生物钟在注意力缺陷多动症（ADHD）中的调节机制研究

Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children, and mainly caused by the imbalance of dopamine level in the brain. While ADHD patients often display circadian abnormalities such as sleep/wake cycle disorders. In our former researches, we found that zebrafish core circadian gene per1b mutant and Per1 knockout mice displayed hyperactivity, impulsivity and attention deficiency, mimic the human ADHD behaviors. Therefore, we generated the first ADHD disease animal model caused by circadian dysfunctions. However, the mechanisms that how circadian clocks regulate the dopamine signaling pathway and the development of dopaminergic neurons are still far from certain. Recently, we did small non coding RNA deep sequencing using small RNA extracted from zebrafish per1b mutant brain, and found that within 19 differential miRNAs, up to 14 miRNAs can target to nurr1 gene, the key protein controlling the dopaminergic neuron’s development, and one miRNA target to dopamine beta hydroxylase gene, dbh. We also found that the PER1 protein is co-localized with NURR1 in the dopaminergic neurons of mice substantia nigra (SN) region. Based on these new findings, we plan to clarify the mechanisms that how circadian controlled microRNAs regulate ADHD, and explore the evidences that the core circadian factors can directly take part in the dopamine signaling pathway; Meanwhile, we also try to develop new diagnosis molecular markers in the ADHD patients. This study will provide new theoretical and experimental basis for the accurate diagnosis, prevention and treatment of ADHD.

注意力缺陷多动症 （ADHD）为儿童期常见神经系统疾病，与大脑多巴胺信号有关，患者常伴有生物节律问题。申请人前期研究中发现斑马鱼钟基因per1b突变体和小鼠Per1突变体均表现出ADHD的症状，从而建立了第一个生物钟相关的ADHD疾病模型，然而生物钟对于大脑多巴胺信号的调控机制不明。前期实验中，我们对斑马鱼per1b突变体大脑进行了早晚两个点的小RNA深度测序，发现19个差异小RNA中有14个均靶向到多巴胺能神经元发育关键基因nurr1, 还有一个靶向到dbh 基因，且发现小鼠中PER1与NURR1在黑质多巴胺能神经元中共定位。本项目拟采用CRISPR基因编辑技术结合分子生物学手段明确钟控小RNA对ADHD的调控机制，寻找生物钟基因直接参与多巴胺信号调控的证据，同时结合临床，发展ADHD分子诊断标记。本项目的实施对阐明ADHD的发病机制具有重要意义，为ADHD的精确诊断与治疗提供理论依据。

生物钟是以大约24小时为周期的生物节律，可发生在分子、生化、细胞、生理及行为等各种水平。生物钟基因突变导致的生物节律紊乱会引起很多精神类疾病. 注意缺陷多动障碍 （ADHD）为儿童期常见的中枢神经系统疾病,在起病过程中常伴随睡眠障碍等生物节律问题。大脑多巴胺水平及多巴胺能神经元发育与ADHD密切相关。已有研究表明部分多巴胺合成代谢基因受到生物节律调控而表现出了多巴胺的节律振荡，核心生物钟基因period1突变体也出现多巴胺神经元发育受损的表型。然而生物节律与多巴胺神经元发育之间的相互作用以及对多巴胺水平调节的分子机制始终不明确。本项目针对这一核心问题展开研究，目前基本完成预期项目任务。共取得了以下几个重要进展。1，在ADHD斑马鱼模型中鉴定到4个小RNA表达量显著改变的分子，miR206, miR216, miR19a, miR153, 实验结果表明这4个小RNA均呈现节律变化，属于钟控基因。且均靶向到多巴胺神经元发育关键基因nurr1/nr4a2, 证明生物钟可能通过调控小RNA的表达来调节多巴胺神经元发育，进而调节大脑多巴胺水平，影响ADHD。2，首次发现，核心生物钟蛋白period1能够与多巴胺神经元发育关键核受体因子Nurr1直接相互作用并调控多巴胺代谢关键基因tyrosine hydroxylase(th)和dopamine β hydroxylase（dbh）的表达，提出了生物钟与多巴胺系统相互作用的新思路。相关研究仍在进一步深入。3，在ADHD治疗方面展开积极探索，发现维生素C聚合物能够有效减轻ADHD斑马鱼模型的相关症状，提升大脑多巴胺水平，同时促进中脑多巴胺能神经元发育，对于治疗ADHD具有潜在的实用价值。4，结合基础研究结果，临床上收集了数百例ADHD初症患者，发现患者血液多巴胺水平与miR206呈显著负相关，ADHD患者血液中miR206水平与正常人相比显著升高，具有统计学意义。因此，miR206可以发展成一个ADHD的分子诊断标记用于临床病例的精确分子诊断。