Browsing by Department "Neuroscience And Experimental Therapeuti"

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Derivation of the Mechanisms Mediating the Adverse Effects of Morphine in a Rodent Model of SCI: Functional Recovery and Neuron Loss
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/443; DOD-Army-Medical Research and Materiel Command
In the critical first few days following spinal cord injury (SCI), many patients will experience distressing pain that arises from the trauma to the cord, the spinal nerves, the spinal fracture, or from accompanying wounds and lacerations. In the battlefield arena, this pain is managed with opioids (morphine and fentanyl) and ketamine. Effective pain management in the emergency setting is essential as under-sedation may allow delirium to develop and may increase the risk of later development of affective disorders (such as post-traumatic stress disorder [PTSD]) and cognitive impairments. Moreover, early management of acute pain is paramount in minimizing the development of chronic pain. Chronic pain is cited as one of the most significant consequences of SCI and one that patients most want cured. Unfortunately, however, our studies indicate that morphine (a potent opioid) has significant adverse effects on recovery of function in a rodent model of SCI. In the rodent model, we discovered that morphine given on the day following injury undermines locomotor recovery, increases long-term pain, and increases tissue loss at the injury site. Opioids may also adversely alter the prognosis for recovery in humans. Despite similar injury severity, Soldiers injured on the battlefield have a lower level of neurological recovery than those injured outside the combat arena. Pain on the battlefield is managed with opioids (morphine, fentanyl), whereas outside of combat pain can be treated with alternative medications (non-steroidal anti-inflammatory drugs [NSAIDs]). Given these data, it is tempting to suggest that morphine should not be used for pain management after a SCI. For the significant proportion (approximately 66%) of SCI patients faced with a lifetime of intractable pain, however, simply removing morphine as a potential analgesic is not an option. Instead, we need to identify the mechanisms mediating the adverse effects of opioids and therapeutic strategies that improve their safety. In preliminary studies, we have found that a single dose of morphine, given on the day following SCI, increases the expression of immune cells (microglia) at injury site. We have shown that the microglia express kappa-opioid receptors (KORs). We hypothesize that activation of KORs on microglia underlie the morphine-induced attenuation of locomotor recovery. Indeed, we found that the adverse effects of a single dose of intrathecal morphine can be blocked with minocycline, which inhibits microglial activation, and norBNI, a KOR antagonist. These studies strongly suggest that microglia play a critical role in the adverse effects of opioids; however, both minocycline and norBNI have off-target effects that may compromise their clinical utility. We need a cell-specific targeted approach to derive the mechanisms mediating the adverse effects of opioids and to develop effective strategies for blocking these effects while preserving the analgesic efficacy of the medications. With the experiments outlined in the current proposal, we will use cutting-edge technologies to target microglia and extend these studies to a more clinically relevant model of pain management (using repeated morphine administrations to treat pain). In Aim 1, we will identify the molecular mechanism underlying morphine’s effects on the immune response and cell death after SCI. We will compare the temporal expression and the functional activity (cytokine expression, ex vivo phagocytosis) of activated microglia in morphine and vehicle-treated SCI subjects. Aim 2 will then test the efficacy of targeting microglia as a future therapeutic intervention. We will test the necessity of microglial activation in the morphine-induced attenuation of recovery (locomotor, sensory, histological) using hM4Di DREADDs to selectively inhibit microglia. We will also test the necessity of microglial KORs using a specific agonist (dynorphin) and determine whether activation of neuronal KORs alone is sufficient to reduce recovery when microglia are inactivated. Protective effects of inhibiting microglia (on recovery of function and cell survival) with opioid administration will provide a strategy that can be rapidly transitioned to the clinical setting. Identifying the critical molecular modulators of the morphine-induced attenuation of locomotor recovery, cell death, and the development of pain has the potential to change our standards of care, and enable the implementation of clinical procedures that protect recovery of function while affording effective pain management in the emergency and early hospital setting. Importantly, our data suggest that an interaction between opioids and a sensitized immune system (that would also be characteristic of traumatic brain injury, stroke and chronic pain per se) could have detrimental effects on recovery, irrespective of the injury type. We must understand the mechanisms that underlie the negative consequences of opioid (morphine) treatment to develop therapies that provide pain relief without harm.
Epigenetic mechanisms of post-traumatic epilepsy
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/531; DOD-Army-Medical Research and Materiel Command
Traumatic brain injury (TBI) is a major cause of mortality and morbidity in military SERVICE members and Veterans, which carries a large cost burden to society. TBI is a highly complex brain condition that inflicts long-lasting pain and suffering to affected persons and their family. As per the U.S. Centers for Disease Control and Prevention, an estimated 1.7 million individuals suffer a TBI annually in the United States. Post-traumatic epilepsy (PTE) with spontaneous seizures often occurs within a few months or years after TBI in up to 50% of cases, especially in penetrating brain injury among the Veterans. PTE is a devastating brain disease characterized by repeated seizures that are often medically uncontrollable. Despite such widespread devastation, the pathophysiology of PTE is poorly understood, and there is currently no drug therapy for preventing PTE in TBI-affected Soldiers and Veterans. A greater insight is required to advance the field of PTE; there is especially an urgent need to identify at least one critical underlying mechanism of PTE. A variety of mechanisms such as excitotoxicity, inflammation, and neuronal apoptosis are proposed, but epigenetic mechanisms are not studied in PTE. Epigenetics refers to specific changes in gene expression mediated by chromatin-based mechanisms. Epigenetic mechanisms, including DNA methylation, histone alterations, and RNA-based transcriptional control, can potentially alter a broader neuronal gene profile and thus regulate TBI pathology. Therefore, epigenetics is a revolutionary and powerful research tool for finding new frontiers on PTE in people at risk. In this project, we seek to identify an epigenetic mechanism by which the brain controls hyperexcitability and seizures following TBI, with the ultimate goal of identifying the common mechanistic link between brain injury and the subsequent development of PTE. A more rational, effective strategy for preventing epilepsy is to target the primary signaling pathways that initially trigger the numerous downstream cellular and molecular mechanisms mediating epileptogenesis, the process whereby the brain becomes progressively epileptic because of injury factors. One signaling pathway, the histone deacetylation (HDAC) pathway, represents a logical candidate for such a mechanism, because diverse HDACs regulate multiple physiological functions in the brain, such as synaptic plasticity and ion channel expression, which may promote epileptogenesis under pathological conditions. Importantly, sodium butyrate, a drug that is tested for gastrointestinal and oncological conditions, can be used to inhibit the HDAC pathway and thus may represent a simple therapy for preventing PTE in at-risk people. The main goal of this project is to investigate the epigenetic-based HDAC signaling pathway as a critical pathophysiological mechanism of PTE. We propose that long-lasting alterations in the epigenetic HDAC pathway, triggered due to TBI, will facilitate critical neuronal mechanisms leading to epileptogenesis. We will test this hypothesis by addressing two specific aims using a validated model of TBI in adult and middle-aged mice: Aim 1: To determine the alterations in the HDAC pathway and the functional consequences of HDAC inhibition on the development of PTE in a mouse CCI model. Aim 2: To determine whether targeted HDAC inhibition attenuates neurodegeneration and neurological dysfunction in a mouse CCI model of PTE. Recently, we identified the epigenetic HDAC pathway in mechanisms of epileptogenesis, and the HDAC inhibitor sodium butyrate has been proposed to have anti-epileptogenic effects in preventing or inhibiting acquisition of epilepsy. In this "Idea Development" project, we propose a proof-of-concept study of whether similar HDAC mechanisms are involved in PTE and whether targeted HDAC inhibition prevents or modifies the development and retention of post-traumatic epileptogenesis with intractable seizures. We propose to use a controlled cortical impact (CCI) model, in which a piston-driven by electromagnetism impacts the head at a controlled angle, velocity, and depth. CCI, which is often delivered by a craniotomy, simulates aspects of concussions, brain contusion, and hemorrhage seen in human TBI. Post-traumatic seizures are monitored using continuous video-EEG (electroencephalogram) recording for 16 weeks following TBI. HDAC inhibitor treatment will be given starting 1 hour post-TBI for 4 weeks. The primary measures of protection include extent of post-traumatic seizures, neurodegeneration, neuroinflammation, mossy fiber sprouting, and cognitive dysfunction. Other key observations include checking seizure threshold, behavior dysfunction, and neurological/motor deficits. Our pilot studies prove the viability of accomplishing these goals. We have assembled a highly qualified team, and we possess all the facilities to implement this research within the project period. This 3-year study will provide critical information on the mechanistic role of the HDAC pathway and the viability of an epigenetic HDAC inhibition strategy for the prophylaxis of TBI-induced epilepsy and neurodeficits in patients with TBI, notably military personnel and Veterans at high risk of epilepsy due to brain trauma. Finally, this application is consistent with the goals of the Fiscal Year 2015 Epilepsy Research Program Idea Development Award mechanism, which is seeking applications in the focus areas of Markers and Mechanisms of PTE to support investigator-initiated research that may be high-risk and/or high-gain.
FMRP in the striatum: mechanisms of early drug reward
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/244; DHHS-NIH-National Institute On Drug Abuse
Project Summary/Abstract: Substance use disorders, affecting approximately 20.1 million individuals in the U.S., are characterized by a shift in voluntary drug-taking to compulsive drug-seeking and -taking behaviors, which persist despite negative consequences and remain prone to relapse after periods of abstinence. Though environmental influences have been identified as risk factors, there are major gaps in understanding of biological factors that contribute to the development of substance use disorders, limiting our ability to provide effective and lasting treatments. Previous work suggests that the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates synaptic plasticity, is required for cocaine-induced synapse elimination in the striatum, a brain region critical to reward function. Moreover, loss of FMRP, either broadly or in the ventral striatum (nucleus accumbens; NAc), is capable of dampening cocaine-induced behaviors that are considered indicative of higher addiction-related risk. The main objective of this R36 application is to determine the mechanism by which FMRP facilitates operant self-administration of intravenous cocaine and reinstatement of drug-seeking behavior. The central hypothesis, based on published and preliminary data, is that FMRP positively mediates cocaine intravenous self-administration and reinstatement of drug-seeking via its regulation of the activity- regulated cytoskeleton-associated protein (Arc) in D1-receptor (D1R) expressing cells of the NAc. This hypothesis will be tested in two specific aims, each utilizing conditional knockdown approaches and an extensive self-administration assay that includes cue- and drug-induced reinstatement paradigms. Aim 1 will determine whether FMRP mediates these phenotypes via its function in specifically D1R or D2R cells of the NAc, while Aim 2 will determine whether FMRP’s regulation of Arc contributes to these phenotypes. This work will complete the applicant’s dissertation, as part of her training for a career in addiction-related science, provide insight into the mechanism by which FMRP mediates drug-related behaviors, providing critical direction for future studies aimed at identifying downstream targets for therapeutic intervention.
FMRP in the striatum: mechanisms of early drug reward
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/244; DHHS-NIH-National Institute On Drug Abuse
Project Summary/Abstract Substance use disorders, affecting approximately 20.1 million individuals in the U.S., are characterized by a shift in voluntary drug-taking to compulsive drug-seeking and -taking behaviors, which persist despite negative consequences and remain prone to relapse after periods of abstinence. Though environmental influences have been identified as risk factors, there are major gaps in understanding of biological factors that contribute to the development of substance use disorders, limiting our ability to provide effective and lasting treatments. Previous work suggests that the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates synaptic plasticity, is required for cocaine-induced synapse elimination in the striatum, a brain region critical to reward function. Moreover, loss of FMRP, either broadly or in the ventral striatum (nucleus accumbens; NAc), is capable of dampening cocaine-induced behaviors that are considered indicative of higher addiction-related risk. The main objective of this R36 application is to determine the mechanism by which FMRP facilitates operant self-administration of intravenous cocaine and reinstatement of drug-seeking behavior. The central hypothesis, based on published and preliminary data, is that FMRP positively mediates cocaine intravenous self-administration and reinstatement of drug-seeking via its regulation of the activity- regulated cytoskeleton-associated protein (Arc) in D1-receptor (D1R) expressing cells of the NAc. This hypothesis will be tested in two specific aims, each utilizing conditional knockdown approaches and an extensive self-administration assay that includes cue- and drug-induced reinstatement paradigms. Aim 1 will determine whether FMRP mediates these phenotypes via its function in specifically D1R or D2R cells of the NAc, while Aim 2 will determine whether FMRP’s regulation of Arc contributes to these phenotypes. This work will complete the applicant’s dissertation, as part of her training for a career in addiction-related science, provide insight into the mechanism by which FMRP mediates drug-related behaviors, providing critical direction for future studies aimed at identifying downstream targets for therapeutic intervention.
From FASD to AUDs: Strategies for Preventing Alcohol Addictions
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/511; DHHS-NIH-National Institute of Neurological Disorders and Stroke
Project Summary/Abstract: Prenatal alcohol exposure (PAE) is a leading cause of intellectual and other brain disabilities, contributing to an estimated prevalence of Fetal Alcohol Spectrum Disorder (FASD) at between 1 and 5% of school-aged children in the US. Despite prevention guidelines, alcohol use during pregnancy continues to be a problem, and consequently, FASD is difficult to prevent. Behind every child with an FASD is an adult with unmet mental health needs that result in risky patterns of alcohol consumption or an Alcohol Use Disorder (AUDs). Therefore, preventing FASD requires preventing risky alcohol consumption in adults. Preventing AUDs is challenging due to the paucity of effective medications. In this application, I propose a transitioning plan in which I complement my passion for the study of FASD (the F99 phase) with the study of AUDs (the K00 phase), with the expectation that the route to preventing FASD lies through preventing AUDs. However, in both phases, I plan to pursue my interests in the mediating biology of non-protein-coding RNAs. In the first phase of my predoctoral studies, I focused on Oct4/Pouf51, a transcription factor that is a key determinant of stem cell identity, and target of ethanol. I also identified a novel pseudo- gene duplication of the Oct4/Pou5f1 locus, encoding a long non-coding RNA that I termed, Oc4pg9 lncRNA. Oct4pg9 lncRNA is upregulated in neural stem cells (NSCs), following ethanol exposure. I found that Oct4pg9 lncRNA mediates many maturational effects of ethanol on NSCs. In the F99 phase, I plan to assess, using single-cell RNA sequencing, whether the expression of Oct4pg9 lncRNA and Oct4/Pou5f1 marks unique non-overlapping NSC subpopulations. Using an in vivo murine model for PAE, I plan to determine the extent to which ethanol exposure disrupts, at the cellular level, the association between Oct4Pou5f1 and Oct4pg9, leading to the emergence of new NSC subpopulations with aberrant maturation signatures. In the K00 phase, I will transition to the field of adult alcoholism and continue studying the regulation and function of lncRNAs in the context of AUDs. This research direction will focus on developing and behaviorally phenotyping mouse models of AUD-sensitive lncRNAs. Additionally, I will utilize transcriptomic signatures and high-throughput behavioral screening to identify and test candidate compounds that show promise in decreasing excessive alcohol consumption. This proposal provides a research and training plan for a transition from predoctoral FASD training to post-doctoral training in the biology of adult alcoholism, with the aim of developing an independent research program in the field of adult alcoholism.
Isolation of Terminal Schwann Cells by Fluorescence-Activated Cell Sorting
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/507; National Institutes of Health
PROJECT SUMMARY The vertebrate neuromuscular junction (NMJ), like all synapses throughout the nervous system, has three cellular components: the presynaptic cell (the motor neuron), the postsynaptic cell (the skeletal muscle fiber) and the glial wrappings (the nonmyelinating terminal Schwann cells (tSCs) that cap the nerve terminal). At the molecular level, we know the least about tSCs. Nevertheless, evidence suggests that mammalian tSCs play important roles in re-establishment of synaptic connections following nerve damage in adult animals, regulate synapse pruning in neonates, and may have key roles at early stages of the neuromuscular diseases amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Compelling as they are none of these studies directly tested whether tSC are necessary for each of these processes and what mechanisms are possibly involved because there is a lack of genetic and molecular tools specifically targeting mammalian tSCs. This scarcity of tools is due to the paucity of proven tSC-specific markers. This R21 application proposes an innovative method for isolating tSCs using fluorescence-activated cell sorting (FACS) that will allow identification of tSC-specific markers at an unprecedented larger scale. The approach is based on comparing the RNA-Seq-generated transcriptomes of myelinating and tSC-enriched cell pools derived by FACS from muscle tissue in which fluorescent reporters of different colors are present in either tSCs or myelinating SCs. The expected outcome of this work is the identification of a largely complete set of those genes expressed in tSCs but not in myelinating SCs. These markers then could be used in future experiments to selectively manipulate tSCs in vivo using molecular genetics and thus determine more definitely their contribution to synaptic homeostasis in normal and pathological situations.
Maternal Circulating miRNA Function In Fetal Alcohol Spectrum Disorders
Neuroscience And Experimental Therapeuti; TAMHSC; https://hdl.handle.net/20.500.14641/511; DHHS-NIH-National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Project Summary Prenatal alcohol exposure (PAE) is the leading non-genetic cause of intellectual and other brain disabilities. However, fetal alcohol spectrum disorders (FASD), estimated to affect ~2-5% of school-aged children in the US, remain difficult to diagnose and to prevent. Our recent work (PMCID 5102408) identified several circulating microRNAs (miRNAs) in heavy alcohol-consuming pregnant women whose expression levels in the 2nd and 3rd trimester predicted adverse infant outcomes including craniofacial anomalies and neurobehavioral and growth deficits1. Specifically, Analysis of Variance (ANOVA) models identified 11 elevated plasma miRNAs in mothers whose infants were severely affected by alcohol consumption. Using Random Forest Analysis (RFA), we were further able to use a distinct group of miRNAs to classify infants apparently unaffected by ethanol exposure with affected infants as opposed to alcohol unexposed infants. Aside from their diagnostic value, it is unknown if these Maternal circulating miRNAs associated with Adverse Infant Outcomes (M-circmiRNA-aAIO) contribute to the developmental pathologies of FASD. Bioinformatic analysis suggests these circulating miRNAs potentially regulate important hub genes for STAT3 and ephrin signaling pathways, which are in turn known to control cycles of epithelial mesenchymal transition (EMT) crucial for normal embryogenesis and placental and fetal development. Therefore, my overarching hypothesis is that PAE impairs early development by interfering with the endocrine action of circulating maternal miRNAs on both the placenta and fetus. In my research proposal, using both human cell lines and in vivo mouse models, I will test the hypothesis that pathogenic levels of circulating miRNAs mediate effects of ethanol on placenta and fetus and that they control embryo growth and survival by regulating EMT-like behaviors of key placental and fetal cells. The long-term goals of this project will be to develop interventional strategies that exploit the biology of endocrine circulating miRNAs in mitigating negative outcomes due to PAE and other teratogens. My immediate goals will be to define the role of maternal miRNAs, which predict FASD outcomes, on placental and embryo growth and development. Aim 1: Determine the impact of M-circmiRNA-aAIOs on trophoblast growth, survival, migration and invasion under basal and ethanol exposed conditions. Aim 2: Determine the impact of M-circmiRNA-aAIOs on embryonic growth, death, and cellular maturation/differentiation under basal and ethanol exposed conditions. Aim 3: Determine the impact of M-circmiRNA-aAIOs on in vivo fetal development and neonatal outcomes. My studies are expected to uncover novel endocrine and pregnancy related functions of FASD-associated maternal systemic miRNAs and may provide novel therapeutic targets and non-invasive modalities to mitigate effects of PAE. Given its translational potential, this project will further my training as a physician-scientist interested in pursuing both clinical duties and research on early developmental disorders.
Novel Water-Soluble Adjunct Anticonvulsants for Nerve Agents
Neuroscience And Experimental Therapeuti; https://hdl.handle.net/20.500.14641/1098; DHHS-NIH-National Institute of Neurological Disorders and Stroke
Project Summary The overall goal of this proposal is to identify novel ‘water-soluble’ neurosteroid anticonvulsants that will control benzodiazepine-resistant seizures and brain injury caused by acute organophosphate (OP) intoxication. Exposure to nerve agents or OP compounds can result in persistent seizures, status epilepticus (SE), and permanent brain injury. Benzodiazepine anticonvulsants are the primary therapy for OP-induced SE but they do not sufficiently protect the brain from SE at later time after exposure. Neurosteroids are robust anticonvulsants against SE induced by a variety of OP agents and hence they can overcome key limitations of benzodiazepines. The objective of this project is to investigate the efficacy and pilot safety of new water-soluble synthetic analogs of brexanolone (FDA-approved) as adjunct anticonvulsants to midazolam therapy for OP intoxication. Test drugs are administered as adjunctive treatment either with midazolam or after midazolam has failed to control SE. The goal is to rapidly stop seizures, reducing the further brain damage. This novel therapy is based on the molecular mechanisms of neurosteroids and cellular changes involved in refractory SE caused by OP agents. The proposed adjunct therapy is based on central hypothesis that synthetic neurosteroids that enhance phasic and extrasynaptic tonic inhibition more effectively control nerve agent-induced SE and neuronal damage than benzodiazepines alone and thereby completely mitigate morbidity. The neurosteroid brexanolone is highly effective for controlling OP-induced SE and neuronal damage in rat models, but has certain limitations for its launch as medical countermeasure. Valaxanolone and lysaxanolone are two lead hydrophilic analogs of the neurosteroid with improved biopharmaceutical and pharmacological (extrasynaptic-preferring) properties. Test drugs can be formulated as dry powder for injection for extended stability and they have promising efficacy as medical countermeasures for OP-induced SE. The key emphasis is to generate requisite data on the efficacy and safety profile of lead candidates and identify at least one lead drug for further development. The proposed goals will be implemented by addressing three specific aims: (Aim 1) To determine the adjunct efficacy of hydrophilic neurosteroid analogs against DFP-induced SE and brain damage; (Aim 2) To determine the adjunct efficacy of hydrophilic neurosteroid analogs against Soman-induced SE and brain damage; and (Aim 3) To determine the preclinical pharmacokinetics and pilot safety of lead drugs. The project will be implemented as per the progressive “go/no-go” milestones plan focusing on three primary outcome measures: (i) anticonvulsant efficacy; (ii) neuroprotectant efficacy; and (iii) prevention of neurodegeneration and behavior dysfunction. The outcome from this project will identify a novel adjunct anticonvulsant to midazolam for OP intoxication and that the “dry-power for injection” system would provide a lengthy shelf-life for stockpiling at the military and civilian centers. Thus, such neurosteroid-midazolam combination will be highly efficient investment for the biodefense program.