Mechanisms of cAMP signaling that drive spontaneous activity in nociceptors

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 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.