Mark R. Opp - US grants

Over the past 15 years, there have been intense efforts focused on determining the structures and biological activities of muramyl peptides (MPs). This interest in MPs is based , in a large part, on the assumption that MPs are tailored from bacterial cell wall peptidoglycan; once thus produced, they then can elicit their central nervous and immune system activities which are involved in host defense as well as physiological responses. However, this assumption has never been experimentally verified. The broad objective of this proposal is to investigate the hypothesis that mammalian macrophages produce and release biologically active MPs during the digestion of bacterial cell walls. This hypothesis is proposed because: a) MPs are present in mammalian tissue, including the brain, and have profound effects on the central nervous and immune systems, yet there are no known mammalian synthetic pathways for MPs; and b) our preliminary data and previously published data suggest that macrophages do, in fact, release biologically active substances of low molecular weight whose effects mimic those of synthetic MPs. However, chemical characterization of substances released by macrophages during peptidoglycan digestion has not been done, nor have the biological activities of such released substances been characterized except in our preliminary experiments. Therefore, to test our hypothesis: Viable bacteria, radioactively marked in their cell walls, will be fed to murine macrophages. The substances released by the macrophages will be fractionated using various chromatographic techniques (e.g., reversed phase C18-HPLC). 2)Fractions thus obtained will be tested for their ability to enhance rabbit sleep and body temperature in vivo.3) Fractions containing biological activity will be purified and retested, and substances will be identified by amino acid analysis and mass spectrometry. The proposed studies will help to evaluate the role MPs may play in the regulation of central nervous system and immune system involvement in host defense and physiological responses.

Sleep is altered in response to psychological stressors; the precise mechanisms whereby such alterations occur are not known. Behavioral responses to most stressors include increased arousal and waking. We hypothesize that corticotropin-releasing hormone (CRH) is a mediator of waking, particularly that which follows periods of exposure to acute stressors. The behavioral, physiological, electrophysiological, and anatomical data currently available support this hypothesis. We have obtained new experimental evidence supporting the hypothesis that CRH is involved in physiological regulation of waking; Lewis/N rats, a strain that exhibits reduced synthesis and secretion of CRH relative to histocompatible Fischer 344/N (F344/N) or Sprague-Dawley rats, exhibit reduced amounts of spontaneous waking compared to these other two strains. Our central hypothesis will be tested within the context of two specific aims. Specifically, we will determine 1) the extent to which CRN contributes to increased wakefulness that follows exposure to acute stressors, by subjecting rats to a cage-switch stressor, in the presence or absence of CRH antagonists. We will also 2) ascertain the potential contribution of CRH to physiological regulation of waking by administering CRH antagonists into normal animals, and by exploiting the "natural" model of the Lewis/N, F344/N, and Sprague-Dawley rats. We will use rats instrumented with EEG electrodes, a chronic ventricular guide cannula, a thermistor to monitor brain temperature, and, in some cases a jugular cannula to allow blood sampling from freely behaving animals. Waking and sleep will be determined by visual inspection of electrophysiological records that have been recorded by a computerized data acquisition system. Upon completion of these experiments we will know the extent to which CRH contributes to stressor-induced alterations in waking, mechanisms of action where, by such responses may be mediated, and the role of CRH in normal patterning of waking and sleep.

DESCRIPTION (Adapted from applicant's abstract): Tissues of the immune and nervous systems are the primary targets of the human immunodeficiency virus (HIV). Sleep, a definable behavior, is regulated by interactions between neural and biochemical mechanisms, and is altered during viral infections, including HIV. It is estimated that as many as 60% of all HIV-infected individuals experience debilitating daytime fatigue and sleepiness. In addition, polysomnographic studies conducted on HIV-infected individuals indicate that nighttime sleep is altered. These alterations occur during chronic HIV infection, prior to the onset of AIDS, and in the absence of substance abuse or psychiatric symptoms associated with anxiety or depression. The precise mechanisms whereby HIV alters sleep are not known. In spite of HIV's inability to infect rodent cells, other laboratories have demonstrated the utility of rodent models for the study of cytokine actions and neurologic effects. The investigators have found, in a rat model of sleep, that HIV products induce alterations in sleep in a manner resembling those observed during HIV infection. Furthermore, in the investigators' model, HIV products increase cytokine mRNA expression within the rat central nervous system (CNS). HIV enters the CNS shortly after infection; cytokine concentrations, including the somnogenic cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), are elevated within the CNS during all stages of HIV infection. The investigators hypothesize that altered sleep during HIV infection is due, in part, to HIV-induced increases within the CNS in concentrations of cytokines that enhance sleep (e.g. IL-1, TNF) as well as those that suppress sleep (e.g. IL-1 receptor antagonist (IL-1ra) and IL-10). The investigators will test this working hypothesis by: determining the components of HIV capable of altering sleep (i.e. HIV envelope glycoproteins [gps]), and by elucidating the roles of IL-1, TNF, IL-1ra, and IL-10 in responses to HIV and HIV gps. Finally, they will determine changes in cytokine mRNA expression in brain regions important for the regulation of sleep, in response to the HIV gps. They will use rats instrumented with EEG electrodes, and chronic guide cannulae directed into lateral cerebral ventricles. Sleep-wake activity, and changes in cytokine mRNA expression in the CNS, will be determined after administration of HIV or HIV gps. These experiments should show not only the extent to which cytokines within the CNS contribute to HIV-induced alterations in sleep, but may also suggest new therapeutic approaches for the debilitating symptoms of fatigue and sleepiness that occur during AIDS.

DESCRIPTION (adapted from applicant's abstract): The fundamental question addressed in this application concerns the mechanisms that mediate bidirectional communication between the CNS and the immune system. The investigators focus on one potential mechanism, the interactions between neurotransmitters and cytokines, as they pertain to sleep. There is ample evidence that serotonin (5-HT) and interleukin-1 (IL-1) modulate sleep. 5-HT and IL-1 influence each other; IL-1 induces serotonergic activation, and 5-HT induces IL-1. Furthermore, the investigators' new data indicate that somnogenic responses to IL-1 are altered in animals in which 5-HT synthesis has been inhibited. Therefore, the somnogenic responses to IL-1 may be mediated, in part, by the serotonergic system. On the other hand, IL-1 may represent one of the sleep factors through which 5-HT has been proposed to influence sleep. The PI will investigate three specific aspects of these interactions: 1) Does 5-HT mediate, in part, IL-1-induced alterations in non-rapid eye movement sleep (NREMS)? 2) Do raphe nuclei mediate this effect? 3) Does IL-1 mediate, in part, the enhancement of NREMS induced by the 5-HT precursor L-tryptophan? They will investigate these questions by a) blocking 5-HT2 receptors prior to central administration of IL-1, b) microinjecting IL-1 into the raphe nuclei, and c) blocking central IL-1 actions with antibodies prior to inducing serotonergic activity with L-tryptophan. They will use rats chronically instrumented to allow administration of substances directly into the CNS with subsequent determination of sleep-wake behavior and serotonergic activity. Although there is evidence that IL-1 and 5-HT interact at multiple levels, the critical experiments necessary to determine the extent of interaction and specific effects on sleep have not been done. Direct evidence that interactions between these two systems may be a critical feature of the regulation of sleep would be obtained if blockade of serotonergic activity alters IL-1 induced enhancement of NREMS, and/or pretreatment with anti-IL-1 alters L-tryptophan-induced enhancement of NREMS. The proposed experiments will provide information useful for understanding the mechanisms whereby sleep is altered in response to an immune challenge. These experiments will also explore one possible brain site for the interactions between 5-HT and IL-1. The successful completion of these experiments will warrant the development of future, more extensive, studies of IL-1/5-HT interactions as they pertain to sleep-wake regulation.

DESCRIPTION (adapted from applicant's abstract): The fundamental question addressed in this application concerns the mechanisms that mediate bidirectional communication between the CNS and the immune system. The investigators focus on one potential mechanism, the interactions between neurotransmitters and cytokines, as they pertain to sleep. There is ample evidence that serotonin (5-HT) and interleukin-1 (IL-1) modulate sleep. 5-HT and IL-1 influence each other; IL-1 induces serotonergic activation, and 5-HT induces IL-1. Furthermore, the investigators' new data indicate that somnogenic responses to IL-1 are altered in animals in which 5-HT synthesis has been inhibited. Therefore, the somnogenic responses to IL-1 may be mediated, in part, by the serotonergic system. On the other hand, IL-1 may represent one of the sleep factors through which 5-HT has been proposed to influence sleep. The PI will investigate three specific aspects of these interactions: 1) Does 5-HT mediate, in part, IL-1-induced alterations in non-rapid eye movement sleep (NREMS)? 2) Do raphe nuclei mediate this effect? 3) Does IL-1 mediate, in part, the enhancement of NREMS induced by the 5-HT precursor L-tryptophan? They will investigate these questions by a) blocking 5-HT2 receptors prior to central administration of IL-1, b) microinjecting IL-1 into the raphe nuclei, and c) blocking central IL-1 actions with antibodies prior to inducing serotonergic activity with L-tryptophan. They will use rats chronically instrumented to allow administration of substances directly into the CNS with subsequent determination of sleep-wake behavior and serotonergic activity. Although there is evidence that IL-1 and 5-HT interact at multiple levels, the critical experiments necessary to determine the extent of interaction and specific effects on sleep have not been done. Direct evidence that interactions between these two systems may be a critical feature of the regulation of sleep would be obtained if blockade of serotonergic activity alters IL-1 induced enhancement of NREMS, and/or pretreatment with anti-IL-1 alters L-tryptophan-induced enhancement of NREMS. The proposed experiments will provide information useful for understanding the mechanisms whereby sleep is altered in response to an immune challenge. These experiments will also explore one possible brain site for the interactions between 5-HT and IL-1. The successful completion of these experiments will warrant the development of future, more extensive, studies of IL-1/5-HT interactions as they pertain to sleep-wake regulation.

DESCRIPTION (Applicant's Abstract): The central nervous system (CNS) and immune system engage in reciprocal communication. Sleep is a fundamental CNS process that is regulated by neurotransmitters and responds to immune challenge, as evidenced by changes in sleep that occur when we are sick. In this application we focus on interactions in brain between serotonin (5-hydroxytryptamine; 5-HT) and interleukin-1 (IL-1) as representatives of neurotransmitters and immune-active molecules that are involved in sleep regulation. We previously investigated IL-1 effects on 5-HT and showed that sleep responses to IL-1 are altered if the 5-HT system is antagonized. Studies proposed in this application focus on the reciprocal interaction, i.e., 5-HT effects on IL-1. Our central hypothesis is that, serotonergic activation stimulates the somnogenic cytokine IL-1, which may be one of the factors through which 5-HT exerts its effects on sleep. This central hypothesis will be addressed within the framework of the following questions. 1) Does serotonergic activation alter the brain IL-1 system? 2) Are 5-HT effects on sleep altered when the brain IL-1 system is antagonized? 3) Is the preoptic area/anterior hypothalamus (POA) an important site for these interactions? These questions will be addressed using behavioral, electrophysiological, and molecular approaches. Our preliminary data from the rat indicate that serotonergic activation increases NREM sleep, IL-1 mRNA expression, and c-fos activity in the hypothalamus. These new results and those of previous studies indicate that interactions between these two systems are functionally relevant to sleep. 5-HT and IL-1 are involved in the regulation in the regulation of a number of physiological functions and behaviors in addition to sleep. As such, although our focus remains firmly on sleep, the integrated experimental approach we propose will result in information relevant to several fields of basic and clinical neuroscience.

DESCRIPTION (provided by applicant): Infection negatively impacts mental health. Sick individuals become lethargic, experience cognitive deficits and malaise, and lose interest in social contact and other usual daily activities. Prominent among the changes in CNS processes during infection are alterations in sleep. Cytokines, such as interleukin (IL)-1, tumor necrosis factor (TNF), and IL-6 are upregulated during infection. Two lines of evidence suggest that infection-induced alterations in sleep are mediated by actions of these cytokines in brain. First, numerous studies indicate IL-1, TNF, and IL-6 regulate/modulate physiological sleep in the absence of immune challenge. Second, experimental models for which alterations in sleep have been determined are associated with increases in these same cytokines. The involvement of IL-1, TNF, and IL-6 in the regulation of sleep, and the alterations in sleep that occur during infections in which these cytokines are upregulated, have led to suggestions that infection-induced alterations of sleep are mediated by cytokines in brain. Although plausible, and based on empirical evidence, studies to directly test this hypothesis have not been conducted. The fundamental goal of this project is to determine how acute infections alter sleep. To achieve this goal we will use a clinically relevant murine model of infection, sepsis induced by cecal ligation and puncture. We propose experiments that focus on cytokines (IL-1, TNF, IL-6) as mediators of infection-induced alterations in sleep. We will: 1) determine the extent infection alters sleep and the impact of prior sleep loss on responses to infection; 2) quantify alterations in cytokine mRNA and protein in brain during infection; and 3) answer the question "Does interfering with cytokine actions in brain impact infection-induced alterations in sleep?" IL-1, TNF, and IL-6 have been the subject of intense investigation with respect to their peripheral roles in sepsis, and are known central regulators/modulators of sleep. As such, there is a strong conceptual framework within which to investigate the mechanistic relationships between sleep and sepsis, and mediators implicated in both processes. We present preliminary data that demonstrate long-term alterations in CNS function following acute peripheral infection. We demonstrate our ability to determine multiple facets of sleep-wake behavior of mice and to target cytokine systems in brain. Successful completion of the proposed studies will provide information critical to understanding how infection impacts CNS function, as evidenced by alterations in sleep.

Acutely ill individuals experience cognitive deficits, malaise, and lethargy. Sick individuals also exhibit profound changes in sleep with reduced motor activity, they become anorexic, and they lose interest in social contact and other usual daily activities. Collectively, this constellation of symptoms is referred to as sickness behavior. Cytokines such as interleukin (IL)-I, IL-6, and tumor necrosis factor (TNF), act in brain to induce many of the sequelae of sickness behavior. Although phylogenetic data and conventional wisdom suggest that sickness behavior during acute infection promotes survival, few studies have tested this hypothesis. The fundamental goal of this project is to determine how immune responses to acute infection impact central nervous system (CNS) function. Many of the behavioral responses to immune challenge described above are manifest during the development of sepsis. Previous work, and studies proposed in other projects of this PPG application, focused on inflammatory mediators in the peritoneum. The central hypothesis to be tested by experiments in this project is that brain responses to sepsis are a critical determinant of clinical outcome. We will use a dual approach to test this central hypothesis: 1) pre-clinical experiments will be conducted in mice using the well-defined model of sepsis induced by cecal ligation and puncture (CLP); 2) patients presenting to the Trauma and Burn ICU will be recruited for continuous EEG recordings. Using this parallel approach, we will: quantify in mice the extent to which behavior is altered by the infectious process (Specific Aim 1), determine how selected cytokines and their regulatory mechanisms in brain promote or inhibit specific aspects of sickness behavior of mice, and how interfering with their actions impacts survival (Specific Aim 2), and determine in both mice and ICU patients the degree to which sleep disruption and sedation alter immune responses, sickness behavior and survival (Specific Aim 3). We have demonstrated our ability to obtain similar and overlapping measures from mice and from acutely ill patients. This capability represents a powerful tool for translational studies of the role of the CNS in responses to sepsis. Given that the brain and the peripheral immune system engage in bi-directional communication, determination of how brain status influences systemic immune responses to sepsis may provide insight into new therapeutic interventions so that sepsis may be more frequently followed by recovery.

DESCRIPTION (provided by applicant): Sleep presents a conundrum for neurobiology: we do not know what function(s) sleep serves for the brain (or the body). We do know, however, that adequate sleep is essential for physical and mental health. In addition to effects on cognition and performance, lack of sleep, or sleep disruption due to sleep disorders may be a contributing factor to multiple pathologies, including but not limited to hypertension, coronary artery disease, cerebrovascular disease, and hyperglycemia. Numerous studies demonstrate that sleep loss impairs immune function and that immune activation from infection alters sleep. Responsiveness to infection varies widely: in the extreme, some will live and others will die in response to the same pathogen(s). Numerous systematic pre- clinical studies demonstrate infection-induced alterations in sleep. Most infections increase non-rapid eye movements sleep (NREMS) and decrease rapid eye movements sleep (REMS). Our functional hypothesis is that the manner in which sleep is altered during infection is a critical determinant of clinical outcome. Indeed, one retrospective study demonstrates that specific sleep patterns of rabbits are associated with survival from infection. To further our understanding of central nervous system responses that result in good clinical outcome, we focus on the cytokine interleukin (IL)-1 as one mediator of altered sleep during immune activation. Data indicate IL-1 increases NREMS and suppresses REMS. We propose in this application to focus effort on IL-1-induced suppression of REMS, an effect that has been universally ignored. IL-1 inhibits ACh synthesis and release. Cholinergic neurons of the laterodorsal tegmental (LDT) and pedunculopontine (PPT) nuclei are involved in EEG desynchronization and thalamocortical activation during REMS. REMS- generating structures are under GABAergic inhibition: IL-1 enhances GABA inhibitory effects at multiple levels. Studies proposed in this application will test the mechanistic hypothesis that IL-1 suppresses REMS by opposed, yet complementary actions on brainstem cholinergic and GABAergic systems. Our preliminary data indicate: IL-1 microinjected into the LDT reduces REMS of rats; IL-1 reduces firing rates of cholinergic neurons in LDT slice preparations; and IL-1 increases the number of c-Fos+ neurons in the ventrolateral periaqueductal grey (vlPAG), a GABA-rich area that projects to the pontine reticular formation and the LDT. In this application, we propose to determine: 1) the impact on sleep of IL-1 microinjection into brainstem cholinergic/cholinoceptive nuclei, 2) in vitro effects of IL-1 on electrophysiological properties of cholinergic neurons, and 3) the impact of IL-1 on REMS-relevant brainstem nuclei and neurotransmitter systems using immuno- fluorescence techniques. Successful completion of these aims will provide novel data critical for our understanding of mechanisms by which REMS is suppressed during infection. Determination of whether alterations in sleep contribute to good clinical outcome will only be possible when the neuroanatomic and neurochemical substrates targeted by immune responses to infection are clearly understood. PUBLIC HEALTH RELEVANCE Some individuals live and others die in response to infections. Sleep is dramatically altered during infection. Evidence suggests the manner in which sleep is altered may contribute to survival. This project will determine effects of immune activation on brain systems responsible for regulating one phase of sleep that is suppressed during sickness. Once we understand how (by what means) sleep is altered during infection, we will be able to study why sleep is altered during infection, i.e., does altered sleep facilitate recovery?

DESCRIPTION (provided by applicant): The factors by which aging predisposes to critical illness are varied, complex, and not well understood. Sepsis annually kills hundreds of thousands of people in the US with associated hospital costs of billions of dollars. Of concern, sepsis incidence is increasing at more than 8% annually. Sepsis is considered a quintessential disease of old age because the incidence and mortality of severe sepsis increases exponentially as we age. Patients 65 and older account for 65% of all sepsis cases and age independently predicts sepsis mortality. Studies suggest that chronic inflammation contributes to increased morbidity and mortality in older adults. Among predisposing factors, two are underappreciated as contributing to chronic inflammation and sepsis outcomes in older adults: sleep fragmentation and blood brain barrier transport. Sleep of older adults is fragmented, and sleep disruption is associated with increased production of cytokines, including tumor necrosis factor (TNF). Aging increases the rate TNF is transported from blood-to-brain across the blood brain barrier (BBB), and TNF transported across the BBB can induce the production of more TNF within the brain. These observations suggest the intriguing hypothesis that aging, sleep fragmentation and alterations in BBB transport synergistically contribute to chronic neuroinflammation. We will test this hypothesis within the context of four Aims. We will use the well-characterized model of cecal ligation and puncture to induce sepsis in mice. We will determine the impact of aging and sleep fragmentation on sepsis outcomes (Aim 1) and quantify changes in cytokine profiles in brain and periphery (Aim 2). We will also quantify effects of aging and sleep fragmentation on the rate of TNF transport across the BBB and its accumulation in brain (Aim 3). Finally, we will inhibit cytokine actions in brain and determine effects on sepsis morbidity and mortality (Aim 4). Outcome measures for most experiments include symptoms of clinical illness (altered sleep, changes in brain temperature, reductions in water and food consumption, loss of body weight) and mortality. We have validated Luminex xMAP(R) technology for multiplex assay of cytokines from mouse brain. We will use this approach to quantify cytokine profiles in plasma and discrete brain regions (hypothalamus, hippocampus, brain stem) from the same animal. We will determine influx rates from blood-to-brain for TNF and ascertain the integrity of the BBB. Finally, we will antagonize TNF directly in brain and interfere with transcriptional regulation of TNF and other cytokines. To our knowledge, effects of aging, sleep fragmentation and alterations in BBB characteristics as determinants of sepsis outcomes have not been studied. Completion of this project will provide critical information that is currently lacking with respect to interactions among dynamic processes (aging, altered BBB parameters, sepsis) and predisposing factors (sleep fragmentation) that may contribute to negative outcomes in response to critical illness or injury.

DESCRIPTION (provided by applicant): Sleep Regulation and Function 2014: The inaugural Gordon Research Conference on Sleep Regulation and Function will be held in 2014. The focus of this conference is Emerging Themes and Paradigm Shifts. During the past two decades we have witnessed a paradigm shift in the conceptualization of how sleep is regulated. In contrast to the traditional view that the brain imposes sleep on the organism, this new paradigm views sleep as a homeostatically regulated, use-dependent process that first emerges at the level of simple neuronal networks. At this level, activity and rest alternate both in neural networks and in invertebrates. In more complex organisms, the alternation between sleep and wakefulness is regulated by neural structures and neuromodulatory pathways that orchestrate and synchronize local sleep processes. The hypothesis that sleep is a local-use dependent process is now reflected in research programs ranging from synaptic scaling and plasticity to oscillating networks in vitro. The importance of gliotransmission and neural-glial interactions for normal brain function and for the regulation of sleep is an emerging theme within the field. Newly characterized sleep phenotypes under conditions of impaired gliotransmission suggest that astrocytes do much more than simply provide support for neurons. Within the context of paradigm shifts and emerging themes, a research program has emerged in which investigators from many different backgrounds ask basic questions about the regulation and function of sleep at a local level as well as at the level of the whole organism. Investigators use a wide range of methodologies developed within the disciplines of molecular biology, genetics, systems biology, neuroscience, mathematical modeling and others. Although the aforementioned paradigm shift and emerging theme will be highlights of the conference, their relatedness to other topics of sleep research will be emphasized in sessions on model systems; the genetics of sleep; and molecular-genetic dissection of complex networks. The objectives of the conference are to; bring together scientists from within and outside the sleep research community who will contribute to a highly integrated conference; provide a forum for discussion of the latest research in the field; and contribute to the training of the next generation of sleep researchers.

? DESCRIPTION (provided by applicant): Sleep research has historically been neuron-centric; much effort has focused on determining neuronal circuits and/or actions of transmitter substances produced by neurons. Historically, glia were thought to be merely passive brain residents, yet new research demonstrates that glial cells, particularly astrocytes and microglia, are active participants in sleep regulation and in sleep-immune interactions. For example, recent studies demonstrate that inhibiting vesicular release from astrocytes reduces EEG slow wave activity and attenuates sleep deprivation-induced increases in non-rapid eye movement (NREM) sleep. However, much remains to be learned about the relative contribution of astrocytes to mechanisms underlying alterations in sleep during immune challenge. The cytokines interleukin-1? (IL-1) and tumor necrosis factor-? (TNF) are involved in the regulation of sleep and in the alterations in sleep that occur during immune challenge. Importantly, IL-1 and TNF are produced by neurons and glia, their receptors are present on neurons and glia, and they constitute an important mechanistic link in neuronal-glial communication. Our knowledge about astrocyte contributions to sleep-immune interactions will be advanced if actions of this cell type can be specifically, independently, and reversibly modulated in vivo. Thus, the overall objective of this R21 Exploratory / Developmental Research Grant Award is to selectively stimulate G protein-coupled signaling cascades in astrocytes to elucidate their contributions to the regulation of sleep during immune challenge. To accomplish this task we will use designer receptors exclusively activated by designer drugs (DREADDs). DREADDs are engineered human muscarinic G protein-coupled receptors that are unable to bind their endogenous ligand, acetylcholine. Instead, they bind a chemically inert synthetic ligand, clozapine-N-oxide (CNO), which then stimulates intracellular signaling. To date, DREADDs have almost exclusively been used to modulate activity of neurons. The central hypothesis to be tested by experiments proposed in this application is that stimulating G protein-coupled signaling cascades in astrocytes will attenuate changes in sleep that are induced by immune challenge. We will use the Cre-loxP system, commercially-available GFAP:Cre mice, and adeno-associated virus vectors to selectively express DREADDs on astrocytes. We will then test our overall hypothesis by conducting experiments within the context of two Aims. In Aim 1 we will stimulate Gq or Gi G protein-coupled signaling in the absence of immune challenge and quantify regional changes in mouse brain cytokine protein. In Aim 2 we will activate these same signaling cascades and determine the impact of this manipulation on lipopolysaccharide (LPS)-induced alterations in mouse sleep patterns and brain cytokine protein. Upon completing these studies we will not only possess a new toolkit, but we will have novel data that will contribute to our understanding of mechanisms and functions for sleep-immune interactions. These achievements will have a prolonged and sustained impact on the field by filling a gap in our knowledge of sleep and gliotransmission during immune challenge.

PROJECT SUMMARY Alzheimer's disease (AD) is a disease of aging. AD is the most common form of dementia, afflicting more than 5 million Americans aged 65 and older. By 2050 it is estimated that more than 14 million Americans will suffer this disease, and that its direct financial impact will exceed $1.1 trillion. AD is particularly burdensome because it impairs memory; it worsens with time; and there is no cure. Sleep disruption in AD is highly prevalent, and changes in sleep architecture and circadian rhythmicity that result in excessive daytime sleepiness and nighttime insomnia are well documented. Less well known is the impact of sleep or circadian disruption on the etiology of the disease. Sleep facilitates A? clearance from brain, and sleep disruption increases A? in cerebrospinal fluid. A? pathology impairs core clock genes and exacerbates neuroinflammation. Collectively, these data suggest that sleep and circadian disruption induce responses that feed forward and contribute to, or exacerbate AD pathology and accelerate disease progression. However, to our knowledge definitive studies to determine the extent to which sleep disruption per se contributes to AD pathology have not been conducted. We will use mice expressing an inducible mutant amyloid precursor protein (APP) transgene to temporally dissociate sleep disruption and mutant APP expression from subsequent A? deposition and AD-like pathology. Specifically, we will: 1) determine how chronic sleep disruption of transgenic mice alters the course of pathology induced by expression of mutant APP; 2) determine if sleep disruption accelerates AD onset; and 3) target a key mediator of innate immune activation and determine effects on responses to sleep disruption and/or mutant APP expression. Outcome measures for each aim include assessments of cognitive performance; synaptic plasticity; differential gene expression; glial activation; cytokine production; neuroinflammatory signaling; and proteinopathy. Our multidisciplinary research team has demonstrated expertise and possesses all requisite skills to successfully complete the proposed project. Successful completion of this project will have a sustained impact on the field because we will elucidate the extent to which, and potential mechanisms by which, chronic sleep disruption alters the progression of AD-like pathology.