Mechanisms of cAMP signaling that drive spontaneous activity in nociceptors

 描述(申请人提供)：由外周或中枢神经系统损伤引起的慢性疼痛(神经病理性疼痛)是出了名的抗拒治疗。将任何类型的神经病理性疼痛维持数月或更长时间的机制尚不清楚。脊髓损伤(SCI)大鼠模型中的慢性疼痛最近被证明依赖于伤害性感觉神经元(伤害性感受器)的过度活动，其中大部分痛觉启动活动产生于细胞体内。疼痛相关的伤害性感受器、超兴奋性和自发活动(SA)在体外的持续表达为将生化机制直接与维持慢性脊髓损伤疼痛至关重要的电生理活动联系起来提供了特殊的机会。初步结果表明，腺苷环化酶(AC)、蛋白激酶A(PKA)和A-激酶锚定蛋白(AKAPs)以及可能的cAMP激活的交换蛋白(EPAC)的复合体的持续信号传递起着至关重要的作用。虽然cAMP信号长期以来一直被认为对持续数小时到数天的急性疼痛很重要，但在维持疼痛持续数月的过程中发挥重要作用是意想不到的。选择性抑制cAMP依赖通路不同步骤的药物可阻断慢性脊髓损伤诱导的SA，包括AKAP5(AKAP79/150)锚定复合体的抑制剂。对脊髓损伤后背根神经节细胞膜的生化研究表明，脊髓损伤后AC调节发生了变化，提示在AC功能水平上存在一种先前未知的机制，从而导致慢性疼痛。这些和相关的观察导致假设脊髓损伤后的慢性伤害性感受器SA和疼痛是通过1)AC调节的改变和2)AKAP5支架大分子复合体促进cAMP依赖的PKA和EPAC对离子通道的调节而维持的。拟议的研究将通过将体外生物化学、细胞生物学和电生理学与脊髓损伤后疼痛相关行为的体内测试相结合，来开发两个PI实验室的互补优势。实验将利用我们的发现，在脊髓损伤水平以下的许多伤害性感受器中，强大的脊髓损伤诱导的SA明显与行为表现的超敏和疼痛有关。这将允许使用分离的伤害性感受器中的电生理和分子变化作为研究的信息终点，以评估cAMP途径中的信号分子的疼痛相关功能。它还将允许汇集脊髓损伤动物的多个神经节，以促进生化和分子研究。体外研究中披露的针对大分子复合体的干预措施的预测行为和细胞效应将使用补充方法在整个动物中进行测试，包括用于在伤害性感受器中选择性表达干扰肽的新型病毒载体、针对特定cAMP信号成分的基因敲除和抑制方法。从这些研究中获得的信息可能会导致重大的机制发现，通过靶向持续的细胞内信号来指导未来治疗慢性疼痛的努力，这种信号维持伤害性感受器的过度活动，从而促进慢性疼痛。

 DESCRIPTION (provided by applicant): Chronic pain caused by injury to the peripheral or central nervous system (neuropathic pain) is notoriously resistant to treatment. The mechanisms that maintain any type of neuropathic pain for months or longer are poorly understood. Chronic pain in a rat model of spinal cord injury (SCI) has recently been shown to depend upon hyperactivity in nociceptive sensory neurons (nociceptors), with much of the pain-initiating activity generated within the cell bodies. The continued expression of pain-linked nociceptor hyper excitability and spontaneous activity (SA) in vitro provides a special opportunity to link biochemical mechanisms directly to electrophysiological activity critical for maintaining chronic SCI pain. Preliminary results indicate that continuing signaling by complexes containing adenylyl cyclase (AC), protein kinase A (PKA), and A-kinase anchoring proteins (AKAPs), and possibly exchange protein activated by cAMP (EPAC) plays a vital role. While cAMP signaling has long been known to be important for acute pain lasting hours to days, a major role in maintaining pain lasting months is unexpected. Agents selectively inhibiting different steps along cAMP-dependent pathways blocked chronic SCI-induced SA, including inhibitors of AKAP5 (AKAP79/150)-anchored complexes. Biochemical studies of membranes from dorsal root ganglia revealed a change in AC regulation after SCI, suggesting the existence of a previously unknown mechanism at the level of AC function that contributes to chronic pain. These and related observations led to the hypothesis that chronic nociceptor SA and pain after SCI are maintained by 1) alterations in AC regulation and 2) AKAP5-scaffolded macromolecular complexes that facilitate cAMP-dependent PKA and EPAC regulation of ion channels. The proposed studies will exploit complementary strengths of the two PIs' laboratories by combining in vitro biochemistry, cell biology, and electrophysiology coordinated with in vivo tests of pain-related behavior after SCI. Experiments will take advantage of our findings that robust SCI-induced SA in numerous nociceptors below the spinal injury level is clearly linked to behaviorally expressed hypersensitivity and pain. This will allow the use of electrophysiological and molecular alterations in dissociated nociceptors as informative endpoints for studies evaluating pain-related functions of signaling molecules within the cAMP pathway. It will also allow pooling of multiple ganglia from SCI animals to facilitate biochemical and molecular studies. Predicted behavioral and cellular effects of interventions targeting macromolecular complexes disclosed in the in vitro studies will be tested in the whole animal using complementary approaches, including a novel viral vector for expression of disrupting peptides selectively in nociceptors, an knockdown and inhibitor methods targeting specific cAMP signaling components. Information gained from these studies may lead to major mechanistic discoveries that could guide future efforts to treat chronic pain by targeting the persistent intracellular signaling that maintains hyperactivity in nociceptors that promotes chronic pain.