基于BDNF/TrkB通路的表观遗传调控探讨叶酸及丰富环境联合治疗孤独症的机制

Our studies and others have shown that in the brain of autism exist the epigenetic dysregultion, the abnormality of the activity of BDNF/TrkB siganl pathway, and the therapeutic effects of folic acid and enriched environment. Therefore, it is a problem deserving of study whether the two methods yield therapeutic effects on autism by correcting the activity of BDNF/TrkB pathway via epigeneitic action. In the present project, focusing on the epigenetic regulation on BDNF/TrkB pathway, we intend to test the expression changes of BDNF/TrkB pathway-related molecules in the brain regions of autistic animal model, to investigate two key epigenetic regulation mechanisms, the CpG island methylation and histon acetylation in the corresponding genes, and to explore the relationship of epigenetic regulation with the activity of BDNF/TrkB pathway. We also intend to observe the therapeutic effects of folic acid combined with enriched environment on the morphological, electrophysiological and behavioral changes in the autistic animal model, and then to investigate the correlation between the therapeutic effects and the activity of BDNF/TrkB pathway. After the successful completion of this project we hope to verify whether the epigenetic regulation on BDNF/TrkB pathway is involved in the pathogenesis of autism, we also hope to clarify the mechanism of folic aicd combined with enriched environment in the treatment of autism, providing new experimental evidences for the prevention and cure of autism.

目前已知，BDNF/TrkB通路及表观遗传机制均参与孤独症的发病，而叶酸及丰富环境有治疗作用。那么，这两种治疗措施是否通过表观遗传途径修正BDNF/TrkB通路活性，进而发挥对孤独症的治疗作用，是值得研究的问题。本项目以BDNF/TrkB通路的表观遗传调控为切入点，观察孤独症模型脑组织BDNF/TrkB通路信号分子的表达变化；研究该通路基因CpG岛甲基化及组蛋白乙酰化等表观遗传修饰，并研究表观遗传修饰与通路活性变化的关系；随后研究叶酸和丰富环境联合干预对孤独症模型BDNF/TrkB通路基因表观遗传修饰的影响及其与通路活性变化的关系，并观察干预对孤独症模型脑形态、电生理及行为等方面的治疗作用，进而探讨BDNF/TrkB活性变化与治疗效应之间的相关性。依此，有望明确BDNF/TrkB通路的表观遗传调控在孤独症发病中的作用，揭示叶酸及丰富环境联合治疗孤独症的机制，为孤独症防治提供新线索。

BDNF/TrkB通路及表观遗传修饰均参与孤独症发病，而叶酸及丰富环境有治疗作用，为明确其机制，我们主要进行了以下主要工作：. 1．研究了孤独症模型大鼠脑组织BDNF/TrkB通路的表观遗传调控及通路活性变化. （1）制作autism大鼠模型。采用Wistar大鼠，在妊娠第12.5天时，腹腔注射丙戊酸制作了autism大鼠模型。. （2）BDNF/TrkB信号通路基因CpG岛甲基化及组蛋白修饰的研究。甲基化特异性PCR （MSP）及硫转化DNA测序结果显示，模型鼠脑组织BDNF/TrkB通路BDNF及TrkB基因去甲基化水平显著高于对照组；染色质免疫共沉淀（CHIP）结果显示：与对照组比较， BDNF及TrkB基因启动子区组蛋白H3和H4乙酰化水平显著增高，BDNF基因乙酰化升高达4.8倍，TrkB基因也增高2.5倍以上。. （3）BDNF/TrkB通路基因表观调控异常与通路活性关系研究。研究结果显示，在autism模型大鼠皮层和海马脑区，BDNF及TrkB基因mRNA及蛋白表达显著高于对照组。BDNF/TrkB通路效应蛋白CREB及其靶基因在autism模型中含量升高，表明BDNF/TrkB通路活性上调。以上结果表明，BDNF/TrkB通路相关基因表观遗传修饰导致BDNF/TrkB通路活性上调。. 2．叶酸和丰富环境联合干预对孤独症模型大鼠脑组织BDNF/TrkB通路表观遗传修饰的影响及治疗作用的研究. （1）叶酸和/或丰富环境干预对脑组织BDNF/TrkB通路表观遗传修饰的影响。研究结果表明上述干预引起BDNF/TrkB通路基因CpG岛甲基化水平增高及乙酰化水平显著降低，BDNF/TrkB通路下调。. （2）叶酸和/或丰富环境干预对孤独症模型治疗作用研究。 观察了对照组、autism模型、叶酸治疗组、丰富环境治疗组BDNF/TrkB通路活性，并检测大鼠模型的学习记忆行为。研究表明叶酸及丰富环境可以改善孤独症大鼠模型症状，与BDNF/TrkB信号通路抑制处理改善大鼠模型的学习记忆能力具有类似效应。表明叶酸治疗及丰富环境通过下调BDNF/TrkB通路活性可以改善孤独症大鼠模型症状。