Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures.
Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures.
复制标题
雷帕霉素对有或没有癫痫发作的大鼠的 S6 磷酸化具有矛盾的影响
作者：
Chen L;Hu L;Dong JY;Ye Q;Hua N;Wong M;Zeng LH
越来越多的数据表明，kainate （KA）或pilocarpine诱导的癫痫发作激活了雷帕霉素（mTOR）通路的哺乳动物靶点，而mTOR抑制剂雷帕霉素可以抑制mTOR的激活，从而具有潜在的抗癫痫作用。然而，一项初步研究显示，当在KA注射前短时间内给予雷帕霉素时，S6磷酸化所反映的mTOR途径活性增加的矛盾加剧。在目前的研究中，我们更详细地研究了雷帕霉素在正常大鼠和注射ka的动物中的这种矛盾作用。在雷帕霉素给药后或癫痫发作后的不同时间点（1h、3h、6h、10h、15h、24h）处死正常大鼠或经不同时间间隔雷帕霉素预处理的ka处理大鼠，进行Western blotting分析。分析Akt、mTOR、Rictor、Raptor、S6K和S6的mTOR信号靶点磷酸化情况。对癫痫发作活动进行行为监测，并根据改进的拉辛量表进行评分（每个时间点n=6）。氟玉B染色检测神经元细胞死亡。在正常大鼠中，我们发现雷帕霉素在注射后3-24小时显示出预期的剂量依赖性抑制S6磷酸化，而在雷帕霉素注射后1小时观察到S6磷酸化的矛盾升高。同样，在KA发作前10小时用雷帕霉素预处理可抑制KA发作引起的mTOR活化。相反，在KA发作前1至6小时给予雷帕霉素会引起KA发作诱导的mTOR激活的矛盾增加。在KA前1小时用雷帕霉素预处理的大鼠表现出癫痫发作的严重程度和持续时间的增加，与给药组相比，更多的神经元细胞死亡。相比之下，在KA前10小时预处理雷帕霉素对癫痫发作和减少神经元细胞死亡没有影响。雷帕霉素对S6磷酸化的矛盾作用与上游mTOR信号传导相关，并通过Akt抑制剂perifosine预处理逆转。这些数据表明S6调控的复杂性及其对癫痫的影响。在临床应用中需要考虑雷帕霉素的矛盾效应，例如对癫痫和其他神经系统疾病的潜在治疗。
Accumulating data have demonstrated that seizures induced by kainate (KA) 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 KA injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA-injected animals. Normal Rats or KA-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 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 KA inhibited the KA seizure induced mTOR activation. In contrast, rapamycin administered 1 to 6 hours 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 pre-treatment of perifosine, an Akt inhibitor. 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 neurological disorders.
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158.5
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