Carl Hirschie Johnson, PhD, Stanford University - US grants

Humans and other eukaryotic organisms manifest circadian (daily) rhythms which are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanisms of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, the nature of the underlying biochemical mechanism remains elusive. Does it use or depend upon a known metabolic pathway, perhaps in a heretofore unsuspected way? Or is the circadian pacemaker driven by a totally unknown system? The biochemical mechanism of circadian oscillators is the most fundamental unanswered question in this field. The long-term objective of this research project is to clarify the cellular/molecular nature of circadian clocks in unicells. The strategic approach might be termed an analysis of the oscillator's inputs and outputs. The input pathway by which the clock is synchronized to the daily light/dark cycle will be identified by action spectroscopy, mutations, and pharmacology. The output pathways of the clock will be determined by a variety of techniques. For example, rhythmic proteins and mRNAs will be assayed by electrophoresis and nucleic acid hybridization. "Second messenger" output will be assayed by microelectrode recording, calcium-sensitive photoproteins, and radioimmunoassay of cyclic nucleotides. In addition, the possibility that these second messengers may be an integral part of the oscillator's mechanism will be tested by pharmacological analyses. Simple unicellular organisms offer technical advantages which should accelerate the identification of cellular processes involved in the circadian oscillators of all eukaryotes.

This award supports a two-year collaborative research project between Professor Carl Johnson of Vanderbilt University and Takao Kondo of the National Institute of Basic Biology (NIBB), Okazaki, Japan. Also involved are Professor Susan Goldin of Texas A&M University and Masahiro Ishiura of NIBB. The goal of the project is to elucidate the molecular mechanism of circadian rhythms. Circadian rhythms are an adaptation of organisms to the regular daily change of their environment. Although much is known about the formal properties of circadian rhythms that allow them to synchronize to the daily light/dark cycles, almost nothing is known about how these rhythms work on the biochemical level. Two approaches will be utilized. First, the investigators will use the simplest organism now known to express circadian rhythms -- prokaryotic cynaobacteria. Second, they will exploit the genetic tools available in this simple cell to analyze its mechanism. A major effort will be to identify the photoreceptor that is involved by action spectroscopy. This study will rely on the use of the Okazaki Large Spectrograph and Dr. Kondo's video camera apparatus to screen hundreds of colonies for specific manifestations of circadian behavior.

Humans and most other organisms manifest circadian (daily) rhythms which are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, the nature of the underlying biochemical mechanism remains elusive. Does it use or depend upon a known metabolic pathway, perhaps in a heretofore unsuspected way? Or is the circadian pacemaker driven by a totally unknown system? The biochemical mechanism of circadian oscillators is the most fundamental unanswered question in this field. This project will elucidate the molecular mechanism of this fascinating behavior by identifying molecular components of the circadian biological clock in a simple unicellular organism which can be manipulated genetically. The control pathway by which the circadian clock regulates the "cab" gene will be analyzed. A reporter gene will be attached to the promoter of the cab gene so as to create an easily-manipulatable assay of this gene's expression. Other genes which are involved in the clock's central mechanism will be identified and cloned. The input photoreceptor pathway will be characterized by investigating the light-induced phase resetting of mutants of the phototransduction pathway. Simple unicellular organisms offer technical advantages which should accelerate the identification of cellular processes involved in the circadian oscillators of an organisms.

Circadian rhythms are an adaptation of organisms to the regular daily change of their environment. Although much is known about the formal properties of circadian rhythms which allow them to synchronize to the daily light\dark cycles, almost nothing is known about how these rhythms work on a biochemical level. This project is two-fold. First, the simplest organism now known to express circadian rhythms will be used namely, prokaryotic cyanobacteria. Second, genetic tools will be used to analyze its mechanism. A reporter gene encoding a bioluminescence enzyme has been attached to an endogenous promoter. Not only does this construct make these cyanobacteria bioluminescent, the glow meets all of the salient criteria of circadian rhythmicity| This is the first "artificial" overt rhythm of a circadian clock yet described. This unique case of circadian gene expression will be exploited to: (1) identify by action spectroscopy the photoreceptor involved in the clock's entrainment; (2) analyze the control pathway by which the circadian clock regulates this "artificial rhythm; and (3) search for genes which are involved in the clock's central mechanism. Using a simple prokaryotic system should accelerate the identification of cellular mechanisms by which circadian oscillators operate. %%% Most organisms respond to the cycles of light and dark by physiological mechanisms that are not well understood. Plants respond to these cycles by turning on and off certain genes. By genetic manipulation these investigators have introduce a light emitting enzyme gene into a gene that is turned on by a daily light-dark cycle. Thus after light exposure these bacteria will begin to glow as the gene is turned on. This novel technique will allow the investigators to better understand the biochemical genetic mechanisms by which organisms respond to light-dark cycles.

This research concerns daily biological clocks, which are an important component of the physiology of humans and other organisms. For example, medical and psychiatric studies have shown that these biological clocks are involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of these clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, the nature of the underlying biochemical mechanism is unknown. The research strategy is to study the molecular and genetic nature of biological clocks in two model organisms: one prokaryotic, and the other eukaryotic. The immediate aims of his research are (I) to manipulate genetically the model organisms so that they express rhythms which can be easily screened, (2) to identify and clone genes which are involved in the timing mechanism, (3) to characterize the input pathway (light) by which this clock is synchronized to environmental time, and (4) to track the output pathways of the clock "upstream" to discover the clock mechanism itself. The long-term research goal is to understand the mechanism of this fascinating clockwork.

INT 9605193 Johnson This U.S.-Czech research project between Carl H. Johnson of Vanderbilt University and Ivana Machackova of the Czech Institute of Experimental Botany will examine the chemical basis of plant flowering. Results should contribute to our basic understanding of the photoperiodic time measurement (PTM) system and the process of information transfer to flowering tissue, meristems, in plants. Specifically, Drs. Johnson and Machackova plan to use the tobacco plant as the model system because much is known about PTM in tobacco and a variety of strains in the same genus display different photoperiodic phenotypes. The researchers will build this inquiry upon recently discovered information about melatonin, a crucial hormone for PTM found animals, and new evidence suggesting that cytosolic free calcium fluxes are involved in the phototransduction pathway. Since melatonin is similarly found in higher plants, this project will attempt to determine the mechanisms controlling the circadian production of melatonin in plants and the roles, if any, played by melatonin and/or cytosolic free calcium in photoperiodic induction of flowering in higher plants. This project in cell biology fulfills the program objective of advancing scientific knowledge by enabling leading experts in the United States and Eastern Europe to combine complementary talents and pool research resources in areas of strong mutual interest and competence. ??

DESCRIPTION (Adapted from applicant's abstract): Humans and most other organisms manifest circadian (daily) rhythms which are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, clues to the nature of the underlying biochemical mechanism are only just beginning to emerge. So far, these clues suggest that the mechanism may have important similarities in organisms as diverse as fungi, fruitflies, and mammals. However, we still do not know whether the circadian pacemaker depends upon a known metabolic pathway, or if it is driven by a totally unknown system. The biochemical mechanism of circadian oscillators is the most fundamental unanswered question in this field. Our approach will elucidate components of the molecular mechanisms of circadian clocks by using model systems that will allow specific technological approaches to this important question. We have found oscillations of the global cellular regulator, ionic calcium, and we will ascertain the role of these oscillations at cellular and subcellular levels using organisms expressing a transgene of a luminescence calcium indicator, the photoprotein aequorin. We will also isolate and characterize the expression patterns of clock genes. These studies will allow us to identify molecular components of the circadian biological clock.

Organisms at all levels of biological complexity manifest circadian (daily) rhythms which are controlled by an endogenous biochemical oscillator. The biochemical nature of these biological clocks has been elusive, but one principle which has emerged is that their salient properties (persistence, temperature compensation, and entrainment) are conserved in all organisms in which circadian behavior has been observed, from bacteria to mammals. This principle has encouraged the hope that the biochemical mechanism has also been conserved- if not in exact homology, then at least in terms of the general components and composition of the clock. In this project the approach to unveiling the mechanism of circadian oscillators focuses on the least complex and most technically approachable organism in which a biological clock has been demonstrated: the cyanobacterium, Synechococcus sp. strain PCC 7942. The technical advantages of this organism are its small genome, which is easily manipulated genetically, and its bioluminescent strain, which offers a highly flexible and facile system of monitoring circadian gene expression.. This cyanobacterial system provides excellent tools for detailed molecular/genetic analyses and for clock investigations. The project will use this system to address three aspects of biological rhythmicity: (i) an assessment of the fitness advantage that cyanobacteria derive from their circadian oscillators, (ii) an analysis of the roles of clock gene products in the central clock mechanism, and (iii) characterization of the mechanism by which the oscillator controls metabolic pathways.

Many physiological and cellular processes, including sleep cycles, body temperature maintenance, homeostatic functions, gene expression, cell division, and enzymatic activities, are regulated by biological clocks. In addition, these clocks are also the timers that measure the daylength in photoperiodic timing of reproductive processes in plants and animals. The biochemical mechanism of circadian clocks is of fundamental biological interest; understanding it may lead to insights which will be useful to society in many ways.

This research concerns daily biological clocks, which are an important component of the physiology of humans and other organisms. For example, medical and psychiatric studies have shown that these biological clocks are involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of these clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders which are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, the nature of the underlying biochemical mechanism is unknown. The research strategy is to study the molecular and genetic nature of biological clocks in two model organisms: one prokaryotic, and the other eukaryotic. The immediate aims of his research are (I) to manipulate genetically the model organisms so that they express rhythms which can be easily screened, (2) to identify and clone genes which are involved in the timing mechanism, (3) to characterize the input pathway (light) by which this clock is synchronized to environmental time, and (4) to track the output pathways of the clock "upstream" to discover the clock mechanism itself. The long-term research goal is to understand the mechanism of this fascinating clockwork.

DESCRIPTION (Applicant's abstract reproduced verbatim): Protein-protein interaction is a key method by which biological events are regulated. We have developed a new method for measuring protein interactions that overcomes limitations of previous assays. This method, which we call Bioluminescence Resonance Energy Transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and an acceptor fluorophore fused to another protein of interest. If the two candidate proteins interact so as to bring the luciferase and fluorophore in proximity, resonance energy transfer can occur. This interaction is measure as a shift in the color of the bioluminescence emission. BRET will be particularly useful for testing protein interactions within the native cells, especially with integral membrane proteins or proteins targeted to specific organelles. We will apply this method to our topic of primary research interest, "biological clocks," and to general applications for the determination of protein-protein interactions within cells and organelles. Biological clocks are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, clues to the nature of the underlying biochemical mechanism are only just beginning to emerge. Recent investigations report that protein interactions play key roles in circadian clock mechanisms in eukaryotes. Using well-characterized interacting proteins and proteins encoded by clock genes, we will use the BRET system to 1) test whether results obtained by previous methods for assessing protein interactions can be confirmed with the BRET method, and 2) assay clock protein interactions in situ over the daily cycle to appraise temporal control of protein interaction.

DESCRIPTION (provided by applicant): Daily biological clocks are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, alertness, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. Despite the importance of clocked phenomena, however, clues to the nature of the underlying biochemical mechanism are only just beginning to emerge. Recent investigations report that protein-to-protein interactions play key roles in circadian clock mechanisms in prokaryotes and eukaryotes. We will investigate the temporal and spatial control of clock protein interaction, primarily using a new method that we developed that is optimal for studies of clock protein interactions. This method, which we call Bioluminescence Resonance Energy Transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and an acceptor fluorophore fused to another protein of interest. If two candidate clock proteins interact so as to bring the luciferase and fluorophore into proximity, resonance energy transfer occurs that can be measured as a shift in the color of the bioluminescence emission. BRET will be particularly useful for testing clock protein interactions and spatial distributions within native cells that exhibit daily rhythms in culture. Using proteins encoded by clock genes for which evidence exists of interaction, we will use the BRET system (and other confirmatory techniques) to assay clock protein interactions in situ, both in short term experiments and also over the daily cycle to appraise temporal control of protein interaction of clock proteins in prokaryotes, Drosophila, and mammals. Our hypothesis is that clock protein interaction will not be simply a function of the protein abundance, but that there will be phase-specific interaction regulated by other factors than merely the proteins' abundance.

DESCRIPTION (provided by applicant): Humans and most other organisms manifest circadian (daily) rhythms that are controlled by an endogenous biochemical oscillator. Many cellular processes, including cell division, enzyme activity, and gene expression, are timed by this oscillator. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, and insomnia. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. The salient properties of circadian clocks-24 hour time constant, high precision, temperature compensation-are presently impossible to explain biochemically. Although recent breakthroughs in the field of circadian rhythms have identified a number of proteins that appear to act as clock components, we have only just begun to understand how these components interact functionally with themselves and the environment. In model systems, it has been possible to reset the phase of circadian rhythms by induction of clock protein synthesis at specific phases. This approach has been difficult to accomplish in mammalian systems. The current project will test hypotheses concerning the significance of rhythmic clock protein abundance in mammals by using new methods to introduce proteins directly into cells by peptide-mediated transduction across cell membranes. This technology will allow us to modulate the intracellular concentration of clock proteins in cells, tissue slices, and intact animals. These studies will yield results of theoretical importance, but also have the potential for designing treatments for jet lag, insomnia, and other clock-related disorders. This project is appropriate for the NIMH Exploratory/Developmental Grant (R21) Program because it fulfills all of the following primary criteria: (1) innovative research directions, (2) exploration of approaches that are new to a substantive area, and (3) development of new technologies and methods.

Abstract for NSF Proposal 0140522 - "Equipment for Automated Acquisition of DNA Sequence and DNA Fragment Size Data"

A grant has been awarded to Dr. David E. McCauley at Vanderbilt University to support the purchase of an automated DNA sequencer, a DNA extraction system, and supporting computer hardware. The BaseStation gathers DNA sequence data from slab gels and stores it in a form that can be interpreted by sequence analysis software. The machine can also gather and process other types of DNA fragment size data such as microsatellite DNA genetic markers. In the case of both DNA sequence and microsatellite analysis, products of polymerase chain reaction (PCR) are labeled with a florescent dye and separated by size during electrophoresis. The advantage of the BaseStation is that its 96 well capacity and automated loading allows for high throughput, and its configuration makes it equally suited for gathering sequence and microsatellite data. The associated DNA extraction system will allow for rapid extraction of genomic DNA from a large number of samples. The data gathered by these machines will be used by five P.I.'s (D. McCauley, J. Burke, D. Funk, C. Johnson and N.O. Pellmyr) in a variety of studies in evolutionary and population biology which rely on large quantities of DNA sequence and/or microsatellite data.
Some of the specific research projects that will make use of data generated by the equipment include the following. 1) A study designed to use both DNA sequence information and polymorphic microsatellite markers to investigate the population genetics of Silene vulgaris, a plant invasive in North America. Comparison of the genetic characteristics of the plant in North America and in its native Europe should indicate the mechanism or route of invasion. 2) A comparative molecular systematics study of several species of yucca plants and of the moths that pollinate them. This phylogenetic study will help document how highly coevolved plant-pollinator systems develop. 3) A study using polymorphic microsatellite loci that will help to map genes associated with the domestication of sunflowers from their wild relatives. 4) A DNA sequence based study of adaptive radiation and host plant shifts in the Neochlamisus bebbianae beetle complex. 5) A study of the adaptive significance of biological clock genes that follows changes in the frequency of molecular markers in model laboratory populations of cyanobacteria.
All of these studies will contribute to understanding basic biological processes such as range expansions, coevolution, host plant shifts and the evolution of the biological clock. However, knowledge coming from these studies will also contribute to the solution of more applied problems such as how invasive species might be controlled, how insects shift feeding from native plant species to plants of economic value, how the genetic manipulation of domesticated species might be made more efficient, or how we might overcome health problems associated with biological rhythm disorders. Finally, the results of all of the studies will contribute to the emerging field of bioinformatics, in that each study will require the P.I. to refine methods of gathering, storing, and analyzing a large volume of genetic information.

DESCRIPTION (provided by applicant): Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro; this remarkable demonstration has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. Moreover, the crystal structures of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of this circadian pacemaker marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by genetic, biochemical, structural, and phylogenetic approaches. Three critically important unanswered questions in chronobiology are to explain how a biochemical mechanism (i) can be temperature compensated, (ii) keep time so precisely over such a long time constant (~24 h), and (iii) modulate chromosomal topology to confer output rhythms of gene expression. This project will face these issues head-on. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants of KaiC, KaiB, and KaiA in vivo. These mutations will be mapped onto the 3-D structures of the proteins to generate specific hypotheses that will be tested by novel in vitro biochemical analyses and targeted mutations. The rate constants and other biochemical data that result from the analyses of these mutants will be integrated with our previous data to generate a mathematical model that accounts for the 24 h time constant of the in vitro oscillator. The hypothesis that KaiC monomer exchange is responsible for the genetic dominance/recessive relationships observed for co-expression studies in vivo will be tested by biophysical/biochemical analyses and visualization of monomer exchange by cryo-electron microscopy. Finally, the linkage between this core clockwork and the global orchestration of gene expression over the daily cycle will be illuminated by isolating the KaiC-containing clock protein complex (chronosome) from intact cells and testing the hypothesis that the biochemical action of KaiC and/or the chronosome is that of a DNA/RNA helicase.

Biological Clocks; CRISP; Clock, Biologic; Computer Retrieval of Information on Scientific Projects Database; Funding; Genes; Genetic Polymorphism; Grant; Human; Human, General; Individual; Institution; Investigators; Man (Taxonomy); Man, Modern; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nurses; Personnel, Nursing; Polymorphism (Genetics); Polymorphism, Genetic; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Testing; United States National Institutes of Health; body clock; day shift; internal clock; night shift; night work; polymorphism; preference; shift work

[unreadable] DESCRIPTION (provided by applicant): Summary fluxes of free calcium ions (Ca++) are a universal regulator of biological processes and signaling in animal, plant, and microbial cells. Quantification of these Ca++ fluxes is crucial for understanding these central regulatory & signaling pathways. Currently, fluorescence-based probes are the preferred methods for measuring Ca++ levels, however these methods suffer from problems associated with fluorescence excitation, such as autofluorescence, phototoxicity, photobleaching, and poor penetration of tissue. Luminescence methods are an alternative approach that avoids these problems. This project will develop and characterize novel luminescence probes that are genetically encodable and quantify [Ca++] ratiometrically. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by Ca++-sensitive linkers so that the spectrum of emission is dependent upon Ca++ concentration. This Ca++ probe will be ideal for applications where autofluorescence, photobleaching, tissue penetration, and undesirable phototransduction & phototoxicity are problems. Using a very sensitive CCD camera coupled to a microscope through a Dual-View, Ca++-fluxes will be imaged from a variety of tissues and cells. In addition to developing and characterizing this new generation of luminescent Ca++ probes, the use of these reporters will be applied to the study of Ca++-fluxes in biological clocks (circadian rhythms). Circadian (daily) rhythms are a crucial component of human mental and physical health that regulates sleep, alertness, hormones, and many other biological processes. These rhythms are strongly implicated in some types of depression and in sleep disorders such as hypersomnia. The new Ca++ probes will be used to study circadian oscillations of Ca++ fluxes. This project is appropriate for the Exploratory/Developmental R21 Bioengineering Program because it proposes to develop new molecular probes for measurement and imaging of function as related to ubiquitous Ca++ signaling. Public Health: This project will develop new probes for Ca++ fluxes that will be optimal for conditions under which currently available methodology is limited. As a test case, these probes will be applied to the topic of biological clocks that have an important influence over mental and physical health. [unreadable] [unreadable] [unreadable]

Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolic activities of various tissues, and many other biological processes. The ultimate explanation for the mechanism of circadian oscillators will require characterizing the structures, functions, and interactions of the molecular components of these clocks. The current project is to elucidate the basic principles of circadian clocks at a biophysical/molecular level in a model system, the prokaryotic cyanobacteria, where genetic/biochemical studies have identified three key clock proteins, KaiA, KaiB, and KaiC. These three proteins can reconstitute a circadian oscillator in vitro. This remarkable observation has led to a re-evaluation of our understanding of circadian clocks in all organisms, including mammals. The crystal structure of the KaiA, KaiB, and KaiC proteins have been reported-these are the first clock proteins to have their 3-D structures determined. The advent of atomic resolution structures of the molecular components of a clockwork marks a dramatic watershed in circadian research by ushering in truly molecular analyses of circadian mechanisms. The current project will determine the molecular basis of the core clockwork by biophysical, genetic, and structural approaches. In particular, the relative roles of (i) the rhythmic formation of a KaiA/KaiB/KaiC complex as compared with (ii) the rhythm of KaiC phosphorylation as the key cogs in the timing mechanism will be assessed. The biophysical/molecular analyses will be coupled with mathematical modeling to determine the essential parameters. Temperature compensation of this biological clock will be investigated by screening for temperature dependent mutants in vivo followed by in vitro analyses. Finally, the linkage between this core clockwork and the global orchestration of gene expression over the daily cycle will be illuminated by testing the novel "oscilloid" model, which proposes that the core clockwork rhythmically regulates promoter activity by modulating chromosomal topology as a function of circadian time. RELEVANCE TO PUBLIC HEALTH: This project will clarify circadian mechanisms at molecular levels that were heretofore unattainable. Biological clocks have been found to be crucial for mental health. In addition, biological clocks are key for optimal performance under shiftwork and "jet-lag" conditions that affect a large proportion of the USA workforce. Knowledge of the circadian mechanism along with the development of therapies to properly phase sleep will allow us to enhance the performance, alertness, and well-being of shiftworkers and travelers in addition to improving the quality of life for depressed subjects.

[unreadable] DESCRIPTION (provided by applicant): Hypersomnia is a sleep disorder in which patients have a prolonged nighttime sleep episode from which it is difficult to awaken and/or excessive daytime sleepiness. Hypersomnia can have a number of underlying causes, some of which are related to improper phasing of the daily biological clock, such as in the cases of Delayed & Advanced Sleep Phase Syndromes. In addition, there are temporary hypersomnias associated with shiftwork and jet lag that are also related to the daily clock. Both recurrent and temporary hypersomnias, while not life threatening, can have serious consequences. This project will discover therapeutics for treating the subset of hypersomnias that are related to the biological clock. At the present time, there are practically no pharmacological treatments that can be used to specifically manipulate these biological clocks. Novel chemical compounds that directly affect the phase and period of biological clocks in mammalian cells for use as "chronotherapeutics" will be identified by using the newly developed system of mammalian fibroblasts that are stably transfected with luminescence reporters so that they glow rhythmically under the behest of their biological clock. These cells will be used in an automated high- throughput screen to identify compounds from the Vanderbilt Institute of Chemical Biology's library of ~150,000 chemically synthesized compounds that reset the phase and/or modulate the activity of the biological clock. Subsequent screens will test the efficacy of candidate compounds on the clock in brain and other tissue slices in vitro and intact mice in vivo. This project is appropriate for the NINDS Exploratory/Developmental Grant (R21) Program in Translational Research because it will develop screens at cellular, tissue, and organismal levels (including high-throughput screens) for discovering candidate therapeutics that will lead directly to the development of therapies for hypersomnias and other sleep disorders that are clock-related. TO PUBLIC HEALTH: This project will provide pharmacological tools for manipulating the phasing of the biological clock systems in mammals that will lead to the identification of a chrono- pharmacopoeia for treating recurrent and temporary hypersomnias (and other sleep disorders) that are caused by improper phasing of the biological clock in humans. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Circadian (daily) rhythms are a crucial component of human health, especially of mental health. The "clocks" underlying these rhythms regulate sleep, alertness, hormones, metabolic activities of various tissues, and many other biological processes. Appropriate daily regulation of these processes to attain optimal phase relationships is crucial for mental health. We have discovered significant associations of polymorphisms in two circadian clock genes with sleep and drug/alcohol abuse phenotypes in humans suffering from unipolar Major Depressive Disorder (MDD). Clock gene polymorphisms can cause atypical phasing of the human circadian system (or of other metabolic pathways) leading to sleep disorders and/or metabolic disorders. The central hypothesis of this proposal is that depression pushes the susceptibilities for hypersomnia and substance abuse closer to a "phenotype threshold," and that the polymorphisms we have identified in these two clock genes modulate the activities of these genes so as to enhance those susceptibilities such that the hypersomnia/substance abuse phenotypes are expressed. We will test that hypothesis by analyzing clock gene polymorphisms in a larger sample of depressed subjects and compare those patterns with data from a characterized control population of non-depressed subjects. In addition, initial studies towards understanding the functional cell/molecular basis for these genetic polymorphisms will be conducted using state-of-the art luminescence assays of clock gene function in mammalian cells. This project is appropriate for the NIH Exploratory/Developmental Research Grant Program because it proposes to develop an novel research area-namely the interface between circadian clocks, depression, and genetics-that will facilitate the prediction, diagnosis, and treatment of phenotypes related to depression. PUBLIC HEALTH RELEVANCE: This project will use genetic techniques to study the relationship between daily biological clocks and depression in humans. In particular, hypersomnia and drug/alcohol abuse are correlated with biological clock genes in depressed humans. This investigation could lead to new genetic methods of diagnosis and/or treatment of sleep disorders and substance abuse in depressed humans.

DESCRIPTION (provided by applicant): Circadian (daily) rhythms are a crucial component of human health. Inappropriate daily regulation/phasing of sleep and other clock-controlled parameters is associated with several types of depression, obesity/metabolic syndrome, and cardiovascular disease. At the cellular level, circadian pacemakers regulate cell division, metabolism, and gene expression networks. The selective pressures that led to the evolution of the special characteristics of circadian rhythms (esp. their precise 24 h time constant and temperature compensation) have not been identified. How can metabolic processes that intrinsically feed back with short time constants be recruited by selective pressures to the establishment of a self-sustained 24 h oscillator that is temperature compensated? The answer to this question has broad significance to our understanding of cell cycles, gene transcriptional networks, Systems biology, evolutionary processes, and chronobiology. This project will undertake an experimental evolution of biological rhythms that have circadian characteristics in a quest to identify relevant selective pressures and whether particular metabolic pathways are predisposed towards the evolution of self-sustained biological oscillators. Single cell organisms with genetic capabilities will be subjected to a variety of environmental cycles (light/dark, UV- B, temperature) to ascertain which conditions can lead to the evolution of biological oscillators with circadian characteristics. The successful evolution of such oscillators will be assessed with luminescence reporters of cell cycle and metabolic events.

Regulation of intracellular acidity (pH) is vital to cells for many reasons. The measurement of pH inside living cells is therefore crucial to the study of many biological processes. The currently available methods for measuring intracellular pH mostly use fluorescence technologies that have limitations such as autofluorescence, phototoxicity, photobleaching, etc. These problems can be particularly serious in green plant tissue. Additionally, the currently available pH methods are useless when measuring light-dependent pH changes (such as those occurring in the chloroplast) because the method can trigger the very responses that are to be studied. The goals of this project is to develop a new method for measuring pH in living cells. The experimental approach that will be employed is based on a technique developed by the Principal Investigator of the project that allows for monitoring of in vivo protein interactions, Bioluminescence Resonance Energy Transfer (BRET). BRET avoids the problems of fluorescence methods. The project will develop a genetically encodable, targetable, ratiometric pH monitoring method. Therefore, pH can be quantified in living plant and animal cells with this method. The usefulness of this transformative technology will be demonstrated by application to important experimental questions in plant physiology, including the measurement of (i) pH changes in the chloroplast in response to light/dark transitions, and (ii) daily rhythms of cellular pH.
Broader impacts.This project will lead to new information about the impact of the environment (for example, light/dark) on cellular activities in plants, thereby enabling new strategies for agricultural adaptation. The project will also provide an optimal training environment for a postdoctoral fellow and a graduate student to learn innovative technology development.

DESCRIPTION (provided by applicant): Humans and most other organisms manifest circadian (daily) rhythms that are controlled by an endogenous biochemical oscillator that regulates the timing of sleep, cardiovascular functions, and metabolism. These "biological clocks" are important to human physiology. For example, psychiatric and medical studies have shown that circadian rhythmicity is involved in some forms of depressive illness, "jet lag," drug tolerance/efficacy, memory, insomnia, and other sleep disorders. Therefore, understanding the biochemical mechanism of circadian clocks may lead to procedures which will be useful in the diagnosis and treatment of disorders that are relevant to sleep, mental health, and pharmacology. The salient properties of circadian clocks-24 hour time constant, high precision, temperature compensation-are presently impossible to explain biochemically. The current Transcription and Translation Feedback Model for circadian rhythms posits that rhythmic clock protein abundance is critical for clock function. In model systems, it has been possible to reset the phase of circadian rhythms by induction of clock protein synthesis at specific phases. This approach has been difficult to accomplish in mammalian systems. The current project will test hypotheses concerning the significance of rhythmic clock protein abundance in mammals by using new methods to introduce proteins directly into cells by peptide-mediated transduction across cell membranes. This technology will allow us to modulate the intracellular concentration of clock proteins in cells, tissue slices, and intact animals. This project is appropriate for the NIH Exploratory/Developmental Grant (R21) Program because it fulfills all of the following primary criteria: (1) innovative research directions, (2) exploration of approaches that are new to a substantive area, and (3) development of new technologies and methods. PUBLIC HEALTH RELEVANCE: Rigorous testing of the current model for circadian oscillators is crucial for our understanding of these biological clocks that control so much of human physiology. These studies will yield results of theoretical importance, but recent evidence also implicates sleep disorders, obesity, cardiovascular disease, and cancer when normal circadian clock function is disrupted. Therefore, these studies also have the potential for designing treatments for sleep disorders, jet lag, insomnia, cardiovascular disease, and other clock-related disorders.

DESCRIPTION (provided by applicant): Addictive drugs change synaptic properties, both at the molecular level and in terms of remodeling circuits. We will develop a novel luminescence-based methodology for monitoring synaptic activity as a new tool for functional neuroscience that can be applied to the science of drug abuse. Optogenetic methods for stimulating neural activity are revolutionizing neurobiological research in vitro and in vivo. Brief exposure to lightof cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the bacteriorhodopsin bR). To date, the impact of optogenetic stimulation has usually been monitored by electrophysiological methods that are accurate and well characterized, but are difficult and expensive to implement in freely behaving animals in vivo and/or in multiple neurons simultaneously. Optogenetic stimulation would optimally be partnered with less invasive optical methods to monitor activity among many cells. Unfortunately, the currently preferred methods for optically measuring synaptic activity are based on fluorescence methods that are poorly matched with ChR2/bR because the fluorescence excitation needed to monitor synaptic activity will trigger ChR2 and/or bR. Our new luminescence methodology will avoid the drawbacks of electrophysiology and fluorescence excitation (esp. reporter stimulation, photo bleaching & tissue auto fluorescence), and will therefore optimally partner with optogenetic methods for in vivo stimulation. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. This project will develop novel luminescence probes for synaptic activity that are genetically encodable and targeted to specific cellular loci that are involved in neural activity. This will be accomplished by using Bioluminescence Resonance Energy Transfer (BRET) between a luciferase and the Venus fluorophore that is modulated by pH or Ca++ so that the spectrum of luminescent emission changes when synapses are activated optogenetically, thereby avoiding the problems associated with fluorescence excitation. These luminescence reporters of synaptic activity will be characterized in a well-studied hippocampal primary neuron culture preparation in vitro in conjunction with optogenetic stimulation before and after treatment with endocannabinoids. Finally, a viral vector encoding these reporters will be used to monitor neural activity of cortex after optogenetic stimulation in freely behaving rats in vivo. This project is appropriate for the Cutting-Edge Basic Research Award (CEBRA) R21 mechanism of the NIDA because it proposes to develop new technologies that will advance drug abuse and related neurobiological research.

DESCRIPTION (provided by applicant): Circadian (daily) rhythms are a crucial component of human health that regulates sleep, alertness, hormones, metabolism, and many other biological processes. The fascination of this phenomenon is to explain how a biochemical mechanism (i) can robustly sustain a long period (~24 h) oscillation whose frequency keeps time so precisely, and (ii) enhance fitness in the natural environment. These questions remain critically important unanswered issues in the circadian rhythms field. For example, the adaptiveness is not clear for the most obvious circadian characteristic-a robust self-sustained oscillation in constant conditions. If anticipation of future temporal events (e.g., dawn, dusk, etc.) is the goal of circadian timekeepers, why is a temperature-compensated hourglass timer that is initiated by dawn or dusk not sufficient? And yet evolution in every case has selected an oscillator that sustains itself in non-natural continuous as the timekeeper for regulating daily processes, and this characteristic forms the core defining factor for circadian rhythms. The overall hypothesis of this project is that circadian pacemakers that are self-sustained in constant environments do provide a fitness advantage in cyclic environments and that multioscillator structure contributes to the maintenance of high amplitude oscillations in vivo. Testing this hypothesis will take advantage of the unique capabilities of the eubacteria Synechococcus elongatus (cyanobacterium) and E. coli by a three-pronged approach-genetic, biochemical, and by tests of adaptive fitness. First, the adaptive value of sustained circadian oscillations will be quantified y competition assays and metabolic patterns that correlate with adaptiveness will be identified as a signature of the advantage conferred by sustained circadian oscillations. Second, the contributions of multioscillator organization will be assessed towards establishing (i) robust, sel-sustained oscillations, and (ii) adaptive competitiveness. Finally, a novel experimental selection approach will identify environmental pressures that can lead to the evolution of self-sustained circadian oscillations. The answers to these questions will help us to understand fundamental circadian organization and rhythmic regulation of metabolism; this understanding can help us to better design therapies for disorders in which circadian clocks are implicated.

? DESCRIPTION (provided by applicant): A novel luminescence-based methodology for monitoring neural activity as a new tool for functional neuroscience will be developed in this project. Optogenetic methods for stimulating neural activity are revolutionizing neurobiological research in vitro and in vivo. Brief exposure to light of cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the bacteriorhodopsin bR) To date, the impact of optogenetic stimulation has usually been monitored by electrophysiological methods that are accurate and well characterized, but are difficult and expensive to implement in freely behaving animals in vivo and/or in multiple neurons simultaneously. Optogenetic stimulation would optimally be partnered with less invasive methods to monitor activity among many cells, such as by optical methods. Unfortunately, the currently preferred methods for optically measuring neural activity are based on fluorescence methods that are poorly matched with ChR2/bR because the fluorescence excitation needed to monitor synaptic activity can trigger ChR2 and/or bR. Moreover, fluorescence can photobleach probes and excite tissue autofluorescence that generates undesirable background. Luminescence is an alternate optical technology that avoids problems associated with fluorescence. This project will develop novel luminescence probes for neuronal activity that are genetically encodable and can be targeted to specific cell types and to specific cellular loci that are involved in neural activity. These probes will respond to neuronal activity by changing their luminescence intensity and/or luminescence spectrum. In the latter case, probes based on Bioluminescence Resonance Energy Transfer (BRET) will be modulated by neural activity so that the spectrum of luminescent emission changes when neurons are activated. Our new luminescence methodology will avoid the drawbacks of electrophysiology and fluorescence excitation (esp. off- target optogenetic stimulation, photobleaching & tissue autofluorescence), and will therefore optimally partner with optogenetic methods for in vitro and in vivo stimulation. These luminescence reporters of neural activity will be characterized in conjunction with optogenetic stimulation of hippocampal primary neurons and of brain slices that reconstitute neural circuits in vitro. In addition, viral vectors encoding these reporters will be used to introduce the probes to the brain in a minimally invasive manner so as to monitor neural activity in freely behaving rodents (i) over the circadian cycle from the hypothalamus, and (ii) before and after optogenetic brain stimulation of the cortex in vivo. This project is appropriate for the R21 Exploratory/Developmental Research Grant mechanism of the NIMH because it will develop new technologies and tools to advance spatiotemporal analyses of complex circuits and cellular interactions in the brains of multiple model animal species.

Project Summary/Abstract: Circadian (daily) rhythms regulate myriad behavioral and molecular processes including locomotor activity, sleep timing, feeding behavior, metabolism, and gene expression. These biological clocks are a crucial component of human health, and improper functioning of this system is associated with sleep/wake disorders, metabolic syndrome, and obesity. The current molecular model for the circadian clock mechanism comprises autoregulatory transcriptional & translational feedback loops of central clock genes that necessitate rhythmic synthesis and degradation of clock gene products. We have found that the effective gene dosage of the Ube3a gene that encodes an ubiquitin ligase (involved in protein degradation) regulates fundamental properties of the circadian clock system in mammals. The expression level of the Ube3a gene is crucial for normal neurodevelopment. For example, reduced dosage of Ube3a leads to Angelman Syndrome (AS) and increased dosage/activity can result in autism. Therefore, the level of expression of Ube3a is critical for normal cognitive development, and the thesis of this project is that balanced expression of Ube3a is key for stable circadian rhythmicity as well. AS is a disorder characterized by cognitive/developmental delays, speech impairment, sleep disorders, and seizures. The paternal allele of Ube3a is imprinted (silenced) in neurons, and most cases of AS result from a deletion of the maternal Ube3a allele that further downregulates Ube3a in neurons. Mouse models have been generated that have (i) a deletion of the maternal Ube3a allele (model of AS), and (ii) extra copies of Ube3a (model of autism); these models enable tests of our hypothesis, which is that Ube3a expression affects the plasticity of circadian rhythms and that environmental, genetic, and/or pharmacological treatments can be identified that compensate for the loss of Ube3a expression. This hypothesis will be tested by manipulating environmental, genetic, and developmental conditions to affect the circadian system in mouse models of AS. Specific pharmacological treatments will be tested for their potential in reversing the circadian phenotypes of AS models to use as a basis for identifying a biomarker to be used with human subjects. Clock proteins will be identified as molecular targets of Ube3a-mediated ubiquitination. Finally, an Ube3a overexpressing mouse model will be tested to determine if Ube3a overexpression has reciprocal effects to the Ube3a null of AS models. This project represents a novel area of investigation that has the potential to enhance health-related research; its overall significance is (i) to elucidate the role of ubiquitination and imprinting in the circadian mechanism, and (ii) to identify treatments that ameliorate the circadian disorders of Ube3a imprinting in mouse models. The answers to these questions will help us to understand fundamental circadian organization and plasticity in this fascinating?and potentially clinically relevant?example of gene X environment interaction.

Project Summary/Abstract: Optogenetic methods for modulating neural activity are revolutionizing neurobiological research. Brief exposure to light of cells expressing channelrhodopsin-2 (ChR2) can elicit excitatory cation fluxes (or inhibitory ion fluxes with the halorhodopsin NpHR). The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring neural activity are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. A novel luminescence-based methodology for monitoring neural activity as a new tool for functional neuroscience will be developed in this project. Luminescence avoids the complications associated with fluorescence because it is an enzymatic reaction that circumvents the need for excitation. The newly developed luminescence sensors for neuronal activity are genetically encodable and can be targeted to specific cell types and to specific cellular loci that are involved in neural activity. These sensors respond to the calcium ion fluxes generated by neuronal activity by changing the intensity and/or spectrum of their luminescence emission. In the latter case, a novel sensor based on Bioluminescence Resonance Energy Transfer (BRET) is modulated by neural activity so that the spectrum of luminescent emission is altered when neurons are activated. This new luminescence methodology avoids the drawbacks of electrophysiology and fluorescence excitation (esp. off-target optogenetic stimulation, photobleaching & tissue autofluorescence), and therefore optimally partners with optogenetic methods for in vitro and in vivo stimulation. These luminescence sensors will be characterized in conjunction with optogenetic stimulation of neuronal activity in vitro, especially while actuating both ChR2 (excited by blue light) and NpHR (excited by orange light) in the same experiment to control positive and negative ion fluxes into cells. In addition, the luminescence sensors will be applied to high-throughput screening of autofluorescent neuroactive compounds. In addition to these in vitro applications, however, the primary goal of this project is to apply these novel sensors for the first time to several new in vivo applications of measuring neural activity in a minimally invasive manner in freely behaving rodents. This project is appropriate for the R21 Exploratory/Developmental Research Grant mechanism of the NIMH because it will develop new technologies and tools to advance spatiotemporal analyses of complex circuits and cellular interactions in the brains of multiple model animal species.