CRMP2, Nav1.7 sodium channel, and chronic pain

摘要 慢性疼痛病症由于其极高的患病率和严重的并发症而对社会造成巨大负担。 缺乏有效的治疗。本申请解决了如何间接调节神经元的兴奋性， 疼痛状况可以通过改变Nav1.7钠通道的表达和功能来实现。的 科学前提是，由于直接封锁Nav1.7通道一直不成功， Nav1.7的调节剂可以提供允许分级镇痛反应的治疗优势。Rajesh医生 该项目的主要研究者卡纳首先发现，Nav1.7在表面的表达是 受一种蛋白质--轴突再生素反应介导蛋白2（CRMP 2）调节，并且CRMP 2的突变体 缺乏小泛素样修饰物（SUMO）翻译后修饰（去SUMO化）， 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通道不受影响。在初步研究中，我们证明CRMP 2 SUMO化的缺失 增加与内吞蛋白的结合，可能是Nav1.7从表面去除的原因。的 外周神经损伤后SUMO化CRMP 2的分数显著增加。令人兴奋的是， 转染CRMP 2-K374 A SUMO无效质粒或模拟CRMP 2 SUMO化基序的肽， 在神经病理性疼痛模型中，总之，这些发现 强烈支持CRMP 2 SUMO化的缺失减少Nav1.7在血浆中的定位的假设 膜，从而降低伤害性神经元的兴奋性和阈值，以热和机械 急性和慢性疼痛的刺激。我们将在三个具体目标中检验这一假设。在目标1中，我们将测试 CRMP 2 SUMO化对Nav1.7电流和神经元兴奋性的一般作用 转基因K374 A Crmp 2敲入小鼠模型，其中CRMP 2的SUMO化位点（K374）已被 替换为丙氨酸突变;这只老鼠是由托马斯Doetschman博士，这方面的合作研究者， 他是亚利桑那大学基因工程小鼠模型中心的主任。的 Nav1.7表面运输和内化发生的机制尚不清楚，将在 该提案的目标2。目的3将评估CRMP 2 SUMO化状态对急性疼痛的贡献 阈值以及实验诱导的疼痛阈值后，使用模型，其中Nav1.7水平 增加;这些研究将与该研究的共同研究者托德范德拉博士合作进行 在临床前疼痛建模方面拥有深厚专业知识的项目。最后，在这些小鼠中，我们还将测量CRMP 2- 对记忆、运动/镇静以及Nav1.7相关行为的依赖性脱靶效应，包括 味道这项拟议的研究将大大提高我们对细胞内运输蛋白质 可以在疾病/损伤中进行修饰，为阐明修饰的机制奠定坚实的基础， Nav1.7在慢性疼痛中的转运，为疼痛研究提供了新的、有选择性的治疗靶点。

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.