Jadwiga M. Giebultowicz - US grants

Circadian rhythms are ubiquitous in the living organisms, synchronizing life functions at the biochemical, physiological, and behavioral levels. The rhythms generating mechanisms. collectively known as circadian clocks, are not fully understood in any organism. Research in the fruit fly Drosophila has led to major insights into the molecular bases of the circadian mechanism. Two clock genes, period and timeless, have been shown to play central roles in the function of the brain-centered clock, which controls behavioral rhythms of adult flies. Using those genes as markers, the PI has identified a new circadian clock in the fly excretory organ, the Malpighian tubules and established that this clock can function in the absence of the brain. The PI plans to study the relationships between the peripheral clock in the excretory organ and pacemakers in the central nervous system. Experiments will involve organ transplants and in vitro cultures, using novel strains of transgenic flies carrying luciferase reporter, which allows real time continuing measurement of mRNA levels of clock genes. The PI wishes to establish the degree of autonomy of the peripheral clock in the Malpighian tubules and to test how other tissues affect its function. The PI will also probe the nature of the photoreceptor that mediate the resetting of the clock by light and investigate the functional role of the clock in the excretory organ. In this pursuit, the PI seek to answer a fundamental question of whether the circadian system in Drosophila is a centralized one, comprised of a single dominant clock that would control all rhythmic functions, or is a decentralized system with several independent clocks located in different tissues and controlling subsets of physiological functions. Malpighian tubules are uniquely accessible to both physiological and molecular genetic investigations. The broad scope of this proposal will allow thorough characterization of the circadian clock in this tissue. providing impetus for future resear ch aimed at understanding how already identified clock genes receive environmental input on one hand, and produce rhythmic outputs on the other.

DESCRIPTION (provided by applicant): As life span increases in the US population and more women choose demanding careers deferring childbearing to later years, age-related health problems become a matter of national concern. There is an urgent need to understand the biological basis of the aging process and the basis of reproductive senescence in humans. Research on model organisms such as Drosophila helped to uncover several lifespan-regulating genes; however, genetic pathways that postpone aging are far from being fully understood. We recently discovered that the gene period (per), a well-known component of the biological clock, affects longevity and fertility in female Drosophila. There is extensive knowledge of this gene and its protein product; therefore, tools are available to research its role in aging. We plan to verify and expand our initial findings and address several new questions: 1) Are per effects on lifespan gender-specific? 2) Does per affect reproduction and life span in both virgin and mated females? 3) Which physiological systems are involved in per action? Results of these proposed studies will provide the first insights into the mechanisms underlying the action of the clock gene period in the investigated phenotype and allow us to develop a research project grant (R01) aimed at understanding the molecular pathways underpinning female reproductive aging. Our goal is consistent with NIA plans to accelerate efforts to discover additional longevity-related genes and to characterize their biological function. Given the conserved functions of per in biological timing from flies to humans, knowledge gained concerning the mechanism of action of this gene may provide important contributions to our understanding of reproductive and chronological aging in humans.

A. PROJECT SUMMARY
Intellectual merit. Circadian clocks are important coordinators of physiological and behavioral rhythms. Drosophila melanogaster serves as an excellent model for investigating clock mechanisms, however little is known about the functional significance of clock genes in the fly physiology. The group led by Dr. Giebultowicz demonstrated that peripheral circadian clocks play multiple roles in reproductive physiology. Recent research revealed that two essential clock genes, period and timeless, are involved in the regulation of female fecundity. Surprisingly, these genes seem to act not as components of the circadian clock but as part of a homeostatic mechanism. An integrated physiological and molecular approach is proposed to investigate this novel fitness-related phenotype. Preliminary data led to a hypothesis that proteins encoded by period and timeless are part of the signaling pathway that modulates the rate of egg production. This hypothesis will be tested in an experimental program divided into four objectives. (1) Several aspects of reproductive physiology will be compared between mutant and wild-type females, to determine if mutant females have altered endocrine functions or other physiological parameters that may account for their failure to increase oogenesis in response to protein-rich diet. (2) A genetic approach will be used to determine whether increased fecundity of flies on high-protein diet is dependent on the function of period and timeless genes in the ovary, or in other organs such as the nervous system. (3) Expression of period and timeless will be investigated at the mRNA and protein level to determine whether it is affected by female age and nutritional status. (4) Biochemical tools will be used to elucidate the role of period and timeless in female fecundity. Accomplishment of these objectives will be aided by the wealth of genetic information and the ease of molecular manipulations in Drosophila, which make it an ideal system for the dissection of multi-component nutrient-fecundity pathway. The results of these studies will help to understand the functional significance of phylogenetically conserved clock genes in a genetic pathway regulating fecundity.
Broader impact. Scientific program outlined here will facilitate partnerships between several faculty members, graduate, and undergraduate students. The proposed research will provide ample opportunities for training students; participants from groups underrepresented in science will be actively recruited to the program. The combination of physiological, molecular and genetic experiments will allow students to gain experience in the interdisciplinary approach to research. Students will be mentored with respect to their academic goals and encouraged to consider career in science. Dr. Giebultowicz has an excellent record of engaging undergraduate female students in her research and helping them to choose science as a career. The PI also integrate research activities into the teaching of science in area schools. She is a co-PI in the NSF-funded K-12 Rural Science Education Program at OSU, mentoring undergraduate and graduate students who help to teach science in selected rural schools in Oregon. She also participate in "Adventures in Learning" program for middle school girls, inviting students to the lab to conduct small experiment, offering hands-on approach to science. Research results obtained with NSF funds will be published in peer-reviewed journals, and research highlights will be posted on the laboratory web-site in a format accessible to the general public.

[unreadable] DESCRIPTION (provided by applicant): Circadian clocks are evolutionary conserved coordinators of behavioral and physiological processes. Malfunctions of circadian clocks in humans lead to serious pathologies such as sleep disorders and cancer. Circadian timekeeping is accomplished by molecular feedback loops that involve several clock genes and their proteins. The role of two genes period (per) and timeless (tim) has been well established in the clock feedback loop, which operates in the model organism Drosophila melanogaster. The products of these two genes, proteins PER and TIM, translocate to cell nuclei and are subsequently degraded; both events are essential for clock function. Surprisingly, in the ovary, these proteins behave differently. Their levels do not cycle; instead, they remain stable and cytoplasmic at all times. Despite such unorthodox behavior, ovarian PER and TIM have important functions in the modulation of egg production. We hypothesize that PER and TIM may undergo different post-translational modifications in the ovary than they do in the clock cells. We propose to use biochemical and genetic tools to test this hypothesis in three specific aims. First, we will study interactions of PER and TIM in the ovary and their functional significance. Second, we will investigate posttranslational modifications of PER and TIM related to phosphorylation. Finally, we will explore the reasons for the lack of TIM degradation in response to light in the ovary. Results obtained in this study will give us important insights into the functional significance of non-circadian expression of clock proteins in the fly ovary. There is increasing evidence that genes that were thought to act exclusively as clock components have other important pleiotropic roles. They act in a non-circadian manner in both Drosophila and mammals. Therefore, understanding the non-circadian functions of clock genes in a model organism should provide valuable insights into human health. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Exploring links between circadian clocks and aging Summary: Age-related decline in the physiological and cognitive functions in humans is of great concern to society and there is an urgent need to identify the biological mechanisms that support healthy aging and longevity. Recent evidence suggests that the biological (circadian) clocks are important for maintaining health during aging. Circadian clocks are endogenous molecular regulators that coordinate daily changes in the level of gene expression, physiological functions and behavior with external day/night cycles. Disruption of circadian clocks in mammals result in accelerated aging and increased age-related pathologies such as cancer and neurodegenerative diseases. Data from our laboratory demonstrated that disruption of circadian clock in the model organism Drosophila melanogaster also leads to premature aging and compromised longevity. The aim of this proposal is to test the hypothesis that deregulation of circadian network is causally linked to aging in Drosophila. We plan to investigate the relationship between circadian systems and aging by focusing on two aims: a) we will attempt to increase the expression of declining clock genes via transgenic manipulations, and determine whether these treatments can enhance the amplitude of per and tim circadian oscillations in old flies. This will allow us to identify the molecular defects that cause age-related decay of circadian network and help us attempt to reverse this decay by genetic interventions b) Test if high amplitude of circadian oscillations support longevity and health during aging. Our results should provide critical information regarding links between strong circadian clocks and longevity. The fruitfly Drosophila is an excellent model to address these links due to its short lifespan (~60 days) and conservation of clock genes and aging mechanisms between flies and humans. Insights obtained from this innovative exploratory research will lead to a better understanding of the mechanisms that link rhythmic oscillations of the circadian system with health and longevity. The outcomes of this exploratory research proposal may point to novel ways to maintain optimal health during aging in humans by enhancement of the circadian systems. PUBLIC HEALTH RELEVANCE: Age-related decline in various life functions in humans is of great concern for society, and there is an urgent need to identify the biological mechanisms that support healthy aging and longevity. Recent evidence suggests that the biological (circadian) clocks play important roles in maintaining health during aging. The proposed studies will uncover the molecular mechanisms that cause age related decay of the circadian clock mechanism. Insights obtained from this work performed on a model organism may lead to novel ways of increasing longevity in humans by enhancing the circadian clock amplitude in aging individuals.

DESCRIPTION (provided by applicant): The long term objective of this research is to define molecular pathways by which the circadian clock regulates neuronal health. Circadian clocks are molecular feedback loops that generate daily cellular rhythms in the brain and various peripheral tissues. Disruption of the circadian clock has been implicated in neurological disorders but the underling mechanisms connecting the clock to neuronal health are not understood. We have recently shown that loss of the circadian clock in Drosophila dramatically increased accumulation of oxidatively damaged proteins, lipids peroxides, and caused neurodegenerative changes in the brain. Furthermore, we identified a daily rhythm in levels of reactive oxygen species (ROS), while ROS was constantly elevated in flies with a disrupted circadian clock. Molecular oxidative damage is a significant risk factor for age-related neurological disorders and glutathione (GSH) is a key antioxidant that protects neuronal cells against oxidative stress. Depleted GSH levels are found in a number of neurological disorders including schizophrenia, Parkinson's, and Alzheimer's diseases as well as in normal aging. To counteract neurological diseases, basic research is needed to understand mechanisms regulating GSH homeostasis. We obtained exciting preliminary data suggesting that GSH synthesis may be controlled by the circadian clock. We revealed a circadian rhythm in the expression of the catalytic (GCLc) and modulatory (GCLm) subunits of glutamate cysteine ligase (GCL), which is the rate-limiting enzyme in GSH biosynthesis, as well as daily rhythmic changes in GSH levels. These rhythms were abolished in flies with a genetically disrupted circadian clock and in older flies, whose circadian clock becomes impaired. We hypothesize that the circadian clock modulates GSH biosynthesis, and that temporal regulation of GSH homeostasis results in efficient prevention/repair of oxidative damage and protection of the nervous system. To test this hypothesis, we propose an interdisciplinary collaboration using the excellent model system Drosophila melanogaster. In aim 1, we will determine roles of circadian clocks in the regulation of glutathione biosynthesis in the brain. We will then explore functional links between rhythms in GSH biosynthesis and neuronal health in aim 2. Finally, in aim 3 we will determine the effect of an aging circadian clock on the GSH system and neuronal health. Public health significance: Insights obtained from this work may lead to novel strategies to avert neurodegeneration in aging humans, which is a critically important medical and societal issue.

DESCRIPTION (provided by applicant): Age-related decline in the physiological and cognitive functions in humans is of great concern to society and there is an urgent need to identify biological mechanisms that support healthy aging and longevity. Recent evidence suggests that the circadian system is important for maintaining health during aging. The circadian system comprises of a central pacemaker regulating behavior and peripheral oscillators in most body organs that coordinate daily oscillations in gene expression, small metabolites, and tissue-specific physiological processes. Multiple observational studies in humans linked disruption of circadian clocks with accelerated aging symptoms, such as neurodegenerative diseases, but the underlying mechanisms are not understood. We recently reported that a mutation in one of the clock genes leads to premature aging and impaired neuronal homeostasis in the model organism Drosophila melanogaster. We also determined that the circadian mechanism decays in peripheral clocks of aging flies due to reduced expression of clock genes. Our preliminary data suggest that specific genetic interventions can improve molecular and behavioral rhythms in the aging organism. We hypothesize that increasing the strength of circadian peripheral clocks may retard aging and promote health span. We will test this hypothesis as a collaborative team between three labs with complementary expertise focusing on three aims: 1) Identify molecular causes of age- related decay of clock gene expression in different tissues; 2) Test whether improvement in clock genes oscillations avert functional decay in aging flies and extend health span and lifespan; and 3) Identify pathways controlled by peripheral clocks that mediate health span benefits in aging flies. Our results should yield critical information regarding functional links between strong peripheral circadian clocks and aging rate. Insights obtained from this in vivo research may have clinical relevance by uncovering novel ways to maintain optimal health during aging in humans by enhancement of the circadian systems.

? DESCRIPTION (provided by applicant): Circadian clocks are important regulators of cellular functions and homeostasis. Age-related alterations in the human circadian system are implicated in neuronal pathologies. Recent evidence in fruit flies and mice suggests a correlation between disrupted rhythms and neurodegeneration; however, very little is known about the mechanisms involved. To address this, we compared the circadian transcriptome in young and old Drosophila heads using RNA- Seq. As expected, we found that several genes that were expressed rhythmically in young flies lose their cycling patterns to become constitutively low or high in old flies. Surprisingly, we also uncovered a novel group of genes which were low and arrhythmic in heads of young flies that became highly expressed and strongly rhythmic in old. This group contains known stress- responsive genes that are induced in response to oxidative stress or hypoxia to protect proteins from damage. Based on our preliminary data, we hypothesize that the circadian system orchestrates rhythmic expression of neuroprotective genes, which we termed late life cyclers (LLCs), in response to intrinsic stress and damage in the aging nervous system. We will test our hypothesis in two Aims. In Aim 1, we will conduct a comprehensive comparison of the circadian transcriptome in the heads of young and old males and females to fully characterize age-related changes in core clock and clock-controlled genes, and determine whether these changes are sexually dimorphic. We will also test whether LLC rhythms are maintained in constant darkness but abolished in flies with clock-disrupting null mutations in core circadian genes. In Aim 2, we will test whether LLCs play neuroprotective roles by comparing biomarkers of aging in wild type and clock mutant flies with LLC rhythms present or absent. We will also test whether exposure to exogenous oxidative stress induces rhythmic LLC expression in young flies. This explorative proposal may reveal clock-controlled pathways that protect the brain from age-related damage. Given the conserved genetic mechanisms of the circadian clock and aging biology, these pathways may also function in mammals.

Aging is the greatest risk factor for the development of Alzheimer's disease and related neurodegenerative conditions. There is increasing evidence that age-associated changes in metabolism play a major role in the onset or progression of these diseases. Both aging and Alzheimer's disease are associated with higher glucose accumulation and its reduced usage in the brain. While correlative data suggest that impaired glucose metabolism promotes Alzheimer's disease, the mechanisms causing these impairments are poorly understood. We recently identified profound age-related changes in the expression of genes associated with glucose metabolism in the heads of a model organism, fruit fly Drosophila melanogaster. Our preliminary NMR study demonstrated age-dependent increase in glucose in fly heads suggesting metabolic dysregulation similar as occurs in Alzheimer's disease. We will take advantage of transgenic flies with Alzheimer's like symptoms to perform mechanistic studies on the links between energy metabolism and neurodegeneration. Our overall aim is to understand factors that impair neuronal energy metabolism in aging and Alzheimer's disease models. Results of these studies may help to reveal ways to prevent or delay neurodegeneration and identify novel avenues for prevention and therapeutic intervention in Alzheimer's disease. We propose three specific aims. 1) Elucidate functional significance of age-related changes in the expression of metabolic genes and insulin signaling in causing neurodegeneration. 2) Determine changes in metabolomic profile in Alzheimer's disease models and investigate how they are controlled. 3) Integrate transcriptomic and metabolomic data to detect putative neurodegeneration-preventing pathways and test functional role of these pathways using genetic and pharmacological approaches. Taken together, proposed experiments will help to reveal mechanistic links between age-related changes in energy metabolism and Alzheimer's disease.