PKC-dependent GluR2 internalization in the spinal cord by persistent pain

描述（由申请人提供）：持续性/慢性疼痛是一个主要的公共卫生问题，标准镇痛药无法充分控制。了解持续性疼痛的机制对于开发新的治疗策略以改善临床治疗非常重要。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。我们有了新的发现，完全弗氏佐剂（CFA）诱导的炎症损伤降低了脊髓GluR 2对GRIP的结合亲和力，脊髓NMDA受体的激活诱导了背角神经元中GluR 2 Ser 880的PKC磷酸化，提示NMDA受体-持续痛时，PKC激活可能导致GluR 2在Ser 880磷酸化，并导致GluR 2内化条件该提案旨在确定CFA诱导的持续性炎症损伤是否以及如何驱动脊髓GluR 2内化，以及这种内化是否有助于CFA诱导的持续性疼痛的中枢机制。在具体目标1中，我们将首先确定CFA诱导的外周炎症损伤是否时间依赖性地增加脊髓Ser 880处的GluR 2磷酸化。使用表面表达测定和免疫电镜方法，然后，我们将研究是否在背角神经元表面和突触表达的GluR 2 CFA诱导的持续性疼痛条件下减少。在具体目标2中，我们将确定NMDA受体触发PKC 1激活是否是CFA诱导的脊髓GluR 2在Ser 880磷酸化及其内化所必需的。我们将研究是否PKC 1或NMDA受体激活产生GluR 2磷酸化Ser 880和减少GluR 2的表面表达，以及这些影响是否可以在体外培养的背角神经元中通过PKC 1抑制来阻断。此外，我们将研究是否脊髓PKC 1抑制或NMDA受体阻滞剂减弱CFA诱导的GluR 2在Ser 880磷酸化的增加和减少GluR 2在背角的表面表达在体内。在具体目标3中，我们将确定CFA诱导的脊髓GluR 2内化是否有助于CFA诱导的持续性疼痛条件下的疼痛超敏反应。使用GluR 2 K882 A基因敲入小鼠，我们将研究GluR 2 PKC 1磷酸化位点的靶向突变是否减弱CFA诱导的热和机械疼痛超敏反应。拟议的研究将为持续性炎症性疼痛的机制提供新的见解，并可能导致治疗或预防持续性疼痛的新治疗策略。项目叙述：持续性疼痛或慢性疼痛是一种常见的临床病症，标准镇痛药如阿片类药物和非甾体抗炎药控制不佳。了解持续性/慢性疼痛的机制对于制定治疗策略非常重要。脊髓NMDA受体和AMPA受体的激活有助于脊髓中枢致敏，这被认为是持续性或慢性疼痛的发展和维持的基本机制。然而，NMDA受体拮抗剂以及AMPA受体拮抗剂在临床环境中使用时产生不可接受的副作用。本研究旨在探讨NMDA受体和AMPA受体在持续性疼痛中的作用机制。这一建议可能为开发选择性靶向受体单亚基和位点特异性药物提供一种有效的策略，可用于治疗持续性/慢性疼痛。

DESCRIPTION (provided by applicant): Persistent/chronic pain, a major public health problem, is inadequately controlled by standard analgesics. Understanding the mechanisms that underlie persistent pain is important for developing novel therapeutic strategies to improve clinical treatment. 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 novel discoveries that Complete Freund's Adjuvant (CFA)-induced inflammatory insult reduced the binding affinity of spinal GluR2 for GRIP and that activation of spinal NMDA receptors induced PKC phosphorylation of GluR2 Ser880 in dorsal horn neurons, suggesting that NMDA receptor-triggered PKC activation might lead to GluR2 phosphorylation at Ser880 and GluR2 internalization in dorsal horn neurons under persistent pain conditions. This proposal seeks to determine whether and how CFA-induced persistent inflammatory insult drives spinal GluR2 internalization and whether this internalization contributes to the central mechanism of CFA-induced persistent pain. In Specific Aim 1, we will first determine whether CFA-induced peripheral inflammatory insult time-dependently increases spinal GluR2 phosphorylation at Ser880. Using a surface expression assay and an immunoelectron microscopic approach, we will then examine whether surface and synaptic expression of GluR2 in dorsal horn neurons is decreased under CFA-induced persistent pain conditions. In Specific Aim 2, we will determine whether NMDA receptor triggering of PKC1 activation is required for CFA-induced spinal GluR2 phosphorylation at Ser880 and its internalization. We will examine whether PKC1 or NMDA receptor activation produces GluR2 phosphorylation at Ser880 and decreases GluR2 surface expression and whether these effects could be blocked by PKC1 inhibition in cultured dorsal horn neurons in vitro. Furthermore, we will study whether spinal PKC1 inhibition or NMDA receptor blockade attenuates the CFA-induced increase in GluR2 phosphorylation at Ser880 and decrease in GluR2 surface expression in dorsal horn in vivo. In Specific Aim 3, we will determine whether CFA-induced spinal GluR2 internalization contributes to pain hypersensitivity under CFA-induced persistent pain conditions. Using GluR2 K882A knock-in mice, we will study whether targeted mutation of the GluR2 PKC1 phosphorylation site attenuates CFA-induced thermal and mechanical pain hypersensitivities. The proposed studies will provide a new insight into the mechanisms of persistent inflammatory pain and may lead to novel therapeutic strategies for treatment or prevention of persistent pain. PROJECT NARRATIVE: Persistent pain or chronic pain, a common clinical condition, is poorly controlled by standard analgesics, such as opioids and non-steroidal anti-inflammatory drugs. Understanding mechanisms that underlie persistent/chronic pain is important in developing therapeutic strategies. Activation of spinal NMDA receptors and AMPA receptors contributes to spinal central sensitization that is thought to be an essential mechanism underlying the development and maintenance of persistent or chronic pain. However, NMDA receptor antagonists as well as AMPA receptor antagonists produce unacceptable side effects when they are used in the clinical setting. This proposal will explore novel molecular mechanisms of both NMDA receptor and AMPA receptor actions on persistent pain. The proposal may provide an effective strategy in developing selective targeting receptor single subunit- and site-specific drugs that could be useful in treating persistent/chronic pain.