Data_Sheet_4_PRG5 Knockout Precipitates Late-Onset Hypersusceptibility to Pilocarpine-Induced Juvenile Seizures by Exacerbating Hippocampal Zinc Signaling-Mediated Mitochondrial Damage.ZIP

IntroductionEpileptogenesis is understood as the plastic process that produces a persistent reorganization of the brain’s neural network after a precipitating injury (recurrent neonatal seizures, for instance) with a latent period, finally leading to neuronal hyperexcitability. Plasticity-related genes (PRGs), also known as lipid phosphate phosphatase-related proteins (PLPPRs), are regulators of mitochondrial membrane integrity and energy metabolism. This study was undertaken to determine whether PRG5 gene knockout contributes to the delayed hypersensitivity induced by developmental seizures and the aberrant sprouting of hippocampal mossy fibers, and to determine whether it is achieved through the mitochondrial pathway. Here, we developed a “twist” seizure model by coupling pilocarpine-induced juvenile seizures with later exposure to penicillin to test the long-term effects of PRG5 knockout on seizure latency through comparison with wild-type (WT) mice. Hippocampal mossy fiber sprouting (MFS) was detected by Timm staining. In order to clarify the mechanism of the adverse reactions triggered by PRG5 knockout, hippocampal HT22 neuronal cultures were exposed to glutamate, with or without PRG5 interference. Mitochondrial function, oxidative stress indicators and zinc ion content were detected.
ResultsPRG5 gene knockout significantly reduced the seizure latency, and aggravated the lowered seizure threshold induced by developmental seizures. Besides, knockout of the PRG5 gene reduced the MFS scores to a certain extent. Furthermore, PRG5 gene silencing significantly increases the zinc ion content in hippocampal neurons, impairs neuronal activity and mitochondrial function, and exacerbates glutamate-induced oxidative stress damage.
ConclusionIn summary, PRG5 KO is associated with significantly greater hypersusceptibility to juvenile seizures in PRG5(–/–) mice compared with WT mice. These effects may be related to the hippocampal zinc signaling. The effects do not appear to be related to changes in MFS because KO mice with juvenile seizures had the shortest seizure latencies but exhibited less MFS than WT mice with juvenile seizures.

引言
癫痫发生（epileptogenesis）指的是在诱发损伤（例如反复性新生儿惊厥）后，伴随潜伏期的脑神经网络持续性重塑的可塑性过程，最终引发神经元过度兴奋。可塑性相关基因（PRGs，亦称为脂质磷酸酶相关蛋白PLPPRs）是线粒体膜完整性与能量代谢的调控因子。本研究旨在探究PRG5基因敲除是否会加剧发育性惊厥诱导的迟发性易感性以及海马苔藓纤维异常出芽，并明确其是否通过线粒体通路实现。
本研究通过将毛果芸香碱诱导的幼年惊厥与后续青霉素暴露相结合，构建了变式惊厥模型，并与野生型（WT）小鼠对比，以验证PRG5基因敲除对惊厥潜伏期的长期影响。采用梯姆（Timm）染色法检测海马苔藓纤维出芽（MFS）。为阐明PRG5基因敲除所引发不良反应的机制，本研究将海马HT22神经元培养物分别置于含或不含PRG5干扰的谷氨酸环境中进行处理，并检测各组线粒体功能、氧化应激指标及锌离子含量。

结果
PRG5基因敲除可显著缩短惊厥潜伏期，并加剧发育性惊厥诱导的惊厥阈值降低。此外，PRG5基因敲除在一定程度上降低了MFS评分。进一步研究发现，PRG5基因沉默可显著升高海马神经元内锌离子含量，损伤神经元活性与线粒体功能，并加重谷氨酸诱导的氧化应激损伤。

结论
综上，与野生型（WT）小鼠相比，PRG5纯合敲除（PRG5–/–）小鼠对幼年惊厥的易感性显著升高，上述效应可能与海马锌信号通路相关。该效应似乎与MFS的变化无关：相较于野生型幼年惊厥小鼠，PRG5敲除幼年惊厥小鼠的惊厥潜伏期更短，但MFS水平更低。