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

项目摘要/摘要： 在颈髓损伤(SCI)的啮齿动物模型中，我们建议检查EphB2基因改变的贡献 浅层NMDAR受体与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)。NP的发展在很大程度上发生在 对受脊髓损伤影响的个人的影响，导致虚弱的，往往是慢性的身体和心理负担。 重要的是，这种病理性疼痛尤其难以治疗，迫切需要确认 既有力地调节病理性疼痛又避免阿片类药物破坏性影响的机械性靶点- 以干预为基础。中枢敏化是诱发NP的主要底物。 SCI。研究表明，NP通过增强NMDAR功能与EphB/ewitinB信号转导有关， 提示EphB-NMDAR的相互作用可能是控制脊髓损伤所致神经痛的一个重要靶点。我们 最近发现，EphB2-NMDAR的相互作用受单个胞外氨基酸的调节 EphB2(Y504)。我们在体外证明了脊髓神经元需要EphB2-Y504的磷酸化 对于EphB-NMDAR相互作用、NMDAR突触定位和兴奋性突触功能。我们还发现 在体内，EphB2与NMDAR结构性地相互作用，转导DH神经元，导致长时间的- 持续性超感痛症。我们假设在背角浅层调控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 颈挫伤后初级感觉神经传入与脱氢酶神经元之间的关系及NP相关行为。 目的1.确定与EphB2的相互作用是否促使NMDA受体在 颈髓损伤后初级伤害性传入和浅层去氢能神经元。我们将决定 在未损伤和颈部挫伤的脊髓损伤小鼠中，是否使用DH神经元亚型击倒EphB2- EphB2-shRNA的特异性表达减少了NMDAR亚单位对兴奋性突触的定位。 目的2.确定EphB2是否调节兴奋性突触传递 颈椎脊髓损伤后的行为。我们将确定DH神经元亚型特异性敲除EphB2 影响：(2)使用全细胞贴片在初级传入和板层I-II神经元之间的突触传递 完整的体外制剂中的钳制记录；以及(2b)NP相关行为的启动和/或持续。 目的3.确定EphB2-Y504磷酸化是否调节EphB2-NMDAR突触 颈椎脊髓损伤后神经功能障碍相关行为的交互作用。通过表达野生型EphB2-Y504或 组成磷酸化(Y504E)或非磷酸化(Y504F)突变体在一个DH神经元亚型中- 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)兴奋性突触的NMDAR水平，(3c)兴奋性突触传递 颈挫伤后初级感觉神经传入和脱氢酶神经元，以及(3D)NP相关行为。

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.