The recent definition of the circadian molecular oscillator has permitted identification of the critical pacemaker neurons underlying circadian timekeeping. That information now presents the possibility to re- examine fundamental questions regarding the cellular basis for circadian regulation of behavior. We are interested in the molecules and signaling mechanisms by which circadian pacemaker neurons transmit information from the clock to the brain and body. A major focus of our studies concerns transmitter signaling by pacemaker neurons. Genetic evidence indicates the neuropeptide PDF (Pigment Dispersing Factor) is a principal circadian transmitter in Drosophila. Our working hypothesis is that PDF is an important synchronizing agent within the pacemaker network. In the previous research cycle, we studied where the receptor for PDF (PDF-R) is expressed, we developed realtime imaging methods to study its activation in vivo, and screened for kinases and phosphatases that participate in its signaling pathways. Those outcomes helped establish a foundation for a cycle of studies we now propose - to analyze PDF-R signal transduction mechanisms and consequences on neuronal activity. PDF-R is a Class B peptide GPCR - that category includes receptors for the mammalian PACAP and VIP peptides. We will pursue two specific aims related to PDF receptor signaling. First, we will analyze the composition of the PDF-R signalosomes, starting with the identification of associated adenylate cyclases. The signalosomes are different for different pacemakers - their molecular definition will greatly advance our understanding of circadian physiology. Second, we will determine how PDF-R signaling and its inactivation are controlled by internalization, via endocytotsis - preliminary genetic evidence suggests this is a key step in proper PDF-R signaling. Finally, in Aim 3, we will exploit novel imaging technology (called OCPI) for fast, realtime measures of neuronal activity in all 150 Drosophila brain pacemakers - once per minute, for up to 24 hours. When performed with genetic and pharmacological manipulations of PDF, this will permit us to relate PDF-R signaling data to normal pacemaker physiology. Biological timekeeping is essential for numerous homeostatic physiological events. Challenges to these mechanisms (from shift-work schedules or seasonal change) cause disruptions severe enough to include clinical conditions. Our studies focus on evolutionarily-conserved molecular mechanisms of circadian clock output and will identify leads for better therapeutic interventions to redress such conditions.
Stroke and cardiac arrest display clear of time-of-day variation, yet circadian biology has a poor understanding of the nature of output signals from the circadian clock to effector organs. Here I propose a research program dedicated to addressing fundamental circadian output mechanisms.
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