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Circadian Rhythms and Sleep
Summary: Amita Sehgal's goal is to understand the molecular basis of behavior. Her studies, which are done largely with the fruit fly, Drosophila melanogaster, are directed partly toward understanding the endogenous mechanisms that confer a circadian (~24-hour) periodicity on many behaviors and physiological processes. Her research is also focused on the regulation and function of sleep, which is controlled by the circadian clock and also by noncircadian mechanisms.
Cycles of sleep and wake in humans are controlled by an endogenous circadian (~24-hour) clock. This clock also drives rhythmic fluctuations of many other aspects of human physiology, such as body temperature, blood pressure, and the release of various endocrine hormones. Since they are controlled by an internal clock, these processes continue to cycle even when people are removed from the cyclic day:night environment. However, any kind of desynchrony between the endogenous clock and the environment, as is caused by travel to a different time zone or by shift work, results in a multitude of physiological disturbances. It is believed that circadian rhythms are also disrupted in some affective disorders.
We are interested in determining at the molecular level how the endogenous clock functions, how it synchronizes to light, and how it interacts with various body systems to drive rhythms of behavior and physiology. We are also interested in the regulation of sleep:wake cycles by a noncircadian homeostatic system that drives the need to sleep.
The Circadian Clock
Drosophila melanogaster display cycles of rest and activity very similar to human sleep:wake cycles. In addition, populations of flies display rhythmic eclosion (adult hatch) behavior such that the hatching of adults from their pupal cases occurs predominantly around the hours of dawn. Work done in several laboratories, including ours, has led to a basic understanding of how the clock that drives these rhythms is generated. Products of specific genes, called clock genes, cycle with a circadian rhythm and negatively regulate the expression of their own genes. The autoregulatory cycle thus generated takes ~24 hours to complete and constitutes the basic clock mechanism. The major components of this cycle are the period (per) and timeless (tim) genes. We found that even when levels of per and tim mRNA are held constant, the proteins continue to cycle and provide time-of-day signals sufficient to drive rest:activity cycles. Our work also showed that post-translational regulation of PER involves the activity of protein phosphatase 2A (PP2A), which directly dephosphorylates PER and affects its stability and nuclear expression. Regulatory subunits of PP2A are expressed with a circadian rhythm, which may impart cyclic control to clock protein expression. We are extending our work on phosphorylation to identify other relevant phosphatases and determine how these contribute to the maintenance of a 24-hour period.
Previously we showed that light exposure produces a reduction in TIM levels that is effected by the circadian photoreceptor cryptochrome (CRY). Both TIM and CRY are degraded by the proteasome in response to light, and the decrease in TIM is required to synchronize the clock to the environmental light:dark cycle. This response can be modulated by serotonin, which acts through the 5-HT1B receptor to decrease the activity of glycogen synthase kinase 3β (GSK3β), an enzyme that normally phosphorylates TIM and increases its sensitivity to light. Recently we have identified the E3 ligase that targets TIM for degradation by light. This molecule, named jetlag because of its role in facilitating adaptation to a new light:dark cycle, can even confer light responsiveness onto TIM expressed in tissue culture cells.
Control of Behavior and Physiology by the Clock
To understand how the clock controls behavior, we have identified pathways that transmit signals from the clock. We found that the Drosophila homolog of the Neurofibromatosis 1 (NF1) gene, the gene mutated in the human disease of the same name, is required for rest:activity rhythms. NF1 signals through the well-known Ras/MAP kinase pathway in a circuit that carries signals away from the clock cells. In collaboration with Thomas Jongens (University of Pennsylvania), we found that flies lacking the dfmr gene, the Drosophila homolog of the gene mutated in the human fragile X disease, also have an intact clock and yet lack rest:activity rhythms. Circadian/sleep disturbances are also reported in fragile X patients, suggesting that the fly model may provide insight into the mechanisms underlying fragile X disease.
We are also investigating how the clock controls other behaviors and physiology. It is clear that in addition to the central clock in the brain there are clocks in other tissues. We have found that the pupal prothoracic gland (PG) contains a clock required for eclosion rhythms. Unlike other clocks in the fly body that are independent of the brain clock, the PG clock requires the brain clock for its function. In fact, a neuropeptide secreted by brain clock cells, which is required for rest:activity rhythms, is also required for PG clock function and for eclosion rhythms.
We have extended our studies of body clocks to mammals, in particular to the analysis of clock proteins in the testes. We found that although the circadian clock proteins are expressed in specific cell types in the testes, their expression does not cycle. Cycling of clock gene expression is also attenuated in the thymus, which, like the testes, contains actively dividing and differentiating cells. In addition, the expression of PER in the testes and the thymus does not appear to be under the control of Clock (CLK), which, in other tissues, is a major transcriptional activator of PER. These studies suggest that robust circadian activity may require an initiating or synchronizing signal that occurs in differentiated cells.
The Drosophila Model for Sleep
Circadian rhythm research in Drosophila provided the framework for studies of mammalian clock function. Mechanisms, as well as the genes themselves, are conserved in mammals. This prompted us to determine whether Drosophila could also be a useful system for sleep research. We and our collaborator, Joan Hendricks (University of Pennsylvania), found that rest in flies shares behavioral and pharmacological attributes with human sleep. Perhaps most importantly, flies, like mammals, show a homeostatic need for rest (sleep), such that they need to compensate for sleep loss and will do so even against their circadian rhythm; e.g., if sleep-deprived at night, they will sleep in the morning, a time at which they are normally most active.
In previous work we showed that components of the cAMP/PKA pathway regulate sleep such that up-regulation of this pathway results in less sleep, and vice versa. We followed up on this work to determine where in the fly brain this pathway can be manipulated to affect sleep and found that the mushroom body (MB) in the fly brain is an important sleep-regulating structure. Although some regions of the MB promote sleep, others inhibit sleep. The MB is the site of learning and memory in Drosophila, and the cAMP/PKA pathway is implicated in learning and memory in all organisms examined. Thus, these studies support the idea that there is a connection between sleep and synaptic plasticity. We have also identified an effect of serotonin on sleep and find that this is mediated in the MB by the 5-HT1A receptor.
We have also examined sleep:wake cycles in older flies and find that the strength of the cycle gets weaker with age. Old flies show increased sleep during the day and less sleep at night, with increased nighttime awakenings. We believe that this sleep fragmentation is caused, at least in part, by buildup of oxidative damage, and that it contributes to the aging process.
Work in our laboratory is supported in part by the National Institutes of Health.
Last updated: July 17, 2008
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