Alan Faden, M.D. - US grants

We have suggested that endogenous opioids and opiate receptors play a role in the pathophysiology of secondary spinal cord injury that follows trauma, based on the therapeutic effects of opiate antagonists in certain models of spinal cord injury. Increasing circumstantial evidence from our laboratory over the past two years has implicated the dynorphin opioid system and the Kappa opiate receptor in the injury process: the goals of the studies are to more specifically address these questions. A series of parallel, multidisciplinary studies in the rat are planned. These includes: (a) measurement of changes in endogenous opioid immunoreactivity in spinal cord after trauma; (2) determining whether dynorphin tolerance reduces the pathophysiological consequences of spinal cord trauma, including changes in somatosensory-evoked responses, blood pressure, spinal cord blood flow, histopathology and neurological function; (3) evaluating whether acute dynorphin infusions, below those necessary to produce paralysis, exacerbate the consequences of traumatic spinal injury; (4) examinaing whether intrathecal infusions of antibody to dynorphin A-(1-17) reduce the severity of spinal injury produced by trauma; (5) investigating the effects of upregulation of opiate receptors (through use of chronic administration of opiate antagonists) on the response to injury; and (6) comparing the effects of a recently developed opiate antagonist (with increased activity at Kappa sites), its dextrostereoisomer and naloxone on outcome after traumatic spinal injury; and determining whether beneficial effects of opiate antagonists are mediated by stemic actions or through local effects in the spinal cord. Taken together, the proposed studies address the hypothesis that a specific endogenous opioid (dynorphin), acting through a specific opiate receptor (Kappa-receptor), mediates certain pathophysiological consequences of spinal cord trauma. Such findings, if demonstrated, would serve to enhance understanding of the mechanism of secondary spinal cord injury and suggest more effective forms of pharmacological therapy.

magnesium; glutamates; bioenergetics; brain injury; sodium; NMDA receptors; potassium; inhibitor /antagonist; mitochondria; calcium; extracellular matrix; cell water; stimulant /agonist; chlorine; neurochemistry; magnetic resonance imaging; laboratory rat;

DESCRIPTION (Adapted from applicant's abstract): Traumatic brain injury (TBI) or traumatic neuronal injury (TNT) in vitro causes neuronal apoptosis, in part, through activation of caspase-3-like proteases. inhibition of caspase-3 in vitro reduces posttraumatic cell death and provides additive neuroprotection to that produced by agents that inhibit necrotic cell death. Caspase-3 activation is modulated by upstream caspases, including caspase-9 (intrinsic pathway) and caspase-8 (extrinsic pathway). Our preliminary data suggests that the caspase-9 pathway appears to be more important in neurotrauma. Akt (protein kinase B) is a well-established anti-apoptotic factor, which may act, in part, by modulating caspase activation. Akt itself can also be modulated by several factors, including the novel tumor suppression protein PTEN. Recent experiments in our laboratory have suggested a role for PTEN in neuronal apoptosis. We propose to examine the role of Akt in neuronal apoptosis after TB! and TNT, and elucidate the critical upstream and downstream signal transduction pathways involved. Specific hypotheses include: 1) caspase-9, but not caspase-8, represents an important upstream modulatory mechanism for caspase-3 mediated apoptosis after trauma; 2) Akt plays an important modulatory role in apoptosis following TBI or TN! in vitro; 3) Anti-apoptotic actions of Akt include inhibition of caspase-3 activation by phosphorylating the pro-apoptotic factor BAD, as well as through other non-caspase mechanisms; 4) The recently identified tumor suppressor factor PTEN is activated after trauma or trophic withdrawal and contributes to neuronal apoptosis, in part, by downregulating Akt activity; and 5) Development of PTEN antagonists may provide a novel neuroprotective treatment strategy for CNS injury. We propose the following specific aims: 1) To examine the relative contributions of the intrinsic (caspase-9) and extrinsic (caspase-8) pathways in modulating caspase-3-induced apoptosis in TB! and TNT, using complementary in vivo and in vitro model systems; 2) To establish an anti-apoptotic role for Akt in TBI and TNT and examine proposed mechanisms, including phosphorylation of BAD. 3) To demonstrate a pro-apoptotic role for PTEN in TBI and TNI, and show that this action results substantially from its ability to downregulate Akt, resulting in activation of caspase-9 and caspase-3; and 4) To show that PTEN antagonists, newly developed by us in collaboration, are neuroprotective following injury in vivo and in vitro.

DESCRIPTION: (Adapted from the Investigator's Abstract) : Brain or spinal cord trauma initiates an endogenous autodestructive process-a cascade of biochemical and metabolic changes that causes delayed neuronal cell death. Pharmacological treatment approaches have generally focused on inhibiting a single injury factor, despite the clear recognition that secondary tissue damage reflects a multifactorial injury process. The failure of any drug treatment strategy to date to improve recovery after human brain trauma supports the conclusion that targeting single components of secondary injury may not be sufficient to substantially modify posttraumatic recovery. The tripeptide, thyrotropin releasing hormone (TRH), modulates multiple components of the secondary injury cascade and treatment with TRH or certain TRH analogs improves outcome across a variety of neurotrauma models and species. In addition, TRH or related analogs have cognitive enhancing effects. However, these compounds also have other substantial physiological actions- including autonomic, endocrine and analeptic effects-that may not be optimal for treatment of severe head injury or for chronic administration (i.e. for cognitive action). For example, pressor effects may serve to increase intraparenchymal bleeding, increased body temperature may limit certain neuroprotective effects; and analeptic actions may compromise ability to utilize pharmacological coma treatments. Based upon extensive structure-activity studies, we have conceptualized and developed novel prototypic analogs of TRH and its dipeptide metabolite that exhibit both neuroprotective and nootropic properties, but appear to be devoid of other key physiological effects of TRH including endocrine, autonomic and analeptic actions. Moreover, using pharmacophore modeling techniques we have identified non-peptide mimics of the effective tripeptides. The proposed studies are intended to extend these preliminary observations by addressing the following hypotheses: (1) small peptide structures, related to TRH but devoid of the primary physiological actions of TRH, provide neuroprotection after traumatic brain injury, (2) neuroprotective actions of these compounds result from modulation of multiple components of the secondary injury cascade, including necrotic and apoptotic pathways; (3) these drugs may also be used to enhance long term cognitive function after brain injury; and (4) non-peptide mimics of these effector compounds may also serve as prototypes for novel drug discovery. Specific aims are to demonstrate that prototype tripeptide and dipeptide derivatives of TRH: (1) inhibit multiple components of the secondary injury cascade after traumatic brain injury; (2) have a high therapeutic index and a broad therapeutic window; (3) reduce both apoptotic and necrotic cell death; and 4) have nootropic properties and enhance cognitive function after chronic brain injury.

Spinal cord injury (SCI)causes neuronal cell death combined with astroglial proliferation and inflammation associated with activation of microglia. Upregulation of cell cycle proteins occurs after central nervous system (CMS) trauma, and appears to contribute to apoptotic cell death of post-mitotic cells such as neurons and oligodendroglia. It also likely contributes to post-traumatic gliosis and microglial activation. Recent studies in our laboratory have shown significantly increased expression of many cell cycle proteins after SCI in rodents, with the proteins co-expressed in neurons showing caspase-3 activation and morphological features of apoptosis. Moreover, in several classical models of caspase-3 dependent apoptosis in primary neuronal cell cultures, injury is associated with upregulation of many of these same cell cycle proteins. In addition, studies by us and others indicate that inhibition of key cell cycle regulatory pathways reduces injury-induced cell death both in vitro and after acute brain injury. We recently found that treatment with a cell cycle inhibitor after SCI in rats markedly reduces lesion volumes and the surrounding glial scar;it also significantly improves motor functions following injury. The proposed studies are intended to address the following hypotheses: (1) SCI up-regulates key cell cycle constituents at both the mRNA and protein levels in neurons, oligodendroglia, astrocytes, and microglia and induces activation of the cell cycle pathways;(2) such upregulation and activation promotes apoptosis in neurons and oligodendroglia;(3) upregulation and activation of cell cycle proteins also contribute to proliferation of astrocytes as well as microglial activation and subsequent release of associated inflammatory factors;and (4) treatment with cell cycle inhibitors is neuroprotective, through mechanisms that include inhibition of caspase-dependent apoptosis in neurons and oligodendroglia, along with reduced glial activation, diminished release of microglial mediated inflammatory factors and attenuation of the SCI- induced immuneresponse. The specific aims are to demonstrate that: (1) a. SCI causes increased expression of critical cell cycle related genes/proteins (including c-myc, cyclin D1, CDK4, Rb and E2F5) and activation of cell cycle pathways (activation of CDKs and phosphorylation of Rb) in neurons and glia in a rat spinal cord contusion model;b. increased cell cycle protein expression/activation is associated with caspase- dependent apoptosis in neurons and oligodendroglia;c. upregulation of cell cycle pathways is also associated with proliferation of astroglia;d. induction of cell cycle pathways after SCI activates microglia and induces the release of related inflammatory factors;(2) a. central (intrathecal) administration of two structurally different cell cycle inhibitors (Flavopiridol or roscovitine) decreases cell cycle activation after SCI, thereby reducing subsequent neuronal and oligodendroglial cell death, reactive gliosis and microglial activation;b. central administration of Flavopiridol or roscovitine reduces lesion size and improves motor recovery following SCI;(3) a. systemic administration of Flavopiridol has dose-dependent effects on functional outcome and the systemic immune response;b. systemic administration of Flavopiridol decreases cell cycle activation after SCI, thereby reducing subsequent neuronal and oligodendroglial cell death, reactive gliosis and immune activation;c. therapeutically relevant, delayed systemic administration of Flavopiridol improves post-traumatic function and histological outcome.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI), or traumatic neuronal injury in vitro, causes neuronal apoptosis, in part through activation of caspases. Inhibition of caspase-3, in both in vivo or in vitro trauma models, reduces post-traumatic apoptosis, and improves functional outcomes in clinically relevant TBI models. However, some of these studies indicate that improvements often reflect only a delay in cell death, which still occurs eventually without the classical apoptotic phenotype. This suggests that caspase-independent pathways might play an important role in determining the final fate of cells. Recent work supports this hypothesis, demonstrating that caspase- independent apoptosis also contributes to neuronal cell death in a variety of in vitro model systems, and that translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus, in association with apoptotic morphological features, occurs after acute brain ischemia or TBI. Moreover, AIF translocation can occur under low energetic conditions, in association with activation of poly-ADP-ribose polymerase I (PARP-1) and reduction of nicotinamide adenine dinucleotide (NAD+). In contrast, caspase activation is generally associated with a more preserved bioenergetic state and requires adenosine 5'-triphosphate (ATP). Thus caspase-independent apoptosis may play a greater role than caspase-mediated cell death after a more severe injury, or within more central regions of the evolving lesion - sites at which cellular bioenergetic state is substantially compromised. AIF-mediated apoptosis may be initiated either by the same mechanisms responsible for intrinsic caspase activation or through PARP-1 activation. In the former, the role played by AIF becomes visible only when caspase activation has been blocked. In the latter, AIF is the main death-inducing factor. PARP-1 inhibition or PARP knockout animals, as well as knockout of the AIF carrier protein cyclophilin A, show reduced AIF translocation. We propose to utilize a well-established, controlled cortical impact (CCI) model of TBI in mouse, as well as selected in vitro models, to compare mechanisms underlying both caspase- dependent and caspase-independent programmed cell death of neurons and their relative roles as a function of injury severity and injury localization. Specific hypotheses include: 1) both caspase-independent and caspase-dependent pathways contribute to post-traumatic cell loss and associated neurological dysfunction after TBI, as well as to apoptotic neuronal cell death in cell culture models associated with DNA damage; 2) caspase-independent apoptosis is induced to a relatively greater degree than caspase-dependent cell death after more severe insults, or at more central regions of the expanding lesion, where bioenergetic state is reduced; 3) cell specific, inducible 'functional knockouts of AIF pro-death domains, as well as models in which AIF translocation is inhibited (PARP knockout, treatment with PARP inhibitors, or cyclophilin A knockout), show reduced apoptotic cell death after TBI or after cell injury in vitro, and; 4) inhibition of both caspase- dependent and caspase-independent cell death improves recovery after CCI in additive or synergistic fashion. We propose the following specific aims: 1) to compare the relative degree and location of caspase-dependent and caspase-independent neuronal cell death after mild, moderate or moderately-severe TBI; 2) to investigate the role of AIF in TBI-induced neuronal death and behavioral recovery by comparing two inducible, neuron-specific, pro-death domain selective AIF transgenic models versus their non-induced controls; 3) to evaluate the effects of cyclophilin A knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models and; 4) to evaluate the effects of two structurally-distinct PARP inhibitors or PARP-1 knockout on AIF translocation, apoptosis and behavioral outcome after TBI and in selected cell culture models, and determine whether such effects are additive or synergistic to that of caspase inhibition. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) represents a major cause of death and disability in the United States. A better understanding of the mechanisms underlying TBI would offer the possibility of improving survival and insuring a more complete recovery.

DESCRIPTION (provided by applicant): According to the recent Institute of Medicine (IOM) report on Relieving Pain in America (2011), chronic pain is a public health epidemic affecting more than 116 million Americans and costing more than $600 billion per year in healthcare expenses and lost work productivity. More Americans suffer from pain than those afflicted with heart disease, diabetes and cancer combined. Despite recent advances in treatment, most people do not obtain adequate pain relief. An important focus has been on understanding the basic biology of chronic pain, so that new mechanistically based therapeutics can be developed. Unfortunately, standard research and development pipelines that start at the bench with several hundred known signaling pathways have not yielded many new targets in pain research. In a recent commentary, the NIH Director Francis Collins argued that these failures may be due to the feed-forward translational continuum from bench to beside that relies on biologically well-understood pathways. The result, in terms of drug development, is prolonged time to clinic, high failure rates and exorbitant cost. In this era of omics research, studies that incorporate geneti and genomic data may yield thousands of new, potentially druggable targets. The purpose of this Center for the Genomics of Pain is to combine rigorous phenotyping of pain and comorbid conditions with cutting edge genomics to more fully understand how individual differences can reduce or amplify pain. The Center's conceptual framework, adapted from Dr. William Maixner's model, incorporates comorbid pain conditions (intermediate risk factors), epigenetics, genomics, environment and gender to explain pain phenotypes. For the first time on the University of Maryland Baltimore Campus, geneticists, genomicists and pain researchers will combine their expertise to identify critical new therapeutic targets that can be exploited to reduce or eliminate chronic pain.

DESCRIPTION (provided by applicant): Chronic pain is a public health epidemic in the U.S., affecting more than 116 million people and costing greater than $600 billion per year to treat. Spinal cord injury (SCI) results not only in debilitating motor, sensory and cognitive deficits, bu also in a chronic, severe and often unrelenting pain that is largely resistant to conventional treatments (SCI-PAIN). Occurring in as many as 85% of SCI patients, pain starts weeks or months after the original insult, and includes increased pain with noxious stimulation (hyperalgesia), pain in response to previously innocuous stimuli (allodynia), and spontaneous pain. This unremitting pain can be diffuse, bilateral, and usually extends to locations caudal to the spinal injury. The delayed expression of SCI- PAIN and the diffuse localization of painful symptoms suggest that the pathophysiology reflects more than the direct effects at the denervated spinal segments. Indeed, these features of SCI-PAIN strongly suggest the occurrence of maladaptive plasticity in the spinal dorsal horn. An important focus for drug development has been to identify new therapeutic targets/molecules that participate in the spinal cord plasticity associated with the persistence of SCI-PAIN. One promising new therapeutic target, Brain-derived Neurotrophic Factor (BDNF), modulates nociception in the spinal cord. BDNF exerts its effects on nociceptive processing by binding to its full-length, cell surface receptor tropomyosin-related kinase B (trkB.FL) and initiating intracellular signaling. In addition to trkB.FL, the trkB locus also produces a widely-expressed alternatively-spliced truncated isoform, trkB.T1, but the function of this receptor isoform in nociception is largely unknown. TrkB.T1 is upregulated in several non-pain and pain related pathological states and we have reported that the genetic deletion of this receptor in mouse provides significant protection from the development of thermal hyperalgesia and mechanical allodynia across several models of chronic pain. Crucial to this proposal, we have preliminary data showing that trkB.T1 deletion results in significantly improved locomoter recovery and reduced allodynia in a moderate contusion injury mouse model of SCI developed by our group. We conducted differential gene expression studies to examine the potential transcriptional mechanisms regulating these improvements, and found that upregulation of key cell cycle genes correlating with neuronal apoptosis after experimental SCI, are not upregulated in the trkB.T1 null spinal cord. These results suggest that trkB.T1 may be an exciting new molecular target. In this study, we will systematically evaluate, using in vitro and in vivo approaches, whether trkB.T1 regulation of cell cycle genes contributes to SCI-PAIN and determine whether targeting cell cycle genes or trkB.T1, separately or in combination for enhanced effectiveness, can be utilized to develop novel therapeutic interventions to reduce or ameliorate SCI-PAIN.

Project Summary Spinal cord injury (SCI) causes not only in sensorimotor deficits, but also in a chronic, severe and often unrelenting pain (SCI-pain) that occurs in as many as 85% of patients. SCI-pain has neuropathic features and is often resistant to conventional pain therapy. The latter may reflect, in part, an incomplete understanding of injury mechanisms. Identifying mechanisms responsible for post-injury neuropathic pain could provide targets for more effective therapeutic interventions. We identified a promising new therapeutic target trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor?tropomyosin related kinase B (trkB). In mouse models of neuropathic pain including SCI, genetic deletion of trkB.T1 reduces both mechanical and thermal hypersensitivity. However, the precise cellular mechanisms underlying this finding are not fully understood. The purpose of this study is to investigate how trkB.T1 drives post-injury neuropathic pain via astrocyte dysfunction and test the hypothesis that astrocytic trkB.T1 functions as a key mechanism in post- injury reactive astrogliosis, through altered transcriptional programming that controls cellular movement and immune function, thus affecting chronic pain after spinal cord injury. We will use astrocytic trkB.T1 knock out (KO), Nox2 KO, and trkB.T1-KFG knock in transgenic mice and in vivo and in vitro innovatively technologies to determine the mechanisms of SCI-triggered trkB.T1 elevation on post- injury hyperpathia. Aim 1 will determine the function and mechanisms of the trkB.T1/[Ca2+]i/Nox2 pathway in astrocytes after SCI. Multiple quantitative assessments of astrogliosis will be combined with a genetic intervention targeting trkB.T1 to test the hypothesis that SCI-triggered trkB.T1 elevation in astrocytes increases [Ca2+]i and Nox2 activity, contributing to neuroinflammation and hyperpathia. Aim 2 will elucidate the role of astrocytic Nox2 signaling in post-injury hyperpathia. We will utilize genetic intervention to delete trkB.T1-dependent up-regulation of Nox2 in astrocytes, and evaluate the effects on astrocytic Nox2 on neuropathic pain after SCI. Aim 3 will determine the role for the KFG domain on trkB.T1 in the regulation of astrocyte function and SCI-Pain. Complimentary cellular, molecular, and genetic approaches will be used to test the hypothesis that KFG domain of trkB.T1 regulates trkB.T1 function in response to BDNF, and mutation of KFG domain abolishes post-injury neuropathic pain. Our study will be the first to implicate astrocytic trkB.T1-mediated [Ca2+]i/Nox2 signaling in the pathophysiology of SCI. Our data should establish that second messenger binding to the intracellular KFG domain on trkB.T1 is a physiologically important mechanism that regulates trkB.T1-mediated pain signaling. These observations may lead to novel therapeutic targets for neuropathic pain in a wide range of disease states.

Project Summary: Traumatic brain injury (TBI) triggers delayed molecular secondary injury cascades, including chronic neuroinflammation, that contribute to progressive tissue loss and neurological deficits, including dementia. We have shown that microglia are chronically activated for months-to-years following experimental TBI in mice, contributing to progressive neurodegeneration associated with cognitive decline. Microglia also undergo changes in their activation profile that may contribute to cognitive decline during neurodegenerative diseases, including Alzheimer?s disease (AD) and dementias of non-AD type. An important component of these pathological states is the maladaptive transformation of microglia from a neurorestorative/neuroprotective phenotype to a persistent, dysfunctional neurotoxic activation state. Our new studies show that microglia isolated from chronically injured brain display deficits in phagocytosis in parallel with elevations of pro-inflammatory cytokines and senescence markers, indicative of a chronic dysfunctional/neurotoxic activation state. Furthermore, we identify specific histone acetylation (H3K9ac) and methylation (H3K27me3) changes in neurotoxic microglia, which implicate intrinsic epigenetic mechanisms as drivers of this chronic phenotype. Importantly, new pilot data show that global removal of microglia from the chronically injured brain by short-term administration of a CSF1R inhibitor (PLX5622) starting at 1-month post-injury results in the repopulation of the injured brain with microglia with an anti-inflammatory phenotype. This process of resetting microglial activation after TBI dampens the chronic neuroinflammatory environment and improves long-term motor and cognitive function recovery. Thus, our data indicates that erasing posttraumatic immunological memory, by removing microglia epigenetically programmed toward a neurotoxic activation state, promotes neuroprotective microglial activation responses and improves long-term neurological recovery. Therefore, we hypothesize that moderate- severe TBI induces specific epigenetic mechanisms in microglia that promote a chronic neurotoxic activation state, causing progressive neurodegeneration and cognitive deficits. Moreover, we predict that strategies that eliminate this microglial phenotype and/or targeted inhibition of pro-inflammatory epigenetic mechanisms, even at highly delayed time points after TBI, can substantially improve long-term cognitive recovery. Here, we will use neurobehavioral, immunological, and molecular approaches to test our novel hypotheses as outlined in following specific aims: 1) To elucidate TBI-induced intrinsic epigenetic changes that lead to chronic microglial dysfunction, with a shift toward a pro-inflammatory, neurotoxic phenotype. 2) To demonstrate that microglia that repopulate the injured brain following delayed administration of CSF1R inhibitor are reprogramed toward a neurorestorative and neuroprotective phenotype that improves cognitive function. 3) To determine whether delayed interventions that target specific epigenetic mechanisms promote the neurorestorative/neuroprotective microglial phenotype and improve long-term functional recovery after TBI.