DNA去甲基化通路表观遗传动态变化与神经发育障碍机制以及药物干预的研究

Increasing evidence suggests that epigenetic dysfunction may account for the alteration of gene transcription present in neurodevelopment disorders such as schizophrenia (SZ), bipolar disorders (BP), major depression and autism. The results from our recent postmortem study show that the DNA demethylation pathway dysfunction may contribute largely to the pathogenesis of neurodevelopment disorders. It has been proposed that the 5-mC mark on promoter CpG rich regions of specific genes can be oxidized to form 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) proteins in mammalian brain. Further, it has been proposed that 5hmC undergoes two successive processing steps: a) a deamination step that is catalyzed by the AID (activation-induced deaminase)/APOBEC (apoliporotein B mRNA-editing enzyme complex) family of cytosine deaminases, turning 5hmC into 5hydroxymethyluridine (5hmU); and b) the base excision repair (BER) pathway, in which 5hmU can be removed and replaced by unmethylated cytosine by a group of glycosylases such as MBD4 and TDG. Rapidly inducible growth arrest and DNA-damage-inducible proteins 45 (GADD45) are thought to be master coordinators of this process by recruiting deaminases and glycosylases to 5mC and 5hmC rich promoter regions. .Although numerous studies of DNA methylation have been carried out in the postmortem brain of psychotic patients, the different components of the DNA demethylation pathway, with the exceptions of GADD45, TETs AID/APOBECs and MBD4 or TDG, remain largely unknown.. Therefore, in this proposal, we are planning to deeply to investigate the mechanisms of DNA demethylation pathway, especially the alterations of the factors modulate the pathway during development. This project will be using a established prenatal stress (PRS) model to carried all proposed experiments to test our hypothesis. Prenatal stress triggers alterations of neuronal plasticity and behaviors. Such changes including anxiety, impaired spatial learning and memory can lead to neurodevelopmental disorders. Studies suggest that offspring behavioral changes induced by PRS could be the outcome of programming changes in the epigenome but not through Mendelian mechanism. Therefore it is a good model for ourto probe the dynamic DNA demethylation pathway with development; and also for us to understand how prenatal stress epigenetically affects offspring. In addition, by using this platform, we can screen more potent compounds which can effectively intervene the aberrant DNA demethylation.

近年的研究显示，表观遗传学与多种神经系统发育异常性疾病关系密切，如精神分裂症、抑郁症和小儿孤独症，我们对神经发育障碍病人脑组织的系列研究结果也符号表观遗传动态调控失调导致精神性病变的理论。神经系统基因甲基化异常与神经发育障碍密切相关，并进而影响胚胎正常发育及生长，因此深入研究神经元中表观遗传动态变化机制对探索神经发育障碍病因，诊断及治疗有重要意义。我们近期的研究显示，神经发育障碍病人脑组织存在DNA甲基化和去甲基化通道异常，而目前国内外尚缺乏对神经系统发育过程中表观遗传调控动态变化，特别是环境刺激后的变化研究。本课题应用我们已建立的神经发育障碍小鼠模型，对DNA去甲基通路中多个关键因子的表达进行深入研究，试图找到调控神经系统去甲基化的关键因子和主要的信号通路，并进一步研究药物干预对其的影响，最后利用这个平台筛选调控甲基化通路有效的药物，为临床防治精神病提供理新的思路和方法。

目的 建立慢性产前应激(Prenatal Stress, PS)动物模型，观察小鼠子代不同时期抑郁相关行为学特点，并探究产前应激对后代子鼠中枢神经系统髓鞘发育的影响，从而探讨产前应激对后代子鼠的行为学影响是否与髓鞘发育有关。同时进一步研究其机制是否与表观遗传学相关。.方法 以KM小鼠为研究对象，将妊娠KM鼠随机分为2组：空白对照组（No Stress, NS）和产前束缚应激组(Prenatal Restraint Stress, PRS)，NS组在妊娠期间不给与任何刺激，PRS组从妊娠第7天开始至生产持续定时定点给与束缚刺激。子鼠长至21天，雌雄小鼠断奶并分笼饲养。应用强迫游泳实验（Forced Swimming Test，FST)和悬尾实验（Suspension Test，TST)两种方法检测子鼠不同时期行为学情况。为探索孕期应激对子代行为变化的机制我们应用Western Blot及QRT-PCR技术检测子代小鼠不同时期髓鞘发育相关的生物分子Mag、Mbp、Mobp、Mog、Olig1、Sox10、Plp变化情况。用Western Blot、QRT-PCR、MeDIP及ChIP试验研究海马区BDNF、HDACs、AcH3K14、DNMT1。.结果 我们的结果表明，来自产前束缚应激组的小鼠后代表现出抑郁样和焦虑样行为；应激组后代与非应激组后代相比，BDNF的表达降低，DNMT1，HDAC1和HDAC2的表达增加，另外海马中AcH3K14的表达降低。同时MeDIP和ChIP数据显示AcH3K14与特异性BDNF启动子的甲基化增加而结合减少。 Pearson分析表明，由产前应激诱导的表观遗传变化与抑郁样和焦虑样行为相关。 .结论 产前应激能导致后代子鼠MDD相关的行为学改变，并且这种改变在青春期至成年期均存在；产前应激影响后代子鼠髓鞘正常发育、成熟分化相关的生物分子Mag、Mbp、Mobp、Mog、Olig1、SOX10、PLP的表达及髓鞘显微结构的形态学变化；产前应激对后代子鼠产生的MDD相关行为学负面改变可能是通过影响PFC髓鞘发育而起作用；同时这种作用机制与甲基化通路相关。