大脑发育窗口期胆碱通过DNA甲基化途径对成年海马神经发生的影响及机制

The effect of choline on brain development occurs during a sensitive time window, choline influences lifelong hippocampal function and risk for central nervous system disorders. However, the underlying mechanisms remain poorly understood. Collective data suggests a prominent role of adult hippocampal neurogenesis in cognitive processes, including learning, memory, and emotion. Our previous findings provide evidence that maternal choline determines embryonic neurogenesis via DNA methylation. And we also found the same DNA methylation pattern of a single gene changed by choline in both embryonic and adult neural stem cells (NSCs). Since adult NSCs originate from embryonic NSCs, so we hypothesize that choline influences the molecular control of adult neurogenesis and cognitive functions throughout life via DNA methylation and gene transcription in the adult NSCs. To test this hypothesis, timed-pregnant transgenic Nestin-CFP and cell lineage tracing mice will be randomly assigned and fed with different choline diets during the sensitive period. First, to investigate the consequences of adult hippocampal neurogenesis in the offsprings from different maternal choline, immunofluorescence will be used. Second, to establish a link between altered DNA methylation pattern and transcriptional activity in NSCs, high-throughput DNA methylation profiling and gene expression profiling will be identified in fluorescence activated cell sorting-sorted CFP-positive NSCs. And significant changed genes which have the same pattern in both embryonic and adult cells will be selected by bioinformatic analysis. Third, to validate the roles for the candidate genes identified in the approaches above in adult neurogenesis, lentiviral delivery of designed shRNA or overexpressed genes will be applied to regulate gene expression in cultured NSCs and mice hippocampus by stereotactic injection. The overall goal of the proposed project is to determine the mechanisms by which the availability of choline during the sensitive period modifies brain structure and function, characterize the changed programs in epigenetics, genetics of the embryonic NSCs which give rise to adult NSCs caused by choline availability. We hope that more basic understanding of lasting modifications in brain plasticity provide preventative or therapeutic interventions that restore normal brain activity and, ultimately, the associated cognitive and behavioral deficits.

大脑发育窗口期胆碱水平影响成年后海马认知功能和相关疾病发生，但机制尚不明确。成年海马神经发生在学习记忆和情感认知功能中非常重要。我们前期研究发现胆碱通过DNA甲基化调控胚胎神经发生，并在胚胎和成年神经干细胞检测到单个基因启动子区同样的甲基化改变模式。鉴于成年神经干细胞起源于胚胎神经干细胞，我们推测胆碱在窗口期通过改变DNA甲基化谱影响成年神经干细胞的基因表达谱，进而决定成年神经发生和海马认知功能。本项目拟给予转基因Nestin-CFP及谱系追踪孕鼠不同胆碱含量食物①免疫荧光法确定对子代成年海马神经发生的影响；②流式分选子代CFP阳性神经干细胞，测定胚胎和成年神经干细胞基因转录组和DNA甲基化谱，生物信息学筛选两个时期表达模式相同的差异甲基化基因；③体内外实验验证基因对成年海马神经发生的影响。目标是阐明窗口期胆碱影响海马长期功能的机制，鉴定调控神经发生的关键基因，为防治认知功能缺陷提供靶标。

大脑发育窗口期胆碱水平影响成年后认知功能和相关疾病发生，但机制尚不明确。我们使用国际公认的胆碱处理剂量在大脑发育窗口期E11-E17天分组饲养Nestin-CFP转基因孕鼠，利用CFP作为荧光标记流式分选神经干细胞，针对不同胆碱水平对胚胎神经干细胞基因表达谱的影响通过RNA测序结合定量RT-PCR进行了确认。再由生物信息学结合基因功能注释筛选表达改变显著的基因，确立候选基因。通过焦磷酸测序分析启动子区CpG岛DNA甲基化改变，分析候选基因甲基化变化是否与基因表达变化匹配，建立DNA甲基化和mRNA表达谱的对应关系。通过表观编辑(dCas9-Dnmt)进行功能验证，慢病毒感染神经干细胞dCas9-Dnmt带有sgRNA靶向候选基因的CGG重复序列，观察神经干细胞的分化、增殖情况。另外，以Ube3a，Bdnf为代表的基因转录水平有显著改变，但DNA甲基化并未受胆碱影响，由于它们在神经发育和认知功能中的重要作用包括影响神经干细胞分化、存活，突触形成和可塑性，我们检测了其组蛋白甲基化状态，并对其中改变最明显的H3K27me3进行了表达调控的研究。我们发现胆碱缺乏时通过DNA甲基化影响基因Atf3和Chac1的表达调控神经元存活和分化，通过组蛋白甲基化影响神经干细胞的关键基因Ube3a和Bdnf的表达调控神经元突触可塑性，进而影响大脑发育和认知功能。在成年后认知相关疾病中，阿尔茨海默氏病(Alzheimer’s disease, AD)严重危害人类健康，目前尚无有效干预措施。胆碱与认知功能密切相关，因此我们在前期工作基础上探索了补充胆碱对AD的作用。从AD小鼠早期2月龄开始补充胆碱至11月龄发现：补充胆碱提高AD小鼠空间学习记忆能力，减轻焦虑，改善了AD病理如Aβ沉积、炎症、胆碱能神经元丢失。其机制一是由于胆碱是合成神经递质乙酰胆碱的前体，所以AD的主要病变部位海马和皮层乙酰胆碱增多；二是胆碱抑制炎性小体活化；三是胆碱促进突触膜修复，而突触丢失是与AD认知功能下降最相关的神经病理改变，并且突触丢失发生在AD早期，持续整个病程，与认知下降程度平行。因此在AD早期补充胆碱可以为防治AD认知障碍、延缓AD发展提供新思路。