Alan I. Faden - 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.

[unreadable] DESCRIPTION (provided by applicant): Release of glutamate and activation of ionotropic glutamate receptors have been strongly implicated in the pathobiology of post-traumatic CNS injury. The role of metabotropic glutamate receptors (mGluR) has been less well studied. Using both in vivo and in vitro trauma models, we have acquired strong evidence that activation of group I mGluR contributes to neuronal necrosis. We have also demonstrated that among group I mGIuR, mGluRl but not mGluR5 activation, exacerbates post-traumatic neuronal injury. Moreover, recent data indicate that activation of both mGluRl and mGIuR5 may attenuate neuronal apoptosis in vitro. Elucidation of the mechanisms involved in these modulatory actions can provide a better understanding of glutamatergic processes involved in secondary neuronal injury, as well as a potential basis for novel treatment strategies. The proposed studies are intended to extend our initial findings by addressing the following hypotheses: (1) activation of mGluR1 contributes to post-traumatic neuronal necrosis by multiple mechanisms that include enhancing the activity of NMDA receptors, modulating Ca2+ channels in a direction favoring necrosis, potentiating the release of arachidonic acid, upregulating cyclic AMP, and stimulating the activation of calpain; (2) activation of either mGluR1 or mGluR5 attenuates neuronal apoptosis, both post-traumatic and biochemically induced, by inhibiting the intrinsic caspase cascade; (3) injury alters expression of mGluR1 and mGluR5 in a cell type specific fashion; and (4) activation of group I mGluR alters cellular bioenergetic state and associated magnesium homeostasis after traumatic brain injury (TBI), in part by modulating NMDA receptors. The Specific Aims propose to address these hypotheses by: (1) delineating potential mechanisms of group I mGluR mediated neurotoxicity, including assessment of the relative contributions of modulation of NMDA receptors, Ca2+ channels, arachidonic acid release, cAMP, and calpain activation; and comparing the relative roles of mGluRl and mGluR5 in this process; (2) examining mechanisms by which activation of group I mGluR inhibits neuronal cell death, and comparing the relative effects of mGluR1 and mGluR5 in this regard; (3) evaluating the effects of injury on cell type-specific alterations in mGluR1 and mGluR5 expression, receptor activity, and cell-cell interactions after neuronal injury; (4) determining the effects of group I mGluR modulation on cellular bioenergetics and intracellular free magnesium concentration after TBI, their relationship to subsequent cell death, and the extent to which this is mediated through modulation of NMDA receptors.

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.

[unreadable] DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) causes neuronal cell death combined with astroglial proliferation and inflammation associated with activation of microglia. Upregulation of cell cycle proteins occurs after CMS trauma, and appears to contribute to apoptotic cell death of post-mitotic cells such as neurons. It also likely contributes to posttraumatic gliosis and microglial activation. Recent studies in our laboratory have shown significantly increased expression of many cell cycle proteins after TBI or spinal cord injury 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 up-regulation of many of these same cell cycle proteins. In addition, pilot studies have indicated that inhibition of key cell cycle regulatory pathways reduces injury-induced cell death both in vitro and in vivo. Thus, treatment with a cell cycle inhibitor after TBI in rats markedly reduces lesion volumes and the surrounding glial scar; it also significantly improves motor and cognitive functions following brain injury. The proposed studies are intended to address the following hypotheses: (1) TBI up-regulates key cell cycle constituents at both the mRNA and protein levels in neurons, astrocytes, and microglia; (2) such an up-regulation promotes apoptosis in neurons and proliferation of astrocytes; (3) up-regulation of cell cycle proteins contributes to microglial activation and subsequent release of associated inflammatory factors; and (4) treatment with cell cycle inhibitors is neuroprotective, through mechanisms that include inhibition of the intrinsic caspase pathway in neurons, as well as reduced glial activation and diminished release of microglial mediated inflammatory factors. Specific aims are to demonstrate that: (1) a. TBI causes increased expression of a number of critical cell cycle related genes/proteins, including cyclin D1, CDK4, CDK5 and Rb in both neurons and glia; b. increased protein expression is associated with caspase-dependent apoptosis in neurons, proliferation of astroglia, activation of microglia and facilitated release of microglia-related inflammatory factors; c. cyclin D1 knockout mice show less intense injury-induced pathobiology including neuronal apoptosis, brain lesion, astroglial scar formation, release of microglial associated inflammatory factors, and post-traumatic neurological deficits; (2) a. structurally different cell cycle inhibitors in dose-dependent manner reduce lesion volumes and improve cognitive as well as motor function in two pathobiologically different TBI models in the rat and mouse; b. cell cycle inhibitors decrease cell cycle activation after TBI, thereby reducing subsequent neuronal cell death, reactive gliosis and microglial activation; c. delayed systemic administration of a cell cycle inhibitor, a more clinically relevant paradigm, is neuroprotective. [unreadable] [unreadable] [unreadable]

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) causes neuronal cell death, combined with astroglial proliferation and inflammation associated with the activation of microglia, which contributes to irreversible tissue damage. Such secondary injury begins within seconds to minutes after the insult and may continue for days, weeks and potentially even months to years. Recent evidence from our laboratory shows chronic inflammation in the CNS lasting at least 6 months after injury. Inflammation may contribute to chronic neurodegeneration after trauma as well as to disorders such as Alzheimer's disease (AD) or Parkinson's syndrome. Pilot studies in our laboratory have indicated that inflammation may be inhibited by activation of mGluR5 receptors in microglia. Furthermore, our data suggest that mGluR5 effects may be mediated by actions on the NADPH oxidase enzyme, which functions to produce reactive oxygen species (ROS) by microglia and may play a significant role in persistent activation of microglia following injury. The proposed studies are intended to address the following hypotheses: (1) NADPH oxidase inhibition reduces microglial activation, production of pro-inflammatory factors and associated neurological dysfunction after TBI;(2) mGluR5 stimulation attenuates microglia activation and secondary neuronal cell death, and improves functional outcomes after TBI;(3) mGluR5, but not mGluR1, modulates microglial activation and neurotoxicity in primary microglia cultures, a microglial cell line, and microglia/neuronal co-cultures, in part through inhibition of NADPH oxidase;and (4) the G1q-protein signal transduction pathway is the critical component of the mGluR5 signal transduction events leading to inhibition of microglial NADPH oxidase and suppression of microglial activation. Specific aims are to demonstrate: (1) the importance of NADPH oxidase in microglial activation and correlated production of pro-inflammatory factors in the chronic neuronal cell loss and associated neurological dysfunction after TBI;(2) that mGluR5 stimulation attenuates microglial activation, decreases neuronal cell death and improves functional outcomes after TBI in contrast to mGluR5 global knockout animals that show greater microglial activation and neuronal cell death. We will also distinguish the relative roles of microglial versus neuronal mGluR5 using conditional/inducible knockout mice;(3) that mGluR5, but not mGluR1, modulates microglial activation and neurotoxicity in multiple cell culture models of microglial stimulation;and (4) that the G1q-protein signal transduction pathway is initiated by mGluR5 stimulation and is critical for attenuation of microglial activity by preventing activation of the NADPH oxidase enzyme complex, using in vitro microglial cell models. PUBLIC HEALTH RELEVANCE There are 1 million incidences of traumatic brain injury (TBI) per year nationally. Patients with TBI demonstrate evidence of prolonged microglial activation that lasts at least 3 weeks post-injury. The proposed work, in which we will investigate the neuroprotective effects of microglial and neuronal mGluR5, will provide initial investigation to a novel therapeutic approach for TBI.

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): This application entitled "Attenuation of Diverse Cell Death Pathways after Traumatic Brain Injury by Multi- drug Combination Therapy" addresses Broad Challenge Area (15): Translational Science and Specific Challenge Topic: 15-HD-104, Multi-drug Combination Therapy for TBI and Stroke Treatment. The purpose of the proposed studies is to evaluate a novel combination treatment strategy in experimental traumatic brain injury (TBI). Traditional neuroprotective treatment strategies for TBI aim to prevent delayed (secondary) neuronal cell death, generally by inhibiting one proposed cell death mechanism. Yet considerable research indicates that multiple pathways and mechanisms of cell death contribute to tissue loss. In focal TBI, the central injury site is thought to largely reflect necrotic cell death, which primarily occurs within the first 6-8 hours after trauma and is associated with severe bioenergetic compromise. Our recent work, however, indicates that in several rodent TBI models -mouse controlled cortical impact (CCI) or rat lateral fluid percussion (LFP)- cell death in the central core region also includes a substantial component of caspase-independent apoptosis, whereas the better known caspase-dependent cell death is detected in the more peripheral regions where ATP and ADP levels are largely preserved. The caspase-independent programmed cell death (PCD) is due in large part to the activation/translocation of apoptosis-inducing factor (AIF). Such cell death occurs relatively late after injury (24-72h) and can be inhibited by delayed treatment hours after the insult. These data raise serious questions about classical assumptions regarding mechanism of post-traumatic brain injury, suggesting the possibility of newer treatment approaches with extended therapeutic windows and explaining why, if used exclusively, caspase inhibitors may have only a partial protective effect. Ideally, treatment for TBI should attempt to inhibit both caspase-independent and caspase-dependent PCD. Recent studies in ischemia, as well as unpublished work from our laboratory, suggest two intriguing approaches for limiting the three major pathways of cell death after injury. One approach is to up-regulate heat shock protein 70 (HSP70), which binds both Apaf-1 and AIF at distinct sites, thereby neutralizing their pro- apoptotic functions by preventing the formation of the apoptosome (and caspase 3) and attenuating AIF mediated actions. Our preliminary data show that TBI causes up-regulation of HSP70 in many neurons within the injury zone;those neurons expressing HSP 70 show neither caspase-3 activation nor AIF translocation. Up-regulation of HSP70 in cerebral ischemia is strongly protective. The other approach is to inhibit PARP-1. PARP-1, through the release of poly ADP ribose (PAR), is a critical upstream activator AIF release from neuronal mitochondria;it also more recently has been shown to be a critical activator of microglia. Inhibition of PARP-1 after TBI strongly attenuates both caspase dependent and independent forms of PCD, as well as microglial activation, with markedly improved outcome. The advantage of each of these strategies is that their therapeutic window should be very broad, at least 24h. By combining these distinct therapeutic strategies, additive or synergistic protective effects may potentially be achieved. Should our hypotheses be supported, concepts regarding treatment of TBI will be markedly altered and target populations for therapeutic intervention considerably expanded. Combination treatment evaluation is proposed in two pathobiologically different models in different species, with the assumption that potent treatment effects duplicated across models and species makes ultimate clinical translation more likely. Specific hypotheses include: 1) HSP-70 inducers or PARP-1 inhibitors attenuate caspase-independent and caspase-dependent PCD after TBI, reducing long-term neurological dysfunction;2) Each treatment approach has a long therapeutic window of at least 24h;3) combined therapy with HSP-70 inducers and PARP-1 inhibitors demonstrate additive and/or synergistic effects in both mouse CCI and rat LFP models. We propose the following specific aims: 1) to compare the efficacy, dose response, and therapeutic window of two structurally distinct HSP-70 inducers with regard to attenuation of post-traumatic neuronal cell death and improved functional recovery after moderate CCI injury in mice;2) to compare the efficacy, dose response and therapeutic window of two structurally distinct PARP-1 inhibitors with regard to attenuation of post-traumatic neuronal cell death and improved functional recovery after moderate CCI injury in mice;3) to determine whether combined multi-drug therapy with the best HSP-70 inducer and best PARP-1 inhibitor, at optimal doses, has additive or synergistic effects on cell death, microglial activation, and neurodegenerative conditions up to 3 months post-injury. The Centers for Disease Control and Prevention (CDC) defines traumatic brain injury (TBI) as craniocerebral trauma associated with a decreased level of consciousness, amnesia, other neurologic or neuropsychological abnormalities, skull fracture, intracranial lesions, or death. It has been reported that the combined incidence of fatal and hospitalized TBI among all age groups has a median annual incidence of 101 per 100,000. Approximately 20% of TBIs cause death either immediately or during acute hospital care, with estimated annual rates of mild TBI treated only in outpatient facilities or hospital emergency departments (EDs) in the United States were 392 and 540 visits per 100,000, respectively. A disability prevalence of 37% has been reported for TBI patients followed more than one year after hospitalization;based on this figure, the CDC has estimated that nearly 2% of the entire US population has TBI-related disabilities. Traumatic brain injury (TBI) may occur in as many as 22% of troops deployed in Afghanistan and Iraq. Recent studies in ischemia, as well as unpublished work from our laboratory, suggest two intriguing approaches for limiting the three major pathways of cell death after injury. One approach is to up-regulate heat shock protein 70 (HSP70), which binds both Apaf-1 and AIF at distinct sites, thereby neutralizing their pro-apoptotic functions by preventing the formation of the apoptosome (and caspase 3) and attenuating AIF mediated actions. Our preliminary data show that TBI causes up- regulation of HSP70 in many neurons within the injury zone;those neurons expressing HSP 70 show neither caspase-3 activation nor AIF translocation. Up-regulation of HSP70 in cerebral ischemia is strongly protective. The other approach is to inhibit PARP-1. PARP-1, through the release of poly ADP ribose (PAR), is a critical upstream activator AIF release from neuronal mitochondria;it also more recently has been shown to be a critical activator of microglia. Inhibition of PARP-1 after TBI strongly attenuates both caspase dependent and independent forms of PCD, as well as microglial activation, with markedly improved outcome. The advantage of each of these strategies is that their therapeutic window should be very broad, at least 24h. By combining these distinct therapeutic strategies, additive or synergistic protective effects may potentially be achieved. Should our hypotheses be supported, concepts regarding treatment of TBI will be markedly altered and target populations for therapeutic intervention considerably expanded. Combination treatment evaluation is proposed in two pathobiologically different models in different species, with the assumption that potent treatment effects duplicated across models and species makes ultimate clinical translation more likely.

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): Despite numerous positive animal studies, clinical neuroprotection trials after traumatic brain injury (TBI) have uniformly failed with regard to primary end points and general populations. However, most such therapies have been directed toward a single proposed injury pathway and have targeted very early biochemical changes. Our prior work has shown that cell cycle activation is a key component of secondary injury following TBI; based upon our research and that from other groups, we hypothesize that E2F 1, 2, and 3 are involved in both cell cycle-related neuronal death (CRND) and microglial activation after brain trauma. In the present project we propose to use highly specific E2F decoy oligonucleotides (ODN) to demonstrate the neuroprotective effect of blocking the E2Fs. We will generate cell specific and inducible E2F 1, 2, and 3 knockouts and Prohibitin-1 knock-in to demonstrate that cell cycle activation in neurons and microglia represent separate and additive secondary injury mechanisms. We will also use the retinoblastoma modulator RRD-251 to confirm the neuroprotective effect of blocking the E2Fs. Moreover, we propose to expand our focus beyond the acute injury and test the hypothesis that delayed cell cycle activation contributes to chronic neurodegeneration and microglial activation months after TBI. Specific aims are to show that: (1) TBI-induced activation of E2F transcription factors is a critical event contributing to acute neuronal cell death and microglial/astrocyte reactivity, and that treatment with highly specific E2F decoy oligonucleotide (ODN) sequences or the Rb modulator RRD-251 attenuates these cellular changes and limits injury-induced pathobiology (2) E2F 1-3 are important contributing initiators of neuronal cell death and activation of microglia, with attenuation of E2F activity in neurons, and microglia showing additive neuroprotective effects; conditional and cell specific triple knockouts (E2F 1-3) will be generated by mating E2F 1-3 loxP mice with our inducible neuronal and microglia specific Cre mice; conditional and cell specific Prohibitin-1 knock-in will be generated by mating Prohibitin-1 loxP mice with our inducible neuronal and microglia specific Cre mice (3) Cell cycle activation after TBI persists beyond the acute period and leads to progressively increased polyploidy of certain neurons in selectively vulnerable brain regions as demonstrated by slide-based cytometry and fluorescence in- situ hybridization (FISH); these changes are not followed by rapid neuronal death but rather serve to predispose to chronic progressive neurodegeneration (4) Induction of conditional, neuron specific or microglial specific triple knockouts (E2F 1-3), or treatment with a the pan-CDK inhibitor CR8, at 1 month after trauma, reduce progressive neuronal hyperploidy, and attenuate chronic progressive neuronal loss and associated neurological impairment detected at 1 year after trauma.

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.

? DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) leads to neuronal cell loss and associated motor and cognitive deficits. The underlying neuronal cell death is mediated through multiple interconnected mechanisms, which include activation of multiple BH3-only and multi BH-domain pro-apoptotic proteins. Our preliminary data show that early up-regulation of these pro-apoptotic Bcl2 family proteins occur in the cortex after TBI and may play a significant role in neuronal cell death. Furthermore, we show that changes in specific microRNAs such as miR- 23a and -27a may be an important regulator of these pathophysiologic events. We propose to use genetically engineered mouse models and central administration of miR mimics to test our central hypothesis that combined modulation of multiple pro-apoptotic BH3-only as well as multi BH-domain molecules via miR- 23a/27a mimics results in superior attenuation of neuronal damage and improved reduction in neurological deficits after TBI, compared to more targeted interventions directed toward individual BH3-only pathways. Specific Aims will show that: Aim 1. To determine the individual and additive neuroprotection provided by inhibiting specific pro-apoptotic Bcl2 molecules after TBI. Study #1.1 Demonstrate the effects of constitutive deletion of Puma, Noxa or Bim on neuronal loss and functional deficits after brain trauma. Study #1.2 Compare the additive neuroprotective effects of Puma-/-/Noxa-/-/Bim-/- triple KO with those of Bax-/- Bak-/- double KO on neuronal loss and functional deficits after brain trauma Aim 2. To examine the mechanisms underpinning the rapid down-regulation of miR23a/27a and identify their key targets in neuronal apoptosis in vitro. Study #2 Identify the regulators of transcription and key targets for miR23a/27a in various models of neuronal apoptosis Aim 3. To demonstrate the relative neuroprotective effects of miR-23a and -27a as well as their additive benefits and therapeutic window; examine their modulation and targets after TBI in vivo. Study #3.1 Examine effects of acute central (icv) administration of miR-23a and/or miR-27a mimics on neuronal loss and functional deficits after brain trauma Study #3.2 Examine the therapeutic window of delayed central (icv) administration of miR mimics on neuronal loss and functional deficits after brain trauma after TBI with an extended therapeutic window.

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 Neuropsychological deficits have been reported after spinal cord injury (SCI) without concurrent head injury; although most such studies reflect patient self-reports, more formal neuropsychological testing has demonstrated performance impairments with an associated high risk of dementia including deficits in learning and memory. On the other hand, patients with dementia, such as that resulting from Alzheimer?s disease (AD), could have higher risk of falls, and therefore increased risk of SCI. Little research has addressed potential mechanisms for such neuropsychiatric changes or their implications for targeted therapy. There is an urgent need for such studies, as posttraumatic dementia such as cognitive and psychiatric changes negatively impact rehabilitation and impair recovery. The purpose of this study is to identify the mechanisms responsible for these less well examined yet important consequences of SCI and test the hypothesis that SCI-triggered release of CCL21 in the brain contributes to spreading neuroinflammation with cognitive dysfunction and depressive-like behavior, which can be improved by targeting specific mechanisms of neuroinflammation. We will use transgenic mice and molecular interventions to delineate the role of CCL21 as a key regulator of brain microglial activation and related down-stream injury mechanisms in SCI. Aim 1 will identify that SCI-induced CCL21 elevation mediates detrimental microglial activation in the brain through NOX2 activity. Multiple quantitative assessments of microglia activation will be combined with a molecular/genetic intervention targeting CCL21 to test the hypothesis that SCI-induced release of CCL21 in key regions of the brain contributes to detrimental microglial activation through NOX2 activity. Aim 2 will demonstrate that genetic depletion or pharmacological inhibition of CCL21/NOX2 reduces detrimental microglial activation, resulting in improved cognitive decline and depressive-like behavior. Complimentary pharmacological, molecular, and genetic approaches will be used to test the hypothesis that brain CCL21/NOX2-mediated inflammation after SCI causes chronic neurodegeneration associated with cognitive decline and depressive-like behavior. Aim 3 will determine that genetic or pharmacological microglial ablation after SCI reduces brain neuroinflammation leading to improved functional recovery. Using genetic or pharmacological microglia-deletion, we will examine the role of resident microglia in SCI-mediated neuroinflammation in the brain and functional outcomes. The information gained from these studies would have an important positive impact by identifying the key mechanisms involved in important yet largely ignored brain changes after SCI and identifying potential therapeutic interventions.

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.