Mechanisms of cAMP signaling that drive spontaneous activity in nociceptors

项目概要： 由外周或中枢神经系统损伤引起的慢性疼痛（神经性疼痛）是 众所周知，对治疗有抗性，而驱动和/或维持慢性疼痛的机制仍然存在， 不清楚我们已经证明，严重损伤后的慢性伤害感受器过度兴奋是由以下因素维持的： cAMP信号通过多个cAMP效应物，包括PKA、EPAC和HCN通道。这些 AKAP介导的与AC的复合物形成增强了通路，并显示出显著的串扰 Ras/MAPK信号通路。cAMP和Ras介导的通路的激活起始于血浆中 膜（PM），并且对PM内的脂质聚集唯一敏感。我们还表明 脊髓损伤减少了Gαi对AC的抑制，导致DRG中阿片类药物的效力降低 神经元这种降低的敏感性可以在未经处理的动物的DRG神经元中通过过夜来模拟 暴露于神经营养因子或通过5分钟，接近放电的适度去极化 严重损伤后DRG神经元的阈值。重要的是，伤害感受器过度兴奋和减少 在阿片样物质效力中，由损伤、神经营养因子或急性去极化诱导， 通过抑制Ras依赖性信号传导或质膜中脂质的重组来逆转。 我们假设，在许多损伤模型中发生的持续去极化驱动改变 在PM脂质组织中，导致ERK信号传导增加和阿片样物质反应减少。释放 的神经营养因子加强这些途径，并结合cAMP信号，驱动 伤害感受器过度兴奋和慢性疼痛状态。为了解决这些问题，我们提出了三个假设。 目标。1)确定C-Raf降低MOR-Gαi抑制AC的机制，2）定义 通过去极化和SCI的Ras激活和伤害感受器过度兴奋的机制，以及3）定义 去极化和cAMP、C-Raf和 埃里克重要的是，我们的模型确定了多种FDA批准的药物，可以同时增强 内源性阿片样物质反应和阻断严重损伤后的伤害感受器过度兴奋。

Project Summary: Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment, while the mechanisms that drive and/or maintain chronic pain remain unclear. We have shown that chronic nociceptor hyperexcitability after severe injury is maintained by cAMP signaling through multiple cAMP effectors, including PKA, EPAC and HCN channels. These pathways are enhanced by AKAP-mediated complex formation with AC and show significant cross-talk with Ras/MAPK signaling. Activation of cAMP- and Ras-mediated pathways initiate at the plasma membrane (PM) and are uniquely sensitive to clustering of lipids within the PM. We have also shown that spinal cord injury reduces AC inhibition by Gαi, resulting in reduced potency of opioids in DRG neurons. This reduced sensitivity can be mimicked in DRG neurons from naïve animals by overnight exposure to neurotrophic factors or by a 5 min, modest depolarization that approaches the firing threshold of DRG neurons after severe injury. Importantly, nociceptor hyperexcitability and reductions in opioid potency, induced by either injury, neurotrophic factors or acute depolarization, can be reversed by inhibition of Ras-dependent signaling or reorganization of lipids in the plasma membrane. We hypothesize that the sustained depolarization that occurs in many injury models drives alterations in PM lipid organization, leading to increased ERK signaling and decreased opioid responses. Release of neurotrophic factors reinforce these pathways and, in conjunction with cAMP signaling, drives nociceptor hyperexcitability and a chronic pain state. To address these hypotheses, we propose three Aims. 1) Determine the mechanism for reduced MOR-Gαi inhibition of AC by C-Raf, 2) Define the mechanism of Ras activation and nociceptor hyperexcitability by depolarization and SCI, and 3) Define functional consequences of interactions among depolarization and cell signaling by cAMP, C-Raf, and ERK. Importantly, our model identifies multiple FDA-approved drugs that could simultaneously enhance endogenous opioid responses and block nociceptor hyperexcitability after severe injury.