Charles Nichols - US grants

The specific aims of the research in this proposal are to identify and characterize genes whose expression patterns are influenced by acute LSD administration in the brains of rats. Candidate genes will be identified from a pool of clones isolated in previous Differential Display PCR experiments. Differential expression within the brain for candidates will be determined and characterized by RNase protection and tissue in situ experiments. Antibodies will be generated to analyze candidate protein expression. Differential expression will be tested after administration of different hallucinogens. The receptor mediated pathways in which the differentially expressed genes act will be determined in experiments employing a variety of receptor antagonists. Ultimately these genes, their products and the pathways that are involved may be investigated for their potential role in animal behavior and in human mental disorders such as schizophrenia. This training and research could provide the basis for future work in establishing an independent research program in an academic environment.

cytomegalovirus retinitis; cidofovir; ganciclovir; AIDS therapy; eye disorder chemotherapy; human therapy evaluation; combination chemotherapy; drug administration routes; clinical research; human subject;

[unreadable] DESCRIPTION (provided by investigator): The fundamental hypothesis of our research is that molecular events initiated by agents acting at serotonin receptors in Drosophila, and other animal models, represent potential candidates for involvement in the development and etiology of neuropsychiatric disorders in humans, and importantly, may be targets for new avenues of discovery for therapeutics. The goal of this project is to develop the fruit fly, Drosophila melanogaster, as a genetically tractable model system to investigate molecular mechanisms underlying human neuropsychiatric disorders that involve serotonin. We propose to treat flies with specific serotonergic agents, including psychotomimetics and antipsychotics, to assess behavioral outcomes, and to develop the genetic tools necessary to elucidate the pathways linking receptor activation with behavior. As shown with most previously studied processes in Drosophila, ranging from development to drug response, the molecular and cellular events underlying serotonergic function are likely to be highly conserved between the fly and humans. The use of the fly to study these conserved neurochemical events brings into play extremely powerful genetic techniques to rapidly elucidate key pathways and molecules that otherwise could take years, at a substantially greater cost, to identify by traditional mammalian-based methods. Importantly, the fly is believed to express a functional ortholog of the mammalian 5-HT2 receptor, as well as orthologs of the mammalian 5-HT1A, 5-HT7, dopamine D1 and D2, GABA, NMDA, and metabotropic glutamate receptors, all of which have been strongly implicated in a variety of human neuropsychiatric disorders. Here, we propose investigations to develop this fly model by characterizing the: 1) neuropharmacology, 2) circuitry, and 3) behaviors of the Drosophila CNS serotonin system using molecular, genetic, pharmacological, and behavioral experiments to lay the foundation and create a knowledge base of serotonergic function in Drosophila, with a view toward developing the fly as a model genetic system to use in concert with ongoing proposed mammalian target identification experiments. Together, these experiments form a novel systems-based approach to explore neurochemical events relevant to neuropsychiatric disorders in humans, and will be of great importance to facilitate the discovery of novel targets for therapeutics. Schizophrenia is a debilitating neuropsychiatric disorder that affects about one out of every 100 Americans at a cost to the U.S. economy of nearly $63 billion/year. New approaches towards understanding underlying schizophrenia mechanisms are urgently needed in order to further understand and treat this disease, as well as other psychiatric disorders. We propose to develop the fruit fly, Drosophila melanogaster, as a model system to study the underlying serotonin neurochemistry of these diseases. The development and utilization of this model system will lead to an enhanced discovery rate of novel targets for therapeutics to treat these conditions. [unreadable] [unreadable]

Project Abstract: The overall goals of this project are to establish and pursue an innovative approach combining discovery studies in mammalian systems with translational and functional studies in the powerful genetic model organism Drosophila melanogaster as a novel strategy to identify and characterize genes and proteins underlying the behavioral changes that occur during the development of psychosis. In the search for new medical therapies, and in particular treatments for disorders of the central nervous system involving serotonin, like schizophrenia, psychosis, and depression, there has been increasing recognition that identification of a single biological target is unlikely to be a recipe for success; a broader perspective is required. Systems biology is one such approach, and has been increasingly recognized as a crucial and dynamic area of research, as it places specific molecular targets within a context of overall biochemical action. Understanding the complex interactions between the components within a given biological system that lead to modifications in output, such as changes in behavior, may be important avenues of discovery to identify new therapies. Within this framework, our underlying hypothesis is that molecular events, such as gene expression changes, influenced by aberrant serotonin receptor activation in specific regions of the brain, represent molecules that directly participate in, or regulate signal transduction networks that underlie normal cognitive processes that when perturbed lead to neuropsychiatric disorders. Here, we propose a series of experiments following a systems based approach to determine the functional and behavioral role of specific genes and proteins that respond to pharmacological activation of specific neurotransmitter receptors in the prefrontal cortex of rat brain in a proposed animal model of the neurochemical and genetic events underlying psychosis and schizophrenia. We will: 1) Identify additional, and perhaps more relevant, genes and proteins in specific regions of the rat prefrontal cortex through functional genomic, proteomic, and gene expression analysis; 2) Examine the functional role of these genes and proteins in behaviors in translational studies in the fruit fly, Drosophila melanogaster. We strongly believe that this multidisciplinary systems-based approach combining mammalian CNS pharmacology and whole animal studies in our powerful genetic Drosophila model, is the best route to follow to achieve our goals. Significantly, our results may lead to novel avenues for therapeutics to treat such devastating diseases as schizophrenia and psychosis.

DESCRIPTION (provided by applicant): The overall goals of this project are to elucidate the molecular mechanisms underlying our recent discovery that serotonin 5-HT2A receptor activation super-potently inhibits TNFa-mediated inflammatory pathways, and to translate our findings to an animal model as a potential novel therapeutic approach to treat and/or prevent diseases like atherosclerosis that involve TNFa-mediated inflammation. TNF- a-mediated inflammatory pathways have been strongly implicated in a number of diseases including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, irritable bowel syndrome and Crohn's disease, and septicemia. Significantly, TNF-a and other cytokine induced inflammatory pathways also have been linked to psychiatric conditions such as depression and bipolar disorder, as well as schizophrenia, and neurodegenerative diseases. As such, inhibitors of TNF-a pro-inflammatory pathways represent potential therapeutics for each of these conditions. Currently, the only available therapeutic inhibitors of TNF-a pathways are monoclonal antibodies against TNF-a (infliximab and adalimumab) and soluble TNF-a receptor (etanercept), and the development of small molecules for this purpose is highly desirable. We have recently discovered that activation of 5-HT2A receptors in cardiovascular tissues including primary aortic smooth muscle, aortic endothelial, and macrophage cells by (R)-DOI, and likely additional 5-HT2A receptor agonists, represents a novel, and extraordinarily potent, therapeutic avenue to develop for the treatment of diseases and disorders involving TNF-a-mediated inflammation. Significantly, pro-inflammatory marker blockade occurs with IC50 drug levels of 10-20 picomolar. With the exception of a few natural toxins no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. The experiments described in this proposal will elucidate the molecular signaling pathways linking activation of 5-HT2A receptors to inhibition of TNF- a-mediated pro-inflammatory process using molecular and biochemical approaches in primary cell culture experiments. Importantly, we will also perform translational studies in rodents to explore the anti-inflammatory effects of (R)-DOI in vivo. Results from these studies may lead to potential therapeutic strategies to not only prevent, but also treat existing pathological conditions like atherosclerosis via 5-HT2A receptor stimulation.

Cell and molecular imaging of tissue/cell preparations stained with histochemical and immunohistochemical markers allows researchers to identify the biochemical components and pathways of specimens at subcellular resolution. The COBRE Imaging and Histology Core has been instrumental in this area and provides investigators with space, facilities, instrumentation, supervision, and training for microscopy of livecells and histological specimens (fixed and frozen), laser capture microdissection, confocal imaging, and a variety of histochemical techniques to investigate cardiovascular biology at the cellular and molecular level. The specific aims of this Core are: 1)To provide skilled personnel to assist and/or perform highly specialized microscopic techniques including deconvolution, confocal microscopy, 3D reconstruction, co-localization, ratio imaging. Total Internal Reflection Fluorescence (TIRF), fluorescent resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP) of both fixed and live cell samples; 2) To assist in the collection, processing and staining of histological samples, including standard and special stains, and immunohistochemical antibody labeling; 3) To conduct experiments and assist investigators with the selection of appropriate special stains as well as interpretation of histological and immunohistochemical data; 4) To mentor young investigators and students, and advise senior investigators, on the potential application of microscopic techniques, histological analysis and immunohistochemistry to enhance their research projects and provide avenues towards developing translational research projects; 5) To guide image analysis methodologies for histological examination. This Core continues to provide COBRE and University investigators, including faculty, postdoctoral fellows, and graduate students, with imaging recourses and technical assistance. Whether investigators are studying cardiovascular biology at the level ofthe cell, tissue, or whole animal, this core has been instrumental to each of our COBRE researchers in examining the structural and/or histological basis for, or confirmation of, different cardiovascular disease states.

? DESCRIPTION (provided by applicant): Abuse of stimulants like cocaine and meth amphetamines is a significant public health problem in the United States. Although the study of traditional mammalian systems has been very informative, there are still no FDA approved therapies for their abuse. The development of new tools and models is urgently needed to address these problems. Incorporation of the fruit fly, Drosophila melanogaster, into the repertoire of models to study drug abuse has been a recent and exciting development for the field. As in mammals, dopamine is a key neurotransmitter in flies that has been found to mediate conserved behaviors to CNS stimulants that include increased locomotor activity and stereotypy. Despite the discoveries made in the fly relevant to human drug action, how stimulants elicit their behaviors in the fly at a fundamental level remain undefined. Little is knon of the neurochemistry and circuitry underlying the effects of cocaine and amphetamines in the fly aside from the fact that dopamine is a key component, and that the mushroom body is involved. In mammals, serotonin has been shown to modulate the behavioral effects of these stimulants. The underlying molecular mechanisms of this modulation at the cellular and circuitry level related to drug abuse, however, remain largely unknown. Although the overall circuitry and structure of the fly brain is different from the human brain, at a basic and fundamental level, processes involving inputs and outputs between individual neurons and larger structures and circuits and how they mechanistically communicate with each other are conserved to a significant degree between fly and mammal. A greater understanding of how these circuitries interact and communicate with each other using neurotransmitters like serotonin and dopamine to produce acute and/or adaptive changes that alter normal behavior in response to stimulants will at a fundamental level translate to a better understanding of how neurons and circuits in mammalian brain that utilize the same neurotransmitters mechanistically respond to the same drugs to produce conserved behaviors. We propose here to elucidate the neural circuitry underlying the response to methamphetamine and cocaine, and to examine the role of serotonin and serotonin receptors in these responses. For our proposed work we will combine pharmacological, genetic, behavioral, and pharmacogenetic DREADD receptor approaches to manipulate circuitry and receptor function. Significantly, our preliminary data indicate that all three serotonin receptor families in the fly (5-HT1A, 5-HT2, and 5-HT7) mediate aspects of the response to stimulant drugs. Ultimately our work may uncover new molecular and/or genetic aspects of stimulant drug abuse and facilitate the development of potential therapeutic strategies.