John R. Bethea, PhD - US grants

developmental neurobiology; neurogenesis; neurotrophic factors; congenital disorders; neural degeneration; clone cells; genetic manipulation; cell type; mammalian embryology; genome; genetic recombination; nucleic acid probes; laboratory mouse; histology; polymerase chain reaction;

Traumatic spinal cord injury (SCI) initiates a plethora of biological responses that are collectively called the secondary injury cascade. Secondary injury responses are thought to be a major mediator of cell death and tissue destruction, both within and remote to the lesion epicenter. It is proposed that immunological and/or inflammatory mechanisms are a critical component in the secondary injury phenomenon. The broad objective of this proposal is to demonstrate that traumatic SCI initiates immunological responses that exacerbate the initial injury and worsen functional outcome. It is also proposed that inhibition of injury induced inflammation by the potent anti-inflammatory cytokine interleukin-10 will be neuroprotective. In this proposal we will use a weight drop apparatus (N.Y.U. Impactor) to induce spinal cord injury in rats. The neuropathology generated by this methodology is reproducible and well documented in the literature. However, the cellular and molecular mechanisms that are generated and ultimately result in: systemic inflammatory responses, infiltration of inflammatory cells into the spinal cord, tissue destruction and cell death are not well understood. In specific aims 1 and 2 we propose to investigate the inflammatory responses initiated by traumatic SCI and determine how they mediate tissue destruction and cell death. In Specific Aims 3 and 4 we propose to study the anti-inflammatory and neuroprotective effects of interleukin-10 in vitro and in vivo. Finally, in Aim 5 we are going to determine if traumatic SCI in humans initiates an inflammatory response. These studies will provide essential information about the immunological consequences of SCI. Furthermore, the results of these studies may aid in the development of novel therapeutic protocols for the treatment of SCI.

Traumatic spinal cord injury (SCI) initiates a plethora of biological responses that are collectively called the secondary injury cascade. Secondary injury responses are thought to be a major mediator of cell death and tissue destruction, both within and remote to the lesion epicenter. It is proposed that immunological and/or inflammatory mechanisms are a critical component in the secondary injury phenomenon. The broad objective of this proposal is to demonstrate that traumatic SCI initiates immunological responses that exacerbate the initial injury and worsen functional outcome. It is also proposed that inhibition of injury induced inflammation by the potent anti-inflammatory cytokine interleukin-10 will be neuroprotective. In this proposal we will use a weight drop apparatus (N.Y.U. Impactor) to induce spinal cord injury in rats. The neuropathology generated by this methodology is reproducible and well documented in the literature. However, the cellular and molecular mechanisms that are generated and ultimately result in: systemic inflammatory responses, infiltration of inflammatory cells into the spinal cord, tissue destruction and cell death are not well understood. In specific aims 1 and 2 we propose to investigate the inflammatory responses initiated by traumatic SCI and determine how they mediate tissue destruction and cell death. In Specific Aims 3 and 4 we propose to study the anti-inflammatory and neuroprotective effects of interleukin-10 in vitro and in vivo. Finally, in Aim 5 we are going to determine if traumatic SCI in humans initiates an inflammatory response. These studies will provide essential information about the immunological consequences of SCI. Furthermore, the results of these studies may aid in the development of novel therapeutic protocols for the treatment of SCI.

[unreadable] DESCRIPTION (provided by applicant): In the progression of spinal cord injury (SCI), the first phase of injury, which involves mechanical tissue destruction, is followed by a phase of secondary injury, due to an impairment of blood supply and release of pro-inflammatory mediators from both invading and resident cells, such as lymphocytes, macrophages, microglia and astrocytes. Astrocytes respond to injury with the induction of reactive astrogliosis, a profound cellular activation whose functional significance is still a matter of debate. If, on one hand, reactive astrocytes release neurotrophins essential for neuronal survival and repair, on the other, they are responsible for production of pro-inflammatory molecules (cytokines, chemokines, growth factors, NO etc) detrimental to functional recovery. Many of the processes occurring in reactive astrocytes are regulated by NF-KB, a key modulator of inflammation and secondary injury. The studies outlined in this proposal are designed to investigate the role of astroglial NF-KB in SCI taking advantage of a transgenic mouse model generated in our laboratory, where NF-KB is functionally inactivated selectively in astrocytes. Based on extensive behavioral studies providing evidence that these transgenic mice display much greater functional recovery than wild type mice after SCI, we hypothesize that activation of NF-KB in astrocytes following SCI initiates transcriptional programs resulting in "deleterious" astrogliosis and ultimately increased damage. This hypothesis will be tested in a series of experiments organized in the following specific aims: 1) Determine the effect of inactivation of astroglial NF-KB on SCI-induced inflammation. 2) Determine the effect of inactivation of astroglial NF-KB on cell death following SCI. 3) Determine the effect of inhibition of astroglial-NF-KB on scar formation in the injured spinal cord. 4) Determine whether pharmacological inhibition of NF-KB activation is therapeutically effective in the treatment of SCI. These studies will contribute to the elucidation of the molecular mechanisms activated by NF-KB in astrocytes following SCI and how they can affect the survival and recovery of both glial and neuronal cells. This will lead to a better understanding of the pathophysiology of SCI and possibly to the development of novel strategies for therapeutic intervention. [unreadable] [unreadable]

Project Summary Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are believed to be initiated by T cell-mediated immune responses to myelin antigens. In recent years, however, a significant body of evidence has been compiled indicating the contribution of various cell populations within the central nervous system (CNS), such as microglia and astrocytes, to the development and progression of the disease. Nevertheless, the role of these cell types is far from being clearly understood. Chronic neuroinflammation and demyelination may also contribute to disease progression and chronic neurological deficits. In all these processes, in MS as well as in many other neurodegenerative diseases, astrocytes have been demonstrated to play an active role. Astrocytes respond to injury by becoming reactive or gliotic, a complex cellular response whose functional significance is still poorly understood. For instance, reactive astrocytes release neurotrophins essential for neuronal survival and repair, and are also responsible for the production of pro-inflammatory molecules (cytokines, chemokines, growth factors, NO etc) growth-inhibitory molecules detrimental to functional recovery. Many of the processes occurring in reactive astrocytes are regulated by NF-kB, a key modulator of inflammation and secondary injury. The studies outlined in this proposal are designed to investigate the role of astroglial NF-kB in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), taking advantage of a transgenic mouse model generated in our laboratory (GFAP-IkBa-dn mice) where NF-kB is functionally inactivated in cells expressing GFAP, such as astrocytes and non-myelinating Schwann cells. Preliminary data indicate that blocking astroglial NF-kB significantly reduces disease severity, improves functional recovery following EAE and reduces neuroinflammation and demyelination. This leads us to hypothesize that reactive astrocytes significantly contribute to disease progression and development of chronic neurological deficits in EAE and MS. This hypothesis will be tested in the four specific aims outlined below. While the results generated in our transgenic mice are very promising, the studies in Aim 1 will compare our GFAP-IkBa-dn mice to two additional mouse lines (described below) to confirm that the results obtained so far in our experimental model are uniquely associated with the astrocyte-specific inhibition of the NF-kB pathway. The first mouse line (73.12xffIKKb) is obtained by breeding a GFAP-Cre line developed in Dr. Sofroniew's laboratory to a floxed (f/f) IKKb line generated in the laboratory of Dr. Michael Karin. The second mouse line (GFAPCreERT2xffIKKb) is obtained by breeding a tamoxifen inducible GFAP-Cre line (CreERT2) developed in Dr. McCarthy's lab to the same floxed (f/f) IKKb line. In Aims 2 and 3 we will use the line(s) that provides the most robust clinical improvement over the corresponding control mice to further investigate the mechanisms at the basis of the protection provided by blocking astroglial NF-kB. Specifically, studies in Aim 2 will determine if there are differences in blood brain permeability and infiltration of leukocytes in the CNS of diseased WT and mutant mice. Studies in Aim 3 will determine the mechanisms through which inhibiting astroglial NF-kB promotes an anti-inflammatory response. Studies in this aim will focus on how inhibiting astroglial NF-kB alters T and B cell responses in the spinal cord. Finally, since demyelination is a hallmark of this disease and could be modulated by neuroinflammation, studies in Aim 4 will investigate the effect of the inhibition of astroglial-NF-kB on oligodendrocyte survival and demyelination. Our experiments will not only give insights into NF-kB signaling mechanisms, but also elucidate astrocyte responses under pathological conditions. Ultimately, our goal is to determine if interfering with these responses could be beneficial as a therapeutic strategy for MS and other neurological disorders.

DESCRIPTION (provided by applicant): Our research program investigates the role of astrocytes and in particular astroglial- NF?B in the pathophysiology of neurodegenerative disorders. The experimental aims proposed in this application will test two objectives and are based upon extensive preliminary data. In support of our first objective we have determined there is a significant reduction in oligodendrocyte death, oxidative injury and NADPH oxidase activity following injury. Furthermore, based upon completed and partially confirmed micro array studies, preliminary flow cytometry and immunostaining we have determined that inhibiting astroglial- NF?B greatly modifies the inflammatory environment in the spinal cord such that potentially toxic immunoregulatory molecules along with infiltrating leukocytes are significantly altered in injured TG mice relative to injured WT mice. With respect to the first objective we hypothesize that astrocyte mediated oligodendrocyte death is dependent upon engineering a robust inflammatory environment as well as complex interactions between oxidative pathways and excitotoxicity. In support of our second objective we have significant preliminary data that are very supportive of enhanced oligogenesis. First, we demonstrate there is significantly more white matter in our TG mice following SCI which could be due to reduced oligodendrocyte death (objective 1) and /or oligogenesis. We have also demonstrated there is enhanced myelin gene/protein expression in TG mice following injury, as well as transcription factors known to be important in oligogenesis. Finally it has been previously demonstrated that CXCL12 (SDF-1) and its receptors (CXCR4 and CXCR7) support oligogenesis and neurite extension on inhibitory substrates. Results from completed microarray studies that have been confirmed by quantitative RT-PCR and Western blotting have determined that specific immune/inflammatory molecules such as chemokines and their receptors (e.g., CXCL12 and CXCR4) are elevated in TG mice following SCI during periods of oligogenesis/remyelination and functional recovery. With respect to our second objective we hypothesize that inhibiting astroglial- NF?B promotes an environment that is favorable for oligogenesis and remyelination. These hypotheses and our experimental objectives will be tested in the following specific aims. Specific Aim 1: Investigate the role of oxidative injury in astrocyte mediated oligodendrocyte death and demyelination. Specific Aim 2: Investigate the role of inflammation in astrocyte mediated oligodendrocyte death and demyelination. Specific Aim 3: Determine what effect inhibiting astroglial- NF?B has on oligogenesis and remyelination following SCI. Specific Aim 4: Investigate the role of CXCL12 and CXCR4 in oligogenesis and remyelination following SCI. PUBLIC HEALTH RELEVANCE: Studies in this application will better define mechanisms of oligodendrocyte death and demyelination following spinal cord injury. In addition we will also investigate the role of astrocytes and secreted factors that may promote remyelination and oligodendrogenesis. Successful completion of these studies may lead to the development of therapies for spinal cord injury, multiple sclerosis and other neurodegenerative disorders.

DESCRIPTION (provided by applicant): Transplantation of cells into a spinal cord injury (SCI) is a promising approach to promote axonal regeneration across the injury in which continuity of fiber tracts has been interrupted. How effective this transplantation will be will depend upon the interfaces that form between the implant and the host tissue. If these interfaces are not permissive for the regenerating axons, then the cell therapy will not be suitably efficacious. Schwann cells (SCs) are clinically relevant because they can be transplanted into humans autologously; in rats they reduce large cysts that form following SCI, protect tissue from secondary damage, promote axonal regeneration across the injury, provide myelin and modestly improve hindlimb movements (Bunge 2008, Fortun et al 2009a). Our rat work so far has demonstrated that descending brainstem axons are able to cross the rostral host/SC interface and extend along the SC bridge following combination treatments or by simply injecting the SCs in fluid matrigel into the complete transection gap without additional interventions (as described in the Progress Report). The extension of GFAP+ processes into the SC bridge is key to creating a permissive rostral interface for brainstem axons to cross. But these regenerated axons do not cross the caudal interface. Regenerative failure is likely due to the intrinsic failure of adult axons to grow across the impenetrable host/SC caudal interface. The goals of this proposal are to: 1) modify the caudal interface to enable brainstem axons to cross it and grow into the spinal cord; and 2) enhance the intrinsic growth properties of injured adult brainstem neurons. To achieve these goals we will test three different strategies to make the caudal interface more permissive to axon growth and clinically relevant deep brain stimulation (DBS) and genetic approaches to enhance the intrinsic growth potential of injured brainstem neurons. First, SCs from adult nerve will be stimulated with a culture medium (developed in the Monje laboratory) that causes them to revert to an earlier precursor stage and then implanted. Second, inhibiting NF-kB and Eph receptor expression in SCs will be tested as a way to modify one side of the caudal interface. Third, the other side of this interface will be specifically modified by reducing NF-kB expression in astrocytes genetically or pharmacologically. Finally, we will combine the most effective strategy to enhance intrinsic growth of injured brainstem axons with the best strategy to modify the caudal interface to seek the most effective therapies. Outcome measures include assessment of SC and astrocyte co-mingling at the interfaces, counting GFAP+ processes that extend into the SC bridge, and counting GFP+ brainstem axons that regenerate across the interfaces. When axons are found to exit the SC bridge, retrograde tracing to identify the responding parent neurons and electrophysiological, locomotor (BBB, catwalk), sensory (mechanical and thermal) and autonomic (blood pressure, heart rate in response to colon distension) testing will be performed. Discovery of translational methods to improve exiting of regenerated brainstem axons from the SC bridge into the cord will have an important impact on fostering spinal cord repair, particularly for the use of SCs to mend human SCIs.

? DESCRIPTION (provided by applicant): Tumor Necrosis Factor (TNF) is a critical mediator of SCI-induced neuroinflammation. TNF exists in two biologically active forms, soluble-TNF (solTNF) and transmembrane-TNF (tmTNF) that preferentially bind to TNFR1 and TNFR2, respectively, and elicit quite distinct biological responses. The overarching goals of this competitive renewal are to: 1) investigate the therapeutic potential of pharmacologically manipulating TNF signaling to develop a therapy for traumatic SCI; and 2) investigate the mechanisms through which TNFR2 signaling on astrocytes and oligodendrocytes is neuroprotective. In support of our first goal we have determined that delivering XPro1595 directly to the injured cord for just 3 days, beginning 1h post-trauma, significantly improved functional recovery and reduced tissue damage, for up to 5 weeks. In contrast, etanercept, an inhibitor of both solTNF and tmTNF, did not improve functional recovery or tissue damage when delivered directly to the cord. The systemic administration of XPro1595 or etanercept did not improve functional recovery or reduce tissue damage. Thus, we hypothesize that solTNF is toxic to neurons and oligodendrocytes. Further, inhibiting solTNF and promoting tmTNF signaling through TNFR2 within the cord is therapeutic and neuroprotective following SCI. In support of our second goal, we are using genetic strategies to selectively delete TNFR2 from astrocytes, oligodendrocytes and OPCs in vivo to investigate what effect this has on functional recovery and tissue damage. Using GFAPcreER-TNFR2f/f mice we provide evidence that deleting TNFR2 expression on astrocytes worsens functional recovery and tissue damage following SCI. Next, utilizing CNPcreTNFR2f/f and PDGFR?creTNFR2f/f we show that oligodendrocyte and OPC TNFR2 are not required for normal myelination during development. However following injury, there is significantly more myelin damage in CNPcreTNFR2f/f mice. Finally, we show in vitro that TNFR2 induced oligogenesis is dependent in part in the IRE1a/XBP1 signaling. Thus, we further hypothesize that TNFR2 signaling on glial cells plays very specific role in reducing damage and promoting functional recovery. These hypotheses and experimental objectives will be tested in the following specific aims: Specific Aim 1: Investigate the therapeutic potential of XPro1595 and TNC-sc-mTNFR2, on functional recovery, histopathology and neuroinflammation following SCI. Specific Aim 2: Investigate the role of TNFR2 signaling on astrocytes in functional recovery, histopathology, and neuroprotection. Specific Aim 3: A) Investigate the role of oligodendrocyte-TNFR2 and OPC-TNFR2 in remyelination, oligogenesis, neuroprotection and functional recovery following SCI and B) Investigate the mechanisms through which TNFR2 induces oligogenesis, in vitro.

Project Summary It is becoming increasingly evident that plasticity within supraspinal networks, induced by therapeutic interventions, is necessary for optimal recovery of function after spinal cord injury. We have developed a novel combination therapy of motorized bike, 5-HT replacement therapy and treadmill training that can restore open-field weight-supported stepping (BBB score >9) in animals with complete spinal transection. Our preliminary data suggest that both supraspinal neuronal and glial plasticity modulated by therapy and that they influence each other. The central hypothesis of this proposal is that therapy combined with strategies to either promote beneficial neural/glial plasticity and/or attenuate deleterious plasticity (e.g., astrogliosis and inflammation) will enhance supraspinal remodeling and improve functional outcome. This Aim will be addressed with two Specific Aims. Aim 1: Investigate the impact of therapy on functional recovery and supraspinal plasticity after SCI as measured by changes in neurons and glial cells and their relationship to functional recovery. Aim 2: Determine if combining NCTherapy with: (A) strategies to enhance supraspinal plasticity (e.g. via brain-machine interface (BMI) training) and/or (B) inhibiting aspects of reactive gliosis (e.g. modulate TNF activity) is more effective than NCTherapy alone in improving functional recovery after SCI. The results of this work will aid in the development of therapies for recovery of volitional control of movement. Moreover, results could be used for translational research to develop assistive devices to maintain balance (e.g. cortical control of an exoskeleton or functional electrical stimulation). Glial plasticity is defined as a change in the number and or ?activation? of astrocytes and microglia in response to SCI or therapy after SCI. Neuronal plasticity includes changes in the organization of sensorimotor cortex and in neuronal firing patterns that carry information about sensory and motor events. The combined Bethea and Moxon labs have extensive experience measuring and manipulating glial and neuronal plasticity after spinal cord injury. By combining expertise, we can address, for the first time, how these two systems, neuronal and glial, interact to promote functional recovery. We will compare results from a series of 9 Experiments in animals with a complete spinal transection to those with a severe spinal contusion. These Experiments will assess electrophysiology changes (Experiments 1-4), the effect of lesioning the reorganized cortex (Experiment 5) and trace the source of this reorganization (Experiment 6). In Experiment 7, the impact of therapy on differences in spared fibers that cross the lesion will be measured. Finally, difference in the proteins/ genes associated with neuroplasticity and inflammation in the brains of animals will be compared between transected and contused animals (Experiments 8 and 9).

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).

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.