Louis F. Reichardt - US grants

A heparan sulfate (HeS) proteoglycan has been identified on the surface of neurons and shown to be a potent inducer of neurite outgrowth by other neurons in an in vitro assay. The purification and characterization of this factor will be completed using monoclonal antibodies as affinity reagents. Biochemical, immunological, and genetic procedures will be employed to identify the domains on the proteoglycan responsible for retention on the cell membrane, attachment to polycationic tissue culture substrata, and induction of neurite outgrowth. These studies will be facilitated by the current availability of separated HeS sidechain and core protein fractions, monoclonal antibodies to each of these components, and a variant of a neuronal cell line that synthesizes an altered and biologically inactive proteoglycan fragment. Attempts will be made to detect and characterize the receptors on responsive neurons that mediate the action of the HeS-proteoglycan. Antibodies will be used to block the interactions of neurites with this factor in order to determine the role of this proteoglycan in directing neurite growth in vitro and in vivo, assaying neurite extension, fasciculation, association with astrocytes, and development of the Superior Cervical Ganglion. A family of other factors, released by many different cell cultures, also acts as potent inducers of neurite growth in vitro. One of these has also been shown by our laboratory to contain a HeS-proteoglycan. Several factors from different sources will be screened by digestion with different enzymes, precipitation with antibodies to the neuron associated HeS-proteoglycan and competition for receptors to determine whether these factors share a biochemical structure, antigenic determinants, or sites of action on responsive neurons.

This grant proposes a series of related studies on the synthesis and processing of the neuronal trophic factor Nerve Growth Factor (NGF) in sites that are important for neuronal development. Using a cDNA probe encoding the mouse beta-NGF gene, studies will be continued on identifying tissues that synthesize NGF in the adult and during development. We will attempt to identify the cell types that synthesize NGF within these tissues by examining NGF gene expression in defined cell types in culture and by doing in situ hybridization on tissue sections using an NGF gene probe. The NGF probe will be used to quantify NGF mRNA levels during the development of tissues innervated by sympathetic and sensory neurons. We will determine whether there are relationships between the synthesis of NGF mRNA and the induction of sumpathetic neurons, the development and extent of innervation in targets, and neuronal cell death. We will determine in adult tissues whether denervation, already known to increase NGF levels, does so by increasing beta-NGF gene expression. In a second set of experiments, we will examine the form of NGF synthesized in tissues and cells that express the gene using antibodies that are specific for amino acid segments in the precursor. Residues between these segments are cleaved during normal processing in exocrine cells in the male mouse submaxillary gland. We will see whether the same processing steps occur in the tissues and cells believed to be the physiological sources of NGF. Some of these cells are known not to process other prohormones, so the precursor may be important. It will be purified and its properties will be examined. If NGF is processed in physiological sources, we will determine whether proteases are able to process the prohormone in vitro are present at appropriate sites in vivo. Antibodies specific for amino acid residues in the precursor will be used to purify the three polypeptides produced in addition to NGF by processing of the prohormone in the mouse submaxillary gland. These purified polypeptides will be tested for biological actions on NGF-responsive cells and will be used to make antibodies for studies on processing of proNGF. These results should provide information on how NGF, an important trophic hormone, controls the development of responsive neuronal cell populations.

A family of neurite outgrowth-promoting factors has been characterized and each factor has been shown to be a complex of laminin and a heparan sulfate proteoglycan, in which laminin funcitons as a potent inducer of neurite outgrowth in an in vitro assay. The characterization of these factors will be completed, using electron microscopy. Possible reasons why laminin and neurite-outgrowth-promoting factors differ in their sensitivities to antibodies will be studied. The sites on laminin that are required for its actions on neurons will be identified and characterized. Efforts will be made to identify the receptors on neurons that mediate the actions of laminin. In particular, the role of a purified neuronal cell surface heparan sulfate proteoglycan that has been shown to bind laminin will be studied. Using a responsive cell line, PC12 cells primed by growth with NGF, some of the intreacellular changes caused by laminin will be identified and compared to those induced by Nerve Growth Factor. Antibodies to laminin, laminin-heparan sulfate proteoglycan complexes, and neuronal receptors will be used to characterize the role of neurite outgrowth-promoting-factors in mediating neuronal axon outgrowth on substrates and cells that growth cones encounter in vitro. Injections of antibodies into chick will be used to identify possible roles for LN in developing and in regenerating nerves.

A family of neurite outgrowth-promoting factors has been characterized and each factor has been shown to be a complex of laminin and a heparan sulfate proteoglycan, in which laminin funcitons as a potent inducer of neurite outgrowth in an in vitro assay. The characterization of these factors will be completed, using electron microscopy. Possible reasons why laminin and neurite-outgrowth-promoting factors differ in their sensitivities to antibodies will be studied. The sites on laminin that are required for its actions on neurons will be identified and characterized. Efforts will be made to identify the receptors on neurons that mediate the actions of laminin. In particular, the role of a purified neuronal cell surface heparan sulfate proteoglycan that has been shown to bind laminin will be studied. Using a responsive cell line, PC12 cells primed by growth with NGF, some of the intreacellular changes caused by laminin will be identified and compared to those induced by Nerve Growth Factor. Antibodies to laminin, laminin-heparan sulfate proteoglycan complexes, and neuronal receptors will be used to characterize the role of neurite outgrowth-promoting-factors in mediating neuronal axon outgrowth on substrates and cells that growth cones encounter in vitro. Injections of antibodies into chick will be used to identify possible roles for LN in developing and in regenerating nerves.

Neurons interact with several different extracellular matrix glycoproteins which help regulate many aspects of their development. Recent work in vertebrates and invertebrates has ascribed a central role in mediating these interactions to a family of heterodimeric receptors named the integrins. This work has shown that integrins have ligands of several classes: secreted ECM glycoproteins and membrane-associated Ig family members, disintegrins, and at least one cadherin. Work has also shown that integrins regulate many aspects of development including cell survival, cell migration, polarity and differentiation, and organogenesis. The overall goal of this project is to understand the roles of integrins in regulating the development of the nervous system. In this grant, we will continue characterization of a novel class of integrins, the beta8 integrins, which we recently showed formed an unsuspected additional set of neuronal receptors for several ECM proteins. We will continue our extensive work on the beta1 integrin family, characterizing their expression patterns, ligands, and essential roles in vivo as assessed by targeted conditional mutation in mice. We will try to understand in more detail how integrin binding to ligand activates intracellular signalling pathways which lead to growth cone motility, assessing the roles of cytoskeletal linker proteins and an integrin-associated tyrosine kinase. We will try to understand the interactions between the NGF and integrin signaling pathways which result in dual regulation of neurite outgrowth by neurotrophins and ECM proteins.

DESCRIPTION (provided by applicant):To train predoctoral students in Neurobiology, 52 cooperating faculty propose continuation of broad, interdisciplinary predoctoral training program with approximately 63 total students in fall, 2001. These faculty members have research interests in molecular, cellular, developmental, systems, and medical neurobiology. They have appointments in several basic science and clinical departments with fully equipped, funded laboratories, mostly on the main UCSF campus at Parnassus Heights. We anticipate approximately 10 additional faculty will join our graduate program during the next five years. An annual retreat, a weekly seminar series, a weekly student-faculty journal club, and student journal/pizza club meetings will promote interactions benefiting trainees. Neuroscience training will be conducted within the broader context of the Boyer Program in Biological Sciences (P.I.B.S.), a consortium of 7 graduate programs with 185 total faculty. A weekly P.I.B.S. journal club, plus seminar series and retreats sponsored by the other P.I.B.S. programs will promote the broader education of our students and their interactions with the faculty and students of these programs. During the next 5 years, the number of our students at the beginning of each academic year is anticipated to be 60-70, increasing slowly from present size. We will continue major efforts to recruit under-represented minorities. The major selection criteria for all trainees will be originality, commitment and scientific potential. In the 1st year, students will take core courses in Neurobiology, Neuroanatomy, and Cell Biology and will rotate in 3 laboratories that will introduce them to major Neuroscience research areas. Later students will take at least four advanced seminar/discussion courses, exploring in depth subjects of particular importance. These will provide students will analytical skills, current knowledge and experience in making formal and informal scientific presentations. Near the end of the first year, trainees will chose Ph.D. thesis research mentors. During the 2nd year, the proposed thesis project will be described in a written proposal. During a qualifying examination, the importance, scope and feasibility of this project will be defended. The exam will also test knowledge and analytical abilities in broader areas of Neuroscience. Training in later years will focus on the research required for a Ph.D. thesis, but will be supplemented by seminars, conferences, and journal clubs. Our goal is to facilitate the intellectual growth and development of scientific skills of new investigators committed to solving major problems in Neuroscience and to making positive contributions to education in the 21st Century.

Funds are requested to support the Gordon Conference on Cell Contact and Adhesion. This conference first met in 1973. It has convened every other year since that time. All together there have been 10 sessions. Each session has been highly oversubscribed, thus demonstrating a sustained interest over the past years in this general field by the scientific community. The 1993 conference has been organized to avoid as much as possible an overlap in topics and speakers of other related conferences. The topics emphasize areas of recent progress and are of interest to a diverse community of scientists including molecular biologists, cell biologists, develop- mental biologists, neurobiologists, oncologists, hematologists, plant biologists, and others. The topics covered will examine the molecular mechanisms controlling cell interactions in several interesting biological systems. They will include discussions of specific cell surface receptors, such as those in the integrin, immunoglobulin, cadherin and selectin families. Adhesion mediated-cell signalling, cell motility, and cytoskeletal interactions will be discussed. Several speakers will discuss the roles of cell interactions in mediating developmental processes in vertebrates, invertebrates, and plants, such as fertilization, cell determination and organ differentiation. Other speakers will discuss their roles in cell division, migration, and regulation of gene expression. Finally, cell contact influences on infection, cancer, and other diseases will be considered. The conference aims to provide scientists and students with an overview of the field's recent advances, current status and future opportunities.

In this grant we will investigate synaptic function and plasticity in projects which depend on shared expertise of the principal investigators and a shared mouse transgenic core. Edwards will characterize transporters important in synaptic vesicle and nerve terminal function. He will identify sequences important in targeting catecholamine transporters to their appropriate organelles, characterize a novel family of vesicular glutamate and GABA transporters, characterize functions of a phosphate transporter, and examine the phenotype of mice deficient in this protein. With Copenhagen, Stryker and Malenka he will study its functions in the retina, visual cortex and hippocampus. Copenhagen will examine regulation of Ca2+ and exocytosis in rod and cone photoreceptors and in retinal ganglion cells, determining the roles of dopamine and, using homozygous mutant or genetic chimeric mice, examining roles of second messenger pathways and neurotrophin signaling. Copenhagen and Reichardt will characterize roles of neurotrophin signaling in retinal development using genetic chimeras. Stryker will use these mutant and genetically chimeric mice to determine the roles of neurotrophin signaling and second messengers in regulating plasticity during the ocular dominance critical period. Malenka will study CA3 LTP. With Copenhagen, he will examine changes in synaptic vesicle exocytosis using the membrane probe FM1-43. With Edwards, he will examine time courses of changes in cAMP and PKA activity and will determine whether proteins implicated in regulated exocytosis are phosphorylated during establishment of LTP. Using mutant mice he will determine how deficiencies in synaptic vesicle and membrane proteins affect CA3 LTP. Reichardt and Copenhagen will study the role of neurotrophin signaling pathways in modulating exocytosis at the developing Xenopus neuromuscular junction and on exocytosis in PC12 cells. Roles of neurotrophin-activated signaling pathways will be defined by mutagenesis of trk receptors and introduction of mutated molecules which act as dominant negative regulators of downstream signaling molecules. Using chimeric synaptic proteins tagged with Green Fluorescent Protein, Reichardt will determine effects of electrical activity and neurotrophins on establishment by retinal ganglion cell axons of synapses in the Xenopus optic tectum. The transgenic core will be used to generate mutant mice and genetically chimeric mice for these studies, using ES cell technologies and the lox-cre recombination system.

The overall goal of this work is to delineate the roles of neurotrophic factors in regulating development, differention, and function of the nervous system. During the past few years, we and others have generated targeted mutations that result in deficiencies in neurotrophins or neurotrophin receptors. Our laboratory has also generated mouse strains where athe coding region of a neurotrophin gene is replaced by lacZ, providing a convenient and sensitive assay for neurotrophin expression within individual cells. In addition, we have prepared function-inhibitory antibodies to some of these molecules which have been invaluable for immunocytochemical studies of their distributions and tests of their functions. In one project, the roles of the neurotrophins will be examined using mice with targeted mutations in and mice expressing ectopically individual neurotrophins. The distributions on the single cell level of neurotrophins in sensory ganglia and the hippocampus will be examined using the lacZ replacement strains of mice and these distributions will be compared to those of the neurotrophin receptors trkA, trkB, and trkC. Consequences of neurotrophin deficiency on neuronal precursor cell proliferation, neuronal fate determination, target innervation pattern, and acquisition of differentiated phenotype will be examined in sensory ganglia and the hippocampus. To avoid pleitrophic effects of neurotrophin deficiency, methods designed to target deficiencies spatially and temporally will be attempted. In the first, injection of Fab fragments of function-inhibitory anti-trkA, B, or C IgG will be used to assess effects of receptor inhibition at key developmental times. In a second approach, Adenovirus vectors will be used to express dominant-negative trk mutants in a few neurons. In a third, efforts will be made to generate conditional mutations by introducing loxP sites flanking essential trk receptor exons. Introduction of cre recombinase by virus or mating to a transgenic mouse strain will be used to eliminate neurotrophin or neurotrophin receptor in defined subsets of neurons at controlled times. This should make it possible to examine consequences of deficiency in a few cells on differentiation and/or function in an otherwise normal environment. Finally, we will extend initial observations indicating that hippocampal neurons express several integrin subunits, expression of some of which are strongly regulated by glutamate receptor activation. We will try to completely characterize athe repertory of integrin subunits expressed by hippocampal neurons. We will examine regulation of mRNA and protein expression by the neurotrophic factors in vitro and in response to neurotrophin deficiency in genetically altered mouse strains in vivo. Effects on expression of agents that affect glutamate receptor activation or LTP will be examined. Investigation of these molecules is potentially important because they are strong candidates to help mediate longer term responses to synaptic activity, involving sprouting, retraction of processes, and other structural changes.

The major objectives of this project are to identify the cell types that require TrkB-mediated signaling for normal development and synapse formation and function in the retina, cerebellum and neocortex. We will continue studies that have documented deficits in synaptic communication from photoreceptors to the inner retina in the absence of trkB signaling, attempting to determine which cells in the retina must express trkB for normal development and function of photoreceptors. We will also characterize the molecular changes that result in the observed synaptic deficit. Within the cerebellum, we will characterize the phenotypes caused by absence of TrkB signaling on development of Purkinje cell spines and expression of GABAergic proteins and development of synapses by Golgi interneurons. Again, we will identify the cells where absence of trkB results in these phenotypes and will attempt to identify proteins and mRNAs altered by absence of TrkB signaling that are candidates to explain these phenotypes. We will characterize further the progressive deficit observed in the cortex of mice in which TrkB has been eliminated from the majority of cortical pyramidal cells. We will determine whether it becomes more and more progressive as animals age, determine whether there are additional secondary effects on neurons not targeted directly, and will try to identify proteins and mRNAs known to be involved in dendrite formation that depend upon TrkB signaling for normal expression. Receptor tyrosine kinase signaling obviously mediates many diverse actions in the central nervous system. It is hoped that studies on this one important tyrosine kinase will result in conclusions applicable to understanding the roles of other tyrosine kinases in addition to the Trk receptors in regulating neuronal development, function, and aging in the central nervous system in both health and disease.

The majority of projects in this program project grant will rely heavily on transgenic mice, some of which have already been generated and others of which need to be generated. During the past five years, existence of this core has greatly facilitated the ability of the laboratories directed by Rob Edwards, David Copenhagen, Louis Reichardt and Michael Stryker to exploit this powerful technology. During the next five years, we anticipate expanded use of this core that will almost certainly be used by all members of our new program project team. The existence of a core has made it possible to hire a senior specialist with more than 15 years of relevant experience in preparing transgenic mice plus a junior technician able to manage the mouse colonies and perform routine genotyping, etc. The former person has helped our laboratories generate more than a dozen transgenic lines and half a dozen knockout strains. The more junior technician has been particularly valuable in helping the labs entering this field. All laboratories will continue to benefit from a transgenic core with a person dedicated to performing well certain tasks of critical importance in generating such animals. To help culture and screen ES cells, provide transgenic animals with targeted mutations and provide help in training postdoctoral fellows and graduate students in this important technology, we propose to maintain a modestly-sized and staffed core facility with a cell culture facility for embryonic stem cells, equipment for injection of single cell embryos and blastocysts, and a mouse colony with fertile and vasectomized males and fertile females for use as single cell embryo or blastocyst donors and pseudopregnant recipients. The Reichardt laboratory will provide the supervision and expertise for setting up and managing this facility under the overall guidance of the Principal Investigator.

DESCRIPTION (from applicant's abstract): We are requesting funds to help defray the major expenses of the fourth Neurotrophin Gordon Conference, to be held in June, 1999. The goal of the Neurotrophin Gordon Conference is to provide a biannual forum to foster the exchange of new information about neurotrophic factors and to provide a forum for the dissemination of new tools and methodologies for their study. Neurotrophic factors are polypeptide growth and differentiation factors that influence the properties and functions of neurons and non-neuronal cells within the nervous system. During vertebrate development they play essential roles in regulating survival, proliferation, differentiation, axon growth and guidance and' synapse formation by neural cells, In the mature nervous system, they continue to function by regulating expression of neurotransmitters, receptors and ion channels as well as by modulating synaptic efficacy and connectivity. They have been shown to protect neurons from a variety of toxic insults and in animal models of human neurodegenerative diseases, raising the possibility that they can be used as therapeutic agents. In the past, studies on these factors have had major conceptual impacts on developmental and cell biology and they continue to motivate and guide much modern research in developmental biology. While the initial definition of neurotrophic factors focused on the functions of Nerve Growth Factor and closely related proteins named neurotrophins (NGF, BDNF, NT-3 and NT-4), more recently this definition has been broadened to include other families, including the neuropoietic cytokines, such as CNTF and LIF, and members of the TGFbeta superfamily , such as glial-derived neurotrophic factor and neurturin. The vast majority of neurotrophic factors and their receptors have only been identified and cloned in the past decade. Their biological functions are being intensively studied in vitro and in vivo by neuroscientists, cell and molecular biologists, and physiologists. Discovery of novel factors continues to be an active and productive endeavor. Novel strategies for discovery and functional characterization have been made possible by new technologies and will be discussed. The expanded list of functions attributed to these factors has required scientists to either adapt existing methods or develop new methods. This meeting provides a forum to facilitate interchanges between scientists familiar with the problems and those with new or unfamiliar methodologies. In addition to their impact on basic science, the past decade has witnessed an explosion of research, carried out in both universities and biotechnology companies, aimed at using these factors to alleviate symptoms of diseases of the nervous system. While not invariably successful, efforts have been made to utilize these factors in experimental treatments of Alzheimer's Disease, Amyotropic Lateral Sclerosis, and Parkinson's Disease. Trials examining possible beneficial roles for these factors in treatment of sensory neuropathies and controlling pain are far advanced. An effort will be made in this Gordon Conference, as in past ones, to ensure that attendees are educated in possible clinical applications of their work and have the opportunity to interact with scientists whose major research focus is on developing therapeutic applications. In summary, knowledge obtained about the biological activities of neurotrophic factors and mechanisms of signal transduction mediated by their receptors will lead to greater understandings of the development, function, and repair of the mammalian central and peripheral nervous systems. This meeting seeks to help advance this work by providing a forum for exchange of new insights and information.

This application requests funding for the support of invited speakers for the Gordon Conference on Basement Membranes. This will be the tenth in a highly successful series of conferences, which have become a major international forum for the dissemination of new ideas and information about the structure and biological functions of basement membranes (Bms). These are complex, three-dimensional, extracellular structures at the interface of epithelial and mesenchymal cells and enveloping mesenchymal cells. They play important roles in the organization and function of most tissues and organs, e.g. blood vessels, lung, kidney, skin, peripheral nerve, and muscle. For example, basement membranes facilitate the migration and organization of cells in the nervous and musculoskeletal systems. Mutations in genes encoding basement membrane components or their receptors are associated with severe inherited disorders in humans (e.g. epidermolysis bullosa of skin, congenital muscular dystrophy, Alport's syndrome of kidney). Acquired defects in basement membranes contribute to the pathogenesis of diabetic microvascular disease, and serve as entry sites for infectious agents, such as leprosy, or metastatic cancer cells. Traditionally, the conference has attracted scientists from a wide range of fields in basic research, including protein and carbohydrate structure, gene expression, cell and developmental biology, and neurobiology. In addition, it has been attended by clinicians and scientists involved in translational research or treatment of human disorders involving BM components of lung, blood vessels, skin, kidney, bone, muscle or the immune system. Basic studies of BM degradation and turnover also interest scientists investigating dynamic processes such as embryo implanatation, involution of the mammary gland or uterus, or cancer metastases. In recent years there has been substantial attendance and interest from clinicians and scientists in the pharmaceutical and biotechnology industries studying the role of Bms in wound healing, nerve regeneration, inflammation and tissue repair. The Conference will present a diverse mixture of sessions on the basic science of basement membrane and extracellular matrix (ECM) structure, biosynthesis, assembly turnover and functions. Comparative studies of BM function in vertebrates and invertebrates, and the role of BM and ECM in embryonic development will also be incorporated into the program. Recent work on mechanism by which BMs affect intracellular signaling pathways, regulate cell survival, and interact with polypeptide growth factor-activated signaling paths will be features of the meeting, as these studies have broad implications for understanding normal function and disease processes. In addition, emphasis will be given to studies on the genetics of BM and ECM function, the characterization of human BM diseases, and the generation of animal models for these diseases.

DESCRIPTION (provided by applicant): Cells in the nervous system interact with several different extracellular matrix proteins that regulate many aspects of their development. Neural development is also regulated by many signaling molecules that are associated with the extracellular matrix. Work over the past decade has shown that integrins mediate many of these interactions. Integrins bind ligands of many classes: extracellular matrix proteins, Ig-family members, disintegrins, a cadherin, proteases and protease inhibitors, and growth factors. Integrins have been shown to regulate many aspects of development including cell proliferation and survival, polarity, differentiation and organogenesis. In the mature nervous system, they have been implicated in the regulation of synaptic function and plasticity. Integrins interact with the cytoskeleton and also regulate signaling through many cytoplasmic pathways including the ERK and Jun kinases, PI-3 kinase, and the cdc-42/rac/rho family of G proteins. The overall aim of this grant is to understand the mechanisms by which integrins and integrin-regulated signaling pathways exert their effects on development of the nervous system. Much of our effort will be focused on an understudied class of integrins, the B8 integrins. We have recently shown that these are essential for normal vascularization of the nervous system. We will try to identify the cellular and molecular pathways that this integrin family regulates to control maturation of the brain vasculature. We will attempt to identify the ligands with which it interacts in the CNS, both inside and outside of the cell. We will determine whether it controls activation of TGF-beta in vivo as has been demonstrated in vitro and will try to determine whether deficits in TGF-B signaling account for the phenotype of the B8 integrin mutant. A foxed B8 integrin allele will be introduced into mice and used to characterize other functions of this integrin in the nervous system. We will also extend our current work on the B1 integrins that, using a foxed allele, we have recently shown are essential for normal formation of the cortex and cerebellum. We will attempt to characterize the signaling pathways controlled by this family of integrins that mediate their effects on normal brain development. In particular, using genetic techniques, we will focus on the role of Focal Adhesion Kinase (FAK) and the signaling pathways controlled by FAK, since FAK is a central mediator of signal transmission by the integrins that have been implicated in cortical development. Among other aims, we will determine whether the essential functions of FAK require its kinase activity or only its function as a scaffold that mediates binding to other signaling proteins.

DESCRIPTION (provided by applicant): We will investigate development, function and plasticity of the synapse in projects that depend on sharing expertise between the principal investigators and upon a shared mouse transgenic core. Edwards and Copenhagen will characterize the molecular pathways controlling the cycling of glutamate, the major excitatory transmitter in the CNS. They will identify the transporters involved in glutamate uptake into glia in the brain and retina, test genetically the role of glutamine synthetase in converting glutamate to glutamine, and examine the roles of glutamine transporters in controlling release from astroglia and reuptake by neurons of glutamine. Edwards will extend his studies to identification of substrates for two additional putative transport proteins that have been shown genetically to be important regulators of synaptic function--the synaptic vesicle constituent SV2 and a mammalian homologue of Drosophila diphthong first identified genetically in the laboratory of Graeme Davis. Identification of their substrates should help characterize the biochemical pathways that are disrupted by their absence. Nicoll will characterize the molecular mechanisms that underlie establishment of long-term potentiation at mossy fiber-pyramidal cell synapses in the hippocampus. He will examine the roles of presynaptic kainate acid receptors, A1 adenosine receptors, and the hyperpolarization-activated nonselective cation channel Ih in expression of LTP at this synapse. Copenhagen, Reichardt and Stryker will characterize essential roles for neurotrophin signaling in synapse establishment, function and plasticity in the retina, cerebellum, and visual cortex. They will identify the cells that require TrkB signaling for synapse development and/or function and will characterize the effects of this signaling pathway on structures of axons and dendrites in addition to those of synapses. Stryker will also characterize the roles of tyrosine kinases and proteins involved in Ca2+-mediated and cAMP-mediated signaling by examining the development of cortical topography by imaging receptive field properties in mutant mice. Dr. Davis will characterize pathways that regulate glutamate receptor clustering and postsynaptic differentiation in Drosophila, focusing on the roles of the protein kinase PAK, the adapter molecule DOCK/NCK and proteins involved in their signaling pathways.

DESCRIPTION (provided by applicant): The overall aim of this grant is to understand the role of a novel extracellular matrix protein named nephronectin in kidney development. In previous work, we have shown that the vast majority of mice lacking the integrin a8 subunit fail to develop the metanephric kidney with the initial phenotype of a deficit in ingrowth of the ureteric bud into the metanephric mesenchyrne, the site of expression of this integrin. We have identified nephronectin as an extracellular matrix protein expressed in the ureteric bud that is associated with the integrin a8bl and is distributed appropriately to activate its ligand-dependent signaling pathways. Because the phenotype during metanephric development of mice lacking a8bl is very similar to those of mice lacking any of the constituents of the GDNF-GFRal-c-ret signaling pathway, where the ligand GDNF is expressed in the mesenchyme and the receptor subunits GFRal and c-ret are expressed in the ureteric bud, we will examine possible interactions between these two signaling pathways and test the hypothesis that absence of a8bl results in reduced efficiency of signaling through this pathway. We will identify the receptors that mediate interactions of cells with nephronectin and determine which of the cell-types in the developing kidney interact directly with this ECM protein. In previous work, ligand engagement of integrins by extracellular matrix constituents has been shown to regulate many aspects of development including cellular proliferation, migration, differentiation and survival through interactions with the cytoskeleton and by activation of cytoplasmic signaling pathways including the ERK and Jun kinases, PI-3 kinase, and the cdc-42/radc-rho family of G proteins. We will attempt to determine which of these pathways are activated by nephronectin-engagement of a8bl and which are compromised by the absence of a8bl in the metanephric mesenchyme.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests partial support for an international meeting on Molecular and Cellular Neurobiology as part of a Gordon Research Conference series to be held at the Hong Kong University of Science and Technology June 8 to 12, 2008. The overall goal of this conference is to increase our understanding of fundamental mechanisms that control development and function of the nervous system in health and disease. In particular we wish to promote scientific interactions between American and Asian scientists to advance this objective. For this purpose, we have identified 30 speakers, including two Nobel Laureates, who will deliver presentations on recent developments in their laboratories to approximately 160 attendees. We have also reserved several time slots for presentations by less established scientists with speaker choice to be based upon submitted abstracts. Poster sessions will provide an opportunity for every attendee to present his/her work. First organized in 1998, this conference has provided a unique bridge between American and Asian neuroscientists. Although initially, much of the information transfer was unidirectional, the interchange is now much more balanced. China, in particular, has initiated scientific efforts in some areas that are unlikely to be duplicated in the United States, such as a massive forward genetic transposon mutagenesis project in mice. This conference will provide an opportunity for leading American and Asian scientists to meet and exchange ideas and hypotheses and establish collaborations. Research into mechanisms of brain function is essential if our society is to conquer neurological diseases that afflict a large portion of our citizens, including autism and mental retardation in children, addiction and mental illness in children and adults, and neurodegenerative diseases that are most prevalent in our senior citizens. The conference covers a broad range of neuroscience from molecules and cells to circuits, behavior, mental illness and neurodegeneration. The conference is directed at communicating exciting new developments in these areas and stimulating discussion among participants from different disciplines and nationalities that will accelerate our efforts to understand mechanisms that control human brain development and function. This conference will identify issues in basic brain research that are limiting our ability to address effectively diseases and disorders that affect brain function. Speakers at the conference will describe recent progress in studies important for understanding afflictions that include autism, mental retardation, addiction, mental illness, and neurodegenerative diseases. The conference venue will provide an opportunity for interactions between scientists of many nationalities who otherwise have few opportunities for exchange of ideas and establishment of collaborations. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We will characterize mechanisms through which TrkB regulates inhibitory synapse formation and maintenance in the cerebellum with the expectation that the insights obtained will prove important for understanding the role of TrkB in many regions of the brain. Extending our prior demonstrations that TrkB controls inhibitory synapse formation throughout the cerebellum and has important pre- and postsynaptic cell- autonomous roles, we will use high resolution stochastic optical reconstruction microscopy (STORM) imaging to characterize the localizations of inhibitory synapse-associated cell surface and synaptic scaffold proteins and determine the effects of TrkB activation and inhibition on their presence at the synapse. Extending our recent observation that adult TrkB activity is required to maintain inhibitory synapses and that reactivation of TrkB signaling after earlier inhibition results in reappearance at the synapse of many synaptic proteins, we will examine the effects of adult TrkB inhibition and reactivation on the molecular composition of these synapses. Using cell culture we will examine in more detail the appearance and disappearance of proteins associated with the synapse following TrkB activation and inactivation. We shall determine whether TrkB functions in part through control of protein synthesis or turnover. We shall also examine the effects of TrkB activity on the kinetics of gephyrin stability, insertion and removal a synaptic sites in cell culture. Finally, we more critically examine our model that TrkB acts in par through control molecular assembly of the proteins that form the synaptic scaffold. We will determine the effects of TrkB activation and inactivation in vivo and in vitro on the distribution f gephyrin and other postsynaptic scaffold proteins in detergent soluble and resistant fractions, the interactions of these proteins with binding partners and on phosphorylation and other post-translational modifications.