PKC-Dependent Spinal GluR2 Internalization Triggered by NMDARs in Chronic Pain

描述(由申请人提供)：了解慢性疼痛的潜在机制对于改进临床治疗和开发新的治疗策略非常重要。AMPA受体GluR2亚单位通过与GRIP和PICK1结合，可能在突触AMPA受体转运中发挥作用，而突触AMPA受体转运对许多形式的神经元可塑性至关重要。GRIP将GluR2锚定在突触上，而PICK1将PKC1带到突触GluR2。在体外，PKC1使GluR2 Ser880磷酸化，使GluR2从GRIP中释放出来，并促进GluR2的内化。我们有了新的发现，完全弗氏佐剂(CFA)诱导的外周炎症和脊髓神经损伤(SNI)导致脊髓GluR2与GRIP的相互作用中断，并且脊髓NMDA受体的激活导致含有内吞的网状蛋白包裹的囊泡的150,000 g旋转部分中GluR2的数量增加，这表明CFA诱导的炎症和SNI诱导的神经病理性损伤可能诱导GluR2 Ser880的PKC磷酸化，并通过激活NMDA受体在体内推动GluR2内化。这一建议试图确定在慢性疼痛条件下，GluR2是否以及如何内化在背角神经元中，以及这种内化是否有助于慢性疼痛的中枢敏化。在特定的目标1中，我们将确定(A)CFA注射和SNI是否随时间依赖地增加Ser880处的GluR2磷酸化水平和来自背角的150,000-g自旋部分中的GluR2蛋白含量，(B)是否降低背角神经元GluR2的表面和突触表达，以及(C)是否导致许多背角神经元上表达的不透钙(含GluR2)的AMPA受体转换为可通透的(缺乏GluR2的)AMPA受体。在特定的目标2中，我们将确定脊髓PKC1或NMDA受体的激活是否会增加体外培养的背角切片中Ser880处的GluR2磷酸化水平和15万g组分中的GluR2的量。此外，我们将检查抑制脊髓PKC1或NMDA受体激活是否减弱CFA或SNI诱导的体内背角Ser880处GluR2磷酸化水平和15万g组分中GluR2的数量增加。在特定的目标3中，我们将确定是否通过抑制脊髓PKC1的激活来阻止GluR2的内化，通过PICK1缺失来阻止PKC1重新募集到GluR2，或者通过靶向突变GluR2 PKC1磷酸化位点来减弱CFA或SNI诱导的热和机械痛过敏。这些研究将为AMPA受体和NMDA受体在慢性疼痛中的作用机制提供新的见解，并为开发新的止痛药治疗或预防慢性疼痛打开大门。

DESCRIPTION (provided by applicant): Understanding the mechanisms underlying chronic pain is important to improving clinical treatment and developing novel therapeutic strategies. The AMPA receptor GluR2 subunit, through its binding to GRIP and PICK1, may function in the synaptic AMPA receptor trafficking that is critical for many forms of neuronal plasticity. GRIP anchors GluR2 at synapses, whereas PICK1 brings PKC1 to synaptic GluR2. PKC1 phosphorylates GluR2 Ser880 to release GluR2 from GRIP and to promote GluR2 internalization in vitro. We have made the novel discoveries that Complete Freund's adjuvant (CFA)-induced peripheral inflammation and spinal nerve injury (SNI) led to the disruption of interaction of spinal GluR2 with GRIP and that activation of spinal NMDA receptors produced an increase in the amount of GluR2 in the 150,000-g spin fraction that contains endocytosed clathrin-coated vesicles, suggesting that CFA-induced inflammatory and SNI-induced neuropathic insults might induce PKC phosphorylation of GluR2 Ser880 and drive GluR2 internalization via NMDA receptor activation in dorsal horn in vivo. This proposal seeks to determine whether and how GluR2 is internalized in dorsal horn neurons under chronic pain conditions and whether this internalization contributes to the central sensitization underlying chronic pain. In Specific Aim 1, we will determine (a) if CFA injection and SNI time-dependently increase the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 protein in 150,000- g spin fraction derived from dorsal horn, (b) if they decrease the surface and synaptic expression of GluR2 in dorsal horn neurons, and (c) if they result in a switch of Ca2+impermeable (GluR2-containing) AMPA receptors expressed on many dorsal horn neurons to Ca2+permeable (GluR2-lacking) AMPA receptors. In Specific Aim 2, we will determine if spinal PKC1 or NMDA receptor activation increases the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 in the 150,000-g fraction in in vitro dorsal horn slices. Furthermore, we will examine if inhibition of spinal PKC1 or NMDA receptor activation attenuates CFA- or SNI-induced increases in the level of GluR2 phosphorylation at Ser880 and the amount of GluR2 in the 150,000-g fraction in in vivo dorsal horn. In Specific Aim 3, we will determine if blocking GluR2 internalization through inhibition of spinal PKC1 activation, blockade of PKC1 recruitment to GluR2 by PICK1 deletion, or targeted mutation of the GluR2 PKC1 phosphorylation site attenuates CFA- or SNI-induced thermal and mechanical pain hypersensitivity. The proposed studies will provide new insights into the mechanisms of both AMPA receptor and NMDA receptor actions on chronic pain and open a door for the development of new analgesics in treatment or prevention of chronic pain.