Toshihiro Kitamoto - US grants

IBN-9723216 Post-transcriptional regulation of the Drosophila cholinergic genes PI: Toshihiro Kitamoto Neurons use a variety of molecules as neurotransmitters to communicate with their target cells. Specification of particular neurotransmitter phenotypes (enzymes required for transmitter synthesis and release) is a fundamental process for orderly construction and operation of neural circuits, and requires coordinated expression of a particular set of neurotransmitter-specific genes. The objective of this proposed research is to understand the regulatory mechanism for two cholinergic neuron-specific genes, encoding choline acetyltransferase (ChAT) for biosynthesis of acetylcholine and vesicular acetylcholine transporter (VAChT) for packaging acetylcholine into synaptic vesicles. It has recently been demonstrated that the ChAT and VAChT genes are organized into a "cholinergic" locus where the latter gene is nested within the former. This unique organization of the cholinergic locus, conserved among distantly related species, suggests that novel post-transcriptional mechanisms are involved in coordinated regulation of these two genes. The project will thus specifically focus on mRNA processing and translation mechanisms of the ChAT and VAChT genes using Drosophila melanogaster as a model organism. First, transgenic animals that carry appropriate reporter genes will be created using P-element mediated transformation technology available for Drosophila. The mechanisms for ChAT and VAChT production will be investigated by qualitative and quantitative analyses of reporter gene expression in the nervous system of the transformants. Second, Drosophila VAChT mutants will be isolated and their phenotypes will be carefully characterized. VAChT mutants together with already available ChAT mutants will be used to examine the effects of altered acetylcholine metabolism on regulation of the cholinergic genes. The proposed research using molecu lar and classical genetics will provide a clearer picture of interesting new mechanisms regulating coordinated expression of the two cholinergic genes.

DESCRIPTION (provided by applicant): The long-term goal of this proposed project is to understand fundamental neuronal mechanisms underlying higher brain functions that control complex behaviors. Disruption of higher brain functions, such as learning and memory, can occur for a number of reasons including brain surgery, chronic alcohol abuse, head injury, anoxia, and various neurodegenerative disorders such as Alzheimer's disease. A basic understanding of how anatomically distinct neurons in the brain communicate with one another to manipulate complex behavior is essential for the prevention and treatment of many disorders affecting higher brain functions. Recent studies indicate that molecules and cellular mechanisms responsible for important biological processes, including learning and memory, are well conserved among distantly related species. In this project, Drosophila male courtship, which consists of a highly stereotypical sequence of activities and also shows considerable experience-dependent plasticity, will be used as a physiological model of higher-order brain functions. Specific aims of the project are to identify the neuronal subsets involved in the learning/memory process of the courtship plasticity and to determine the temporal requirements of neuronal activity during different phases of the memory formation. To accomplish these aims, a novel molecular genetic approach has been established. In this approach a temperature-sensitive allele of the Drosophila shibire gene (shi") is expressed in restricted neuronal subsets using the GAL4IUAS system. Then, synaptic transmission of the targeted neurons is blocked rapidly and reversibly by a mild temperature-shift in intact animals. By taking advantage of the large collection of available GAL4 lines that are specific to restricted brain regions, the significance of particular neuronal subsets in the courtship plasticity will be determined. A combination of genetic and morphological analysis will be applied to further investigate the neuronal subsets whose functional significance is revealed. The neuronal subsets involved in the genetically determined, stereotypical aspects of male courtship will be also identified using the same approach. The anticipated results, together with the accumulated information of Drosophila behavioral genetics, will provide new insight into the neuronal mechanisms of higher-brain functions in flies, and will contribute to the development of conceptual frameworks for the study of complex behaviors in higher vertebrates including humans.

[unreadable] DESCRIPTION (provided by applicant): Lithium has been used as the highly effective therapy for bipolar disorder (BPD), a chronic and disabling mental illness that affects more than 2 million people in the US. Understanding the therapeutic action of lithium would provide important insight into the etiology and pathophysiology of BPD, and help to develop better treatment for this serious disease. The goal of the proposed research is to elucidate molecular and cellular mechanisms involved in the lithium-responsive neurological pathway using the fruit fly as a model organism. A Drosophila mutant Shudderer (Shu) is an X-linked dominant mutant that exhibits various neurological phenotypes, including hyperactivity, uncoordinated movements, sporadically occurring jerks and anesthesia-induced seizure. Interestingly, many of these phenotypes are greatly suppressed by lithium with the internal concentrations used for treatment of BPD patients. In addition, Shu may functionally interact with the glycogen synthase kinase 3 gene that has been implicated in the lithium therapeutic action. Specific aims of this proposal are to: 1) identify the Shu gene and determine the molecular nature of the Shu mutation and; 2) identify genes that functionally interact with Shu. To accomplish the Aim-1, the location of the Shu mutation will be mapped in a small genomic region (<50 kb) using a high-resolution recombination-based mapping approach with molecularly defined P element insertions. The Shu gene will be identified and the molecular nature of the mutation will be determined by sequencing the genomic DNA isolated from the fully isogenized Shu mutant flies. Transformants carrying either a wild type or a mutant form of the Shu gene will be used to confirm the identity of the gene. For Aim-2, the molecularly defined deficiencies and mutations will be introduced into the Shu mutant background to identify suppressors and enhancers for the Shu mutation. The research proposed in this application is significant, because studies of the Shu mutant are expected to provide novel knowledge of genetic components underlying the lithium-responsive process in the nervous system. Based on the fact that the fundamental molecular and cellular mechanisms are well conserved between the fruit fly and vertebrates, the findings are also expected to lead to the recognition of uncharacterized players or processes responsible for the lithium action in the vertebrates, which would open up the future possibility to develop novel and improved therapies for BPD. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Lithium has been used as the highly effective therapy for bipolar disorder (BPD), a chronic and disabling mental illness that affects more than 2 million people in the US. Understanding the therapeutic action of lithium would provide important insight into the etiology and pathophysiology of BPD, and help to develop better treatment for this serious disease. The goal of the proposed research is to elucidate molecular and cellular mechanisms involved in the lithium-responsive neurological pathway using the fruit fly as a model organism. A Drosophila mutant Shudderer (Shu) is an X-linked dominant mutant that exhibits various neurological phenotypes, including hyperactivity, uncoordinated movements, sporadically occurring jerks and anesthesia-induced seizure. Interestingly, many of these phenotypes are greatly suppressed by lithium with the internal concentrations used for treatment of BPD patients. In addition, Shu may functionally interact with the glycogen synthase kinase 3 gene that has been implicated in the lithium therapeutic action. Specific aims of this proposal are to: 1) identify the Shu gene and determine the molecular nature of the Shu mutation and; 2) identify genes that functionally interact with Shu. To accomplish the Aim-1, the location of the Shu mutation will be mapped in a small genomic region (<50 kb) using a high-resolution recombination-based mapping approach with molecularly defined P element insertions. The Shu gene will be identified and the molecular nature of the mutation will be determined by sequencing the genomic DNA isolated from the fully isogenized Shu mutant flies. Transformants carrying either a wild type or a mutant form of the Shu gene will be used to confirm the identity of the gene. For Aim-2, the molecularly defined deficiencies and mutations will be introduced into the Shu mutant background to identify suppressors and enhancers for the Shu mutation. The research proposed in this application is significant, because studies of the Shu mutant are expected to provide novel knowledge of genetic components underlying the lithium-responsive process in the nervous system. Based on the fact that the fundamental molecular and cellular mechanisms are well conserved between the fruit fly and vertebrates, the findings are also expected to lead to the recognition of uncharacterized players or processes responsible for the lithium action in the vertebrates, which would open up the future possibility to develop novel and improved therapies for BPD. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuropathic pain is a type of chronic pain caused by injury or disease of the nervous system. This disorder has proven particularly difficult to treat clinically, because the fundamental mechanisms for its development and maintenance are not well understood. As a result, there is an obvious need to identify novel molecules or biological processes responsible for this pathological pain, against which new therapeutic strategies can be developed. The long-term goal of this research project is to understand the mechanisms of gene regulation critically important for the manifestation of neuropathic pain. The objective of this particular application is to identify microRNAs and their regulatory targets that play key roles in the chronic neuropathic pain condition. MicroRNAs are a new class of non-protein-coding small RNA molecules that are encoded by the genome, which regulate gene expression mainly at the post-transcriptional level and play a variety of biological functions. In particular, microRNAs have recently been implicated in the plasticity of the nervous system. Because neuropathic pain can be considered as an unwelcome consequence of the neural plasticity in the pain transmission pathways, the involvement of microRNAs in neuropathic pain is highly anticipated. The central hypothesis for the proposed research is that nerve injury induces changes in expression of particular microRNAs, which in turn regulate expression of a set of pro-nociceptive and anti-nociceptive proteins, contributing to long-lasting chronic pain states. The rationale for the proposed research is that, once particular microRNAs are identified as regulators of pain-related proteins in the neuropathic pain conditions, it would become possible to control neuropathic pain by modulating the levels of those microRNAs either pharmacologically or through molecular biological strategies. Thus, the proposed research is expected to lead to the development of fundamental knowledge that will potentially help to reduce the burdens of human disability. Based on positive preliminary data, two specific aims will be pursued. They are to: 1) identify microRNAs involved in the development and maintenance of neuropathic pain;and 2) identify target genes for the neuropathic pain-related microRNAs. For the first aim, the microarray technology and other molecular biological methods will be used to find microRNAs that show significant differences in expression levels after spinal nerve injury that causes neuropathic pain. For the second aim, the computational methods as well as histochemical approaches will be used to identify genes that are potentially regulated by particular microRNAs in the pain transmission pathways in response to nerve injury. This proposal is novel, because the possible functions of microRNAs in neuropathic pain have never been investigated. It is also significant, because it is expected to trigger a new field of research focusing on microRNA function in chronic pain, which would advance understanding of gene regulation associated with the chronic pain conditions. PUBLIC HEALTH RELEVANCE: This proposed project concerns an unexplored area of research that is expected to lead to a better understanding of the fundamental mechanisms responsible for the development and maintenance of chronic neuropathic pain. The project has relevance to public health, because once microRNA's functions in chronic neuropathic pain are identified and characterized, they would be excellent new therapeutic targets for this serious disorder.

DESCRIPTION (provided by applicant): Although lithium displays remarkable mood-stabilizing properties and has served as one of the most effective therapies for bipolar disorder (BPD), the mechanisms underlying its actions on the nervous system remain unclear. The long-term goal of this project is to understand-at the molecular and cellular levels-how lithium affects nervous system function. The objectives of this proposal are to identify genes that are involved in the lithium-responsive neurological process and to determine their roles in lithium's therapeutic action. To accomplish these objectives, we will utilize genetic tools that are uniquely available in the fruit fly Drosophila melanogaster. These include a neurological mutant Shudderer (Shu), whose phenotypes are largely rescued by lithium administration at therapeutic concentrations. Our central hypothesis, which is based on strong preliminary data, is that lithium reduces the severity of neurological defects caused by up-regulation of the Ca2????dependent phosphatase calcineurin, and that it does so by suppressing the innate immune response. The rationale for the proposed research is that, once the genes involved in lithium-responsive neurological processes in Drosophila have been identified and their roles have been revealed, this information should be readily translated into the vertebrate system, which is known to use signaling mechanisms that overlap extensively with those in Drosophila. Thus, our study is expected to provide novel and important insights into lithium's therapeutic action-with respect not only to BPD, but also other neurological disorders. Our central hypothesis will be tested by pursuing three specific aims: 1) Determine how up-regulation of calcineurin causes the lithium-responsive neurological defects;2) Delineate the mechanisms responsible for lithium's therapeutic action on the Shu phenotype;and 3) Identify novel genes whose up- or down-regulation mimics lithium's actions in the nervous system. For the first aim, particular cell types and developmental timing of the calcineurin overexpression leading to the Shu phenotype will be defined by genetically manipulating calcineurin activity in a spatially and temporally specific manner. For the second aim, various genetic variants will be utilized to determine the extent to which the innate immune system is involved in manifestation of lithium-responsive neurological defects. For the third aim, molecularly defined mutants and RNAi will be employed to confirm the involvement of novel genes (from candidates that have been identified in genome-wide screens) in the lithium-responsive neurological processes. The proposed research is innovative in that it capitalizes on the power of Drosophila genetics to identify and characterize genes that are involved in lithium's actions in the nervous system. The proposed research is significant because it is expected to lead to the recognition of molecules and molecular interactions that are responsible for lithium's actions in the vertebrate nervous system. This would open up new avenues toward a better understanding of the etiology and pathophysiology of BPD, and result in improved therapies for BPD and other disorders of the nervous system. PUBLIC HEALTH RELEVANCE: The proposed studies aim at understanding the evolutionarily conserved neurological processes that are affected by lithium. This research is of high relevance to public health because lithium is an effective drug for the treatment of mood disorders, as well as potentially being useful for the treatment or prevention of brain damage. Thus, the findings are expected to contribute to significant future improvements in human health.

DESCRIPTION (provided by applicant): Steroid hormones play a critical role in various neurological and psychiatric conditions. In addition to the genomic mechanisms involving nuclear steroid hormone receptors, biological effects of steroids are also mediated by nongenomic mechanisms that occur rapidly and independently of new mRNA synthesis. The functions and molecular mechanisms for such unconventional steroid signaling, particularly in the nervous system, are still poorly understood. This gap in the knowledge base represents an important unmet need for a mechanism-based understanding of nervous system disorders related to steroid hormones. The long-term goal is to understand how steroid hormones modulate nervous system functions and thereby control behavior. The objective of this particular application is to elucidate the fundamental mechanisms for nongenomic steroid signaling that controls ethanol-induced behaviors using the fruit fly Drosophila. Recent genetic studies in the applicant's lab demonstrate that DopEcR, a G-protein coupled receptor (GPCR) for the insect steroid hormone ecdysone, plays a significant role in behavioral responses to ethanol. This new finding provides an unprecedented opportunity to investigate the underpinnings for a novel GPCR-mediated steroid signaling that is important for alcohol-induced behavior, by taking advantage of Drosophila genetics. The central hypothesis is that DopEcR is a unique dual receptor for ecdysone and dopamine, and that it modulates resistance to the sedative effects of ethanol by negatively regulating epidermal growth factor receptor (EGFR) signaling. To test this hypothesis and attain the objective of this application, we will pursue the following two specific aims: 1) Determine the expression pattern of DopEcR and sites of its actions; and 2) Identify molecular components involved in DopEcR-mediated signaling. Under the first aim, immunological and genetic methods will be used to determine the anatomical localization of DopEcR protein in the adult brain, whereas rescue and phenocopy experiments will be performed to identify brain neurons involved in DopEcR-mediated steroid actions. Under the second aim, roles of two ligands (ecdysone and dopamine) and potential downstream signaling (EGFR signaling) in DopEcR actions will be investigated using pharmacological and genetic methods. The genetic approach is innovative, because it utilizes not only a null allele, but also various genetic tools uniquely available in Drosophila to study the mechanisms responsible for nongenomic steroid signaling. The expected outcomes will form an essential foundation for understanding the novel nongenomic steroid signaling that plays critical roles in regulation of alcohol-induced behavior. This study is significant, because it is expected to provide strong mechanism-based in vivo evidence for a novel steroid signaling in the nervous system, which may ultimately contribute toward the development of innovative strategies for the prevention and treatment of alcohol use disorders as well as other common diseases that are affected by steroid hormones.

Sleep and feeding are both indispensable for life and are strictly controlled for their own need. However, these essential behaviors are mutually exclusive, and therefore their regulatory processes significantly influence one another. Sleep loss leads to stimulation of appetite, and food deprivation leads to a suppression of sleep. Elucidation of the basic mechanisms underlying these interactions between sleep and feeding is highly significance, as it is expected to provide insights into fundamental principles by which animals efficiently adapt their behaviors in response to changes in external environments and internal states. The focus of the study is on steroid hormones and their involvement in starvation-induced sleep suppression (SISS), an evolutionarily conserved behavioral response to starvation. The outcome of this research is expected to make an important general contribution to the field of behavioral neuroendocrinology, given the universal importance of the molecular components studied (steroids, dopamine and G-protein coupled receptor), and the adaptive behavior of interest (SISS). The project will also provide unique training opportunities for undergraduate, with an emphasis on broadening participation through involvement of underrepresented minorities, as well as graduate students and promote outreach activities targeting local students and general public.

This project uses the fruit fly, Drosophila melanogaster as a model system and investigates how the endocrine system regulates the nervous system to control sleep and feeding. The research will elucidate how the G-protein coupled receptor DopEcR interacts with neuromodulators to control sleep under the stress associated with starvation. The central hypothesis is that dopamine acts on DopEcR in the context of starvation, counteracting the effects of ecdysone on DopEcR by reducing the strength of epidermal growth factor receptor (EGFR) signaling, and that this in turn induces SISS. This hypothesis will be tested through two specific aims: 1) Identify the critical sites of DopEcR action in regulating SISS, and 2) Determine the roles of ecdysone, dopamine and EGFR signaling in DopEcR-mediated SISS. Preliminary studies in the PI's group have revealed that Drosophila DopEcR, a unique G-protein coupled receptor (GPCR) that responds to both the major insect steroid hormone ecdysone and the catecholamine dopamine plays a critical role in SISS. The study will identify the critical anatomical sites of DopEcR action in regulating SISS, and determining the roles and mechanisms of ecdysone and dopamine in DopEcR-mediated SISS.

Epilepsy is one of the most common neurological disorders affecting more than 65 million people worldwide. Although effective anti-epileptic drugs exist, a significant portion of patients (~30%) do not properly respond to currently available drugs. Therefore, there is an urgent need for new strategies to combat such refractory forms of epilepsy. Notably, previous studies have demonstrated that severity of epilepsy is significantly modified by a variety of genetic and environmental factors, raising the exciting future possibility for better management of epilepsy by appropriately manipulating these phenotypic modifiers. The long-term goals of this project are to 1) identify the genetic and environmental modifiers of epilepsy, and elucidate their action mechanisms. New knowledge about such ?epilepsy modifiers? is expected to lead to alternative strategies for prevention and treatment of refractory epilepsy. To attain these goals, the current small project will particularly focus on identification and initial characterization of genetic modifiers for the Drosophila voltage-gated sodium (Nav) channel mutant, Shudderer (Shu) ? a fly model of human epilepsy. In preliminary studies, an unbiased forward genetic screen for modifiers of Shu revealed that severity of the seizure-like phenotypes of Shu is significantly reduced when function of the Glutathione S-transferase S1 (GstS1) gene is suppressed. GstS1 mutations were found to activate antioxidant signaling and increase the levels of the inhibitory neurotransmitter GABA in the Shu brain. Furthermore, it was unexpectedly discovered that a simple dietary modification during development drastically suppresses Shu phenotypes and leads to increased GABA levels in the brain. These intriguing findings has led to the hypothesis that reduced GstS1 function results in suppression of the seizure-like phenotypes of Shu through activation of antioxidant response pathways to modify neural development and enhance GABAergic inhibitory tone in the adult brain. To investigate this hypothesis, this project is designed to collect basic information about genetic modifiers of Shu by pursing the following three specific aims: 1) Determine spatiotemporal requirements of GstS1-dependent modification of Shu's seizure-like phenotypes; 2) Identify additional genetic modifiers of Shu and determine if they affect antioxidant signaling and the GABAergic system. Successful completion of these aims is expected to provide the foundation to understand the role and action mechanisms of genetic modifiers for a fly epilepsy models. Based on evolutionary conservation of the basic neurobiological processes between flies and humans, the outcomes of the proposed experiments will provide fundamental insights into genetic modifiers of human epilepsy, which is expected to lead to the future development of novel strategies for preventing and treating epilepsy that does not respond to conventional therapies. As Shu is a mutant for the evolutionarily conserved Nav channel gene, the future broader impact of this study will be a contribution toward a better understanding of etiology and pathophysiology of other disorders that are linked to abnormal Nav channel functions.

PROJECT SUMMARY/ABSTRACT Commensal bacteria in the gut have a significant influence on the development and physiology of the brain, indicating their critical involvement in human neurological disorders. The long-term goal of the proposed research is to obtain a basic understanding of the key molecular and cellular processes responsible for the interactions between the gut microbiota and the brain. The current project is based on unique findings in a Drosophila voltage-gated sodium (Nav) channel mutant Shudderer (Shu). Shu mutants display severe neurological phenotypes including abnormal wing posture, neuronal hyperexcitability, spontaneous tremors and heat-induced seizures. Unexpectedly, these phenotypes were found to be drastically rescued when the mutants were treated with antibiotics. In addition, Shu mutants showed increased innate immune signaling and abnormalities in GABAergic neurons. The fruit flies carry relatively simple commensal bacterial communities and are amenable to versatile genetic approaches, providing a powerful experimental system to uncover the fundamental biological processes important for host/microbiota interactions. The strong microbiota-brain interactions identified in Shu thus offer an exciting opportunity to advance knowledge regarding the roles and action mechanisms for bacteria-dependent modulation of neural function and behavior. Guided by preliminary results, the central hypothesis for the current project is that severity of the neurological phenotype of Shu is increased by particular bacteria species in the gut, which causes aberrant activation of the host immune system to modify neural development and function. This hypothesis will be tested by pursuing the following two specific aims: 1) Determine the effect of particular gut bacteria on severity of the phenotypes of Shu, and 2) Identify the mechanisms by which the gut bacteria modulate the neurological phenotypes of Shu. For Aim 1, Shu mutants mono-associated with particular bacteria species will be generated and their neurological phenotypes will be examined. For Aim 2, immune pathways are genetically manipulated in Shu mutants and the effect on the mutant phenotypes will be investigated. As a consequence of this project, particular bacterial species that significantly exacerbate Shu phenotypes is expected to be identified. A causative relationship between gut bacteria, immune signaling and neural development and/or function is also expected to be revealed. Given that the molecular and cellular mechanisms underlying the basic biological processes are highly conserved between flies and mammals, the proposed study in Drosophila should provide valuable insights into the effect of the gut microbiota on neural development and function in humans. Such new information is expected to contribute to a better understanding of the role of the gut microbiota in etiology and pathophysiology of neurological disorders that are linked to abnormal Nav channel functions, such as epilepsy, autism, migraine, ataxia and pain syndromes, leading to the future development of novel bacteria-related strategies that reduce the burden of patients with these neurological disorders.