RNA Editing Alterations in Spinal Cord Injury

脊髓损伤（SCI）通常伴随着肌肉痉挛的发展 从受伤部位下方的脊髓受神经支配。超过80%的SCI患者有痉挛 以及痉挛状态，其显著地破坏残余运动功能，引起使人衰弱的疼痛，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，或不耐受的 因为它有副作用比如嗜睡和虚弱 5-羟色胺（5-HT），脑干源性神经递质，在调节兴奋性方面起着关键作用 通过促进持续性钙电流（Ca 2 + PIC）放大和延长脊髓运动神经元 对突触输入的反应。当SCI消除了5-HT的主要来源时，脊髓运动神经元最初 处于抑郁状态，不能产生足够的肌肉收缩。在那之后的几周里 然而，损伤后，脊髓运动神经元中的Ca 2 + PICs自发恢复，有助于恢复脊髓运动神经元的功能。 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 研究表明，Ca 2 + PIC的恢复是由一种称为RNA编辑的分子机制提供的。月后 SCI后，5-羟色胺受体（2C受体或5-HT 2CR）之一的mRNA编辑发生改变， 导致在没有5-HT的情况下有活性的5-HT 2CR同种型的表达增加。这种组成型受体 活性与受损大鼠运动神经元中大Ca 2 + PIC的恢复相一致。 编辑由作用于RNA的特异性酶-腺苷脱氨酶（ADAR 1和ADAR 2）催化。 ADAR 2），其调节机制知之甚少。它们中的大多数编码受体或通道（例如，5-HT2CR， 离子型谷氨酸受体，Kv1.1钾通道和Cav 1钙通道），其表达于 遍布大脑和脊髓的神经细胞。这些分子的编辑调节它们的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， SCI诱导的5-HT 2CR编辑变化与ADAR 2（而不是ADAR 1）mRNA的下调有关， SCI与对照动物中的表达。因此，我们假设，除了5-HT 2CR，SCI触发器 编辑其他受体和通道的变化，其mRNA由ADAR 2编辑， 有助于运动神经元兴奋性的恢复和痉挛的并发发展。我们也 假设ADAR 2的下调是由特定的调节途径介导的， 通过使用全基因组测序和新的 生物信息学工具。为了验证这些假设，我们提出以下目标： 具体目的1：研究AMPA和红藻氨酸谷氨酸受体的RNA编辑，电压门控 钾通道Kv1.1和/或L型钙通道Cav 1在SCI动物的脊髓中改变。 具体目标2：识别调控下调的网络、途径和单个分子 SCI后的ADAR 2。我们将使用全基因组转录组测序技术（RNA-Seq）， 加权基因共表达网络分析（WGCNA），以研究ADAR 2的转录调控 及其在SCI后的变化。 具体目标3：确定SCI诱导的编辑和ADAR 2相关调节的改变， 对运动神经元有特异性虽然目标1和2中的研究将在整个脊髓制备中进行， 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。 总之，拟议的研究将确定其编辑被SCI改变的ADAR 2底物 以及鉴定ADAR 2表达和功能的调节剂。这些研究将推动我们的 了解脊髓损伤和痉挛状态的神经病理学。最重要的是，他们会建议可能的目标 用于开发新型解痉药物疗法。

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 calcium 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.