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： 在颈脊髓损伤 (SCI) 的啮齿动物模型中，我们建议检查改变的 EphB2 的贡献 受体-NMDA 受体 (NMDAR) 与浅表兴奋性突触神经传递的相互作用 背角 (DH) 和持续性神经性疼痛 (NP) 大部分发生 NP。 受 SCI 影响的个体，导致衰弱且往往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以治疗，迫切需要识别 既能强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目标 DH 回路的过度兴奋（“中枢敏化”）是 NP 的主要基础。 SCI。研究表明，NP 通过增强 NMDAR 功能与 EphB/ephrinB 信号传导相关， 表明 EphB-NMDAR 相互作用可能是控制 SCI 诱导的 NP 的重要靶点。 最近发现EphB2-NMDAR相互作用是由单个胞外氨基酸调节的 EphB2 (Y504)。我们在体外证明脊髓神经元需要 EphB2-Y504 磷酸化。 我们还发现了 EphB-NMDAR 相互作用、NMDAR 突触定位和兴奋性突触功能。 用与 NMDAR 组成型相互作用的 EphB2 在体内转导 DH 神经元，导致长- 我们勇敢地调节浅表背角的 EphB2-NMDAR 相互作用。 (DH) 神经元将影响 NMDAR 的突触定位和功能、兴奋性突触传递 初级感觉传入神经元和 DH 神经元之间的关系，以及颈挫伤 SCI 后 NP 相关行为。 目标 1. 确定与 EphB2 的相互作用是否会驱动 NMDA 受体到达之间的突触 我们将确定颈椎 SCI 后的初级伤害性传入神经元和浅表 DH 神经元。 是否使用 DH 神经元亚型在未受伤和颈挫伤 SCI 小鼠中敲低 EphB2 EphB2-shRNA 的特异性表达减少了 NMDAR 亚基对兴奋性突触的定位。 目标 2. 确定 EphB2 是否调节 DH 和 NP 相关的兴奋性突触传递 我们将确定 DH 神经元亚型特异性敲除 EphB2 后的行为。 影响：(2a) 使用全细胞贴片在初级传入神经元和层 I-II 神经元之间进行突触传递 完整的离体制剂中的钳记录；和（2b）NP相关行为的启动和/或持续。 目标 3. 确定 EphB2-Y504 磷酸化是否调节 EphB2-NMDAR 突触 通过表达野生型 EphB2-Y504 或颈椎 SCI 后 DH 和 NP 相关行为的相互作用。 DH 神经元亚型中的组成型磷酸化 (Y504E) 或不可磷酸化 (Y504F) 突变体 具体方式，我们将确定调节 EphB2-Y504 磷酸化是否影响：(3a) EphB2- NMDAR 相互作用，(3a) 兴奋性突触处的 NMDAR 水平，(3c) 兴奋性突触传递 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。