Allan I. Pack, MD, PhD - US grants

respiratory center dysfunction; respiratory airflow disorder; REM sleep; sleep disorders;

This Program represents a renewal application to be a Special Center of Research for Cardiopulmonary Disorders in Sleep. An important goal of the basic science studies of the SCOR is to establish the neural mechanisms that lead to hypotonia of the upper airway during sleep. It is our view that pharmacological therapies for sleep apnea are most likely to come from elucidating these basic mechanisms. These neurobiological investigations involve complementary studies using electrophysiological techniques (Project 2), neuroanatomical approaches (Project 1),and studies in chronic, normally sleeping animals (Project 4). These studies are first addressing (Project 2) the overall hypothesis that disfacilitation is the major mechanism for reduction in motor activity in the upper airway during sleep. Attention is directed at the aminergic systems in the brainstem. Studies will be done to examine the role of the different relevant neurotransmitters. Such studies direct attention to complex neural systems such as the caudal raphe. In Project 1, the neurochemical and neuroanatomical organization of this system will be studied using a variety of techniques-tract tracing, double-labeling, immunohistochemistry, and receptor autoradiography. Apneas in REM sleep are associated with the phasic events of this state. In Project 4, we propose that an area in the rostral pons, the pedunculopontine tegmental nucleus (PPT), mediates these phasic respiratory phenomena and a series of studies are elucidated to test this postulate. These basic investigations lead to studies in the animal model of sleep apnea that we identified (Project 3), in which we will study neuromuscular adaptations to the presence of a chronically obstructed upper airway. This project is based on the premise that upper airway obstruction leads to an adaptive increase in activity of upper airway dilator muscles in wakefulness that prevents airway obstruction and results in secondary changes in the muscles themselves. As part of the studies being conducted in Project 3, novel imaging techniques, that we have pioneered, will be used. Efficacy of CPAP will be addressed in Project 5, but here the emphasis will be on patterns of use and impact on daytime sleepiness. Our previous studies have raised concerns that many patients prescribed CPAP are functioning at sub-optimal levels. The results of the studies proposed here are likely to have a major impact on how sleep apnea patients are managed. Thus, this is a comprehensive program incorporating both basic neurobiological and clinical research.

DESCRIPTION: (Adapted from Applicant's Abstract). Insomnia is a common complaint in elderly subjects. There is general agreement that optimal management of the problem should be directed at the underlying cause. But currently there is no universally agreed on approach, so that elderly with this complaint are commonly simply treated with sedatives/hypnotic medications. This application is based on the belief that a conceptual framework is needed to tackle insomnia that is based on physiology. It is hypothesized that age-related changes in sleep/circadian systems in the elderly are the core of the problem. Such changes can produce insomnia on their own and augment the effect of sleep disorders that interrupt sleep (apnea or myoclonus) or somatic disorders (pain, chronic cough, etc.). This provides a testable set of hypotheses that have important treatment implications. These concepts lead to a case-control study of insomnia which is ideally suited to the proposed multi-factorial origins of the complaint. In this study, major streams of investigation in this area will be united, an epidemiological approach, based on self-report of symptoms and physiological study of age-related changes in sleep in small numbers of subjects, often with no complaints of insomnia. In our study we will identify controls as those with no or rare trouble sleeping in the previous one year, while cases will have current, frequent trouble sleeping that is at least of three weeks duration. The status of case-controls will be confirmed by objective testing so that cases and controls can be analyzed with only subjective definitions or with both subjective and objective definitions. Individuals with depression will be excluded by a careful two-stage screening process since depression is a risk factor for insomnia and insomnia is a risk factor for depression. This complex interaction will confound our analysis. Measures include: sleep characteristics, the 24 hour temperature and melatonin rhythms and the response to sleep deprivation. The plans will specifically address the following questions: 1) what is the relative role of phase advance of the biologic clock, low melatonin levels and decreased sleep process(s) in the genesis of insomnia?; 2) are sleep apnea and nocturnal myoclonus risk factors for insomnia in the elderly?; and 3) do age-related changes in the sleep/circadian system augment the effect of sleep apnea, nocturnal myoclonus, and somatic disorders that can interrupt sleep in terms of the risk of insomnia? The approach is aimed at the development of a new diagnostic framework for insomnia and new treatment approaches.

Major changes take place in the output to respiratory pump and upper airway muscles in association with the phasic events of REM sleep. These phasic changes have adverse consequences and initiate apneas in REM sleep. The overall goal of this proposal is to investigate the neural mechanisms producing these phasic respiratory phenomena. Our attention is directed to the pedunculopontine tegmental (PPT) nucleus in the pons. Lesions of this area abolish ponto-geniculo-occipital (PGO) waves that occur phasically in REM sleep. PGO waves are temporally linked to the phasic changes in respiration in REM sleep. Our goal is to determine the role of neurons in this area in generation of the phasic respiratory phenomena in REM sleep. The first protocol involves lesioning the area by neurotoxic chemicals and examining the effect on PGO waves and respiratory phasic phenomena. If, as we postulate, this area is critical to generating the phasic-changes in respiratory motor output, they should be abolished or markedly reduced in frequency by such lesions. PGO waves and alteration in diaphragm output can also be produced by alerting stimuli; this has led to the concept that there is endogenous activation of the startle system in REM sleep. Thus, in this protocol we will also determine whether lesions of the PPT abolish the PGO and respiratory response to alerting stimuli. In a second series of studies, we will apply complimentary techniques. These will be based on manipulating the neurochemical environment of the region. There are different theories as to what modulates burst neurons that generate PGO waves. One theory implicates serotonin from the dorsal raphe, another serotonin and noradrenaline from locus coeruleus, while a third proposes that GABA from substania nigra plays the major role. The studies we propose will directly test each of these hypotheses. In each case we will administer antagonists in non-REM states to see if we can induce PGO waves and associated phasic respiratory phenomena. Agonists will be administered in REM sleep to see if PGO wave production and phasic respiratory changes can be blocked. Such studies will both enhance our understanding of the role of this area in generating phasic respiratory phenomenon and the fundamental mechanism controlling these phasic generators. Activity of these neural generators must be relayed to the respiratory motor outputs. There are both ascending and descending projections from the area. In order to begin to establish which of these is likely to mediate the respiratory effects, we will, in a third series of protocols, study the effect of interrupting the descending projections on phasic respiratory events. Because activity of neurons in the PPT may also be affected by sleep disruption, the final protocol will address whether a vicious cycle exists whereby interruption of REM sleep leads to more phasic respiratory phenomena. These studies are, therefore, a comprehensive investigation of the mechanism generating phasic respiratory change in REM sleep, their neurochemical control and their relevance to sleep apnea syndrome.

The Computer and Electronics Core is an integral part of this SCOR program. It provides support to all the projects of the SCOR and to the Human Assessment and Biostatistics Core (Core C). The Core fulfills the following roles: a) development of specialized software; b) support and training of users in use of specialized, custom-made software and specialized packages; c) provision of continuing maintenance of laboratory devices; d) provision of bioengineering liaison; e) new research to develop new technology to support the SCOR's goals. The Core has an experience computer analyst as it major staff member who is thoroughly familiar with the projects of the SCOR and has been involved in developing technology that makes our projects feasible. These include, in particularly, techniques for sophisticated image analysis that are used in Projects 01 and 03 and techniques for on-line monitoring for sleep in Project 04. The Core works closely with the Human Assessment and Biostatistical Core in support of the human clinical projects (01 and 05).

Sleep apnea is associated with excessive daytime sleepiness and hence an increased risk of vehicular crashes. The basic mechanism underlying this sleepiness is unknown. Currently it is proposed that increasing sleepiness results from accumulation of molecules that promote sleep. While several such molecules have been identified, perhaps the clearest evidence is for adenosine. We do not known, however, how adenosine levels in critical brain regions are regulated in relation to the sleep/wake cycle nor whether there is within the brain a regional specificity to this regulation. This proposal is based on the fundamental notion that regulation of enzymes involved in adenosine metabolism (and/or nucleoside transporters) in relationship to the circadian system and to sleep homeostasis play a major role in setting adenosine levels in relation to sleep need. This postulated mechanism provides a powerful, novel method to achieve interaction between the sleep and circadian systems. This hypothesis will be addressed in a complementary series of studies with investigators at two universities (University of Pennsylvania and University of Manitoba) who have complementary skills. In one series of studies, we will directly measure total adenosine levels in several different brain regions relevant to sleep, and how these levels changes across the day and following different durations of sleep deprivation. In other studies, we will determine the diurnal changes in the relevant enzymes (adenosine kinase, adenosine deaminase, and 5'-nucleotidase) in brain regions relevant to sleep. We will study the relative role of the circadian and sleep homeostatic system in mediating such changes. To address how the adenosine enzymes might themselves be regulated, we will address, in another series of studies, whether changes in enzyme activity are correlated with alterations in mRNA abundance for the relevant enzymes. mRNA analysis will also be used to assess whether similar temporal changes occur in nucleoside transporters. All of these studies will be done in rats. Our hypothesis would predict, however, that animals with different diurnal distributions of their sleep/wake cycles will have different diurnal variations in adenosine and its enzymes in brain regions relevant for sleep control. Such an animal is the Octodon degus and we will address in our final protocol whether there is predicted differences in the diurnal changes in adenosine and its enzymes of metabolism in this species as compared to rat. Taken together, these studies will provide a comprehensive picture about how brain adenosine is regulated in relation to sleep need.

This study examins the effects of an experimental drug, Modafinil, in the treatment of Narcolepsy. The study is being carried out in approximately 26 sites around the US. Subjects with a history of Narcolepsy are screened to assume absense of other significant medical problems and drug-free status. They are then brought into the CRC and sleep lab for overnight sleep studies and daytime tests to confirm the diagnosis of Narcolepsy and to establish the level of baseline functioning. Subjects are then treated in a nine week, double-blind, placebo-controlled paradigm with Modafinil, 200mg or 400mg. Follow up studies of sleep and daytime functioning are carried out at two other times during the protocol.

This project is directed at changes in the sleep-promoting system with age. The investigator is an established investigator who is beginning a new line of investigation into the changes with age in molecular mechanisms that promote sleep. Sleep is thought to be determined by the interaction between two processes-the circadian process and the sleep- promoting process. The later (process S) increases in relationship to the duration of prior wakefulness. There is considerable evidence that the strength of this sleep-promoting process declines with age. Changes in this may, at least in part, play an important role in the high prevalence of insomnia in the elderly. Sleep promotion is thought to be the result of accumulation during wakefulness of molecules that promote sleep. Among such molecules, cytokines have a prominent role. Both interleukin-1 (IL-1) and tumor necrosis factor (TNF) enhances sleep and increases in the levels of these molecules and their mRNA occur with sleep deprivation. Currently we do not know, however, whether changes in this sleep-promoting system occur with age. Thus, one of the goals of our studies is to determine, as we predict, the increase in IL-1 and TNF with sleep deprivation is less in older as compared to younger rates in relevant brain regions. It is unlikely, however, that IL-1 and TNF are the only cytokines involved. There is powerful positive feedback between these prototypical "pro-inflammatory" cytokines. Thus, there must be other counter-regulating cytokines involved. We predict that IL-4, IL-10 and IL-13, and possibly IL-16, will be unregulated in relevant brain regions with sleep deprivation and act to limit the increases in IL-1 and TNF. We will determine whether this is so. Thus, this pilot project is designed to assess whether changes in cytokine response to sleep deprivation occur with age and to assess more globally the various cytokines that are involved. The results of this pilot project will allow us, in the future a more in-depth study of changes in the brain cytokine response to sleep deprivation.

This SCOR in Sleep Neurobiology of Sleep and Sleep Apnea is focused on understanding the pathogenesis of sleep apnea and its consequences and in improving treatment for this common disorder. It is estimated that obstructive sleep apnea affects 2-5% of middle-aged adults and produces both neurobehavioral (excessive sleepiness) and cardiovascular consequences. There are several components to the pathogenesis of the disorder. First, there are structural risk factors that reduce the size of the upper airway even in wakefulness. In Project 01 (Richard Schwab, M.D.) we argue that there several structural risk factors that are likely to have an important genetic basis. We address this in a case control study in which we also study sibs of probands and controls. Imaging techniques are used to identify intermediate traits and we week family aggregation of such traits that are not explained by shared environmental risk factors. A second component of the pathogenesis is the neural mechanism that leads to hypotonia during sleep. We propose that two brainstem systems are critical, i.e. the serotonergic and noradrenergic neurons. The former, we propose, plays a critical role in mediating the reduction in motor done during sleep, while the latter plays a large role in mediating changes in the effect of afferent inputs and hence in apnea termination. This proposed relative role of these two neural systems is addressed in Project 02 (Leszek Kubin, Ph.D), while in Project 03 (Joan C. Hendricks, V.M.D.., Ph.D.) we address whether manipulating the serotonin system will increase the motor tone of the airway and hence represent a pharmacotherapy for the disorder. While these studies address the pathogenesis of the disorder, in Project 04 (Allan I. Pack, M.D., Ph.D.) we turn our attention to arguable the most important consequence of the disorder, sleepiness. Here we propose that adenosine could play a major role and we address how adenosine levels might be controlled at a molecular level in different brain regions in relation to sleep need. In the final project of the SCOR, we address shorter-term clinical needs and address, using new technology and a concept based on assessment of efficacy, the most important management problem for this disorder, the high rates of non-adherence to therapy (Project 05; Terri E. Weaver, Ph.D., R.N.). These five projects are supported by three cores (Administrative, Computer, and Human Assessment and Biostatistics Cores).

sleep apnea; sleep; disease /disorder proneness /risk; sleep disorders; circadian rhythms; human old age (65+); case history; clinical research; human subject;

To conduct a study which will obtain a relatively precise estimate of the prevalence of sleep apnea in a population of commercial motor vehicle operators who are at risk for sleep apnea. To determine whether the drivers with sleep apnea are more likely to be impaired than those without sleep apnea. In addition, we will endeavor to develop a cost effective way of screening for obstructive sleep apnea and driving impairment.

Excessive sleepiness that interferes with daytime functioning is a common complaint in the elderly. The case-control study of excessive sleepiness in the elderly, that we have been conducting, shows that sleep apnea is an important risk factor for excessive sleepiness. In sleep apnea syndrome there are repeated breathing episodes, i.e., pauses or decrements, during sleep that interrupt sleep and lead to daytime sleepiness. Specifically, we find that individuals with more than 15 such episodes per hour have a statistically significant increased risk of sleepiness, and this risk increases with more such episodes per hour. Our study suggests that about 6 percent of the elderly might therefore benefit from treatment for sleep apnea syndrome. While there are treatments with known efficacy that have been used, and studied, in middle-aged populations, we know nothing about how well such treatments might work in the elderly. This is primarily because elderly with excessive sleepiness seek clinical evaluation for this symptom less frequently. Thus, studies of sleep apnea in the elderly cannot be based on clinic populations. The major current treatment for sleep apnea is use of pressurized air applied through the nose to maintain the airway open (continuous positive airway pressure, CPAP). There are problems with adherence to this cumbersome therapy but we know nothing about adherence in the elderly. We, therefore, propose an intervention study in which we compare, in elderly individuals with sleepiness and documented sleep apnea, the effects of treatment with CPAP and no therapy. Subjects will be randomly assigned into these treatment arms. Since elderly subjects with sleepiness do not commonly seek help at clinical facilities, we will employ a community-based recruitment strategy using lists of individuals in Medicare in geographic regions close to our Sleep Center. This will provide us with the opportunity to determine the efficacy of the case finding strategy that we propose. Particular attention will be paid to recruitment of minority populations, specifically African- Americans. The goals of this study will therefore be the following: evaluating our ability to identify and recruit from the community elderly subjects with problems with sleepiness who have sleep apnea syndrome; evaluating the effect of treatment of sleep apnea on activity of daily living and other outcomes; and determining the patterns of use of CPAP in comparison to published data in middle-aged subjects. These data will give novel definitive information both about identification and treatment of sleep apnea in the elderly.

human therapy evaluation; central nervous system stimulants; nervous system disorder chemotherapy; drug screening /evaluation; narcolepsy; functional ability; circadian rhythms; sleep; clinical trials; clinical research; human subject;

disease /disorder etiology; epidemiology; sleep disorders; aging; human old age (65+); sleep apnea; physiology; myoclonus; circadian rhythms; melatonin; clinical research; human subject; electroencephalography;

PROPOSED PROGRAM (Adapted from the Applicant's Abstract) The theme of this application is examining gene/environment interactions in rodent models of human disease using cDNA microarrays to link phenotype to genotype. Disease phenotypes arise from complex interactions of organisms with their environments. While the investigators have a long history of associating genes and gene defects with a large array of disease phenotypes, a growing body of data suggests that many disease phenotypes arise from the interactions of genes with their environments, including the genetic background in which the genes are expressed. The goal is to begin an exploration of these interactions using rodent models of human disease and cDNA microarray assays to elucidate patterns of gene expression. This PGA brings together biologists, statisticians, computer scientists, engineers, and physicists who are will lend their expertise to the achievement of the common goals. The proposal builds on existing expertise at TIGR in the analysis of gene expression using cDNA microarrays, the mouse mutagenesis and phenotyping programs that have been developed by The Jackson Laboratories and their collaborators at Penn, Duke, and Boston Universities, and the efforts in rat genomics underway at the Medical College of Wisconsin, including the generation of phenotypically characterized consomic and congenic rat strains. Linking these programs are coordinated efforts in informatics that will both facilitate data exchange between consortium members and will make that data easily accessible to the wider research community. Underlying this is a commitment to continuing to develop and improve the reagents and assays to provide a firm statistical basis for any inferences that the assays provide. Finally, the investigators maintain a commitment to community service and will provide reagents, software, and data generated as part of this PGA to the wider research community. The investigators will develop high-density mouse and rat cDNA microarrays and use these to characterize gene expression in mouse and rat models of heart, lung, and blood diseases as well as sleep disorders. The rodent models to be surveyed will be ascertained by measuring phenotypic response of congenic, consomic, and mutagenized animals to environmental challenges known to elicit responses relevant to these diseases. Gene expression data will be integrated with genotype and phenotype data and analyzed to begin to develop a more complete understanding of the mechanistic basis for these diseases as well as the modifying and mitigating factors contributed by the environment.

? DESCRIPTION (provided by applicant): This proposal is for a training program in the area of sleep and its 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 structure at the University of Pennsylvania for support of research in sleep and its disorders (the Center for Sleep and Circadian Neurobiology). The proposal describes four specific training aspects that are intended to complete the matrix for training opportunities at the University of Pennsylvania in the area of research in sleep/sleep disorders. These four aspects are the following: a) a 5-year graduate program in neurobiology of sleep and circadian rhythm. This will be based on the highly successful and well developed Neuroscience Graduate Program. We will utilize, where appropriate, structures, courses and other resources developed by this group; b) a 5-year graduate track in genomics/computational biology; c) a targeted MD/PhD program to train physician-scientists in sleep research (this component is 5 years). This aspect of our program will be based on the outstanding institutional MD/PhD program at the University of Pennsylvania; and d) a 2-year 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 Sleep Center and the School of Nursing 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 that have been developed by the Center for Sleep and Circadian Neurobiology at the University of Pennsylvania.

sleep apnea; human therapy evaluation; psychic activity level; central nervous system stimulants; drug screening /evaluation; chemotherapy; respiratory therapy; clinical research; human subject;

This program of research is directed at elucidating the fundamental mechanisms which lead to alterations in the circadian and sleep promoting system with age. Such changes with age contribute to the high prevalence of sleep disorders in the elderly. We propose to address this issue at the most fundamental level and determine the age-related changes in the molecular mechanisms for these processes. Studies will both address changes with age in known processes, as well as address hypotheses to further our understanding of the mechanisms with age in known processes, as well as address hypotheses to further our understanding of the mechanisms involved. We plan studies in both Drosophila and rats. The former are studied since knowledge of the molecular basis of the circadian block is more complete, and there is growing evidence for an analogy of their rest-like period and sleep. Rats are used since the data on the neuronal circuitry are most complete, and we will investigate molecular mechanisms within the framework of the circuitry involved. In Project 1, we plan to study changes in the molecular mechanisms of the circadian clock in Drosophila. Studies will involve both investigating in vitro and in vivo, using new transgenic technology, alterations with age in the circadian rhythm of expression of the period and timeless genes. These studies will also investigate control of the rest period in Drosophila as an analog of sleep. The effect of manipulating the adenosine system pharmacologically on rest/activity will be studied. Adenosine, a sleep promoting molecule will be the major focus of Subprojects 0002 and 0002 which will be conducted in rats. Using unique facilities, we will study the levels of adenosine in key brain regions in young and old rats and address whether adenosine changes with age and/or in relationship to the duration of prior wakefulness. Adenosine levels are controlled by a complex metabolic system. Enzymes and transporters for adenosine could themselves change with age and/or diurnal effects. This will be addressed in these two projects by studying changes in the various components of the adenosine system t a molecular level Subproject 0002 and how changes in these components alter adenosine in the brain regions relevant to sleep (Subproject 0002). Thus, this Program Project is a comprehensive, integrated series of novel studies to address the changes with age in the sleep and circadian system at a fundamental level. The projects are supported by two cores- an Administrative and Rodent Behavioral and Assessment and Biostatistical Core. Common behavioral assessment protocols and biostatistical approaches facilitate a unique approach for this program of research.

DESCRIPTION: (provided by applicant) There is family aggregation of obstructive sleep apnea (OSA) as has been shown in the United States, Europe and recently in Iceland. Iceland represents a unique opportunity for genetic research. It is a community that was settled by founders in the 9 th Century, and has developed in relative isolation since that time to its present size of 285,000 persons. Moreover, there is a commitment to record keeping that has allowed deCODE Genetics, who are collaborators on this grant, to develop a computerized genealogy data base that permits the ancestry of individuals to be traced over centuries. This tool, together with the founder nature of the population, makes possible a unique genealogy-driven approach to study the genetics of complex disorders, an approach that has already been successful. We propose in this application to study the genetic basis of the common disorder---obstructive sleep apnea--using this genealogical approach. The study will be built on patients with the disorder, who have already been diagnosed in Iceland and where large family pedigrees have been identified. The proposed study involves a genome-wide family linkage investigation. This will be conducted with an affected only approach examining allele sharing between affected individuals using 1,100 markers spaced across the genome. We plan to oversample the relatively non-obese subjects providing us the opportunity to evaluate linkage in both relatively non-obese and obese subjects. The linkage study will be complemented with an association study, with unrelated cases and controls, matched for age, gender, and menopausal status. In the association study, we will, as a primary aim, test candidate genes arising from the linkage study and, as a secondary aim, evaluate candidate genes that we believe will be identified in the ongoing Cleveland Family Study. A subset of subjects in both the family linkage and association study, will have in-depth phenotyping to determine whether there are sub-phenotypes for this complex disorder and, if so, whether they aggregate in families. This in depth phenotyping will involve upper airway magnetic resonance imaging to evaluate upper airway soft tissue and craniofacial structures, acoustic rhinometry to quantify nasal resistance, a known risk factor for the disorder, and insulin resistance. We will explore whether there are distinct patterns of linkage for the different sub-phenotypes. To accomplish this large genetic study, we have put together the resources of three major organizations--the University of Pennsylvania, the University of Iceland Hospitals, and deCODE Genetics. We propose to leverage the truly unique infrastructure developed by deCODE Genetics, the clinical research programs in sleep apnea at the University of Iceland Hospitals, and the in-depth phenotyping expertise at the University of Pennsylvania to accomplish our goals.

Our major hypothesis is that regulation of sleep and wakefulness is coupled to regulation to cerebral energy stores and production of ATP. At the onset of wakefulness, we propose mechanisms are activated to increase energy supply and production and meet the increased energy demands. With continued wakefulness, changes at the transcriptional level decrease energy production and reduce ATP levels. As a result, adenosine accumulates and promotes sleep. This intuitively appealing idea has until recently been lacking experimental evidence, but preliminary data that we present supports this concept. In Specific Aim 1, we will test our hypothesis in both mouse and Drosophila with regard to the regulation of glycogen storage. We believe that early in wakefulness, the regulation of the activity of both the degradative and synthetic enzyme for glycogen (glycogen phosphorylase GP and glycogen synthase GS) are regulated in a coordinated way by phosphorylation to increase glycogen degradation and enhance energy supply. With more prolonged wakefulness, regulation at the transcriptional level alters the mRNA for both genes (GP, GS) as well as for protein targeting to glycogen, such that less glucose is made available from glycogen. In Specific Aim 2 we will broaden our evaluation of other sources of energy and of ATP-producing molecules. This aim is based on a hypothesis derived from microarray studies conducted by us. We propose that there is coordinated up-regulation of genes involved in the production of ATP, but this upregulation is only transient, returning towards baseline after a few hours. We will combine the high-throughput technology of real-time RT-PCR to evaluate, in multiple brain regions, specific candidate genes in relationship to sleep/wakefulness as suggested by our microarray studies. We will also quantify the protein products of selected genes and will study enzyme activity for a key mitochondrial enzyme that we have found is altered during wakefulness. These studies thus include comprehensive gene profiling; extensive temporal profiles; spatial localization within the brain; and studies of molecular regulation from the transcriptional to the post-translational level. These studies will be conducted in mice (Specific Aim 2). Finally, we will test the hypothesis that the transcriptional regulation of energy genes is mediated, at least in part, by the transcription factor cyclic-AMP response element binding protein (CREB). In Specific Aim 3, we will determine whether the "sleepy" Creb-deficient mice have deficits in expression of relevant energy genes that temporally correlate with their recently demonstrated inability to stay awake. Together with the results from Project 04, we will begin to establish whether--and how--the dynamic regulation of these genes could be part of the signaling mechanism for sleepiness and sleep promotion.

bioengineering /biomedical engineering; computer program /software; computer system design /evaluation

The overall question that motivates this SCOR in Neurobiology of Sleep and Sleep Apnea is the question of differential sleepiness in patients with obstructive sleep apnea. There is a marked disparity in the degree of sleepiness between patients with similar degrees of respiratory disturbance during sleep. Individuals are found with severe sleep apnea who are not excessively sleepy during the day and have limited benefit from therapy. Differential sleepiness is more marked in those with mild to moderate sleep apnea, a condition which affects millions of Americans. This issue is, hence, of fundamental import to the major clinical questions--who will benefit from treatment of sleep apnea, and how can such individuals be identified? To address this question, we propose a comprehensive, coordinated set of projects. Project by Weaver addresses differential susceptibility in patients with sleep apnea. We consider whether differential sleepiness is the result of the following: co-morbidities, in particular obesity; variations in sleep duration; night-to-night variability in respiratory disturbance during sleep. Fundamental is the concept that differential susceptibility reflects biological differences between individuals with respect to the robustness of their sleep promoting system (sleep homeostasis) in a similar way to trait differences between normal individuals in the response to sleep deprivation. Our hypothesis is that sleep homeostatic differences will be, in large part, genetically determined, based on recent observations of the genetic determination of sleep homeostasis in mice. Hence, we propose a study evaluating the heritability of sleep homeostasis in humans using a design based on monozygotic and dizygotic twins (Project by Kuna). These human projects are complemented by two projects in mice to evaluate the genes involved in determining sleepiness and sleep homeostasis, that will provide candidate genes for likely future studies of the mechanisms of differential sleepiness in humans. Project by Pack addresses whether there is coordinated regulation of genes involved in ATP production, in part mediated by cyclic-AMP response element binding protein (CREB), with wakefulness and sleep and whether the dynamic nature of this regulation is likely part of the signaling mechanisms for sleepiness. Complimentary studies are proposed at the mRNA, protein and enzyme activity levels, and the issue of site-specific regulation in brain is addressed. The final project Abel more directly examines the role of CREB and determines, using immunohistochemical and mutant mice with reporter genes, the time course and spatial distribution of CREB activation in relationship to sleep/wake, the role of noradrenaline in this process, and likely CREB target genes using both microarray approaches and hypothesis-driven investigation. These four closely interconnected projects are supported by three Cores.

Description (provided by applicant): This application is for renewal of a postdoctoral training grant that is focused primarily on sleep/circadian biology and sleep disorders, in particular sleep-disordered respiration. Sleep disorders are increasingly recognized as common disorders that have a significant morbidity and even mortality. Sleep apnea results in excessive daytime sleepiness and an increased risk of vehicular crashes; it is also associated with an increased risk of hypertension, stroke and myocardial infarction. Although sleep disorders are very common, there is limited knowledge about the basic mechanism of disease or about outcomes of therapy. This is largely related to the paucity of investigators in this area. This has been documented in the recent Institute of Medicine report, Sleep Disorders and Sleep Deprivation: An Unmet Public Health Problem. Currently, however, few institutions have a critical mass of investigators in this area to mount a training program. There is, at the University of Pennsylvania (Penn), a large number of established investigators in different departments studying sleep and its disorders. Penn has the largest number of research grants in sleep and its disorders in the United States. These investigators are both in basic science and in patient- oriented research. They have, and are, collaborating in programs that include this training program, a new Program Project and a Special Center of Research (SCOR) in Neurobiology of Sleep and Sleep Apnea. Thus, this group has the ability to mount a major training program to meet what is perceived to be a major national need. The training program, which is a three year program, has the following three major tracks: a) basic science; b) translational research; and d) patient-oriented research. In each track trainees are given the opportunity to do relevant course work and some will pursue a Master's degree. Much of their research training is research conducted under a mentor. The program uses a group mentorship strategy with a mentorship committee put in place for each fellow to oversee the training program and provide advice to the trainee and primary mentor(s). The training program proposed takes advantages of many of the highly established academic entities and training mechanisms at Penn. The training program includes structures to help with grant writing and provides career counseling. There is an active minority recruitment program with minority investigators collaborating with the Program Directors in this effort.

gene environment interaction; sleep; twin /multiplet; circadian rhythms; clinical research; human subject;

sleep apnea; epidemiology; human old age (65+); patient oriented research; human subject; clinical research;

epidemiology; sleep disorders; human old age (65+); pathologic process; psychological aspect of aging; clinical research; human subject;

ABSTRACT The Administrative Core provides infrastructure support for overall direction of the programs, organization of external and internal advisory committee meetings, and appropriate fiscal management of the program. The Core has the following functions: 1) Oversight of Research Program: This will include monthly meetings of the Steering Committee of all investigators. There will be an Internal Advisory Committee of relevant established investigators in Philadelphia who will meet regularly with the Program Director and project faculty and staff. In addition, this Core will establish an External Advisory Committee and there will be an annual site visit to review progress. 2) Preparation of Manuscripts/Maintenance of Records: This Core will be responsible for preparation of manuscripts and for maintaining all records relevant to this Program Project. The Core will also assist investigators in preparation of audio-visual material, slide presentations, etc. 3) Seminar Series: The Core will organize a seminar series with outside speakers. The names of speakers will be suggested by faculty in the Program Project. The goal of this seminar series is to keep faculty in the Program Project current, both with respect to new concepts, and also with respect to new experimental approaches. 4) Financial Management of the Program: This Core will be responsible for financial management of this Program Project. It will maintain financial records, do all purchasing and provide investigators with monthly reports about expenses from their component of the program. 5) Liaison with Other Entities: The Core will facilitate smooth interaction between the Program Project and other relevant entities at the University of Pennsylvania. 6) Liaison with Research Training Efforts: We have a major commitment to research training for the next generation of investigators. The Administrative Core will facilitate interaction between the programs of research being conducted as part of this Program Project and the postdoctoral and junior faculty research training efforts. 7) Data Sharing/Sub-studies and Ancillary Studies. The Core will oversee management of data sharing and the ability to do sub-studies and ancillary studies. A specific oversight group will be established for this purpose.

DESCRIPTION (provided by applicant): This proposal outlines a program to train physician scientists and Ph.D. scientists in genetic/genomic approaches to lung disease. The program builds on the substantial resources in this area at the University of Pennsylvania (Penn). These include the following: the recently established Center for Applied Genomics;the recently established Center for Genetic Analysis of Complex Traits;the Penn Genomics Institute;Penn Center for Bioinformatics;Institute for Translational Medicine;the recently funded Clinical and Translational Science Award;the Center for Sleep and Respiratory Neurobiology;the vibrant research programs of the Pulmonary, Allergy and Critical Care Division at the Hospital of the University of Pennsylvania;and the Pulmonary Division at the Children's Hospital of Philadelphia. The program is a joint one between the School of Medicine at Penn and the Children's Hospital of Philadelphia. The program is directed by an Executive Committee of Dr. Allan Pack (Principal Investigator), Dr. Steven Albelda, Dr. Jason Christie and Dr. Hakon Hakonarson. Faculty involved have expertise in a variety of lung diseases (obstructive sleep apnea, asthma, COPD, interstitial disease, lung cancer, sarcoidosis, lung transplantation) and for many of these diseases there are already DMA samples in a large well characterized patient populations facilitating the research of the trainees. The faculty also include individuals with methodological expertise from the areas of bioinfomatics, genetic epidemiology and statistical genetics. The proposed program for each trainee is flexible and will be based on the trainee's previous educational experience. It will involve a core curriculum of courses, elective courses, laboratory rotations and a mentored research project. A new specific track for genetic/genomic approaches is being established as part of the recently created Master's in Translational Research program. It is envisaged that during the support provided by this K12 award, trainees would apply for their own K/R award. The program is designed to be state of the art and to train the diverse scientific workforce that is required for the interdisciplinary teams of the future. (End of Abstract)

The overall goal of this project is to address the postulate that the optimal molecular signature for the common disorder obstructive sleep apnea (OSA) is change in relevant biomarkers during the sleep period. In sleep apnea, events lead to sleep fragmentation and cyclical deoxygenation/reoxygenation. It is proposed that these changes will lead to molecular consequences can be detected by assessing biomarkers in blood. To determine which changes are due to OSA and which to circadian/sleep mechanisms, studies will be done in patients with OSA before and after effective treatment with CPAP and also in controls of similar visceral adiposity without OSA.Multiple assessments of biomarkers will be made before, during and after sleep. Since it is proposed that the magnitude of these dynamic changes across the sleep period will be affected by degree of visceral obesity and be greater in OSA subjects with cardiovascular comorbidities, studies will be done in 4 groups of subjects: lean and obese with and without such morbidities. In assessing biomarkers the primary outcome variables will be: urinary isoprostanes (oxidative stress); plasma TNFa (inflammation); plasma norepinephrine (sympathetic activation); and free fatty acids. Secondary biomarkers will be: IL-6, urinary norepinephrine; urinary normetanephrine; glucose, ICAM, leptin. To complement assessment of circulating biomarkers, an approach utilizing a cellular window will be used. Monocytes will be separated from each blood sample (before, during and after sleep) and RNA extracted. Expression of key genes will be assessed by RT-PCR and microarray studies will be performed in a subset of subjects to assess changes in expression of all genes as a resuft of OSA.A particular focus will be investigating differences between individuals with OSA with and without cardiovascular comorbidities. Three aspects will be evaluated: a) whether individuals with comorbidities have more oxidative stress and inflammatory change for equivalent degrees of OSA than individuals without such comorbidities; b) whether individuals with comorbidities have lower levels of protective mechanisms melatonin (an anti-oxidant secreted during sleep), IL-10 (anti- inflammatory); c) different gene variants based on a genetic association study using a recently developed CV SNP array. Finally, data will be used to determine whether there is a diagnostic urine and/or blood test for OSA.

This Program Project Grant (PPG) is focused on the common problem of obstructive sleep apnea (OSA) and its relationship with obesity. Patients with OSA not only develop excessive sleepiness, but are at increased risk for hypertension, insulin resistance and cardiovascular events. Obesity is the major risk factor for OSA. Patients with OSA have oxidative stress, sympathetic activation, and increased inflammatory state that are independent of obesity. Since obesity is also postulated to produce identical effects, and OSA and obesity common coexist, it is important to consider the relative role of these two pathogenetic processes. The program of research has three projects and five cores. Project 01 (PL, Dr. R. Schwab) is directed at the question as to why obesity leads to OSA. It is proposed that the major pathogenetic mechanism is fat infiltration of tongue and other upper airway structures that increase their size, thereby reducing airway size and affecting the function of muscle. This will be addressed in a human case-control study, in a longidutinal study of individuals loosing weight after bariatric surgery, and in rat models of obesity. The project will use novel, state-of-the-art MRI techniques to assess fat in tongue and other structures. In Project 02 (PL, Dr. S. Kuna), a multidisciplinary team has been assembled to address whether the presence of obesity attenuates benefits of treatment of OSA on insulin resistance, hypertension and CV function, since obesity in the absence of OSA produces these effects. The study is powered to separately assess treatment effects in individuals with low and higher amounts of visceral fat. The study also assesses changes in biomarkers of the relevant processes-oxidation, sympathetic activity, proflammatory cytokines, adhesion molecules and free fatty acids. Controls without OSA are included to assess whether OSA leads to irreversible changes in clinical end-points. Project 03 (PL, Dr. A. Pack) proposes to assess temporal changes in biomarkers during sleep in subjects with OSA both before and after effective treatment with CPAP. The concept that movitates this project is that studying change in these processes across the sleep period provides a molecular signature of OSA. Studies are done in obese and lean individuals with OSA with and without cardiovascular consequences and in controls. It is argued that obesity will alterthe nature of the biomarker response to OSA, and individuals with OSA who develop comorbidities will have greater oxidative stress and inflammatory state than those who do not. The PPG is supported by five cores (A: Administrative; B: Sleep Study and Recruitment: C: Imaging; D: Biomarker; and E: Biostatistical and Data Management). Thus, this PPG is focused on a common clinical problem. The program will lead to defining who with OSA benefits from therapy and the magnitude of benefit for different end-points. It will lead to a new molecular signature of OSA that could transform the practice of medicine in this area in a new, cost-effective way.

DESCRIPTION (provided by applicant): Sleep deprivation is common and increasing in prevalence in the American population. Loss of sleep has a number of adverse consequences. These include behavioral changes, impairment of cognition with an increased risk of motor vehicle crashes, and errors in the workplace. Moreover, sleep loss leads to metabolic changes with insulin resistance, increased appetite and is a risk factor for development of obesity. There are, however, substantial individual differences in the degree of impairment produced by sleep loss. This is heritable, i.e., a large part of the difference between individuals is genetic in oriin. Elucidating the genetic basis of the response to sleep loss will enable a more rationale scheduling in operations that require 24/7 activity, and will likely identify novel pathways that could be the basis of future pharmacological approaches to alter the consequences of sleep loss. Further study to elucidate genes involved in humans is, however, challenging since examination of the response to sleep deprivation requires expensive studies with a high protocol burden (four days in a laboratory). In this application an alternative approach to identify responsible genes is proposed, based on studies in mice. Response to sleep deprivation in mice varies between inbred strains but the gene variants responsible for this difference are unknown. This proposal plans to take advantage of the recently created diverse outbred strain of mice which are all genetically different. This strategy requires study of a large number of mice (in this application, 800) in a high throughput fashion. Hence, response to sleep deprivation will be assessed by a novel high throughput strategy based on digital video analysis. Video analysis not only provides accurate estimates of sleep and wake, but also of the stages of sleep-rapid-eye movement (REM) sleep and non-rapid-eye movement (NREM) sleep. All mice will not only be phenotyped but will be genotyped based on a new genotyping chip with 7,000 single nucleotide polymorphisms. This approach allows identification of a small region of the mouse genome associated with this quantitative trait. In addition, the normal duration of wakefulness that a mouse can sustain will also be simultaneously assessed. This, too, is a heritable trait that is highly relevant to the common problem of sleeping difficulty. Validation studies in relevant mice from the collaborative cross lines will be employed for both quantitative traits. Future studies will also extend this investigation into human populations using samples of well characterized individuals who have been studied with sleep loss and whose DNA is already available.

DESCRIPTION (provided by applicant): Epigenetics is rapidly evolving as an important area of inquiry. There are already data that epigenetic mechanisms play an important role in linking metabolism to the functioning of the circadian clock. Currently, however, there is very limited information as to the role of epigenetics in regulation of sleep and in the pathogenesis of sleep disorders. This conference proposal is designed to address this void. The conference title is Epigenetics: Opportunities for Sleep and Circadian Research. It is proposed to bring together investigators from sleep and circadian research and from epigenetics. The conference is designed to bring investigators in sleep/circadian research up to date with epigenetic concepts and techniques. Further, it is constructed in such a way that investigators in epigenetics will become familiar with current concepts and questions in sleep/circadian research and study of sleep disorders. The conference is designed to stimulate future research collaborations between these two scientific communities and to excite new investigators in getting involved in this area. The conference has initial overview sessions to familiarize epigenetic investigators about sleep and sleep disorders and then investigators in sleep/circadian research about epigenetics. Thereafter there are multiple sessions each on a specific topic with investigators presenting from the sleep/circadian background and from epigenetics. The specific topic areas are as follows: a) Synaptic Plasticity: Sleep & Epigenomics; b) Aging & Neurodegeneration; c) Metabolism, Sleep & Epigenomics; d) Psychiatric & Neurodevelopmental Disorders; and e) Techniques. Travel awards will also be provided to new investigators and a specific session on the program will be for relevant oral presentations from new investigators. A workshop summary of the conference will be published in the leading sleep research journal, Sleep. This will contain information about the website, which will have slides from all presenters; thus, talks will be available to anybody interested on this website. The topic of the conference is timely since it is one of the new areas of inquiry proposed in the recently announced NIH Strategic Plan for Sleep Disorders Research, as well as research priorities within the National Institute on Aging. PUBLIC HEALTH RELEVANCE: A two and 1/2 day conference to identify research opportunities to further elucidate epigenetic mechanisms in sleep processes and leading to sleep disorders will be held on October 3-5, 2012 in Lafayette Hills, PA, a suburb of Philadelphia. This conference grant application to the National Institute on Aging, with support from other Institutes, requests partial support for the conference that will be used primarily to cover the travel and hotel expenses of the invited speakers and student awards.

There are major changes in sleep and wake that occur with age in all species studied. The major changes are in the ability to sustain state, in particular wakefulness, and reduced amounts of sleep. These physiological changes with age make older adults vulnerable to conditions that lead to difficulty sustaining sleep and wakefulness. This program of research investigates mechanisms for this at a fundamental molecular level. The projects focuses on two key groups of neurons: a) orexin cells in the lateral hypothalamus that stabilize behavioral state and promote wakefulness and b) galanin cells in the ventrolateral preoptic (VLPO) area that are active during sleep and promote sleep. We propose that there will first be decreased transcriptional response to neuronal activation in these neural populations with cell loss occurring at a later age. A major hypothesis being addressed is that changes in these neuronal populations with age are the result of age-related changes in the ER stress response pathway. To address these key hypotheses, we have three specific aims. In Aim 1, we will assess the temporal association between 3 measures of neuronal function of orexin cells and a key behavioral variable that is affected by age, i.e., the ability for mice to sustain long bouts of wakefulness. In Aim 2 we will have a similar approach but studying galanin VLPO cells with behavioral measures being reduction in NREM sleep across 24 hours and the amount of NREM in the first 24 hours of recovery sleep following 6 hours of sleep deprivation. Aim 3 will focus on the ER stress response pathway. Changes in expression of key genes in this pathway with age in these neuronal populations will be assessed. Moreover, we will determine, using a transgenic approach, whether reductions in the master regulator of the ER stress response pathway in BiP leads to changes with age being accelerated both in terms of function and number of cells in these neuronal groups and also in sleep/wake behavior. Finally, a discovery aim is proposed to use the new technology, RNA sequencing, to assess changes with age in the transcriptome of these two neuronal populations. RELEVANCE (See instructions): Fragmentation of sleep and wake occur with age. There is a reduction in the ability to sustain wakefulness and reductions in sleep. This has adverse consequences and reduces the quality of life. The mechanisms underiying these changes are unknown. This project takes a mechanistic approach focusing on specific brain neurons that control sleep and wake. This study will identify new molecular pathways to target to reduce this key effect of aging on an important behavior.

DESCRIPTION (provided by applicant): Sleep disorders are increasingly recognized to be common in the American population and to have important adverse consequences, not only on behavior with an increased risk of crashes but also on the cardiovascular system. Thus, sleep disorders are a major public health problem. This application is for renewal of a postdoctoral research training program for sleep research and research in sleep disorders at the University of Pennsylvania. The overall goals of the program are to train a cadre of independent investigators on these topics with a particular focus on physician-scientists. The training program has three distinct tracks: a) basic research, b) translational research, and c) patient-oriented research. Each track combines didactic training with an intense research experience working with a mentor(s). The program utilizes established Masters Degrees at the University of Pennsylvania to amplify what is provided by this particular program. The relevant Masters degrees are the Masters in Translational Research (Track 2) and the Masters in Clinical Epidemiology (Track 3). The program utilizes the infrastructure for research in sleep and its disorders that has been developed at the University of Pennsylvania by the interdisciplinary Center for Sleep and Circadian Neurobiology. This infrastructure is for both basic and clinical research and includes a dedicated 4-bedroom sleep research facility for studies in humans, including patients with specific sleep disorders. The program utilizes a group mentorship model and a mentorship committee is established for each trainee. Trainees are given specific training not only in sleep research but also in survival skills, including how to write grants. Career counseling is provide to each trainee and many of the trainees go on to successful academic careers as independent investigators. The strengths of the program include: a) a stable, dedicated leadership; b) a large group of productive research-oriented faculty who work in a very collaborative fashion; c) strong institutional support; and d) major research funding for faculty to pursue research. (End of Abstract)

DESCRIPTION (provided by applicant): There are profound changes in sleep and wake in older adults. This is found in all species studied from Drosophila to humans. The major changes are in the ability to sustain state, in particular long bouts of wakefulness. Moreover, the amount of sleep is decreased. In humans, this has adverse consequences that are amplified in the presence of comorbidities. While the phenomena are well known, the mechanisms producing these clinically relevant changes are not. In this program of research we propose a new approach to this question by investigating changes in molecular mechanisms using the mouse as a model system. The overall program addresses alternative hypotheses and uses novel transgenic mice to address our overall question. The first project (T. Abel, PL) addresses the role of the cyclic AMP response element binding protein (CREB) in age-related changes in the ability to sustain wakefulness. The investigators argue that changes in this mechanism across the lifespan affect the ability to sustain wakefulness in older mice. They propose studies to localize the neuronal group responsible for the effect and also to rescue this effect of age on wakefulness by transgenically manipulating this pathway. Project 02 (N. Naidoo, PL) addresses the role of one particular CREB target, i.e.. Homer 1a. Recent data have shown that Homer 1a is essential to sustain long bouts of wakefulness. This project seeks to determine the following: in which neuronal groups this effect is mediated; the mechanism of action based on a specific hypothesis about interaction with mGLuR5; and how the pathway is altered by aging. Project 03 (A. Pack, PL) takes a complementary strategy and focuses on neuronal groups known to be involved in wake/sleep control and stabilization of state, i.e., orexin neurons and galanin cells i the ventrolateral preoptic area. Changes in function of these neurons with age are assessed as is reduction of cell number with aging (neurostereology). A major focus of this project is the ER stress response pathway. The hypothesis is that changes in this pathway lead to the age-related changes in neuronal function and cell number. These three projects are supported by three cores; a) Administrative Core; b) Mouse Behavioral Core; and c) Biostatistics and Bioinformatics Core.

instmctions); The Mouse Behavioral Assessment and Breeding Core is an extension of a core in the previously supported program. The Core has a number of specific tasks that support all of the projects in the program. The specific tasks undertaken by the experienced Core staff are the following: a) behavioral assessment of wake and sleep stages in mice using high throughput video assessment of behavior. In the previous period of funding we developed a novel methodology to assess wake and stages of sleep from video analysis; 2) recording of wake and sleep using electroencephalogram and electromyogram. This involves implantation of electrodes and careful scoring of records. Core staff also perform spectral analysis of the EEG signal; 3) these studies generate a considerable amount of behavioral data. Thus, the Core also does data reduction and, using customized software we have developed, provides summary data to investigators in an easy to access format; 4) this new program of research now involves breeding and genotyping of a number of specific transgenic mice. It is proposed that this is best done by a central facility and therefore this Core now provides breeding of mice and genotyping. While these are ongoing and enhanced services that the Core will provide, the Core is also engaged in innovative activities. First, Core staff will conduct a study of changes in sleep/wake across the lifespan in C57BL/6NIA mice. These mice are used in Projects 01 and 02. Thus, these data will inform the science of these two projects. The Core is also addressing the capability to create new transgenic mice. As a first step, we proposed to create conditional transgenics where expression of Homer l a can be increased in older animals. This is an immediate future direction for Project 02. Finally, the Core will work to extend, in collaboration with staff in Core C, the video-based technology so that wake and stages of sleep can be assessed in individual mice among a group of mice that are raised together. RELEVANCE (See Instmctions): Mice are a powerful model system to study sleep and aging effects. Mice show the same changes in wake and sleep as humans. This Core provides the ability to study sleep and wake in mice of all ages to provide the behavioral assessments needed for this program of research. It also will create mutant mice that provide the capability to study mice with alteration in a particular gene and be responsible for mouse breeding.

DESCRIPTION (provided by applicant): Sleep medicine is a relatively new discipline. Hence, few institutions have the critical mass of faculty to mount a single institutional training grant. he lack of research training is a major barrier for the field of sleep medicine to take advantage of current scientific opportunities. One area where there are substantial opportunities for new science is in the field of genetic/genomic approaches. These are needed to develop personalized sleep medicine. Many sleep disorders are heritable, although the gene variants that confer risk are largely unknown. Moreover, recently sleep-like states have been identified in many species such as worms, fruit flies and zebra fish as well as mice. These model systems can be used to determine the functional significance of gene variants identified in human studies. To capitalize on these opportunities, a novel national effort is proposed. The basis of this is a multi-institutional training grant involving three institutions-Johns Hopkins, Stanford University and University of Michigan. The program will be directed by the University of Pennsylvania with fellows at each of these three institutions. The University of Pennsylvania has the most experience in research training in sleep and has significant administrative and training infrastructure to support this program. The program will focus on training the next generation of physician-scientists. Trainees will be given three years of training supported by this program. There will be a core curriculum that will be taken by all trainees. This will use new video-based IT technology that is already operational. The core curriculum will involve the following: lectures on genetics/genomics of sleep and its disorders by faculty at all participating institutions; caree development; grants workshop; journal club; research-in-progress talks by trainees. Each trainee will have co- mentors at their home institution-one expert in sleep research and one in genetics/genomics. They will also have a mentorship committee with experts in sleep research and genetics. For the mentorship committee, mentors from the various institutions in this program, as well as from institutions that currently have funded training programs in sleep research (Harvard, Penn, Pittsburgh) will be involved, if appropriate. Trainees who pursue genetic/genomic research at these other institutions will also be considered part of this national effort. There will be an annual retreat of all trainees and faculty. This will orient new trainees o research opportunities and allow more advanced trainees to present their research. There will also be keynote speakers selected by trainees. This retreat will facilitate interaction between the trainees at all the different sites. The goal of this national effort is to develop a cadre of new investigators in genetic/genomic approaches to sleep and its disorders.

ABSTRACT Obstructive sleep apnea (OSA) is a common disorder whose prevalence is increasing. Sleep apnea is a systemic disorder, with recurrent falls in oxygen (hypoxia) and reoxygenation (oxidative stress) affecting every tissue. Thus, sleep apnea is an independent risk factor for cardiovascular disease (heart attack and stroke), hypertension, cognitive impairment and dementia, and cancer. However, there are major individual differences, with some individuals even with severe OSA not developing any of these consequences. While it has been known for two decades that OSA aggregates in families, no convincing gene variants conferring risk have been identified in human studies. This suggests that a new approach is required. The study we propose introduces the following new approaches: a) it will be conducted in mice as a model system. Mice have proven to be a powerful model system to identify gene variants for human disease; we will use the recently described Diversity Outbred strategy that identifies quantitative trait loci of the order of three genes. This requires high throughput phenotyping of large numbers of genetically heterogeneous mice in combination with genotyping by a universal mouse SNP chip; b) we will study key endophenotypes that confer risk to OSA?tongue fat and ventilatory responses to hypoxia and hypercapnia. We will assess heritability of each of these traits and determine if tongue fat is a unique fat distribution; and c) we will assess the blood pressure response to cyclical intermittent hypoxia that simulates the pattern of deoxygenation/reoxygenation that occurs in patients with OSA. For all phenotypes assessed, functional assessment of genes identified will be performed. Thus, the study introduces an entirely novel approach to identifying gene variants and performing a functional assessment of genes for all phenotypes assessed. Large numbers of human samples with well phenotyped individuals are already in hand to translate findings from these mouse studies to humans, and in particular the phenotypes being assessed in these mouse studies are being assessed in a quantitative manner in the two human projects (Projects 01, 02). Sequencing of genes identified will be performed in the samples from the other projects in the Program Project Grant.

ABSTRACT The Molecular Assessment Core will be primarily located in the Translational Research Laboratory (TRL), the same building which houses the Center for Sleep and Circadian Neurobiology. The functioning of the Molecular Assessment Core will take advantage of existing facilities and the sophisticated equipment already available within the Translational Research Laboratory. The Molecular Assessment Core will act as a central facility to collect, process and analyze the large number of incoming blood and urine samples generated by the Projects 02 and 03. Importantly, the Molecular Assessment Core will ensure quality control of all samples collected by the proposed projects. The objectives of the Molecular Assessment Core are to: 1) standardize blood and urine collection between sites, 2) ensure proper handling and storage of samples, 3) code, record and monitor incoming samples, 4) ensure quality control of assays and 5) provide a centralized facility for assays. The Molecular Assessment Core will be under the leadership of Dr. Allan I. Pack, with support staff consisting of two experienced research specialists.

ABSTRACT Sleep disturbances with reduced amounts of sleep and sleep fragmentation are common in Alzheimer?s disease. These sleep abnormalities occur before the onset of cognitive impairments. This also occurs in mouse models of Alzheimer?s disease in mice in whom the mutations of genes that are responsible for familial Alzheimer?s disease are knocked in. These sleep abnormalities are not just a consequence of the disease process but likely also accelerate disease progression. The responsible A?42 is released from neurons in the brain during wakefulness. This reduction in sleep and increase in wakefulness give more time over the 24 hour period for increased production of A?42. Moreover, the protein ? amyloid that aggregates in plaques in Alzheimer?s disease is more rapidly cleared from the brain during sleep. Thus, study of sleep abnormalities in Alzheimer?s disease are important to understanding the pathogenesis of disease. While much has been learned about Alzheimer?s disease from studies of mouse models, these models lack the genetic heterogeneity found in the human population. Recently a new approach in mice has been developed. Mice carrying mutations that are responsible for Alzheimer?s disease are bred with a panel of recombinant inbred lines creating multiple different lines of mice that are genetically driven but all carry the same mutations for Alzheimer?s disease. Some of these mouse lines are very resistant to developing cognitive deficits, while others are very sensitive. Moreover, initial studies show that there is also a variation in the onset of sleep abnormalities in these diverse mouse lines. This variation gives the opportunity to identifying modifier genes that alter the timing of the onset of sleep abnormalities. One potential modifier gene has already been identified. Building on this initial observation we propose to study sleep in more of these lines. We will conduct in-depth phenotyping of multiple lines at different ages and assess sleep amounts, sleep fragmentation, vigilance (latency to sleep in a new environment), and circadian behavior using wheel running. These data will be used to identify additional modifier genes. Studying an increasing number of these lines increases the power of the study and provides more precision for mapping and identifying genes. In addition, we will assess with EEG/EMG recording whether one modifier gene already identified does indeed affect sleep. Identifying modifier genes that slow the rate of progression of disease could provide new targets for pharmacological intervention.

ABSTRACT Obstructive sleep apnea (OSA) is recognized as an extremely common disorder that results in excessive daytime sleepiness and an increased risk of automobile crashes. It is also an independent risk factor for insulin resistance, hypertension, cardiovascular events, increased cancer rates and cancer mortality, and accelerates the progression of neurodegenerative disorders. OSA is a systemic disorder and the cyclical intermittent hypoxia that occurs during sleep affects every organ system. OSA is heritable, with first degree relatives of individuals with OSA having a two-fold increased risk of the disorder. Although this has been known for over two decades, there are very limited data on gene variants conferring risk or protection for OSA. This is likely the result of underpowered studies and GENETIC HETEROGENEITY. There are multiple pathways to OSA. Obesity is a major risk factor, as are craniofacial dimensions. The relative risk of these different risk factors varies by ethnic group. There are also neuronal mechanisms and other physiological risk factors. This proposal seeks to directly address the issue of genetic heterogeneity using a very large sample of patients and state-of- the-art analysis techniques. Towards this end, we have assembled by far the largest sample of well characterized and genotyped patients with OSA. This is the result of collaboration with key sites in the eMERGE Network [Geisinger, Vanderbilt, Mayo Clinic, Northwestern], Kaiser Permanente Southern California, and the University of Pennsylvania. We propose to carry out a robust genome-wide association studies (GWAS) using an electronic health record (EHR) derived case definition and quantitative trait analysis of OSA severity. We will use novel machine learning approaches to specifically address genetic heterogeneity. The GWAS approach will be complimented by a PheWAS strategy as another approach to identifying common variants and related clinical phenotypes. Specifically, we will look for variants that associate with OSA and other clinical diagnoses in the EHR. It is likely that gene variants for OSA will have pleiotropic effects. Gene variants identified from GWAS and/or PheWAS will be further assessed. First, we will use available whole exome sequencing data to see if there are relevant rare variants in these genes, as well as to perform exploratory discovery analyses to look for novel rare variation. The functional role of the genes identified will be studied by functional network analysis. Finally, we will assess associations with quantitative data on intermediate traits identified by high throughput craniofacial and intra-oral photography. These data are currently available within the Sleep Apnea Global Interdisciplinary Consortium (SAGIC). Future directions will assess the function of the genes identified in model systems and the clinical utility of utilizing variants as part of risk stratification for OSA.

ABSTRACT There is growing evidence of the role of sleep and circadian disorders in cardiovascular disease, metabolic disorders, neurodegeneration and cancer. This reflects that sleep and circadian disorders have systemic effects affecting multiple organs. While there is a growing and rapidly developing view as to the major public health significance of sleep and circadian disorders, there is a paucity of investigators in the area, particularly those employing state of the art approaches. This postdoctoral fellowship research training program is addressing this issue. The program is housed at the University of Pennsylvania (Penn). It is part of the comprehensive effort in research training by the Center for Sleep and Circadian Neurobiology at this institution. Penn was the first institution in the United States to develop in 1991 a comprehensive medical school-wide center for this activity recognizing the very multidisciplinary nature of sleep and circadian research field. Since 1991 Penn has played, and continues to play, the leading role in research training in this field. Penn has the most faculty investigators in the area of any institution nationally and the most research support. While the research training program is well established, it continues to evolve to meet changing scientific direction and needs. The program is now organized into 4 specific tracks: a) basic research; b) clinical and translational research; c) behavioral science and ?basic human research?; and d) implementation and comparative effectiveness research. The program takes advantage of the infrastructure that has been developed at Penn for research training. This includes the Office of Biomedical Postdoctoral Programs (BPP), the Masters of Science in Clinical Epidemiology, the Masters of Science in Translational Research, and the Masters of Science in Health Care Policy Research.

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.

ABSTRACT Sleep homeostasis maintains the balance between sleep and wakefulness. Homeostatic sleep regulation is essential for cellular health and sleep disorders are implicated in many neurological disorders and age-related diseases. Understanding sleep homeostatic mechanisms is necessary for developing new therapies for sleep disorders. The preoptic area (POA) of the hypothalamus is essential for sleep homeostasis. Multiple nuclei of POA, including the ventrolateral preoptic area (VLPO) and the median preoptic nucleus (MnPO), contain sleep- active neurons that display increased activity during sleep compared with wake. The numbers of c-Fos positive neurons in VLPO and MnPO increase under high sleep pressure, e.g. after sleep deprivation and during recovery sleep following sleep deprivation. The complete makeup of the sleep-active neurons in POA is unknown. The galanin-expressing GABAergic neurons in VLPO are the most widely studied sleep-active neurons. However, not all c-Fos positive sleep-active neurons express galanin and not all galanin neurons are c-Fos positive during sleep at any given time in POA. Given the heterogeneous molecular and functional makeup of POA, it is important to comprehensively characterize the sleep-active neurons in POA at the individual cell level in an unbiased way. Towards this end, we will apply the recently advanced single-nucleus RNA sequencing (snRNA- seq) technique to POA and compare gene expression changes in individual cells between mice during recovery sleep following sleep deprivation (high sleep pressure) and mice after long periods of spontaneous sleep (low sleep pressure). Aim 1 will comprehensively map all neuronal groups that are activated under high sleep pressure based on a panel of activity-regulated genes. We expect to find that specific subtypes of galanin- expressing inhibitory neurons, as well as non-galanin expressing inhibitory neuronal groups that express other neuronal markers, are activated with high sleep pressure. Aim 2 will reveal the transcriptional changes regulated by homeostatic sleep pressure in all cell groups, including neurons and non-neuronal cells. For example, astrocytes play key roles in maintenance of sleep homeostasis. However, little is known about transcriptional regulation of astrocytes involved in sleep homeostasis in POA. Given the recent discovery of the molecular and regional specificity of astrocytes, we hypothesize that we will reveal region-specific and cell-specific changes in astrocytes. Aim 3 will use multiplex fluorescent in situ hybridization (RNAscope) to characterize the anatomical localization of the identified sleep-active neurons based on the molecular markers identified by snRNA-seq. This combination of molecular and spatial characterization of the sleep-active neurons in POA will enable future dissection and manipulation of the sleep circuit.