Effects And Mechanism Of Regulated MTOR Signaling Pathway In Epilepsy

The mammalian target of rapamycin(mTOR)siganling pathway is an important regulator of protein synthesis,cellular growth and proliferation.In previous study,the over-activated mTOR pathway is involved in kainic acid-induced seizure in rats and promotes a series of pathological characteristic.The mTOR inhibitor rapamycin can inhibit mTOR activation,which has potential antiepileptic effects.Rapamycin acts on Raptor protien and impoves postepileptic pathological symtom,reduces the frequecy of spontaneous seizures.Thus,to clarify the effects of rapamycin on mTOR signaling pathway in epilepsy,we firstly examined the effect of regulated mTOR siganling in both normal rats and KA-injected rats after rapamycin administration.Then we generated the mice with conditional knockout(CKO)of the Rptor gene to study the mechanism of mTOR in regulating epileptogenesis and comorbidity.Stage 1 Rapamycin has paradoxical effects on mTOR signaling pathwayObjective:A previous study showed a paradoxical exacerbation of increased mTOR siganling pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before KA injection.In present study,we examined this paradoxical effect of rapamycin in more detail,both in normal rats and KA-injected animals.Methods:Normal rats pretreated with rapamycin at different dosage(0.3,1,3,and 10mg/kg)and different time intervals(1,3,6,15,and 24h)to study the dose-and time-dependent effects.Then KA-treated rats were killed at various time points(1,3,6,10,15,and 24 h)after rapamycin administration or seizure onset for western blotting analysis.Seizure activity was monitored behaviorally and neuronal death was detected by Fluoro-Jade B staining.Finally,phosphorylation of mTOR,Akt,Raptor,Rictor,S6K,S6 and the pretreatment of perifosine,an Akt inhibitor were analyzed.Results:1.In normal rats,we found that rapamycin had a dose-dependent inhibition of S6 phosphorylation 3?24 h after injection,whereas a paradoxical increasing of S6 phosphorylation was detected 1 h after rapamycin treatment.2.Pretreatment with rapamycin 10 h or longer before KA inhibited the KA-induced mTOR activation.In contrast,rapamycin administered 1-6 h before KA caused a paradoxical increase in the KA seizure-induced mTOR activation.3.Rats pretreated with rapamycin 1 h before KA injection exhibited an increase in seizure stage and duration of seizures,and more neuronal death when compared to KA-treated groups.In contrast,rapamycin pretreated 10 h before KA injection had no effect on the seizure activity and decreased neuronal death.4.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:Rapamycin induced S6 activation within a short time and abolish the antiepileptic activity of rapamycin.However,our results demonstrate that there is a high level of complexity associated with the regulation of S6 phosphorylation induced by rapamycin.These paradoxical effects may result in upstream signals.Stage2 Effects of neural progenitor cells Rptor ablation on mTOR signaling pathwayObjective:Raptor protein encoding by Rptor gene is a regulator-associated protein of the mTOR signaling pathway.Previous study suggests that mTOR signaling pathway may play an important role in epileptogenesis.To explore the contribution of Raptor protein to the development of epilepsy and comorbidity,we generated the conditional knockout(CKO)of the gene Rptor in neural progenitor cells.Methods:Mice were generated by Lox-P technology by cross-bred Rptorflox/flox mice with nestin-CRE mice.The General appearance and development of Rptor CKO were observed.Meanwhile,the mTOR signaling pathway of Rptor CKO mice in naive or after epileptic status were analysis by western blotting.Neuronal death and mossy fiber sprouting were detected by FJB staining and Timms staining,respectively.Spontaneous seizures were recorded by EEG-video.Morris water maze,open field test and excitability test were used to study the behaviors of Rptor CKO mice.Results:1.Rptor CKO mice had decreased mTORC1 signaling with normal appearance.2.Rptor CKO mice decreased body weight,brain weight and cortical thickness.3.Over-activation of mTOR signaling after kainic acid-induced seizure was markedly inhibited in Rptor CKO mice and the seizure activity had no significant alteration.4.Epilepsy-associated manifestation is attenuated and low frenquency of spontaneous seizures and neuronal cell death in Rptor CKO mice.5.Additionally,cognitive deficit and anxiety-like behavior after KA-induced seizure were improved in Rptor CKO mice.Conclusions:Although knockout of the Rptor gene in mice neural progenitor cells affects the development in young age,these still have some benefits in the postepileptic behavior.It also suggests that Raptor protein plays an important role in epilepsy.