Michael P. Kavanaugh - US grants

The basic amino acid transporter (system y+) is the primary mechanism for cellular uptake of arginine and lysine; it is the first mammalian amino acid transporter to be cloned, and the first to be expressed and studied by electrophysiological methods in Xenopus oocytes. The y+ molecule also functions as the receptor that mediates attachment and infection of ecotropic host-range murine retroviruses that produce leukemia. The specific aims of this proposal involve structure-function studies on the y+ molecule and isolation and characterization of genes related to it. Aim 1 is to express y+ in oocytes and to determine its transport functions using voltage clamp methods, both with microelectrodes and with the 'cut- open' oocyte technique. A biophysical of model time-, voltage-, and concentration-dependence of substrate uptake and efflux will be developed. Aim 2 is to clone and express genes encoding other transporters. Using a partial-length cDNA encoding the y+-related gene Tea, the full length cDNA will be cloned and expressed, and its transport functions characterized. Other genes homologous to y+ will be identified by screening cDNA libraries and similarly tested. Another strategy for isolating amino acid transporter genes will utilize an oocyte expression cloning scheme to screen for expression of radiolabeled substrate uptake. Aim 3 involves structure-function studies on the system y+ protein; comparisons with related transporters will allow chimera construction and site-directed mutagenesis to map functional domains in the y+ molecule. In Aim 4 studies will be done to analyze the structural features of y+ important for its role in infection and in the natural resistance of non-murine species. Site-directed mutants and chimeric molecules made in aim 3 will be expressed in mammalian cells and analyzed for abilities to bind virus and to mediate infection. The results will be of significance in cell biology because they provide information about a critical class of transporter molecules, in virology because they will suggest how retroviruses invade cells, and in medicine because they promise to provide new understanding of common inherited diseases that result from abnormal amino acid transport.

glutamate transporter; neurotransmitter transport; glutamates; protein structure function; protein isoforms; chimeric proteins; electrophysiology; chemical binding; biophysics; Xenopus oocyte; tissue /cell culture; transfection; Xenopus; voltage /patch clamp; site directed mutagenesis; radiotracer;

glutamate transporter; protein structure function;

DESCRIPTION (provided by applicant): Glutamate is the predominant excitatory transmitter in the central nervous system, and its cellular uptake is mediated by 5 excitatory amino acid transporters (EAAT1-5) that have distinct distribution patterns in mammalian brain. There are fundamental unanswered questions concerning the structure and function of glutamate transporters and their role in influencing the spatiotemporal profile of synaptically released glutamate. This application proposes to elucidate the molecular mechanisms underlying transporter gating and interaction with substrates, and to characterize the influence of the primary neuronal transporter (EAAT3) on synaptic transmission and synaptic plasticity in the hippocampus. The specific aims are: 1. To elucidate the mechanism and molecular pharmacology of glutamate transport by identifying and characterizing the structural determinants involved in controlling substrate selectivity and pore access. 2. To test the hypothesis that the neuronal transporter EAAT3 limits activation of extrasynaptic receptors at the Schaeffer collateral-CA1 synapse in hippocampus and influences postsynaptic responses, plasticity, and behavior. The results of these studies will be of significance in neurobiology and medicine because they will provide new tools and insights into the molecular mechanism of glutamate transport and its roles in the brain. Elucidating the transporters' structure, mechanism, and physiological functions is necessary for understanding how they function in normal and pathophysiological processes and for developing new therapeutic strategies for diseases like amyotrophic lateral sclerosis, epilepsy, and stroke.

Address; Ammon Horn; Autoregulation; Biochemistry; Brain; CRISP; Cell Communication and Signaling; Cell Signaling; Central Nervous System; Chemistry, Biological; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Computerized Models; Cornu Ammonis; Encephalon; Encephalons; Funding; Glia; Glial Cells; Gln; Glutamates; Glutamine; Grant; Hippocampus; Hippocampus (Brain); Homeostasis; Institution; Intracellular Communication and Signaling; Investigators; Kolliker's reticulum; L-Glutamate; L-Glutamine; Mathematical Model Simulation; Mathematical Models and Simulations; Membrane Transport Proteins; Membrane Transporters; Modeling; Models, Computer; Molecular; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Nervous System, Brain; Nervous System, CNS; Neural Cell; Neural Transmission; Neuraxis; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Non-neuronal cell; Pharmacology; Physiologic; Physiological; Physiological Homeostasis; Physiology; Play; Process; Q. Levoglutamide; Range; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Signal Transduction; Signal Transduction Systems; Signaling; Simulation, Computer based; Source; Synaptic Transmission; Synaptic Vesicles; Synthesis Chemistry; Synthetic Chemistry; System; System, LOINC Axis 4; Testing; United States National Institutes of Health; biological signal transduction; computational modeling; computational models; computational simulation; computer based models; computerized modeling; computerized simulation; excitotoxicity; hippocampal; in silico; inhibitor; inhibitor/antagonist; interdisciplinary approach; nerve cement; neuronal; novel; pharmacophore; reuptake; social role; spatiotemporal; virtual simulation

DESCRIPTION (provided by applicant): Endocannabinoids are released in the brain in response to synaptic activity and they interact with presynaptic receptors in a retrograde fashion to inhibit release of transmitter. They play key roles in modulating signaling between neurons. As with most other neurotransmitters, the termination of endocannabinoid action is thought to involve a transport activity that mediates reuptake into cells. Several putative inhibitors of reuptake have been shown to modulate endocannabinoid signaling, suggesting that the transporter may be an important target for gaining an understanding of endocannabinoid actions as well as for potential therapeutic applications. However, there is a great deal of controversy and a number of conflicting accounts concerning the mechanism and molecular basis of endocannabinoid transport. Because of this, we propose to use a novel fluorescent cannabinoid substrate as a selective tool to study endocannabinoid transport. This will allow us to 1) study the mechanism and kinetics of transport, 2) determine its spatiotemporal distribution in brain, and 3) to identify it by molecular cloning. Characterization and identification of the transporter will provide new strategies for developing potential therapeutic agents that modulate endocannabinoid levels for a wide range of applications including analgesia, anti-emesis, hypertension, and neurodegeneration. PUBLIC HEALTH RELEVANCE: Endocannabinoids produced in the brain interact with specific receptors to modulate signaling between neurons. As with most other neurotransmitters, the termination of endocannabinoid action is thought to involve a transport activity that mediates reuptake into cells. Because the molecular basis of endocannabinoid transport is unknown, we propose to use a novel fluorescent cannabinoid to 1) study its functional properties, 2) to determine its locations in the brain, and 3) to identify it by molecular cloning. Characterization and identification of the transporter will provide potential therapeutic strategies to modulate endocannabinoid levels for a wide range of applications including analgesia, anti-emesis, hypertension, and neurodegeneration.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. After meeting with Dr. Taylor to discuss strategies for the Year 9, Dr. Kavanaugh decided with the approval of the SAC to allocate the funds resulting from the graduation of a junior PI to a new subproject intended to 1) provide modest bridge-funding to Center investigators who are resubmitting scored but unfunded applications and 2) provide funding for new and promising "seed projects" designed to generate data in support of publications and/or grant submissions. The overall aim of this project is to facilitate Center investigators'ability to conduct necessary preliminary studies for grant applications or in some cases to complete studies for publication with the aim of strengthening future grant submissions. Qualifying investigators submitted brief (1-3 page) research proposals to a committee consisting of the Director, the Associate Director, and, if applicable, the mentor of the investigator. When appropriate, input was also be solicited from select members of the SAC. The decision was then be made as to whether to fund the projects, and at what level. It was anticipated that these awards will not exceed $15-20K, and we required progress reports from all PI's. The program announcement for the upcoming year is appended below: CSFN CALL FOR PILOT PROJECT PROPOSALS The aim of this program is to support pilot projects for members and affiliates of the University of Montana NIH COBRE Center for Structural and Functional Neuroscience. The program is designed to support the generation of preliminary data for grant proposals and/or to develop a new direction of innovative or promising research. Faculty, postdoctoral, and predoctoral fellows are eligible to apply. Fellows must have faculty sponsorship. A faculty member can sponsor more than one project at a time, but recipients are eligible to receive funding for only one project at a time. The CSFN anticipates being able to fund 8-10 projects annually. Selected applications will be funded at a maximum of $15,000 for one year for projects of faculty and postdoctoral fellows and a maximum of $7500 per year for projects of graduate students. There is a possibility for renewed support depending on demonstrated productivity and need. The deadline for new applications and renewal of previously awarded grants is May 1, 2009. Applications should be limited to five pages or less and include an abstract, statement of the hypothesis or aim, a brief statement of background information and/or preliminary data, a description of the experimental design and methods, and references. A one page budget (not included in the page limit) should be included. Proposals will be reviewed by two senior Center members in the program area together with outside reviewers including members of the CSFN external advisory committee. The criteria for review will be based upon: Relevance/Scientific Merit: Is the proposed research project cutting edge and does it have the ability to break new research barriers and address an issue relevant to neuroscience? Significance: Will the project make a significant contribution to the general knowledge and the literature;does it have potential for development into a larger, funded project of some significance? Quality: Are the research questions/aims clearly specified;is the design appropriate;are the measurements/data collection/analysis appropriate? Feasibility: Can the project be done? Have adequate time and resources been allocated? Potential: Will the project be likely to result in an application to NIH or NSF and lead to one or more peer-reviewed publications? Awardees will be asked to provide an interim (6 month) and final progress report. An electronic version of the proposal should be submitted to both michael.kavanaugh@umontana.edu and jesse.hay@umontana.edu. Questions can be directed to Mike (406 243 4398) or Jesse (406 243 2381).

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Administrative (Admin) Core is intended to provide organizational and infrastructure support to CSFN investigators. This takes many forms and includes support for the Center's Financial Officer (K. Stewart, M.S.), Program Coordinator (J. Geist, M.S.) and two work study students. Funding for these administrative personnel is enhanced by additional match funds from the State of Montana and the University of Montana's Office of Research. The Admin Core supports the COBRE mission by assisting with pre- and post-award grant management, providing community outreach, and coordinating Center activities (weekly research meetings, Summer Undergraduate Research opportunities, travel to scientific meetings, etc.). To increase the competitiveness of individual CSFN investigators, as well as to strengthen the overall research environment of the Center, the Core also provides salary support to post-docs, graduate and undergraduate students engaged in research with Center laboratories. In summary, the Admin Core provides strategically needed support necessary to build and maintain the strong research environment required to foster the development of the junior investigators, as well as maintain the competitiveness of the senior investigators. Lastly, the Admin Core coordinates the mentoring activities within the COBRE program including the annual meeting with Scientific Advisory Committee, statewide neuroscience conference, travel of advisors and collaborators to Montana and increasing the opportunities for CSFN investigators to participate in grantsmanship training and scientific conferences.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major goal of the COBRE project is to enhance research competitiveness by increasing the access investigators have to state-of-the-art equipment through the development of core facilities. An additional aim of the core environment is to foster interaction and collaboration between core users and core directors and to promote the exchange of technical information. Investigators have access to six critically needed Core Research Facilities (CRFs) on campus. Four of these CRFs are directly funded by the COBRE CSFN: Molecular Histology and Fluorescence Imaging, Molecular Computation and Modeling, BioSpectroscopy, and Molecular Biology and Viral Production. In the past award period, the Mass Spectroscopy and Proteomics Core was integrated and subsumed into two new cores, the Core Laboratory for Neuromolecular Production (CLNP) and the Molecular Biology and Virus Production Core (MBVP). These new cores are described below. The Center CRFs are supported not only by the present COBRE award, but also by the University Research Office and State of Montana match funding, as well as by NIH and NSF grants awarded to UM. The CRFs have continued to develop and, as planned, have served not only to enhance individual projects but also to promote collaborations. Projects emerging from these cores have begun to produce publications and to be included as a critical resource in a number of funded R01 applications by CSFN investigators as well as in submitted applications. It should be noted in this regard that in almost every instance the summary statements from grants submitted through the Center characterize the research environment as excellent/very supportive of the proposed projects.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this subproject is to fill an Assistant/Associate Professor position within the College of Arts &Sciences at the University of Montana. This represents the fourth and final recruitment of our first renewal period. This is a collaborative recruitment with matching funds from the University and the College of Arts and Sciences. It is expected that this recruit will have a tenure-track position in the Division of Biological Sciences and will have a strong research program in the area of computationally-focused Neurobiology.

DESCRIPTION (provided by applicant): Glutamate is released by presynaptic neurons and stimulates an electrical signal in postsynaptic neurons. In order for recurrent signaling to occur, the neurotransmitter must be removed from the synapse soon after release. In the normal brain, glutamate is efficiently removed by glutamate transporters, known as excitatory amino acid transporters (EAATs). These molecules are found on the surface of neurons and other brain cells. In abnormal states, however, high amounts of glutamate can lead to overexcitation of the receiving nerve cell, resulting in damage or death. This process, known as glutamate excitotoxicity, is thought to contribute to neuronal loss seen in cerebral ischemia and head trauma, and is involved in neurodegenerative conditions such as Huntington's and Alzheimer's diseases. Therefore, treatments aimed at returning glutamate transporters to normal levels of expression and function may be therapeutic. Both antagonists and agonists of EAATs could be neuroprotective under certain conditions, and so development of such compounds is of significant biomedical importance. Development of effective inhibitors is impeded by lack of a recombinant expression system for EAAT3. Current methods to assay the transporter activity of EAATs are cumbersome, and the lack of high-resolution structural information for mammalian EAAT3 makes rational inhibitor design challenging. The goal of this project is to develop an insect cell expression system to produce pure, active human EAAT3 as a stable detergent complex in milligram quantities. The purified protein will be used for ligand screening, biophysical characterization and crystallization for high resolution X-ray structure determination. A novel fluorescence-based assay will be created, based on site-directed mutants of EAAT3, to measure binding activity in a microplate format for screening of glutamate transporter inhibitors synthesized at the University of Montana. A variety of experimental approaches will be taken to produce and optimize crystals of EAAT3 suitable for high-resolution structural analysis, in order to elucidate the mechanism of human EAAT3 transport activity and facilitate the design of compounds to block glutamate excitotoxicity. PUBLIC HEALTH RELEVANCE: In stroke, head trauma, Huntington's and Alzheimer's diseases, levels of glutamate become too high, causing the permanent loss of neurons. In the normal brain, glutamate levels are kept low by glutamate transporters, known as excitatory amino acid transporters (EAATs). This research will help explain how these proteins work and possibly aid in the development of new drugs that will help prevent the effects of high glutamate.

DESCRIPTION (provided by applicant): The overarching goals of the The Big Sky Brain Project are to increase the neuroscience literacy of tens of thousands of Montana K-12 students and to enhance their opportunities for careers in the STEM field. Montana - with its vast and predominantly rural landscape, high rates of poverty, and large Native American population - requires novel science education approaches that are appropriate to the unique geographical, financial, and cultural challenges of the state. The Big Sky Brain Project team is made up of state and national leaders in the fields of neuroscience, science education, and Montana Indian education. In order to be responsive to these unique regional factors while providing a rigorous educational experience, the University of Montana's Center for Structural and Functional Neuroscience and the spectrUM Discovery Area, the University's hands-on science museum, will collaborate with the Exploratorium to create a world-class neuroscience exhibition called the Brainzone. This exhibition will be housed in the new spectrUM Discovery Area, which will open in late autumn 2011 in a major regional shopping mall near the University. In a state with a population of less than a million, Southgate Mall has seven million annual visits - more than double the number of Montana's Glacier and Yellowstone National Parks combined. The Brainzone will be an integral part of spectrUM's popular field trip program for local K-12 students and will also be used in its mobile science program, which brings hands-on exhibits to isolated, underserved rural and tribal K-12 schools. The Brainzone will feature four exhibits and a computer learning lab to implement grade-specific curricula centered on neuroscience core themes ranging from gross neuroanatomy to cellular neurophysiology to learning and memory. Unlike similar educational exhibitions, the Brainzone will also include a working laboratory, called the BrainLab, that will house research-grade EEG instrumentation and a wet lab for Drosophila melanogaster neurobiology. The BrainLab, which will be in full public view, will be staffed by a graduate-level lab manager, and it will be utilized for K-12 student research demonstrations as well as by researchers ranging from high school interns to K-12 teachers to neuroscience faculty and clinicians from the University of Montana and Community Hospital in Missoula. In partnership with the Missoula County Public Schools, the Brainzone will host field trips for K-12 students to participate in grade-specific hands-on neuroscience exhibits and bench research. At other times it will be open to the public. In the final two years of the five-year project, the Brainzone exhibition will travel with spectrUM's mobile science program to bring core neuroscience curriculum to thousands of K-12 students living throughout Montana including those in isolated rural and tribal communities. Beyond the five-year duration of the grant, committed institutional support from the University, the Center for Structural and Functional Neuroscience, and spectrUM will ensure the long-term value, care, and use of these unique educational resources. PUBLIC HEALTH RELEVANCE: The Big Sky Brain Project will develop and implement world-class educational programming to increase public awareness of neuroscience. The project will involve exhibits and laboratory facilities in a high-profile retail mall location in the local community. K-12 students and visitors will learn about basic and applied neuroscience research, including research relating to public health issues such as brain trauma, epilepsy, and neurodegenerative disease. The project will provide opportunities to tens of thousands of Montana K-12 students to learn about careers in neuroscience and related STEM fields.

DESCRIPTION (provided by applicant): Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. Glutamate is released by presynaptic neurons and signals primarily through postsynaptic ionotropic and metabotropic receptors. Excitatory amino acid transporters (EAATs) play critical roles in neuronal physiology and pathophysiology. In order for high frequency signaling to occur, neurotransmitter must be removed from the synapse soon after release. Additionally, low levels of ambient glutamate are required to prevent chronic excitotoxic activity of high-affinity receptors. In pathological circumstances, changes in glutamate transporter function and/or surface density can occur. Disruption of alkali ion gradients caused by metabolic impairment after stroke or traumatic brain injury cause transporter slowing or reversal, contributing to glutamate elevation and excitotoxicity. Bidirectional changes in transporter surface expression occur in both normal and pathophysiological conditions, causing ambient glutamate levels to change. Development of selective new ligands for modulating and imaging glutamate transporter density changes is an important goal. We propose to determine the first high-resolution atomic structure of a human neuronal glutamate transporter. This will provide new understanding of transporter function and facilitate development of selective and high- affinity EAAT ligands for potential therapeutic and diagnostic uses.