Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are focal malformations of cortical development (FMCDs) that are highly associated with intractable epilepsy. TSC and FCD are mTORopathies caused by a spectrum of pathogenic variants in the mechanistic target of rapamycin (mTOR) pathway genes leading to differential activation of mTOR signaling. However, whether the degree of mTOR hyperactivity influences disease severity remains unclear. Here, we examined the effects of differential mTOR hyperactivity levels on epilepsy and associated neuropathology in a mouse model of TSC and FCD. Constitutively active Rheb (Rheb CA), the canonical activator of mTOR complex 1 (mTORC1), was expressed in mouse embryos of either sex via in utero electroporation at low, intermediate, and high concentrations to induce different mTORC1 activity levels in developing cortical neurons. We found that Rheb CA expression induced mTORC1 hyperactivation and increased neuronal soma size and misplacement in a dose-dependent manner. No seizures were detected in the low Rheb CA mice, whereas the intermediate and high Rheb CA mice displayed spontaneous, recurrent seizures that significantly increased with higher Rheb CA concentrations. Seizures were associated with a global increase in microglial activation that was notably higher in the regions containing Rheb CA-expressing neurons. These data demonstrate that neuronal mTOR hyperactivity levels influence the severity of epilepsy and associated neuropathology in experimental TSC and FCD. Overall, these findings highlight the importance of evaluating the outcome of individual variants on mTOR activity levels and support personalized medicine strategies based on patient variants and mTOR activity level for TSC, FCD, and potentially other mTORopathies.

Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway during fetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development (FMCD) and intractable epilepsy. Recent evidence suggests a role for dysregulated cap-dependent translation downstream of mTOR in the formation of FMCD and seizures. However, it is unknown whether modifying translation once the developmental pathologies are established can reverse neuronal abnormalities and seizures. Addressing these issues is crucial with regards to therapeutics since these neurodevelopmental disorders are predominantly diagnosed during childhood, when patients present with symptoms. Here, we report increased phosphorylation of the mTOR effector and translational repressor, 4E-BP1, in patient FMCD tissue and in a mouse model of FMCD. Using temporally regulated conditional gene expression systems, we found that expression of a constitutively active form of 4E-BP1 that resists phosphorylation by mTOR in juvenile mice reduced neuronal cytomegaly and corrected several neuronal electrophysiological alterations, including depolarized resting membrane potential, irregular firing pattern, and aberrant expression of HCN4 channels. Further, 4E-BP1 expression in juvenile FMCD mice after epilepsy onset resulted in improved cortical spectral activity and decreased spontaneous seizure frequency in adults. Overall, our study uncovered a remarkable plasticity of the juvenile brain that facilitates novel therapeutic opportunities to treat FMCD-related epilepsy during childhood with potentially long-lasting effects in adults.

Hyperactivation of mTOR signaling during fetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development (FMCD) and intractable epilepsy. Recent evidence suggests increased cap-dependent translation downstream of mTOR contributes to FMCD formation and seizures. However, whether reducing overactive translation once the developmental pathologies are established reverses neuronal abnormalities and seizures is unknown. Here, we found that the translational repressor 4E-BP1, which is inactivated by mTOR-mediated phosphorylation, is hyperphosphorylated in patient FMCD tissue and in a mouse model of FMCD. Expressing constitutive active 4E-BP1 to repress aberrant translation in juvenile mice with FMCD reduced neuronal cytomegaly and corrected several electrophysiological alterations, including depolarized resting membrane potential, irregular firing pattern, and aberrant HCN4 channel expression. This was accompanied by improved cortical spectral activity and decreased seizures. Although mTOR controls multiple pathways, our study shows that targeting 4E-BP1-mediated translation alone is sufficient to alleviate neuronal dysfunction and ongoing epilepsy.