Phillip Popovich, Phd - US grants

This research will investigate to what extent the reparative potential of macrophages exists within the traumatically injured spinal cord. Historically, macrophages have been considered inflammatory scavenger cells in the CNS, capable only of removing cellular debris at sites of injury or infection. Now, many functions have been attributed to these cells including the ability to promote blood vessel growth, myelination, and neurite sprouting/regeneration. Unfortunately, these same cells can promote demyelination and cell injury. How and under what conditions macrophages effect such a broad range of biological functions is not clear, but may result from changes in the lesion microenvironment. It is the major hypothesis of this proposal that the inherent ability of macrophages to promote tissue repair and functional recovery changes as a function of time after spinal cord injury (SCI) and is negatively influenced by the accumulation of blood-borne elements (cells and proteins) at the injury site. Using radiation bone marrow chimeric rats in combination with macrophage and complement-depletion protocols, we will examine how these vascular constituents influence resident (microglia) and recruited (blood-derived) macrophage activation and function. Specifically, the neurotrophic and oxidative capacity of each macrophage subpopulation will be evaluated in vivo using immunohistochemistry and in vitro using cytokines and other factors present in the injury site to trigger macrophage effector functions. Cells exhibiting neurotrophic secretory profiles will be transplanted into injured rat spinal cords to test their ability to promote regeneration and functional recovery. By learning more about the factors that influence macrophage function in the injured CNS, we may be able to harness the innate reparative potential of the inflammatory response (specifically macrophages) to promote functional regeneration. Moreover, we will be determining the feasibility of manipulating an intrinsic component of the injury site to promote tissue repair. Such an approach may be biologically and clinically advantageous and could eliminate the need for chronic drug therapy.

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) triggers a neuroinflammatory reaction that can exacerbate tissue damage and promote repair of injured neurons and glia. Exploitation of immune-mediated repair mechanisms and antagonism of degradative immunological cascades has therapeutic value. Unfortunately, these mechanisms and cellular/humoral cascades remain enigmatic. To date, studies of neuroinflammation after SCI have focused largely on neutrophils, microglia and/or macrophages. However, T-lymphocytes also infiltrate the traumatized spinal cord; yet, their roles in processes of secondary degeneration and repair are poorly defined. Given that T-cells directly influence blood-brain barrier integrity, axonal conduction, extracellular matrix composition, macrophage/microglial function and neuronal/glial survival, activated T-cells undoubtedly affect recovery from SCI. We have demonstrated that SCI primes the activation (i.e., proliferation and cytokine production) of peripheral T-cells and that activated T-cells infiltrate the injury site. How and to what extent these cells influence recovery from SCI is not known. Studies in Aim 1 will evaluate T-cell influences on the normal progression of SCI pathology and functional recovery. A systematic manipulation of all T-cells will be accomplished using nude rats and antibody-mediated depletion of T-cells. In Aim 2, we will determine whether recovery from SCI can be improved by inhibiting CNS myelin-reactive T-cells -- cells that we have previously shown exacerbate pathology and impair functional recovery after SCI. Selective depletion of myelin-reactive T-cells will be accomplished using a clinically feasible oral tolerance paradigm. Newer preliminary data has prompted us to also consider whether other (non-myelin reactive) T-cells can be exploited for therapeutic purposes. Accordingly, studies in Aim 3 will determine whether heat shock protein-reactive T-cells can convey neuroprotection and improve recovery from SCI by suppressing acute neuroinflammation. Studies in Aim 4 will explore a suspected mechanism of T-cell mediated injury after SCI, i.e., activation of recruited macrophages. This will be accomplished by macrophage depleting animals with enhanced myelin-reactive T-cell function. The primary hypothesis to be tested in this proposal is that T-cells exert pathological and neuroprotective effects within the injured spinal cord. This functional diversity depends on the phenotype and antigen-specificity of recruited T-cells as well as the cellular and biochemical milieu at the injury site.

DESCRIPTION (provided by applicant): Catecholamine "storms" and spikes in circulating glucocorticoids (GCs) are immune suppressive and are elicited by physical or psychological stress. In tetraplegics and high-level paraplegics, it is common for recurrent bouts of autonomic dysreflexia (AD) to develop weeks or months after spinal cord injury (SCI). AD is a profound physiological stress that elicits marked dysregulated release of norepinephrine (NE) and GCs. This might explain why those with high-level SCI exhibit signs of systemic immune suppression and are at increased risk for infection. The immune suppressive effects of AD also would minimize or block the efficacy of prophylactic vaccines designed to boost host-defense. This application will test the novel hypothesis that AD is a potent and recurrent mechanism of post-SCI immune suppression. Two specific aims are proposed to test this hypothesis. In both aims, mice will receive high (T3) or low level (T9) SCI. In Aim 1, we will compare immune function between T3 SCI mice that experience spontaneous AD and T9 SCI mice that do not develop AD. We will monitor episodes of AD in awake, freely moving animals by telemetric monitoring of blood pressure. The frequency and duration of AD will be determined from BP traces obtained during the previous 24 hours. For those mice that develop at least two bouts of AD in the preceding 24 hours, blood will be collected to measure circulating antibodies and leukocytes. Time matched bleeds from T9 SCI mice will be collected for comparison. Using purified lymphocytes from spleen, we will also complete terminal analyses (42 dpi) of lymphocyte function. In Aim 2, we will intentionally trigger AD in T3 SCI mice then subsequently immunize them with prophylactic vaccines designed to elicit production of anti-viral or anti-bacterial antibodies. Again, comparisons will be made between T3 and T9 mice. This will test whether AD adversely affects the ability of the immune system to respond to pathogens that cause morbidity/mortality after SCI. If successful, we will have identified a mechanism underlying idiopathic SCI-induced immune suppression and subsequent increased susceptibility to infection. Also, we will have identified therapeutic targets for bolstering the efficacy of influenza or pneumonia vaccines. PUBLIC HEALTH RELEVANCE: This proposal will determine whether autonomic dysreflexia, a serious complication suffered by patients with high level spinal cord injury, suppresses immune function. Data from these studies will be used to develop novel clinical therapies to treat spinal cord injury in humans.

This contract is to identify one of two NINDS [unreadable]Facility of Research Excellence in Spinal Cord Injury (FORE-SCI) sites to conduct research to replicate promising studies that could lead to new and effective treatments for SCI. The objective of this contract is to independently review and replicate novel treatments for SCI and to compare the efficacy of different treatments in a standardized environment with a minimum of variability in surgery, animal care, outcome evaluation and cellular analyses.

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) activates immune cells that cause tissue damage in the CNS. To date, most studies in this area have focused on the injurious effects of macrophages and T cells. However, antibodies that bind CNS proteins accumulate in sera and cerebrospinal fluid of people with SCI, suggesting that activated B cells may contribute to post-traumatic inflammatory damage. During the last five years, we have shown that SCI triggers B cell activation and autoantibody synthesis. Importantly, mice without B cells have smaller lesions and improved recovery after SCI. In this renewal application, we will test the hypothesis that antibodies produced after SCI exacerbate neuron and glial pathology thereby limiting functional recovery. Three specific aims are proposed. In Aim1, we will use proteomics to reveal the identities of proteins that activate B cells after SCI. In Aim 2, we will investigate the biological effects of purified antibodies obtained from SCI mice at different times after injury. This will be accomplished by injecting purified antibodies into specific regions of the spinal cord followed by behavioral and electrophysiological analysis of spinal cord function. Antibody-mediated changes in neuron/glial survival, axon pathology and/or demyelination will be documented using standard immunohistochemical techniques. Also in Aim 2, we will evaluate the mechanisms responsible for any detrimental effects caused by intraspinal antibody injection. This will be accomplished by injecting antibodies into mice that have been genetically modified such that they lack key proteins that known to mediate the effects of antibodies. In Aim 3, we will determine if two key B cell survival factors are responsible for the chronic intraspinal activity of B cells after SCI. Specifically, we will document spatiotemporal induction patterns and sources of BAFF and APRIL after SCI. To determine if these growth factors can be manipulated to stem B cell and antibody-mediated pathology after SCI, we will infuse the injury site with decoy receptors that will block BAFF and APRIL signaling. Collectively, the experiments in this proposal will provide novel information about the contributions of antibody-producing B cells to the tissue damage caused by SCI. Moreover, these studies will likely reveal novel therapeutic targets for treating SCI.

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) triggers a neuroinflammatory reaction that can aggravate tissue injury (e.g., neuronal death, axonal injury, demyelination) and promote repair (e.g., axon regeneration, remyelination, revascularization). Recently, functionally distinct subsets of macrophages, i.e., M1 (pro-inflammatory) and M2 (anti-inflammatory) cells have been identified at sites of SCI which may underlie the functional dichotomy. M1 macrophages are neurotoxic and dominate the injured spinal cord for several weeks post-injury. In contrast, M2 macrophages promote axon growth, even in the presence of inhibitory molecules (e.g. CSPG and myelin), without concomitant neurotoxicity. Unfortunately, M2 macrophages populate the injury site for only a few days, eventually becoming overwhelmed by M1 macrophages. Thus, the high M1:M2 ratio might explain why repair of the injured CNS is slow and inefficient relative to tissues in the periphery. We predict that the efficiency and magnitude of spinal cord repair will be improved by modulating the phenotype and function of macrophages that respond to the injury. We will test this hypothesis using genetic loss-of-function (knockout) and gain-of-function (lentiviral) techniques to manipulate expression of the triggering receptor expressed on myeloid cells-2 (TREM2) on resident microglia and myeloid precursor cells, i.e., cells that give rise to monocyte-derived macrophages (MDMs), since overexpressing TREM2 in macrophages induces an M2 phenotype. In Aim 1 we will examine how TREM2 overexpression or selective knockout on microglia vs. MDMs effects inflammation, motor recovery and anatomical indices of repair after contusive spinal cord injury. By using lentiviral constructs to overexpress TREM2 on MDMs in a model of dorsal spinal hemisection injury in Aim 2, we will determine if TREM2 manipulation influences macrophage effects on myelin/axon phagocytosis, axon regeneration and axonal retraction or die-back of injured axons. Using a model of focal intraspinal demyelination (lysolecithin) in conjunction with the gain of function protocols, we will determine if manipulating macrophage TREM2 affects OPC differentiation and remyelination within the spinal cord in Aim 3. The current proposal outlines proof-of-principle experiments that will advance our understanding of how a distinct molecular signaling pathway, i.e., TREM2, influences the natural course of CNS macrophage function after SCI. Importantly, if data from these studies indicate that manipulating TREM2 confers anatomical or functional benefits with minimal or no adverse effects on CNS structure or function, then it should be feasible to develop similar protocols for human clinical trials. Indeed, intravenous delivery of bone marrow cells (the primary technique to be used in the proposed studies) has already been tried in SCI patients and without adverse effects. Moreover, enzyme replacement therapies, using lentiviral transduction of autologous peripheral blood mononuclear cells, were shown to be safe and effective in human subjects.

DESCRIPTION (provided by applicant): After spinal cord injury (SCI), M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages populate the lesion site. M1 macrophages, which cause neurotoxicity and hamper neuroregeneration, persist in SCI lesions. In contrast, M2 macrophages, which support axon growth and are not neurotoxic, disappear after a few days. This progressive loss of M2 macrophages is thought to be due to the conversion of newly activated microglia and infiltrating monocytes into M1 cells as they respond to pro- inflammatory cues in the acute SCI environment. This phenomenon has so far impeded attempts to harness the regenerative power of M2 macrophages to improve SCI recovery. Therefore, therapeutic strategies that suppress M1 and enhance M2 macrophages in the highly inflammatory SCI environment are actively sought. We have recently identified a specific microRNA (miRNA) required for M1 differentiation. miRNA are small RNAs that post-transcriptionally regulate gene expression networks, contributing to cell differentiation and lineage choice. We predict that specific modulation of this miRNA will reduce the ratio of M1/M2 macrophages after SCI, thereby improving the efficiency and extent of tissue repair after SCI. This hypothesis will be tested in an in vivo model of SCI, using mice deficient for this specific mRNA, and developing a pharmacologic strategy to modulate macrophage phenotype with specific miRNA inhibitors. Since miRNA can be manipulated to treat human disease, these studies will provide the basis for development of promising new therapies. In addition, understanding how this specific miRNA controls inflammation through regulation of the M1/M2 balance will have important implications to regulation of macrophage-mediated inflammation (or immune suppression) in other conditions, such as atherosclerosis, wound healing and cancer. PUBLIC HEALTH RELEVANCE: These studies investigate the role of the small RNA miR-155 in spinal cord injury inflammation, pathology and functional recovery. The effects of macrophage-targeted miR-155 loss in control of inflammation and enhancement of neuroregeneration will be assessed in a rodent model of spinal cord injury. These studies will likely impact therapies for spinal cord injury and other conditions such as atherosclerosis, wound healing and cancer.

DESCRIPTION (provided by applicant): Autonomic dysreflexia (AD) is a potentially fatal clinical syndrome that develops after high-level spinal cord injury (SCI) and results in uncontrolled hypertension. Early symptoms may include throbbing headache, profuse sweating or nasal congestion but if left untreated, seizure, pulmonary embolism, stroke or death can occur. The symptoms and frequency of AD can be minimized by removing the stimulus that triggers AD (e.g., full bladder/bowel) but there currently is no way to prevent AD from developing. New data show that besides causing potentially fatal hypertension, recurrent bouts of AD also suppress immune function. This may explain why people with high-level SCI are more susceptible to infection - a leading cause of morbidity and mortality in this patient population. Three Aims are proposed to answer two main questions. First, is it possible to restore immune function in SCI mice despite persistent AD (Aim 1)? Second, is it possible to block or minimize AD and indirectly improve immune function (Aims 2&3)? Experiments in Aim 1 will use a novel combination of drugs designed to promote immune cell survival in SCI mice with AD. Experiments in Aims 2&3 will try to prevent the development of AD by inhibiting post-injury synaptogenesis in the spinal cord. This will be accomplished using genetic loss of function and pharmacologic techniques (Aim 2) or, alternatively, by using a fetal neural stem cell graft to restore communication between injured supraspinal axons and spinal autonomic circuitry below the injury. If successful, data from Aim 1 could be used to develop new drug regimens that would reduce post-SCI immune suppression, especially in people that experience frequent AD. Aims 2 and 3 are basic science experiments, designed to reveal novel molecular targets (Aim 2) or establish the feasibility of using neural repair strategies (Aim 3) to prevent the development o AD and restore immune function. Collectively, experiments in this proposal address an unmet need for people living with a SCI, i.e., improving or reversing problems associated with autonomic dysfunction. If successful, data from these experiments could significantly improve quality of life for SCI people and provide significant savings to national health care costs.

The Sixteenth International Symposium on Neural Regeneration (ISNR) is planned for November 30- December 4, 2015 at the Asilomar Conference Center in Pacific Grove, California. Similar symposia have been held every two years at Asilomar beginning in 1985. NIH has continuously co-sponsored the symposia since 1987. The Craig H. Neilsen Foundation has already donated funds for the 2015 meeting and applications for support have been or will be submitted to the Paralyzed Veterans of America, the Rick Hansen Institute and several other private foundations that support neural regeneration or spinal cord injury research. The primary purpose of this meeting is to present cutting- edge research in neural regeneration, especially in areas where notable recent progress has been made. A secondary purpose is to foster an atmosphere that is both stimulating and conducive to a free interchange of ideas among all conference attendees. The longer-range plan is to continue to hold these symposia on alternate years at the same site, and to vary the programs of successive symposia so that broad coverage of regeneration biology is achieved. The ISNR have become established, regularly occurring events with high attendance by both students and internationally recognized experts in the field of neural regeneration.

Abstract Spinal cord injury (SCI) disrupts the autonomic nervous system (ANS), impairing the ability of the ANS to coordinate organ function throughout the body. Emerging data indicate that systemic pathology resulting from ANS dysfunction contributes to intraspinal pathology and neurological impairment. For example, the post-injury onset of neurogenic bowel and immune suppression can cause gut dysbiosis ? a pathological state where beneficial symbiotic bacteria (probionts) in the GI tract become outnumbered by aggressive bacteria (pathobionts). Recent data from our lab show that SCI triggers gut dysbiosis, which impairs functional recovery and exacerbates lesion pathology. Since different types of gut bacteria exert unique effects on the host and these effects can vary by sex, it is important to understand how gut ecology changes as a function of time, spinal injury level and injury severity in both males and females. Accordingly, experiments in Aim 1 will use state-of-the-art PhyloChip technology to profile post-SCI changes in gut microbial communities in male and female mice as a function of injury severity, time post-injury and injury level. The primary goal is to identify post-injury changes in gut microbe populations that could be manipulated for therapeutic gain. Gut microbe manipulation is clinically feasible and can profoundly affect mammalian physiology. Indeed, we found that functional recovery is improved and lesion pathology reduced in mice treated post-SCI with a medical-grade probiotic (VSL#3). Aim 2 will explore the mechanisms underlying VSL#3-mediated neuroprotection. SCI can affect the gut microbiome but the altered microbiota can in turn affect the immune system and spinal cord. Aim 3 will examine how SCI-induced gut dysbiosis influences macrophage phenotype and function. Emerging data indicate that gut microbes can cause transcriptional and epigenetic changes in macrophage precursors in bone marrow. Such changes can render mature macrophages more or less responsive to subsequent inflammatory stimuli, including those found in the injured spinal cord. Using germ-free mice (devoid of any commensal microbe) and fecal transplantation to selectively recolonize mice with control or SCI microbiota, we will test whether gut dysbiosis adversely affects macrophage function. Rather than ?treat the spinal cord?, this proposal seeks new ways to treat SCI as a systemic disorder caused by breakdown of the spinal cord-gut-immune axis.

PROJECT SUMMARY/ABSTRACT This project will study how glucocorticoids (GCs) interact with glucocorticoid receptors (GRs) in sensory neurons and glia to effect the development and maintenance of pain after nerve injury. The majority of studies examining nerve-injury induced pain do not examine where GC/GR interactions are important for pain development and or how they contribute to the changes that arise after injury (e.g. reactive gliosis). Thus, this project is relevant to the NIH mission because it examines the fundamental importance of GRs in various nervous system cell types and will elucidate where GR expression is required for the development of neuronal hyperexcitability and pain after injury. Even though GR expression in the spinal cord increases after injury, whether GR activity is necessary for downstream responses has not been demonstrated. Thus we will use Cre-Lox systems to evaluate GR loss in sensory neurons, microglia and astrocytes with and without elevated corticosterone levels to determine where GC/GR interactions are influencing pain and neuronal excitability. Extensive sensory behavior testing will determine if loss of GR in each cell type effects reflexive and spontaneous pain development and maintenance. Three-dimensional tissue clearing techniques will provide an overarching view of neuronal excitability changes using cFOS in the spinal cord and brain, as well as facilitate the visualization of changes in sensory process innervation in the periphery and in the CNS. Changes in sensory neuron excitability will be directly measured using patch-clamp recording. Glial activation, gene expression, and morphology will be extensively examined in each condition. Together, these data will provide fundamental insight into glucocorticoid receptor biology in sensory neurons, microglia, and astrocytes as well as discern the importance of GR actions in neurons versus glia in an injury environment.

The singular goal of most spinal cord injury (SCI) research programs is to repair the injured spinal cord and restore locomotor function. Unfortunately, restoration of walking is a low priority for most SCI individuals 1. In addition to impaired mobility, SCI causes slow and steady pathological changes in organ systems throughout the body. Failure to recognize and treat multi- organ system pathology as a standard of care may explain why survival rates have not improved for SCI patients (relative to able-bodied individuals) over the past 30 years 2. Emerging data indicate that after SCI, the loss of sympathetic tone and the development of aberrant spinal autonomic reflexes that control immune organs (e.g., spleen) and the major organs that control metabolism (e.g., liver, adrenal gland, muscle, adipose tissue and gut) cause immune dysfunction and multi-organ pathology. Thus, mitigating the onset and downstream consequences of post-injury dysautonomia could improve immune and metabolic homeostasis. Since immune and metabolic processes are normally tightly coupled and are essential for life 3,4, it is likely that most, if not all, co-morbidities that affect SCI individuals (e.g., spontaneous infections in lung or skin, impaired wound healing, non-alcoholic fatty liver disease (NAFLD), chronic depression, atherosclerosis, type 2 diabetes, fatigue and anxiety), can be explained by impaired immunometabolism. Experiments in this proposal are designed to study SCI as a disease of the entire body and will test the overall hypothesis that post-injury dysautonomia breaks neuro-immune homeostasis creating a state of ?neurogenic meta-inflammation?. This proposal is an integration of currently funded NINDS R01 grants and new ideas. All experiments and concepts will build on my lab's past successes using both ?macroscopic? (systems and networks) and ?microscopic? (cells to molecules) tools to study the pathophysiological significance of neuro-immune interactions. Just as recent NIH initiatives have emphasized that cures for human brain disease are likely to arise from better understanding of brain networks or circuits, rather than defects in a single brain region, a cure for SCI is unlikely to originate from a focus only on repairing the injured spinal cord.

Neuroimmunology is the interdisciplinary study of interactions between the immune system and the central nervous system in health and disease. The Ohio State Wexner Medical Center is a unique training environment in this context because the faculty mentors of the proposed training program have expertise that covers all major aspects of basic and clinical Neuroimmunology. The goal of this training grant is to provide comprehensive training across key areas of relevance within the field of neuroimmunology including neurotrauma, autoimmunity, stress, neural development, aging, and biological rhythms. Students will enter the program either soon before or immediately after successful completion of their candidacy exam. These students will have an opportunity to work in a highly collaborative research environment with established mentors and clinical partnerships. Thus, we are unique nationally in terms of crossing the boundaries of traditional neuroscience and immunology training, and therefore are positioned to offer students an exceptional training experience in Neuroimmunology that will extend beyond the conventional training experiences that currently exist within ?umbrella? graduate training programs. Trainees will engage in unique program-specific learning and professional development experiences including course-work, clinical experiences/mentorship, and professional networking with scholars in the Neuroimmunology field. These trainees will be a benefit to the national neuroscience and immunology communities based on their advanced graduate training experience that is augmented by a translational experience with the clinical pairing. Our TPNI students will be positioned to excel as post-doctoral fellows and will be competitive for future faculty positions.