mTOR Hyperactivity Levels Influence the Severity of Epilepsy and Associated Neuropathology in an Experimental Model of Tuberous Sclerosis Complex and Focal Cortical Dysplasia

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 ofmTORcomplex 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 onmTORactivity levels and support personalized medicine strategies based on patient variants and mTOR activity level for TSC, FCD, and potentially other mTORopathies.