Circadian rhythms are daily molecular oscillations that strongly influence physiology and behavior in organisms from cyanobacteria to humans. These rhythms are entrained by and synchronize to environmental light cycles but are also maintained in constant darkness, demonstrating the function of a biological clock. Circadian clock dysfunction has been identified as an important factor in sleep and mood disorders. The phenomenon of jet-lag occurs while the circadian clock is attempting to re-synchronize to a new light cycle resulting in sleep disturbances and reduced capacity for both physical and mental tasks. In insects and humans, the molecular oscillations of the core clock are generated and maintained in distinct cell groups in the brain. Studies in the fruit fly Drosophila melanogaster and in mouse have revealed that signaling among these cells plays a critical role in entrainment. In Drosophila, the neuropeptide Pigment Dispersing Factor (PDF) and its receptor (PDFR) play a key role in synchronizing the cell-autonomous oscillators. We have recently identified PKA as the primary signaling molecule acting downstream of PDFR to mediate effects on the core molecular clocks in- vivo. This proposal aims to determine the role of PDF signaling through PKA in light-entrainment of the circadian clock and elucidate the mechanism by which PKA directly influences the core molecular clock in circadian cells. The first specific aim will determine the PDFR- and PKA-dependent components of entrainment by assaying behavioral re-entrainment to shifted light cycles and examining the molecular clocks in circadian cells during this process.
The second aim will determine the potential PKA phosphorylation sites on the core clock proteins PERIOD (PER) and/or TIMELESS (TIM) that are important for responding to PDF, as well as characterizing the cellular substrates of PDF by determining the PDFR expression pattern. Third, I will Determine the degree to which PKA effects on PER or TIM are direct by performing functional luciferase assays testing specific PER and TIM mutants, co-immunoprecipitation with PKA, and in-vitro phosphorylation. Relevance: Failure to entrain or maintain synchrony among the body's many circadian clocks has deleterious effects ranging from learning deficits to sleep and mood disturbance to metabolic disorders. While this proposal is directed toward the molecular basis of light signaling to the clock, we believe that the results will aid the development of new approaches in the study and treatment of sleep and circadian disorders, as well as treatment of circadian consequences of neurodegenerative disease.
In insects and mammals, circadian rhythms are molecular oscillations generated and maintained in a network of cells in the brain. The communication among the cells in these networks is critical for maintaining robust molecular and behavioral rhythms. The experiments proposed here will elucidate the biochemical link between the cell-surface receptor PDFR and the core molecular clock that underlies rhythmic behavior, thus revealing key regulatory steps in the entrainment of the circadian rhythm in Drosophila.
| Seluzicki, Adam; Flourakis, Matthieu; Kula-Eversole, Elzbieta et al. (2014) Dual PDF signaling pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian behavior. PLoS Biol 12:e1001810 |
