RNA Editing Alterations in Spinal Cord Injury

脊髓损伤(SCI)通常伴随着肌肉衰弱痉挛的发展。 从损伤部位下方的脊髓开始神经支配。超过80%的脊髓损伤患者有痉挛 和痉挛，严重扰乱残余运动功能，导致虚弱的疼痛，并中断睡眠。 用传统的抗痉挛药物(如巴氯芬)治疗痉挛通常是不够的，或者是不耐受的 因为有嗜睡和虚弱等不良副作用。 5-羟色胺(5-HT)是脑干来源的神经递质，在调节兴奋性方面起着关键作用。 通过促进放大和延长的持续钙电流(Ca~(2+)PIC)对脊髓运动神经元的影响 对突触输入的反应。当脊髓损伤消除这种5-羟色胺的主要来源时，脊髓运动神经元最初是 处于抑郁状态，不能产生足够的肌肉收缩。在接下来的几周里 然而，损伤后，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，但也会导致痉挛。最近，大卫·班尼特博士和他的同事们 表明，钙离子PICS的恢复是由一种称为RNA编辑的分子机制提供的。几个月后 脊髓损伤，其中一种5-羟色胺受体(2C受体或5-HT2CR)的mRNA编辑发生变化，这是 导致在没有5-羟色胺的情况下活性的5-HT2CR亚型表达增加。这种构成受体 活动与损伤大鼠运动神经元中大量的钙离子图的恢复相一致。 编辑由特定的酶催化-作用于RNA(ADAR1和ADAR1)的腺苷脱氨酶 (ADAR2)，人们对其监管知之甚少。它们中的大多数编码受体或通道(例如，5-HT2CR， 离子亲性谷氨酸受体、Kv1.1钾通道和Cav1钙通道)在 遍布大脑和脊髓的神经细胞。这些分子的编辑可以调节它们的钙离子 通透性和动力学，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 脊髓损伤诱导的5-HT2CR编辑改变与ADAR2(而不是ADAR1)基因表达下调有关 在脊髓损伤和对照动物中的表达。因此，我们假设，除了5-HT2CR外，脊髓损伤还会触发 编辑其他受体和通道的变化，这些受体和通道的mRNA由ADAR2编辑，它们共同 有助于运动神经元兴奋性的恢复和痉挛的并发发展。我们也 假设ADAR2的下调是由可以识别的特定调控途径介导的 通过使用全基因组测序和新颖的方法筛选脊髓损伤的全球转录反应 生物信息学工具。为了检验这些假设，我们提出了以下目标： 具体目的1：研究电压门控条件下AMPA和海人酸谷氨酸受体的RNA编辑 脊髓损伤后，脊髓内钾通道Kv1.1和/或L型钙通道Cav1发生改变。 具体目标2：确定调节下调的网络、途径和单个分子 在脊髓损伤后的ADAR2。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)研究ADAR2转录调控 以及它在SCI后的变化。 具体目标3：确定脊髓损伤引起的编辑改变和与ADAR2相关的调节 特定于运动神经元。虽然AIMS 1和AIMS 2的研究将在全脊髓准备中进行，但 在这个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。 总之，拟议的研究将确定其编辑被SCI改变的ADAR2底物 以及确定ADAR2的表达和功能的调节因子。这些研究将推动我们的 对脊髓损伤和痉挛的神经病理学的理解。最重要的是，他们将提出可能的目标 用于开发新的抗痉挛药物疗法。

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.