutic effects of mTOR inhibition across both systems support downstream targeting of the phosphoinositide 3-kinase–AKT–mTOR pathway as a rational therapeutic strategy for AKT3 -related epilepsy.