Amita Sehgal - US grants

9319623 Sehgal All organisms display biological rhythms that are controlled by endogenous clocks. These endogenous oscillators often have a period of about 24 hours, i.e. they are "circadian", and they control important functions such as sleep-wake cycles, maintenance of body temperature, and secretion of hormones. In this proposal Dr. Sehgal will investigate the molecular mechanisms underlying rhythmic behavior. The organism studied will be the fruit fly, where genetic technology has already been used to dissect some components of the circadian clock. One regulator of activity rhythms in the fruit fly is the period (per) gene. When this gene undergoes mutation, the period of circadian rhythms may lengthen, shorten or even be abolished. Dr. Sehgal's group will study a gene called timeless (tim) which appears to operate by influencing the products of the per gene. A combinmation of molecular biological and genetic methods will be used to isolate and characterize tim and to determine how it interacts with per. Recent findings suggest that genes such as per are present in the brains of many animals. Therefore studying per and tim in this system may yield important insights into the molecular architecture of the clock that times all living organisms.***

DESCRIPTION (Adapted from applicant's abstract): The goal of this proposal is to understand the molecular basis of circadian (approx. 24 hour) rhythms. Circadian rhythms are a central component of normal physiology and are displayed by organisms across the phylogenetic tree. This proposal will test the hypothesis that circadian rhythms in Drosophila depend upon the appropriate regulation and interaction of the period (per) and timeless (tim) proteins. Products of the per gene are known to be controlling elements of the central pacemaker in Drosophila. The arrhythmic clock mutation, timeless (tim), eliminates oscillations of per RNA and protein and reduces the overall levels of per protein. We recently isolated the tim gene and demonstrated that the per and tim proteins interact directly with each other. We also determined that the tim gene displays cyclic expression that requires per protein. Available data indicate that the per and tim proteins regulate each other and interactions between them control the phase and the periodicity of behavioral rhythms. Experiments are proposed to: (1) test the effects of the per mutants on tim protein expression, (2) address the mechanisms by which the per and tim proteins regulate each other's expression, (3) test the prediction that interactions between per and tim regulate the periodicity of circadian behavioral rhythms, (4) understand how per and tim proteins mediate the resetting of the circadian clock by light. Our preliminary data show that levels of tim protein are reduced rapidly by a pulse of light, suggesting that tim protein is the light-responsive element of the central pacemaker. This proposal is unique in its emphasis on the interaction between two circadian rhythm proteins. The mechanisms that regulate the per and tim proteins are likely to be conserved across species. Thus far the two organisms that have allowed extensive molecular analysis of circadian rhythms, Drosophila and Neurospora, show remarkable conservation of underlying mechanisms.

Sehgal, Amita IBN-9723035 Sleep-wake and other activity cycles, the maintenance of body temperature, and the secretion of several hormones are just a few examples of physiological processes that are periodic and tied to the 24 hour daily cycle. The timing of all these processes is determined by a master circadian (24 hour) clock within the body. The body clock is usually set by the day:night cycle of light and dark. The clock is genetically determined, and some of the genes that are part of the clock have been identified. This project focuses on one of these genes, the "timeless" gene, which was recently isolated and shown to control a component of the body's clock function. The protein made by the timeless gene is destroyed by light, which is part of the mechanism by which the body clock synchronizes to the day:night light cycle. This project identifies the important parts of the timeless protein and determines how they mediate the clock function's sensitivity to light. These studies provide insight into the mechanisms used by organisms to adapt to their environment, and provide information important to understanding the basis for daily rhythms in normal physiology, cognition, and behavior.

DESCRIPTION (provided by applicant): The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called clock genes) whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock's response to light, which has its basis in the control of protein stability. Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability; (2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above; (3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.

The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called "clock genes") whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock’s response to light, which has its basis in the control of protein stability. Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability;(2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above;(3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.

The goal of this project is to determine the mechanisms underlying age-associated fragmentation of sleep:wake cycles and the relevance of this fragmentation to the aging process. The organism that will be used for these studies is the fruit fly,Drosophila melanogaster. In preliminary studies, we have found that the strength of the sleep:wake rhythm declines with age, such that the duration of sleep bouts decreases while the number of sleep bouts and brief awakenings increases. Using a longitudinal study design we are able to document these changes in individual Drosophila over the course of their lifespan. We have also found that an increase in oxidative stress produces changes in the sleep:wake cycle that are similar to those caused by changing;conversely, decreasing oxidative stress strengthens the cycle. We hypothesize that the deterioration of sleep:wake cycles contributes to the determination of lifespan and is caused, at least in part, by a buildup of oxidative damage. To address this hypothesis wewill: (i) Determine the relationship between sleep:wake cycle strength and lifespan. We will assay changes in sleep:wake cycles throughout life in flies with altered lifespan and test the hypothesis that these changes are associated with physiological, rather than chronological, age. (2) Examine oxidative stress as a possible mechanism for the age-related breakdown in sleep:wake cycles. We will determine how increased and decreased oxidative stress affect the changes that occur in sleep:wake cycles with age. We will also identify specific behavioral and molecular effects of oxidative stress on the sleep-regulating circadian and homeostatic systems and compare these with the effects of age. (3) Manipulate sleep and assay effects upon lifespan. We will alter environmental conditions or use genetic or pharmacological interventions to increase or decrease sleep consolidation, and assay effects upon lifespan. These experiments will indicate if sleep fragmentation contributes to the aging process. In the long-term, the knowledge gained from these studies should help to develop strategies for treating sleep problems in the elderly. Treatment ofthese problems may also alleviate other symptoms ofaging.

? DESCRIPTION (provided by applicant): We request partial support for the 19th Chronobiology Gordon Research Conference (GRC) and the accompanying now 2nd Gordon Research Seminar (GRS), which will be held in Girona (Spain) from June 27 to July 3, 2015. Funds received from the NIH will be used to support registration fees for GRC and GRS participants, especially postdoctoral fellows and graduate students. The conference will focus on key research areas in chronobiology - a very interdisciplinary field continuously and increasingly attracting scientists from many diverse research areas including but not limited to neuroscience, behavior (including sleep), metabolism, aging, cancer and immunology. The subtheme of the conference is Biological rhythms: mechanisms - functions - implications for health emphasizing not only the integration of diverse research fields and organisms (from bacteria to humans) but also the more and more recognized critical importance of circadian clocks for health and disease. All speakers and discussion leaders work at the forefront of science and have been selected in consultation with renowned figures in our field representing the breadth of chronobiology research, leading to an extremely high-quality program. Special attention has been given (in the speaker selection process) and will be given (in the registration process) to cultural diversity, gender balance as well as to incorporating young upcoming scientists (~35% of invited speakers of the GRC are at the Assistant Professor level). Training will be further integrated in the program by (i) assigning four additional Late-Breaking Topic talk to students and postdoctoral fellows based on submitted abstracts and (ii) reserving four talk slots for young scientists at the student and postdoctoral level selected from the preceding GRS taking place for the second time after being highly successful in 2013.The specific objectives of the 2015 conference are: 1. To provide a forum for free, unhampered discussion of the latest (unpublished) chronobiology research. This conference will feature several new measures towards this objective. 2. To bring together scientists from diverse research levels ranging from those focusing more on molecular aspects of chronobiology over physiological and organismal levels up to translational researchers studying the importance of circadian clocks for health and disease. We expect representatives from industry in attendance, which will help in this process. 3. To identify priority themes for future research (neuroscience (including sleep), aging, metabolism, immunology, pathologies etc.) by openly debating knowledge gaps and especially controversies. 4. To continuously foster interactions between trainees and senior researchers by providing a high-quality and accessible arena for scientists at very different stages of their careers.

? DESCRIPTION (provided by applicant): Our overall goal is to determine how circadian rhythms of behavior are generated. In previous work on this project we identified and characterized molecular mechanisms of the endogenous circadian clock in Drosophila. We also initiated studies to identify the mechanisms that carry time of day cues from the clock and transmit them through the brain. Specifically, we identified output neurons that are required for rhythmic rest:activity and connect anatomically to central clock neurons. These output neurons include the Dorsal Neuron 1 (DN1) group, which contains a clock, and two non-clock clusters in the pars intercerebralis (PI), a region of neurosecretory cells equivalent to the mammalian hypothalamus. The two PI clusters that regulate circadian rhythms of rest:activity secretes the neuropeptides DH44 and SIFamide respectively. We found that DH44 is required for behavioral rhythms, but a role for SIFamide has not been determined yet. Interestingly, the DN1s have also been associated with the function or expression of known circadian output molecules, narrow abdomen and unpaired. In addition, our preliminary data indicate that another output molecule, Neurofibromatosis 1 (NF1), is required in DN1s for rest:activity rhythms. We also find that neural activity cycles with a 24 hour rhythm in DN1 and DH44 cells. Thus, we have started to place molecular components in the cellular substrates we identified, and develop assays to monitor the transmission of rhythmic signals through the network. We hypothesize that time-of-day cues generated in central clock cells are transmitted through DN1s to drive rhythmic activity in the PI, which then controls rest:activity rhythms through release of specific neuropeptides. We propose to: (1) Address the significance of rhythmic activity in the DN1s and determine which clock cells and output molecules are required for this rhythm. (2) Identify the upstream components in clock cells that drive rhythmic activity in the DH44 cells, and determine if DH44 acts rhythmically. (3) Address a role for SIFamide in rest:activity rhythms and identify other PI molecules relevant for rest:activity rhythms. Together these studies are expected to provide a comprehensive understanding of the molecular network and cellular circuit that generates a behavioral rhythm. Given known conservation of clock mechanisms and molecules from flies to humans, these studies will likely be relevant for our understanding of human rhythms, which are critical for normal behavior and physiology. These studies will also provide general insight into the maintenance and function of neural circuits, which are impaired in several neurological disorders.

Project Summary We request partial support for the 20th Chronobiology Gordon Research Conference (GRC) and the accompanying 3rd Gordon Research Seminar (GRS), which will be held at Stoweflake Conference Center, Vermont from July 15th to July 21st, 2017. Funds received from the NIH will be used to support registration fees and/or travel costs for GRC and GRS participants, especially postdoctoral fellows and graduate students. The conference will focus on key research areas in chronobiology - a very interdisciplinary field that continuously and increasingly attracts scientists from many diverse research areas including neuroscience, sleep, metabolism and aging and uses approaches ranging from molecular genetics to theoretical biology. Given its emphasis on brain circadian circuits that regulate behavior and physiology, the interactions of clocks with metabolic factors, and the relevance of circadian regulation for aging as well as for age-related neurological disorders, the conference fits with the missions of NINDS, NIA and NIDDK). The subtheme of the conference is ?Circadian regulation across physiological systems, lifespan and pathological conditions? emphasizing not only the integration of diverse research fields and organisms, but also the increasingly recognized critical importance of circadian clocks for health and disease. All speakers and discussion leaders work at the forefront of science and have been selected in consultation with renowned figures in our field representing the breadth of chronobiology research, leading to an extremely high-quality program. Special attention has been given (in the speaker selection process) and will be given (in the registration process) to cultural diversity, gender balance as well as to incorporating upcoming young scientists. Training will be further integrated in the program (i) through the GRS taking place for the third time; (ii) assigning eight additional short talks to students and postdoctoral fellows based on submitted abstracts and (iii) providing many opportunities for trainees to be involved in discussions with senior researchers. The specific objectives of the 2017 conference are: 1. To provide a forum for free, unhampered discussion of the latest (unpublished) chronobiology research. This conference will feature several measures towards this objective. 2. To bring together scientists from diverse research levels, ranging from molecular aspects of chronobiology to physiological and organismal rhythms to translational studies highlighting the importance of circadian clocks for health and disease. The latter will be helped by industry representatives, who are expected. 3. To identify priority themes for future research (e.g. impact of circadian regulation on neural disorders, aging, and metabolism) by openly debating knowledge gaps and especially controversies. 4. To continuously foster interactions between trainees and senior researchers by providing a high-quality and accessible arena for scientists at very different stages of their careers. We are also taking specific steps to encourage participation of trainees in Discussions, and to provide them with ready access to faculty.

Project Summary We request partial support for the 21st Chronobiology Gordon Research Conference (GRC) and the accompanying 4th Gordon Research Seminar (GRS), which will be held at the Rey Don Jaime Grand Hotel, Castelldefels, near Barcelona, Spain from June 23-28th 2019. Any funds that we receive from the NIH will be used to partially support the registration fees for predominantly younger researchers, postdoctoral fellows and graduate students who will attend the GRC and associated GRS. The Nobel Prize in Medicine or Physiology awarded in 2017 to our colleagues Jeff Hall, Michael Rosbash and Mike Young for their pioneering work in deciphering the molecular basis of the circadian clock ensures that both GRC/GRS will attract a wide-range of researchers working at many different levels with diverse methodologies and model organisms. The GRS will focus on training and mentorship, while the GRC will present the latest unpublished work from the senior and emerging stars of the field. The GRC subtheme is `Clocks in model organisms: Circadian Networks, Physiology and Health' and emphasizes the diverse range of organisms that are studied from many different perspectives, from the molecular, cellular, physiological to behavioral and population layers. This also includes the application of these findings to the clinic and workplace. Consequently there is a wide range of topics covering most aspect of chronobiology using a diverse range of organisms and research methodologies included within the program. In higher organisms, the master circadian clock is in the brain, and chronic circadian disruption by modern lifestyles has important implications for human behavior, metabolism and healthy ageing, so the conference fits well within the sphere of NINDS, NIA and NIDDK. The speakers and discussion leaders represent senior established figures and younger emerging stars of the field and have been selected by consultation with an informal committee consisting of both senior and more junior figures in the field. There is an almost equal number of male and female speakers/session chairs as well as a range of nationalities invited to speak, from the Americas, Europe and the Far East ensuring a cultural and gender diversity. Apart from the mentorship, the GRS will also have 8 junior speakers and the GRC will further select 9 abstracts from younger scientists for presentation as short talks at the main meeting, providing an important learning experience for the trainees.

PROJECT SUMMARY Sleep loss is increasingly recognized as a significant risk factor in numerous metabolic diseases such as diabetes, obesity, metabolic syndrome, cancer, and cardiometabolic disorders. Clinical and model studies have confirmed negative metabolic effects of sleep loss. Intriguingly, sleep loss also dampens peripheral rhythms in human populations, which raises the question of whether the metabolic-sleep connection is mediated through circadian rhythms. Time-dependent analysis of metabolic changes has revealed large-scale oscillations in metabolite pools through the course of the circadian day in humans and other model systems such as rodents and flies. Disruption of the molecular clock, either genetically or through dietary intervention such as high-fat diet or mis-timed restricted feeding, causes insulin resistance and a lack of so-called `metabolic flexibility', phenotypes shared with sleep loss. We hypothesize that effects of reduced sleep on metabolism are mediated through changes in rhythms of energetic and redox metabolic pathways. One major limitation in gleaning mechanistic understanding of the sleep-circadian-metabolism connection is difficulty in measuring metabolic flux at different times of day in vivo. Our team has developed an innovative model of circadian flux using Drosophila melanogaster (fruit fly). Furthermore, dietary manipulations, such as time-restricted feeding in the active period or caloric restriction, maintain amplitudes in metabolic cycles in face of circadian disruption and have been associated with cardio-metabolic health in flies. In this proposal, we will exploit the genetic flexibility of D. melanogaster to test the above hypothesis in the following related but independent aims: ? Aim 1: Determine the impact of sleep loss on metabolic rhythms. Impact: An impact of sleep loss on metabolic oscillations will clarify the approach towards understanding how circadian rhythms and sleep, each of which is currently studied independently, affect metabolism. ? Aim 2: Determine if nutritional challenge exacerbates the metabolic effects of sleep loss in a time-of- day specific manner. Impact: These studies will provide direct mechanistic insights into the origin of metabolic imbalance which has only been inferred in studies to date. Future pharmacological or behavioral interventions can be targeted accordingly. ? Aim 3: Determine if time-restricted feeding can mitigate effects of sleep deprivation on metabolism. Impact: Demonstration that dietary manipulation mitigates negative metabolic consequences of sleep loss has the potential for interventional applicability in at risk real-world human populations.

ABSTRACT This proposal is for a training program in the area of sleep and circadian research and the related disorders. There is growing evidence of the prevalence of sleep disorders in the American population, and that problems related to inadequate sleep have a major impact on many aspects of our society. At a basic level, little is known about the fundamental mechanisms that control sleep and the function(s) of sleep. Thus, there is a major opportunity for scientific discovery. One of the barriers that is recognized to advancing the knowledge base in this area is the paucity of investigators, both those engaged in basic research and in patient-oriented research. This application describes a training program that is based on the relatively unique faculty resources and structures at the University of Pennsylvania for support of research in sleep and its disorders. The proposal describes specific training aspects that are intended to complete the matrix for training opportunities at the University of Pennsylvania (Penn) in the area of research in sleep/sleep disorders. These aspects are the following: research training for graduate students. This is based on training provided by 3 graduate groups at Penn. Each graduate program has a similar structure, albeit with different required coursework. The graduate groups are: a) the Neuroscience Graduate Program. This is the Graduate Group that has been involved in this program since its inception. We will utilize, where appropriate, structures, courses and other resources developed by this group; b) a graduate track in genomics/computational biology; c) graduate group in cell and molecular biology that offers our graduate students training in areas such as metabolism, genetics, and epigenetics; d) a targeted MD/PhD program to train physician-scientists in sleep/circadian research. This aspect of our program will be based on the outstanding institutional MD/PhD program at the University of Pennsylvania. We also have a postdoctoral training program for nurse investigators. This will be based on the preeminent School of Nursing at the University of Pennsylvania. The strong, well-established collaboration between the School of Medicine and the School of Nursing in this area provides a unique opportunity to develop a much needed national program to train nurse investigators in this area. All of these components of the program will utilize the extensive resources for research in sleep/circadian that have been developed at the University of Pennsylvania.