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: 10487467
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10487467
• 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.

项目概要/摘要： 在啮齿动物模型的颈脊髓损伤（SCI），我们建议检查改变EphB 2的贡献， 受体-NMDA受体（NMDAR）相互作用，既兴奋性突触神经传递在表面 背角（DH）和持续性神经性疼痛（NP）。NP的发展在很大程度上 这是一种严重的创伤性疾病，导致衰弱和经常是慢性的身体和心理负担。 重要的是，这种病理性疼痛特别难治疗，迫切需要识别 既能有力地调节病理性疼痛，又能避免阿片类药物的破坏性影响的机制性靶点， 基于干预。DH回路的超兴奋性（“中枢敏化”）是NP的主要底物， SCI.研究表明，NP通过增强NMDAR功能与EphB/ephrinB信号传导相关， 这表明EphB-NMDAR相互作用可能是控制SCI诱导的NP的重要靶标。我们 最近发现EphB 2-NMDAR相互作用受单个胞外氨基酸调节， EphB2（Y504）。我们在体外证明EphB 2-Y504磷酸化是脊髓神经元所必需的， 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 用与NMDAR组成性相互作用的EphB 2在体内转导DH神经元导致长时间的 持续异常性疼痛我们假设调节背角浅层EphB 2-NMDAR相互作用 (DH)神经元将影响NMDAR的突触定位和功能，兴奋性突触传递 初级感觉传入与DH神经元之间的关系，以及颈挫伤后NP相关行为。 目标1.确定与EphB 2的相互作用是否驱动NMDA受体进入突触， 脊髓损伤后初级伤害性传入神经元和浅层DH神经元。我们将确定 是否使用DH神经元亚型在未受伤和颈部挫伤SCI小鼠中敲低EphB 2- EphB 2-shRNA的特异性表达减少了NMDAR亚基在兴奋性突触的定位。 目标2.确定EphB 2是否调节DH和NP相关的兴奋性突触传递 颈脊髓损伤后的行为。我们将确定是否DH神经元亚型特异性敲低EphB 2 影响：（2a）使用全细胞贴片的初级传入和板层I-II神经元之间的突触传递 在完整的离体制备物中的钳记录;和（2b）NP相关行为的起始和/或持续。 目标3。确定EphB 2-Y504磷酸化是否调节EphB 2-NMDAR突触 脊髓损伤后DH和NP相关行为的交互作用。通过表达野生型EphB 2-Y504或 DH神经元亚型中的组成型磷酸化（Y504 E）或非磷酸化（Y504 F）突变体- 具体而言，我们将确定调节EphB 2-Y504磷酸化是否影响：（3a）EphB 2-Y504磷酸化。 NMDAR相互作用，（3a）兴奋性突触处的NMDAR水平，（3c） 初级感觉传入和DH神经元，以及（3d）颈挫伤SCI后的NP相关行为。