RNA Editing Alterations in Spinal Cord Injury

脊髓损伤中的 RNA 编辑改变

• 批准号: 8974362
• 负责人: STELLA DRACHEVA
• 金额: --
• 依托单位: JAMES J PETERS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974362
• 关键词: ADAR1; Adverse effects; Alberta province; Animals; Baclofen; Bioinformatics; Brain; Brain Stem; Calcium; Calcium Channel; Cells; Code; Collaborations; Control Animal; DRADA2b protein; Data; Depressed mood; Development; Down-Regulation; Enzymes; Genes; Glutamate Receptor; Health; Incidence; Individual; Injury; Kinetics; L-Type Calcium Channels; Lasers; Lead; Left; Massive Parallel Sequencing; Mediating; Messenger RNA; Middle Insomnia; Military Personnel; Molecular; Motor; Motor Neurons; Muscle; Muscle Contraction; Neurons; Neurotransmitters; Pain; Pathway Analysis; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacotherapy; Potassium; Potassium Channel; Preparation; Protein Isoforms; Proteins; Quality of life; RNA; RNA Editing; Rattus; Recovery; Regulation; Regulatory Pathway; Research; Residual state; Role; Serotonin; Serotonin Receptor 5-HT2C; Site; Source; Spasm; Specimen; Spinal; Spinal Cord; Spinal cord injury; Synapses; Technology; Testing; Transcript; Transcriptional Regulation; United States; Universities; Veterans; Voltage-Gated Potassium Channel; Weight; dsRNA adenosine deaminase; genome-wide; improved; injured; kainate; mRNA Expression; member; neuropathology; novel; novel therapeutic intervention; receptor; research study; response; restoration; screening; spasticity; tool; transcriptome sequencing

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is often followed by the development of debilitating spasms in the muscles innervated from the spinal cord below the site of injury. More than 80% of individuals with SCI have spasms and spasticity that significantly disrupt residual motor function, cause debilitating pain, and interrupt sleep. Treatment of spasms with conventional antispastic drugs (e.g., baclofen) is often not adequate, or not tolerated because of adverse side effects such as lethargy and weakness. Serotonin (5-HT), the brain stem-derived neurotransmitter, serves a critical role in tuning the excitability of the spinal motoneurons by facilitating persistent calcim currents (Ca2+ PICs) that amplify and prolong responses to synaptic input. When SCI eliminates this major source of 5-HT, spinal motoneurons are initially left in a depressed state and are not able to produce adequate muscle contractions. Over the weeks after injury, however, Ca2+ PICs in spinal motoneurons spontaneously recover, helping with restoration of rudimentary motor functions, but also contributing to spasms. Recently, Dr. David Bennett and his colleagues showed that the recovery of Ca2+ PICs is afforded by a molecular mechanism called RNA editing. Months after SCI, there are alterations in mRNA editing of one of the serotonin receptors (2C receptor or 5-HT2CR), which lead to increased expression of the 5-HT2CR isoforms that are active without 5-HT. Such constitutive receptor activity coincides with a restoration of large Ca2+ PICs in the motoneurons of the injured rats. Editing is catalyzed by specific enzymes-adenosine deaminases that act on RNA (ADAR1 and ADAR2), whose regulation is poorly understood. Most of them encode receptors or channels (e.g., 5-HT2CR, ionotropic glutamate receptors, Kv1.1 potassium and Cav1 calcium channels) that are expressed in the neuronal cells throughout the brain and spinal cord. Editing of these molecules modulates their Ca2+ permeability and kinetics, thus influencing intrinsic excitability of neurons. In our preliminary studies we found that SCI-induced 5-HT2CR editing changes are related to downregulation of ADAR2 (but not ADAR1) mRNA expression in SCI vs. control animals. We, therefore, hypothesize that, in addition to 5-HT2CR, SCI triggers editing changes in other receptors and channels whose mRNA is edited by ADAR2, which collectively contribute to the recovery of motorneuron excitability and the concurring development of spasticity. We also hypothesize that downregulation of ADAR2 is mediated by specific regulatory pathways that can be identified by screening the global transcriptional response to SCI using genome-wide sequencing and novel bioinformatics tools. To test these hypotheses, we propose the following Aims: Specific Aim 1: To investigate if RNA editing of AMPA and kainate glutamate receptors, voltage gated potassium channel Kv1.1, and/or L-type calcium channel Cav1 is altered in the spinal cord of SCI animals. Specific Aim 2: To identify networks, pathways, and individual molecules that regulate downregulation of ADAR2 following SCI. We will use genome-wide transcriptome sequencing technology (RNA-Seq) and weighted gene coexpression network analysis (WGCNA) in order to investigate ADAR2 transcriptional control and its alteration following SCI. Specific Aim 3: To identify SCI-induced alterations in editing and ADAR2-related regulation that are specific for motoneurons. While studies in Aims 1 and 2 will be performed in whole spinal cord preparation, the experiments proposed in this Aim will use laser microdissected spinal motoneurons. To summarize, the proposed research will identify ADAR2 substrates whose editing is altered by SCI as well as identify regulators of ADAR2 expression and function. These studies will advance our understanding of the neuropathology of SCI and spasticity. Most importantly, they will suggest possible targets for the development of novel antispastic drug therapy.

描述(由申请人提供)： 脊髓损伤(SCI)后，损伤部位以下的脊髓神经支配的肌肉经常发生衰弱痉挛。超过80%的脊髓损伤患者有痉挛和痉挛，这些痉挛和痉挛显著扰乱残余运动功能，导致衰弱疼痛，并中断睡眠。传统的抗痉挛药物(如巴氯芬)对痉挛的治疗通常是不够的，或由于嗜睡和虚弱等不良副作用而无法耐受。5-羟色胺(5-HT)是脑干来源的神经递质，通过促进持续钙电流(Ca~(2+))放大和延长对突触输入的反应，在调节脊髓运动神经元的兴奋性方面发挥关键作用。当脊髓损伤消除了5-羟色胺的这一主要来源时，脊髓运动神经元最初处于抑制状态，不能产生足够的肌肉收缩。然而，在损伤后的几周里，脊髓运动神经元中的钙离子图像会自发恢复，有助于基本运动功能的恢复，但也会导致痉挛。最近，大卫·贝内特博士和他的同事们证明，钙离子PIC的恢复是由一种名为RNA编辑的分子机制提供的。脊髓损伤后几个月，其中一种5-羟色胺受体(2C受体或5-HT2CR)的mRNA编辑发生改变，导致5-HT2CR亚型表达增加，这些亚型在没有5-羟色胺的情况下是活跃的。这种结构性受体活性与损伤大鼠运动神经元中大量钙离子PIC的恢复相一致。编辑是由特定的酶催化的-作用于RNA(ADAR1和ADAR2)的腺苷脱氨酶，其调节机制尚不清楚。它们大多编码受体或通道(如5-HT2CR、离子型谷氨酸受体、Kv1.1钾通道和Cav1钙通道)，这些受体或通道在大脑和脊髓的神经细胞中表达。这些分子的编辑调节它们的钙离子渗透性和动力学，从而影响神经元的内在兴奋性。 在我们的初步研究中，我们发现与对照组相比，脊髓损伤诱导的5-HT2CR编辑变化与ADAR2(而不是ADAR1)mRNA表达下调有关。因此，我们假设，除5-HT2CR外，脊髓损伤还可触发其他受体和通道的编辑改变，这些受体和通道的mRNA由ADAR2编辑，共同促进运动神经元兴奋性的恢复和痉挛的并发发展。我们还假设ADAR2的下调是由特定的调控途径介导的，这些调控途径可以通过使用全基因组测序和新的生物信息学工具筛选对SCI的全球转录反应来识别。为了验证这些假说，我们提出了以下目标：具体目标1：研究脊髓损伤动物脊髓AMPA和海人藻氨酸受体、电压门控钾通道Kv1.1和/或L型钙通道CaV1的RNA编辑是否发生改变。具体目标2：确定调控脊髓损伤后ADAR2下调的网络、途径和单个分子。我们将使用全基因组转录测序技术(RNA-Seq)和加权基因共表达网络分析(WGCNA)来研究ADAR2的转录调控及其在脊髓损伤后的变化。具体目标3：确定脊髓损伤引起的编辑改变和ADAR2相关的运动神经元特有的调节。虽然目标1和目标2的研究将在整个脊髓准备中进行，但该目标中提出的实验将使用激光显微解剖的脊髓运动神经元。综上所述，这项拟议的研究将确定其编辑被脊髓损伤改变的ADAR2底物，以及确定ADAR2表达和功能的调节因子。这些研究将促进我们对脊髓损伤和痉挛的神经病理学的理解。最重要的是，他们将为新的抗痉挛药物疗法的开发提供可能的靶点。