David M. Raizen - US grants

DESCRIPTION (provided by applicant): Impeding our understanding of behaviorally quiescent states such as sleep and their relationship to development is a lack of an animal model that has a well-understood anatomy in addition to powerful tools for genetic analysis of quiescence. Caenorhabditis elegans is a genetically tractable animal with an exquisitely well-understood neuroanatomy and has been used to model several processes of human disease relevance. A quiescent state occurs during lethargus, a developmentally controlled period during which there is extensive synaptogenesis in the nervous system. Dr. Raizen, the principal investigator, plans to investigate the processes and neurochemicals that control the timing and execution of this quiescent state. Specifically, he will test the roles of serotonin, dopamine, and adenosine in the control of quiescence and characterize the homeostatic control of this state. In Aim 2, he will test the hypothesis that the LIN-42 protein, the C. elegans orthologue to the circadian protein PERIOD, plays a role in the control of the timing of quiescence. In Aim 3, he will determine whether or not quiescence is required for synaptogenesis during development. These studies will compare and contrast behavioral quiescence in C. elegans to that seen in other animals and will answer the question: Can quiescence in C. elegans be considered a sleep like state? Regardless of the answer, the study will address fundamental problems in behavioral state control and its relationship to nervous system change. The studies will be performed in the laboratories of Dr. Allan Pack in the Center for Sleep and Neurobiology, and of Dr. Meera Sundaram, in the Department of Genetics. This training grant will support Dr. Raizen while he learns the scientific field of sleep and chronobiology and develops C. elegans as a model system for the study of behavioral quiescence and its relationship to development.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the genetic regulation of sleep and sleep-like states. Sleep-wake regulation consists of clock timing and of sleep onset and offset signals. Execution of the sleep state requires sensory gating, which refers to the phenomenon of reduced responsiveness during sleep. Sensory gating is a poorly-understood yet fundamental property of sleep that distinguishes it from quiet wakefulness. This proposal aims to advance our mechanistic understanding of sensory gating using the model organism Caenorhabditis elegans. The approach is to study lethargus, a sleep-like period that occurs during the life cycle of C. elegans. In this grant period, the global hypothesis to be tested is that EGL-4/PKG and cAMP signaling act antagonistically in sensory neurons to regulate sensory input during lethargus, and that reduced sensory input in turn facilitates sleep-like behavior. This global hypothesis will be tested through four specific aims. Specific aim 1 will test the hypothesis that the molecular mechanism that regulates sensory responsiveness during lethargus is the same molecular mechanism that regulates chemosensory adaptation. This hypothesis will be tested by assessing for an association between defects in sensory adaptation and defects in sensory gating during lethargus. Additionally, we will test whether animals adapt more readily during lethargus than outside of lethargus. Specific aim 2 will test the hypothesis that cAMP signaling acts in sensory neurons to antagonize sensory gating during lethargus. This hypothesis will be tested by expressing the gene pde-4, which normally degrades cAMP, in sensory neurons. In addition, we will assess the gene order relationship between egl-4 and pde-4 is regulating sensory gating. Specific aim 3 will test the hypothesis that sensory input regulates sleep-like behavior. This third aim will be tested by examining the effects on sleep-like behavior of dampening sensory input during lethargus and of mutants and operations that reduce sensory function. The final specific aim will perform a genetic screen to identify genes required for the enhanced sleep- like behavior in egl-4 gain of function mutants. Given the phylogenetic conservation of sleep and sleep-like states and the conservation of cGMP-and cAMP-dependent signaling pathways, it is likely that these experiments will shed light on sleep regulation in other species. Improved understanding of sleep regulation will enhance the diagnosis and treatment of people with sleep-disorders. PUBLIC HEALTH RELEVANCE: Sleep disorders and sleep deprivation are major unmet public health problems. This proposal aims to add to our understanding of sleep regulation, in order to enhance the diagnosis and treatment of patients with sleep disorders.

DESCRIPTION (provided by applicant): A major cause for age-related debilitation is the loss of motor power, which is partially explained by muscle loss or sarcopenia. Identifying interventions that slow the loss of power or frailty are of high interest because they may lead to reduced morbidity and improved quality of life. The nematode C. elegans is often used as a model biological system to study aging, and has led to fundamental advances in our understanding of the process. The C. elegans body wall muscle is analogous to human skeletal muscle in several respects including aging-associated sarcopenia/frailty. One of the few human interventions proposed to attenuate sarcopenia and improve motor function is exercise. Yet the mechanism of the beneficial effect of exercise is incompletely understood. We propose a method to study the effect of exercise on frailty in C. elegans. We will develop and characterize a kind of nematode infinity pool, and use this device to test the effect of age and exercise regime on nematode propulsive power. The device will consist of a tapered conduit filled with aqueous solution. The conduit will be subjected to pressure-driven flow directed from its narrow end. The nematode will be inserted at the conduit's wide end and stimulated with a weak DC electric field to deliberately swim upstream. The nematode's response to the electrical field (electrotaxis) is sensory and does not involve any electrostatic forces. As the nematode progresses towards the narrowest end of the conduit, the adverse fluid velocity and the corresponding adverse hydrodynamic force acting on the nematode will increase. Eventually, the nematode will arrive at an equilibrium position, at which its propulsive force will balance the viscous drag force. At its equilibrium position, the animal will swim while maintaining a nearly fixed spatial position. The conduit's width at the equilibrium position will correlate with the nematode's propulsive power. The further upstream the nematode progresses, the larger its propulsive power will be. The propulsive power will be quantified with the aid of direct numerical simulations of the flow field around the nematode. By applying the electric field at predetermined frequencies and durations, the exercise of the animal will be controlled. Experiments will be carried out to correlate the propulsive power with the animal's age and exercise regime. The device will be fabricated with soft lithography. Many conduits will be accommodated on a single substrate to enable high throughput studies. In addition to quantifying the nematode's propulsive power as a function of age and exercise regime, our method can be used, in the future, to study the effect on propulsive power of genetic nutritional, pharmacological, or other environmental perturbations.

? DESCRIPTION (provided by applicant): Despite the importance of sleep to our well-being and its impact on disease and work productivity, our cellular and molecular understanding of sleep regulation is limited. Sleep/wake regulation arises via the interaction between neurons. We are investigating the regulation of a sleep behavior during larval transitions called lethargus in the roundworm C. elegans. Lethargus shows both behavioral and molecular genetic similarities to sleep in mammals and Drosophila. Since identifying the neuronal circuit regulating sleep is the next step in the analysis of this behavior, we propose to identify sleep-regulating neurons in C. elegans. In Aim 1 we will conduct an optogenetic screen for interneurons that modulate feeding and locomotion rate, as sleep is characterized by cessation of these behaviors. In Aim 2 we will test whether candidate sleep-modulating interneurons identified in Aim 1 affect sleep-like sensory arousal thresholds and behavioral quiescence. We will also establish the order of action of sleep/wake active neurons. Our studies will make use of innovative optogenetic stimulation, optical imaging, and image processing technologies for perturbing neurons, quantifying feeding movements, measuring quiescence behavior, and measuring arousal thresholds. Our overall goal of identifying every interneuron modulating sleep/wake in an animal is a goal never before accomplished in any animal, yet it is feasible in C. elegans due to the simplicity of its nervous system. Our experiments will provide a knowledge base for mechanistic studies of genes affecting C. elegans sleep-like behavior as well as for physiological studies of sleep-regulating neurons.

? DESCRIPTION (provided by applicant): The timing of sleep behavior is controlled by a feedback loop involving expression of genes of the `molecular clock'. How this clock regulates sleep behavior is poorly understood. This project is focused on understanding how the timing mechanism regulates sleep behavior at a single neuron resolution. We use the lethargus stage in the roundworm C. elegans as a model for sleep. Lethargus is a quiescent state that shows both behavioral and molecular genetic similarities to mammalian sleep. We have identified a sleep-inducing neuropeptide called NLP-22, which is expressed in one pair of neurons, the RIAs. We will use a combination of genetic, optical, and behavioral tools to study the mechanism by the RIA neurons integrate timing molecular signals with sleep signals from other neurons and how they ultimately affect peripheral target organs. In Aim 1, we focus on understanding the role of the RIA neurons in sleep regulation. In Aim 2, we investigate the mechanism by which NLP-22 induces feeding quiescence. And in Aim 3 we use a discovery approach to identify new somnogenic signals. Our project makes use of the powerful genetic and optical tools available in C. elegans to illuminate the molecular mechanism of sleep regulation within a defined neural circuit.

PROJECT SUMMARY The complaint of fatigue is common among patients receiving radiation therapy (RT) for the treatment of cancer, yet the mechanism of this fatigue is unknown. Many patients report drowsiness with RT, suggesting that sleepiness is contributing to their complaint of fatigue. But drowsiness, like fatigue, is a subjective complaint, which may not correlate with objective measurements of sleepiness and activity. Identifying objectives correlates of fatigue, including sleepiness and reduced activity, will be critical to advancing our understanding of underlying mechanisms. Understanding these mechanisms can lead to new therapies. The studies proposed in this Exploratory/Developmental Research Grant (R21) project will fill a gap in our understanding of the biological mechanisms of fatigue associated with radiation therapy for cancer. The hypothesis motivating this proposal is that sleepiness, reduced activity, and impaired vigilance associated with EGF cytokine elevation contributes to the fatigue experienced by patients receiving radiation therapy for prostate cancer. To test this hypothesis, we plan to measure before, and during RT the following: (1) Levels of the cytokine HB-EGF, (2) activity, (3) subjective sleepiness, (4) objective sleepiness, and (5) fatigue. We will make the same measurements also in a cohort of subjects with prostate cancer who are not treated with RT. All measurements except for objective sleepiness will be repeated at least three months after RT is completed. In each of these five measurements, we hypothesize a change on RT from baseline values. We will test our over-arching hypothesis that the pathway leading to the complaint of fatigue on RT treatment involves an elevation of HB-EGF, reduced activity, and increased sleepiness.

PROJECT SUMMARY/ABSTRACT The high prevalence of coexistent sleep and metabolic disorders suggest that these processes are integrated at the molecular level, but mechanisms of this integration are unknown. The recent finding that the AMPK family member SIK3 is a phylogenetically conserved sleep drive regulator combined with our preliminary data showing both reduced sleep and elevated energy stores in animals mutant for the C. elegans SIK homolog kin- 29, suggests that SIKs are key nodes connecting sleep and energy homeostasis. The model motivating this proposal is that SIKs are responsive to the energy level in particular neurons; low energy (i.e. low ATP levels) result in the movement of SIK into the nucleus where, via phosphorylation of a class II HDAC it de-represses genes that signal to promote sleep and energy reserve mobilization. We will test this model using the nematode Caenorhabditis elegans and with the following hypotheses: (1) Cellular energy charge is lower under conditions of increased sleep drive. (2) KIN-29/SIK signals under conditions of low energy to mobilize energy stores and restore cellular ATP levels and sleep. (3) KIN-29/SIK functions acutely in metabolically-responsive sensory neurons that regulate the sleep-inducing ALA and RIS neurons; It functions in the same neurons to regulate fat stores. (4) KIN-29/SIK sleep-promoting activity is controlled by nuclear import, which is regulated by the upstream kinases LKB1 and PKA. Finally, (5) we will pursue an exploratory aim by performing a pilot genetic screen to discover new genes that are required for the reduced sleep phenotype of kin-29 mutants. Experiments in aims 1-4 will illuminate the molecular and cellular mechanism by which SIKs function to regulate animal sleep and energetic stores. Aim 5, in which we will identify new sleep genes, will provide a bridge into the next set of hypotheses regarding mechanisms of sleepiness. Lessons gained from the nematode can motivate focused experiments in mammals, and will inform our understanding of patients with disorders of sleep regulation.

Project Summary In the nematode C. elegans as in other animals, sickness is associated with reduced movement, reduced feeding, and increased sleep. Sickness behavior in worms is induced by environmental stressors including heat shock, ultraviolet light, and infection. This behavioral program during sickness is regulated by worm central neurons that are activated by the cytokine epidermal growth factor (EGF). EGF causes reduced activity in worms, flies, fish, and mice, but the mechanisms of EGF activation itself during sickness are not clear. We will use C. elegans to study the mechanism of EGF regulation during sickness. In Aim 1, we will determine where (from which cells) and when EGF is released to promote sickness behavior. To identify other regulators of EGF activation, in Aim 2, we will perform genome-wide discovery screens for genes required for sickness behavior. In Aim 3, we will test the hypothesis that sickness behavior supports survival during sickness. A long- term goal of these studies is to identify candidate signaling molecules for developing diagnostics and treatments of sleepiness during sickness and to understand the importance of sleep in promoting recovery from illness.

ABSTRACT This proposal is for a training program in the area of sleep and circadian research and the related disorders. There is growing evidence of the prevalence of sleep disorders in the American population, and that problems related to inadequate sleep have a major impact on many aspects of our society. At a basic level, little is known about the fundamental mechanisms that control sleep and the function(s) of sleep. Thus, there is a major opportunity for scientific discovery. One of the barriers that is recognized to advancing the knowledge base in this area is the paucity of investigators, both those engaged in basic research and in patient-oriented research. This application describes a training program that is based on the relatively unique faculty resources and structures at the University of Pennsylvania for support of research in sleep and its disorders. The proposal describes specific training aspects that are intended to complete the matrix for training opportunities at the University of Pennsylvania (Penn) in the area of research in sleep/sleep disorders. These aspects are the following: research training for graduate students. This is based on training provided by 3 graduate groups at Penn. Each graduate program has a similar structure, albeit with different required coursework. The graduate groups are: a) the Neuroscience Graduate Program. This is the Graduate Group that has been involved in this program since its inception. We will utilize, where appropriate, structures, courses and other resources developed by this group; b) a graduate track in genomics/computational biology; c) graduate group in cell and molecular biology that offers our graduate students training in areas such as metabolism, genetics, and epigenetics; d) a targeted MD/PhD program to train physician-scientists in sleep/circadian research. This aspect of our program will be based on the outstanding institutional MD/PhD program at the University of Pennsylvania. We also have a postdoctoral training program for nurse investigators. This will be based on the preeminent School of Nursing at the University of Pennsylvania. The strong, well-established collaboration between the School of Medicine and the School of Nursing in this area provides a unique opportunity to develop a much needed national program to train nurse investigators in this area. All of these components of the program will utilize the extensive resources for research in sleep/circadian that have been developed at the University of Pennsylvania.