生物钟蛋白Period3在睡眠中的调节机制

While the sleep-wake cycle is the most significant circadian rhythm, the molecular genetic mechanisms underlying how the circadian clock regulates sleep are still unclear. By the analysis and examination of the TALEN-generated period3 mutant zebrafish, the Han Wang laboratory recently found that loss of Per3 results in significant sleep disorders. The objective of this project is to elucidate the genetic mechanisms underlying Per3 regulation of sleep and the evolution of Per3 sleep function in three specific aims: 1) to establish and examine per3-driven GFP, Luciferase, and GCamp6 transgenic lines to determine its expression, rhythmicity and cellular activities in different regions of the brain; 2) to determine whether Per3 contributes to sleep via γ-aminobutyric acid (GABA) and norepinephrine signaling through generating whole-body and tissue-specific gene knockout and overexpression zebrafish lines, optogenetics-mediated neuronal activation, chemogenetics-mediated neuronal ablation as well as high-throughput behavioral recording and analytic techniques; and 3) to determine the evolution of Per3 sleep function by establishing and examining overexpression of wild-type and mutated PER3 of human and mouse in wild-type and per3 homozygous zebrafish, respectively. This study should systematically elucidate the genetic mechanisms of Per3 regulation of sleep.

睡眠-觉醒周期是最为显著的生物节律，然而生物钟在睡眠中调控的遗传机制尚不清楚。申请人前期通过对斑马鱼period3突变体的分析发现缺失Per3导致显著的睡眠障碍。本项目旨在阐明Per3对睡眠调控的遗传机制及其睡眠功能的演化规律：1）通过建立和检验per3驱动的GFP、Luciferase和GCamp6的转基因品系确定其在大脑中不同区域表达、振荡和细胞活动的特征；2）通过建立全身和组织特异基因敲除和过表达、光遗传学介导的神经元激活、化学遗传介导神经元杀死和高通量行为追踪分析等技术确定Per3是否通过γ-胺基丁酸（GABA）和去甲肾上腺素（Norepinephrine）信号在睡眠中起作用；3）通过建立和检验在正常和per3纯合突变体斑马鱼中表达人和小鼠正常PER3和携带突变的PER3的品系，确定生物钟蛋白Per3睡眠功能的演化规律。本研究将系统地阐明Per3对睡眠调节的遗传机制。

斑马鱼(Danio rerio)是研究生物钟和睡眠的脊椎动物模型。众所周知，内稳态过程(S)和昼夜节律过程(C)都有助于调节睡眠内稳态。虽然我们对昼夜节律调节有了很好的了解，但我们对睡眠稳态的昼夜节律调节和/或昼夜节律系统与睡眠之间的相互作用的分子机制知之甚少。本项目，我们报告了由TALEN生成的斑马鱼period3 (per3) 无效突变体的特征。行为试验表明，在恒定黑暗条件下，per3突变体的周期比野生型缩短1.1小时，相位提前约3小时;在恒定光照条件下，per3突变体的周期比野生型缩短近3小时;per1a、per1b和per2在per3突变鱼中下调;这表明per3对斑马鱼的昼夜节律调节至关重要。有趣的是，per3突变鱼在睡眠剥夺后表现出更少的睡眠时间、更低的唤醒阈值和更难恢复睡眠。ELISA检测结果显示，per3突变体鱼夜间GABA水平降低，GABA处理可部分恢复per3-/-突变体的睡眠缺陷，提示per3突变体鱼睡眠模式紊乱可能是内源性GABA水平改变所致。基于深度测序的转录组分析使我们关注两个候选基因，谷氨酸脱羧酶基因gad1b和gad2，它们在per3突变鱼中都下调了。综上所述，这些结果确定了Per3在斑马鱼昼夜节律系统中的重要作用，证明了Per3通过GABA信号通路作用于睡眠调节，并为药物筛选和发病机制分析提供了理想的睡眠障碍脊椎动物模型。