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，标准止痛药无法充分控制。了解持续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。我们有新的发现，CFA诱导的炎症损伤降低了脊髓GluR2对GRIP的结合亲和力，脊髓NMDA受体的激活诱导背角神经元中GluR2 Ser880的PKC磷酸化，这表明NMDA受体触发的PKC激活可能导致持续疼痛条件下背角神经元中GluR2 Ser880的磷酸化和GluR2内化。本研究旨在确定cfa诱导的持续性炎症性损伤是否以及如何驱动脊髓GluR2内化，以及这种内化是否有助于cfa诱导的持续性疼痛的核心机制。在Specific Aim 1中，我们将首先确定cfa诱导的外周炎症性损伤是否会时间依赖性地增加脊髓GluR2 Ser880位点的磷酸化。使用表面表达测定和免疫电镜方法，我们将检查在cfa诱导的持续疼痛条件下，背角神经元中GluR2的表面和突触表达是否减少。在Specific Aim 2中，我们将确定NMDA受体触发PKC1激活是否需要cfa诱导的脊髓GluR2 Ser880位点磷酸化及其内化。我们将在体外培养的背角神经元中检测PKC1或NMDA受体激活是否会产生GluR2 Ser880位点磷酸化并降低GluR2表面表达，以及这些作用是否可以通过PKC1抑制来阻断。此外，我们将研究脊髓PKC1抑制或NMDA受体阻断是否会减弱cfa诱导的GluR2 Ser880位点磷酸化的增加和体内背角GluR2表面表达的降低。在Specific Aim 3中，我们将确定cfa诱导的脊髓GluR2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GluR2 K882A敲入小鼠，我们将研究GluR2 PKC1磷酸化位点的靶向突变是否会减弱cfa诱导的热和机械疼痛超敏性。这些研究将为持续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疼痛的新治疗策略。项目描述：持续性疼痛或慢性疼痛是一种常见的临床症状，标准镇痛药如阿片类药物和非甾体抗炎药控制不良。了解持续/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，这被认为是持续或慢性疼痛发生和维持的重要机制。然而，NMDA受体拮抗剂和AMPA受体拮抗剂在临床使用时会产生不可接受的副作用。本研究将探索NMDA受体和AMPA受体作用于持续性疼痛的新分子机制。该建议可能为开发选择性靶向受体单亚基和部位特异性药物提供有效策略，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.