糖酵解酶GAPDH对钠通道基因的转录后调控及其在生酮饮食抗癫痫中的作用

Abnormal expressions of voltage-gated sodium channels are associated with epilepsy, Post-transcriptional expression regulations of sodium channel genes have not yet been reported. Ketogenic diet (KD) is an effective treatment for children with refractory epilepsy. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional enzyme, is involved in the antiepileptic activity of KD. Our previous studies found that GAPDH, functioning as an RNA-binding protein, bound to one of the conserved regions in the 3'untranslated regions of sodium channel α subunit SCN1A, SCN2A and SCN3A genes, which down-regulated SCN1A and SCN2A expression and up-regulated SCN3A expression at post-transcriptional level. These results suggest that the post-transcriptional regulations of sodium channel genes by GAPDH may be associated with epilepsy. This project will investigate how the bindings of GAPDH to the elements in the 3'UTRs of sodium channel genes are regulated by the factors (GAPDH modifications, oxidation conditions, etc.) and their roles in regulating gene expression at post-transcriptional level using RNA Electrophoretic Mobility Shift Assay (RNA EMSA),RNA Immunoprecipitation (RNA RIP), mRNA stability analysis,RNA in situ hybridization and Immunohistochemistry, and so on. We will further investigate the regulations of GAPDH binding to the cis-element by GAPDH modifications and other factors, mRNA stabilities and subcellular localizations of sodium channel α subunits in the brain tissues of normal, epilepsy and KD-treated mice. These studies will demonstrate the post-transcriptional expression regulations of sodium channel genes by GAPDH and their roles in the epileptogenesis and the antiepileptic effect of KD.

电压门控型钠通道的异常表达与癫痫密切相关，钠通道基因的转录后调控还未见报道。生酮饮食（KD）是治疗儿童难治性癫痫的一种有效措施，其中甘油醛-3-磷酸脱氢酶GAPDH在KD的抗癫痫中发挥重要作用。我们前期研究发现，GAPDH作为RNA结合蛋白与钠通道α亚基SCN1A、SCN2A和SCN3A基因3'非翻译区的一个共同保守序列相结合，在转录后水平上下调SCN1A和SCN2A表达，上调SCN3A表达，提示GAPDH对钠通道基因的表达调控很可能与癫痫相关。本项目拟通过RNA EMSA及RIP、mRNA稳定性分析、原位杂交与免疫组化等实验分析GAPDH与钠通道基因调控元件相结合的影响因素及其转录后调控作用，比较正常、癫痫及KD处理小鼠脑组织中GAPDH与调控元件结合状况及影响因素、钠通道基因mRNAs稳定性及蛋白的亚细胞定位情况，以期揭示GAPDH对钠通道基因的调控在癫痫发生发展和KD抗癫痫中的作用。

电压门控型钠离子通道的异常表达与癫痫发生密切相关。本研究发现糖酵解酶GAPDH可作为RNA结合蛋白与钠通道SCN1A与SCN3A基因3′UTR的保守序列相结合，在转录后水平上负性调控SCN1A表达而正性调控SCN3A表达，在癫痫发作小鼠中GAPDH表达上调及磷酸化修饰可影响该蛋白与RNA调控元件结合能力增强，从而下调Scn1a基因表达，并上调Scn3a基因表达；而生酮饮食（KD）处理后可回复这一系列过程，从而揭示了GAPDH参与调控钠通道基因表达及其在癫痫发生及KD抗癫痫中的作用机制。首次发现苹果酸脱氢酶MDH2的一种新功能：作为RNA结合蛋白与钠通道SCN1A基因3′UTR的保守区相结合，在转录后水平上负性调控SCN1A基因表达；在癫痫小鼠中MDH2表达上调，同时脑组织中的氧化环境增强了MDH2与SCN1A基因3′UTR的结合，从而导致该基因表达下调，这可能是癫痫发生发展的重要因素之一。另外，本研究还发现抗癫痫药物丙戊酸钠（VPA）可通过诱导Scn3a基因启动子上-39C位点的甲基化，导致其启动子活性下降从而下调Scn3a基因的表达，而去甲基化酶FTO和MBD2均参与了这一过程；这一条表观调控途径在VPA处理的癫痫小鼠中得到证实，从而揭示VPA通过表观遗传途径下调Scn3a表达的分子机制及其在抗癫痫作用中发挥重要作用。上述研究结果提示：能量代谢过程及表观遗传途径参与钠通道基因的异常表达调控与癫痫发生发展过程密切相关，可望作为新的抗癫痫靶点开展后续研究，为癫痫的诊治提供新的思路。