Exploring the EphB2-NMDA receptor interaction in spinal cord injury-induced neuropathic pain

探索 EphB2-NMDA 受体在脊髓损伤引起的神经性疼痛中的相互作用

• 批准号: 10245041
• 负责人: MANUEL L COVARRUBIAS
• 金额: $ 50.94万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245041
• 关键词: 3-Dimensional; Affect; Amino Acids; Anatomy; Animal Model; Behavior; Calcium; Cells; Cervical; Cervical spinal cord injury; Chemosensitization; Chronic; Complex; Contusions; Development; EphB2 Receptor; Ephrin B Receptor; Event; Excitatory Synapse; Extracellular Domain; Future; Heterogeneity; In Vitro; Individual; Intervention; Link; Mediating; Modeling; Molecular; Mus; N-Methyl-D-Aspartate Receptors; Neurons; Nociception; Opioid; Pain; Pathologic; Persistent pain; Pharmacology; Phenotype; Phosphorylation; Population; Posterior Horn Cells; Preparation; Refractory; Rodent Model; Sensory; Signal Transduction; Spinal Cord; Spinal Cord Contusions; Spinal cord injury; Surface; Synapses; Synaptic Receptors; Synaptic Transmission; Synaptic plasticity; Tactile; Testing; Thermal Hyperalgesias; Viral Vector; Work; allodynia; base; central sensitization; chronic pain; dorsal horn; effective therapy; extracellular; in vivo; innovation; knock-down; mutant; neurotransmission; novel; painful neuropathy; patch clamp; prevent; psychologic; receptor; receptor function; side effect; small hairpin RNA; synaptic function; targeted agent; targeted treatment; therapeutic target; transmission process; vector

Project Summary / Abstract: In a rodent model of cervical spinal cord injury (SCI), we propose to examine the contribution of altered EphB2 receptor-NMDA receptor (NMDAR) interaction to both excitatory synaptic neurotransmission in the superficial dorsal horn (DH) and persistent neuropathic pain (NP). The development of NP occurs in a significant portion of individuals affected by SCI, resulting in debilitating and often chronic physical and psychological burdens. Importantly, this pathological pain is particularly refractory to treatment, urgently calling for the identification of mechanistic targets that both robustly regulate pathological pain and avoid the devastating effects of opioid- based interventions. Hyperexcitability of DH circuitry (“central sensitization”) is a major substrate for NP after SCI. Studies have shown that NP is linked to EphB/ephrinB signaling through potentiation of NMDAR function, suggesting that the EphB-NMDAR interaction may be an important target for control of SCI-induced NP. We recently discovered that the EphB2-NMDAR interaction is regulated by a single extracellular amino acid of EphB2 (Y504). We demonstrated in vitro that EphB2-Y504 phosphorylation is required in spinal cord neurons for EphB-NMDAR interaction, NMDAR synaptic localization, and excitatory synapse function. We also found that transduction of DH neurons in vivo with EphB2 that constitutively interacts with the NMDAR results in long- lasting allodynia. We hypothesize that modulating the EphB2-NMDAR interaction in superficial dorsal horn (DH) neurons will impact synaptic localization and function of NMDARs, excitatory synaptic transmission between primary sensory afferents and DH neurons, and NP-related behaviors after cervical contusion SCI. Aim 1. Determine whether interaction with EphB2 drives NMDA receptors to synapses between primary nociceptive afferents and superficial DH neurons following cervical SCI. We will determine whether knocking down EphB2 in both uninjured and cervical contusion SCI mice using DH neuron subtype- specific expression of EphB2-shRNA reduces the localization of NMDAR subunits to excitatory synapses. Aim 2. Determine whether EphB2 regulates excitatory synaptic transmission in DH and NP-related behaviors after cervical SCI. We will determine whether DH neuron subtype-specific knockdown of EphB2 impacts: (2a) synaptic transmission between primary afferents and laminae I-II neurons using whole-cell patch clamp recording in an intact ex vivo preparation; and (2b) initiation and/or persistence of NP-related behaviors. Aim 3. Determine whether EphB2-Y504 phosphorylation regulates EphB2-NMDAR synaptic interaction in the DH and NP-related behaviors after cervical SCI. By expressing wild-type EphB2-Y504 or constitutively-phosphorylated (Y504E) or non-phosphorylatable (Y504F) mutants in a DH neuron subtype- specific manner, we will determine whether modulating EphB2-Y504 phosphorylation impacts: (3a) EphB2- NMDAR interaction, (3a) NMDAR levels at excitatory synapses, (3c) excitatory synaptic transmission between primary sensory afferents and DH neurons, and (3d) NP-related behaviors after cervical contusion SCI.

项目摘要 /摘要： 在宫颈脊髓损伤（SCI）的啮齿动物模型中，我们建议检查改变EPHB2的贡献 在表面上与两种兴奋性突触神经传递的受体-NMDA受体（NMDAR）相互作用 背角（DH）和持续性神经性疼痛（NP）。 NP的发展发生在很大一部分 受SCI影响的个体，导致衰弱，通常是慢性的身体和心理伯恩斯。 重要的是，这种病理疼痛特别是对治疗的难治性，紧急要求鉴定 机械靶标既可以牢固地调节病理疼痛，又避免了阿片类药物的毁灭性影响 基于干预措施。 DH电路（“中心灵敏度”）的过度兴奋性是NP之后的主要底物 科学。研究表明，NP通过NMDAR功能的潜在作用与Ephb/Ephrinb信号传导有关， 表明EPHB-NMDAR相互作用可能是控制SCI诱导的NP的重要目标。我们 最近发现EPHB2-NMDAR相互作用受到单个细胞外氨基酸的调节 EPHB2（Y504）。我们在体外证明了脊髓神经元中需要EPHB2-Y504磷酸化 用于EPHB-NMDAR相互作用，NMDAR突触定位和兴奋性突触功能。我们还发现 与NMDAR相互作用的EPHB2体内DH神经元的翻译导致长期相互作用 持久的异常性。我们假设调节浅表背角EPHB2-NMDAR相互作用 （DH）神经元将影响NMDAR的突触定位和功能，兴奋性突触传播 在主要的感觉传入和DH神经元之间以及颈挫伤后NP相关的行为之间。 目标1。确定与EPHB2的相互作用是否驱动NMDA接收器之间的突触 宫颈科学后的原发性伤害性传入和浅表DH神经元。我们将确定 是否使用DH神经元亚型中的未受伤和宫颈挫伤小鼠击倒Ephb2 Ephb2-shRNA的特定表达将NMDAR亚基的定位降低到兴奋性突触。 AIM 2。确定EPHB2是否调节DH和NP相关的兴奋性突触传播 宫颈科学后的行为。我们将确定dh神经元亚型特异性EPHB2是否敲低 影响：（2a）使用全细胞斑块之间的主要传入和I-I-II神经元之间的突触传播 在完整的离体制备中记录夹具； （2B）与NP相关行为的主动性和/或持续性。 目标3。确定EPHB2-Y504磷酸化是否调节EPHB2-NMDAR突触 宫颈SCI后DH和NP相关行为的相互作用。通过表达野生型EPHB2-Y504或 DH神经元亚型中的组成型磷酸化（Y504E）或非磷酸化（Y504F）突变体 具体方式，我们将确定调节EPHB2-Y504磷酸化是否会影响：（3A）EPHB2-- NMDAR相互作用，（3A）兴奋性突触时的NMDAR水平，（3C）兴奋性突触传播 原发性感觉传入和DH神经元，以及（3D）NP相关的行为后，颈挫伤后SCI。