Ronald L. Davis - US grants

The long range objectives of this research program are to understand the structure, function, evolution and regulation of the cyclic nucleotide phosphodiesterase (PDE) enzymes and the genes which encode them. These enzymes hydrolyze cyclic nucleotides and therefore serve to inactivate or modulate the biological signals produced by hormones, neurotransmitters or other agents which function by increasing intracellular Cyclic nucleotide levels. Studies of three mammalian genes, each homologous to the Drosophila dunce gene, and each coding for a cAMP PDE are proposed. Nucleic acid and antibody. probes specific for each will be used to reveal the populations of cells expressing the mRNAs and proteins in the rat brain. This will allow determination of which of the mammalian PDEs are concentrated in specific neuronal processes, like the dunce PDE in flies. To evaluate the physiological role(s) of one or more of the PDE genes, mutant mice will be obtained and analyzed phenotypically, especially with respect to potential neurological problems. This is particularly important, since one major phenotype associated with dunce mutation in flies is a pronounced memory deficit. Since existing cDNA clones and Northern blotting suggest that the PDE genes code for several proteins by alternative splicing of RNAs, additional mouse brain cDNAs will be isolated for a representative PDE gene to reveal the protein product diversity and to isolate full-length copies of the mRNAs. The cDNA clones will be expressed in yeast, to characterize the biochemical properties of the enzymes and to determine whether they are inhibited by the antidepressant rolipram, like the existing representatives. The cDNA clones, in turn, will be used to isolate genomic clones for the gene. The structure of the gene will be elucidated to compare its structure with the remarkably complex counterpart from Drosophila. The transcription start site(s) will be mapped and the promoter region(s) characterized to potentially understand the signals responsible for spatial expression patterns in the brain. We expect these experiments to contribute critical structural and functional information about these members of an important family of enzymes, the cyclic nucleotide PDEs.

The long range objectives of this research program are to define the molecular and cellular processes underlying animal learning and memory. In Drosophila, the dunce gene is the best characterized of the set of genes involved in olfactory learning and memory, causing an abbreviated memory when mutated. Recent work has demonstrated that the product of dunce, cAMP phosphodiesterase, is particularly concentrated in mushroom bodies, a collection of about 2500 neurons and their associated neuropil in each brain hemisphere. This observation along with several others, led to the development of a new model envisioning mushroom body cells as the primary cells serving olfactory learning and memory and those cells which require the dunce gene product for their normal physiology in this role. The proposed research will test whether the expression of dunce in mushroom body cells is sufficient for normal learning and memory. Flies carrying chromosomal rearrangements which preserve or delete the specific dunce promoter (one of several) responsible for mushroom body expression will be tested for normal olfactory learning/memory, to determine whether behavioral abnormalities correlate with the loss of this promoter. And, the enhancer/promoter elements responsible for normal mushroom body expression of dunce and other mushroom body-specific genes will be defined and used to drive dunce minigenes in transgenic flies, to determine whether this expression is sufficient for rescue of dnc mutation. Other experiments will determine whether dunce activity is required physiologically or developmentally for normal learning and memory. Transgenic lines, carrying a dunce minigene driven by the hsp70 promoter which appears to rescue dnc mutants, will be used to determine when during development and/or adulthood that heat shock is required to effect rescue. In addition, temperature-sensitive alleles of dnc will be isolated and used to define the phenocritical period for dunce activity. Finally, mushroom body-specific enhancer/promoter elements will be used to promote the expression of a temperature-sensitive toxin gene in mushroom bodies, to determine whether the cells are required for learning and whether they are required for the long-term storage of information after training. These experiments will enhance our understanding of learning and memory in Drosophila, specifically providing evidence for when the activity of "learning and memory" genes is required and whether mushroom bodies are indeed, the true neuroanatomical substrate for olfactory learning and memory.

Some of the most exciting recent advances in the neurosciences are being made in studies of the fruit fly Drosophila melanogaster, where the combined power of genetics, molecular biology, developmental biology and behavioral analysis can be realized. This meeting is planned to bring together major practitioners in the field of Drosophila molecular neuroscience to discuss topics that include neurogenesis, sensory system development and function, and behavior. The participation of younger scientists will be encouraged.

DESCRIPTION (provided by applicant):The goal of this research is to define the molecular, cellular, and systems neuroscience that provides the basis for active forgetting. This area of learning and memory research has been overlooked and yet, there is every reason to believe that the processes underlying active forgetting are as complicated and important as learning itself and the stabilization of memories by consolidation. The research project utilizes the model system Drosophila melanogaster because of the ease with which the fly can be conditioned using olfactory cues, the numerous genetic and molecular tools available, and the ability to peer into the brain of living animals and watch the activity of different sets of neurons. The latter approach, functional cellular imaging, employs flies carrying transgenes that express reporters for calcium influx, synaptic transmission, or other neuronal events, to monitor changes in neuronal response properties among the expressing neurons before and after conditioning. Our prior studies using this technique demonstrated that dopamine neurons exhibit ongoing activity after the learning event itself and that this activity likely provides a forgetting signal to the postsynaptic mushroom body neurons. We will extend these studies in several different ways to help understand the mechanistic basis for active forgetting. There is a rich medical importance to this research given the well- documented problems of cognition associated with numerous neuropsychiatric disorders.

DESCRIPTION (provided by applicant): The goal of this research project is to define the logic by which the brain organizes different types of memories among its component neurons. The project contrasts how the brain organizes olfactory memories learned in association with a rewarding cue and those learned in association with an aversive cue, and delves into some of the underlying mechanisms. The research project utilizes the model system Drosophila melanogaster because of the ease in conditioning the fly using olfactory cues and because of the ability to peer into the brain of living animals and watch the activity of different sets of neurons. The latter approach, functional optical cellular imaging, employs flies carrying transgenes expressing reporters for calcium influx, synaptic transmission, or other neuronal events, to monitor changes in neuronal response properties among the expressing neurons before and after conditioning. To date, six different cellular memory traces have been defined using aversive olfactory conditioning. These traces form in different sets of neurons in the olfactory nervous system and occur with differing temporal dynamics. The memory traces that occur within these same neurons after a rewarding olfactory conditioning event will be examined along with the molecular mechanisms underlying memory trace formation. Since nearly every neuropsychiatric disorder affects memory formation, these studies will aid in understanding memory formation in the normal brain as well as in the diseased brain.

DESCRIPTION (provided by applicant): The long-term goals of this research project are to detail the possible role of the cAMP signaling system in Bipolar Disorder. A genetic association study of polymorphisms in twenty different candidate genes representing the cAMP signaling components in humans is proposed, using well-characterized populations of Bipolar Type I Disorder and Bipolar Type II Disorder. These genes were selected because of existing evidence that cAMP signaling may be perturbed in Bipolar Disorder and because cAMP signaling underlies many types of behavior, including learning and memory, in model organisms. Researchers from the Massachusetts General Hospital and the Baylor College of Medicine will collaborate on this project, with researchers from MGH facilitating the exchange of DNA samples from well-characterized Bipolar Disorder patients and assisting with the interpretation of clinical relevance, and researchers from the Baylor College of Medicine performing the genetic analyses. These translational studies are designed to systematically and critically test the hypothesis that alterations in one or more cAMP signaling genes are involved causally in the behavioral abnormalities associated with Bipolar Disorder. Public Health Relevance: Bipolar Disorder is a prevalent brain disorder. The studies proposed here will help to elucidate the genetic basis for the disease.

DESCRIPTION (provided by applicant): This application addresses the thematic area, "Applying Genomics and Other High Throughput Technologies." The goal of this research project is to identify all genes that have fundamental roles in the neurobiological processes underlying learning, short-term memory, and retrieval. The project will capitalize on recent developments in "genomics" by using libraries of reagents (transgenic RNAi) that interfere with the expression of individual genes. The model system employed will be the fruit fly, Drosophila, because of the ease of working with this organism genetically, the availability of libraries of reagents that interfere with the expression of individual genes, and because Drosophila genes are known, in general, to have conserved functions in mammals. Flies carrying interfering RNAi transgenes for each individual Drosophila gene will be constructed and then trained and tested for odor learning. This will identify the set of genes required for acquisition of information, memory stability over a short period, and/or the retrieval of that stored information. The knowledge that emerges from this project will help reveal the molecular/cellular machinery for learning in Drosophila and will be tremendously useful for uncovering learning mechanisms in mammalian organisms including humans. Given that Drosophila "learning" genes are conserved both structurally and functionally, with some already implicated in human brain disorders, the knowledge gained from this project will also catalyze discoveries about the genetics of human neurological and psychiatric disorders and contribute to the development of drugs to treat impairments in cognition. PUBLIC HEALTH RELEVANCE: The majority of human neurological and psychiatric disorders involve impairment in learning and memory. This project will identify all genes in the genome of a model organism required for learning and memory. Since genes that are involved in learning are conserved between species, the knowledge from this project will contribute to understanding the genetics of human brain disorders and the development of drugs to treat impairments in cognition.

DESCRIPTION (provided by applicant): Despite an overwhelming need for effective CNS therapeutics, little progress has been made. Improving CNS drug discovery efforts is an urgent goal, as an estimated 1.5 billion people suffer from a CNS-related disease or disorder worldwide. We believe that a major roadblock toward more effective CNS therapeutics is the lack of neuron-based probe discovery platforms cable of supporting HTS-level screening. It seems logical that CNS disease targets should be assayed in neurons instead of cell-lines, though the use of neurons in HTS screening campaigns is uncommon. We argue that development of a flexible and scalable neuron-based assay development platform that is compatible with HTS would facilitate probe development, while also perhaps spurring drug discovery efforts aimed at treating a variety of brain diseases. One of the significant barriers preventing R01-driven investigators from interacting with screening centers is the inability of these centers to miniaturize and scale-up neuron-based assays. We have developed an innovative approach for migrating neuron-based benchtop assays to an HTS-ready platform in order to make HTS more accessible to neurobiologists interested in probe discovery. The Neuroscience Department at Scripps Florida has engaged the Screening Center and the Lead ID group at TSRI in collaborative efforts to overcome this barrier and we have identified ways to alter current procedures so that neuron-based benchtop assays can be miniaturized and automated to produce turnkey assays capable of supporting biological and chemical screens with tens-of-thousands of molecules. We propose that the neuroscience field would benefit tremendously from a system that enabled a migratory route for bench-top neuron-based assays to be miniaturized and then scaled, leading to their use in HTS probe discovery campaigns. Importantly, the system that we have developed will also provide investigators with a toolset to develop novel assays for neuron-based HTS. Our proposal details plans to develop novel reagents, instrumentation and workflows that will demonstrate that a bench-top assay developed in primary neurons can be migrated to an HTS-compatible assay. As a proof of principle, we will migrate a bench-top synaptogenesis assay to this HTS-enabled system. We then propose to take this HTS-ready, neuron-based assay through a screen of ~25,000 compounds. Successful achievement of a screen of this magnitude in primary neurons would demonstrate that primary neurons can be used as a common platform for drug/probe discovery and that our approach could serve as a general assay development system for neuron-based HTS. Thus, the outcome of this project is expected to provide researchers and Screening Centers with an assay development platform capable of supporting HTS-level screens in a neuronal environment. Thus, funding this proposal will result in novel probes to regulate synaptogenesis, but will also generally serve the Neuroscience community as a whole by providing an assay development platform that can be used by anyone interested in scaling up bench-top assays for HTS campaigns.

DESCRIPTION (provided by applicant): The goal of this research is to relate the biology of a single type of neuron in the Drosophila brain to its function in memory processes and aging to meet the directives of Program Announcement PA-11-320: Develop research on single cell biology to enhance the understanding of the mechanisms of normal aging and of age-related diseases. The neuron under study, the dorsal paired medial neuron (DPM), is unique in morphology and function. The fly brain has but one DPM neuron per hemisphere and prior studies have shown that this neuron functions specifically in specific temporal phases of memory, including intermediate- and long-term memory (ITM and LTM). Moreover, the function of this neuron degrades with aging, leading to poor ITM and LTM in aged flies. This neuron thus offers a singular opportunity to relate the biology of a single type of neuron to aging and memory impairment due to age. Our studies will detail the synaptic connections in young and aged flies to determine whether connectivity underlies the cognitive impairment with age. They will also profile the RNA population of the DPM neurons across age to reveal how gene expression within this neuron type is altered with age, and how specific genes that are altered may underlie possible connectivity changes and the age-related memory impairment. Overall, the studies will contribute significantly to an understanding of how aging alters the structure and physiology of a specific type of neuron that is involved in specific phases of memory. There is a rich medical importance to this research given the well-documented problems of cognition associated with aging.

PROJECT SUMMARY / ABSTRACT There is a pressing need to develop new classes of therapeutics for the treatment of psychiatric and neurological disorders. Despite this need, little progress has been made towards this goal over the last two decades. One explanation for this failure is that the cell-based screens designed to identify new lead compounds have traditionally utilized immortalized, non-neuronal cell lines. It becomes understandable, in retrospect, why past screens have failed to yield the hoped-for plethora of new and important classes of compounds given the extraordinary architecture and physiology of neurons compared to other cell types. A second possible reason for this failure is that most cell-based screens have utilized a target-based approach, in which a specific protein target is identified that may hold promise for the development of new therapeutics. However, the brain and how brain disorders influence its function remains a large mystery. This fact argues that the alternative of phenotypic-based screens may offer more promise. Phenotypic screens search for influences on particular cell biological phenotypes without knowledge of the specific molecular targets that influence the phenotype. We have developed a novel screening platform that employs primary cultured neurons and cell biological, phenotypic readouts to identify the influences of small molecules. The methodology is flexible, scalable, and offers numerous advantages over traditional approaches. In this project, we propose to develop assays using this platform for the processes of mitochondrial dynamics, including biogenesis, fission, fusion, protein import, branching, and damage. We will use fluorescent markers for mitochondria to follow mitochondria for such assays, and conduct three screens of small molecules for influences on mitochondrial dynamics once the assays are optimized. Mitochondria dysfunction is associated with multiple psychiatric disorders like mood disorders, schizophrenia and autism; and neurodegenerative disorders as well. Thus, this project promises a rich new resource that can be used to survey small molecules, RNAi, or gene overexpression effects on mitochondrial dynamics in neurons.

Project Summary This proposal is for a long-term and flexible research program designed to obtain key insights into the biology of learning and memory. Although flexibility is inherent in its design, such that novel observations made over the course of the research can and will be pursued without delay, the program is grounded in three major lines of research: (1) the molecular, cellular, and systems neuroscience that underlie the process of active forgetting, (2) the logic by which the brain organizes different types of olfactory memories among its component neurons, and (3) the identification and characterization of protein-coding and microRNA genes that function to suppress the process of memory formation. The active forgetting component stems from the recent identification of a signaling system that removes previously formed memories and is modulated by internal states of arousal and sleep, and by external sensory stimulation. This represents an unstudied area in the neuroscience of memory formation and offers tremendous opportunities for discovery in the molecular biology and systems neuroscience of the process. The second component is founded on innovative discoveries that allow the visualization of cellular memory traces ? changes in the response properties of neurons due to learning ? that offer a window into the logic behind how memories are organized in the brain. This component contrasts, as one example, how the brain organizes olfactory memories learned in association with a rewarding cue and those learned in association with an aversive cue, and delves into the underlying mechanisms. The third component derives from recent genetic screens that have provided a plethora of new genes, both protein- coding and microRNA-coding, which enhance memory when suppressed, thus representing new memory suppressor genes. The proposed behavioral, functional cellular imaging, and molecular genetic experiments will dissect the roles for these genes in different temporal forms of memory: short-, intermediate-, and long- term memory; as well as different operational phases of memory formation: acquisition, memory stability, or forgetting. The results will offer an unprecedented view of the constraints the brain uses to limit memory formation. There is a rich medical importance to this research given the well-documented problems of cognition associated with numerous neurological and psychiatric disorders.

There is a pressing need to develop therapeutics that can promote brain health with aging. Such therapeutics would postpone the deterioration in cognition and mental health that occurs with age. The proposed research will identify, optimize, and then test in laboratory animals such therapeutics as steps towards future clinical use. The research focus is on mitochondria, the organelle in brain neurons that provides energy and many other functions for neurons to function and remain healthy. Mitochondria become impaired with age; mitochondrial impairment is one of the well-known hallmarks of aging. In pilot studies, we have identified a few new drugs that have the potential to enhance mitochondrial function across age. We will search for and identify five times more to enlarge the set of potential ?mitotherapeutics.? These drugs will then be tested in laboratory animals to determine whether they can protect mitochondria from the insults associated with age and enhance brain function; postponing or eliminating the cognitive and mental health issues that occur with age. Drugs with the potential to postpone or eliminate these issues would have an enormous impact on brain health for the late adult and elderly in the U.S. population.