RNA Editing Alterations in Spinal Cord Injury

描述（由申请人提供）： 脊髓损伤（SCI）后通常会出现损伤部位以下脊髓神经支配的肌肉痉挛。超过80%的SCI患者有痉挛和痉挛状态，严重破坏了残余运动功能，导致衰弱性疼痛，并中断睡眠。用常规解痉药治疗痉挛（例如，巴氯芬）通常是不充分的，或者由于不良副作用如嗜睡和虚弱而不能耐受。 5-羟色胺（5-HT）是脑干源性神经递质，通过促进持续性钙电流（Ca 2 + PIC）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中起关键作用。当SCI消除了5-HT的主要来源时，脊髓运动神经元最初处于压抑状态，不能产生足够的肌肉收缩。然而，在受伤后的几周内，脊髓运动神经元中的Ca 2 + PIC自发恢复，有助于恢复基本的运动功能，但也有助于痉挛。最近，大卫班尼特博士和他的同事表明，Ca 2 + PIC的恢复是由一种称为RNA编辑的分子机制提供的。SCI后几个月，5-羟色胺受体（2C受体或5-HT 2CR）的mRNA编辑发生变化，导致5-HT 2CR亚型的表达增加，这些亚型在没有5-HT的情况下也有活性。这种组成性受体活性与受损大鼠运动神经元中大Ca 2 + PIC的恢复相一致。 编辑是由特定的酶催化的-作用于RNA的腺苷脱氨酶（ADAR 1和ADAR 2），其调控机制知之甚少。它们中的大多数编码受体或通道（例如，5-HT 2CR、离子型谷氨酸受体、Kv1.1钾和Cav 1钙通道），其在整个脑和脊髓的神经元细胞中表达。这些分子的编辑调节其Ca 2+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SCI诱导的5-HT 2CR编辑变化与SCI与对照动物中ADAR 2（而不是ADAR 1）mRNA表达的下调有关。因此，我们假设，除了5-HT 2CR，SCI触发编辑的其他受体和通道的mRNA被ADAR 2编辑的变化，共同有助于运动神经元兴奋性的恢复和痉挛的并发发展。我们还假设ADAR 2的下调是由特定的调控途径介导的，这些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SCI的全局转录反应来确定。为了验证这些假设，我们提出了以下目标：具体目标1：研究脊髓损伤动物脊髓中AMPA和红藻氨酸谷氨酸受体、电压门控钾通道Kv1.1和/或L型钙通道Cav 1的RNA编辑是否发生改变。 具体目标2：确定SCI后调节ADAR 2下调的网络、途径和单个分子。我们将使用全基因组转录组测序技术（RNA-Seq）和加权基因共表达网络分析（WGCNA）来研究脊髓损伤后ADAR 2的转录调控及其变化。 具体目标3：确定SCI诱导的运动神经元特异性编辑和ADAR 2相关调节的改变。虽然目标1和2中的研究将在整个脊髓制备中进行，但本目标中提出的实验将使用激光显微切割的脊髓运动神经元。 总而言之，拟议的研究将识别SCI改变编辑的ADAR 2底物，并识别ADAR 2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状态的神经病理学的理解。最重要的是，它们将为开发新型解痉药物治疗提供可能的靶点。

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.