CRMP2, Nav1.7 sodium channel, and chronic pain

CRMP2、Nav1.7 钠通道和慢性疼痛

基本信息

批准号：
10113570
负责人：
Rajesh Khanna
金额：
$ 38.29万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10113570
关键词：
Accounting Action Potentials Acute Pain Address Afferent Neurons Alanine American Analgesics Arizona Axon Behavior Binding Budgets Calcium Channel Cell membrane Cells Clathrin Clinical Collaborations Core Facility Department of Defense Disease Endocytosis Excision Foundations Generations Genes Genetic Genetically Engineered Mouse High Prevalence Human Injury Institute of Medicine (U.S.)Knock-in Mouse Link Locomotion Maintenance Measures Mechanics Mediating Membrane Memory Modeling Modification Mus Mutation Neurons Nociception Pain Pain Research Pain Threshold Pathogenesis Peptides Peripheral nerve injury Pharmaceutical Preparations Plasmids Play Post-Translational Modification Site Post-Translational Protein Processing Principal Investigator Proteins Rattus Recycling Regulation Research Research Personnel Role Sedation procedure Site Smell Perception Societies Sodium Channel Solid Specificity Spinal Cord Spinal Ganglia Stimulus Sumoylation Pathway Surface Syndrome Testing Therapeutic Thermal Hyperalgesias Transfection Transgenic Organisms Ubiquitin Universities Work chronic pain chronic painful condition collapsin response mediator protein-2 cost density design effective therapy gabapentin gain of function mutation improved in vivo inflammatory pain mechanical allodynia mouse model mutant neuronal excitability novel novel therapeutic intervention overexpression pain behavior pain model pain signal painful neuropathy pre-clinical pregabalin protein protein interaction protein transport recruit response side effect success therapeutic target trafficking voltage

项目摘要

Abstract Chronic pain conditions cause an immense burden on society due to their astonishingly high prevalence and lack of effective treatments. This application addresses how indirect modulation of the excitability of neurons in pain conditions can be achieved by altering the expression and function of the Nav1.7 sodium channel. The scientific premise is that because direct blockade of Nav1.7 channels has been unsuccessful, targeting regulators of Nav1.7 may offer therapeutic advantages allowing for a graded analgesic response. Dr. Rajesh Khanna, Principal Investigator on this project, first discovered that expression of Nav1.7 at the surface is regulated by a protein, axonal collapsin response mediator protein 2 (CRMP2), and that a mutant of CRMP2 lacking the small ubiquitin-like modifier (SUMO) post-translational modification (deSUMOylation) reduces Nav1.7 surface expression and currents. Importantly, the related Nav1.1, Nav1.3, Nav1.5, Nav1.6, Nav1.8, and Nav1.9 channels are unaffected. In preliminary studies, we demonstrate that loss of CRMP2 SUMOylation increases binding to endocytic proteins, potentially accounting for removal of Nav1.7 from the surface. The fraction of SUMOylated CRMP2 increases significantly following peripheral nerve injury. Excitingly, in vivo transfection of a CRMP2-K374A SUMO-null plasmid or a peptide mimicking the CRMP2 SUMOylation motif, into the spinal cord reversed mechanical allodynia in a model of neuropathic pain. Together, these findings strongly support the hypothesis that loss of CRMP2 SUMOylation reduces Nav1.7 localization at the plasma membrane, thereby decreasing nociceptive neuron excitability and thresholds to thermal and mechanical stimuli in acute and chronic pain. We will test this hypothesis in three specific aims. In Aim 1, we will test the general role of CRMP2 SUMOylation on Nav1.7 currents and neuronal excitability using a recently created new transgenic K374A Crmp2 knock-in mouse model where the SUMOylation site (K374) of CRMP2 has been replaced with an alanine mutation; this mouse was made by Dr. Thomas Doetschman, a co-Investigator on this project and Director of the Genetically Engineered Mouse Models Core facility at the University of Arizona. The mechanism by which Nav1.7 surface trafficking and internalization occur is unknown and will be examined in Aim 2 of this proposal. Aim 3 will evaluate the contribution of the CRMP2 SUMOylation state to acute pain thresholds as well as after experimentally induced pain thresholds using models in which Nav1.7 levels are increased; these studies will be performed in collaboration with Dr. Todd Vanderah, a co-Investigator on this project with deep expertise in preclinical pain modeling. Finally, in these mice, we will also measure CRMP2- dependent off-target effects on memory, locomotion/sedation, as well as behaviors linked to Nav1.7, including smell. The proposed study will considerably improve our understanding of how intracellular trafficking proteins can be modified in diseases/injuries, lay a solid foundation for unraveling mechanisms of the modification and trafficking of Nav1.7 in chronic pain, and offer novel and selective therapeutic targets for pain research.

抽象的慢性疼痛状况导致社会巨大负担，因为他们的患病率惊人而缺乏有效的治疗方法。该应用程序解决了神经元兴奋性如何间接调节可以通过改变NAV1.7钠通道的表达和功能来实现疼痛条件。这科学前提是，因为直接阻止NAV1.7频道的封锁不成功，针对性 NAV1.7的监管机构可以提供治疗优势，允许分级镇痛反应。 Rajesh博士该项目的首席研究员Khanna首先发现NAV1.7在表面的表达是由蛋白质调节，轴突折叠蛋白反应介质蛋白2（CRMP2），而CRMP2突变体缺少小泛素样修饰剂（SUMO）翻译后修饰（Desumoylation）会减少 NAV1.7表面表达和电流。重要的是，相关的NAV1.1，NAV1.3，NAV1.5，NAV1.6，NAV1.8和 NAV1.9频道不受影响。在初步研究中，我们证明了CRMP2 Sumoylation的丧失增加与内吞蛋白的结合，可能考虑从表面去除NAV1.7。这外周神经损伤后，Sumoypated CRMP2的比例显着增加。令人兴奋的是，体内转染CRMP2-K374A SUMO-NULL质粒或模仿CRMP2 Sumoylation基序的肽，在神经性疼痛模型中，脊髓逆转了机械性异常性。在一起，这些发现强烈支持以下假设：CRMP2 Sumoylation的损失降低了NAV1.7等离子体的定位膜，从而降低伤害性神经元的兴奋性，并阈值对热和机械急性和慢性疼痛的刺激。我们将以三个具体目标来检验这一假设。在AIM 1中，我们将测试 CRMP2 Sumoylation在NAV1.7电流和神经元兴奋性上使用最近创建的新作用的一般作用转基因K374A CRMP2敲入小鼠模型，其中crmp2的sumoylation位点（K374）已经用丙氨酸突变取代；这只鼠标是由托马斯·多埃茨曼（Thomas Doetschman）博士制作的，亚利桑那大学的项目和基因工程鼠标核心设施的项目和主管。这 NAV1.7表面运输和内在化发生的机制尚不清楚，并将在该提案的目标2。 AIM 3将评估CRMP2 Sumoylation对急性疼痛的贡献使用NAV1.7级别的模型，阈值以及实验诱导的疼痛阈值增加；这些研究将与托德·范德拉（Todd Vanderah）博士合作进行。具有临床前疼痛建模方面具有深厚专业知识的项目。最后，在这些小鼠中，我们还将测量CRMP2- 依赖目标对记忆，运动/镇静以及与NAV1.7相关的行为，包括闻。拟议的研究将大大提高我们对细胞内运输蛋白的理解可以在疾病/伤害中修改 NAV1.7在慢性疼痛中的运输，并为疼痛研究提供新颖和选择性的治疗靶标。