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

描述（由申请人提供）：了解慢性疼痛的机制对改善临床治疗和开发新的治疗策略非常重要。AMPA受体GluR2亚基，通过其与GRIP和PICK1的结合，可能在突触AMPA受体运输中起作用，这对许多形式的神经元可塑性至关重要。GRIP将GluR2固定在突触上，而PICK1将PKC1带到突触的GluR2上。PKC1磷酸化GluR2 Ser880，从GRIP中释放GluR2，并促进GluR2体外内化。我们已经有了新的发现，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相互作用的中断，脊髓NMDA受体的激活导致150,000 g自旋部分GluR2的数量增加，其中包含胞吞的网格蛋白包被囊泡。这表明cfa诱导的炎症和sni诱导的神经性损伤可能诱导PKC磷酸化GluR2 Ser880，并通过NMDA受体激活在体内背角驱动GluR2内化。本研究旨在确定慢性疼痛条件下GluR2是否内化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。在特定目标1,我们将决定(a)如果CFA注入和SNI时间增加GluR2磷酸化水平在Ser880 GluR2蛋白质的数量在150000 - g旋转部分来源于背角,(b)如果他们降低表面在背角神经元突触GluR2的表情,和(c)导致开关Ca2 +不透水(GluR2-containing) AMPA受体表达在许多背角神经元钙离子渗透(GluR2-lacking) AMPA受体。在特异性目的2中，我们将确定脊髓PKC1或NMDA受体激活是否会增加体外背角切片中150,000 g部分中GluR2 Ser880磷酸化水平和GluR2数量。此外，我们将研究脊髓PKC1或NMDA受体激活的抑制是否会减弱CFA-或sni诱导的GluR2 Ser880磷酸化水平的增加以及体内背角150,000 g部分GluR2的数量。在Specific Aim 3中，我们将确定阻断GluR2内化是否通过抑制脊柱PKC1激活，通过PICK1缺失阻断PKC1对GluR2的募集，或者GluR2 PKC1磷酸化位点的靶向突变来减弱CFA或sni诱导的热和机械疼痛超敏反应。本研究将为AMPA受体和NMDA受体在慢性疼痛中的作用机制提供新的见解，并为开发治疗或预防慢性疼痛的新型镇痛药打开大门。

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.