Abstracts

Rationale: Temporal lobe epilepsy (TLE) is the most common form of refractory epilepsy. TLE is due to aberrant electrical activity within hippocampal, peri-hippocampal or temporal cortical networks. Rodent TLE models have implicated mTOR dysregulation in the development of chronic epilepsy. However, little is known whether this pathway is deregulated in human TLE. We hypothesized that, similar to animal models, mTOR signaling would be significantly upregulated in both hippocampal and neocortical neurons from therapy-resistant TLE patients.Methods: Hippocampal and neocortical tissue was obtained following brain surgery for therapy-resistant TLE at the Hospital of the University of Pennsylvania. Subjects ages 20-56 years (mean 35.7) were prospectively enrolled in the study after undergoing a comprehensive pre-surgical evaluation (n=10). Hippocampus samples were only available for 8 out of the 10 patients. Mesial temporal sclerosis (MTS) was diagnosed in 5 out of the 8 hippocampus samples by standard neuropathological examination. Malformations of cortical development were excluded in all cases. Age and region matched autopsy samples from cases with normal neurological history ages 20 to 58 (mean=39.9) were obtained from the NICHD Maryland Brain and Tissue Bank and were used as controls (n=5). The study was approved by the local Institutional Review Board. Markers of mTOR activation including phospho-S6 Ser235/236 and Ser240/244, and phospho-Akt Thr308 and Ser473 were assessed by Western blotting and expressed as ratios to total S6 and Akt, respectively. Immunohistochemistry for phospho-S6 Ser240/244 was also performed.Results: Compared to controls, hippocampus from TLE patients demonstrated significant overactivation of mTOR Complex 1 (mTORC1) signaling, as demonstrated by increased phosphorylation of S6 at both Ser235/236 (p<0.0001) and Ser240/244 (p<0.0001). In addition, we found increased phosphorylation of Akt at both Thr308 (p<0.0001) and Ser473 (p<0.001), consistent with activation of the PI3K/Akt and mTOR Complex 2 (mTORC2) pathways, respectively. There were no significant differences between TLE with MTS versus TLE without MTS for any of these markers. Neocortex showed a significantly increased phosphorylation of S6 at Ser235/236 (p<0.01), and of Akt at Thr308 (p<0.05) and Ser473 (p<0.001). Immunohistochemistry demonstrated robust pS6 (Ser240/244) expression in the hippocampus, most prominent in the CA4 region and dentate gyrus, while in the neocortex there was more widespread labeling throughout all layers.Conclusions: Although mTOR signaling has been mostly studied in malformations of cortical development, we now show a robust activation of both the PI3K/Akt/mTORC1 and mTORC2 pathways in neocortical and hippocampal tissue from therapy-resistant TLE patients. These results validate previous experimental literature, while further investigation of these pathways in human tissue will facilitate the development of new therapies for refractory TLE. NIHR01NS031718, NICHD Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD, contract HHSN275200900011C.