Joseph S. Takahashi, B.A., Ph.D. - US grants

Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. A fundamental question in the field concerns the molecular mechanism of circadian oscillators. How are circadian oscillations generated, and what components at the tissue, cellular or subcellular level are required for circadian properties? Our long-term objectives are to understand the cellular and biochemical events that underlie circadian rhythms among the vertebrates. The mechanisms that generate circadian rhythms will be studied in vitro using cultured vertebrate pineal tissue as a model system. The isolated pineal gland of birds and lizards contains circadian oscillators, with photoreceptive input, which regulate the rhythmic synthesis of melatonin. Immunocytochemical methods will be used to identify cell types that compose the gland. Photoreceptor-and melatonin-specific determinants will be used to identify cell types and to correlate structural information with functional properties. The biochemical events mediating the effects of light on melatonin biosynthesis will be defined with emphasis on the role of cyclic nucleotides. Light and pharmacological agents will be used to determine whether an oscillation of cyclic AMP controls the melatonin rhythm. To test whether cyclic AMP is a molecular component of the circadian oscillator itself, perturbations of cyclic AMO levels will be used to perturb parameters of the oscillator. If a molecular component is part of the timing mechanism, then a minimum of two conditions must be fulfilled: the level of the component must oscillate and perturbation of its level must perturb the circadian oscillation in a determinate fashion. Finally, the mechanism generating the circadian oscillation of intracellular cyclic AMP will be studied by measuring the enzymes that synthesize and degrade cyclic AMP. In two model systems, the vertebrate pineal and the molluscan eye, there exists compelling evidence for important regulatory roles of cyclic nucleotides in circadian function. The elucidation of the role of cyclic nucleotides in these systems should aid ultimately in the search for the molecular components of the circadian clock. An understanding of the biological basis of circadian rhythms in vertebrates may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunctions such as sleep disorders, mental health and endocrine abnormalities.

Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. A fundamental question in the field concerns the molecular mechanism of circadian oscillators. How are circadian oscillations generated, and what components at the tissue, cellular or subcellular level are required for circadian properties? Our long-term objectives are to understand the cellular and biochemical events that underlie circadian rhythms among vertebrates. The mechanisms that generate circadian rhythms will be studied in vitro using dissociated check pineal cells as a model system. Chick pineal cells contain circadian oscillators, with photoreceptive input, which regulate the rhythmic synthesis of melatonin. The phase-shifting effects of light on the circadian oscillator will be used as a physiological stimulus to study this pathway. Identification of the steps in the photic entrainment pathway should ultimately aid in identifying components of the oscillating mechanism because the pathway must end upon some component of the biological clock. The potential entraining effects of norepinephrine will be analyzed to determine whether an additional input pathway to the oscillator exists. If adrenergic input entrains the rhythm, then tracing this pathway will also aid in identifying components of the oscillator. The role of "G- proteins" in controlling the melatonin rhythm will be investigated. These regulatory proteins are in a pivotal position to regulate the spontaneous oscillation in cyclic AMP in chick pineal cells and therefore are prime candidates for generating these oscillations. The hypothesis that single pinealocytes are circadian pacemaker cells will be tested directly using single-cell measurement. These experiments will determine for the first time whether circadian oscillations in vertebrates are a cellular property. The possible role of calcium in mediating various processes within pineal cells will be explored. Finally, we will attempt to develop cell strains and cell lines of pinealocytes. These cell lines would provide a powerful new model system for studying the cell biology of circadian rhythms. Establishment of cell lines would enable biochemical, molecular and genetic approaches to be feasible in a vertebrate preparation. An understanding of the biological basis of circadian rhythms in vertebrates may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunctions such as sleep disorders, mental health and endocrine abnormalities.

Circadian rhythms regulate a number of diverse physiological functions in the vertebrate eye. Inspite of the significant body of information clearly documenting important physiological roles for circadian rhythms in the retina, very little is known about the molecular mechanisms that generate circadian oscillations in the eye. Evidence from a diverse set of experiments suggests that visual sensitivity, photoreceptor metabolism and turnover, retinomotor movements, indoleamine biosynthesis, and gene expression of photoreceptor transduction proteins all vary with a circadian rhythm. What accounts for these 24-hour rhythms that persist in the absence of environmental timing cues? Research in circadian biology has established that these rhythms are the manifestation of a physiological timing mechanism with the properties of a self-sustained oscillator. Physiological experiments have shown that circadian oscillators are located in three diencephalic structures: the suprachiasmatic nucleus, the pineal gland and the retina. Our long-term objectives are to understand the cellular and biochemical events that underlie the temporal regulation of rhythmic photoreceptor metabolism. Primary cultures of embryonic chick retina and human retinoblastoma cell lines will be studied to determine if either of these preparations exhibit properties of a circadian oscillator in vitro. The phase-shifting effects of light on melatonin release will be studied because identifying the photic entrainment pathway should ultimately aid in identifying the cellular components of the oscillator. The potential entraining effects of dopamine will also be analyzed to determine whether an additional input pathway to the oscillator exists. Using purified photoreceptor cultures, we will determine whether photoreceptors are the melatonin-synthesizing cells and whether these cells are also the locus of the retinal oscillator. These experiments will determine if circadian oscillations are a cellular property of retinal photoreceptors. In recent work we have discovered that retinoblastoma cell synthesize melatonin. We propose to define the biochemical events regulating melatonin in these cells. In addition we will explore the intriguing possibility that retinoblastoma cells express circadian rhythms. The identification of a human retinoblastoma cell line that oscillates would provide a novel system in which to study the cell biology of ocular clocks using biochemical, molecular and genetic probes. Ultimately, an understanding of the biological basis of ocular circadian rhythms may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions in the retina.

Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. A fundamental question in the field concerns the molecular mechanism of circadian oscillators. How are circadian oscillations generated, and what components at the tissue, cellular or subcellular level are required for circadian properties? Our long-term objectives are to understand the cellular and biochemical events that underlie circadian rhythms among vertebrates. The mechanisms that generate circadian rhythms will be studied in vitro using dissociated check pineal cells as a model system. Chick pineal cells contain circadian oscillators, with photoreceptive input, which regulate the rhythmic synthesis of melatonin. The phase-shifting effects of light on the circadian oscillator will be used as a physiological stimulus to study this pathway. Identification of the steps in the photic entrainment pathway should ultimately aid in identifying components of the oscillating mechanism because the pathway must end upon some component of the biological clock. The potential entraining effects of norepinephrine will be analyzed to determine whether an additional input pathway to the oscillator exists. If adrenergic input entrains the rhythm, then tracing this pathway will also aid in identifying components of the oscillator. The role of "G- proteins" in controlling the melatonin rhythm will be investigated. These regulatory proteins are in a pivotal position to regulate the spontaneous oscillation in cyclic AMP in chick pineal cells and therefore are prime candidates for generating these oscillations. The hypothesis that single pinealocytes are circadian pacemaker cells will be tested directly using single-cell measurement. These experiments will determine for the first time whether circadian oscillations in vertebrates are a cellular property. The possible role of calcium in mediating various processes within pineal cells will be explored. Finally, we will attempt to develop cell strains and cell lines of pinealocytes. These cell lines would provide a powerful new model system for studying the cell biology of circadian rhythms. Establishment of cell lines would enable biochemical, molecular and genetic approaches to be feasible in a vertebrate preparation. An understanding of the biological basis of circadian rhythms in vertebrates may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunctions such as sleep disorders, mental health and endocrine abnormalities.

Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. The long- term objectives of our research are to understand the cellular and molecular events that underlie circadian rhythms among the vertebrates. We have extensively studied the photoreceptive system that mediates entrainment to light in the golden hamster. Using these quantitative behavioral experiments as a framework, we have recently shown that a number of cellular immediate-early genes (IEGs) are induced by light in the suprachiasmatic nucleus (SCN) and that the induction of these IEGs is correlated with circadian behavior. Light exposure of hamsters during the subjective night causes a profound induction of c-fos and jun-B mRNA levels as well as AP-1 DNA binding in the SCN. the induction of c-fos mRNA is directly correlated with the phase-shifting effects of light on the circadian behavior of the animal in two ways. First, the photic induction of c-fos is phase-dependent: the ability of light to stimulate c-fos is restricted to those phases at which light can reset the circadian oscillator. Second, the photic induction of c-fos and the phase-shifting response have the same photic threshold. Taken together these experiments suggest that photic entrainment in mammals may involve transcriptionally regulated signal transduction processes. In this proposal we will investigate the role of IEGs in the SCN. Specifically, our goals are: 1) To characterize of the effects of light upon immediate-early gene expression int he SCN; 2) To identify neurotransmitters that regulate c-fos in the SCN; and 3) To describe how circadian gating of c-fos induction by light is regulated. The results from the proposed experiments should provide new information on the role of gene expression in the entrainment and generation of circadian rhythms in mammals, and should ultimately lead to an understanding of the molecular components of the mammalian circadian clock. An understanding of the biological basis of circadian rhythms in mammals may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions associated with circadian rhythm dysfunctions such as sleep disorders, mental health and endocrine abnormalities.

Circadian rhythms regulate a number of diverse physiological functions in the vertebrate eye. Inspite of the significant body of information clearly documenting important physiological roles for circadian rhythms in the retina, very little is known about the molecular mechanisms that generate circadian oscillations in the eye. Evidence from a diverse set of experiments suggests that visual sensitivity, photoreceptor metabolism and turnover, retinomotor movements, indoleamine biosynthesis, and gene expression of photoreceptor transduction proteins all vary with a circadian rhythm. What accounts for these 24-hour rhythms that persist in the absence of environmental timing cues? Research in circadian biology has established that these rhythms are the manifestation of a physiological timing mechanism with the properties of a self-sustained oscillator. Physiological experiments have shown that circadian oscillators are located in three diencephalic structures: the suprachiasmatic nucleus, the pineal gland and the retina. Our long-term objectives are to understand the cellular and biochemical events that underlie the temporal regulation of rhythmic photoreceptor metabolism. Primary cultures of embryonic chick retina and human retinoblastoma cell lines will be studied to determine if either of these preparations exhibit properties of a circadian oscillator in vitro. The phase-shifting effects of light on melatonin release will be studied because identifying the photic entrainment pathway should ultimately aid in identifying the cellular components of the oscillator. The potential entraining effects of dopamine will also be analyzed to determine whether an additional input pathway to the oscillator exists. Using purified photoreceptor cultures, we will determine whether photoreceptors are the melatonin-synthesizing cells and whether these cells are also the locus of the retinal oscillator. These experiments will determine if circadian oscillations are a cellular property of retinal photoreceptors. In recent work we have discovered that retinoblastoma cell synthesize melatonin. We propose to define the biochemical events regulating melatonin in these cells. In addition we will explore the intriguing possibility that retinoblastoma cells express circadian rhythms. The identification of a human retinoblastoma cell line that oscillates would provide a novel system in which to study the cell biology of ocular clocks using biochemical, molecular and genetic probes. Ultimately, an understanding of the biological basis of ocular circadian rhythms may lead to procedures useful in the diagnosis and treatment of pathophysiologic conditions in the retina.

Circadian rhythms regulate the function of living systems at virtually every level of organization - from molecular to organismal. Previous studies on this project have demonstrated that the photic threshold for phase-shifting a behavioral circadian rhythm in ld hamsters is increased about 20-fold, and similarly, the photic induction of a transcription factor, Fos, and the light-induced phosphorylation of CREB are also attenuated in the central circadian pacemaker located in the suprachiasmatic nucleus (SCN). Studies in the present project will build on these findings to examine in depth the extent of these age-related changes in response to light, and the chronology for the development of these changes. Other studies will determine if age-related increases in photic threshold are specific to the Fos/phase-shifting pathway, whether these changes influence the ability of animals to entrain to light cycles, and whether the age-related changes are due to changes in the expression of glutamate receptors in the SCN/ On addition, the laboratory mouse will be utilized as a model system to further study the mechanisms underlying age- related changes in the photic entrainment of mammalian circadian rhythms. The recent isolation of the Clock mutant mouse opens up a new set of questions that can be addressed with respect to interactions between the circadian system and aging. Of particular interest is the finding that the Clock mutation in the heterozygous condition has effects on rhythmicity that are similar to those observed in old wild-type mice. Thus, studies in the mouse will characterize in detail circadian changes associated with aging in mice, determine whether the Clock mutation affects these changes, either in degree or in time of appearance, and whether the clock mutation and aging alter the circadian clock system through similar mechanisms. The results from the proposed experiments should provide new information on the influence of aging on gene expression and its role in the entrainment and generation of circadian rhythms in mammals, and should ultimately aid in an understanding of the molecular components of the mammalian circadian clock.

The overall objectives of this proposal are to create a Center that will focus on large-scale ENU mutagenesis screens in five phenotypic domains relevant to the nervous system and behavior. We have carefully chosen to focus upon five phenotypic screens: 1) circadian rhythms, 2) fear conditioning, 3) vision, 4) neuroendocrine hormones, and 5) response to psychostimulants. In order for us to include a screen, we have established the following set of criteria: * the biological context of the phenotype must be mature and of significance to neuroscience; * the characterization of mutants in the phenotypic class is well established; * the phenotypic screen must be amenable to automation and scaling; * the initial screen must be capable of a throughput of at least 10,000 mice per year; * the investigators involved in the screens and their follow up must be leading experts in the field. Our aims are: 1. To conduct a large-scale, genome-wide, phenotype-driven ENU mutagenesis screen for recessive mutations that targets five domains influencing the nervous system and behavior. 2. To screen, isolate and characterize mutations that alter the circadian phenotype of mice. 3. To screen, isolate and characterize mutations that alter context- dependent and cued fear conditioning in mice. 4. To screen, isolate and characterize mutations that alter vision using three different methods: electroretinogram (ERG), visually evoked potentials (VEP) and fundus photography. 5. To screen, isolate and characterize mutations that alter the hypothalmic-adrenal (HPA) axis and the hypothalamic-thyroid (HPT) axis. 6. To screen, isolate and characterize mutations that alter the response of mice to psychostimulant treatment. 7. To act as a national resource for mouse mutants by providing rapid access to phenotypic screening analyses "online" so that mice are accessible to the greater scientific community. As the human genome project progresses and the sequences of more human and mouse genes are determined, the function of a large number of genes will not be predictable by sequence and expression alone. Phenotype-driven mutagenesis screens provide an important approach to understand the function of these genes.

DESCRIPTION (provided by applicant): The last decade has witnessed a revolution in our understanding of the molecular mechanism of circadian clocks in animals, including the identification of at least seven different genes that are essential elements of the circadian clock mechanism (Clock, Email, Perl, Per2, Cryptochromel, Cryptochrome2 and Casein kinase 1 epsilon). With the initial discovery of these clock genes came the realization and documentation that the capacity for circadian expression is widespread throughout the body. Most peripheral organs and tissues can express circadian oscillations in isolation, yet still receive and may require input from the dominant circadian pacemaker in the suprachiasmatic nucleus (SCN) in vivo. The existence of both central and peripheral circadian oscillators raises a number of novel questions and hypotheses concerning the integration of the system to control the behavioral state of the organism. These discoveries on the clock mechanism and the organization of the circadian system have provided a unique opportunity to apply cutting-edge technology to discover both chemical and genetic tools to manipulate circadian rhythms both in vitro and in vivo. Tissue-specific and conditional genetic tools will be used to determine the consequences of central vs. peripheral circadian rhythm dysfunction on behavioral state (Takahashi project). The discovery of small molecules (McKnight project) and molecular targets (Hogenesch project) will provide new tools for manipulating circadian rhythms. New alleles of the central clock components will be identified and characterized (Green project) which will provide new insight into the clock mechanism and will provide new ways to analyze the chemical and genetic tools identified in the McKnight and Hogenesch projects. These tools will also be analyzed at the cellular and organismal level (Menaker and Block project). Finally, circadian clock gene variants and circadian disorders in humans will be utilized to cross validate and analyze the chemical and genetic tools developed in the molecular, cellular and animal studies (Ptacek project). The discovery of molecules (small chemicals and genetic tools) should provide new opportunities for the eventual translation of our knowledge of circadian clocks to human behavior and disease.

[unreadable] DESCRIPTION (provided by applicant): Partial support is requested for the 15th Gordon Research Conference on Chronobiology, to be held on May 6- 11, 2007, at the Centre Paul Langevin, Aussois, France. In recent years, the significance of internal body clocks (circadian clocks) in regulating and adjusting temporal physiology has become increasingly clear and as a result, much effort has been devoted to the analysis of the mechanisms underlying circadian processes. Recognition of the key role played by circadian rhythms in human health and disease, combined with recent landmark discoveries regarding the molecular mechanisms that both generate and regulate the circadian system, has led to a remarkable expansion of the field. Today, chronobiological research attracts a host of researchers from a broad range of disciplines, including neuroscience, molecular genetics, photobiology, computational biology, and metabolism, as well as sleep medicine, psychiatry, oncology, and gerontology. The aims of the Gordon Conference on Chronobiology are: 1) To provide a forum for discussion and for identifying general principles in the rapidly expanding field of chronobiology. 2) To stimulate interactions among researchers working in the various sub-disciplines of chronobiology and thus enhance the coherence of the research field as a whole. 3) To intensify interactions between young and established researchers, and thus sustain high-quality chronobiological research. 4) To promote both national and international collaboration by bringing together scientists from many different research groups and countries. 5) To promote translational research by bringing together investigators employing model organisms with those studying human subjects in circadian research. The conference will achieve these goals by assembling approximately 150 scientists encompassing individuals from different countries, age groups, genders, and using different research models. The program will focus on new and emerging developments in key research areas (see Tentative Program). In accordance with the highly successful Gordon Conference format, lectures will be presented in 8 morning and evening sessions with ample time for discussion. Four poster sessions will allow participants to present additional data. Special attention has been given to achieving gender balance (see discussion leaders) and to including experts from very diverse disciplines to achieve a broadly-based understanding of the frontier of our science and insights as to future opportunities. Outcomes will have health relevance for ameliorating dysfunctions that manifest as sleep, affective, cognitive, neurological, metabolic, endocrine and oncological disorders and occupational health. Partial support is requested for the 15th Gordon Research Conference on Chronobiology, to be held on May 6-11, 2007, at the Centre Paul Langevin, Aussois, France. Conference objectives are to bring together a diverse group of leading researchers for a program focused on new and emerging developments in key research areas, in order to increase interactions, promote collaboration, and promote translational research. Outcomes will have health relevance for ameliorating dysfunctions that manifest as sleep, affective, cognitive, neurological, metabolic, endocrine and oncological disorders and occupational health. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Over the past decade, tremendous progress has been made in our understanding of the molecular mechanism of circadian clocks in mammals. Circadian oscillations are generated by a set of genes that form a transcriptional autoregulatory feedback loop. In mammals, there are at least seven different genes that have been proposed as 'core' circadian elements of the clock mechanism. Despite this remarkable progress, it is clear that a significant number of genes that regulate circadian rhythms in mammals remain to be discovered and identified. Forward genetic screens have been the most effective tool for circadian gene discovery, and we have used this approach to screen the mouse genome for circadian rhythm mutants in the Neurogenomics Project in the Center for Functional Genomics at Northwestern University. Over 60 new circadian mutants were identified as part of a large-scale ENU mutagenesis screen to create a mutant mouse resource for nervous system and behavioral phenotypes. Our specific aims are: 1. To identify the gene responsible for the 'Overtime' circadian period mutant in mouse. We will test the hypothesis that the 'Overtime' mutant represents a novel circadian rhythm gene. 2. To determine the genetic map position of twenty of the most robust circadian mutants isolated in our large- scale mutagenesis screen. To provide a more valuable research tool for the circadian research community, we propose to map 20 new circadian mutants. 3. To characterize and identify the 'swing-shift' mutant which is a novel entrainment mutant in mice. We will clone the 'swing-shift' mutant and test the hypothesis that 'swing-shift' mice have either a molecular or an anatomical lesion in the photic input pathway into the suprachiasmatic nucleus. Characterization of these novel circadian mutants and the identification of the causative genes should provide new insight into the mechanism of the mammalian circadian clock. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Partial support is requested for the 16th Gordon Research Conference on Chronobiology, to be held on July 19-24, 2009 at Salve Regina University, Newport, Rhode Island. The significance of circadian clocks in regulating temporal physiology has become increasingly clear and as a result, much effort has been devoted to the analysis of the mechanisms underlying circadian processes. Recognition of the key role played by circadian rhythms in human health and disease, combined with recent landmark discoveries regarding the molecular mechanisms that both generate and regulate the circadian system, has led to a remarkable expansion of the field. Today, chronobiological research attracts a host of researchers from a broad range of disciplines, including neuroscience, molecular genetics, photobiology, computational biology, and metabolism, as well as sleep medicine, psychiatry, and oncology. The aims of the Gordon Conference on Chronobiology are: 1) To provide a forum for discussion and for identifying general principles in the rapidly expanding field of chronobiology. 2) To stimulate interactions among researchers working in the various sub-disciplines of chronobiology and thus enhance the coherence of the research field as a whole. 3) To intensify interactions between young and established researchers, and thus sustain high-quality chronobiological research. 4) To promote both national and international collaboration by bringing together scientists from many different research groups and countries. 5) To promote translational research by bringing together investigators employing model organisms with those studying human subjects in circadian research. The conference will achieve these goals by assembling approximately 150 scientists encompassing individuals from different countries, age groups, genders, and using different research models. The program will focus on new and emerging developments in key research areas (see Tentative Program). In accordance with the highly successful Gordon Conference format, lectures will be presented in 8 morning and evening sessions with ample time for discussion. Four poster sessions will allow participants to present additional data. Special attention has been given to achieving gender balance (see discussion leaders) and to including experts from very diverse disciplines to achieve a broadly-based understanding of the frontier of our science and insights as to future opportunities. PUBLIC HEALTH RELEVANCE: Partial support is requested for the 16th Gordon Research Conference on Chronobiology, to be held on July 19th-July 24th, 2009, at Salve Regina University, Newport, Rhode Island. Conference objectives are to bring together a diverse group of leading researchers for a program focused on new and emerging developments in key research areas, in order to increase interactions, promote collaboration, and promote translational research. Outcomes will have health relevance for ameliorating dysfunctions that manifest as sleep, affective, cognitive, neurological, metabolic, endocrine and oncological disorders and occupational health.

DESCRIPTION (provided by applicant): Circadian rhythm is a fundamental property of all eukaryotic and some prokaryotic organisms, allowing the organism to adapt its cellular metabolism and physiology to the ~24 hr light-dark cycle of the earth. In mammals, circadian rhythm is generated by an autoregulatory transcription-translation feedback loop, the core components of which are CLOCK, BMAL1, Cryptochrome (CRY), and Period (PER) proteins. The heterodimeric CLOCK:BMAL1 transcriptional factor activates the transcription of genes Cry and Per, and is in turn inhibited by their protein products CRY and PER, ultimately creating rhythmic gene expression patterns. Circadian clock is estimated to affect the expression of at least 10% of all genes in human genome, thus influences many aspects of metabolism, physiology, and behavior. Currently, little is known about molecular interactions between CLOCK:BMAL1 and its negative regulator CRY and PER at biochemical and structural levels, and how such interactions modulate the activity of CLOCK:BMAL1. The goal of the proposed study is to elucidate molecular interactions between CLOCK:BMAL1, CRY, and PER, through rigorous in vitro biochemical, biophysical and structural investigations, and to delineate the structural mechanisms by which the transcriptional function of CLOCK:BMAL1 is modulated by PER and CRY. We will first generate various functionally relevant constructs of clock proteins based on functional and structural considerations (Aim 1). We have in hand the over-expressed and purified recombinant constructs of the structured regions of individual clock proteins and have determined the complex structure of CLOCK:BMAL1 in our preliminary studies. We will include in our investigation additional functional domains/motifs of clock proteins. CRY is the main repressor of CLOCK:BMAL1. We have demonstrated that CRY forms stable complex with CLOCK:BMAL1 in vitro. In Aim 2, we will determine the structure of CRY:CLOCK:BMAL1 complex to learn how CRY interacts with and represses the transactivation function of CLOCK:BMAL1. In the last Aim (Aim 3), we will investigate the interactions of PER with other clock components, CRY:PER and PER:CLOCK:BMAL1, by in vitro and in vivo protein-protein interaction assays, structure determination of functionally relevant complexes, as well as mutagenesis and functional analysis. We hope to learn the distinct role of CRY and PER in the mammalian clock mechanism and how they work together with CLOCK:BMAL1 to generate and maintain circadian rhythm in mammals.

DESCRIPTION (provided by applicant): Caloric restriction (CR) is one of the few regimens that enhances longevity in mammals, yet the mechanisms underlying this beneficial effect have been elusive. A confound in the majority of CR studies is that temporal restriction of food intake almost always accompanies caloric restriction. Temporal restriction or restricted feeding (RF) is known to be a potent entraining stimulus for circadian rhythms in mammals and can shift the entire circadian metabolic profile of peripheral organ systems such as the liver. Interestingly, CR combined with RF has been shown to be an even more potent entraining signal and can reset both the central pacemaker in the suprachiasmatic nucleus (SCN) as well as circadian oscillators in peripheral tissues. Given that deterioration of circadian rhythms is one of the hallmarks of aging and given that circadian disruptions in both human and animal models lead to cardiovascular and metabolic disorders, such temporal disruptions of circadian order (coherence and synchrony of internal rhythms) could contribute significantly to aging and longevity. In preliminary data we have found that RF has wide-ranging and complex effects on genome-wide circadian transcriptional architecture, gene expression and RNA polymerase II recruitment and initiation as well as chromatin modifications involving histone methylation and acetylation. These changes result in large shifts in the phases of rhythmic gene expression patterns and impact pathways that are central to the aging process including energy utilization and metabolic pathways, insulin signaling, mTOR signaling, xenobiotic detoxification, and ubiquitin mediated proteolysis. Lowered circadian amplitude and inappropriately phased rhythms are hallmarks of aging, and treatments that improve circadian function have been linked to well-being and longer lifespan. Therefore, it is possible that the beneficial effects of caloric restriction paradigms originates partially or fully from the temporal restriction of food intake, rather than the reduction in calories. Thus, we hypothesize that synchronization of central and peripheral oscillators during caloric restriction improves hormonal, biochemical and physiological functions, which can then lead to attenuation of aging and increased life span. In these experiments we will generate comprehensive circadian profiles of circadian clock transcription factor binding, gene expression and chromatin modifications to assess genome-wide changes that occur during the aging process. We will use an experimental design that distinguishes the contributions of caloric restriction from those of temporal restrictions. Analyse of the circadian transcriptional landscape as a function of aging using Hidden Markov Models and correlation matrices will provide a foundation for discovery of genomic and epigenomic signatures and mechanisms critical to circadian rhythms, aging and longevity.

In mammals, behavioral and physiological processes display 24-hr rhythms that are controlled by circadian oscillators located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN acts as a master pacemaker at the top of a hierarchy of circadian oscillators distributed throughout the body. Although the SCN is a relatively small nucleus in the brain containing about 10,000 neurons on each side, it is composed of many cell types. Two major classes of neuropeptide-containing neurons, VIP (vasoactive intestinal polypeptide) and AVP (arginine vasopressin), are enriched in the ?core? and the ?shell? regions of the SCN, respectively. It is known that the VIP and AVP neurons can subserve different functions, however, it has not been possible to study genetically identified cell types in the SCN in real time at the cellular level. We have developed a new generation of bioluminescent circadian reporter mice that are Cre-lox recombination dependent. Using cell-type-specific Cre drivers, these Cre-lox dependent reporters can be activated in restricted and genetically defined cell populations so that circadian properties of these cells can be studied separately from other cell types. In this proposal, we will analyze the cell-type specific circadian properties of VIP and AVP neurons within the SCN neuronal network by using a ColorSwitch PER2::LUCIFERASE reporter that has a click beetle red (CBR) luciferase fused to PER2 that switches to a click beetle green (CBG) luciferase upon Cre-lox recombination. With the cell-type restricted reporter, we can study for the first time the circadian properties of each of these neuropeptide classes of neurons in the SCN. Here we will study the role of VIP and AVP neurons in controlling circadian behavioral phenotypes using both loss-of-function and gain-of-function circadian mutations in these cell classes. We will also use optogenetic control of VIP and AVP neurons to analyze the dynamics of resetting within the SCN neuronal network. Finally, we will use single-cell RNA-seq of SCN cells to classify SCN cell types and in labeled VIP and AVP neurons in order to determine the molecular signatures and pathways characteristic of these two cell types. Together, these experiments will provide critical new information on the circadian properties and dynamics of the SCN in order to promote our understanding the circadian system in mammals, which is critical for understanding how circadian disruption in humans contributes to morbidity associated with neurological disorders, cognition, mental health, obesity, type 2 diabetes and cancer.

Project Summary This is a resubmission of a new competitive proposal for an NHLBI T32 training grant. Sleep/wake and circadian rhythms involve state changes of multisystem biological function that may not in themselves be vital to life but the systems under this modulatory control are vital to life. Further, optimal function of these systems, required for healthy living, is severely compromised in the absence of these intricately organized state changes. The regular and/or rhythmic occurrence of state changes is evolutionarily preserved and adverse outcomes in a variety of organ systems, including CNS, cardiovascular, respiratory, immune, and metabolic systems are associated with their disruption. However, the mechanisms, the mechanisms? integration and both qualitative and quantitative impact of the state-change related modulation and overall health of the organism (including humans) is not well understood. An overall goal of this T32 proposal is to prepare a group of pre-doctoral and post-doctoral researchers to engage in the challenging endeavor of effective investigation to help remedy this vitally important gap in our biological understanding. Not only will the trainees receive in depth exposure and experience in effective application of multiple and integrated state of the art technologies and experimental approaches from world leaders, but additionally, strong integrative and collaborative interaction and facilitation of this interaction will be generated from our proposed structure within the newly developed Peter O?Donnell Brain Institute. This will enhance the trainee?s ability to engage in independent development of successful research programs that truly span the usual gulf from bench to bedside in a manner that integrates multiple biological systems across behavioral states as demanded by effective sleep/wake and circadian research. Finally, trainees will receive mentored experience in grant writing and in the communication and publication of integrative form of research and career counseling to enabling successful pursuit of a sleep and circadian research oriented career.

Cellular pathways that have been implicated in aging and longevity are regulated by the circadian clock. In addition, we have found that overall circadian gene expression in the mouse declines significantly with aging, both in the number of significantly cycling genes as well as the amplitude of their oscillations. Since longevity pathways are under circadian regulation, we hypothesize that age-related changes in circadian function can lead to a decline in multiple longevity pathways. Thus, interventions that could reduce or rescue the decline in circadian rhythmicity would be expected to maintain healthy longevity pathways. Since the circadian clock regulates many longevity pathways, interventions at this level could rescue or reverse the effects of aging on many independent longevity pathways which could have additive or synergistic benefits by targeting a single nodal point (the circadian clock). Thus, the overall theme of this application is that the key pathways that have been implicated in aging and longevity are under circadian regulation and that the master transcription factors, CLOCK:BMAL1, regulate these circadian cycles. We propose to use two different types of ?circadian interventions? in order to test whether these interventions can increase lifespan and healthspan in mice. In order to use a more genetically diverse mouse model, we will use UM- HET3 (HET3) mice that are being employed in the NIA Intervention Testing Program. First, we will characterize the circadian phenotypes of HET3 mice as well as the four parental inbred strains that contribute to HET3 mice. Then we will use two different types of circadian interventions: 1) Time-restricted feeding (12-hr and 8-hr); and 2) Loss of function of the Clock gene (Clock knockout mice) vs. over-expression of wild-type Clock gene expression using Clock BAC transgene mice. Because we have shown a profound decrease in the number cycling genes and their amplitude with aging, and because CLOCK:BMAL1 regulates these cycling genes, we will test the hypothesis that enhancement Clock gene expression can lead to a rescue of the age-related decline in circadian gene expression and that enhancement of Clock gene expression can extend healthspan and lifespan.

PROJECT SUMMARY People and other animals learn many of their complex and socially oriented behaviors by imitating more experienced individuals in their environment. Vocal imitation is one of the more striking and readily quantifiable examples of this type of learning, but the genetic basis of this complex trait is still poorly understood. The goal of this research is to determine the genetic basis of vocal imitation abilities by establishing the first mutagenesis screen in a vocal learning species and the genetic tools for independently testing the function of the identified genes by developing novel transgenic models using germline gene targeting technologies. Humans are the only primate and one of only a handful of mammalian species to have evolved the facility for vocal imitation. Aside from humans, songbirds, and in particular zebra finches, are the best studied vocal learning species and they provide the only practical platform for systematically identifying the genes involved in this important social behavior. Like speech, zebra finch song is a culturally transmitted behavior learned via imitation. Moreover, functional, genetic and molecular parallels underscore the use of zebra finch for identifying genes essential for vocal imitation. We hypothesize that a forward genetic dominant screen, followed by the detailed genetic mapping and manipulations developed through this proposal, will identify convergent and divergent genetic signatures for this polygenic trait. Establishing a forward genetic screen and the genetic tools for verifying gene function in zebra finches will provide a novel, comprehensive, and broadly impactful approach for trying to understand the genetic basis of vocal and social communication.