注意缺陷多动障碍的斑马鱼发育模型

Attention deficit hyperactivity disorder (ADHD）is the most prevalent neurodevelopmental disorder in childhood, with a prevalence rate of 3%-5%。It has poor prognosis without proper treatment. Although there are some treatment drugs, almost all target neurotransmitter systems. There are limited variety of drugs, and the effect is not satisfying. Deep understanding of the etiology of ADHD and eatablish animal model that acturally imitate the disorder are very important for the discovery and validation of new biological targets, as well as development of new targeted medication. This project was based on our previous work that decreasing the expression of neurodevelopmental gene, MICALL2, induced hyperactive impulsive zebrafish model. We will use multiple genetic techniques, including CRISPRi gene expression regulatory technique, CRISPR/Cas9 and TALENs gene knock out technique, as well as transgenic technique, to culture expression reduced, knock-out, and mutant ADHD genetic developmental model in zebrafish, which will mimic the behavioral phenotype of ADHD from hyperactive, impulsive behavior to inattention, to evaluate the face validity. We will also study the construct validity on the basis of both the new neurodevelopmental theory and the traditional neurotransmitter imbalance theory for ADHD pathophysiology, and further use drug and gene rescue experiment to investigate predictive validity. Afterwards, we will screen more neurodevelopmental genes. The ultimate objective is to establish new ADHD developmental model in zebrafish, to support future pathogenic mechanism and drug screening.

注意缺陷多动障碍（ADHD）是最常见的儿童神经发育障碍，患病率高达学龄儿童的3%-5%，不治疗成年预后不良。临床有限的药物主要作用于神经递质系统。深度理解ADHD病因学建立能真实模拟人类疾病的动物模型对发现、确证新的生物学靶标及开发新的靶向治疗药物十分重要。本项目在前期神经发育基因MICALL2表达降低诱导的斑马鱼多动模型基础上，采用CRISPRi基因表达调控技术、CRISPR/Cas9和TALENs基因敲除技术、转基因技术等多种方法在斑马鱼中深入探索敲低、敲除和突变的遗传发育模型，从运动活动、冲动行为、注意力三方面模拟ADHD的行为学表型，进行表面效度评价；在最新的ADHD神经发育病理机制理论和传统的神经递质失调理论基础上进行结构效度的研究；并通过药物/基因挽救探讨预测效度。以此为基础进一步开展更多神经元发育基因的筛选。新的ADHD斑马鱼发育模型为新药筛选精准治疗提供支撑。

注意缺陷多动障碍（ADHD）是最常见的儿童神经发育障碍，患病率高达学龄儿童的3%-5%，不治疗成年预后不良。临床有限的药物主要作用于神经递质系统。深度理解ADHD病因学建立能真实模拟人类疾病的动物模型对发现、确证新的生物学靶标及开发新的靶向治疗药物十分重要。本项目采用多种技术方法在斑马鱼中深入探索敲低、敲除和突变基因的遗传发育模型，从运动活动、冲动行为、注意力三方面模拟ADHD的行为学表型，进行表面效度评价；在最新的ADHD神经发育病理机制理论和传统的神经递质失调理论基础上进行结构效度的研究；并通过药物/基因挽救探讨预测效度，开展神经元发育基因的筛选。项目共筛选了四个基因：MICALL2、KCNA3、RBFOX1和CNTNAP2，其中前三个基因通过CRISPR/Cas9打靶技术获得了突变体。两个micall2b突变体micall2b(-52,+8)，micall2b(-36)均呈现多动表型，Nkx2.2a和Gad1b阳性细胞显著减少。抑制cntnap2 表达后dbh 表达降低，gad1b、snap25、dat 表达增高，斑马鱼出现多动冲动表型，托莫西汀可以改善多动冲动行为。kcna3 突变体[kcna3(-17)] th、dbh、snap25、shha、glyt2, vglut2, stx1a, st x1b, sypa和sypb、drd2b、htr1b、serta、gad1b表达降低，netrin1b 表达增加, 听觉/前庭觉功能异常，突变体表现多动，注意力不集中和学习记忆障碍，托莫西汀可以改善多动冲动行为。Rbfox1突变体对光、声/震动刺激的适应性下降（学习记忆障碍），并由其下游基因如snap25和grin1等介导，Rbfox1对大量下游基因的调控均呈现剪切异构/转录后调节，rbfox1突变体呈现注意力不集中，但无多动和冲动症状，该项工作发表于本专业排名前列的Biological Psychiatry子刊Global Open Science，并获批发明专利一项。项目累计发表论文5篇，SCI引用4篇，会议交流5次，获得国际学术奖励2项。项目建立了ADHD基因的高通量筛选平台，验证了micall2b与多动行为的关系并深化了机制研究，在此基础上进一步建立了三个基因的遗传模型。本项目的完成为ADHD易感基因的功能基因组学研究及未来药物研发奠定了基础。