Amita Sehgal's major goal is to understand the molecular and cellular networks that drive behavior, in particular rhythmic behaviors such as sleep. Her studies are done largely with the fruit fly, Drosophila melanogaster, and are directed towards elucidating the mechanisms that confer a circadian (~24-hour) periodicity on much of behavior and physiology as well as understanding how and why the need to sleep is generated.
What drives sleep:wake cycles? Understanding the Timing, the Need, and the Impact of Sleep
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 the process of sleep, why a sleep state exists, and how the need to sleep builds up with prolonged wakefulness.
Circadian Rhythms of Behavior and Physiology: The Molecular CLock and its Entrainment to Light
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 feedback to negatively regulate their own expression. In Drosophila, the major genes that act in this manner and generate an autoregulatory loop are the period (per) and timeless (tim) genes. We and our collaborators identified and cloned the tim gene, which was followed by isolation of several other components of such molecular loops. Post-translational regulation is critical for the generation and maintenance of such loops and occurs on several levels. Both proteins are rhythmically phosphorylated, and we identified an essential role for phophatases PP1 and PP2A in the cyclic expression of TIM and PER. We have also uncovered mechanisms that underlie nuclear localization of the two proteins, which occurs at a specific time of night and is an important part of the timing mechanism.
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 and CRY degradation by light.
Control of Behavior and Physiology by the Clock
To determine how the clock controls behavior, we have identified pathways that transmit signals from the clock. For instance, we identified a microRNA, miR-279, which acts through unpaired (upd), a ligand of the JAK-STAT pathway, to produce rhythmic rest:activity patterns. In recent work, we have traced a neural circuit that acts downstream of the clock and appears to link the clock to rhythmic behavior. Within this circuit, a major relay station is the pars interebralis (PI), which is the fly equivalent of the hypothalamus. We found that a specific peptide produced by PI cells, Diuretic hormone 44 (DH44), ortholog of mammalian corticotrophin releasing factor (CRF), is required for rest:activity rhythms. Together these studies are providing an account of how time-of-day signals are transmitted through the brain to generate behavioral and physiological rhythms.
Efforts are underway to determine to place molecular circadian components we identified previously in the aforementioned circuit. These include the Drosophila homolog of the Neurofibromatosis 1 (NF1) gene, the gene mutated in the human disease of the same name, which we showed 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.
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 showed that neuronal and metabolic clocks are 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. Clock mechanisms and most fly clock genes are conserved in mammals, and even implicated in human circadian disorders. 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 to identify the functions served during sleep. To this end, we have identified sleep-regulating cells and molecules in the fly, and also discovered a function for sleep in early life.
Molecules and Circuits that Drive Sleep
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. In yet another screen we identified a mutation we termed redeye (rye), which disrupts an alpha subunit of the nicotinic acetylcholine receptor. We found that the SSS protein controls the expression and activity of the Shaker potassium channel, which also regulates sleep, as well as activity of the RYE protein. Thus, our findings are identifying interacting molecules and pathways that regulate sleep. Importantly, these mechanisms appear to be conserved in humans, as human studies are revealing effects of the same or similar proteins on sleep amount.
We have also initiated studies of mammalian models. We recently identified effects of sleep loss on the peripheral blood metabolome in rats and humans, and are currently extending some of our findings regarding specific circadian/sleep genes to mouse models.
Function for Sleep in Early Life
We are also very interested in the function in the function of sleep. Driven by findings that virtually all animals sleep more at young ages, we sought to address a function for sleep in development. We identified the mechanism that maintains high sleep levels in young flies and then showed that these high levels of sleep are required for the development of courtship circuitry. Thus, when flies are sleep-deprived in early life they do not mate as well and display impairments in their olfactory system.
Clocks, Sleep and Aging
We find that short-sleeping mutants are compromised in terms of lifespan and 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. However, clocks in peripheral tissues are dampened and contribute to aging physiology. Recently, we and our colleagues found that specific clock genes are required to lengthen lifespan in response to dietary restriction.
Together these studies are providing a comprehensive understanding of how internal clocks drive body rhythms, how and why a sleep state occurs, and the extent to which clocks and sleep impact general physiology and aging.
Work in this laboratory is supported in part by the National Institutes of Health.
As of March 11, 2016