The role of the AP2 adaptor complex in inflammatory pain

项目摘要 炎症状态的基本特征之一是通常无害的刺激会产生疼痛。当前 止痛药包括非类固醇抗炎药，其目的是阻断 前列腺素生产、皮质类固醇和阿片类药物。然而，在某些情况下，副作用和成瘾 与这些药物相关的潜在因素限制了它们的长期使用，特别是在慢性炎症疼痛期间。至 开发新的、非成瘾的止痛药，仍然迫切需要了解炎症是如何 产生伤害感受器放电的变化，这是痛觉的基础。在这项建议中，我们的目标是提供 在背根神经节(DRG)神经元中，适配素2网状蛋白介导的内吞作用(AP2- CME)是炎症诱导伤害性感受器敏化的主要促进剂。我们之前已经 研究表明，作为对蛋白激酶A(PKA)刺激的响应，Slack KNA通道通过 AP2-CME从DRG神经细胞膜上分离出来，这引起了超兴奋性。此外，我们还证明了 抑制AP2-CME可抑制PKA诱导的神经元超兴奋性。初步研究现在表明，在 AP2α亚单位AP2A2在DRG神经元中的体内敲除显著减少 炎症性疼痛行为。在这里，我们将应用蛋白质化学，免疫组织化学， 电生理学、脊髓生理学、疼痛行为分析和一种新的体内基因敲除方法 验证AP2-CME是伤害性感受器敏化的关键调节因子的假说。具体目标是：1) 确定AP2-CME是否控制基础兴奋性和神经传递2)以证明 减少AP2-CME可减轻炎症性疼痛。这项研究项目将揭示AP2-CME的核心作用 在疼痛信号中起作用。

Project Summary One of the cardinal features of inflammatory states is that normally innocuous stimuli produce pain. Current pain-relieving drugs include nonsteroidal anti-inflammatory drugs, which are aimed at the interdiction of prostaglandin production, corticosteroids and opioids. However, the side effects and some cases addiction potential associated with these drugs limit their long-term use especially during chronic inflammatory pain. To develop novel, non-addictive analgesics, there remains an urgent need to understand how inflammation produces the change in nociceptor firing that underlies pain perception. In this proposal, we aim to provide proof of principal that in dorsal root ganglion (DRG) neurons, adaptin 2 clathrin-mediated endocytosis (AP2- CME) is a principal facilitator of inflammatory-induced nociceptor sensitization. We have previously demonstrated that in response to protein kinase A (PKA) stimulation, Slack KNa channels are internalized via AP2-CME from DRG neuronal membranes and this caused hyperexcitability. Furthermore we showed that inhibiting AP2-CME prevented PKA-induced neuronal hyperexcitability. Preliminary studies now indicate that in vivo knockdown of the AP2 alpha subunit AP2A2 specifically within DRG neurons, substantially reduces inflammatory pain behavior. Here, we will apply a combination of protein chemistry, immunohistochemistry, electrophysiology, spinal cord physiology, pain behavior assays and a novel in vivo gene knockdown approach to test the hypothesis that AP2-CME is a key regulator of nociceptor sensitization. The specific aims are 1) to determine whether AP2-CME controls basal excitability and neurotransmission 2) To demonstrate that reducing AP2-CME mitigates inflammatory pain. This research project will reveal the central role AP2-CME plays in pain signaling.