Abstract：
Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood.
Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma.
Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day.
Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.
Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma.
Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day.
Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.
摘要：
目的：转录后机制越来越被认为是癫痫过度兴奋网络形成的重要因素。信使RNA（mRNA）聚腺苷酸化是通过增强mRNA稳定性和翻译来控制蛋白质表达的关键调节机制。先前的研究表明，癫痫发展过程中小鼠海马中mRNA多聚腺苷酸化的大规模变化。发现细胞质聚腺苷酸化元件结合蛋白CPEB4可驱动癫痫诱导的poly（A）尾巴变化，缺乏CPEB4的小鼠会出现更严重的癫痫发作和癫痫表型。控制CPEB4功能的机制和影响癫痫自发发作复发的下游途径仍然知之甚少。
方法：通过杏仁核内微量注射海藻酸，在野生型和CPEB4缺陷的雄性小鼠中诱导癫痫持续状态。CLOCK与CPEB4启动子的结合通过血浆中染色质免疫沉淀测定和褪黑激素水平通过高效液相色谱法进行分析。
结果：在本文中，我们显示在小鼠癫痫持续状态期间，CLOCK与CPEB4启动子区识别位点的结合增加，在过表达CLOCK的N2A细胞中Cpeb4mRNA水平增加。对癫痫期间poly（A）尾巴发生变化的CPEB4依赖性基因的生物信息学分析发现，与昼夜节律调节有关的基因特别丰富。时钟转录本在癫痫持续状态后和癫痫期间的小鼠海马中显示更长的poly（A）尾巴长度。此外，CLOCK在小鼠癫痫持续状态后和癫痫期间的海马中表达增加，以及耐药颞叶癫痫患者切除的海马和皮质。Further,CPEB4是癫痫持续状态后CLOCK表达所必需的，与野生型小鼠相比，CPEB4缺陷型小鼠的水平较低。最后,CPEB4缺陷小鼠表现出改变的昼夜节律功能，包括褪黑激素水平的改变和白天自发性癫痫发作的聚集性改变。
结论：我们的结果揭示了一个涉及CPEB4和CLOCK的新的正转录-翻译反馈环（TTFL），这可能有助于癫痫期间睡眠-觉醒周期的调节。
方法：通过杏仁核内微量注射海藻酸，在野生型和CPEB4缺陷的雄性小鼠中诱导癫痫持续状态。CLOCK与CPEB4启动子的结合通过血浆中染色质免疫沉淀测定和褪黑激素水平通过高效液相色谱法进行分析。
结果：在本文中，我们显示在小鼠癫痫持续状态期间，CLOCK与CPEB4启动子区识别位点的结合增加，在过表达CLOCK的N2A细胞中Cpeb4mRNA水平增加。对癫痫期间poly（A）尾巴发生变化的CPEB4依赖性基因的生物信息学分析发现，与昼夜节律调节有关的基因特别丰富。时钟转录本在癫痫持续状态后和癫痫期间的小鼠海马中显示更长的poly（A）尾巴长度。此外，CLOCK在小鼠癫痫持续状态后和癫痫期间的海马中表达增加，以及耐药颞叶癫痫患者切除的海马和皮质。Further,CPEB4是癫痫持续状态后CLOCK表达所必需的，与野生型小鼠相比，CPEB4缺陷型小鼠的水平较低。最后,CPEB4缺陷小鼠表现出改变的昼夜节律功能，包括褪黑激素水平的改变和白天自发性癫痫发作的聚集性改变。
结论：我们的结果揭示了一个涉及CPEB4和CLOCK的新的正转录-翻译反馈环（TTFL），这可能有助于癫痫期间睡眠-觉醒周期的调节。
