John L. Bixby - US grants

The goal of the proposed experiments is to understand, at a molecular level the role of the target muscle in promoting differentiation of the presynaptic neuron. Motor neurons in culture with muscle grow and contact the muscle, synthesize synapse-specific proteins, and concentrate these proteins at sites of nerve-muscle contact, mimicking processes that occur during embryonic development. A quantitative assay for the synaptic vesicl protein p65 will be developed, and used to test effects of muscle on the regulation of specific protein synthesis. Second, specific antibodies to cell adhesion molecules and extracellular matrix protein receptors will be used to test the role of these proteins in triggering synaptic differentiation. Third, monoclonal antibodies will be raised that recogniz embryonic or denervated but not adult innervated muscle, to find novel molecules that may be involved in synaptogenesis. Finally, the intracellular events involved in presynaptic differentiation will be examined by focusing on perturbations in common intracellular "second messengers." An understanding of the molecules and processes involved in the normal differentiation of synapses is important in order to understand how these processes go awry in disease states. Besides being directly valuable for understanding motor neuron trophic disorders (such as amyotrophic lateral sclerosis) and neuromuscular diseases (such as Eaton- Lambert syndrome), the information obtained will bear in general on developmental disorders of the brain and spinal cord.

Dr. Bixby will examine the molecular basis of neuronal process growth by addressing two questions: First, what cell surface and extracellular matrix molecules act as growth and guidance cues for axons? Second, what are the intracellular (second messenger-related) events induced by these extracellular molecules that influence axonal growth? To answer the first question, a tissue culture system will be employed in which axons grow on sections of embryonic limb. Antibodies to extracellular matrix and cell surface adhesion molecules will be added to the cultures to determine which molecules specifically affect axonal growth. To answer the second question, neurons will be grown on artficial substrates consisting of certain purified substrate molecules. Axon growth will be assayed in the presence and absence of pharmacological agents that specifically affect the function of particular intracellular message systems. These experiments will provide evidence relating to the cascade of events stimulated by the binding of extracellular matrix and adhesion molecules to their neuronal receptors. The results will enhance knowledge of both the extrinsic regulatory molecules that influence growth during development, and the intracellular signaling systems triggered by these regulatory molecules.

9309526 Bixby A critical feature of the development of the nervous system is the ability of individual nerve cells to grow cellular processes, or axons. These axons often must grow over long distances over complicated pathways to contact their target cells. This research is aimed at an understanding of the molecular signals underlying axon growth. In the previous grant period, it was found that one important mechanism regulating axon growth involves the modification of proteins in the axons by enzymes. In particular, a rare class of enzyme that phosphorylates proteins on tyrosine residues appears to be involved. Several distinct members of this class of enzyme are localized to the exact region of the growing axon that must make "pathfinding" decisions further pointing to the importance of this class of enzyme. At this point, it is unclear which enzyme performs which function in axonal growth and guidance. The present study is aimed at understanding this issue. This study will test the role of individual kinases and phosphatases in axonal growth and other aspects of development. Both known and novel enzymes have been identified in the early developing nervous system. Several of these kinases and phosphatases will be tested functionally, using the techniques of molecular biology. Finally, the interaction of these intracellular signaling molecules with neuronal receptors mediating cell-cell recognition will be examined biochemically. The results of these experiments should allow a more precise knowledge of the molecular basis of cell-cell communication in the developing nervous system, and an elucidation of the intracellular signals that regulate the ability of nerve cells to extend long process to reach their target cells. *** : ; <> ? A B C D E F G H I J L M N O P Q R S T U V W X Y Z \ ^ ` a b c d e f g h i j k l m n o p q r s t u v w x y z { | } ~ 9309526 Bixby A critical feature of the development of the nervous system is the ability of individual nerve cells to grow c o q j o q ! ! F q q ( Times New Roman Symbol & Arial " h e C @ bixby William Proctor, IBN William Proctor, IBN

The long-term goal of this project is to develop a molecular description of the action of N-cadherin and laminin (LN), representatives of two important classes of proteins that promote axonal growth during development and regeneration. The goals of this proposal are to elucidate the role of Ca2+ signals in neurite growth induced by N-cadherin and LN, clarify the relationship between the Ca2+ signals induced by N-cadherin and those induced by LN, and begin to delineate the role of other intracellular signalling systems in the induction of neurite growth. A strength of the experimental approach is the combination of high resolution single cell imaging, patch clamping, and molecular biology/biochemistry. First, both soluble and bound forms of N-cadherin and LN will be used to effect Ca2+ signals in cultured neurons, and various manipulations and pharmacological reagents will allow the definition of the sources of Ca2+ involved in the induced responses, both in neurons and in non-neuronal cells (where the pathways are expected to differ). Secondly, experimental alterations of the Ca2+ signals, as well as of other second messenger pathways hypothesized to be involved in growth signalling, will be performed for neurons growing on substrates of LN or N-cadherin or neurons contacting artificial LN or N-cadherin "guideposts". These experiments will allow a determination of the relationship between the messengers and the induction of neurite growth. Finally, cultured neurons and a preparation of isolated embryonic growth canes will each be used to assess the binding interactions of receptors for N-cadherin and LN, and oocytes expressing N-cadherin via injected mRNA will be used to study the function of N-cadherin as its own receptor. The results obtained in this project will provide important information concerning the molecular mechanisms underlying the regulation of axon growth by substrate-bound growth promoting proteins. In addition, they will provide insight into the signaling pathways employed by cadherins and integrins, two classes of cell surface receptor found throughout the developing and adult vertebrate body. A detailed understanding of the normal control of axon growth is necessary in order to understand developmental disorders in which abnormal axon growth is implicated (microcephaly, anencephaly, mental retardation, megacolon, etc.), as well as the regulation of axonal regeneration. Further, advances in our knowledge of intracellular signalling pathways used by cadherins and integrins in neurons is relevant to a broad array of developmental disorders of the nervous system (e.g., spina bifida).

DESCRIPTION The long term goal of the proposed research is to understand the molecular mechanisms underlying motor nerve terminal differentiation. Regulated differentiation of nerve terminals is essential for the formation of appropriate connections in the developing nervous system. One important signal leading to terminal differentiation appears to come from the extracellular matrix protein agrin, previously known as an inducer of postsynaptic differentiation. Agrin can induce selective adhesion of motor neurons, inhibit outgrowth of motor neurites, and promote clustering of synaptic vesicles; these properties are expected of a presynaptic inducer. Inhibition of agrin function, in vivo or in vitro, leads to a failure of presynaptic differentiation. The proposed research is designed to answer several questions about agrin's role in presynaptic differentiation. First, how does agrin regulate motor neurons' growth and differentiation. The activities of neural vs. muscle agrin on motor neurons will be compared using transfected muscle cells and transgenic mice. Ca2+ signals induced in motor neurons by agrin will be characterized, and the relationship between these signals and agrin's activities will be examined. Second, what domains of agrin are required for interactions with neurons? Agrin isoforms and fragments will be used to determine the structural basis of agrin's actions on neurons. Finally, what are the neuronal receptors for agrin? The binding properties and localization of neuronal agrin binding proteins will be characterized, as a first step in the identification of functionally relevant neuronal agrin receptors. Additionally, the potential for various cell adhesion molecules to act as agrin receptors will be examined. The proposed experiments will provide key information concerning the mechanisms underlying nerve terminal formation by focusing on agrin, the single best candidate for a target-derived inducer of presynaptic differentiation. Information about the molecular basis of synaptogenesis may be useful in the diagnosis and treatment of developmental disorders of the brain and spinal cord. In addition, these results will bear on the mechanism of regeneration after nervous system injury.

DESCRIPTION (Verbatim from the Applicant's Abstract): The regulation of tyrosine kinase phosphorylation is critical for the growth and guidance of axons. Evidence suggests that the CAM-like receptor-tyrosine phosphatases (RPTPs) play key roles in the signaling processes underlying axon growth. This project tests the hypothesis that RPTPs, expressed on the surface of developing neurons regulate axon growth during embryogenesis, through the binding of specific ligands to their extracellular domains. The focus here is on 2 specific RPTPs -- CRYP-2 and PTP-deltadelta -- that are excellent candidates for involvement in the regulation of axon growth. Evidence implicating these two RPTPs includes structure, expression pattern, and preliminary analysis of function in vitro. The major questions concerning these RPTPs are: What are the regulatory ligands for CRYP-2 and PTP-delta?, and How are CRYP-2 and PTP-delta involved in axon growth? These questions will be addressed through correlation of expression of ligands for CRYP-2 and PTP-delta with expression of the RPTPs themselves, characterization of these ligands, and examination of the function of CRYP-2 and PTP-delta in axon growth, both in vitro and in vivo. There are three specific aims. First, potential regulatory ligands for the RPTPs will be localized, and identified through immunoprecipitation, affinity chromatography and expression cloning. Identification of ligands will allow key biochemical experiments on adhesive and signaling functions of these RPTPs. Second, the extracellular domains of the RPTPs will be used as regulators of axon growth and guidance in vitro, in an analysis of the mechanisms involved in this regulation. These results will provide detailed mechanistic evidence concerning how these RPTPs function. Finally, two different in vivo assay systems (retroviral expression of mutants and gene knockout) will be employed to assess the role of these RPTPs in neuronal development during embryogenesis. These assays will provide direct tests of the hypothesis that the specific PTPs under study are functionally linked to axon growth and guidance in vivo. The proposed experiments will move the field closer to a molecular understanding of the regulation of vertebrate axon growth. This understanding is critical for rational approaches to developmental disorders of the nervous system, as well as the problem of spinal cord regeneration.

DESCRIPTION (provided by applicant): The regulation of tyrosine phosphorylation is critical in the growth and guidance of axons during the development of the vertebrate nervous system. Accumulating evidence suggests that receptor-type tyrosine phosphatases (RPTPs) play key roles in the signaling processes underlying axon growth. Several major classes of RPTP possess extracellular domains (ECDs) that are structurally similar to those of cell adhesion molecules (CAMs). Our overall hypothesis is that these CAM-like RPTPs, which are expressed on the surfaces of developing neurons, are involved both as regulatory ligands and as neuronal receptors in the regulation of vertebrate axon growth. Work in the previous granting period has established that 2 vertebrate RPTPs, PTP-delta and PTPRO, are strong candidates for axon growth regulation. In vitro, the ECD of PTP-delta is a neurite-promoting neuronal cell adhesion molecule that mediates attractive growth cone steering. PTP-delta can bind homophilically, and may be both a regulatory ligand and a neuronal receptor for these attractive cues. In contrast, the ECD of PTPRO is a neurite-inhibitory repulsive guidance cue in vitro. Knockdown of PTPRO or of PTP-delta expression in embryonic motor neurons leads to changes in axon growth. This proposal is designed to build on these observations. In Aim 1, site-directed mutagenesis will be used to determine the regions of the PTP-delta and PTPRO ECDs responsible for functional binding. In Aim 2, chimeric receptor production, neuronal transfection, and growth cone migration assays will be used to identify and characterize the receptor functions of PTP-delta. In Aim 3, biochemical and functional approaches will be used to characterize PTPRO/Trk interactions and characterize novel PTPRO substrates. Finally, Aim 4 will use both the mouse and chick systems to characterize the in vivo roles of PTP-delta, PTPRO, and other CAM-like RPTPs in specific vertebrate axon guidance decisions. Our experiments will move the field closer to a molecular understanding of the role of tyrosine phosphorylation in vertebrate axon growth, by elucidating the roles of RPTPs. This is a critical background for efforts to understand how these processes go awry in developmental disorders of the brain and spinal cord.

DESCRIPTION (provided by applicant): A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin debris and the glial scar. Functional recovery after CNS injury requires that this inhibition be overcome. Recent studies suggest that changes in cAMP, along with increases in PKC, EGFR, and RhoA activities, are important aspects of inhibitory signaling. However, we still lack knowledge about the number/identity of inhibitory proteins associated with inhibition at injury sites, the detailed signaling mechanisms employed by inhibitory receptors, and the cell type-specific responses of damaged axons. Further, there are problems associated with current pharmacological strategies, including lack of specificity, uncertain toxicities, and the targeting of pathways with pleiotrophic functions. To overcome these difficulties, we have initiated a phenotype-based unbiased screen of a novel chemical compound library chosen for its favorable chemical properties rather than known biological function. The screen is based on the ability of compounds to increase neurite outgrowth from CNS neurons challenged with inhibitory myelin substrates. Initial results have produced 4 "hit compounds" capable of strongly increasing neurite growth. Subsequent investigations indicate that the hit compounds a) act on different neuronal types, b) selectively overcome inhibition rather than promote growth, c) are highly potent, d) overcome inhibition in distinct assays relevant to injury, e) do not affect cAMP levels, PKC activity, or EGFR activation, f) alter microtubule dynamics, and g) promote regeneration in vivo. Because the compounds are potent and selective, and may act through novel mechanisms, they are exciting candidates for therapeutic development and for mechanistic studies of regeneration inhibition. The proposal is to 1) investigate the signaling mechanisms and protein targets of the 4 hit compounds, 2) examine the ability of 1 hit compound to promote regeneration after spinal cord injury or optic nerve crush in vivo, and 3) screen the full 4000 compound library on a novel inhibitory (proteoglycan) substrate. These experiments could provide key insights into regeneration inhibition, and pave the way for a novel approach to CNS injury. PUBLIC HEALTH RELEVANCE: The proposed experiments will investigate the mechanisms of action of novel compounds promoting regeneration, and elucidate their ability to increase axonal regrowth after CNS injury.

DESCRIPTION (provided by applicant): Non-arteritic anterior ischemic optic neuropathy (NAION) is the most common cause of optic nerve-related acute loss of vision in the US; there is no effective treatment. NAION causes injury to optic nerve axons, leading to dysfunction and death of retinal ganglion cells (RGCs). Interventions to enhance RGC regeneration could be applied before RGC death, to reverse dysfunction by allowing RGCs to reconnect with their targets in the brain. Enhancement of optic nerve regeneration is a major goal for patients with NAION and other neuropathies. The lack of regeneration-promoting therapies in NAION and other diseases reflects barriers to regeneration in the injured central nervous system (CNS), including growth-inhibitory proteins associated with myelin and the glial scar. Strategies to promote regeneration by overcoming these barriers have shown efficacy in animal models, but novel strategies and translation to the clinic are needed. We have performed a phenotypic screen using a library of novel drug-like triazine compounds on primary mammalian neurons, and have identified 4 compounds capable of increasing neurite growth on a substrate of inhibitory CNS myelin. These compounds a) act on different neuronal types, including RGCs, b) are potent, c) overcome inhibition in several assays relevant to CNS injury, and d) may act by novel mechanisms. We have now shown that one compound, AA4F05, promotes regeneration in an animal model of retinal injury, as well as in a model of spinal cord injury. AA4F05 and its relatives are exciting candidates to lead to novel drugs for promoting regeneration of RGCs and other CNS neurons. Although AA4F05 has favorable chemical properties and is active both in vitro and in vivo, there has been no attempt to optimize its activity or pharmacokinetics. The present proposal will use AA4F05 as a starting point for the development of new compounds with the potential to substantially improve regeneration of damaged axons from RGCs. Derivatives will be tested in primary neurons in vitro (primary and secondary screens), and the best candidates will be tested in 2 models of optic nerve injury.

DESCRIPTION (provided by applicant): This application seeks funds to develop RegenBase - a novel information system to seamlessly integrate diverse data that are produced by neuroscientists and cell biologists studying nervous system injury, disease and cell motility with other resources, such as the Neuroscience Information Framework and the BioAssay Ontology. Over the past decade the NIH has funded the development of informatics tools and ontologies to allow the integration and interrogation of the massive and diverse data sets that have been produced by the human genome project. In the area of neuroscience the most advancement have been made in the creation and annotation of large anatomical data sets that reveal patterns of gene expression and connectivity. Genesat and the BrainMaps are excellent examples and are easily searched using the Neuroscience Information Framework (NIF) portal. But it is still surprisingly difficult to search for information related to repairing the injured nrvous system. To overcome this road block it is critical to build the essential tools that allow semantic web approaches to link diverse data repositories with ontologies that allow them to be interpreted and analyzed. The success of this initiative critically relies on an effective informatcs solution to integrate the various (current and future) data types generated by neuroscientists working on nervous system injury, as well as large-scale screening efforts (such as the Molecular Libraries Probe Center Network, MLPCN) into coherent data sets and to make them accessible, interpretable, and actionable for scientists of different backgrounds and with different objectives. We propose to develop a novel knowledge-based, extensible information system of interconnected components that leverages semantic-web technologies and domain level ontologies. This system is tentatively called RegenBase (Regeneration dataBase). Tremendous progress has been made during the last decade developing semantic web technologies with the goals of formalizing knowledge, linking information across different domains, and integrating large heterogeneous data sets from diverse sources. To develop RegenBase on a fast-track with limited resources, we will leverage technologies and tools from the National Center for Biological Onotology and the recently launched BioAssay Ontology. The long-term goal of the RegenBase system is seamless on-the fly data integration and analysis via a semantic Linked Data approach that is scalable with respect to information volume and complexity. RegenBase will incorporate biomedical domain-level ontologies, including our recently developed BioAssay Ontology (BAO), to semantically associate related data types and to provide a knowledge context of the underlying experiments and screening outcomes. The overarching goal of this proposed RegenBase system is to allow bench scientists to link data and results from studies on nervous system injury and disease to data and knowledge from other domains with an emphasis on molecular targets and the small molecules that perturb their function to speed the development of novel therapeutics.

DESCRIPTION (provided by applicant): A major impediment to recovery after central nervous system (CNS) injury is the failure of axons to regrow effectively. A variety of extrinsic and intrinsic factors contribute to this problem. Extrinsic factors include inhibitory proteins found i and around the injury site such as those from the glial scar as well as those associated with intact or damaged myelin. Regarding intrinsic factors, a key finding motivating this work is that Dorsal Root Ganglion (DRG) neurons can respond to peripheral injury with changes in gene expression that promote CNS regeneration, even in the inhibitory environment around the injury site. In contrast, CNS neurons typically fail to regenerate axons through such inhibitory regions. This implies that CNS neurons have inherent molecular differences that limit CNS regenerative capacity. The recent discovery of PTEN, SOCS3, KLF4 and KLF7 as important intrinsic regulators of CNS axon regeneration validates this hypothesis. However, the small fraction of CNS axons able to regenerate after injury, even in animals in which these genes have been manipulated, indicates that additional regulators remain to be discovered. The present proposal is to use high throughput technologies to identify genes regulating CNS regeneration by examining two related hypothesis about intrinsic factors. The first is a direct continuation of the hypothesis driving the preceding grant; i.e., that DRG neurons express RNAs that are expressed at significantly lower levels in CNS neurons and allow DRG axon regeneration. The second is that DRG neurons that have experienced a conditioning peripheral lesion express RNAs that allow regeneration and are missing (or very lowly expressed) in lesioned CNS neurons, such as the corticospinal neurons. Aim 1 will identify these molecular differences using RNA-Seq combined with bioinformatics approaches. These methods are effective at identifying rare and potentially novel isoforms, allowing identification of targets that are important but may not be abundant or previously identified; examples include miRNAs and specific transcription factor isoforms. Candidates will be tested using phenotypic analysis in vitro. Aim 2 will use viral vectors to transduce corticospinal tract neurons and test candidates from Aim 1, alone and in combination, using a pyramidotomy model of axonal growth. These experiments will provide novel information about the genes expressed in DRG neurons that allow them regenerate in the injured CNS. The identification of these targets and the testing of multiple candidates both in vitro and in vivo should lead to potential treatments not only for SCI, but also for other CNS disorders such as traumatic brain injury and stroke.

Title: Targeting Multiple Kinases to Treat Experimental Spinal Cord Injury Project Summary: Spinal cord injury (SCI) patients experience limited functional recovery, owing in part to the paucity of axon regrowth from injured CNS neurons. Effective treatments are lacking, likely because of multiple factors, intrinsic and extrinsic, that inhibit axon growth. Thus we require agents that target more than one source of regeneration failure. Kinases are ubiquitous signal transducers that regulate most cellular processes, including axon growth. To begin to identify compounds that positively regulate axon growth, we screened 1600 small-molecule kinase inhibitors (KIs) in an in vitro CNS neurite outgrowth assay and identified ?hit? KIs that reproducibly and strongly promote outgrowth. Due to homology of catalytic domains, KIs typically inhibit multiple kinases. This makes it difficult to identify the kinase(s) that mediate a KI's effects on cells. We used information theory and machine learning to analyze the inhibition profiles of KIs in relation to their effects on neurite outgrowth. This enabled us to identify, and later validate via siRNA knockdown in primary neurons, multiple kinase targets (i.e. kinases that should be inhibited to promote neurite outgrowth). These included previously known targets that regulate intrinsic and extrinsic inhibitor factors, in addition to several novel candidates. Conversely, we identified kinases whose activity is critical for neurite outgrowth, and whose inhibition must be avoided (anti-targets). We discovered several KIs that inhibit multiple targets and no anti-targets. These KIs strongly promoted neurite outgrowth in vitro. We tested the KI, RO48, that had the largest effect in vitro in two in vivo models. Our preliminary experiments indicate that RO48 is remarkably effective in vivo. It promoted robust axonal growth of the corticospinal tract (CST) in three separate models of CST injury (pyramidotomy, funiculotomy, dorsal hemisection), and in the dorsal hemisection model, improved forelimb function. We propose to build on these remarkable results to test the working hypothesis that the simultaneous inhibition of RO48's five target kinases (ROCK, PKC, PRKG1, PRKX, and RPS6K) promotes sprouting and regeneration of CST axons. This will be accomplished using viral vectors to knock down expression of the different target kinases individually and in combination. We will do knockdown in CST neurons in the cortex. We will assess CST axon growth at the injury site using light microscopy. We will also perform experiments to determine if RO48-induced CST axon growth promotes axon sprouting, regeneration, or both, and whether RO48 improves behavioral outcomes such as grasping and walking after a contusion injury. These experiments will 1) validate novel kinases as in vivo targets for future development of SCI therapeutics 2) determine whether these kinases regulate CST axon sprouting, regeneration, or both, and 3) confirm whether the substantial stimulation of axon growth induced by treatment with RO48 improves motor outcomes in a clinically relevant contusion model.

PROJECT SUMMARY Spinal cord injury (SCI) patients experience limited functional recovery, owing in part to the paucity of axon regrowth from injured CNS neurons. Effective treatments are lacking, likely because of multiple factors, intrinsic and extrinsic, that inhibit axon growth. Thus, we require agents that target more than one source of regeneration failure. Kinases are ubiquitous signal transducers that regulate most cellular processes, including axon growth. To begin to identify compounds that positively regulate axon growth, we screened 1600 small-molecule kinase inhibitors (KIs) in an in vitro CNS neurite outgrowth assay and identified ?hit? KIs that reproducibly and strongly promote outgrowth. Due to homology of catalytic domains, KIs typically inhibit multiple kinases. This makes it difficult to identify the kinase(s) that mediate a KI?s effects on cells. We used information theory and machine learning to analyze the inhibition profiles of KIs in relation to their effects on neurite outgrowth. This enabled us to identify, and later validate via siRNA knockdown in primary neurons, multiple kinase targets (i.e. kinases that should be inhibited to promote neurite outgrowth). These included previously known targets that regulate intrinsic and extrinsic inhibitor factors, in addition to several novel candidates. Conversely, we identified kinases whose activity is critical for neurite outgrowth, and whose inhibition must be avoided (anti-targets). We discovered several KIs that inhibit multiple targets and no anti-targets. These KIs strongly promoted neurite outgrowth in vitro. We tested the KI, RO48, that had the largest effect in vitro in two in vivo models. Our preliminary experiments indicate that RO48 is remarkably effective in vivo. It promoted robust axonal growth of the corticospinal tract (CST) in three separate models of CST injury (pyramidotomy, funiculotomy, dorsal hemisection), and in the dorsal hemisection model, improved forelimb function. We propose to build on these remarkable results to test the working hypothesis that the simultaneous inhibition of RO48?s five target kinases (ROCK, PKC, PRKG1, PRKX, and RPS6K) promotes sprouting and regeneration of CST axons. This will be accomplished using viral vectors to knock down expression of the different target kinases individually and in combination. We will do knockdown in CST neurons in the cortex. We will assess CST axon growth at the injury site using light microscopy. We will also perform experiments to determine if RO48-induced CST axon growth promotes axon sprouting, regeneration, or both, and whether RO48 improves behavioral outcomes such as grasping and walking after a contusion injury.These experiments will 1) validate novel kinases as in vivo targets for future development of SCI therapeutics 2) determine whether these kinases regulate CST axon sprouting, regeneration, or both, and 3) confirm whether the substantial stimulation of axon growth induced by treatment with RO48 improves motor outcomes in a clinically relevant contusion model.