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善临床治疗和开发新的治疗策略非常重要。AMPA受体GluR 2亚基通过与GRIP和PICK 1结合，可能在突触AMPA受体运输中发挥作用，这对许多形式的神经元可塑性至关重要。GRIP将GluR 2锚定在突触上，而PICK 1将PKC 1带到突触GluR 2上。PKC 1在体外磷酸化GluR 2 Ser 880以从GRIP释放GluR 2并促进GluR 2内化。我们已经做出了新的发现，即完全弗氏佐剂（CFA）诱导的外周炎症和脊神经损伤（SNI）导致脊髓GluR 2与GRIP的相互作用的破坏，并且脊髓NMDA受体的激活导致含有内吞的网格蛋白包被的囊泡的150，000-g自旋级分中GluR 2的量增加，提示CFA诱导的炎性损伤和SNI诱导的神经病性损伤可能诱导体内背角中GluR 2 Ser 880的PKC磷酸化并通过NMDA受体激活驱动GluR 2内化。该提案旨在确定慢性疼痛条件下GluR 2是否以及如何在背角神经元中内化，以及这种内化是否有助于慢性疼痛的中枢敏化。在具体目标1中，我们将确定（a）CFA注射和SNI是否时间依赖性地增加Ser 880处GluR 2磷酸化的水平和源自背角的150，000- g自旋级分中GluR 2蛋白的量，（B）它们是否降低背角神经元中GluR 2的表面和突触表达，以及（c）它们是否导致在许多背角神经元上表达的Ca 2+不可渗透的（含GluR 2的）AMPA受体转换为Ca 2+可渗透的（缺乏GluR 2的）AMPA受体。在特定目标2中，我们将确定脊髓PKC 1或NMDA受体激活是否增加Ser 880处GluR 2磷酸化水平和体外背角切片中150，000 g级分中GluR 2的量。此外，我们将检查是否抑制脊髓PKC 1或NMDA受体激活减弱CFA或SNI诱导的增加在Ser 880的GluR 2磷酸化水平和量的GluR 2在150，000-g级分在体内背角。在具体目标3中，我们将确定是否通过抑制脊髓PKC 1激活来阻断GluR 2内化，通过PICK 1缺失阻断PKC 1向GluR 2的募集，或GluR 2 PKC 1磷酸化位点的靶向突变来减弱CFA或SNI诱导的热和机械性疼痛超敏反应。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，并为开发治疗或预防慢性疼痛的新镇痛药打开大门。

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.