Abstracts

Rationale: Accumulating data have demonstrated that seizures induced by kainate or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and mTOR inhibitor rapamycin can inhibit mTOR activation which subsequently has potential anti-epileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before kainate injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and kainate-injected animals. Methods: Normal Rats or kainite-treated rats pretreated with rapamycin at different time interval were sacrificed at various time points (1h, 3h, 6h, 10h, 15h and 24h) after rapamycin administration or seizure onset for Western blotting analysis. Phosphorylation of mTOR signaling target of Akt, mTOR, Rictor, Raptor, S6K and S6 were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n=6 for each time point). Neuronal cell death was detected by Fluoro-Jade B staining in kainite-treated rats. Results: In normal rats, we found that rapamycin showed the expected dose-dependent inhibition of S6 phosphorylation 3-24 h after injection, while a paradoxical elevation of S6 phosphorylation was observed 1 hour after rapamycin. Similarly, pretreatment with rapamycin over 10 h prior to kainate inhibits the kainate-induced mTOR activation. In contrast, rapamycin administered 1 to 6 hours before kainate causes a paradoxical increase in the kainate-induced mTOR activation. Rats pretreated with rapamycin 1h prior to kainate showed an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle treated groups. In contrast, rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and reversed by pre-treatment of perifosine, an akt inhibitor. Conclusions: These data indicate the complexity of S6 regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications, such as potential treatments for epilepsy and other neurological disorders.