Vance Lemmon - US grants

The neural retina develops from an epithelia of undifferentiated cells into a stratified structure with a highly organized appearance and several different types of cells. While there is a great deal of morphological and physiological information about the mature retina, very little is known about the cellular interactions and biochemical events that result in the retina's normal development. One of the major reasons for this is that techniques that allow identification of specific cell types and the purification of these populations have not been available. As a result, many in vivo and in vitro experiments designed to address developmental quesitons are presently impossible to perform or interpret. We have developed a number of monoclonal antibodies that bind to specific classes of cells in the chick retina, including ganglion cells, amacrine cells, photoreceptors, and Muller cells. One of our goals is characterize the cell specific antigens defined by these antibodies using both morphological and biochemical techniques. We also propose to use recently develop0ed approaches to produce antibodies specific for bipolar cells and horizontal cells. Finally, these antibodies will be used in in vitro experiments to study how cell-cell interactions and various types of substrates ionfluence the differentiation of specific classes of cells in the retina.

DESCRIPTION: (Applicant's abstract) Learning how axons of retinal ganglion cells grow from their origins in the retina to their targets in the brain is an important goal for understanding how the visual system develops. A number of cell adhesion molecules (CAMs) have been found in the optic fiber layer and optic nerve and are thought to be important in axon growth and guidance. We have found that one CAM, called L1 (also referred to as NILE, Ng-CAM, 8D9 or G4), is present on axon of retinal ganglion cells and is a potent substrate for growth of axons of retinal ganglion cells in vitro. Moreover, preliminary experiments suggest that L1 may be able to support axon regeneration in the adult nervous system. The experiments described in this proposal will provide important new information about how L1 participates in visual system development and will extend our knowledge about how CAMs can function to promote and guide axon growth following damage to the visual system. Biochemical, immunological and molecular biological experiments will examine how L1 binding functions in axon adhesion. In vitro experiments will investigate how L1 mediated axonal adhesion functions in guiding axons along visual pathways. In vivo experiments will evaluate the ability of purified L1 to promote axon growth in the visual system.

Learning how axons of retinal ganglion cells grow from their origins in the retina to their targets in the brain is an important goal for understanding how the visual system develops. A number of cell adhesion molecules (CAMs) have been found in the optic fiber layer and optic nerve and are thought to be important in axon growth and guidance. A basic idea in the field has been that CAMs mediate cell-cell interactions via cell- cell binding. Over the past few years a more complex concept has evolved, i.e., that CAMs not only mediate cell adhesion but are also capable of signaling, perhaps by influencing intracellular second messenger systems. One CAM, called L1 (also referred to as NILE, Ng-CAM, 8D9 or G4) is present on axons of retinal ganglion cells. We have shown that L1 is a potent substrate for growth of retinal ganglion cell axons in vitro and that L1 binds to L1 in a homophilic manner. Using two different techniques, we have found that L1 is a strongly adhesive substrate and produces distinctive behavior by growth cones. Other labs have directly implicated L1 in regulating intracellular Ca++ and phosphorylation events, while we have identified a novel S6 kinase activity associated with L1. We have cloned and sequenced the human L1 cDNA and found that the cytoplasmic domain of L1 is highly conserved in mammals. The cytoplasmic domain of L1 in brain is 114 amino acids long and is identical in mice and humans. Using our cDNA clones, other laboratories have shown that human X-linked hydrocephalus (HSAS is due to a defect in L1 expression. Patients with this syndrome die around the time of birth or are severely retarded, have strabismus and other visual system defects. In at least one HSAS family this is doe to a mutation affecting the cytoplasmic domain of L1. L1 is an excellent CAM to study in order to understand how CAMs influence growth cone behavior by simultaneously mediating growth cone-substrate adhesion and influencing second messenger systems. The experiments in this proposal will use biochemical, immunological, molecular biological and cell biological experiments to focus on the cytoplasmic domain of L1. Site directed mutagenesis will be done on the cytoplasmic domain of L1 to define regions critical for L1-L1 homophilic binding and neurite outgrowth. We will also examine how phosphorylation of L1 is regulated and how phosphorylation of L1 influences L1 function. These experiments will provide new information about how cell adhesion modulates intracellular processes and also how changes in intracellular second messenger systems can alter cell adhesion. Finally, these experiments will provide a detailed description of how one cell adhesion molecule regulates growth cone behavior.

9315395 Lemmon This grant will purchase equipment for use in presenting a program about the brain to elementary school children in the Cleveland area. A group of university based brain scientists are developing this program. These scientists are very interested in having the program adapt for use by scientists in all parts of the country. It is clearly important to introduce young children to the brain and how it functions from the standpoint of understanding the nature of substance abuse and the significance of neurological injuries. This could also have an impact on the infrastructure of neuroscience research by encouraging young people to seek a career in science and developing a lay public that appreciates science. *** . ? o = 8 ^& B !/ , D o& E Õ $ B H% D J L N ' W =& O g& Q t S u. f & X p+ 6+ ^ + _ + e U & ;F u S V F V9315395 Lemmon This grant will purchase equipment for use in presenting a program about the brain to elementary school chi h a f h ! ! F h h 8 Times New Roman Symbol & Arial " Univers (WN) " h + E ( s 8 Steve McLoon, IBN William Proctor, IBN

9318068 Lemmon The aim of this grant proposal is to obtain a Confocal Microscope Imaging System for a group of neuroscientists who use light microscopic techniques as our principal methods of data analysis. Over the past few years confocal microscopy has been used in striking ways to localize subcellular structures and to image growing cells in complex tissues, providing new insights into cell cell interactions. The investigators in this user group are interested in the formation of the complex 3 dimensional organization of the nervous system. Four of the users examine axons, growth cones or synapses in living tissues. The fifth user studies the formation of the blood brain barrier and how glia participate in this. The acquisition of a confocal microscope would greatly enhance the capabilities of the members of this group. Not only would we increase the resolution of the data we obtain, we would be able to address questions that are presently unapproachable due to the inherent limitations of our current imaging facilities. Confocal microscopy eliminates stray light from the image focal plane. This dramatically increases the resolution compared with conventional epifluorescence microscopic techniques. This permits optical sectioning of a fairly thick biological specimen. When coupled with a computer/image analysis system, data from a series of optical sections can be assembled to form a 3 dimensional reconstruction of the fluorescent emission pattern from the specimen. Subcellular structures can be localized within or on a cell using antibodies or other fluorescent probes, even in a complex environment such as a t issue slice or an intact embryo or animal. For most of the user group, this technique will be applied to make time lapse movies of live cells in tissues or whole animals. Recently confocal microscopy has been adapted to the technique of interference reflection microscopy (IRM), permitting much larger areas of a specimen to be examined at lower illumination levels. This greatly facilitates time lapse video of IRM of live cells. It is the confocal microscope's ability to produce high resolution, time lapse images of dynamically changing structures, such as developing or aging nervous tissue, that is our major technical requirement at this time. List of Participants Principle Investigator Vance Lemmon, Ph.D. Associate Professor Department of Neurosciences Lynn Landmesser, Ph.D. Professor Department of Neurosciences and Genetics Norman Robbins, M.D./Ph.D. Professor Department of Neurosciences and EVHS Jerry Silver, Ph.D. Professor Department of Neurosciences Dennis Landis, M.D. Professor Departments of Neurology and Neurosciences ~CRD2122TMP ?Ss ~MF2E06 TMP Ys ~CAL2522TMP WZs ~CRD3953TMP YZs ~CAL1F21TMP hzs ~CRD1B5ATMP jzs ~CAL3017TMP

Recent advances in the fields of neural cell adhesion molecules (CAMs) and human genetics have provided a unique opportunity to begin a careful cell biological study of the causes of two inherited X-linked diseases that produce mental retardation; X-linked hydrocephalus and MASA syndrome. It has been demonstrated that these syndromes are the result of mutations in the neural cell adhesion molecule L1. The possibility has also been raised that other X-linked forms of mental retardation, such as some cases of X-linked a specific mental retardation, are also due to mutations in L1. While the primary genetic defects in L1 are now known in several families, the reasons for the alterations in brain development are not clear. What is intriguing is that subtle differences in the form of L1 expressed appear to produce different phenotypes. L1 has been implicated in neuronal migration and in axon guidance. In most parts of the nervous system L1 is expressed on axons and growth cones and is believed to play an important role in providing a substrate for growth cone attachment as well as in mediating axon fasciculation. L1 is expressed by the vast majority of projection axons in the CNS and PNS and is also expressed by Schwann cells in the PNS. Due to its widespread expression in the developing nervous system it is likely to be an important player in the establishment of major fiber tracks and nerves. In vitro and in vivo experiments have shown that disruption of L1 function leads to significant alterations in the formation of both the CNS and PNS. Cell biological studies of L1 have revealed that this CAM has complex interactions with several intracellular and extracellular proteins. It has been reported that L1 binds extracellularly to L1, axonin-1, F3/F11, laminin and NCAM. Additionally, L1 is also capable of influencing intracellular second messenger systems. Crosslinking L1 with antibodies leads to change in intracellular pH, IP3 and CA++. L1 has at least two associated kinases that could be the initial players in initiating second messenger systems. The complex nature of L1, with its many functional domains, may explain the different phenotypes expressed by families with L1 defects. Therefore, we propose to examine in detail, using different cell biological assays, the functional capabilities of mutated L1 forms from human families and to correlate this with the human phenotypes. We will examine mutant L1-L1 binding and neurite outgrowth, mutant L1 interactions with their heterophilic binding partners and the ability of mutant L1 to mediate alterations in intracellular Ca++. This should provide a completely new level of understanding of these X-linked forms of mental retardation.

phenotype; biomedical facility; stainings; image processing; perfusion; sectioning; transmission electron microscopy; scanning electron microscopy; confocal scanning microscopy; light microscopy;

The Program Project is designed to use modern genetic approaches in the study of key components and events in the development, function, and repair of the vertebrate central nervous system. Major areas of molecular neuroscience to be studied include cell-cell interactions (Project 1), signaling via intracellular second messenger systems (Project 2), and genes that contribute to nervous system pattern formation (Project 3). The approaches to be used include creation of animals with specific germ line mutations by homologous recombination in embryonic stem cells, analysis of the fate of individual mutant precursor cells in a normal tissue environment, and identification of specific disease genes by newly devised methods utilizing the mapping of overlapping deletions. This program requires a synergistic and innovative application of the expertise represented by several investigators, with integration of this expertise at both the technical and interpretive stages of each project. Integration is facilitated through three Cores: Administrative, Gene Targeting, and Phenotypic Analysis, that are utilized by each project and provide for unusual economy of effort and expense in this type of research . The combination of results obtained in analysis of the different systems described will provide for a more comprehensive understanding of molecular systems at the tissue level in the intact animal. This body of fundamental information on the assembly and function of the central nervous system is of direct relevance to human health problems concerning defects in brain development and the resulting dysfunction and mental retardation.

DESCRIPTION (From the Applicant's Abstract): The neural cell adhesion molecule L1 is found on some classes of migrating neuronal precursors in the developing nervous system and on almost all projection axons in both the central nervous system and peripheral nervous system. Not surprisingly, it has been implicated in the fasciculation of axon bundles and in migration of some neural precursors in various in vitro systems. In the early 1990's it was shown that mutations in the L1 gene in humans cause severe mental retardation (corpus callosum hypoplasia, adducted thumbs, spastic paraplegia, and hydrocephalus). We have analyzed individuals with different mutations in the L1 gene and discovered that mutations that lead to a loss of L1 expression are much more severe than mutations that only alter the cytoplasmic domain of L1. However, mutations of the cytoplasmic domain are sufficient to cause axon guidance failures and mental retardation. Recently, we and others have analyzed the L1 knock-out mouse and discovered that it has a phenotype remarkably similar to humans with X-linked hydrocephalus. This includes hydrocephalus, abnormal development of the corticospinal tract, and hypoplasia of the cerebellar vermis and corpus callosum. In this project we propose to test the hypothesis that L1 mediated adhesion is essential for normal development of the cerebellar vermis and that the function of the L1 cytoplasmic domain is essential for development of the corticospinal tract. To do this we will generate new mouse lines with specific alterations in the L1 cytoplasmic domain. We will also analyze mice in which the 6th Ig domain of L1 has been removed, deleting the RGD sequence in L1, allowing us to evaluate the difference between L1 homophilic binding and L1-integrin interactions during brain development. Finally, we will undertake the first careful analysis of cerebellar development in mice with altered or absent L1.

animal breeding; genetic manipulation; biomedical facility; laboratory mouse; laboratory rat; genetically modified animals; veterinary science; eye; genetic strain; vision; cryopreservation; biotechnology;

[unreadable] DESCRIPTION (provided by applicant): A major impediment to recovery after spinal cord injury (SCI), traumatic brain injury (TBI), or stroke is the failure of central nervous system (CMS) axons to regenerate effectively through white mater over long distances. A variety of factors are believed to contribute to this problem. These include inhibitors in glial scars, inhibitory material associated with myelin or damaged myelin and molecular changes in neurons during development that reduce their potential for axon growth. Over the past several years it has been shown that Dorsal Root Ganglion (DRG) neurons can send axons very long distances in white mater if they are transplanted into the CNS using techniques that minimize damage. In contrast, transplanting CNS neurons the same way does not produce the same result; i.e. they fail to send out long axons through white matter tracks. This implies that DRG neurons and CNS neurons have inherent molecular differences that limit CNS regenerative efficiency. We propose to test a specific hypothesis that DRG neurons express different genes than CNS neurons, which permit DRG neurons to regeneration in the CNS. In specific aim 1 we will identify these molecular differences using serial subtraction of cDNA libraries. We will look for unique genes in DRG neurons that might enhance regeneration and unique genes in hippocampal neurons and corticospinal neurons that might inhibit regeneration This method is extremely effective at identifying rare and perhaps novel cDNAs, ensuring identification of important targets, such as transcription factors. We will also search public microarray databases to search for additional candidates. In specific aim 2 candidate genes will be tested using a well-established in vitro assay where neurons are grown on myelin or proteoglycans and the lengths of their neurites measured. CNS neurons will be transfected with DRG specific genes or use RNAi of CNS specific genes in order to evaluate the target genes roles in axon growth on inhibitory substrates. Specific aim 3 will use neuronal transfection and microtransplantation in vivo. This will permit us to directly test the role of each candidate gene in the most relevant assay, regeneration in the mammalian central nervous system. These experiments will provide entirely new information about the proteins expressed in DRG neurons that allows them to extend long axons in the CNS. The identification of these targets and testing multiple candidates using in vitro methods and subsequently a refined subset using in vivo approaches should lead directly to potential treatments for SCI, TBI and stroke. Lay Summary: These experiments are designed to identify genes involved in controlling regeneration in white matter in the adult brain. The genes will be tested in neurons that cannot normally grow axons using cell culture and transplantation into animals to determine if the genes promote regeneration. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Increasingly large and diverse data sets are being generated by publically funded screening centers using various high- and low-throughput screening technologies. Much of this data is accessible. The largest public repository of small molecule screening results is PubChem, currently covering over 1,500 assays for 370,000 compounds. The number of publically available assays is expected to grow more than 10 fold during the next five years. The utility of this invaluable resource is currently limited, because the knowledge contained in complex and diverse bioassay data sets is not formalized and therefore cannot be accessed for comprehensive computational analysis or integration with other data sources. This proposal is to attack this limitation. For the past ten years ontologies have been developed by biologists to facilitate the analysis and discussion of the massive amounts of information emerging from the various genome projects. An ontology is a controlled vocabulary representation of the objects and concepts and their properties and relationships. The purpose is to model and share domain-specific knowledge so that software agents can automatically extract and associate information. The aim of this proposal is to develop a bioassay ontology, software tools, and to demonstrate their utility. The bioassay ontology will coherently describe diverse biological assays (such as those in PubChem) with a focus on complex cell-based assays and in particular high-content screening data. Software support and development includes modules to build ontology terms and to curate data sets, tools to map the ontology onto screening experiments and other ontologies, and tools to standardize, reformat, and aggregate data sets in the context of the ontology. We will demonstrate the utility of our approach by creating a PubChem-derived database and making it available to the community via a search interface. The ontology and software tools will facilitate the analysis of bioassay screening data in various contexts, for example signaling or metabolic pathways and indirectly human disease. The tools will enable one to extract data sets for modeling specific interactions between perturbing agents and biological targets (or pathways), or to model assay technology-dependent interferences. End user software needs to provide ease of use for biologists and chemical biologists to utilize the ontology in the context of their own and external data sets. It will be modular and open source. We will develop various collaborations to disseminate the bioassay ontology and software in the community and to facilitate their ongoing development. PUBLIC HEALTH RELEVANCE: This project will develop a bioassay ontology to coherently describe the hundreds of different assays used to study how perturbing agents, such as drugs, alter cell function. Along with new software to search existing assay databases, this will enable scientists to more effectively identify and prioritize chemicals for further development into chemical probes or starting points for therapeutics.

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. PUBLIC HEALTH RELEVANCE: Diseases of the optic nerve (optic neuropathies) are a leading cause of impaired vision in the US. This proposal is designed to develop and test novel therapeutic chemicals that can later be developed into drugs for treatment of optic neuropathies.

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. PUBLIC HEALTH RELEVANCE: Public and private organizations are generating diverse data sets as they attempt to develop therapies for nervous system injury and disease. One reason therapy development is slow lies in the difficulty of collecting, analyzing and displaying information from the thousands of different experiments done on nervous system injury and interpreting it based on knowledge from other areas, such as genomics, cell biology, cancer, immunology and drug discovery. We propose to develop a novel information system that will help neuroscientists working on nerve regeneration to access and use information generated by scientists around the world.

ABSTRACT Poor regeneration and reconnection of retinal ganglion cell (RGC) axons is a major obstacle for treating ocular trauma and diseases including glaucoma. There are as yet no therapies to repair optic nerve once the damage is done. Our new studies have discovered cohorts of RGCs that have a very high regenerative capacity. Furthermore, we now uncover previously unrecognized ability of distinct lipids to promote axon growth. In Aim 1, we will use High Content Screening and functionally test various candidate genes for their ability to promote neurite growth. In Aim 2, we will determine lipid profiles in RGCs, and functionally test neurite growth-promoting effects of select lipids. In Aim 3, we will use in vivo optic nerve injury model to determine RGC axon regeneration receiving various treatments. Identifying novel targets that further increase RGC axon regeneration to the brain represents a critical future study. Results obtained from these studies will provide invaluable information on developing future therapies to repair degenerated optic nerve.

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/ABSTRACT Trauma to the central nervous system (CNS: spinal cord and brain) together affect more than 2.5 million people per year in the US, with economic costs of $80 billion in healthcare and loss-of-productivity. Yet, the precise pathophysiological processes impairing recovery remain poorly understood. This lack of knowledge is exacerbated by poor reproducibility of findings in animal models and limits translation of therapeutics across species and into humans. Part of the problem is that neurotrauma is intrinsically complex, involving heterogeneous damage to the central nervous system (CNS), by far the most complex organ system in the body. This results in a multifaceted CNS syndrome reflected across heterogeneous endpoints and multiple scales of analysis. Multi-scale heterogeneity makes traumatic brain injury (TBI) and spinal cord injury (SCI) difficult to understand using traditional analytical approaches that focus on a single endpoint for testing therapeutic efficacy. Single endpoint-testing provides a narrow window into the complex system of changes that describe SCI and TBI. Understanding these disorders involves managing datasets that include high volume anatomy data, high velocity physiology decision-support data, the high variety functional/behavioral data, and assessing correlations among these endpoints. In this sense, neurotrauma is fundamentally a data management problem that involves the classic ?3Vs of big data? (volume, velocity, variety). Of these, variety is perhaps the greatest data challenge in neurotrauma research for reproducibility in basic discovery, cross-species translation, and ultimately clinical implementation. For the proposed Data Repositories Cooperative Agreement (U24) we will build on our prior work managing data variety in the Open Data Commons for SCI (odc-sci.org) and TBI (odc-tbi.org) to make neurotrauma data Findable, Accessible, Interoperable, and Reusable (FAIR). The milestone-driven aims will: 1) further develop and harden our data lifecycle management system with end-to-end data version control and provenance tracking, data certification, and data citation; 2) develop in-cloud data dashboards and visualizations to monitor data quality and to promote data reuse, exploration, and hypothesis generation; 3) establish a pan- neurotrauma (PANORAUMA) data commons that brings together separate data assets currently supported by our multi-PI (MPI) team by aligning a patchwork of governance structures and policies. The goal of the proposed project is to develop a pooled repository for preclinical discovery, reproducibility testing, and translational discovery both within and across neurotrauma types. Our team is well-positioned to execute this project given that we developed some of the largest multicenter, multispecies neurotrauma data repositories of neurotrauma to-date (N>10,000 subjects 20,000 curated variables); the Neuroscience Information Framework (NIF); data terminologies and standards for these fields (MIASCI, NIFSTD); and policy work with the International Neuroinformatics Coordinating Facility (INCF). The PANORAUMA cooperative agreement is highly responsive to PAR-20-089, leveraging early successes in SCI and TBI data sharing to improve quality and sustainability.

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.