Investigating the role of Ephrins and Eph receptors in circadian physiology

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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.