Paul H. Taghert - US grants

The mechanisms that establish cell-specific patterns of neurotransmitter expression are largely unknown. This research will examine (i) the factors that govern the initial expression of particular neuropeptides during embryogenesis; and (ii) the factors that modulate that expression during post-embryonic life. These studies focus on the simple nervous system of the moth in which neurons and their precursor cells are individually identifiable and can be examined with a variety of cellular and molecular approaches throughout their development. In particular, two previously generated monoclonal antibodies recognize cardioactive neuropeptides that are expressed by different identified neurons at various stages of embryonic and postembryonic development. Using these and other cellular and immunological techniques, the potential contributions of cellular environment and invarient cell lineages in directing the fate of these individual peptidergic neurons can be elucidated with great precision. In order to study the developmental regulation of neuropeptide expression in greater molecular detail, the monoclonal antibodies will be used to purify these neuropeptides. Recombinant DNA techniques will then be used to isolate the genes that encode them. Structural information derived from these experiments will facilitate the study of the cell-specific patterns of neuropeptide gene expression in a relatively simple and highly accessible developing central nervous system.

Neuropeptides are signals that are released by nerve cells to transmit information to other nerve cells and to other cells in the body, like muscle, glands and skin. Very often within a single neuron, neuropeptides are released along with specific neurotransmitters and modulate their subsequent action. Indeed, the nervous system is designed to received and transmit information and neuropeptides are central to those basic processes. While it is estimated that 100's of neuropeptides are made by nerve cells, only 1% of all nerve cells express a given neuropeptide gene. Dr. Taghert will use an innovative molecular genetic approach to examine the fundamental mechanisms underlying the expression and release of neuropeptides. He will create and recover loss-of-function mutations in a specific gene for a precursor protein that encodes 13 related neuropeptides. By studying individuals that are chronically deficient in the production of these molecules, Dr. Taghert will determine function and whether other neuropeptides share overlapping roles. In addition, he has developed methodology to put genes back and thus, will assess the specific capacity of subsets of encoded neuropeptides to restore lost functions. These experiments will lead to a better understanding of the roles played by the neuropeptide gene in individual neurons. Since Drosophila is used as the model system, demonstrating the lethal nature of mutations in the neuropeptide encoding gene may help stimulate research into insecticides that act by inhibiting neuropeptide synthesis or neuropeptide stimulated neurotransmission.

This award will support the participation of 11 undergraduate students each year in the Research Experiences for Undergraduates program offered by Washington University School of Medicine in St. Louis, MO. Each student participant will spend ten weeks engaged in developmental biology research under the direction a faculty mentor of his/her choice, selected from among ca. 40 faculty members who have active, well-funded research programs and who study developmental mechanisms in a wide variety of experimental systems. To permit students to view their research projects in a broader context, five types of supplemental activities will be provided: (i) a beginning-of-the-summer retreat at a state park, where participants will meet with each other, explore the issue of research ethics, and become introduced to the study of developmental biology; (ii) workshops dealing with important model organisms, methods, and paradigms in contemporary developmental biology research; (iii) faculty seminars designed specifically for the students; (iv) participation in a student developmental biology journal club; and (v) and end-of-the-summer research symposium at which students will make presentations summarizing their research accomplishments. Opportunities for frequent, informal social interactions will also be provided. This award will foster the continued education and training of individuals who will be part of the next generation of basic researchers in the developmental biology.

9730003 Taghert Neural communication leads to information processing and ultimately to the command of behavior. The chemistry of the brain is the currency by which communication is exchanged by brain cells. That chemistry is complex and includes several hundred different compounds that are used at different times and different places in the brain. In particular one class of "neural transmitters" are small peptide hormones called neuropeptides. These are very important for the direct regulatory control they appear to have on a variety of specific behaviors including appetitive and reproductive behavior, mood, stress, and anxiety. Dr. Taghert uses a model invertebrate system, the fruit fly Drosophila, to employ genetics to study the relation between neuropeptides and behavior. This use of genetics is important because it provides the means to manipulate gene expression in the animals and Drosophila offers the most comprehensive and sophisticated set of genetic tools. Indeed by manipulating these mechanisms genetically, Dr. Taghert can address neuropeptide functions in vivo. Given that neuropeptides rely on proper post-translational modifications for their correct structure and hence for their functions, he will examine a specific enzyme that is necessary for the synthesis of nearly all neuropeptides, and only neuropeptides. Since animals that are deficient in this enzyme die early indicating the importance of neuropeptides for development and survival, Dr. Taghert has devised two independent means to make animals that are mosaic for the enzyme. Using biochemical, anatomical and behavioral tools, he will fully evaluate these mosaics. The results from these studies will shed new light towards understanding the role that neuropeptides play in modulating how neurons communicate information.

In this proposal, I outline a set of three related yet independent studies of circadian neural output. Recent advances in imaging and data analysis capture information regarding network phenomena with increasing spatial and temporal precision. The circadian pacemaker system we study is advantageous in that it produces physiological activity both spontaneously and rhythmically. In the previous cycle, we used planar illumination methods to perform 24 hr in vivo brain-wide scans of the circadian neural circuit. That work introduced a new concept to theories of how the circadian network encodes time: we showed that the molecularly synchronous pacemaker network displays sequential activation by different identified pacemaker groups across the day. Further we found pacemaker cell interactions, in the form of neuropeptide-mediated delay, represents a key mechanism to effect sequential pacemaker activation. The scientific premise for this project rests on the need to extend those observations on circadian pacemaker neuronal plasticity and to understand how these activity patterns are transmitted to downstream centers. Here I propose work that continues real-time in vivo studies of neuronal activity patterns for the core Drosophila circadian pacemaker neurons. It also continues the focused analysis of neuropeptide modulatory mechanisms that critically regulate the specific timing of pacemaker activity. To extend the scope of our initial studies, and to provide a better understanding of neuronal properties of pacemakers and pacemaking networks, this program will pursue three Aims. Aim 1 will better define daily Ca2+ dynamics in pacemakers by (i) performing in-depth, high frequency sampling, and (ii) by determining the sub-cellular mechanisms underlying these fluctuations. Aim 2 will pursue a Structure-Function analysis of the PDF receptor (PDFR), especially its C-terminus, to understand the regulatory mechanisms that control the quantitative extent of daily PDF signaling. It also seeks to identify key PDFR regulatory proteins. Aim 3 seeks to extend the scope of our work beyond the circadian pacemaker network to identify downstream circuit elements: we will focus on subsets of neurons in the Central Complex for which preliminary evidence suggests an involvement in daily rhythmic physiology associated with locomotion. Jet-lag, shift-work and disturbances in sleep-activity cycles all contribute to degrade mental and physical well-being. Two major causes of death (stroke and cardiac arrest) display clear time- of-day variation, yet, we have little understanding of the causal links between circadian clock functions and disease mechanisms. This research program is dedicated to a better understanding of fundamental circadian output mechanisms.

The Optical Imaging Core of the Washington University Center for Translational Neuroscience (WUCTN) will[unreadable] allow members of the Neuroscience community to expand already successful projects and initiate new[unreadable] projects, especially in the area of bioluminescence. While there are presently a variety of optical imaging[unreadable] facilities within the medical center and main campus, these have not been available to the larger[unreadable] neuroscience community because they have only limited technical support. To accomplish its goals, the[unreadable] Core will coordinate already existing resources at Washington University for confocal and two-photon[unreadable] microscopy by neuroscientists and also establish new low-light level imaging capabilities. Access to the[unreadable] Optical Imaging Core Facilities will be extended to Washington University neuroscientists at three separate[unreadable] locations. Two facilities will be housed at the Medical Campus (WUMS) and one at the Undergraduate[unreadable] campus (WU). All three facilities will offer instruction and supervision for the different microscopy methods[unreadable] supported. The Optical Imaging Core will offer facilities for:[unreadable] 1. Confocal microscopy for high resolution imaging of fluorescent probes. This includes the imaging[unreadable] and differentiation of variants of GFP (such as YFP, CFP, eGFP), and more red-shifted dyes (Cy5) in[unreadable] both living and fixed preparations.[unreadable] 2. Two-photon microscopy for deep tissue imaging, including in vivo and in vitro time-series measures.[unreadable] Use of the 2-photon technique will minimize damage due to repeated imaging.[unreadable] 3. Low-light imaging of fluorescent and bioluminescent reporters. This includes real-time imaging of[unreadable] gene activity using luciferase constructs and of other cellular events using novel transgenic reporters[unreadable] in cultured cells, in tissue slices and in whole small animals.[unreadable] 4. Technical support for the neuroscientists to ensure the efficient creation of high quality images for[unreadable] quantitative image analysis and ultimately publication. This includes training, education, software[unreadable] development, data management and maintenance of imaging equipment and materials.[unreadable] 22 of the 54 projects described involve clinically-relevant studies, and most of the others address hypotheses[unreadable] that directly inform translational research. Significantly several projects involve new experimental directions[unreadable] and collaborations that will be enabled and facilitated by access to these Core facilities.

DESCRIPTION (provided by applicant): We request renewal of the Systems and Molecular Neurobiology Training Program (T32 GM08151) in the Division of Biology and Biomedical Sciences at Washington University. The objective of the grant is to provide critical support for graduate education in the Neuroscience Program, which is one of the finest in the world. This Program will offer three year appointments to at least 45 trainees who have already completed the Qualifying Examination to achieve candidacy to the Ph.D. At its heart, the Program manifests long- standing commitments to excellence in research, to interdisciplinary education, and to providing its students with superb training in their courses and in the laboratory. It is also an especially broad program, combining expertise in molecular, cellular and systems-level approaches to the study of neural function and dysfunction. Continued diversification has allowed it to remain at the forefront of developments on many different research areas. Continued innovation and a commitment to diversity make this educational program exciting and forward-looking. RELEVANCE: Progress in Neuroscience research is making positive impact in numerous clinical, educational and socially- important arenas. This Program will continue to pursue excellence in training the next generation of Neuroscientists who will engage in studies of the brain that range from the molecular level all the way to the systems level.

DESCRIPTION (provided by applicant): This research program addresses how a neuropeptide modulates olfactory processing of volatile pheromone information and shapes downstream courtship behavior. We are approaching this issue as a Multi-PI team using the model genetic system, Drosophila. The CG4395 gene encodes a Class B peptide G protein-coupled receptor (GPCR) highly related to mammalian receptors for Calcitonin and Calcitonin Gene Related Peptide. CG4395 specific antibodies and CG4395-promoter constructs reveal that CG4395 is prominently expressed in olfactory receptor neurons that respond to pheromone as well as in higher regions of the brain that have been implicated in courtship behavior and plasticity. Significantly, mutant male flies that lack CG4395 gene expression exhibit aberrant courtship behavior. Furthermore, this behavioral defect can be restored to mutant animals by expressing a CG4395 transgene in olfactory sensory neurons. These findings lead us to hypothesize that peptide neuromodulation via CG4395 regulates courtship-relevant pheromone processing. The CG4395 GPCR is an orphan, but it responds potently to a peptide factor in Drosophila head extracts which we have greatly enriched by three HPLC steps and now propose to purify to homogeneity. We also propose to use the power afforded by Drosophila genetics to study the CG4395 receptor and peptide ligand, separately and together, in vivo to learn where and when receptor signaling occurs. We will combine a battery of molecular genetic tools with detailed behavioral analyses to understand the roles of the receptor and ligand in courtship. In addition we will employ a novel genetically-encoded real-time reporter of cAMP levels that uses in vivo FRET measurement regulated by CG4395 as revealed by a novel genetic reporter. To achieve these goals demands skills greater than can be assembled in any current Drosophila laboratory. Therefore, this research program represents the collaborative efforts of three independent groups, each of which contributes specific expertise and technical experience. Neuromodulatory transmitter systems regulate numerous higher brain functions including attention, memory, mood, appetite, social behavior and aggression. When transmitter systems go awry, clinical problems often arise. Identifying modulatory peptides and cognate receptors is therefore a key endeavor in studying the mechanisms underlying behavior. GPCRs and their signaling pathways are highly conserved in evolution, thus the molecular mechanisms of neuromodulation we aim to describe will offer potential avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE Neuromodulation is fundamentally important for higher brain functions and regulates diverse brain-based phenomena including attention, memory, mood, appetite and aggressive behaviors. The normal actions of monoamine and peptide transmitters systems are keys to such modulation and when these transmitter systems go awry, these processes are compromised and clinical problems arise. This research program uses biochemical and genetic approaches to explore fundamental mechanisms underlying modulation of the neural circuits that regulate reproductive behaviors (courtship) in the model system Drosophila.

DESCRIPTION (provided by applicant): To support pursuit of the NINDS mission, here we propose to introduce a high-impact novel technology for Imaging to the Washington University Neuroscience community. We intend to provide Core Instrumentation to perform Planar Illumination Microscopy (also known as light sheet microscopy) to catalyze new imaging efforts that aim to monitor large-scale neuronal ensembles. The recent Federal announcement of the Brain Activity Map targets neurophysiology for rapid growth and development in the coming decade. One of the most promising techniques for high-throughput neurophysiology is fluorescence imaging using light sheets. Our implementation, Objective-Coupled Planar Illumination (OCPI) microscopy, achieves imaging speeds that are hundreds or thousands of times faster than two-photon microscopy by simultaneously illuminating all pixels in the objective's focal plane, thereby eliminating the need to collect images by scanning one pixel at a time. The method achieves fast imaging simultaneously with high sensitivity and low phototoxicity and is therefore particularly well-suited to long-term recording periods. The custom-built OCPI instrument will be a Core offering within an existing Imaging Center, the Bakewell Neurolmaging Facility. Therefore, this proposal describes a plan and a schedule by which the new OCPI microscope will be introduced, and the oversight we will establish to ensure that its use is made available fairly and broadly. We also emphasize that we have recent experience in precisely these community-based efforts: we successfully introduced earlier version of this instrumentation as a Core facility and now can foresee the need for more access by a larger community of Neuroscientists to a faster and more flexible version. We plan Facility support for OCPI users that includes a facility manager to provide maintenance and training, a data center for image analysis, and a data manager to help with customized data analysis. We provide a series of quantifiable milestones by which to evaluate progress in offering this new high-impact technology.' By the end of the 4-yr period, we anticipate a steady base of at least 20 different users from across the diverse WU Neuroscience community, and a usage rate at or near full-time.

DESCRIPTION (provided by applicant): The cell biology of secretory pathways has evolved into an exciting but complex field that provides insight into a staggering array of human diseases including Alzheimer's, drug addiction, autism, diabetes, obesity, and various cancers. Initially, this GRC provided much emphasis on processing enzymes - those that catalyze the maturation of hundreds of bioactive peptides, but in addition membrane-associated proteolytic systems, particularly the matrix metalloproteases and the secretases, that control tissue remodeling and membrane shedding in normal cells, as well as the extracellular activation and/or inactivation of a variety of molecules such as growth factors and anti-microbial peptides. The focus has expanded to a cell biological perspective, including the regulation of protein sorting and traffic, vesicle formation and the formation of dense-core secretory granules in stimulus-coupled endocrine and neuroendocrine cells. The cooperative roles of the secretory pathway and other protease systems, taken in the context of the underlying cellular trafficking machinery that contributes to complex, prevalent diseases, provide urgent reasons to continue this multi- disciplinary approach to examine the interactions of secretory pathway components in homeostasis and in disease. The Protein Processing, Trafficking and Secretion Gordon Research Conference are unique in convening scientists from many biological disciplines to join in a forum for just this purpose. This recurring Conference has remained innovative because it works to identify the most exciting and fundamental questions. In large part, this involves developments in the study of pathophysiological states that affect secretory pathways: A notable example is the potential clinical significance of PCSK9 to help control serum cholesterol levels. PCSK9 belongs to a small processing enzyme family that has been extensively studied by this GRC over the years, and drugs for which are now entering the public sphere with considerable anticipation. Likewise, the 2016 Program will present further novelty by featuring increased emphasis on the mechanisms underlying endocytosis and exocytosis. This emphasis jibes with renewed interest by neurobiologists and endocrinologists in the cell biology underlying release of bioactive peptides and peptide hormones. The Program is organized by a diverse committee that represents Academic, Industrial and Governmental science. The GRC is associated with a GRS (Gordon Research Seminar) that is specially designed for young trainees and which has enjoyed success as a scientific and mentoring adjunct. The support we request will permit participation in the GRS and GRC by trainees and junior stage colleagues.

General Abstract

The biological mechanisms that generate biological rhythms are critical for life, as most organisms on earth have to adapt to cyclical changes in their environments. The time-scales associated with these changes vary widely, and thus so do the mechanisms that allow organisms to use them to predict their environments and survive. Identifying the fundamental principles associated with such diversity requires a broad range of intellectual inputs. The conference supported by this award will therefore bring a diverse group of investigators together from across the United States and Latin America, including senior scientists who are leaders in the field of biological rhythms and young investigators just beginning their training, to foster the exchange of ideas and to provide training and mentorship opportunities for the young investigators from Latin America. Senior US investigators will run in-depth discussion groups with young scientists from Latin America during a one-day workshop, and pairs of senior-junior scientists will then interact one-on-one throughout the following week at an associated conference. This not only provides excellent mentorship opportunities for the young scientists, but also the potential for new ideas for the senior scientists that come from fresh perspectives. A high proportion of the US scientists that will be supported are women, and the international collaborations between US and Latin American scientists will promote growth in the field for investigators across the Americas.

Technical Abstract

This is a small meeting that will pair senior US and Latin American scientists with graduate students and postdoctoral associates from Latin America during an intense, one-day workshop in Valparaiso, Chile, followed by continuing interactions between mentors and trainees during a subsequent meeting on the topic of biological rhythms. There will be four roundtable discussions on 1) Neural Circuits Associated with Sleep; 2) Sleep and Human Performance, 3) Neural Models of Rhythmic Physiology, and 4) Mechanisms of Entrainment. Each will be jointly chaired by US and Latin American senior scientists, and attended by graduate students and postdoctoral associates from Latin America. The senior scientists will briefly discuss their research, followed by intense discussion groups with the students. Each senior scientist will be paired with a young scientist and expected to have follow-up interactions with that student during a subsequent meeting Chronobiology that all participants will attend. In the past, these interactions have led to multiple, collaborative papers that include both the senior and junior scientists. This meeting, like those, is expected to greatly benefit both senior and junior scientists across the Americas and lead to significant discoveries in the field of biological rhythms.

Abstract Jet-lag, shift-work and disturbances in sleep-activity cycles all contribute to degrade mental and physical well-being. To begin addressing the chronobiological bases for such pathophysiologies, this proposal seeks to describe the neural basis to generate and refine the timing signals that organize and trigger daily rhythmic physiology. Here I outline proposals for three related yet independent studies of circadian neurophysiology. Recent advances in imaging and data analysis can record network phenomena with increasing spatial and temporal precision. The circadian pacemaker system produces physiological activity both spontaneously and rhythmically, which promotes an in-depth analysis. We have introduced planar illumination methods to perform 24 hr in vivo brain-wide scans of the Drosophila circadian neural circuit. That work outlines a new framework for how the circadian network encodes time: a pacemaker network whose internal clocks are strongly synchronized, which nevertheless displays sequential activation by different identified pacemaker groups across the day. Furthermore pacemaker cell interactions, principally in the form of multi-hour neuropeptide-mediated delays, appear to be the preponderant mechanism by which the sequential activities of pacemakers are organized. Therefore, the scientific premise for this project rests on the need to better understand the neural basis for the operations of this timing circuit and its modulation. Here I propose work to further real-time in vivo studies of the brain network that is composed of the core ~150 Drosophila circadian pacemaker neurons. To provide a better understanding of neuronal properties of the pacemaking network, and to extend the scope of our initial studies, we will pursue three Aims. Pacemaker cell interactions are the keys to understanding the dynamic relationships that govern the sequence and tempo of network outputs, and to-date our knowledge is limited to only a few such signals. Thus Aim 1 will systematically test pacemaker cell interactions across the network with chemogenetic control agents, using Ca2+ as a reporter. Aim 2 seeks to extend the scope of our work beyond Ca2+ signals by employing a genetic realtime reporter for cyclic nucleotides, which are established 2nd messengers in the circadian circuit but whose in vivo dynamics are poorly defined. Finally, Aim 3 will study dopamine signaling within the circadian circuit ? we will define spontaneous 24 hr patterns of dopamine cell activity in vivo and test two hypotheses concerning the putative functions of dopamine signals in the pacemaker network. Together these efforts will provide multi-layered information regarding the dynamic state of the pacemaker system network-wide in vivo for the course of the entire day.