Signaling pathway dysregulation in epilepsy

描述（由申请人提供）：长时间的持续癫痫发作活动（癫痫持续状态，SE）与癫痫并发海马硬化和合并症（如长期认知障碍）的风险增加有关。SE后这些变化的分子机制尚不清楚。在实验模型中，SE触发哺乳动物雷帕霉素靶蛋白（mTOR）通路的即时和长期失调，这与癫痫发生有关。在生理条件下，mTOR调节树突形态和离子通道、突触可塑性和记忆，但mTOR在SE后行为缺陷中的作用尚未得到评估。先前已有研究表明，转基因啮齿动物中mTOR通路的过度激活与行为缺陷、癫痫发作、苔藓纤维发芽和树突异常有关，这些在使用mTOR抑制剂雷帕霉素治疗后会逆转。基于这些研究，我们假设mTOR通路失调有助于海马树突的形态和分子改变，以及相关的海马依赖性学习和记忆缺陷。我们的试点数据表明，雷帕霉素治疗可以改善树突形态和离子通道失调，并挽救早期SE后海马依赖的空间学习和记忆缺陷。我们的试点数据还表明，行为表型的拯救是不可持续的，因为效果不会持续到慢性癫痫期。因此，短暂的雷帕霉素治疗不足以阻止SE后的长期行为改变，这表明可能需要雷帕霉素的慢性治疗或更有效的mTOR抑制剂治疗。在本文提出的研究中，我们将进一步评估SE后mTORC1和2下游信号传导的变化。此外，我们将调查翻译率是否在SE后发生变化。为此，我们将采用多聚体分析并监测PP242与雷帕霉素的有效性。我们将使用抑制剂方案，包括SE后的早期和晚期治疗。我们将使用生物化学、树突形态评估、行为和脑电图作为结果测量。由于这些抑制剂已经在临床使用或正在肿瘤学领域进行广泛的研究和开发，因此有可能迅速转化为人类癫痫和合并症的治疗。本研究的目的如下：1)进一步表征mTOR异常信号，评估SE后mTORC1依赖性mRNA翻译的失调；2)评估mTOR通路失调是否有助于SE后树突结构和分子的改变；3)评估mTOR通路失调是否与SE后出现的学习和记忆缺陷有关；4)比较雷帕霉素与PP242对癫痫样活性的影响。

DESCRIPTION (provided by applicant): Prolonged continuous seizure activity (status epilepticus, SE) is associated with an increased risk for developing epilepsy with hippocampal sclerosis and comorbidities such as long-term cognitive impairments. The molecular mechanisms underlying these changes following SE are not well-understood. In experimental models, SE triggers immediate and long-lasting dysregulation of the mammalian target of rapamycin (mTOR) pathway, which has been implicated in epileptogenesis. Under physiological conditions mTOR modulates dendritic morphology and ion channels, synaptic plasticity, and memory, but the role of mTOR in behavioral deficits following SE has not been evaluated. Previously it has been shown that excessive activation of the mTOR pathway in transgenic rodents is associated with behavioral deficits, seizures, mossy fiber sprouting, and dendritic abnormalities, which are reversed following treatment with the mTOR inhibitor, rapamycin. Based on these studies, we hypothesize that mTOR pathway dysregulation contributes to the morphological and molecular alterations in hippocampal dendrites and the associated hippocampal-dependent learning and memory deficits that occur following SE. We have pilot data demonstrating that treatment with rapamycin improves dendritic morphology and ion channel dysregulation and rescues hippocampal- dependent spatial learning and memory deficits early following SE. Our pilot data also suggest that the rescue of the behavioral phenotype is not sustained as the effect does not last into the period of chronic epilepsy. Thus, transient rapamycin therapy is not sufficient to block long-term alterations in behavior following SE, suggesting that chronic treatment with rapamycin or more potent mTOR inhibitor therapy may be required. In the studies proposed here we will further evaluate changes in the downstream signaling of mTORC1 and 2 following SE. Furthermore, we will investigate whether translation rates are altered following SE. To this end, we will employ polysome profiling and monitor the effectiveness of PP242 compared to rapamycin. We will use inhibitor regimens that include early and late treatments following SE. We will use biochemistry, dendritic morphological assessments, behavior, and electroencephalography as outcome measures. Since these inhibitors are already in use clinically or are being extensively studied and developed in oncology, there is the potential to rapidly translate to treatments for epilepsy and comorbidities in humans. The aims of this proposal are as follows: 1) To further characterize aberrant mTOR signaling and evaluate dysregulation of mTORC1- dependent mRNA translation following SE; 2) To evaluate whether mTOR pathway dysregulation contributes to dendritic structural and molecular alterations following SE; 3) To evaluate whether mTOR pathway dysregulation contributes to learning and memory deficits that occur following SE; and 4) To compare the effect of rapamycin versus PP242 on epileptiform activity.