title: Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures.
abstract: 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 S6 phosphorylation 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, and 24 h) 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.
  
  
  RESULTS
  In normal rats, we found that rapamycin showed the expected dose-dependent inhibition of S6 phosphorylation 3-24 h after injection, whereas a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin. Similarly, pretreatment with rapamycin over 10 h before KA inhibited the KA seizure-induced mTOR activation. In contrast, rapamycin administered 1-6 h before KA caused a paradoxical increase in the KA seizure-induced mTOR activation. Rats pretreated with rapamycin 1 h 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 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 was reversed by pretreatment 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 for potential treatment for epilepsy and other neurologic disorders.
authors.affiliation: School of Medicine, Zhejiang University City College, Hangzhou, Zhejiang, China
authors.forename: Linglin
authors.initials: L
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Hu
authors.forename: Jing-Yin
authors.initials: JY
authors.lastname: Dong
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.forename: Nan
authors.initials: N
authors.lastname: Hua
authors.forename: Michael
authors.initials: M
authors.lastname: Wong
authors.forename: Ling-Hui
authors.initials: LH
authors.lastname: Zeng
mesh_terms: D000818:Animals; D008297:Male; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012640:Seizures / Q000188:drug therapy* / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology* / Q000627:therapeutic use; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D016896:Treatment Outcome
chemical_list: C546845:mTOR protein, rat; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus