Veronica Jean Tom - US grants

CORE E - Surgery Core - Veronica Tom, Ph.D., PI The Research Projects outiined in this Program Project will elucidate our understanding of several therapeutic approaches to spinal cord injury. All of the Projects rely heavily upon animal models of spinal cord injury. Thus, for the success of each Project and for the Program Project as a whole, reliability of surgical procedures is key. The Surgery Core will focus on measures that would increase the consistency of the surgical procedures. Such consistency is integral to the success of the experiments outiined in each of the Research Projects and will improve confidence in the results obtained. We will train personnel for uniformity and identify individuals dedicated to performing specific procedures. To facilitate and standardize training, we have developed a manual describing our Surgery procedures and a videotape is now available for those learning a technique. We will also provide any necessary surgical supplies. Dr. Veronica Tom will direct the Surgery Core and Dr. Takayami Yamagami will supervise its daily operation. Since animal models of spinal cord injury are involved in all of the Research Projects, the Surgery Core will be utilized by each of the individual Projects.

DESCRIPTION (provided by applicant): While the mature central nervous system has an overall diminished capacity for regrowth compared to an embryonic nervous system, axonal regeneration of some populations is possible when they are provided with a potently growth-permissive environment, such as one conferred by a peripheral nerve graft (PNG). Still, axons that are capable of regenerating into the graft fail to grow beyond the PNG to reinnervate spinal cord, at least partly because of the presence of the inhibitory glial scar that is present at the graft-host interface. Digesting the inhibitory extracellular matrix within the scar with chondroitinase ABC (ChABC) allows some axons to traverse the distal interface, synapse upon distal neurons, and mediate some behavioral recovery. Nonetheless, most of the axons remain in the graft. Thus it is important to develop strategies that enhance the ability of axons to regenerate out of a graft in order to increase their potential to improve function. We have preliminary data indicating that expressing constitutively active Rheb (caRheb) - a GTPase that is a critical regulatory component of protein synthesis - in adult neurons results in robust axonal outgrowth in an in vitro glial scar model and in vivo after SCI. In Aim 1, we will test the hypothesis that expressing caRheb after SCI will enhance regeneration into and beyond a PNG. We will use histological, physiological, and behavioral analyses to examine if caRheb augments axonal regeneration - especially following ChABC treatment of the distal graft interface - and if these regenerated axons form functional synapses that facilitate behavioral recovery. Any regeneration is irrelevant if these axons do not integrate into circuits. Though ChABC enhances plasticity, ChABC-induced sprouted/regenerated fibers do not always form synapses to impact function. Thus, it is also important to devise strategies to improve integration of these axons (i.. synaptogenesis) in order to exploit regrowth that we encourage. We recently demonstrated the proof-of-principle that providing a single exogenous factor enhances the integration of axons that regenerate out of a PNG. Recent work indicates that secreted glypican is sufficient to promote functional, excitatory synapse formation in vitro and behavioral recovery in a cerebral ischemia model in vivo. In Aim 2, we will test the hypothesis that providing exogenous glypican will enhance synaptogenesis of regenerated axons following SCI. We will graft a PNG into a SCI site that has been treated with ChABC to promote axonal regeneration. We will determine if infusing exogenous glypican into tissue surrounding the SCI site enhances synaptogenesis of these regenerated axons upon neurons distal to the PNG. We will gauge improvements in functional synapse formation physiologically and histologically by examining the colocalization of pre- and postsynaptic markers. We will also assess if enhanced synapse formation correlates with behavioral recovery. Because the strategies to be tested in Aims 1 and 2 take different approaches - enhancing the growth response and synaptogenesis, respectively - to promote functional repair after SCI, it is possible that even if the results in the previous Aims are incremental, combining the two approaches will lead to greater recovery than either alone. In Aim 3, we will use a novel, multipronged strategy to fill the lesion cavity with a growth-promoting substrate (PNG), enhance the growth response (caRheb), modulate the inhibitory properties of the glial scar (ChABC) and promote synaptogenesis (glypican). We will use histological and physiological analyses to examine if glypican enhances the functional integration of axonal regeneration beyond a ChABC-treated PNG interface that is induced by expressing caRheb and if this results in more robust behavioral recovery than what occurs with either caRheb or glypican treatment separately. These data will identify new therapeutic strategies that may eventually be translated to clinical use to improve the quality of life of persons with SCI.

PROJECT SUMMARY Cardiovascular disease and susceptibility to infection are two leading causes of morbidity and mortality for individuals with spinal cord injury (SCI). One of the major contributors to SCI-associated cardiovascular disease and immune deficiency is the syndrome autonomic dysreflexia (AD), an amplified reaction of the autonomic nervous system in response to sensory stimuli below the injury that manifests in 70%-90% of people who have sustained a high SCI. AD is hallmarked by extreme, sudden bouts of hypertension and reflexive bradycardia (i.e. heart rate decrease). Over time, AD events become more severe. These chronic, frequent episodes of hypertension are thought to lead to peripheral vascular dysfunction and immune suppression that contribute to cardiovascular disease and susceptibility to infection, respectively. Merely limiting AD intensity may have significant therapeutic value and improve SCI patients' overall health. The gradual exacerbation of AD is thought to be driven by plasticity of circuits below the lesion that results in an exaggerated spinal sympathetic reflex. Unfortunately, the mechanisms that trigger this maladaptive plasticity are still poorly understood, limiting the development of prophylactic treatments. Interestingly, an activated neuroimmune system is thought to be an underlying factor in aberrant plasticity and hyperexcitable circuits correlated with other pathologies. The pro-inflammatory, soluble form of the cytokine tumor necrosis factor ? (sTNF?) has been implicated in initiating inflammation in many contexts. Furthermore, sTNF? is associated with various forms of plasticity that could increase neuronal excitability. We hypothesize that sTNF? in spinal cord below a SCI plays a crucial role in triggering aberrant plasticity that leads to hyperactivity of the spinal sympathetic circuit after SCI and the secondary, intensification phase of AD. Moreover, this proposal will focus on the hypothesis that sTNFa/TNFR1 signaling in neurons involved in the circuit is central to the exacerbation. We will test our hypotheses using an established adult rodent spinal cord thoracic level 3 transection model that reliably results in AD. The overall goals of this multi-PI proposal are to: 1) further interrogate the therapeutic potential of inhibiting sTNF? to reduce AD (Aim 1); 2) investigate the mechanisms underlying how neuronal sTNF?/TNFR1 mediates AD (Aims 2 and 3).

PROJECT SUMMARY The lack of successful regeneration after injury in the mature central nervous system is due to intrinsic and environmental obstacles. Though progress in the field has been made, overcoming these barriers to result in robust functional axon regeneration and recovery is still a significant challenge. We build upon preliminary data and hypothesize that recurrent DREADD-mediated neuronal activation after injury is a means to enhance functional axon regeneration. Additionally, we will test the hypothesis that neuronal activation increases axon regeneration via both mTOR activation and increasing dynamic microtubules. We will these our hypotheses both in vitro using adult dorsal root ganglion neuron cultures and in vivo using dorsal root crush, dorsal columns spinal cord injury (SCI)/peripheral nerve graft (PNG), and incomplete cervical SCI/PNG models. In Aim 1, we will elucidate how repeated, chemogenetic neuronal activation enhances axon regeneration. In Aim 2, we will assess if neuronal activation ? alone or in combination with manipulations that increase dynamic microtubules and mTOR activation ? promotes functional axon regeneration. Collectively, we will: 1) elucidate mechanisms behind how neuronal activation enhances growth, potentially identifying new therapeutic targets; 2) determine the extent to which neuronal activation enhances regeneration in different injury models (dorsal root crush, SCI); 3) determine if neuronal activation has disparate or similar effects in different populations of neurons (DRG vs. CNS); 4) test whether meaningful axonal regeneration is facilitated using a unique, multi-faceted approach that: a) uses chemogenetic neuronal activation ? possibly along with further increasing dynamic microtubules and/or mTOR activation ? to enhance the axonal growth response; b) provides a more growth-permissive environment after an injury (i.e. mitigation of the inhibitory matrix of the glial scar with chondroitinase; transplantation of a PNG into an SCI cavity; chondroitinase digestion of plasticity-limiting perineuronal nets). After the completion of these experiments, we will have identified novel therapeutic avenues to foster functional repair after SCI.

Abstract Individuals suffering from chronic neurological disorders, such as spinal cord injury (SCI), are at greater risk of serious life-threatening complications from infections, including Influenza A virus (IAV) and pneumonia. Infections are the leading cause of re-hospitalization and mortality in patients living with chronic SCI. Therefore, reducing complications from infections is critical for improving the health and life span of SCI patients. Several groups, including ours, have endeavored to uncover the mechanisms underlying SCI-induced immune depression. For example, high-thoracic (T3) SCI disrupts sympathetic regulation of lymphoid organs and leads to impaired antibody synthesis and increased splenocyte apoptosis. Elegant studies by Ueno and colleagues demonstrated that high-thoracic SCI-induced immune dysfunction is due, in large part, to massive reorganization of the spinal sympathetic reflex circuit, e.g. the recruitment of glutamatergic interneurons, that results in increased sensitivity of this circuit. Silencing these glutamatergic interneurons restored immune balance, in the absence of pathogen challenge, demonstrating that immune balance can be affected by neurogenic mechanisms. However, the mediator(s) of pathological plasticity and glutamatergic interneuron activation post-SCI have not been established. We have exciting preliminary data suggesting that inhibiting soluble Tumor Necrosis Factor (sTNF) in the spinal cord following SCI: attenuates neuroinflammation and aberrant neuronal plasticity and activation, reduces immune dysfunction, and improves antiviral immunity (reduced viral load, increased specific CD8 T cells). Collectively, these data provide for a strong scientific premise to explore the role of sTNF in SCI- induced immune dysfunction. We hypothesize that heightened levels of sTNF in the spinal cord after injury play a crucial role in triggering robust neuroinflammation (e.g., NF-kB activation) and aberrant plasticity that, in turn, lead to hyperactivity of sympathetic circuitry after SCI and peripheral immune dysfunction. These important findings highlight the role of local sTNF signaling in influencing peripheral immunity. Based upon these data, in the following specific aims, we will: Aim 1: Determine the extent that sTNF/TNFR1 signaling in neurons contributes to immune depression following SCI. Aim 2: Investigate the contribution of sTNF to extrinsic (peripheral) factors of impaired antiviral immune responses in chronic SCI mice.