Michael S. Beattie - US grants

We propose to add a video image analysis computer (the Joyce-Loebl Magiscan 2) to the morphometrics laboratory in the Department of Anatomy at The Ohio State University in order to enhance our abilities to quantitate the results of neuroanatomical and biochemical experiments. The instrument will be used by a major user group from seven (7) currently funded NIH projects to analyze data obtained from fluorescence microscopy, histochemistry, immunocytochemistry, electron microscopy, and bright and darkfield microscopy. The Magiscan computer will also serve as an additional input to our currently operating three-dimensional reconstruction system. A research quality light and fluorescence microscope is also requested to support the system.

A combination of physiological and anatomical studies are proposed to investigate the role of the medullary raphe and the hypothalamus in controlling gastro-intestinal eliminative reflex circuits. The effects of electrical and chemical stimulation of nucleus raphe obscuras/pallidus (nRO/nRP) and the paraventricular nucleus (PVN) of the hypothalamus on the external anal sphincter (EAS) will be examined in the cat. Microinjection of peptides (oxytocin [OXY]) and vasopressin [AVP]), thyrotropin releasing hormone (TRH) and serotonin (5-HT) into Onuf's nucleus will be used to assess the effects of endogenous ligands of the descending systems on EAS tone, recto-anal reflexes, and EAS motoneuron (MN) excitability. Antagonists to 5-HT, OXY and AVP will be used to attempt to reverse the effects of stimulation. Descending projections from nRO/nRP (and adjacent structures) an PVN to Onuf's nucleus in the sacral spinal cord, which contains the MNs innervating the EAS, will be studied with tracing techniques, electron microscopy (EM), and immunohistochemistry (IHC). Our hypothesis is that these descending inputs make direct synaptic contacts with the proximal dendrites and somata of sphincter MNs. Anatomical data strongly suggest that these spinal cord parasympathetic/somatic networks share a number of structural and functional similarities with parasympathetic/somatic control circuits in the medulla. Together, these studies should provide an anatomical and functional analysis of some of the central circuits responsible for forebrain/brainstem descending control of distal gut eliminative reflexes and may allow comparisons with such control of proximal gut reflexes with which we are very familiar. In addition, the proposed studies may yield insights into the etiology of incontinence associated with gastrointestinal diseases, neurological disorders (such as autonomic failure stroke, and spinal cord trauma), and aging, and will add information concerning a unique motoneuron type resistant to motoneuron diseases like ALS.

We have shown in the previous grant period that fibers from the nucleus raphe obscurus (nRO) and nucleus paragigantocellularis lateralis (lPGi) contact the motoneurons that innervate the external anal sphincter (EAS MNs). The nRO and one of its neurotransmitters, serotonin (5-HT) may inhibit reflex activity of EAS. Spinal cord lesions, or lesions of the nRO result in hyperactivity of the EAS, similar to the spasticity seen in man after spinal cord injury (SCI). We know that after partial lesions of the cord, eliminative function gradually improves in our rat SCI model, and the hyperreflexia of the EAS declines. Since 5-HT has been shown to sprout after spinal cord lesions, we propose that the disruption of 5-HT inputs into the EAS motor nucleus or other regions of the cord, results in the release of the reflex from descending control, and that improvement in function is related to the re-establishment of connections that come from spared descending fibbers from the brainstem. We will use a number of interrelated measures to assess the role of sprouting and 5-HT on recovery after SCI. EAS hyperreflexia and its decline will be measured along with a standardized locomotor outcome measure. Sprouting of 5-HT fibers will be measured anatomically in the cord nuclei containing the MNs that produce EAS contractions. Fibers from the brainstem (nRO and lPGi) will be labeled with anterograde tracers and their projects will be quantified to test for sprouting. The effects of nRO stimulation and 5-HT agonists/antagonists on EAS reflexes will be tested in normal and spinal injured rats. The role of putative increases in 5-HT fibers in the recovery process will be measured by determining whether removal or blockade of 5-HT input reinstates hyperreflexia, and by determining whether increases in axons, and decreases in reflex activity, are correlated with an increased in the 5- HT released in the spinal cord by nRO stimulation. Together, these convergent tests of the central hypothesis should yield strong affirmation or denial of the role of 5-HT in changes in eliminative reflex function after SCI. The possible role of other descending systems will be considered. Together, the results may lead toward the identification of drugs that can enhance eliminative function after SCI in man.

DESCRIPTION (provided by applicant): This is a revised submission of a request for continuation of a project aimed at studying functional recovery of penile and external anal sphincter ('sacral') spinal cord reflexes after contusion injuries in rats meant to model human spinal cord injury (SCI). Recovery of sacral reflexes was compared to recovery of locomotion. Serotonergic (5-HT) descending systems are thought to be involved in this recovery, and in the previous funding period, evidence was provided for denervation and reinnervation of sacral MNs by 5-HT after SCI. The source of the 5-HT sprouting is thought to be the small number of axons that remain in the spared rim of fibers after severe contusion SCI. We have shown that glial restricted precursor cells (GRPs) can be transplanted into contusion injuries, and that they alter the lesion environment by reducing glial scarring. The revised submission focuses on the hypothesis that the transplantation of GRPs into the contusion lesion cavity after SCI will result in more 5-HT sprouting and will enhance the recovery of sacral reflexes and locomotion. In aim 1, we will compare the long-term recovery of sacral and locomotor functions in rats receiving contusion lesions with and without GRP transplants given at 9-10 days following injury. In aim 2 we will compare the amount of serotonergic input to identified external anal sphincter (EAS) and bulbospongiosis (BS) motoneurons (MNs) in rats with and without GRP transplants. In aim 3, we will examine the source of the sprouting into the sacral cord by labeling brainstem sources of 5-HT with anterograde tracers combined with 5-HT IHC. This will also allow us to test the hypothesis that the reinnervation of the cord by spared descending systems maintains the specificity of the original connections. These studies represent a collaboration between laboratories studying SCI and developmental biology, and are intended to provide guidance on the possible therapeutic value of glial precursor transplantation in SCI.

DESCRIPTION (provided by applicant): The demographic of spinal cord injury (SCI) is changing, with more and more aged individuals showing up at emergency rooms with spinal cord injuries, especially cervical SCIs related to falls (Ho et al, 2007). In addition, aging is associated with spondylosis and narrowing of the cervical spinal canal. The resultant chronic compression is thought to be associated with slow neurodegenerative changes including demyelination, resulting in loss of function even without a precipitating acute injury. Spondylosis and stenosis also increase the probability of contusion injury associated with falls. Aging also affects myelination and the capacity for remyelination (e.g. Peters and Sethares, 2003) and there is evidence that the CNS itself may be more vulnerable to injury with aging and may have a less plastic response to injury. Both factors could lead to less recovery. Here we propose that that increased vulnerability may be due in part to changes in the reparative response to injury mounted by endogenous adult glial progenitor cells of the oligodendrocyte lineage, which are characterized by the expression of the PDGFR1 and the proteoglycan NG2 (see Horner et al, 2002;Polito and Reynolds, 2006;Rhodes et al, 2006). In Aim 1. we will establish a model of chronic cervical compression that produces progressive neurological deficits and demyelination in young, adult Fischer 344xBN hybrid rats. Rats will be followed with a panel of neurological outcome measures, including forelimb motor function tests and tests for the development of altered sensation (e.g. allodynia and pain). Histological characterization of cord damage, demyelination and remyelination will be made post-mortem. In Aims 2 and 3, we will use the model developed in aim 1 to test the hypothesis that aging results in a greater susceptibility to chronic cervical compression, and ask if the effects of aging are due to increased susceptibility to oligodendrocyte and axonal death, and reduced progenitor cell repair and remyelination, or a combination. We also hypothesize that differences in the inflammatory response in young vs. old rats may play a role. We will compare the responses of young (3 months), middle aged (9 months) and old (15 months) rats to chronic compression injury using both behavioral and histological outcome measures. If the repair response is found to be deficient or reduced in aging, in future studies we will test treatments to reinvigorate or replace the progenitor cell population to ameliorate the deficits. PUBLIC HEALTH RELEVANCE Spinal cord injury (SCI) is now occurring more and more in the aged population, and can be due to acute injury, or the kinds of chronic cervical spinal cord myelopathy that occurs due to age-related arthritis and cord compression. The work proposed here will examine the role of a population of adult stem cells, oligodendrocyte progenitor cells (OPCs) in the development of myelopathy, and in recovery after SCI in aging using rat models of injury. It is expected that the results will contribute to the development of therapies that will improve neurological function in aging humans.

Inflammation and excitoxicity appear to be pivotal in CNS trauma, but effective strategies for targeting these aspects of secondary injury have been elusive. Proinflammatory cytokines appear to have complex and sometimes contradictory roles in CNS injury and repair. Similarly, glutamate receptors are responsible for excitotoxic death of neurons and glia in injury, but are also essential for normal CNS function, and have been implicated in recovery after injury. The pro-inflammatory cytokine tumor necrosis factor-alpha (TNFa) has recently been shown to have a unique and critical role in the modulation of normal neuronal glutamate synaptic transmission (Beattie et ai, 2002;Stellwagen and Malenka, 2006;Aizenman and Pratt, 2008), and also to exacerbate excitotoxic cell death (Hermann et ai, 2001;Beattie, 2004). We have identified TN Fa-mediated trafficking of GluR2-lacking, Ca++-permeable AMPA receptors (CP-AMPARs) as a novel and perhaps 'nodal'link between injury-induced inflammation and excitotoxicity. TN Fa increases excitotoxic cell death in vitro, and enhances neuronal death after spinal cord injury (SCI). Further, reducing GluR2- lacking AMPAR-insertion into neuronal membranes by blocking TNFa after SCI, results in reduced neuronal death, reduced white matter damage, and better outcomes in a cervical injury model of SCI.

DESCRIPTION (provided by applicant): The innate immune response is clearly an integral part of the secondary injury cascade in CNS trauma, but the role of glial and monocyte derived pro-inflammatory cytokines is complicated by multiple downstream effects mediated by multiple concentration-dependent receptors, and a rapidly changing and evolving microenvironment. We have new findings that strongly support the hypothesis that TNF and AMPAR changes are critical in both secondary injury and recovery after CNS injury. Sequestering TNF using soluble TNF receptor protein (sTNFR1) reduces damage after cervical SCI, and, in a highly dose-dependent manner, improves neurological outcomes. We propose to extend our studies of TNF and AMPAR trafficking as a therapeutic target for SCI using a multivariate approach to test preclinical efficacy. Etanercept (ETAN) is a TNF-sequestering protein biologic used clinically in rheumatoid arthritis; topiramate (TPM) is a neuroprotective, anti-epileptic drug that has AMPAR antagonism. We will use these drugs to target AMPAR-trafficking and AMPAR activity, separately and in combination. We will systematically evaluate the biological responses to these drugs using early post-injury biomarkers that have predicted neurological outcomes in our prior work. We will use the biomarker data to plan preclinical dose and timing regimens to evaluate effectiveness in both cervical and thoracic rat SCI, and evaluate their effects on autonomic, sensory and motor outcomes. These efforts are aimed at moving towards clinical application of anti-TNF therapies for SCI, and may be applicable to other CNS degenerative disorders as well. We propose three specific aims: Aim 1: We will evaluate the effects of ETAN and TPM on the time course and extent of biomarkers of AMPAR surface expression, cell death and the production of pro-inflammatory cytokines after cervical SCI. We predict that these treatments will reduce the feed-forward cascade of cell death. Aim 2. Guided by these data, we will optimize dose and timing of single and combination therapies to maximize six week recovery after unilateral cervical SCI using a variety of forelimb functional tests (grooming, paw placement, Catwalk and IBB). Aim 3: We will test the effects of optimized drug regimens on recovery from thoracic contusion lesions using a battery of tests that includes autonomic, sensory, and motor outcomes. This will establish whether efficacy extends to multiple models of SCI. TNF and AMPARs are also involved in the production of chronic hypersensitivity after nerve injury (Choi et al, 2010), and we will test whether this occurs after SCI as well. TPM is already used to treat chronic SCI pain. We predict that acute treatments with ETAN and TPM that result in better motor outcomes will also result in reductions in long term allodynia and in tonic, aversive central pain (King et al, 2009), the latter measured by place preference tests.

PROJECT SUMMARY/ABSTRACT This is a new multi-PI proposal from our California Spinal Cord Injury (SCI) consortium to continue to translate exciting results from the transplantation of neural stem cells (NSCs) from rodent to primate, and to evaluate efficacy and safety in our non-human primate (NHP) cervical contusion SCI model. Our multi-center consortium has examined recovery of function and its anatomical correlates in a series of studies using a cervical hemisection model. We have discovered spontaneous and extensive plasticity of the corticospinal tract (CST) system that had not been appreciated in previous rodent studies. We have developed the first large NHP model of cervical hemicontusion SCI together with an open-field scoring system and novel in-cage forelimb activity and hand function tests to evaluate functional outcomes. The wealth of new information and directions speak to the value of this shared approach to using the very valuable primate model. This project focuses on translation of our NHP stem cell work. We now report that neural stem cells (NSCs) derived from human spinal cord grafted early to hemisection sites in NHP SCI, extend very large numbers of axons over very long distances, and that these transplants appear to enhance long-term recovery of hand function, and support CST regeneration into the graft. NSCs derived from the approved human embryonic stem cell H9 (H9 hESCNSCs) also support CST regeneration into spinal cord grafts in the NHP after SCI. Further, we have advanced our cell therapy strategy to produce the first H9 hESCNSCs caudalized to move them towards a spinal cord fate, and have shown that transplants of these cells in rodents promote much more vigorous regeneration of CST axons7. Therefore, in this proposal in NHPs, we will transplant caudalized hESCNSCs into a contusion lesion at a more chronic and clinically relevant six week time point. We hypothesize that these grafts will support robust CST regeneration and enhance recovery of forelimb function, and provide a relay for CST axons to influence forelimb circuitry in the C8-T1 cord. We will use anterograde and retrograde tracing, IHC and transfection of graft cells and correlate the connectional data with recovery, and test the long-term survival, safety, and functional effects of these transplants.

This is a collaboration between experts at UCSD, UCLA, UCSF, UC Davis, UCI and the Ecole Polytechnique Federale de Lausanne (EPFL) to examine plasticity and regeneration in the non-human primate spinal cord. Our goal is to enhance knowledge and translational relevance of research on spinal cord injury (SCI). This project will map the motor cortex ?connectome? to better understand the primate motor network in both the intact state and after recovery from SCI, and will focus on promoting therapeutic growth of the most important motor control system in primates, the corticospinal projection. Aim 1: The Intact Primate Corticospinal Connectome Aim 1 will map the intact connectome for motor control of the hand using new generation, highly specific and highly sensitive viral tools. These findings will be correlated with motor cortex recordings and forelimb EMG activity in awake, behaving subjects. These tools will allow an unprecedented understanding of hand motor control in the primate, thereby revealing novel mechanisms of motor control and identifying new targets for therapy. Aim 2: The Lesioned Primate Corticospinal Connectome How does injury affect the corticospinal connectome? How does the corticospinal system adapt to injury and alter its set of outputs, and how does this influence functional recovery? We will use the elegant and novel tools of Aim 1 to map the injured, reorganized corticospinal connectome, motor cortex dynamics, and forelimb EMG after C7 hemisection lesions. Aim 3: Spinalized Neural Stem Cell Grafts to Enhance Corticospinal Repair Work in Aim 3 will build on recent progress in neural stem cell technology. We will use ?spinalized? neural stem cells to augment growth of the injured corticospinal tract and form trans- lesion relays that enhance recovery of forelimb function after SCI. Further, we will compare the connectome of the corticospinal system after therapeutic stimulation to its intact and lesioned state. This work will reveal how new corticospinal growth after injury impacts the projections and connections of this vitally important motor system in humans. 1

PROJECT SUMMARY Spinal cord injury (SCI) is a devastating condition that dramatically alters the lives of the patients who suffer from it. Although the first diagnosis of SCI occurred more than 4,000 years ago, there is still no efficient treatment for people suffering from SCI. In addition, the field of SCI research lacks biomarkers that can be utilized i) towards assessing the initial severity of the injury, a major factor in determining the downstream course of action, and/or ii) for predicting the long-term neurological recovery of the patient. Our group and others have shown that SCI generates a systemic inflammatory response that is detectable at cellular and molecular levels. However, putative biomarkers of SCI in blood or cerebrospinal fluid (CSF) have generally failed to replicate in validation studies. These findings are analogous to other fields (e.g. psychiatry) that initially pursued unsuccessful hypothesis-driven studies of candidate genes before turning to less biased high-throughput methods such as genomics, transcriptomics, and proteomics. This project will test the hypothesis that the transcriptomes of white blood cells (WBCs) contain important information about the severity and progression of SCI. We seek to decipher this ?encrypted? information by utilizing high-throughput RNAseq technology in a preclinical rat model of SCI. We will test our hypothesis with the following Specific Aims: Aim 1: We will use RNAseq to quantify the expression levels of all genes in rat WBCs at different acute and sub-acute timepoints after SCI. Aim 2: We will use advanced bioinformatics to discover gene modules in WBCs that are affected by the severity of SCI and/or the long-term neurological recovery. We expect that this study will yield novel biomarkers of SCI in a rat model system, and will subsequently serve as the basis for validation studies in humans.

PROJECT SUMMARY: Trauma to the spinal cord and brain (neurotrauma) together impact over 2.5 million people per year in the US, with economic costs of $80 billion in healthcare and loss-of-productivity. Yet precise pathophysiological processes impacting recovery remain poorly understood. This lack of knowledge limits the reliability of therapeutic development in animal models and limits translation across species and into humans. Part of the problem is that neurotrauma is intrinsically complex, involving heterogeneous damage to the central nervous system (CNS), the most complex organ system in the body. This results in a multifarious CNS syndrome spanning across heterogeneous data sources and multiple scales of analysis. Multi-scale heterogeneity makes spinal cord injury (SCI) and traumatic brain injury (TBI) 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 the holistic syndromes of SCI and TBI. In this sense, complex neurotrauma is fundamentally a problem that requires big- data analytics to evaluate reproducibility in basic discovery and cross-species translation. For the proposed TOP-VISION cooperative agreement we will: 1) integrate preclinical neurotrauma data on a large-scale; 2) develop novel applications of cutting-edge multidimensional analytics to make sense of complex neurotrauma data; and 3) validate bio-functional patterns in targeted big-data-to-bench experiments in multi-PI single center (UG3 phase), and multicenter (UH3 phase) studies. The goal of the proposed project is to develop an integrated workflow 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 with prior NIH funding we built one of the largest multicenter, multispecies repositories of neurotrauma data to-date, housing detailed multidimensional outcome data on nearly N=5000 preclinical subjects and over 20,000 curated variables. We will leverage these existing data resources and apply recent innovations from data science to render complex multidimensional endpoint data into robust syndromic patterns that can be visualized and explored by researchers and clinicians for discovery, hypothesis-generation and ultimately translational outcome testing.

PROJECT SUMMARY The role of the p75 neurotrophin receptor (p75ntr, NGFR1/TNFRSF16) in injury and repair is complex; p75ntr is associated with both cell survival and cell death, depending upon its interactions with multiple co-receptors 1. P75ntr is important for neural development, but is only sparsely expressed in the adult brain. But after CNS injury, p75ntr is re-expressed in both neurons and glia 1. We showed years ago that the pro-form of NGF (pro- NGF), which binds to p75ntr, can induce oligodendrocyte (OL) apopotosis, and that p75ntr deletion protects OLs after spinal cord injury (SCI) 2. There are now many studies demonstrating that deletion or antagonism of p75ntr after CNS trauma can improve outcomes after SCI and traumatic brain injury (TBI)3,4, although protection is not always found 5. We recently found that a novel p75ntr antagonist provided highly significant protection from neuronal and OL cell death, and improved neurological outcomes when given for 7 days after TBI in both rats 6 and mice 7. Treatment reduced the number of pro-inflammatory monocytes in the blood, and reduced the invasion of CCR2+ monocytes into the lesion site after cortical contusion injury (CCI)-TBI in mice 7. P75ntr is expressed in many peripheral tissues including immune cells 8-11. We found that the p75ntr antagonist reduced the pro-inflammatory effects of LPS on mouse leukocytes in vitro 7. In addition, we found that the p75ntr antagonist inhibited LPS-induced production of inflammatory monocytes, suggesting that p75ntr is involved in myeloid cell differentiation and peripheral inflammation after TBI. We want to test that novel hypothesis. There are two aims: AIM 1. We will assess the role of p75ntr in peripheral inflammatory responses by stimulating monocytes from wild-type (wt) vs p75ntr null mice with LPS in vitro. We will test the effects of the TLR4 agonist lipopolysaccharide (LPS) on myeloid cell differentiation in vitro in wild type (wt) C57Bl/6 myeloid cells vs myeloid cells from littermates with p75ntr deletion. We predict that KO of p75ntr will reduce NFkB signaling. As we are testing the in vitro effects of p75ntr deletion in the p75ntr KOs, we will generate mice with selective conditional knock-down of p75ntr in peripheral myeloid cells for the in vivo studies proposed for aim 2. AIM 2. We will cross available p75ntr floxed, and myeloid cell-specific CRE/ERT mice to yield a new mouse line for testing the role of peripheral immune cell p75ntr in TBI. We will use a conditional genetic approach to examine the role of p75ntr in myeloid cells by crossing the p75ntr floxed mouse12 with available transgenics with cre expression in myeloid cells to produce myeloid cell-specific deletion of p75ntr (Csfr1cre/ERT aka CD115creER). These mice will be given tamoxifen or vehicle prior to CCI TBIs to reduce p75ntr expression in peripheral myeloid cells, and we will compare the effects on the differentiation of mono/MAC lineage cells in the circulation and their trafficking to the injured brain. Together, these studies focus on peripheral effects of p75ntr signaling after TBI & test the novel hypothesis that p75ntr signaling in peripheral myeloid cells is an important part of the post-TBI injury/repair cascade.

PROJECT SUMMARY: Trauma to the spinal cord and brain (neurotrauma) together impact over 2.5 million people per year in the US, with economic costs of $80 billion in healthcare and loss-of-productivity. Yet precise pathophysiological processes impacting recovery remain poorly understood. This lack of knowledge limits the reliability of therapeutic development in animal models and limits translation across species and into humans. Part of the problem is that neurotrauma is intrinsically complex, involving heterogeneous damage to the central nervous system (CNS), the most complex organ system in the body. This results in a multifarious CNS syndrome spanning across heterogeneous data sources and multiple scales of analysis. Multi-scale heterogeneity makes spinal cord injury (SCI) and traumatic brain injury (TBI) 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 the holistic syndromes of SCI and TBI. In this sense, complex neurotrauma is fundamentally a problem that requires big- data analytics to evaluate reproducibility in basic discovery and cross-species translation. For the proposed TOP-VISION cooperative agreement we will: 1) integrate preclinical neurotrauma data on a large-scale; 2) develop novel applications of cutting-edge multidimensional analytics to make sense of complex neurotrauma data; and 3) validate bio-functional patterns in targeted big-data-to-bench experiments in multi-PI single center (UG3 phase), and multicenter (UH3 phase) studies. The goal of the proposed project is to develop an integrated workflow 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 with prior NIH funding we built one of the largest multicenter, multispecies repositories of neurotrauma data to-date, housing detailed multidimensional outcome data on nearly N=5000 preclinical subjects and over 20,000 curated variables. We will leverage these existing data resources and apply recent innovations from data science to render complex multidimensional endpoint data into robust syndromic patterns that can be visualized and explored by researchers and clinicians for discovery, hypothesis-generation and ultimately translational outcome testing.