Vincent Cassone - US grants

DESCRIPTION (Adapted from the Abstract): The vertebrate circadian clock is composed of multiple circadian oscillators whose interactions are critical for stable overt rhythmicity. These components, whose relative roles vary among species, include the suprachiasmatic nucleus (SCN), the pineal gland and eyes, which influence circadian rhythms via the hormone melatonin. In birds, melatonin plays a particularly critical role. Removal of the melatonin signal dramatically affects the circadian rhythms of many physiological functions. Recent research has identified the sites of melatonin's action in the avian SCN, the vSCN, and other structures within the visual system using autoradiography of iodinated melatonin (IMEL). Binding is rhythmic, with peak values in the late day. (The Principal Investigator and his associates have characterized two melatonin receptors, CKA and CKB, in the Gi-protein group of membrane receptors whose pharmacological profiles are identical to IMEL binding and which are distributed in avian brain similarly.) Finally, visual system function is regulated by the clock by way of melatonin and is sensitive to the hormone primarily during the late subjective day. Based on these observations, the Investigator hypothesizes that: (1) the avian visual system is rhythmic in its response to light; (2) this rhythm is due to the rhythm of melatonin; (3) brain and retinae are sensitive to melatonin during the late subjective day; (4) this rhythm of sensitivity to melatonin is due to a parallel rhythm in the number of melatonin receptors; (5) the rhythm of melatonin sensitivity is derived from oscillations in the vSCN; and, finally, (6) at least one cellular site of melatonin action in the brain is within astrocytes containing the CKB melatonin receptor subtype. The Investigator proposes to determine whether a rhythm exists in retinal and brain photic sensitivities by recording electroretinograms (ERG) and visually evoked activity (VEP) from visual centers of the chick brain at different times of day. He and his associates will determine whether the anticipated rhythms derive from pineal and/or retinal melatonin rhythms by determining the effects of melatonin removal, by way of pinealectomy, or replacement by way of infusion and injection. They will determine whether the anticipated rhythm in the sensitivity to melatonin derives from a circadian regulation of receptor protein and/or mRNA. They will determine whether the rhythms of visual sensitivity, melatonin sensitivity, receptor protein and mRNA derive from the vSCN by comparing these rhythms in animals whose vSCN have been destroyed versus those with sham surgeries. Finally, they will study the effects of melatonin on astrocytes in vivo and in vitro. In these studies they will address a novel form of neuromodulation in an important sensory system-vision. They will provide an excellent model for the study of the molecular mechanisms of melatonin action, which is important in many species of vertebrates including humans.

The biological clocks that regulator daily and circadian patterns of daily behavior, physiology and biochemistry are fundamental properties of biological organization. In recent years, great strides have been made in elucidating the anatomical localization of clock structures, the physiology of clock function and the biochemical/molecular events underlying circadian rhythmicity. These advances have been made in diverse organisms, which share many common formal and molecular properties but which differ in the level at which clocks are understood and at which they function. Among the best-studied model systems are the cyanobacterium Synechococcus sp., the filamentous fungus Neurospora crassa, the avail pineal gland and the mammalian suprachiasmatic nucleus (SCN), which have all provided disparate pieces of a very large, complex puzzle. However, a complete picture of this puzzle, of clock input, oscillation and output, is not apparent, because divergent systems provide dissimilar views. This may be due to real biological differences in experimental systems and/or diverse approaches. The present Program Project Grant combines the efforts of 4 established laboratories, which have a history of cooperative education and research, devoted to different aspects of biological clock function in these well-studied model systems. Dr. D.J Earnest (Project #2) will investigate mechanisms of circadian oscillation and its output in a unique immortalized cell line derived from the rat SCN. Dr. D. Bell-Pedersen (Project #3) will explore the relationship of developmental and metabolic processes with circadian rhythm function in Neurospora. Dr. V.M. Cassone (Project #4) will study the molecular mechanisms of rhythm generation and coupling to melatonin biosynthesis in the chick pineal and of cellular mechanisms of melatonin action in cultured glia. Two Technical Cores and an Administrative/Integrative Core will enable will enable each laboratory to extend the boundaries of their own research: 1) The Genomics (Core A), directed by Dr. T. Thomas, will perform modern genomics analyses including high density transcriptional profiling, DNA microarray technology and EST analyses. 2) The Cell Physiology/Imaging Core (Core B), directed by Dr. Mark Zoran, will perform electrophysiological and real-time imaging of gene expression. 3) The Administrative and Integrative Core (Core C), directed by Dr. Cassone, will maintain records, facilitate interaction of laboratories by scheduling of regulate meetings, and coordinate meetings with advisory committees. In addition, this ore will facilitate data analysis of the entire data set collected by all laboratories compiling sequence and functional data, archiving these data and analyzing phylogenetic relationships among these diverse systems. All four projects are bonded by common technological approaches to diverse biological systems and by a commitment to interdisciplinary, comparative analyses of biological clock function.

The avian pineal gland is an important model systemfor the study of cellular and molecular bases of circadian clocks and sleep. However, although the mechanisms of the biosynthesis of the major output of this gland, melatonin, are largely understood, the molecular bases for the rhythm generating mechanism has been elusive, and the cellular/molecular basis for its pacemaker properties is completely unknown. Transcriptional profiling and the development of new cell culture methods in the original P01 grant identified several candidate genes that were likely to be important in rhythm generation and showed that melatonin broadly affected metabolic and ionic homeostasis without affecting clock gene rhythms. The present proposal seeks to test the hypothesis that pineal pacemaker activity regulates CNS and peripheral metabolic rhythms via mechanisms that are independent of or at least differentially regulated from output regulation through clock gene rhythms in chicks. (1) We will determine whether surgical disruption of circadian organization by pinealectomy and retinectomy differentially affects glucose metabolism and clock gene rhythms (mRNA and protein) in CNS and peripheral tissues in vivo. We will also ask whether administration of exogenous melatonin differentially affects glucose metabolism and clock genes (mRNA and protein) in CNS and peripheral tissues in vivo by employing both quantitative PCR and DNA microarrays. (2) We have developed a new experimental system in which primary cultured pinealocytes are co-cultured with CNS and peripheral target cells. We will determine the dynamics of pacemaker properties by monitoring melatonin rhythms in the media, and both metabolic activity and clock gene rhythms in pacemaker and target cells simultaneously. Finally, (4) we will determine the roles played by clock genes and other genes identified in our transcriptional profiling by up-regulating and knocking down expression of these and other genes in pacemaker and target cells by lentiviral transduction. Lay Summary: The molecular mechanisms by which the avian pineal generates rhythms of melatonin and by which peripheral tissues respond to melatonin will be studied. The results will provide the first molecular analysis of pineal pacemaker activity. These studies will build a bridge between metabolic and clock gene regulation in sleep-wake cycles in a species whose metabolic demands are more similar to diurnal humans than are those of nocturnal rodents.

DESCRIPTION (provided by applicant): Partial support is requested for the 12th Gordon Research Conference (GRC) on Pineal Cell Biology to be held January 29 - February 3, 2012 at Hotel Galvez in Galveston, Texas. The major objectives and long- term goals of the 2012 GRC will be to increase our understanding of the molecular networks and biochemical pathways linking the pineal gland and its hormone melatonin with the circadian clock timing system, sleep/wake processing and metabolism. Subtitled: Links to clocks, sleep and metabolism, the 2012 GRC will focus on the very recent advances in understanding the underlying mechanisms and networks linking these functions as well as the interaction between metabolic disorders, sleep and circadian disturbances in health and disease. The role of the pineal gland and melatonin, in these interactions will be specifically addressed. Recent research has demonstrated clear links between circadian clocks and metabolism, between sleep deprivation and metabolic disorders and between circadian rhythms, light and sleep. The specific aims of the GRC are to: 1) Provide a forum for communication of recent research advances linking circadian rhythms, melatonin, sleep and metabolism, thus leading to identification of key opportunities for future study, 2) Stimulate interaction among the researchers trained in these different disciplines, so as to promote understanding of the potential for interdisciplinary and collaborative research, 3) Promote research collaboration by bringing together scientists from different research areas and countries, with different levels of training, including groups typicaly underrepresented in science and 4) Promote translational research by bringing together investigators studying human subjects with those investigating other species. The GRC will achieve these aims by providing a unique and timely opportunity for researchers from these diverse areas to meet, debate and discuss. To date no conference has been dedicated entirely to this newly emerging supra- disciplinary field of metabolism, sleep and circadian rhythms. Topics for discussion will range from genes to behavior and will cover areas from basic science to translational research using different model systems. Bringing together world leading experts from these related fields, from academia, government and industry, will allow a unique cross-fertilization of ideas, "out-of-the box" thinking and new research networking opportunities for attendees. The GRC format of lecture sessions in the morning and evening, with ample time for discussion is perfect to promote these scientific interactions. There will also be opportunities fo junior scientists and graduate students to present their work in poster format and exchange ideas with leaders in the field. The benefits for the attendees and for this emerging supra-disciplinary field will be far-reaching. Metabolic disorders (metabolic syndrome, diabetes, obesity), sleep restriction and circadian rhythm disorders (e.g. shift work sleep disorder) are increasing in today's society thus it is crucial that new knowledge of the processes linking these functions is generated which will ultimately benefit the nation's health and economy. PUBLIC HEALTH RELEVANCE: Recent research has demonstrated interactions between the circadian timing system, pineal melatonin, sleep and metabolism. The Conference objective is to bring together for the first time, world leading scientists in these different disciplines to discuss new developments, to increase the level of interaction and cross-fertilization between the disciplines and thus promote collaboration and translational research. Increased understanding of the underlying biological mechanisms and interactions between these processes will ultimately provide novel treatment strategies for circadian rhythm sleep disorders and metabolic disease (such as diabetes, shift work disorder). Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of th individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

DESCRIPTION (provided by applicant): Biological circadian clocks are fundamental properties of all living organisms studied in any detail. In animals, clocks regulate the timing of sleep: wake cycles, as well as the coordination of internal body functions, such as temporal variations in temperature, hormone levels, aspects of disease and gastrointestinal physiology. The molecular bases for biological rhythms are at least in part regulated by the expression of clock genes, a group of genes whose complex interactions govern 24-hour rhythms. Work in our lab has demonstrated that clock genes are important in the regulation of colonic motility: the mouse colon possesses a functional circadian clock that regulates rhythms of a wide array of genes as well as intestinal motility. Further, we have shown that the colonic clock regulates multiple downstream processes associated with normal intestinal motility, absorption, cell proliferation and cell signaling as well as potential pathological processes. In addition, in mammals, there is a hierarchical organization in which a central pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) coordinates multiple peripheral processes as well as entrains the entire system to the light: dark cycle (LD) through specialized photic pathways. Our general hypothesis is that the global regulation of gastrointestinal circadian rhythms involves the complex, bi-directional interaction of central SCN clockworks and intestinal clocks themselves, and that disruption of any of these predisposes us to disease. The overall objective of this proposal is to determine the underlying mechanism through which colonic motility varies with the time of day under normal circumstances and to determine the consequences of aging on circadian patterns of colonic motility. Our hypothesis is that one factor mediating changes in intestinal function during aging is the circadian secretion of the hormone melatonin by the pineal gland and by intestinal tissues themselves. We will therefore ask what aspects of gastrointestinal rhythms are affected by normal aging (Specific Aim 1). We will then ask whether pineal melatonin influences gastrointestinal rhythmicity in young vs aged mice at several levels of biological organization. Finally, we will ask whether interference in the production of melatonin affects aging aspects of gastrointestinal rhythmicity by comparing melatonin deficient strains of mice (C57/Bl6) with strains of mice capable of producing melatonin cycles (C3H) and whether these processes are dependent the known melatonin receptor molecules by comparing wild-type with MT1 receptor, MT2 Receptor and MT2/MT2 receptor knockout mice.

Circadian rhythms are fundamental features of many if not all organisms on Earth. Among animals, the molecular mechanisms underlying these rhythms are remarkably conserved. Yet, outside animals, known mechanisms among the few model systems are disparate, which is not surprising due to the billions of years of separation among these taxa. Among prokaryotes, only one species has been shown to exhibit circadian rhythms, the cyanobacterium Synechococcus elongatus, whose clock derives from cycles of autophosphorylation and phosphatase activity in key clock proteins Kai A,B and C. We have discovered a new, biomedically important model bacterium, Enterobacter aerogenes, which expresses a temperature-compensated circadian clock, which is synchronized by the gut hormone melatonin and is entrained by cycles of temperature, using swarming behavior and motA-driven luciferase bioluminescence. We propose to fully characterize this exciting new clock system. We will determine whether the clock system regulates multiple physiological processes in parallel by asking whether gene expression with different promoter elements and luciferase are expressed rhythmically and/or respond to melatonin similar to motA-driven rhythms. We will determine whether melatonin is a true Zeitgeber for this clock, and we will characterize the effects of temperature cycles, which entrain this clock, testing the hypotheses that host melatonin secretion and/or TB synchronize this enteric bacterium. Secondly, we will knock out candidate genes that bear molecular similarity to Kai proteins, characterize these knockouts, and complement those that exhibit clock phenotypes. In addition, we will conduct a broad mutant screen, searching for clock mutants. Thirdly, we will characterize the biological activity, clock function and melatonin sensitivity of all knockout and mutant strains discovered here. If this clock does indeed share a common ancestor with the cyanobacterial clock, the data have broad and important evolutionary significance, placing the evolution of the circadian clock before the emergence of O2 generating photosynthesis some 3.5 billion years ago. In addition, these observations point to an integrated circadian organization in which master pacemakers in the brain synchronize peripheral oscillators, which in turn may regulate aspects of the microbiota through entrainment of the bacterial clock. This has important implications for biomedical science and therapeutics.