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Dissection of a novel 'periphery to brain' circuit that synchronizes Drosophila's circadian clock with temperature cycles

Reference	BB/H001204/1
Principal Investigator / Supervisor	Professor Ralf Stanewsky
Co-Investigators / Co-Supervisors
Institution	Queen Mary University of London
Department	Sch of Biological and Chemical Sciences
Funding type	Research
Value (£)	369,887
Status	Completed
Type	Research Grant
Start date	01/01/2010
End date	14/06/2013
Duration	41 months

Abstract

Circadian rhythms in flies are controlled by clock neurons in the brain. Clock gene transcriptional feedback loops operate within these neurons, which can be synchronized with the natural environmental rhythms of daylight and temperature. In flies, synchronization to light:dark cycles is mediated by the blue-light photoreceptor Cryptochrome (Cry), a protein expressed within the clock neurons of the fly brain. Clock neurons therefore contain a circadian photoreceptor that can directly synchronize the clock gene oscillations, explaining why fly brains can be entrained to light dark cycles in culture. In contrast, we found that isolated brains can not synchronize their clock to temperature cycles. We have strong preliminary evidence that they require temperature input sensed by peripheral sensory organs, either the chordotonal organs under surface of the cuticle, or external sense organs (or both). Reducing the expression of the 'nocte' gene, which we had identified in a genetic screen for mutants with defects in temperature synchronization, specifically disrupts this mode of clock-resetting. We plan to accurately map the responsible sensory tissues by further restricting the downregulation of Nocte, and by application of specific sensory organ mutants. Furthermore, we want to investigate if PhospholipaseC (PLC) is also expressed and required in the identified sensory structures, which would indicate the signalling process involved, and explain why PLC mutants can not synchronize to temperature cycles. We have preliminary evidence for the requirement of Trp-channels (previously shown to function as 'environmental sensors') for temperature synchronization and plan to identify which channels are involved and if they function in the same sensory structures as Nocte. Finally, we will apply a proteomics approach in order to identify proteins that interact with Nocte, which should help us to further elucidate the mechanisms underlying temperature synchronization.

Summary

Circadian clocks are biological timers that tick in most organisms, including humans. These clocks control a wide array of biological processes, including our sleep/wake cycle, appetite, or body temperature. They function without any input from the outside, meaning they are true clocks that tick with an approximate 24 hr rhythm, even when the organism is kept in total darkness. In other words, human beings keep their daily sleep wake cycles, even when kept in total isolation from the outside world. In nature on the other hand, our circadian clocks are strongly influenced by the environment, as for example by the daily light/dark and temperature changes. As a consequence, circadian rhythms are synchronized with the environment. An impressive example for both the independence of circadian clocks and their ability to communicate with the environment are the phenomena associated with travel across time zones (jetlag) or shift work. If you are 'jetlagged', your circadian clock is still ticking according to the time where you boarded your plane and is telling you to be awake in the middle of the night. Gradually though, your internal clock will adjust (synchronize) to the new time zone and you will feel comfortable again. Molecularly, circadian clocks are assembled by several so called 'clock genes', which are active in certain neurons in the brain and control important biological rhythms. The activity of these clock genes itself is regulated in a rhythmic fashion-they become active in 24 hr periods. The time of maximal or minimal gene activity is determined by the natural light-dark and temperature cycles an organism is exposed to. In other words, the way our clocks are synchronized with the outside world is mediated by directly changing clock gene expression in response to light/dark or temperature changes. The current proposal is aimed to investigate how temperature cycles can synchronize the circadian clock of fruit flies. We are very interested to solve this question, because we discovered that the mechanism involved must be very different from that described for light-synchronization. In flies, the latter is mainly mediated by the blue-light photoreceptor Cryptochrome (Cry), a protein that is expressed within the clock neurons in the fly's brain. As a consequence, the circadian clock of fly brains can be synchronized by light:dark cycles, even when the brains are taken out of the fly and cultured in a dish. Although (in analogy with Cry) we initially expected that the clock neurons also contain a temperature receptor, we found that 'brains in a dish' can not synchronize their clock to temperature cycles! This was a big surprise, indicating that the clock neurons in the brain receive the temperature information from somewhere else in the fly. In this proposal we want to identify these cells or organs, and we already have some promising preliminary findings: Previously we had isolated 'nocte' as temperature synchronization mutant. When we reduce the function of the 'nocte' gene in peripheral sensory organs, we can destroy the fly's ability to synchronize to temperature cycles. This means we now have a gene and candidate sensory structures at hand, which will allow us to start unravelling the temperature synchronization pathway. We will also investigate a class of ion channels (Trp channels), that has been shown to function as 'environmental sensors' in both vertebrates and insects. They can, for example, mediate responses to extreme temperatures, touch, or hot chilli peppers. We will test if these channels are important for temperature synchronization by analysis of available Trp channel mutants. Finally, we want to identify proteins that interact with Nocte by applying a modern proteomics purification approach involving Mass-spec analysis. By doing this, we hope to identify additional factors that will help to resolve the temperature synchronization pathway in flies.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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