Rapamvcin Has Paradoxical Effects On S6Phosphorylation In Rats With And Without Seizures

Objective:Accumulating data have demonstrated that seizures induced by kainate (KA) or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and that mTOR inhibitor rapamycin can inhibit mTOR activation,which subsequently has potential antiepileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6phosphorylation when rapamycin was administrated within a short period before KA injection.In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA-injected animals.Methods:Normal rats or KA-treated rats pretreated with rapamycin at different time intervals were sacrificed at various time points (1,3,6,10,15, and24h) after rapamycin administration or seizure onset for western blotting analysis.Phosphorylation of mTOR signaling target ofAkt, mTOR, Rictor, Raptor, S6K, and S6were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n=6for each time point). Neuronal cell death was detected by Fluoro-Jade B staining.Key Findings:In normal rats, we found that rapamycin showed the expected dose-dependent inhibition of S6phosphorylation3-24h after injection, whereas a paradoxical elevation of S6phosphorylation was observed1h after rapamycin. Similarly, pretreatment with rapamycin over10h before KA inhibited the KA seizure-induced mTOR activation. In contrast, rapamycin administered1-6h before KA caused a paradoxical increase in the KA seizure-induced mTOR activation. Rats pretreated with rapamycin1h prior to KA exhibited an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle-treated groups. In contrast, rapamycin pretreated10h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6phosphorylation was correlated with upstream mTOR signaling and was reversed by pretreatment of perifosine, an Akt inhibitor.Significance:These data indicate the complexity of S6regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications,such as for potential treatment for epilepsy and other neurologic disorders.