Investigating the role of Ephrins and Eph receptors in circadian physiology

昼夜节律系统调控着许多内源生理功能，这些功能与环境的明暗周期同步，决定了生物体的正常功能。众所周知，昼夜节律由分子转录-翻译反馈环发出，并且与nychhemes的同步性依赖于主要的内源性时钟(即哺乳动物的下丘脑[SCN]的视交叉上核)。然而，时钟分子反馈回路向输出系统发出信号的方式大多仍不清楚。这个程序的一般假设是，时钟分子结构通过控制形成突触功能的元件的表达和活动来调节昼夜节律。*肾上腺素/Eph系统是一种细胞黏附系统，被证明参与突触的可塑性。我们最近观察到，这个系统中的一个成员EphA4的突变导致了睡眠/觉醒结构的改变，这表明昼夜睡眠调节发生了变化。此外，EphA4在SCN中表达，其基因包含E-box序列，表明受到时钟分子机制的调控。因此，本项目旨在研究EphA4及其配体(如EfnA3、EfnB3)的时钟元件和节律性表达的转录调控，以及这些元件在调节小鼠昼夜节律功能中的作用。该计划有三个与实验方案相关的特定目标：*目标1：验证核心时钟元件调控EphA4及其配体的转录；使用体外荧光素酶分析评估时钟转录因子对EphA4及其配体的转录激活；利用SCN针孔上的染色质免疫沉淀以及每4小时开始(恒定暗条件：DD)处死的小鼠的定量PCR，以研究时钟转录因子的节律结合和体内时钟突变小鼠的表达。*目标2：确定EphA4在SCN和其他与昼夜节律调节有关的脑区的表达和活性是否存在昼夜变化；使用荧光原位杂交和免疫组织化学方法，从活动开始(DD)开始，每隔4小时在大脑上采集抗EphA4和磷酸化EphA4的抗体样本。*目标3：测试EphA4参与活动和温度节律的昼夜节律调节；使用连续遥测测量EphA4基因敲除小鼠和通过脑siRNA注射获得EphA4下调的小鼠的DD的活动和温度节律。*该程序将揭示时钟机械如何控制EPhin/Eph系统元素的表达，以及该系统对温度和活动的昼夜节律的贡献。它代表了一种创新的方法，可以解开昼夜节律和神经生理学之间复杂的分子联系。

The circadian timing system governs numerous endogenous physiological functions, which synchrony with the environment light-dark cycle determines the proper functioning of organisms. It is known that circadian rhythms emanate from a molecular transcriptional-translational feedback loop and that the synchronisation with the nychthemeron depends on the main endogenous clock (i.e., suprachiasmatic nucleus of the hypothalamus [SCN] in mammals). Yet, the manner by which the clock molecular feedback loop signals to output systems remains mostly unknown. The general hypothesis of this program is that the clock molecular machinery regulates circadian rhythmicity by controlling the expression and activity of elements shaping synaptic function. ***The Ephrins/Eph system is a cell adhesion system that was shown to be involved in synaptic plasticity. We recently observed that a mutation in one member of this system, EphA4, resulted in alterations in sleep/wake architecture that are indicative of changes in circadian sleep regulation. Moreover, EphA4 is expressed in the SCN and its gene contains E-box sequences that suggest regulation by the clock molecular machinery. The present program will thus aim to study the transcriptional regulation by clock elements and rhythmic expression of EphA4 and its ligands (e.g., EfnA3, EfnB3), as well as the role of these elements in regulating mouse circadian functions. The program has three specific aims each associated to an experimental scheme:******Aim 1: Verify that core clock elements regulate the transcription of EphA4 and ligands; using in vitro luciferase assays to evaluate transcriptional activation of Epha4 and ligands by clock transcription factors, and chromatin immunoprecipitation on SCN punches as well as quantitative PCR in mice sacrificed every 4h starting at activity onset (constant dark condition: DD) to investigate the rhythmic binding of clock transcription factors and expression in Clock mutant mice in vivo.***Aim 2: Determine if the expression and activity of EphA4 vary in a circadian manner in the SCN and other brain areas implicated in circadian rhythm regulation; using fluorescent in situ hybridization and immunohistochemistry with antibodies against EphA4 and phospho-EphA4 on brain sampled every 4h starting at activity onset (DD).***Aim 3: Test that EphA4 participates in circadian regulation of both activity and temperature rhythms; using continuous telemetry measurements of activity and temperature rhythms in DD in EphA4 knockout mice and in mice submitted to downregulation of EphA4 achieved by brain siRNA infusion. ******The program will reveal how the clock machinery controls the expression of elements of the Ephrin/Eph system and the contribution of this system to circadian rhythms of temperature and activity. It represents an innovative way to disentangle the complex molecular connections between circadian and neuronal physiology.