Kyunghee Koh - US grants

DESCRIPTION (provided by applicant): Function of SLEEPLESS in Sleep Regulation Summary Sleep serves essential biological functions, and is conserved from flies to humans. The overall goal of the proposed research is to elucidate the molecular genetic basis of sleep regulation. Through an unbiased forward-genetic screen for short-sleeping mutant flies, we identified a novel gene, sleepless (sss), which is required for normal sleep as well as recovery sleep after deprivation. sss encodes a small, brain-enriched protein expressed on the cell surface. Expression of the Shaker (Sh) potassium channel, previously shown to be important for sleep regulation, is markedly reduced in sss mutants. Several key questions need to be addressed to understand how SSS regulate sleep. Does Sh mediate the effect of sss on sleep? Does SSS act directly on Sh by forming a complex or does it act indirectly via a signaling pathway? Does SSS act on Sh cell-autonomously or non-cell-autonomously? Do circadian and homeostatic processes affect SSS and Sh expression? To address these questions, we propose the following specific aims: (1) Test whether SSS promotes sleep by acting on Sh and whether they physically interact. To confirm a potential genetic interaction between sss and Sh, we will look for a synergistic effect on sleep of reducing both sss and Sh activity. We will also examine whether overexpression of Sh can rescue the short-sleeping phenotype of sss mutants. In addition, we will test whether SSS and Sh physically interact. (2) Identify the locus of action of sss. Identification of sss, which has profound effects on both baseline and rebound sleep, allows us an opportunity to explore the cellular substrates of homeostatic sleep regulation. We will examine the expression pattern of the SSS protein, and carry out rescue experiments using a collection of tissue-specific drivers and an sss transgene. Cellular substrates of sleep under normal and deprived conditions will be compared. (3) Determine if SSS acts on Sh cell-autonomously. To understand how SSS acts on Sh to regulate sleep, it is important to determine the locus of action of Sh as well as that of sss. We will determine the Sh expression pattern and tissue requirement using tissue-specific rescue and targeted RNAi knockdown experiments. In addition, we will test whether SSS can be secreted and whether secreted SSS can rescue the sleep phenotype of sss mutants. PUBLIC HEALTH RELEVANCE: Lack of good-quality sleep is a common problem that can lead to accidents and reduced productivity, and there is growing awareness among healthcare professionals and the public that sleep is a critical health issue. Elucidation of molecular mechanisms underlying sleep regulation may provide us with important clues as to how and why we sleep, and may ultimately lead to novel treatments for sleep disorders.

? DESCRIPTION (provided by applicant): Sleep is an essential biological process that is conserved from flies to humans. Studies of sleep regulation have focused on two main mechanisms: the circadian mechanism that controls when to sleep and the homeostatic mechanism that regulates how much to sleep. Nevertheless, other factors, such as gender, also affect sleep, but the mechanism underlying gender-dimorphic sleep regulation is poorly understood. This proposal aims to investigate a novel Drosophila neural circuit involved in male-specific regulation of sleep. We recently identified a small number of neurons in the brain whose activation leads to markedly reduced sleep in male flies, but not in female flies. This is exciting because although several sleep-regulatory neuronal centers have been identified in Drosophila, this is the first example of a gender-dimorphic arousal center. Our preliminary data suggest that the newly identified arousal center promotes wakefulness in the presence of females and makes synaptic contacts with neurons that express the male-specific transcription factor FruitlessM, which is required for male courtship behavior. To understand how this novel arousal center regulates sleep in a gender-dimorphic manner, several key questions need to be addressed. What are the input signals and behavioral outputs of this neural center? What other neuronal populations does the neural center connect to and which neurotransmission system does it employ? We will address these questions using an array of genetic tools that are available for circuit analysis in Drosophila. This work will investigate a novel neural circuit involved in male-specific regulation of sleep and will produce new research avenues for understanding sex differences in sleep. Further, this work may provide valuable insights into the fundamental problem of how the brain balances competing needs to decide on appropriate behaviors.

? DESCRIPTION (provided by applicant): Sleep serves essential biological functions, and is conserved from flies to humans. Sleep disturbance is a common health problem that impinges on quality of life, workplace productivity, and public safety. Sleep usually occurs at specific tims of day and lasts for certain amounts of time. These two features of sleep are controlled by distinct molecular mechanisms. Whereas the molecular and anatomical basis of the circadian clock, which controls when we sleep, has been investigated extensively, the molecules and neural circuits underlying sleep homeostasis that regulates sleep duration are not well understood. Identification of novel genes and circuits that control sleep duration would facilitate elucidation of this mysterious biological process. The Drosophila model for sleep is well suited for discovery of new sleep-modulating genes through unbiased genetic screens. Using a forward-genetic screen for short-sleeping mutants, we isolated a novel sleep gene, taranis (tara). Mutations in tara result in a marked (up to 80%) reduction of sleep duration. [[Importantly tara mutants exhibit decreased levels of REDEYE (RYE), whose expression is regulated by homeostatic sleep drive. Thus isolation of TARA provides an exciting opportunity to investigate the molecular mechanisms underlying sleep homeostasis, a critical process that is poorly understood. Previous findings suggest that TARA and its mammalian homologs are involved in transcriptional regulation and cell cycle progression, and contain a Cyclin A (CycA)-binding homology domain. Notably, CycA, another cell cycle protein, was recently shown to be a sleep-promoting factor, but the molecular function of CycA in sleep is not well understood. Our preliminary studies suggest that TARA promotes sleep by two complementary pathways: 1) by upregulating protein expression of CycA and inhibiting Cdk1 (a Cyclin-dependent kinase that binds CycA and negative regulator of sleep), and 2) by upregulating transcription of dawdle (daw), an Activin-like signaling molecule and positive regulator of sleep. Further, our data identify ~14 CycA expressing cells in the pars lateralis (PL), which is analogous to the mammalian hypothalamus, as a novel sleep center. Building on these preliminary data, we propose to (Aim 1) determine how TARA interacts with other cell cycle proteins to regulate sleep, (Aim 2) how TARA interacts with daw to regulate sleep, and whether DAW acts as a sleep-inducing homeostatic signal, and (Aim 3) determine where and when TARA is required for sleep, and how the PL neurons connect to other sleep centers. The proposed experiments will yield significant mechanistic insights into sleep homeostasis.]]

Sleep is essential for healthy mind and body. It is regulated by homeostasis, by which accumulated sleep drive during wakefulness leads to increased propensity toward sleep. However, we can stay up late if we have something important or interesting to do, for example, take care of a baby or read a fascinating book. In other words, sleep is also regulated by motivational states. Thus sleep drive competes with other motivational drives to decide whether we sleep or engage in other important or interesting activities. We have recently shown that sex drive profoundly affects male sleep in Drosophila and that a subset of octopaminergic neurons (octopamine is analogous to human norepinephrine) mediates sleep suppression by male sex drive. Previous studies have shown that female flies sleep less and lay more eggs after mating. Thus both males and females adjust their sleep patterns to meet reproductive needs. However, the neural mechanisms of how sleep and reproductive behaviors are balanced are not well understood. In preliminary studies for this proposal, we identified several previously uncharacterized neuronal populations for balancing sleep and reproductive behaviors in males and females. Two of the newly identified populations are dopaminergic, suggesting that octopamine and dopamine signaling pathways cooperate to integrate sleep and reproductive behaviors. We will determine the anatomical and physiological connectivity among the various neuronal populations, and investigate their role in sleep and reproductive behaviors using a combination of behavioral analysis, calcium imaging, circuit tracing, immunohistochemistry, and generation of new drivers for precise manipulation of specific neurons. Our proposed studies offer an excellent opportunity to discover basic neural mechanisms and general organizing principles that underlie the balance between competing drives. Investigating how sleep regulatory mechanisms communicate with neural mechanisms for sexually-dimorphic behaviors using neuromodulators such as octopamine and dopamine may lead to a better understanding of how the human brain integrates sleep drive and other motivational states.