Nicolas G. Bazan - US grants

The purpose of this proposed research is to investigate the metabolism and role of prostaglandins, thomboxanes, prostacyclin, hydroxyeicosatetraenoic acids and leukotrienes in the retina. These potent chemical mediators of intercellular and intracellular signals are called eicosanoids. Specifically, we will test the hypothesis that eicosanoid synthesis and release are correlated with neurotransmission in the retina. Studies have been designed to explore retinal eicosanoid metabolism in relation to the release of chemical mediators and the postsynaptic action of neurotransmitters. Our preliminary studies have shown that the retina is able to synthesize a wide variety of eicosanoids and that a K+ evoked increase in the formation and release of 12-hydroxyeicosatetraenoic acid takes place. Hence, depletors of neurotransmitters, receptor agonists and antagonists, and light stimulation will be surveyed along with drugs that selectively inhibit steps of the metabolic pathways of eicosanoids. The following objectives will be addressed: a) a study of the activation, esterification, and release of arachidonic acid; how phosphoinositides and other phospholipids are involved as sources; and how eicosanoids are subsequently synthesized in the retina; b) the use of drugs as tools to understand the pathways of eicosanoid metabolism in the retina in vivo and in vitro, as well as the retinal subcellular sites involved; c) the examination of the activation mechanism of hydroxyeicosatetraenoic acid and subsequent incorporation into retinal lipids; d) the testing of the hypothesis that eicosanoid synthesis and release are correlated with neurotransmission in the retina by characterizing the K+ evoked stimulation of the synthesis and release of lipoxygenase reaction products and the effect of neurotransmitters on these processes; and e) the definition of the cellular localization of some eicosanoids and their participation in the scheme of information flow in the neural retina. To investigate retinal eicosanoid metabolism, we will inject precursors intravitreally into the rat eye, and follow the metabolic pathways and release upon stimulation by a combination of biochemical methods such as TLC, GLC, HPLC, radiochemical procedures, subcellular fractionation, and spectrophotometry. In addition, electrophysiological techniques and light microscopic and ultrastructural autoradiography will be used.

The metabolism of arachidonic acid, lipoxygenase-reaction products, prostaglandins and phospholipids in rat brain will be investigated during experimentally-induced seizures. Phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, which are enriched in arachidonoyl groups, and their metabolites, inositol-1,4,5-trisphosphate and diacylglycerols, will be studied, to test the hypothesis that these particular components of neuronal cell signaling systems are the membrane target affected by convulsions. The accumulation of endogenous arachidonoyldiacylglycerol, known to occur as a result of seizures, is proposed to be a consequence of enhanced breakdown of polyphosphoinositides mediated by phospholipase C. The effects of the muscarinic cholinergic antagonist, atropine, and the inositol phosphatase inhibitor, lithium, on the phosphoinositide cycle, will be evaluated. The hypothesis to be tested also includes convulsion-induced changes in phospholipase A2, resulting in the release of arachidonic acid from synaptic membrane phospholipids, through a receptor-related mobilization of calcium. The role of calcium will be evaluated using calcium channel modulators. As a result of phospholipase A2 activation, subsequent production of oxygenated metabolites of arachidonic acid, particularly hydroxyeicosatetraenoic acids, may be enhanced. Emphasis will be placed on the membrane lipid sources, neuroanatomical and subcellular distribution, and fate of the rapidly released arachidonic acid and arachidonoyl-diacylglycerols that occur in the rat brain during bicuculline-induced status epilepticus and after electroconvulsive shock. Convulsions will be induced in mechanically ventilated, well-oxygenated animals. In some experiments 32P or [14C]arachidonic acid will be injected intraventricularly to follow precursor-product relationships. This proposal will employ in vivo models and subcellular fractions. Very rapid fixation of the tissues within 1 second will be achieved by high-powered, head-focused microwave irradiation. Powerful analytical techniques, such as high performance liquid chromatography, gas-liquid chromatography, and gas chromatography-mass spectrometry, will be used to examine biochemical changes in phospholipids and fatty acids of neuronal membranes. The results of this study will have application in the management of epileptic seizures and will provide data on the use of drugs that are potentially capable of halting or reversing membrane lipid breakdown, consequently preventing or limiting the brain damage caused by epilepsy.

The metabolism and physiological significance of docosahexaenoic acid, a major fatty acid of photoreceptors, remain largely unknown. The present proposal is a continuation of our ongoing studies focusing on several aspects of the cell biology of docosahexaenoic acid metabolism. The hypothesis to be tested is that the supply, uptake and metabolism of docosahexaenoic acid in the visual cell plays a central role in the development, renewal and function of photoreceptor membranes. The proposal follows a postnatal developmental approach to study docosahexaenoic acid metabolism in dissociated rod photoreceptor cells prior, during and after outer segment biogenesis in normal mice and in mice with retinal degeneration. Since docosahexaenoic acid is derived from linolenic acid, an essential fatty acid supplied by the diet, the origin of retinal docosahexaenoic acid, its synthesis from linolenic acid in the liver and its transport via the blood to the retina, will be studied. Another goal of this proposal is to correlate docosahexaenoic acid metabolism with photoreceptor shedding and phagocytosis in the frog. The retina synthesizes docosanoids (22-carbon, leukotriene- like compounds). This proposal will test the hypothesis that these metabolites are involved in signaling mechanisms involved in photoreceptor shedding. Docosanoids formed in response to shedding and the enzymes responsible for docosanoid production will be characterized. The possibility that the docosanoid-producing enzymes are distinct from lipoxygenases involved in arachidonic acid metabolism will be explored. In addition, the subcellular localization of docosanold production will be determined. The approach proposed for these studies combines cell biological techniques (dissociated rod photoreceptor cells, autoradiography and eye cup preparations) with highly sensitive and specific biochemical procedures (high-performance liquid chromatography, capillary gas-liquid chromatography, gas chromatography-mass spectrometry) and should contribute to a better understanding of the role of docosahexaenoic acid in visual cells.

Our goal is to investigate the biochemistry and physiological significance of the arachidonic acid cascade, particularly the lipoxygenase-leukotriene pathway, and the inositol lipid cycle in the retina. The focus is on visual cell - pigment epithelium interactions during photoreceptor renewal. In other systems, metabolites arising from receptor-stimulated turnover of phosphoinositides and from the arachidonic acid cascade are potent mediators of intracellular and intercellular signals. The proposed experiments will: 1) correlate changes in arachidonic acid metabolism in rod outer segments and pigment epithelium with particular stages (times) of photoreceptor shedding; 2) study the effect of inhibitors of leukotriene and prostaglandin synthesis on photoreceptor disk shedding in an eyecup preparation; 3) investigate the involvement of inositol polyphosphates and the inositol lipid cycle as a transmembrane signaling system for modulation of shedding and/or phagocytosis; and 4) characterize the involvement of the arachidonic acid cascade and inositol lipids in the modulation of phagocytosis in pigment epithelium cells in culture. Powerful analytical procedures, such as high performance liquid chromatography, capillary and open column gas-liquid chromatography, gas chromatography-mass spectrometry and radioimmunoassay, will be used to examine biochemical changes and to establish correlates with a histological assessment of disk shedding and phagocytosis. The results obtained will define the involvement of the arachidonic acid cascade and the inositol lipid cycle in the modulation of the interactions between visual cells and retinal pigment epithelium.

This proposal is the continuation of our studies on second messenger systems derived from membrane lipids involved in retinal pigment epithelium - visual cell interactions during photoreceptor renewal. We have developed new approaches and a hypothesis that entails a chain of events triggered at the initiation of shedding and phagocytosis. The focus is on the testing of the hypothesis that an initial step in shedding and phagocytosis is the activation of a phospholipase C that generates inositol trisphosphate and diacylglycerol in the retinal pigment epithelial cells. These second messengers then promote intracellular Ca2+ mobilization and protein kinase C activation, respectively, which in turn modulate several cell functions. The ionized Ca2+ then activates both a phospholipase A2, leading to arachidonic acid release, and lipoxygenase enzymes, resulting in formation of leukotrienes (e.g. leukotriene C4) and other metabolites (e.g. 12-hydroxyeicosatetraenoic acid, lipoxins). These messengers diffuse to the interphotoreceptor matrix and are prime candidates as intercellular signals possibly acting on photoreceptor cells. Phospholipase A2 also gives rise to another lipid mediator, platelet-activating factor, which may contribute to the regulation of long-term events in the retinal pigment epithelium. The proposed experimental design will use powerful now analytical procedures, such as high-performance liquid chromatography-particle beam-mass spectrometry and high-performance liquid chromatography-capillary gas-liquid chromatography. These methods will also allow the isolation and identification of storage sites at membranes of lipid mediators, such as 1 -alkyl-2-- arachidonoyl-glycerophosphorylcholine (the precursor of platelet-activating factor) and of the second messengers of the enzymatic oxygenation of arachidonic acid. Frogs will be used because RPE - visual cell interaction can be experimentally manipulated by altering the light-dark cycle, and shedding can be triggered at the onset of light. A rapid procedure to isolate intact retinal pigment epithelial cells will also allow the direct biochemical study in vivo of each step of the hypothesis. This information will be correlated with the histological assessment of shedding and phagocytosis. Moreover, eyecups will be incubated in vitro with selective antagonists or inhibitors of the second messenger systems involved in these events, as well as receptor antagonists to study the shedding response. In addition, human retinal pigment epithelial cells in culture will be used to explore the various steps of the hypothesis. The results will define the involvement of second messengers in the interactions between visual cells and retinal pigment epithelium during photoreceptor renewal.

? DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) and other retinal degenerative diseases remain among the greatest challenges to public health, and innovative approaches to understand disease mechanisms are needed. The functional integrity of the retinal pigment epithelial and photoreceptor cells (RPE/PRC) are tightly interrelated and regulated to ensure vision. Docosahexaenoic acid (22: 6n-3, DHA), which attains its highest concentrations in the body in PRC outer segments, in which it is avidly retained and conserved, plays a critical role in sustaining RPE/PRC integrity. Under conditions of uncompensated oxidative stress (OS), neuroprotectin D1 (NPD1), a cell survival mediator, is made on-demand from DHA when disruptors of homeostasis evolve, and the initial inflammatory response needs to be modulated to protect RPE/RPC integrity. Our broad goal is to understand the molecular principles underlying DHA retention/conservation and NPD1-mediated protection under stress. Results during the recent funding period provide some strong clues to these principles, as they reveal an unexpected mechanism critical for DHA retention/conservation, as well as a novel connection between NPD1 and transcriptional regulation. Thus, our immediate goal is to further define the genetic and cellular mechanisms that govern pro-survival mechanisms, as well as the proteins that carry out these important protective actions. The three Aims for the next grant period are directed at testing specific predictions based on the following hypothesis: RPE/PRC integrity is sustained via the pivotal role of the protein adiponectin receptor 1 (AdipoR1, hereto unrecognized in these cells) in the retention and conservation functions of DHA. More specifically, AdipoR1 facilitates the synthesis of very long chain-polyunsaturated fatty acid and NPD1 availability that, in turn, modulate RPE preconditioning, Alu-RNA- induction of the NALP3 inflammasome, and cRel/BIRC3 transcription to sustain RPE/PRC integrity. Aim 1: To test the prediction, and define the molecular principles, whereby AdipoR1 is decisive for DHA retention and function in RPE and photoreceptor cells. We propose that a mechanism retains/conserves DHA, and that its ablation leads to retinal degeneration. This will allow us to address how RPE/PRC functional integrity is sustained. Aim 2: To test the prediction that Alu-induction of the NALP3 inflammasome is modulated by AdipoR1-dependent and NPD1-mediated cRel-BIRC3 expression, which are decisive for RPE cell integrity. Aim 3: To test the hypothesis that AdipoR1 mediates NPD1 induction of preconditioning (pre-C) and RPE cell survival. Thus NPD1 induces preconditioning and selectively upregulates BIRC3 expression upon oxidative stress and cREL mediates NPD1 signaling activation of BIRC3 expression and preconditioning-mediated RPE/PRC survival. The outcomes of these studies may lead to novel strategies for enhancing the intrinsic potential of RPE/PRC to protect and repair them and for promoting long-term expression of proteins involved in RPE/PRC survival.

DESCRIPTION: (Applicant's Abstract) The long-term objective of this proposal is to define the role of PAF (platelet-activating factor), a mediator of neuronal injury, and of phospholipase A2 activation in epileptic brain damage and in epileptogenesis. In experimental models of epilepsy, increased excitability, aberrant synaptic reorganization, and neuronal death take place. However, the specific messengers involved in these events are not known. We have discovered that PAF enhances excitatory neurotransmitter release and is an activator of gene expression. The goal of this proposal is to test the hypothesis that a) PAF contributes to increased excitability, seizure generation, and seizure-induced hippocampal damage; b) PAF-triggered gene expression participates in aberrant synaptic reorganization; c) PAF antagonists active at the presynaptic ending are neuroprotective in epileptic damage and PAF antagonists active on gene expression inhibit epileptogenesis; and d) over expression of PAF acetylhydrolase activity slows or prevents kindling development in transgenic rats. We will define the involvement of phospholipase A2 activation in seizure-induced damage and in kindling development by measuring the pool size and metabolism of PAF and the arachidonic acid cascade in hippocampus. We will use a rapid kindling model, kainic acid-induced seizures, and perforant path stimulation of hippocampal slices to ascertain how injury mediators participate in aberrant synaptic reorganization. We will identify PAF-responsive elements in the promoter of the inducible prostaglandin synthase gene in the hippocampus in models of epilepsy. Powerful analytical procedures, such as HPLC-mass spectrometry, will be used to study the biochemistry of second messengers. These will be combined with electrophysiological, histological and molecular biological studies and, in some cases, Ca2+ imaging. These studies will a) define metabolic pathways and events in epileptic damage that could be halted or slowed by novel neuroprotective mechanisms, b) characterize signal transduction pathways involved in aberrant synaptic reorganization, and may c) identify new drug strategies to prevent damage and circuitry reorganization in epilepsy.

DESCRIPTION (provided by applicant): The LSU-HSC leads a partnership of four Louisiana institutions in a COBRE on neuroscience research. The Program Director and established scientists are mentors to selected, highly promising junior faculty who propose four research projects that have been designed for the common goal of mentoring to build new nationally competitive research. The scientific focus is on a central issue of neurobiology: to understand the cellular and molecular basis of synaptic plasticity and neuronal survival critical to clarify the pathophysiology of neurological disorders such as: stroke, neural trauma and neurodegenerative diseases. This multidisciplinary program involves cellular neurophysiology, molecular biology and behavioral neuroscience. To support the COBRE projects, core resources include facilities for imaging , neurochemistry of lipid messengers and molecular neurobiology. A recruitment plan for years 2 to 5 further benefits the collaborating institutions by actively attracting new research faculty who will work under the guidance of established neuroscientists as mentors. A relatively small administrative core funding is requested. The specific aims to attain these objectives are: 1) to promote faculty development through research projects; 2) to further develop a critical mass of competitive extramurally funded investigators by the recruitment, start up, mentoring and retention of new faculty members; 3) to enhance the infrastructure critical for expanding neuroscience capability in Louisiana by developing three core research modules at LSUHSC; 4) to provide scientific and grantsmanship mentoring and strengthen the support network that promotes interactions; and 5) to implement interim and outcome evaluations so as to keep this COBRE program on track. This partnership rests on existing expertise and in our firm decision to build a scientifically successful neuroscience alliance in Louisiana. The four target faculty and the four to-be-recruited faculty are the critical building blocks to achieve these goals. The core resources are vital to the overall success of this consortium, not only in neuroscience but in all the biomedical sciences. The plans for mentoring junior faculty and the recruitment plan will ensure a steady stream of new nationally competitive neuroscientists in Louisiana.

DESCRIPTION (provided by applicant): The long-term objective of this proposal continues to be focused on defining the significance of platelet-activating factor (PAF), phospholipases and arachidonic acid as steps in the neurosignaling events in epileptic brain damage and epileptogenesis. Studies during the previous five years have made significant discoveries pertinent to understanding signaling relevant to epileptogenesis and neuroprotection. This includes the construction of new molecular and genetic tools that can be used as probes to test specific steps of our hypotheses about key signals that maintain neuroprotection in the hippocampus and in pathways engaged in epileptogenesis. The specific aims for the next grant period examine these key pathways using mice deficient in the genes that we have found to be critical in seizures (arachidonoyl-diglyceride-selective diglyceride kinase (DGK) epsilon and PAF receptor) using several approaches including kindling epileptogenesis. The central hypothesis to be tested in this proposal is that the lipid messengers PAF, arachidonic acid (AA), inositol 1,4,5-triphosphate (IP3) and arachidonic acid 1,2-diacylglycerol (AA-DG) mediate the initiation and progression of epileptogenesis. PAF, initially, enhances glutamate release and then activates protein kinases and transcription factors that lead to the activation of cyclooxygenase-2 (COX-2) expression and other genes. AA, AA-DG and IP3 potentiate phospholipase A2 (PLA2) activation and PAF synthesis during epileptogenesis Our specific aims are to 1) test the hypothesis that deficiency in PAF-receptor and/or in arachidonyl-diglyceride-selective diglyceride kinase (DGK)-epsilon result in decreased susceptibility to epileptogenesis, 2) identify upstream signaling modified as a consequence of deficiency in PAF-receptor and in DGK-E in neurons in culture and hippocampal slices; 3) define downstream signaling events in epileptogenesis triggered by synaptic activation that leads to gene transcription modulation by bioactive lipids - these studies will test the hypothesis that COX-2 gene overexpression leads to aberrant synaptic plasticity, and this hypothesis is supported by our finding that COX-2 is overexpressed in a kindling model of epileptogenesis; and 4) use pharmacological agents to clarify the mechanisms of synaptic-triggered lipid signaling in epileptogenesis and to test their potential usefulness as neuroprotectants that selectively block critical events in epileptogenesis. This work is expected to provide new insights into neuronal signaling in epileptogenesis, to uncover novel molecular pathways in seizure-induced neuronal injury and define events for neuroprotective pharmacological interventions.

DESCRIPTION (provided by applicant): Recent studies supported by this grant have begun to fill a major gap in our knowledge regarding neuroinflammation, survival signaling, and protection of the penumbra in experimental stroke. We have found that docosahexaenoic acid (DHA), an omega-3 essential fatty acid-family member, which is selectively enriched and avidly retained in the central nervous system (CNS), upregulates cellular and molecular events when systemically administered that counteract injury and, in turn, exert protection after transient middle cerebral artery occlusion (MCAo). We have discovered that DHA is the precursor of the stereospecific and potent mediator, neuroprotectin D1 (NPD1), and very recently we uncovered during MCAo in the penumbra a novel DHA-derived mediator, which we named neuroprotectin D2 (NPD2). These two docosanoids, through synergizing of their bioactivity, may be critical in driving the protective beneficial effects of DHA. In addition, we have demonstrated that a low molecular weight platelet-activating factor (PAF)-receptor antagonist, LAU-0901 (developed in our laboratory), downregulates neuroinflammation and elicits protection. In both instances, protection has relatively ample windows of post-occlusion, is long-lasting, and correlates with neurobehavioral improvement. Our central hypothesis for the next funding period is that upregulation of DHA survival signaling elicits sustained cellular and behavioral protection of the penumbra after MCAo. We will test this hypothesis in experiments where we drive upregulation of survival signaling by DHA-derived mediators (NPD1 and NPD2) and by neuroinflammatory antagonism (LAU-0901) using MCAo, high resolution magnetic resonance imaging (MRI), primary cell cultures, and LC-PDA-ESI-MS/MS-based mediator lipidomic analysis. We will test the following specific aims: Specific Aim 1 - To test the prediction that the novel DHA-derived mediator, NPD2, which is generated in the penumbra during brain ischemia-reperfusion, elicits neuroprotection; Specific Aim 2 - To test the prediction that NPD1 and NPD2 target survival signaling and pro-inflammatory genes; Specific Aim 3 - To test the prediction that blockage of the PAF receptor elicits penumbra protection after MCAo, downregulating neuroinflammatory signaling. Specific Aim 4 - To test the hypothesis that neuroinflammatory antagonism in combination with docosanoid-mediated survival signaling promotes sustained protection of the penumbra. These findings have the potential to open a new translational avenue for clinical therapies of cerebrovascular diseases.

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 Neurochemistry of Lipid Messengers Core Resource Module (NCLM) is directed by Victor Marcheselli, an investigator who has extensive experience in analysis of lipid second messengers. The NCLM allows core participants to measure by radiochemical and chromatographic techniques (TLC, HPLC and GLC) quantities of endogenous lipid messengers as well as to follow the metabolism of lipid mediatorss after stimulation. The core also provides technical support and assistance in interpretation of the data. The core houses a new LC-MS/MS that was purchased in part with funds requested in the COBRE and in part by matching funds from LSU. This instrument allows identification of new bioactive lipids identified and studied in the experiments of the proposed projects.

DESCRIPTION (provided by applicant): In testing the central hypothesis of our previous proposal, that ROS phagocytosis holds in check pro-inflammatory/cell injury signaling in the RPE, we discovered a lipid mediator that accumulates under those conditions and whose bioactivity includes inhibition of cytokine induction of NF-kappaB and COX-2 expression. The novel lipid (that we named neuroprotectin D1, NPD1) also inhibits oxidative stress-triggered RPE apoptosis and provides potent protection against oxidative stress (H2O2/TNFalpha or A2E). NPD1 formation is initiated by phospholipase A2-mediated release of DHA. We also developed a novel PAF antagonist, LAU-0901, that is cytoprotective, and at the same time, up-regulates expression of prosaposin, one of the differentially expressed genes in RPE in the presence of this PAF antagonist. We now propose to test the hypothesis that in the RPE response to pathological insults, there is coordinated/ concerted survival signaling that involves the synthesis of neuroprotectins, expression of prosaposin, and NFkB modulation. ROS phagocytosis upregulates these responses, and while initially activating these events, oxidative stress, if persistent, can overcome survival signaling. We will use cell, molecular, and biochemical approaches that include liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS-MS)-based lipidomic analysis. By using primary human RPE cells, ARPE-19 cells, and maculae from rhesus monkeys of various ages, the results may be assessed in the context of both normal function and macular degeneration. We will (1) test the prediction that RPE cell integrity is maintained by growth-factor regulation of the synthesis of NPD1during ROS phagocytosis. These studies will determine how PEDF and other growth factors regulate enzyme-mediated DHA-oxygenation pathways in the RPE and thus contribute to defining mechanisms through which ROS phagocytosis is a trigger of NPD1 synthesis in the RPE; (2) Test the prediction that NPD1 promotes RPE survival during photooxidative damage. A2E and A2E oxiranes (epoxides) are known to accumulate in the aging RPE and to mediate apoptosis in Stargardt disease and AMD. These experiments will specifically test the prediction that A2E-mediated RPE cell apoptosis can be down-regulated by NPD1; (3) Test the hypothesis that NPD1 synthesis is down-regulated in the aging macula. We will study the macula from Rhesus monkeys during aging as well as during ischemia to define its ability to synthesize NPD1 and additional DHA-derived messengers; and (4) Test the prediction that PAF antagonism promotes RPE cell survival. This will include defining the mechanism of prosaposin gene induction in RPE cytoprotection, making use of LAU-0901. We aim for a better understanding of AMD and to contribute to the development of effective therapies.

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, Mentoring, Recruitment and Evaluation Core will provide centralized coordination and oversight to all the activities of this COBRE. The LSU-HSC leads a partnership of four Louisiana institutions in a COBRE on neuroscience research. The Program Director and established scientists are mentors to selected, highly promising junior faculty who execute four research projects that have been designed for the common goal of mentoring to build new nationally competitive research. The scientific focus is on a central issue of neurobiology: to understand the cellular and molecular basis of synaptic plasticity and neuronal survival critical to clarify the pathophysiology of neurological disorders such as stroke, neural trauma and neurodegenerative diseases. This multidisciplinary program involves cellular neurophysiology, molecular biology and behavioral neuroscience. To support the COBRE projects, core resources include facilities for imaging, neurochemistry of lipid messengers and molecular neurobiology. A recruitment plan for years 2 to 5 further benefits the collaborating institutions by actively attracting new research faculty who will work under the guidance of established neuroscientists as mentors. A relatively small administrative core funding is requested. The specific aims to attain these objectives are 1) to promote faculty development through research projects;2) to further develop a critical mass of competitive extramurally funded investigators by the recruitment, start up, mentoring and retention of new faculty members;3) to enhance the infrastructure critical for expanding neuroscience capability in Louisiana by developing three core research modules at LSUHSC;4) to provide scientific and grantsmanship mentoring and strengthen the support network that promotes interactions;and 5) to implement interim and outcome evaluations so as to keep this COBRE program on track. This partnership rests on existing expertise and in our firm decision to build a scientifically successful neuroscience alliance in Louisiana. The four target faculty and the four to-be-recruited faculty are the critical building blocks to achieve these goals. The core resources are vital to the overall success of this consortium, not only in neuroscience but in all the biomedical sciences. The plans for mentoring junior faculty and the recruitment plan will ensure a steady stream of new nationally competitive neuroscientists in Louisiana.

[unreadable] DESCRIPTION (provided by applicant): Our COBRE, "Mentoring Neuroscience in Louisiana: A Biomedical Program to Enhance Neuroscience in an IdeA State," has established a culture of scientific excellence at LSUHSC, Tulane University, and other Louisiana institutions of higher education. Neurosciences research performed in this environment is greatly synergized with respect to both quality and productivity; this new culture is playing a critical role in innovative and fundamentally important research breakthroughs in the neurosciences at our institutions. Recognizing the significance of the mentoring process for developing Promising Junior investigators (PJI) and for strengthening the research infrastructure, we now propose a mechanism to further invigorate the mentoring process in order to solidify a self-sustaining system that will enhance individual and collective research effectiveness. Through our "culture of mentoring" approach, we propose to (1) involve all participating scientists in the COBRE grant in the development of a comprehensive Louisiana Center for Neuroscience;(2) support four main research projects and five pilot projects by PJIs; (3) continue to support and develop five vital Core facilities; and (4) continue to strengthen the quality of neuroscience research in Louisiana. In the context of our scientific focus - Cellular and Molecular Bases of Synaptic Plasticity, Regulation, and Function - the PJIs' projects are designed to contribute insights into dendritic function, neural information processing, and mechanisms of neuroprotection relevant to epilepsy, ischemia-reperfusion, deafness, and neurodegenerative diseases. Our seven COBRE Core resources - Imaging of Cell Physiology, Molecular Biology, Computational Neurosciences, Lipidomics, Biostastitics, Cell Biology/Tissue Culture and Proteomics- are selected to support this scientific focus. Our COBRE mentoring plan - teams of mentors including junior mentors (successful former PJIs) - aims to produce R01 applications by the PJIs within the second year of the award, and is coupled with a recruitment plan to attract new research faculty at the participating institutions over Years 3 to 5. Our 'grantsmanship-plan' is designed to guide the PJIs to become competitive in the application and peer-review process. We view the four target faculty, the four to-be-recruited faculty, and additional PJIs as the critical building blocks to reinforce and energize a strong neuroscience research environment throughout the State of Louisiana. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Public interest has resulted in increased consumption of omega-3 fatty acids as dietary supplements (derived mainly from fish-oil) based upon purported effects that include: reduction in cardiovascular morbidity and mortality, reduction in cerebrovascular disease and stroke, preservation of vision, and slowing of cognitive decline during aging. Over the past several years, there has been a dramatic increase in the scientific scrutiny of omega-3 fatty acids and their impact on health. Results indicate that omega-3 fatty acids have potent anti-inflammatory actions. Inflammation is at the root of many chronic diseases, including coronary heart disease, diabetes, arthritis, obesity, macular degeneration, dry eye, osteoporosis, Alzheimer's disease and mental health disorders, and there is evidence from clinical and experimental studies that omega-3 fatty acids are endowed with preventive and therapeutic benefits. Omega-3 fatty acids (docosahexaenoic acid-DHA particularly), are uniquely concentrated in the central nervous system and are involved in neuroprotection, cognition, and other brain and retinal functions. However, the mechanism of action for omega-3 fatty acids and their significance for public health are not completely understood. In this Grand Opportunity, we have assembled a team of three accomplished investigators from complementary fields to propose the foundation for a new field of investigation -- signaling mechanisms of omega-3 essential fatty acids in inflammatory disease control. We will do this by beginning with a collaborative two-year project focused upon novel omega-3 fatty acid mediators at the stroke penumbra that will bring a new understanding to the mechanism of action of omega-3 fatty acid-derived mediators in vivo and will provide guideposts for further studies on how omega-3 fatty acids can be harnessed to prevent or control chronic processes in other diseases and disorders. Together, the PIs have all the required methods and laboratory resources readily available for immediate project implementation. This Grand Opportunity proposal is a large-scale research project that will accelerate critical breakthroughs on the biology of omega-3 essential fatty acids and create cutting-edge technologies to produce novel lipid bioactive mediators, with a high likelihood of enabling growth and investment in biomedical research and development, public health, and health care delivery. Results will serve as the basis for start-up companies or for deployment in established pharmaceutical companies. Moreover, the project can be accomplished using the proposed two years of funds without continued NIH funding beyond the award period. This project can be viewed as a trans-NIH effort because there will be an impact on NCCAM, NINDS, NHLBI, NIA, NEI and others. The specific research question to be tackled is based upon the innovative concept that the recently identified DHA-derived mediators, neuroprotectin D1 (NPD1) and aspirin-triggered (AT)-neuroprotectin D1 (AT-NPD1), inhibit ischemia-reperfusion mediated leukocyte infiltration and pro-inflammatory gene expression, as well as elicit neuroprotection in the stroke penumbra. Since NPD1 and AT-NPD1 are both produced during reperfusion injury, our plan consists of four interrelated Research and Development Innovation goals, which include defining the potential synergism of these DHA derivatives and related mediators in experimental stroke. The isolation and characterization of these novel mediators will serve as a guide leading to new insights on how omega-3 fatty acids can be used as supplements that, in turn, may be used in preventive medicine. Also, the total organic chemical synthesis of these mediators will be the foundation for the development of novel therapeutic agents that will mimic how the brain attempts to protect itself when confronted with injury. In addition, these mediators may be used as novel omega-3 fatty acid biomarkers throughout life, but particularly during aging and in brain injury conditions. As of yet, the components of the AT-NPD1 pathway have not been characterized, their chemical synthesis has not been performed, and their bioactivity has not been studied. To accomplish our objectives, we will use powerful tandem mass spectrometry, medicinal chemistry, high-resolution magnetic resonance imaging, and in vivo experimental models of stroke in rats and 12/15-lipoxygenase (LOX)-deficient mice. Public Health Relevance: This proposal is based upon the innovative concept that this three-Principal Investigator project will uncover novel signaling that will make a difference in the understanding and significance of omega-3 fatty acids in brain injury and the central nervous system. Identification of the new mechanisms and biomarkers could serve as a guide leading to insights on how omega-3 fatty acids could be used as a supplement that, in turn, may possess preventative actions in chronic inflammatory diseases and stroke.

Abstract The mission of the Administrative Core is to focus efforts towards accomplishing the sustainability goals of our COBRE Transitional Center. As described in the Overall Center Organization and Management Plan, the two major, intertwined, sustainability goals of our Center are to: 1) achieve at least one major P01, P50 or U54 Center award from an NIH Institute, one major Louisiana Board of Regents Award, and an NINDS T32 award; and 2) achieve a critical mass of R01-funded PIs by acquiring new R01s for the remaining COBRE mentees, sustaining existing R01s and achieving new R01s for existing PIs, attracting new faculty who have R01 funding, and attracting new faculty who will rapidly achieve R01 funding. To accomplish these goals, during the COBRE III transitional period, the Administrative Core will provide organizational, financial and facilities oversight to the Core Facilities and to the Research Pilot Project Program. The Administrative Core will manage the day-to-day activities of the COBRE Transitional Center. Although administratively located at LSUHSC, the Administrative Core will stimulate and sustain the involvement of neuroscientists at all of the COBRE current participating institutions - Tulane, Xavier, Nicholls State, LSUHSC - ensuring access to COBRE III Core Facilities, ensuring a level playing field in competition for Research Pilot Project funding, and serving as an advocate for access to needed resources at all sites for all members of the COBRE III neurosciences investigator group. The Administrative Core will also oversee the Strategic Planning and Program Evaluation processes that will ensure our ability to achieve our sustainability goals.

DESCRIPTION (provided by applicant): N-methyl-D-aspartate (NMDA) receptors constitute the major subtype of glutamate receptors and normally participate in rapid excitatory synaptic transmission throughout the CNS. To date, a variety of NMDA receptor subunit proteins (NR1, NR2A-D) have been cloned. Native NMDA receptors appear to be heteroligomeric complexes consisting of an essential NR1 subunit and one or more regulatory NR2 subunits (NR2A-D) and possibly the more recently identified NR3 subunit. Activation of NR2A and NR2B receptor channels are permeable to Na+ and K+ and also to Ca2+, which triggers multiple intracellular catabolic processes, leading to the irreversible death of neuronal cells. Recently, we have used reverse phase nano-LC-MS/MS mass spectrometry to analyze protein components in the NR2B receptor complex from forebrain of mice that had been subjected to sham or focal cerebral ischemia. We have shown that cerebral ischemia recruits death-associated protein kinase (DAPK1) into the NR2B receptor complex. DAPK1 is one member of Ca2+/calmodulin (CaM)-dependent serine/threonine kinase family and functions as a critical mediator of cell death. Whole-cell patch clamp recordings have demonstrated that activation of DAPK1 increases the NR1/NR2B receptor-mediated currents. Subsequently, we have generated genetically modified mice (cdDAPK1), in which catalytic domain of DAPK1 is selectively deleted. We have found that neurons in the forebrain of the cdDAPK1 mutant mice are resistant to ischemic insults. Thus, we hypothesize that DAPK1 physically and functionally interacts with NR2B receptors and this interaction contributes to neuronal injury in ischemic stroke. Proposed studies will address this question. To date, all clinical stroke trials targeting glutamate receptors (AMPA or NMDA) have failed, possibly because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of NR2B receptors by targeting DAPK1. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy. PUBLIC HEALTH RELEVANCE: Stroke is a third leading cause of death in the United States. A critical feature of the disease is selective degeneration of neurons in the brain by activation of glutamate receptor channels. To date, all clinical stroke trials targeting glutamate receptors have, however, failed, because receptor antagonists block the physiological actions of glutamate as well. This proposal describes, for the first time, a molecular approach to selectively block the pathological effects of glutamate receptors by targeting DAPK1 enzyme. Thus, this approach should not affect the physiological actions of glutamate receptors in the brain, thereby defining a promising target for stroke therapy.

PROJECT SUMMARY / ABSTRACT Stroke is a leading cause of death and permanent disability, with an estimated impact on public health of $73.7 billion per year in the United States. Ischemic stroke accounts for over 85% of stroke cases. Therapeutic options for ischemic stroke are limited. Early treatment with recombinant tissue-plasminogen activator (tPA) only benefits a fraction of patients. In addition, this treatment does not target immuno-inflammatory events during stroke. Ischemia-reperfusion damage is associated with dysregulation of multiple neuroinflammatory signaling pathways that causes irreversible damage to neuronal circuits resulting in the pathologies that affect stroke survivors. Our multi-PI team with extensive, complementary, and unique expertise and access to multiple research tools will use a rat model to investigate the efficacy of a novel approach to pharmacologically resolve neuroinflammatory disruptions triggered by experimental ischemic stroke and thus preserve neuronal network integrity and promote neurologic recovery. Our central hypothesis is that blocking pro- inflammatory platelet activating factor receptor (PAFR) together with administration of docosanoids will lead to sustained neurological recovery and protect neuronal circuits in the primary motor cortex after ischemic stroke. Compelling preliminary data support this hypothesis. We have identified a low molecular weight PAFR antagonist, LAU-0901, which will be administered together with the aspirin-triggered (AT) isomer, AT-NPD1 (aspirin-triggered neuroprotectin D1), our lead docosanoid, in the studies proposed for this application. We predict that our new experimental combination therapy that targets mechanisms of motor circuit damage by blocking pro-inflammatory PAF signaling will reduce damage and enhance survival by ensuring the availability of pro-resolving and neuroprotective lipid mediators following middle cerebral artery occlusion (MCAo). We propose two specific aims: 1) To test the hypothesis that combined blocking of pro- inflammatory PAF plus treatment with docosanoids after MCAo will lead to sustained neurological recovery, and 2) Test the prediction that pro-homeostatic lipid mediator pathways are restored by combination treatment for experimental ischemic stroke. The scientific premise of the proposed research is to identify key network processes in adaptive brain plasticity, which may help to predict functional outcome and may also lead to development of therapeutic interventions to support and promote recovery after stroke. This innovative therapeutic approach may also be applicable to the treatment of other neurological diseases with an inflammatory component such as Alzheimer's disease, Parkinson's disease and others.

PROJECT SUMMARY / ABSTRACT Although neuroprotective strategies have shown promise, no treatment has demonstrated efficacy after stroke. This project focuses on the neuroprotective bioactivity of docosanoid (DOC) mediators: Neuroprotectin D1 (NPD1), Resolvin D1 (RvD1), and their combination (NPD1+RvD1) against ischemic and embolic experimental stroke. These lipid mediators are biosynthesized ?on demand? in response to the onset of stroke to resolve neuroinflammation and restore homeostasis. Our preliminary studies show that administration of NPD1 after OGD in neuronal cell cultures modifies clusters of genes and upstream potential master regulators that decrease neuronal apoptosis and neuroinflammation, improve cell homeostasis, and that beneficially impact genes expressed in ischemia-reperfusion. In addition, we show that DOC provide neurological/behavioral recovery, reduce infarct size, increase neurogenesis, and promote cell survival after ischemic stroke. The overall goal of our studies is to uncover a mechanistic understanding of DOC action in MCAo. Our central hypothesis is that DOC foster neuronal and astrocyte integrity by targeting selective gene clusters preceding neuronal protection and by the homeostatic signaling integration regulated by the mesencephalic astrocyte-derived neurotrophic factor (MANF) and by the ring finger protein 146 (Iduna). Specific aim 1 will test the hypothesis that DOC regulate pro-homeostatic and cell survival bioactivity after MCAo by modulating specific gene clusters. We will define the doses and therapeutic window, as well as their effect on the ischemic penumbra and we will define whether the lipid mediators are neuroprotective in embolic stroke with or without tissue plasminogen activator of thrombolysis. We selected genes from our data on neuronal cultures, including, some encoding lncRNAs, and propose to define by RT-qPCR high-throughput microfluidics workflow their expression after MCAo with and without DOC. Specific aim 2 will test the hypothesis that MANF and Iduna enhanced abundance by DOC integrates homeostatic signaling restoration and proliferation of neural stem cells leading to neuroprotection. Both are pro- survival proteins that target different neuroprotective mechanisms. Since NPD1 biosynthesis is stimulated by neurotrophins, we will explore the relationship DHA?DOC (NPD1, RvD1 and NPD1+RvD1) ?MANF?Iduna? protection by unfolded protein response pro-survival signaling outputs. A DOC or combinations will outline outputs of the unfolded protein response driven by MANF and Iduna. Since ischemic stroke engages UPR signaling we will define lncRNAs as regulators and effectors of UPR that fine-tune the output of the stress signaling pathways and identify also which specific gene signatures are MANF and or Iduna dependent. The scientific premise of the proposed studies is that identification of the most effective DOC or combination of DOC to target gene clusters necessary for neuroprotection/neurorestoration modulated by the lipid mediators which will provide the basis for future clinical studies on potential interventions to reduce the immediate and long-term consequences of stroke.