Circadian Rhythms and Sleep
Summary: Amita Sehgal's major goal is to understand the molecular and cellular basis of behavior. Her studies are done largely with the fruit fly, Drosophila melanogaster, and are directed, in part, toward understanding the endogenous mechanisms that confer a circadian (~24-hour) periodicity on many behaviors and physiological processes. She also uses a Drosophila model to understand how and why the need to sleep is generated.
Circadian (~24-hour) clocks endogenous to most organisms drive daily rhythms of sleep:wake and of most physiological processes. Since they are controlled by internal clocks, these processes continue to cycle even when people are removed from the cyclic day:night environment. However, any kind of desynchrony between endogenous clocks and the environment, as is caused by travel to a different time zone or by shift work, results in a multitude of physiological disturbances. Circadian rhythms are also disrupted in some affective disorders.
We are interested in determining on a molecular and cellular level how the endogenous clock is generated, 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 sleep per se—specifically, why a sleep state exists and how the need to sleep builds up with prolonged wakefulness.
Control of Behavior and Physiology by Circadian Clocks
Drosophila melanogaster display cycles of rest and activity very similar to human sleep:wake cycles. 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. In Drosophila, the major autoregulatory loop is composed of the period (per) and timeless (tim) genes. In a second, linked autoregulatory loop, a transcriptional activator of per/tim, Clock, is alternately activated and repressed by two of its target genes, vrille and Pdp1. Although the loops are transcription based, we found that even when levels of per and tim mRNA are held constant, the proteins cycle and drive rest:activity cycles. These data do not altogether exclude a role for transcriptional regulation in the clock mechanism but indicate that posttranslational regulation can drive cyclic expression of PER and TIM. Both proteins are rhythmically phosphorylated, and we identified an essential role for phophatases PP1 and PP2A in the rhythmic expression of TIM and PER. Codependent nuclear localization of the two proteins, which occurs at a specific time of night, is also a critical aspect of the loop.
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. Subsequently, we identified the E3 ligase that targets TIM for degradation by light, a molecule we named jetlag (jet) because of the extended jetlag phenotype of flies that lack it. Using JET, we developed a cell culture assay for TIM degradation by light. We also developed an assay for CRY that lends itself to high-throughput analysis and, through a genome-wide RNAi (RNA interference) screen, 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 showed that the Drosophila homolog of the Neurofibromatosis 1 (NF1) gene, the gene mutated in the human disease of the same name, acts through the Ras/MAPK pathway to drive rest:activity rhythms. In collaboration with Thomas Jongens and Janice Fischer, we also identified circadian phenotypes in flies lacking the Drosophila homologs of the fragile X (fmr) and Angelman syndrome genes, respectively. Interestingly, many patients with these disorders report sleep disturbances, which could result from circadian dysfunction.
In recent work, we found that a microRNA, miR-279, must be expressed at appropriate levels for normal rest:activity rhythms. Very high or very low levels of miR-279 disrupt rhythms. Effects of miR-279 on rhythms are mediated by unpaired (upd), a ligand of the JAK-STAT pathway, and manipulations of this pathway also disrupt rhythms. Since the clock is normal in mutants of this pathway, we believe that the pathway serves to transmit time-of-day signals from the clock and produce rhythmic rest:activity patterns. Analysis of this pathway also provides insight into cells that are part of the rest:activity rhythm circuit.
We are also investigating how clocks outside the brain control physiology. We found that the pupal prothoracic gland contains a clock required for eclosion (hatching of adult flies from pupae) rhythms. We identified a clock in the fat body (fly equivalent of the liver) that regulates a rhythm of feeding and 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. We found that neuronal and metabolic clocks can be desynchronized when flies are fed at the wrong time of day because metabolic clocks are reset by food, but brain clocks are not. Such desynchrony reduces reproductive fitness.
The Drosophila Model for Sleep
Circadian rhythm research in Drosophila provided the framework for studies of mammalian clock function. The mechanisms and most fly clock genes are conserved in mammals. Because of 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 how the brain signals the need to sleep and identify the functions served during sleep. To this end, we have identified sleep-regulating cells and molecules in the fly.
We showed that components of the cAMP/PKA pathway regulate sleep, at least in part, through the mushroom body (MB), the site of learning and memory in Drosophila. These studies support a connection between sleep and synaptic plasticity. We have also found that serotonin acts through the MBs to promote sleep. On the other hand, octopamine released by specific neurons in the dorsal part of the fly brain promotes wake and its effects on sleep:wake are mediated by the OAMB octopamine receptor in insulin-producing cells of the fly brain. These studies reveal a circuit that underlies sleep and suggest an interaction between sleep and metabolism.
As an unbiased approach toward identifying molecules required for sleep, we have conducted forward genetic screens for short-sleeping flies. One such screen identified a novel mutation in a dopamine transporter. This is expected to result in increased extracellular dopamine, which increases arousal in flies, as it does in mammals. Through a different 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 gene mutated in these flies encodes a small protein required for silencing neuronal activity. sss flies are short-lived, suggesting that they are compromised by their lack of sleep. We found that the SSS protein controls the expression and activity of the Shaker potassium channel, which also regulates sleep. The predicted structure of SSS resembles that of select toxins, suggesting that it is an endogenous toxin-like molecule that regulates neural activity through its effect on ion channels.
In related work, we have determined the effects of age on sleep:wake cycles and initiated studies of a different, inert behavioral state—anesthesia. We find that sleep:wake cycles grow weaker with age in Drosophila. Sleep becomes fragmented, and circadian parameters change so that the period gets longer, and both period and phase (daily distribution) of activity are unstable. Investigation of underlying mechanisms reveals that the brain clock cycles robustly in old flies, and so output from the clock is compromised. Manipulating environmental cycles or lowering the level of PKA strengthens rhythms in old flies. With respect to anesthesia, we are interested in the mechanisms underlying the response to anesthesia and the extent to which anesthetics act through sleep-regulating circuits.
Work in this laboratory is supported in part by the National Institutes of Health.
As of May 30, 2012