James O. McNamara - US grants

The objective of this proposal is to carry out a comprehensive research program focused on the study of basic mechanisms of limbic epilepsy. The proposal brings together investigators with expertise in anatomy, biochemistry, pharmacology, physiology, and clinical epilepsy. These skills will be focused on studies of both the kindling and kainic acid models of epilepsy in the rat. The proposal also includes characterization of some physiologic processes in hippocampal formation, namely dendritic inhibition and spike frequency adaptation, and will assess whether alterations of these fundamental processes are involved in epilepsy. Biochemical and morphologic studies will be performed on hippocampal formation surgically removed from humans with medically intractable complex partial epilepsy. The ultimate goal of all components of this proposal is to further our understanding of the basic mechanisms of epilepsy from study of model systems. This information should provide a rational basis for new pharmacologic approaches designed to more effectively treat or prevent human epilepsy.

Kindling, an animal model of termporal lobe spilepsy, is a phenomenon in which repeated administration of an initially subconvulsant electrical stimulus eventually results in intense limbic and motor seizures. Once established, the kindling effect is permanent. The mechanisms responsible for this phenomenon are unknown. Modulation of chemical synaptic transmission is likely an important determinant of this permanent state of abnormal neuronal excitability. Among the putative chemical transmitters studied to date, endogenous norepinephrine (NE) has been demonstrated to exert a powerful inhibitory effect on kindling development. The adrenergic receptor subtype with which NE interacts to inhibit kindling development is unknown. In preliminary studies we have found that systemic treatment with alpha2 adrenergic receptor antagonists markedly facilitates the rate of kindling development. We have also found that systemic treatment with an alpha2 receptor agonist markedly retards the rate of kindling development. The objective of this proposal is to employ pharmacological, electrophysiological, and radiohistochemical techniques to test three hypotheses related to these findings: (1) the inhibitory effects of endogenous NE are produced through interactions with alpha2 adrenergic receptors residing on targets of NE neurons; (2) part of the molecular basis of kindling is a reduction of alpha2 adrenergic receptors and/or recrptor coupled responses; and (3) the site of kindling stimulation is the site at which NE acts to inhibit kindling development. Insights derived from understanding the role of this transmitter may serve as a model for understanding the role of other transmitters in the kindling model. Such insights may lead to an understanding of the mechanisms underlying kindling itself. Finally, and perhaps most importantly, the kindling-inhibitory effects of the alpha2 agoinst, clonidine--a drug in current clinical use as an antihypertensive ageng--may lead to novel strategies for prophylaxis againts epilepsy arising as a late sequel to brain injury.

Kindling, an animal model of termporal lobe spilepsy, is a phenomenon in which repeated administration of an initially subconvulsant electrical stimulus eventually results in intense limbic and motor seizures. Once established, the kindling effect is permanent. The mechanisms responsible for this phenomenon are unknown. Modulation of chemical synaptic transmission is likely an important determinant of this permanent state of abnormal neuronal excitability. Among the putative chemical transmitters studied to date, endogenous norepinephrine (NE) has been demonstrated to exert a powerful inhibitory effect on kindling development. The adrenergic receptor subtype with which NE interacts to inhibit kindling development is unknown. In preliminary studies we have found that systemic treatment with alpha2 adrenergic receptor antagonists markedly facilitates the rate of kindling development. We have also found that systemic treatment with an alpha2 receptor agonist markedly retards the rate of kindling development. The objective of this proposal is to employ pharmacological, electrophysiological, and radiohistochemical techniques to test three hypotheses related to these findings: (1) the inhibitory effects of endogenous NE are produced through interactions with alpha2 adrenergic receptors residing on targets of NE neurons; (2) part of the molecular basis of kindling is a reduction of alpha2 adrenergic receptors and/or recrptor coupled responses; and (3) the site of kindling stimulation is the site at which NE acts to inhibit kindling development. Insights derived from understanding the role of this transmitter may serve as a model for understanding the role of other transmitters in the kindling model. Such insights may lead to an understanding of the mechanisms underlying kindling itself. Finally, and perhaps most importantly, the kindling-inhibitory effects of the alpha2 agoinst, clonidine--a drug in current clinical use as an antihypertensive ageng--may lead to novel strategies for prophylaxis againts epilepsy arising as a late sequel to brain injury.

The objective os this proposal is to continue a comprehensive research program focussed on study of mechanisms of limbic epilepsy. The proposal brings together investigators with skills in molecular neurobiology, electrophysiology, anatomy, pharmacology, and clinical epilepsy. These molecular neurobiology, electrophysiology, anatomy, pharmacology, and clinical epilepsy. These skills will be focused on tests of alternative hypotheses advanced to explain the hyperexcitability characteristic of limbic epilepsy. These hypotheses will be tested in hippocampal slices isolated from normal rodents and from rodents in which epilepsy has been induced. The work is centered in particular on the excitability of two principal neuronal populations, the dentate granule cells and CA3 pyramidal cells. Successful completion of the work proposed promises to shed light on the mechanisms underlying limbic epilepsy. Such insights will hopefully lead to new and more effective therapies of this disorder. Insights into normal hippocampal circuitry and how it is modified following brain injury and pathologic activity will also emerge from this work; this promises to aid understanding a diversity of nervous system disorders in limbic epilepsy.

Epilepsy arising months or years after a head injury is a common and often devastating complication, accounting for at least 10,000 new cases of post-traumatic epilepsy annually in the U.S. alone. Although anti- convulsants are commonly prescribed for anti-epileptogenic purposes, no clinically effective anti-epileptogenic agents have been identified. Understanding the molecular basis of epileptogenesis could lead to a pharmacology aimed at prevention of epilepsy. Insight into the mechanisms underlying development of kindling may unravel part of the molecular basis of epileptogenesis. Two necessary conditions have been identified for the development of kindling: the periodic elicitation of brief electrical seizures (afterdischarges [ADs]) and activation of the NMDA receptor during the AD. How fleeting activation of glutamate receptors during AD produces the lifelong hyperexcitability of kindling is unclear. The unifying hypothesis of this application proposes that glutamate receptor activation during stimulation-evoked afterdischarge triggers a signaling cascade which culminates in expression of immediate early genes (IEG) and target genes including neurotrophins and their receptors, thereby inducing structural modifications of neurons in multiple sites, and that these structural modifications contribute to kindling development The objective of this proposal is to test several aspects of this unifying hypothesis, focusing on the dentate granule cells as a model in which to probe the relationship between glutamate receptor regulation of gene expression, structural neuronal modifications, and kindling development. We have established primary cultures of dentate gyrus neurons from post-natal rats and will test aspects of this hypothesis in parallel investigations in vitro and in the kindling model in vivo. This hypothesis will be tested with 3 specific aims: 1. To investigate part of the signaling pathways by which the Ca++ influx signals triggered by glutamate receptor activation regulate lEG expression in dentate gyrus and cortical neurons in vitro. 2. To examine the morphoregulatory and neuroprotective effects of the neurotrophins on dentate gyrus neurons. 3. To determine whether genetic and pharmacologic interventions at various sites in the signaling cascade modify molecular (lEG and neurotrophin gene expression), structural (axonal sprouting of granule cells), and functional (kindling development) events in vivo. Identification of pharmacologic interventions that inhibit kindling development may provide novel therapies for prophylaxis of post-traumatic epilepsy. Fundamental insights emerging from this work may also shed light on signal transduction in a diversity of physiologic and pathologic settings.

animal care; disease /disorder model; biomedical facility; animal colony; epilepsy; kindling; hippocampus; histology; genetically modified animals; laboratory rat; laboratory mouse;

genetics; epilepsy; biomedical facility; clinical research;

Understanding partial epileptogenesis in cellular and molecular terms may provide clues as to how to intervene pharmacologically to prevent this disorder. A commonly studied animal model of epileptogenesis is kindling in which pathological activity in the form of focal seizures induces epilepsy. The neurotrophins represent a family of molecules implicated in linking fleeting changes in activity to long term changes in neuronal phenotype. Striking increases in transcription of two neurotrophins, BDNF and NGF, occur in hippocampal neurons in the kindling model, but whether either or both of these neurotrophins promote epileptogenesis is uncertain. We have also obtained immunohistochemical evidence of enhanced activation of trk receptors in the hippocampus in multiple models of epileptogenesis. The objective of this proposal is to test different aspects of our unifying hypothesis which predicts that glutamate receptor activation during seizures triggers the transcription, translation, and release of BDNF, resulting in enhanced activation of trkB. Activation of trkB in turn activates diverse signal transduction pathways which trigger morphological and/or functional plasticities which culminate in a hyperexcitable state. The specific aims are fivefold: a) to further characterize the role of neurotrophins in limbic epileptogenesis; b) to characterize the molecular nature of the phophotrk immunoreactivity; c) to determine which neurotrophin(s) mediate the increased phosphotrk immunoreactivity; d) to investigate the signal transduction pathways by which trkB activation promotes epileptogenesis; 3) to determine the cellular locale of the kindling-induced increase of phosphotrk immunoreactivity. These specific aims will be accomplished by pharmacological, biochemical, and immunohistochemical studies of rats and mice, including mice in which a single gene has been deleted or modified at a single residue. Accomplishing these specific aims may provide novel therapeutic approaches aimed at prevention of epilepsy.

DESCRIPTION: Rasmussen's syndrome (RS) is a rare disease of unknown cause characterized by progressive destruction of a single cerebral hemisphere. The disease typically begins in the first decade of life and is manifested by severe seizures and progressive loss of functions subserved by the involved hemisphere. Recent evidence suggests that an immune response targeting a glutamate receptor of the AMPA subtype, GR3, contributes to RS. The occurrence of epileptic seizures and inflammatory histopathology in two rabbits immunized with a GR3 fusion protein led to the discovery of anti-GR3 in patients with active RS. The investigators have confirmed and extended their original findings by demonstrating that a subset of rabbits immunized with a GR3 fusion protein develop a neurologic disorder that includes epileptic seizures and inflammatory histopathology. Anti-GR3 isolated from GR3 immunized rabbits promote destruction of rat cortical cells in vitro. The objective of this proposal is to test one aspect of the unifying hypothesis of the etiology and pathogenesis of RS, namely that access of anti-GR3 to antigen in the central nervous system produces seizures and cell death. A diversity of electrophysiologic, anatomic and molecular neurobiologic techniques will be employed in both in vivo and in vitro studies. Specific aim 1 To characterize the animal model of RS observed in our preliminary studies. Specific aim 2 To determine whether GR3 immunization-induced circulating antidestruction of a single cerebral hemisphere. Specific aim 3 To determine whether passive transfer of anti-GR3 in vivo can induce epileptic seizures and cell death. Specific aim 4 To characterize the anti-GR3 raised in specific aims 1 and 2 with respect to titer, subunit specificity, and both agonist and cytotoxic properties. These experiments will facilitate study of mechanisms operative in RS in humans and may lead to novel therapies for the disorder.

The epilepsies comprise a common and devastating collection of disorders that affect about 1% of the U.S. population. This study is aimed at identifying one or more mutant genes that predispose individuals to juvenile myoclonic epilepsy and related forms of idiopathic generalized epilepsies. Large pedigrees continue to be identified throughout India in which there are at least four affected persons. Detailed history, neurological examination and EEG are being obtained and DNA isolated to perform a genomic screen for linkage/association analysis. Both model-based likelihood analyses and model-free analyses have been performed on the first pedigree in order to better define the disease as a genetic entity and to determine chromosomal locations of the genes involved. So far three chromosomal locations have been tentatively identified as suggestive of linkage. Further pedigrees are bing identified in order to confirm one or more of these findings. [unreadable]

The objective of this proposal is to continue a comprehensive research program focused on study of mechanisms of limbic epilepsy. The proposal brings together investigators with skills in molecular neurobiology, electrophysiology, anatomy, pharmacology, and clinical epilepsy. These skills will be focused on tests of alternative hypotheses advanced to explain the hyperexcitability of limbic epilepsy. These alternative hypotheses will be tested in a diversity of preparations including animal models in vivo as well as a diversity of reduced in vitro preparations, some of which have been isolated from the rodents in which epilepsy has been induced. The work is centered in particular on the excitability of two principal neuronal populations, the dentate granule cells and CA3 pyramidal cells. Successful completion of the work proposed promises to shed light on the mechanisms underlying limbic epilepsy. Such insights will hopefully lead to new and more effective therapies and perhaps prevent development of this disorder. Insights into normal hippocampal circuitry and how it is modified following brain injury and pathologic activity will also emerge from this work, this promises to aid understanding of a diversity of nervous system disorders in addition to limbic epilepsy.

DESCRIPTION (provided by applicant): Rasmussen's syndrome (RS) is a rare neurodegenerative disease of childhood in which a single cerebral hemisphere undergoes progressive destruction. The disease is characterized by gradual loss of functions sub-served by the involved hemisphere as well as severe epileptic seizures. During the current funding period, study of an animal model of RS and of brain tissue excised from children with RS provided evidence that inappropriate activation of complement may contribute to the disease. In particular, immunocytochemical evidence of membrane attack complex formation on neurons in RS brains was obtained. Despite evidence of membrane attack complex immunoreactivity in RS brains and in several neurodegenerative diseases including Alzheimer's, whether inappropriate activation of complement in mammalian brain exerts any deleterious effects is unknown. We have obtained preliminary evidence that inappropriate activation of complement, in particular the membrane attack complex, produces seizures and cell death in rats in vivo. The objective of this proposal is to investigate a series of hypotheses related to the pathological consequences of activation of the membrane attack complex in the cortex of mammalian brain. Parallel investigations will be performed in awake, freely moving rats in vivo and in organotypic explant cultures of rat hippocampus and in primary cultures of embryonic rat cortical cells in vitro.

DESCRIPTION (provided by applicant): This renewal application requests support for six trainees for Postdoctoral Training in Fundamental and Translational Neuroscience. The close integration of fundamental, translational and clinical training is enhanced at Duke by having its ten schools (including medicine, engineering, and arts and sciences) and its main hospital on a single contiguous campus. Translational training has long been a hallmark of Duke's research community which was awarded one of the first NIH Clinical Translational Science Awards. The program has 32 participating faculty from 12 departments including 5 clinical departments. All participating faculty are funded, with average funding being $575,000 per year. All except two assistant professors hold R01s and ten hold M.D. or M.D. / Ph.D. degrees. Five are practicing clinicians. The environment for neuroscience research was bolstered last year by the creation of a new interdisciplinary institute, the Duke Institute for Brain Science (DIBS). To enhance postdoctoral training, DIBS will provide two additional postdoctoral stipends each year from institutional funds to supplement those requested here. Postdoctoral fellows, regardless of their source of support, will be vetted and trained equivalently. Training grant support will be for one year while trainees develop research programs that will enable them to compete for independent extramural funding. RELEVANCE: The program seeks to accomplish the mission of NINDS- "to reduce the burden of neurological disease" by training the next generation of neuroscientists. Specifically, this program integrates translational and basic science training to further the overall goal of NIH- to promote translational medicine and bring new discoveries and technologies closer to implementation in the real world. Advances in neuroscience rests on young scientists trained to understand and contribute to the field.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a common and often devastating form of human epilepsy that lacks preventive therapy. Evidence from clinical and preclinical studies supports the idea that an episode of prolonged seizures (status epilepticus or SE) contributes to development of TLE. Defining the molecular mechanisms by which SE transforms a normal brain into an epileptic brain is essential for identifying molecular targets fo preventive therapies. Work accomplished during the current funding period revealed that transiently inhibiting the brain-derived neurotrophic factor (BDNF) receptor tyrosine kinase, TrkB, commencing following SE prevented SE-induced TLE. In preliminary studies we have discovered that global inhibition of TrkB signaling exacerbates SE-induced death of neurons, a detrimental outcome that must be mitigated. This led us to seek the downstream signaling pathway by which TrkB promotes epileptogenesis, our preliminary evidence implicating phospholipase Cy1 (PLCy1) as the dominant pathway. Our objective is to define the molecular mechanism by which SE induces TLE. To accomplish this objective, we propose to examine the effect of pharmacological uncoupling of TrkB from PLCy1 on cell death induced by SE in an adult mouse and on TLE induced by SE in a neonatal animal. We also propose to determine precisely when following SE that TrkB must be uncoupled from PLCy1 in order to prevent TLE in adults. Successful completion of the work proposed may pave the way to prevention of a subset of TLE, a common disorder of the human nervous system.

DESCRIPTION (provided by applicant): This renewal application (years 21 through 25) seeks support for early stage training of doctoral students in the neurosciences at Duke University. The mission of the program is to train the next generation of leading scientists who will discover how the nervous system normally functions to enable a myriad of complex processes, including learning, memory, movement, behavior, sensation, and perception, and how to restore normal function of the nervous system in the face of trauma and disease. The major objective of this program is to endow young scientists with the intellectual breadth and technical flexibility to grow and adapt to the demands of contemporary research, and thus enable them to successfully navigate the variety of scientific career paths that will span the neuroscience research enterprise of the future. The application seeks funding for 6 predoctoral students each year, typically 3 in their first year and 3 in their second year. The program is directed by a Steering Committee in concert with the program director, James O. McNamara, MD and co-director, Richard Mooney, PhD. Preceptors are drawn from a wide range of departments across the university and represent a broad diversity of fields, spanning molecular to translational neuroscience. Currently, thirteen primary faculty in the Department of Neurobiology and twenty-one faculty from other departments in the School of Medicine, School of Engineering, and the College of Arts and Sciences participate as faculty of the Graduate Program in Neurobiology. This diverse organization gives graduate students access to faculty spanning the entire breadth of molecular, cellular, systems, cognitive and translational neuroscience. A large and accomplished applicant pool-averaging over 100 candidates per year-permits recruitment of a talented, diverse class of 5-8 new students each fall. Half of the matriculants are supported in their first two years by the Duke Graduate School, which along with support from this training grant enables the program to recruit the most qualified international and domestic candidates. Extensive efforts are made to recruit and retain minority students, and the program has awarded PhDs to three URM students in the most recent funding cycle. Students undergo extensive intellectual and technical training, including demanding coursework addressing the depth and breadth of fundamental and translational neuroscience. Additional training and course work emphasizes the human nervous system in health and disease, oral presentation of scientific research, grant writing, teaching, and career development. Each student's progress is carefully monitored throughout the doctoral training period, with close attention paid to the time-to-degree and scientific publication. Upon completion of postdoctoral fellowships and clinical training (where applicable), approximately half of the program's graduates secure tenure-track faculty positions in research institutions and half work in industry, medical practice, and non-tenured faculty positions.

21+ years old; Adult; Alleles; Allelomorphs; Animal Model; Animal Models and Related Studies; Area; Articulation; Arts; Body Tissues; Cancer Center; Center Core Grants; Collaborations; Complex; Development; Disease; Disorder; EXTMR; Effectiveness; Environment; Equipment; Extramural; Extramural Activities; Fostering; Funding; Genes; Genetic; Genetic analyses; Genetically Engineered Mouse; Genie; Genome; Genomics; Human; Human, Adult; Human, General; Improve Access; Individual; Infrastructure; Institutes; Investigators; Ion Channel; Ionic Channels; Joints; Knock-in; Knock-in Mouse; Knockout Mice; Laboratories; Lead; Mammals, Mice; Man (Taxonomy); Man, Modern; Medicine; Membrane Channels; Mice; Mice, Knock-out; Mice, Knockout; Mice, Transgenic; Modeling; Molecular; Murine; Mus; NRVS-SYS; Nerve Cells; Nerve Unit; Nervous System; Nervous System Physiology; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Organ System; Neurologic function; Neurological function; Neurons; Neurosciences; Neurosciences Research; Null Mouse; Operation; Operative Procedures; Operative Surgical Procedures; Overexpression; P30 Mechanism; P30 Program; Pathway interactions; Pb element; Physiologic; Physiological; Plant Embryos; Population; Predisposition; Predisposition gene; Protein Overexpression; Proteins; R01 Mechanism; R01 Program; RPG; Research; Research Grants; Research Infrastructure; Research Personnel; Research Project Grants; Research Projects; Research Projects, R-Series; Research Resources; Researchers; Resources; Role; Salaries; Science Policy; Science of Medicine; Seeds; Services; Support of Research; Surgical; Surgical Interventions; Surgical Procedure; Susceptibility; Susceptibility Gene; Synapses; Synaptic; Technology; Time; Tissues; Transgenic Mice; Universities; Wages; Zygotes, Plant; adult human (21+); base; cell type; cost; design; designing; disease/disorder; gene function; gene product; gene replacement; genetic analysis; genetic manipulation; genome, mouse; heavy metal Pb; heavy metal lead; human disease; improved; in vivo; interdisciplinary approach; model organism; mouse genome; nervous system function; neuronal; overexpress; pathway; predisposing gene; seed; social role; surgery; tool

In this application for an NINDS Center Core Grant, we seek support for a Neurotransgenic core laboratory at Duke University. This core provides powerful tools for studying the nervous system, where the vast diversity of cell types and synaptic connections require tools that exceed those needed to study simpler, more homogenous tissues. This core will support individual neuroscience research projects by providing necessary resources and performing required services that would be difficult or impractical to provide in individual labs. Institutional commitments of space and seed funds for equipment and salaries have already been obtained and have enabled the creation of this core within the past two years, the understanding being that extramural support would be obtained for their continued support. This core will enrich the effectiveness of ongoing NINDS-supported research at Duke, promote new directions for both basic and translational neuroscience research, and foster a cooperative and interactive research environment through which multidisciplinary approaches to neuroscience problems and joint research efforts will be stimulated. Moreover, this provides a cost-effective way for investigators to exploit cutting-edge, specialized technologies that would be difficult to setup or justify for an individual laboratory. This core has been developed in partnership with other administrative units within Duke University including the Cancer Center. This core will be utilized by neuroscientists residing in multiple departments, thereby fostering a cooperative and interactive research environment. This core represents a substantial infrastructure support for genetic analysis in support of the neurosciences.

DESCRIPTION (provided by applicant): Understanding the mechanisms of limbic epileptogenesis may lead to novel disease modifying therapies. We have discovered that epileptogenesis is associated with enhanced activation of TrkB in the mossy fiber pathway of hippocampus. We have also discovered that the neurotrophin receptor, TrkB, is required for epileptogenesis in the kindling model. The objective of this application is to address two key questions: In what population of neurons within the mossy fiber pathway does the enhanced TrkB activation reside? Is the powerful antiepileptogenic effect of TrkB in the kindling model generalizable to other clinically relevant models? Morphological and electrophysiological studies of novel lines of genetically modified mice will be used to address these questions. We propose three Aims. To determine the cellular localization within the mossy fiber pathway of a surrogate measure of increased TrkB activation associated with limbic epileptogenesis. To determine whether inhibiting TrkB signaling prevents epileptogenesis in the pilocarpine status epilepticus model. To determine whether inhibiting TrkB signaling prevents epileptogenesis in the Kv1.1 -/- mouse. Successful completion of these Aims will provide valuable information for elucidating a cellular mechanism by which deletion of TrkB limits epileptogenesis. These experiments may also identify a novel molecular target for development of specific and effective anti-epileptogenic therapies. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms of limbic epileptogenesis may lead to novel disease modifying therapies. We have discovered that epileptogenesis is associated with enhanced activation of TrkB in the mossy fiber pathway of hippocampus. We have also discovered that the neurotrophin receptor, TrkB, is required for epileptogenesis in the kindling model. We seek to determine whether inhibiting TrkB signaling prevents other types of epileptogenesis in animal models. This information will guide efforts aimed at exploiting TrkB as a molecular target for anti-epileptogenic therapies.

DESCRIPTION (provided by applicant): The neurotrophin receptor, TrkB, serves a pivotal role in neuronal survival and differentiation as well as in synaptic structure, function, and plasticity. TrkB signaling has also been implicated in diverse psychiatric and neurological disorders. Notably, TrkB is activated during and is required for limbic epileptogenesis. Understanding the mechanisms by which TrkB is activated will provide insight into its pleiotropic functions in health and disease. Transactivation refers to the process whereby a given receptor and its downstream signaling is activated by a stimulus that does not interact directly with the receptor, a mechanism distinct from activation of TrkB by neurotrophins such as BDNF. We recently discovered that the divalent cation, zinc, can transactivate TrkB in cultured neurons by an activity dependent and neurotrophin independent mechanism. Whether zinc transactivates TrkB in vivo and, if so, it's physiological and pathological consequences are unknown. This application centers on the hypothesized transactivation of TrkB by zinc, both in vivo and in slices ex vivo. Three levels of analysis will be assessed: biochemical and immunohistochemical measures of pTrkB and TrkB in membranes and tissue ex vivo; potentiation of the hippocampal mossy fiber-CA3 pyramid synapse in slices ex vivo; and epileptogenesis in the kindling and pilocarpine models in vivo. We will determine whether vesicular zinc transactivates TrkB in vivo; whether vesicular zinc is required for LTP of the mossy fiber-CA3 pyramid synapse; and whether vesicular zinc is required for limbic epileptogenesis in vivo. Successful completion of the proposed work will clarify the role of zinc in transactivation of TrkB in vivo and thereby shed light on the activation of TrkB by both neurotrophin and non-neurotrophin ligands. Understanding how TrkB is activated is important because of the seminal role of TrkB signaling in the mammalian nervous system in development and adulthood and in both health and disease.

DESCRIPTION (provided by applicant): Epilepsy is a serious common neurological disorder, afflicting an estimated 1% of the population worldwide, and temporal lobe epilepsy (TLE) is the most severe and refractory epilepsy in adults. There is no effective prevention. Clinical observations as well as studies of diverse animal models underlie the idea that febrile status epilepticus (FSE) in childhood can cause TLE later in life. Therefore, FSE-related TLE is one epilepsy syndrome for which preventive therapies could be examined. Our overarching goals are to identify (a) a candidate compound and a backup for a clinical prevention trial of TLE following FSE, and (b) predictive biomarker(s) for individuals at high risk that should be targeted for intervention. To achieve these goals, we have assembled a team of preclinical investigators with research focus on mechanisms of epileptogenesis. The objectives of this planning grant are: 1) To create a compact administrative infrastructure that will promote interactions among our core team, NIH and our academic and pharmaceutical company consultants as well as draw additional investigators into the project it develops; 2) To conduct pilot studies aimed at identification of biomarkers across animal models aligned with the clinical phenotype. Our intent is to submit a subsequent application for an Epilepsy Center without Walls on Disease Modification and Prevention (CWW). The following Aims will enable the objectives of the planning phase of the CWW. Aim 1 to develop an administrative structure that oversees and coordinates the activities during the planning grant and prepares for a Center without Walls. Aim 2 To identify a cytokine bio fluid analyte profile that correlates strongly with imaging biomarkersafter prolonged FSE. Aim 3 to establish a multi-institutional team to coordinate study of the utiliy and predictability of MRI imaging following induction of seizures induced by hyperthermia or KA. Aim 4 to directly examine the predictability of these biomarkers for epilepsy, late hippocampal injury, and cognitive impairments in humans, by studying the FEBSTAT cohort.

Disease; Grant; Modification; Prevention; programs; Temporal Lobe Epilepsy;

DESCRIPTION (provided by applicant): This renewal application requests support for six trainees for Postdoctoral Training in Fundamental and Translational Neuroscience. The close integration of fundamental, translational and clinical training is enhanced at Duke by having its ten schools (including medicine, engineering, and arts and sciences) and its main hospital on a single contiguous campus. Translational training has long been a hallmark of Duke's research community which was awarded one of the first NIH Clinical Translational Science Awards. The program has 32 participating faculty from 12 departments including 5 clinical departments. All participating faculty are funded, with average funding being $575,000 per year. All except two assistant professors hold R01s and ten hold M.D. or M.D. / Ph.D. degrees. Five are practicing clinicians. The environment for neuroscience research was bolstered last year by the creation of a new interdisciplinary institute, the Duke Institute for Brain Science (DIBS). To enhance postdoctoral training, DIBS will provide two additional postdoctoral stipends each year from institutional funds to supplement those requested here. Postdoctoral fellows, regardless of their source of support, will be vetted and trained equivalently. Training grant support will be for one year while trainees develop research programs that will enable them to compete for independent extramural funding. RELEVANCE: The program seeks to accomplish the mission of NINDS- to reduce the burden of neurological disease by training the next generation of neuroscientists. Specifically, this program integrates translational and basic science training to further the overall goal of NIH- to promote translational medicine and bring new discoveries and technologies closer to implementation in the real world. Advances in neuroscience rests on young scientists trained to understand and contribute to the field.

DESCRIPTION (provided by applicant): Staphylococcus aureus (S. aureus) infections are a major public health problem, responsible for debilitating and life-threatening conditions including osteomyelitis and infectious arthritis. Technologies that enable the detection and localization of S. aureus in animals provide a means to assess the life-cycle dynamics, host interactions and antibiotic susceptibility of these bacteria in their natural environment and thus have great value as tools for scientific discovery and medical diagnostics. Current methods for imaging unmodified, naturally occurring S. aureus in animals are limited by the fact that they produce signal prior to encountering their target and most are non-specific with respect to bacterial species. An ideal molecular imaging probe for S. aureus would produce signal only upon encountering the targeted, unmodified bacteria or material derived from it. Such probes would enable the in vivo dynamic imaging of naturally occurring S. aureus strains with superior target-to-background ratios over existing technologies and would facilitate the clinical diagnosis and treatment evaluation of S. aureus infections in humans. The long-term goal of this line of investigation is to develop a novel class of nucleic acid-based activatable imaging probes for the specific non-invasive imaging of various bacterial species in animals and humans. The objective of this application is to demonstrate the utility of nucleic acid-based activatable imaging probes for the detection of S. aureus in vitro and in mice. The central hypothesis of this proposal is tha oligonucleotide-based nuclease substrates with fluorophore-quencher pairs (fluorophore is unquenched upon nuclease digestion), can be tailored via chemical modification to specifically detect nucleases of S. aureus and can thus serve as specific and sensitive probes for detection of the bacteria themselves. Specific aims are: 1) Generate nuclease-activated probes that can specifically detect micrococcal nuclease (MN) of S. aureus. Preliminary studies provide examples of chemically modified oligonucleotide probes that can differentiate between MN and mammalian serum nucleases. The working hypothesis for this aim is that oligonucleotides with the appropriate chemical modifications will be readily digested by MN, but resistant to both mammalian and various bacterial nucleases. Several distinct bacterial and mammalian nucleases will be included in these in vitro experiments. 2) Demonstrate the detection of focal S. aureus infections in mice with nuclease-activated probes. The working hypothesis for this aim is that nuclease- activated probes with quencher/fluorophore pairs that are susceptible to digestion by MN will enable the non- invasive detection and localization of focal S. aureus infections in mice. In summary, the proposed work is expected to result in the development of a robust activated imaging probe-based approach for the non-invasive detection and localization of S. aureus in animals. This contribution is significant because activated imaging probes have critical advantages over existing technology for this problem, and may prove to be generally useful for research and clinical diagnostic applications involving S. aureus.

To fulfill tlie new NCATS vision for the CTSA Consortium and accelerate scientific discoveries into improved outcomes for patients, academic health and science systems must invest, transform, and innovate to optimize their unique strengths. Our vision, aligned with that of NCATS, is to create a research environment at Duke that stimulates the translation of scientific discovery from bench to bedside by 1) linking discovery science to a creative engine that efficiently accelerates development of new technologies; and 2) integrating clinical trials, registries, and electronic health records in a learning health system where research and practice form a continuum. Our research environment will be driven by scientific merit and societal need, agnostic to disease or specialty discipline, aligned with our updated institutional framework for research oversight and quality, and continually evaluated for academic productivity, efficiency, and cost. To achieve these goals, we will create an Integrated Home for clinical and translational research by providing infrastructure and resources to serve investigators and trainees across the research spectrum. We will offer resources based upon common needs among our researchers, including education, biostatistics, biobanking, regulatory expertise, ethics, pilot funding and recruitment assistance. We will also tailor our offerings to specialized needs across research communities that include early translation, proof of concept, site-based research and population based research, which includes multi-site trials, outcomes, health services, implementation science and community engaged research. Integrating these resources will require a new tool, a portal for all trainees and investigators, MyResearchHome@Duke, and its human counterpart, MyResearchTeam@Duke. These tools will provide a single point of entry for all clinical and translational research at Duke, regardless of their department or school. Thus, we will enhance our Integrated Home for clinical and translational research with a combination of sophisticated information technology and mentoring and navigation; provide access to common and specialized resources for all of our translational research communities and train the next generation of researchers in our educational and training programs. RELEVANCE (See instructions): The CTSA will foster the translational research process, ensuring that new discoveries are developed and evaluated more quickly and that clinical research is done with high quality, efficiency, safety and cost- effectiveness.

DESCRIPTION (provided by applicant): There is currently an unmet clinical need for diagnostic tests that rapidly provide direct evidence and localization of foreign-body infections (e.g., catheter and prosthetic-joint infections). Staphylococcus aureus (S. aureus) is the second most common bacterial species responsible for foreign-body infections and it is responsible for much of the morbidity and mortality that result from such infections due to its highly virulent nature. This proposal is based on a novel molecular imaging approach that rapidly and specifically detects S. aureus infections with a fluorescent probe that is activated by an S. aureus-specific nuclease. The long-term goal of this line of investigation is to translate this approach into clinical practice. The objective of this application is to carry out preclinical evaluation of an optimized S. aureus-specific activatable probe in a murine foreign-body S. aureus infection model. The central hypothesis of this proposal is that an optimized probe will exhibit low toxicity and high sensitivity and specificity for imaging S. aureus foreign-body infections in mice. The Specific Aims are: 1) Identify a Next Generation TT probe (NGTT probe) by incorporating a fluorophore/quencher combination that is optimal for in vivo imaging. The spectral characteristics and activated/unactivated fluorescence ratio of the first generation TT probe will be optimized with the design, generation and comparison of several probe variants with red-shifted fluorophores and appropriately matched quenchers. These probes will be evaluated in vitro and in a pyomyositis S. aureus infection model. The best- performing probe will be selected as the NGTT probe. 2) Image S. aureus foreign-body biofilm infections in mice with the NGTT probe. Foreign-body biofilm infections are a large and problematic category of S. aureus infections. The utility of the NGTT probe will be tested in a mouse model of such infections in which S. aureus biofilms grow on catheters implanted in the mice. 3) Assess the safety of the NGTT probe in mice. Various measures of toxicity, including complete blood counts will be taken in mice after intravenous administration of the NGTT probe. Doses of the probe, 10-fold greater than those found to yield robust infection imaging, will be administered. The proposed work is expected to result in the optimization and thorough pre-clinical testing of this approach for imaging S. aureus foreign-body biofilm infections. This contribution is significant because it will provide an essential foundation for the translation of a diagnostic imaging approach for S. aureus infections, and thus constitutes critical progress towards addressing an unmet clinical need. The work proposed is novel in several respects. This will be the first thorough preclinical assessment of a nuclease-activated oligonucleotide probe for the non-invasive diagnostic imaging of any medical condition. This work also represents the first evaluation of efficacy and specificity of our imaging approach in a model of a common clinical problem, an important step towards clinical translation. Finally, the application of this approach to foreign-body biofilm infections constitutes an innovative solution to a challenging problem.

Epilepsy arising from the temporal lobes is particularly devastating because it is both common and commonly resistant to symptomatic therapy with anticonvulsants. Preclinical and clinical studies support the idea that an episode of status epilepticus followed by recovery contributes to development of temporal lobe epilepsy months or years later. Insight into the mechanisms by which status epilepticus induces temporal lobe epilepsy may facilitate developing preventive and/or disease modifying therapies. We recently discovered a molecular signaling pathway by which status epilepticus induces temporal lobe epilepsy, namely activation of the brain- derived neurotrophic factor receptor, TrkB. A major unresolved question, to be addressed in this application, is how this signaling is transformed into the cellular and circuit modifications that underlie temporal lobe epilepsy. The anatomic locale at which status epilepticus induced activation of TrkB provides a valuable clue to the cellular consequences. The principal site was the synaptic boutons of the mossy fiber axons of hippocampal dentate granule cells. The objective of this application is to test three facets of our unifying hypothesis: a) that status epilepticus induces plasticities of mossy fiber synapses with both CA3 pyramidal cells and inhibitory interneurons; b) that status epilepticus induction of these plasticities requires TrkB; and c) that transmitter release from the mossy fibers underlies expression of temporal lobe epilepsy.

The mission of the Duke Clinical and Translational Science Institute is to catalyze the translation of scientific discoveries into population health benefits through collaborative research. With previous Clinical and Translational Science Award (CTSA) funding, we have successfully developed infrastructure and provided resources to support investigators across the clinical and translational scientific and workforce spectrums. Our achievements have facilitated high-impact discoveries, increased research efficiency, and nurtured a more capable and diverse workforce. We have also ignited a new era of collaborative science at Duke. Several new transdisciplinary programs have been launched, connecting trainees and faculty from our Schools of Medicine, Nursing, Engineering, Business, and Arts and Sciences to enhance translation. In the next phase, Duke proposes to capitalize on new opportunities with three broad initiatives. First, we will optimize clinical and translational science national networks? methods and processes. Building on Duke?s leading experience coordinating multi-center clinical trials including its national Duke-Vanderbilt NCATS Trial Innovation Center, we will develop methods to optimize ?local hub,? ?hub-to-network? and ?network-to-network? processes for the NCATS Trial Innovation Network. We will also help investigators leverage a number of additional NIH network resources and provide methods to disseminate best practices throughout the CTSA network. Second, we will deepen, nurture, and extend stakeholder collaborations. We will strengthen existing partnerships and foster new ones with stakeholders at both the institutional and investigator levels. Partnerships and programs will expand the breadth and transparency of CTSI activities. We will also support investigators? greater engagement with a broad range of patients, community groups, clinicians, health care systems, industry, venture capitalists, payers, policy makers, and others. Third, we will catalyze further science integration at Duke. An invigorated professional workforce will facilitate major transdisciplinary science initiatives. A new University-wide coordination infrastructure will further amplify collaboration and bridge science siloes. We will also support teams by improving their skills in team science, studying team processes, and by promoting the recognition of team contributions at our institution. Collectively, these activities will amplify the innovation and impact of our research nationally, regionally, and locally. We will measure outcomes that reflect improvement in the translation of discoveries, use of national best practices, institutional and investigator teamwork, research transparency, and community trust. We will share all best practices with the CTSA network. We believe our achievements will meaningfully enhance the impact of clinical and translational science on health across the United States.

Abstract of overall Duke CTSA grant: The mission of the Duke Clinical and Translational Science Institute is to catalyze the translation of scientific discoveries into population health benefits through collaborative research. With previous Clinical and Translational Science Award (CTSA) funding, we have successfully developed infrastructure and provided resources to support investigators across the clinical and translational scientific and workforce spectrums. Our achievements have facilitated high-impact discoveries, increased research efficiency, and nurtured a more capable and diverse workforce. We have also ignited a new era of collaborative science at Duke. Several new transdisciplinary programs have been launched, connecting trainees and faculty from our Schools of Medicine, Nursing, Engineering, Business, and Arts and Sciences to enhance translation. In the next phase, Duke proposes to capitalize on new opportunities with three broad initiatives. First, we will optimize clinical and translational science national networks? methods and processes. Building on Duke?s leading experience coordinating multi-center clinical trials including its national Duke-Vanderbilt NCATS Trial Innovation Center, we will develop methods to optimize ?local hub,? ?hub-to-network?, and ?network-to-network? processes for the NCATS Trial Innovation Network. We will also help investigators leverage a number of additional NIH network resources and provide methods to disseminate best practices throughout the CTSA network. Second, we will deepen, nurture, and extend stakeholder collaborations. We will strengthen existing partnerships and foster new ones with stakeholders at both the institutional and investigator levels. Partnerships and programs will expand the breadth and transparency of CTSI activities. We will also support investigators? greater engagement with a broad range of patients, community groups, clinicians, health care systems, industry, venture capitalists, payers, policy makers, and others. Third, we will catalyze further science integration at Duke. An invigorated professional workforce will facilitate major transdisciplinary science initiatives. A new University-wide coordination infrastructure will further amplify collaboration and bridge science siloes. We will also support teams by improving their skills in team science, studying team processes, and by promoting the recognition of team contributions at our institution. Collectively, these activities will amplify the innovation and impact of our research nationally, regionally, and locally. We will measure outcomes that reflect improvement in the translation of discoveries, use of national best practices, institutional and investigator teamwork, research transparency, and community trust. We will share all best practices with the CTSA network. We believe our achievements will meaningfully enhance the impact of clinical and translational science on health across the United States.

The mission of the Duke Clinical and Translational Science Institute is to catalyze the translation of scientific discoveries into population health benefits through collaborative research. With previous Clinical and Translational Science Award (CTSA) funding, we have successfully developed infrastructure and provided resources to support investigators across the clinical and translational scientific and workforce spectrums. Our achievements have facilitated high-impact discoveries, increased research efficiency, and nurtured a more capable and diverse workforce. We have also ignited a new era of collaborative science at Duke. Several new transdisciplinary programs have been launched, connecting trainees and faculty from our Schools of Medicine, Nursing, Engineering, Business, and Arts and Sciences to enhance translation. In the next phase, Duke proposes to capitalize on new opportunities with three broad initiatives. First, we will optimize clinical and translational science national networks? methods and processes. Building on Duke?s leading experience coordinating multi-center clinical trials including its national Duke-Vanderbilt NCATS Trial Innovation Center, we will develop methods to optimize ?local hub,? ?hub-to-network? and ?network-to-network? processes for the NCATS Trial Innovation Network. We will also help investigators leverage a number of additional NIH network resources and provide methods to disseminate best practices throughout the CTSA network. Second, we will deepen, nurture, and extend stakeholder collaborations. We will strengthen existing partnerships and foster new ones with stakeholders at both the institutional and investigator levels. Partnerships and programs will expand the breadth and transparency of CTSI activities. We will also support investigators? greater engagement with a broad range of patients, community groups, clinicians, health care systems, industry, venture capitalists, payers, policy makers, and others. Third, we will catalyze further science integration at Duke. An invigorated professional workforce will facilitate major transdisciplinary science initiatives. A new University-wide coordination infrastructure will further amplify collaboration and bridge science siloes. We will also support teams by improving their skills in team science, studying team processes, and by promoting the recognition of team contributions at our institution. Collectively, these activities will amplify the innovation and impact of our research nationally, regionally, and locally. We will measure outcomes that reflect improvement in the translation of discoveries, use of national best practices, institutional and investigator teamwork, research transparency, and community trust. We will share all best practices with the CTSA network. We believe our achievements will meaningfully enhance the impact of clinical and translational science on health across the United States.