Abstracts

Rationale: Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been demonstrated in human cortical dysplasia (CD) as well as animal models of epilepsy. Although inhibition of mTOR signaling early in epileptogenesis suppresses 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 have not been investigated in this model. Here, we evaluated whether mTOR inhibition suppresses epileptiform activity and other neuropathological correlates in older NS-Pten KO mice with severe and well-established epilepsy. Methods: The progression of mTOR pathway dysregulation and hippocampal gliosis were evaluated using western blotting in 2, 4, 6, and 8-9 week-old NS-Pten KO mice. Antibodies against p-S6 (S240/244) and p-AKT (S473) were used as markers for mTORC1 and mTORC2 activation, respectively. GFAP and IBA1 were used as markers for astrocytes and microglia, respectively. NS-Pten KO mice were treated with the mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) starting at postnatal week 9 and monitored with video-electroencephalography (EEG) recordings for epileptiform activity. Western blotting was also performed to evaluate the effects of rapamycin on the expression of the glial markers. Results: In parallel to the previously reported progressive epilepsy phenotype, we found elevated protein levels of p-S6, p-AKT, GFAP, and IBA1 in NS-Pten KO mice that became increasingly different from age-matched WT mice with age (p<0.05; n=6-15). Treatment with rapamycin significantly suppressed epileptiform activity (p<0.001; n=5-6) and increased survival (p<0.05; n=7-20) in severely epileptic NS-Pten KO mice compared to untreated controls, suggesting that aberrant mTOR signaling may play a crucial role in the maintenance of epilepsy in this model. At the molecular level, rapamycin treatment was associated with a reduction in the levels of p-S6 and p-AKT as well as GFAP and IBA1 (p<0.05; n=7-14). Conclusions: Late inhibition of mTOR suppresses established, severe epilepsy and associated hippocampal gliosis in NS-Pten KO mice. These findings reveal a wide temporal window for successful therapeutic interventions with mTOR inhibition in the NS-Pten KO mouse model and further support mTOR inhibition as an effective treatment for late-stage epilepsy associated with CD. (Funding: NIH R01 NS81053; 39943, Vivian L. Smith Foundation Research Grant, CURE Challenge Award, Epilepsy Foundation Predoctoral and Postdoctoral Fellowships, and in part by NIH P30HD 024064)