Investigating the role of Ephrins and Eph receptors in circadian physiology

昼夜节律系统控制着许多内源性生理功能，与环境光暗周期的同步性决定了生物体的正常功能。已知昼夜节律源自分子转录-翻译反馈环，并且与昼夜节律的同步取决于主要内源性时钟（即，哺乳动物下丘脑视交叉上核[SCN]）。然而，时钟分子反馈回路向输出系统发出信号的方式仍然是未知的。这个程序的一般假设是，时钟分子机制通过控制塑造突触功能的元素的表达和活动来调节昼夜节律。 Ephrins/Eph系统是一种细胞粘附系统，其被证明参与突触可塑性。我们最近观察到，该系统的一个成员EphA 4的突变导致睡眠/觉醒结构的改变，这表明昼夜睡眠调节的变化。此外，EphA 4在SCN中表达，并且其基因含有E-box序列，表明通过时钟分子机制进行调节。因此，本项目旨在研究EphA 4及其配体（例如，EfnA 3，EfnB 3），以及这些元素在调节小鼠昼夜节律功能中的作用。该方案有三个具体目标，每个目标都与一个实验方案相关联： 目标1：验证核心时钟元件调节EphA 4和配体的转录;使用体外荧光素酶测定来评估时钟转录因子对Epha 4和配体的转录激活，并使用SCN穿孔上的染色质免疫沉淀以及定量PCR在活性开始时每4小时处死的小鼠中进行（恒定黑暗条件：DD）以研究体内时钟突变小鼠中时钟转录因子的节律性结合和表达。 目标二：确定EphA 4的表达和活性是否在SCN和涉及昼夜节律调节的其他脑区域中以昼夜节律方式变化;使用荧光原位杂交和免疫组织化学，用针对EphA 4和磷酸化-EphA 4的抗体对从活动开始（DD）每4小时取样的脑进行。 目标3：测试EphA 4参与活动和温度节律两者的昼夜节律调节;使用EphA 4敲除小鼠和通过脑siRNA输注实现的EphA 4下调的小鼠中DD中的活动和温度节律的连续遥测测量。 该计划将揭示时钟机制如何控制Ephrin/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式来解开昼夜节律和神经元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.