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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. We showed that post-translational regulation of PER includes 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. Recently, we found that PER and TIM are also dephosphorylated and stabilized by protein phosphatase 1 (PP1).
Several years ago we showed that synchrony of this clock to light is mediated by light-induced degradation of TIM. This is mediated by the circadian photoreceptor cryptochrome (CRY), which is then itself degraded. Using classical genetics to isolate a gene that affects the circadian light response, we identified the E3 ligase that targets TIM for degradation by light. This molecule, which we 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. In more recent work, through a cell culture RNAi (RNA interference) screen, we identified components required for the CRY response to light.
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. For instance, we 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. Recently, we identified a clock in the fat body (fly equivalent of the liver and adipose tissue) that regulates a rhythm of feeding and also promotes the storage of energy reserves, in particular glycogen. Neuronal clocks have opposite effects on energy stores and feeding, indicating that the activity of neuronal and metabolic clocks is coordinated to provide energy balance.
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. Based upon the utility of the Drosophila model for determining the molecular basis of the clock, we developed it as a model for sleep. Our goal is to use the fly to address two major questions about sleep: How does the brain signal the need to sleep? What functions are served during sleep? To this end, we have identified cells and molecules that regulate sleep in the fly.
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 found that these PKA effects are mediated in the mushroom body (MB) of the fly brain. 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 found that serotonin promotes sleep and that this is mediated in the MB by the 5-HT1A receptor. Conversely, octopamine promotes arousal through regions outside the MB.
As an unbiased approach toward the identification of molecules required for sleep, we conducted a forward genetic screen for short-sleeping flies. Through this screen we identified a mutant that has a >80 percent reduction in daily sleep time. We named the mutant sleepless (sss) and found that the reduced sleep is caused by a mutation in a single gene. This gene normally produces a small protein required for the silencing of neuronal activity. The protein is lacking, however, in sss flies, which lack coordination and are short-lived, suggesting that they are compromised by their lack of sleep. Identification of the sss mutant, which to our knowledge is the mutant with the shortest sleep time ever to be identified, promises to provide insight into the mechanisms underlying sleep. Thus far we have found that sss controls the expression of the Shaker potassium channel, which was previously implicated in the regulation of sleep.
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 this laboratory is supported in part by the National Institutes of Health.
Last updated March 27, 2009
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