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.

Summary Objective Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in human cortical dysplasia (CD) as well as in animal models of epilepsy. While inhibition of mTOR signaling early in epileptogenesis suppressed epileptiform activity in the neuron subset-specific Pten knockout (NS-Pten KO) mouse model of CD, the effects of mTOR inhibition after epilepsy is fully established were not previously examined in this model. Here, we investigated whether mTOR inhibition suppresses epileptiform activity and other neuropathological correlates in adult NS-Pten KO mice with severe and well-established epilepsy. Methods The progression of epileptiform activity, mTOR pathway dysregulation, and associated neuropathology with age in NS-Pten KO mice were evaluated using video-electroencephalography (EEG) recordings, western blotting, and immunohistochemistry. A cohort of NS-Pten KO mice was treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., five days/week) starting at postnatal week 9 and video-EEG monitored for epileptiform activity. Western blotting and immunohistochemistry were performed to evaluate the effects of rapamycin on the associated pathology. Results Epileptiform activity worsened with age in NS-Pten KO mice, with parallel increases in the extent of hippocampal mTORC1 and mTORC2 dysregulation and progressive astrogliosis and microgliosis. Rapamycin treatment suppressed epileptiform activity, improved baseline EEG activity, and increased survival in severely epileptic NS-Pten KO mice. At the molecular level, rapamycin treatment was associated with a reduction in both mTORC1 and mTORC2 signaling and decreased astrogliosis and microgliosis. Significance These findings reveal a wide temporal window for successful therapeutic intervention with rapamycin in the NS-Pten KO mouse model and support mTOR inhibition as a candidate therapy for established, late-stage epilepsy associated with CD and genetic dysregulation of the mTOR pathway.

The causative link between focal cortical malformations (FCMs) and epilepsy is well accepted, especially among patients with focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC). However, the mechanisms underlying seizures remain unclear. Using a mouse model of TSC- and FCDII-associated FCM, we showed that FCM neurons were responsible for seizure activity via their unexpected abnormal expression of the hyperpolarization-activated cyclic nucleotide–gated potassium channel isoform 4 (HCN4), which is normally not present in cortical pyramidal neurons after birth. Increasing intracellular cAMP concentrations, which preferentially affects HCN4 gating relative to the other isoforms, drove repetitive firing of FCM neurons but not control pyramidal neurons. Ectopic HCN4 expression was dependent on the mechanistic target of rapamycin (mTOR), preceded the onset of seizures, and was also found in diseased neurons in tissue resected from patients with TSC and FCDII. Last, blocking HCN4 channel activity in FCM neurons prevented epilepsy in the mouse model. These findings suggest that HCN4 play a main role in seizure and identify a cAMP-dependent seizure mechanism in TSC and FCDII. Furthermore, the unique expression of HCN4 exclusively in FCM neurons suggests that gene therapy targeting HCN4 might be effective in reducing seizures in FCDII or TSC.