Mark Wu - US grants

[unreadable] DESCRIPTION (provided by applicant): Emerging data point to an important role for sleep in human health. Despite its importance, the function of sleep remains one of neurobiology's great mysteries. Our ability to gain fundamental insights into the function of sleep as well as potentially identify novel targets for the diagnosis and treatment of sleep disorders is hindered by our lack of understanding of the molecular genetic mechanisms underlying sleep. To identify novel genes required for sleep regulation, Dr. Wu, the principal investigator, has carried out a forward genetic screen in Drosophila. By screening approximately 3,000 mutant lines, he has identified 10 short-sleeping mutant strains. One of these mutants carries a novel mutation in a dopamine transporter known to regulate sleep, a finding which validates this approach. The goal of this proposal is to identify the gene mutated in one of the shortest sleepers, named catnap, and to determine the behavioral and signaling mechanisms underlying its phenotype. Catnap mutants have severely reduced and fragmented baseline sleep. Mapping experiments localize the catnap gene to an approximately 600 kb interval. In Aim 1, he will test the hypothesis that the short-sleeping phenotype seen in catnap is caused by changes in arousal threshold or sleep homeostasis. Because the phenotype of catnap mutants resembles that seen with manipulation of cAMP signaling, in Aim 2, he will test the hypothesis that catnap acts via cAMP signaling to regulate sleep. In Aim 3, he will clone the gene mutated in catnap, to identify a novel molecular component required for sleep regulation. These studies will be carried out in the laboratory of Dr. Amita Sehgal, a pioneer in circadian and sleep research. The training environment provided by the Division of Sleep Medicine at the University of Pennsylvania, led by Dr. Allan Pack, is ideal for the development of academic sleep physicians. This training grant will provide an excellent vehicle for Dr. Wu to learn about the design, execution, and analysis of behavioral experiments and facilitate his transition towards an independent career as a physician-scientist studying the molecular mechanisms underlying sleep. Lay Description: Our ability to design new diagnostic tools and treatments for disorders of sleep is impaired by our lack of understanding of the molecular mechanisms underlying sleep. To identify molecules important for sleep regulation, this proposal will characterize a novel short-sleeping fly mutant. [unreadable] [unreadable]

Many physiological processes and behaviors are under circadian clock control, including sleep. However, the molecular and circuit mechanisms underlying how the circadian clock regulates these behaviors remain poorly understood. We recently identified a novel molecule in Drosophila named WIDE AWAKE (WAKE) that plays a key role in mediating the circadian timing of sleep onset. In our original grant studying this molecule, we determined that WAKE is rhythmically expressed in arousal-promoting clock neurons and acts to upregulate GABAA receptors, thus cyclically suppressing the activity of these cells to promote sleep. Interestingly, growing evidence from our group and others suggests that WAKE-related molecules broadly function to spatially organize signaling complexes in a time-dependent manner. Moreover, there is a single homolog of WAKE in mammals, including humans, which is enriched in the circadian pacemaker suprachiasmatic nucleus. Thus, insights gained from studying WAKE in flies may help unravel how the circadian system regulates sleep in mammals as well. In this renewal of our previous grant, we propose to further our understanding of the mechanisms underlying the circadian modulation of sleep, by studying additional circuit and molecular mechanisms by which WAKE modulates this process. Specifically, we plan to carry out the following aims: 1) study the role of WAKE in regulating additional WAKE-expressing circadian clock neurons, and how this regulation impacts downstream arousal circuits; 2) identify and characterize additional proteins that interact with WAKE to modulate sleep; and 3) examine how glia may interact with these WAKE-expressing circadian clock circuits to regulate sleep. We will use a multidisciplinary approach, including cell biological, genetic, behavioral, and electrophysiological assays, to perform these studies. Circadian dysregulation of sleep is estimated to impact millions of people in the U.S. and has been implicated in adverse effects on health and productivity. Developing a better understanding of how the circadian clock regulates sleep could pave the way for identifying novel therapies to treat these disorders.

? DESCRIPTION (provided by applicant): Dopamine (DA) is a critical neurotransmitter, conserved from C. elegans to man, that regulates a wide variety of behaviors, including learning and memory, courtship behaviors, reward-seeking, and sleep/wake rhythms. Dysfunction of DA neural circuits in turn contributes to disorders such as Parkinson's disease, depression, and drug addiction. Yet how DA neural circuits contribute to these behaviors or disorders is not well-understood. The goal of this work is to develop, characterize, and utilize novel genetic reagents for the dissection of the DA neural network in Drosophila melanogaster. Drosophila is an ideal system to carry out these studies, as fruit flies have only ~250 DA neurons to drive many of the same DA-dependent behaviors found in mammals. Moreover, flies are highly tractable to neural circuit analyses, as they have a short generation time, well- developed genetic techniques and resources, and can be easily maintained in large numbers. In previous work, we generated novel transgenic fly lines that allowed us to manipulate distinct subsets of DA neurons. Using these lines, we identified a single pair of DA neurons that promote arousal by projecting to and directly inhibiting a sleep-promoting circuit. In addition, we have recently developed a novel genetic method, CLAMP (Cell Labeling Across Membrane Partners), which allows for identification, morphological characterization, and functional manipulation of neurons based solely on connectivity patterns. Here, we propose to generate novel transgenic DA driver lines, which will be used for the identification, characterization, and connectivity mapping of the DA neural network in the fly brain. First, we will generate new DA transgenic fly lines, based on the genomic enhancers from different genes that express in DA cells. Second, we will screen established Gal4 lines for expression in subsets of DA cells. By using a combinatorial genetic intersectional approach, these fly lines will collectively generate ~22,600 distinct labeling patterns containing small subsets of DA neurons. These lines will be made available to the scientific community to facilitate functional analyses of the DA neural network. Third, we will create a comprehensive database of DA neurons in the fly brain by 1) identifying and naming individual DA cells and 2) by using computer tracing techniques combined with registration to a standard brain model to label projection patterns. Fourth, by using the CLAMP method to systematically map the connectivity of these DA neurons, we will develop a detailed model of the DA neural network in Drosophila. Understanding how DA circuits in Drosophila function to regulate different behaviors would provide insights into related mechanisms in mammals, including humans, and thus set the stage for circuit-based therapeutic interventions for specific neurological and psychiatric diseases.

Project Summary Sleep is regulated by a circadian process that modulates the timing of sleep and a homeostatic process that adjusts the amount and depth of sleep in response to sleep need. Studies over the past few decades have delineated many of the molecular mechanisms underlying the core circadian clock. However, the mechanisms by which this core clock regulates sleep remain poorly understood. Recently, using Drosophila as a gene discovery system, we identified a novel molecule named WIDE AWAKE (WAKE) that mediates the circadian timing of sleep onset. WAKE is expressed in arousal-promoting clock neurons and upregulates GABA signaling in a time-dependent manner to promote sleep in Drosophila. Strikingly, we find a single homolog of WAKE in mice (mWAKE) and have determined that mWAKE is specifically enriched in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals. The overall goal of this proposal is to characterize the molecular and cellular mechanisms underlying the circadian timing of sleep, by investigating the function of mWAKE in mice. To do this, we will employ an array of approaches, including molecular, biochemical, genetic, electrophysiological, and behavioral studies. Specifically, we propose to 1) determine the molecular mechanisms underlying mWAKE function in cultured cells and SCN slices, 2) examine the sleep and circadian phenotypes of mice genetically lacking mWAKE, and 3) characterize and identify the function of specific mWAKE circuits in mice. These studies will be carried out with an outstanding collaborative team, with expertise in mouse genetics, large-scale in situ hybridization experiments, SCN slice physiology, and circadian/sleep behavioral analyses and should yield new insights into SCN function and the molecular and cellular pathways mediating the timing of sleep. ~15 million Americans have to work alternate shift schedules, and emerging evidence suggests that dysregulated sleep/wake cycles in humans can have significant adverse health consequences. Thus, there is an increased urgency to understand the circadian timing mechanisms underlying sleep. The proposed studies should lead to a better understanding of these mechanisms and thus may facilitate the potential development of novel therapeutic targets for the treatment of circadian sleep/wake desynchrony.

Circadian rhythm disturbance, impaired sleep, and agitation in Alzheimer's disease Neuropsychiatric symptoms (NPS) in AD are a major cause of burden to patients, caregivers, and society. One of the most troubling of these symptoms is agitation (which we will term Agit-AD), typified by a variety of problem behaviors including irritability, emotional distress, combativeness, yelling, pacing, lack of cooperation with care, insomnia, and restlessness. Agit-AD is associated with significant caregiver stress. There is a consensus in the field that we need better treatments for Agit-AD and that to do so we need better understanding of the brain mechanisms underlying Agit-AD. Families and clinicians frequently note that Agit-AD occurs cyclically, with patients often becoming more agitated later in the day or in the middle of the night. These clinical observations have led to the hypothesis that Agit-AD is associated with impaired circadian rhythms (i.e., a disordered ?internal clock?). Indeed, there are data suggesting that the severity of Agit-AD is associated with greater circadian phase delay and reduced rhythm amplitude and is a potential target for treatment. Thus, improvement of the strength and phase of circadian rhythms may be a potential approach for treating Agit-AD but medication trials to date have been of little to no benefit. These failures may reflect an incomplete understanding of the precise relationship between circadian rhythms and agitation and the potential need for finer grained analysis of Agit-AD. We propose to examine the association of circadian rhythm and sleep disturbance with Agit-AD in a cross-sectional design comparing 50 subjects with Agit-AD vs. 50 control subjects with AD and minimal NPS. SPECIFIC AIMS Aim 1: Compare phase and amplitude of circadian rhythms in 50 Agit-AD (+) subjects vs. 50 AD subjects with minimal NPS. Aim 2: Compare phase and amplitude of circadian rhythms in affective vs. executive subtypes in 50 Agit-AD (+). Aim 3: Compare sleep amount/quality and presence of SDB in 50 Agit-AD (+) subjects vs. 50 AD subjects with minimal NPS PUBLIC HEALTH SIGNIFICANCE: These data could be essential for the development of chronobiotic or sleep interventions for Agit-AD. If circadian rhythm disturbance (CRD) or poor sleep is associated with Agit-AD then these results would allow investigators to use actigraphy, core body temperature, or sleep measures as a surrogate biomarker in proof-of-concept studies of interventions targeting CRD for treatment of Agit-AD. If CRD or poor sleep is associated with a subgroup of patients with Agit-AD then actigraphy, core body temperature, or sleep measures could help investigators target that subgroup for intervention research.

Project Summary Homeostasis is a fundamental physiological property, maintaining crucial state variables within a specific range for the viability and fitness of animals. Homeostatic mechanisms are also important for motivated behaviors, such as feeding or sleep. For example, prolonged wakefulness increases sleep drive (sleep pressure), leading to an increase in sleep amount and/or depth (sleep rebound). The cellular and molecular mechanisms underlying the homeostatic regulation of sleep remain unclear, and we have recently identified a novel neural circuit that encodes sleep drive. Although sleep drive is widely assumed to inevitably increase with greater wakefulness, exceptions to this rule exist in nature, particularly under conditions of high arousal. We hypothesize that specific signaling mechanisms act on this homeostatic circuit to suppress the accumulation and/or release of sleep drive. The overall goal of this proposal is to leverage these new findings to unravel the molecular and cellular mechanisms underlying sleep homeostasis. In addition, emerging data suggest an important role for glia in the homeostatic regulation of sleep. Thus, we propose to carry out the following aims: 1) characterize the signaling pathways acting on this novel circuit to regulate sleep drive; 2) characterize the circuit mechanisms acting downstream of this sleep homeostatic circuit to promote sleep; and 3) investigate a specific role for glia in the homeostatic regulation of sleep. To carry out these studies, we will use a variety of approaches, including behavioral assays, molecular genetic analyses, immunohistochemistry, functional imaging, and patch-clamp electrophysiology. These studies should reveal new insights into how sleep is homeostatically regulated. Insomnia and disorders of pathologic sleepiness are common and often associated with substantial morbidity. A better understanding of the mechanisms underlying sleep homeostasis may help in our search for novel treatments for both pathologic sleepiness and insomnia.

Summary Sleep is an enigmatic behavior that is conserved across the animal kingdom. The mechanisms underlying the regulation of sleep remain poorly understood, and the function of sleep is even more elusive. Yet, dysregulation of sleep is a major cause of human morbidity, and understanding why we sleep is a fundamental question in neuroscience research. We propose that gaining a deep understanding of sleep requires studying this process across molecular, circuit, neurophysiological, and behavioral levels. To accomplish this, our group has been using a multidisciplinary, cross-species approach (Drosophila and mice) to study sleep. The recent work of our group has coalesced around two main themes: how circadian time organizes sleep and arousal and how dedicated neural circuits encode homeostatic drive. Our investigations into these processes have led to new insights into 1) the conserved molecular mechanisms mediating the circadian regulation of sleep and arousal; 2) how different types of neural codes impact plasticity and behavior; and 3) how sleep drive is generated, released, and persists in time. In this research program project, we will build upon our prior studies of the circadian and homeostatic regulation of sleep and develop new approaches to investigate the neurophysiology and function of sleep. First, we will address whether the temporal coding mechanisms found in Drosophila clock neurons are also conserved in the mammalian suprachiasmatic nucleus. Compared to our understanding of the circadian clock, much less is known about the homeostatic regulation of sleep. Nevertheless, we predict that the nature of neural circuits underlying sleep homeostasis will be conserved, and we will seek to identify a sleep homeostatic integrator circuit in mice. Our studies of the cellular mechanisms mediating sleep homeostasis have led us to examine the role of astrocytes in sleep behavior. We plan to determine whether sleep-regulating molecular pathways in these cells are conserved and will perform systematic investigations into the role of astrocytes in regulating neuronal physiology and behavior. To fully exploit the power of the Drosophila model for studying sleep, we will develop a new multimodal imaging method for characterizing and quantifying sleep. Our research has largely focused on how sleep is regulated, but in the future will also address the function of sleep in neural plasticity using a simple, defined circuit and new electrophysiological methods. Finally, we are extending our studies into human disorders, including studying the genetic basis of familial sleepwalking. Our goal is to not only delineate conserved mechanisms underlying sleep and its function, but also to uncover fundamental neurobiological principles governing these processes.