Derek C Molliver, PhD - US grants

Nociceptive sensory neurons, which convey environmental stimuli indicating real or potentially tissue-damaging events, undergo plastic changes in phenotype in response to prolonged or intense noxious stimulation. These changes represent an essential component of the chronic painful hypersensitivity of pathological pain. Particularly important is the possibility that non- nociceptive neurons may also undergo changes in phenotype in response to tissue injury, resulting in the perception of even light touch as painful. Currently, it is not clear how noxious stimuli result in changes in sensory neuron gene expression. Efforts to address these issues are hampered by the difficulty of determining the modalities of sensory neurons in preparations in which the effects of stimulation can be effectively addressed. Several techniques have been established in this laboratory to identify and isolate specfic populations of nociceptors and non- nociceptive mechansensors. Experiments described here will examine the ability of different qualities of noxious stimulation to activate transcription in sensory neurons both in vivo and in vitro. Finally, the ability to identify innocuous mechanoreceptors will allow us to definitively determine whether noxious stimulation is capable of activating gene expression in non-nociceptive sensory neurons.

DESCRIPTION (provided by applicant): Extracellular ATP is a messenger in the transduction of noxious stimuli by pain-sensing neurons (nociceptors). The cloning of the sensory neuron-specific ATP-gated ion channel P2X3 generated intense investigation into the ability of ATP to directly activate nociceptors. However, we have new evidence that sensory neurons also express members of a family of G protein-coupled receptors (P2Y receptors) that respond to ATP or related compounds and contribute to nociceptive signaling. These receptors are poorly characterized in neurons: of the 8 known family members only P2Y1 and P2Y2 have been evaluated in sensory neurons and both have been implicated in nociceptive signal transduction. P2Y receptors can be divided into 2 groups based on their coupling to signal transduction pathways;we hypothesize that Gq-coupled receptors are proalgesic while Gi- coupled receptors are analgesic. This proposal consists of 3 Specific Aims designed to identify which P2Y family members contribute to nociceptive signaling and may be useful targets for therapeutic intervention in pain. Specific Aim 1 will use quantitative PCR, immunohistochemistry and Wester blotting techniques to determine which P2Y receptors are expressed in sensory neurons and whether expression changes in response to inflammatory injury. Specific Aim 2 will characterize excitatory and inhibitory actions of Gq-coupled and Gi-coupled P2Y receptors in dissociated sensory neurons using calcium imaging and post-hoc immunocytochemistry. Specific Aim 3 will examine the contribution of P2Y ADP receptors to behavioral pain response thresholds in vivo and will determine whether pharmacological or genetic manipulation of P2Y receptors is analgesic in models of acute and persistent inflammatory pain. These studies will demonstrate the contributions of a new family of receptors to sensory neuron signaling and will provide valuable insight into the mechanisms through which nucleotides act as signaling molecules in the setting of persistent pain.Experiments in this proposal will test the hypothesis that members of the P2Y family of nucleotide receptors are powerful regulators of nociceptor sensitivity that play an important role in the maintenance of persistent pain. We will directly test the possibility that manipulation of these receptors, including the use of antagonists already in development for clinical treatment of non-pain-related syndromes, is an effective analgesic treatment in animal models of persistent pain.

DESCRIPTION (provided by applicant): Chronic pain affects more than 50 million Americans per year, resulting in extraordinary personal and societal costs in diminished quality of life, los productivity and health care consumption. Improved treatments of acute and chronic pain conditions require a thorough understanding of the processes underlying the transmission and perception of painful stimuli. Discovery of the molecular and cellular mechanisms underlying acute and chronic pain is critical to the development of new treatments, particularly in mechanisms regulating the transition from acute to chronic pain. Recent studies from our laboratory have identified an endogenous analgesic mechanism that is present in peripheral sensory neurons conveying pain and is mediated by G protein-coupled receptors (GPCRs) for adenosine diphosphate (ADP). This project will investigate the role of ADP receptors signaling through Gi/o in hyperalgesia resulting from inflammatory injury, the regulation of P2Y signaling by GPCR kinases GRK2 and GRK3 during hyperalgesia, and will explore the hypothesis that these receptors are highly effective in ameliorating hyperalgesia mediated by adenylyl cyclase, a fundamental component of inflammatory pain. Specific Aim 1 will investigate the distribution of GRK2 and GRK3 in dorsal root ganglion (DRG) neurons and their regulation of P2Y signaling in vitro. Specific Aim 2 will determine whether GRK2 and GRK3 are co-regulated in the setting of inflammatory injury, and will examine the impact of altered GRK signaling on P2Y receptor inhibition of adenylyl cyclase using real-time cAMP imaging in vitro. Specific Aim 3 will determine the extent to which changes in the regulation of P2Y signaling contribute to the resolution of inflammatory hyperalgesia in vivo and whether manipulation of these pathways may provide a novel approach to the treatment of inflammatory pain. This proposal will examine the value of Gi/o-coupled P2Y receptors as targets for novel analgesic drugs, and will advance our understanding of fundamental mechanisms regulating GPCR function and nociceptive processing in the peripheral nervous system.

Phase 1 COBRE support resulted in the successful initiation of the Histology and Imaging Core and its development into a critical resource for COBRE investigators and the research community at the University of New England. The Core?s histology instruments include paraffin embedding and section processing equipment, as well as microtomes, vibratomes and cryostats. Imaging equipment includes bright-field, epifluorescent, stereologic and confocal microscopes. Technical services include immunohistochemistry and antibody optimization. In Phase I, the H&I Core provided services to 18 investigators (8 UNE-COBRE, 6 UNE- non-COBRE, 2 external contracts, 2 external COBRE), trained over 60 laboratory personnel, developed 11 new stains and optimized 60 antibodies. In Phase II, the Histology and Imaging Core will maintain existing capabilities and its current outstanding level of performance and expand those capabilities significantly while beginning to prepare for post-award sustainability. Proposed expansion includes the addition of antibody validation services and the acquisition of a two-photon microscope configured for in vivo imaging. The superior abilities of the two-photon system to penetrate tissues and detect faint signals with minimum noise and phototoxicity will enable COBRE investigators to develop innovation in their experimental approaches. These capabilities will have a regional impact, as such an instrument does not currently exist anywhere in southern Maine. Finally, the H&I Core will provide an ambitious program of professional development for Core and COBRE personnel. Critical training opportunities have been identified, including a summer program offered by External Advisory Committee Member Dr. Yves De Koninck?s Neurophotonics Centre at Laval University in nearby Quebec City, Canada. In order for the Core services to be sustained into the future, a fee-for-service plan will be implemented, and an extensive outreach effort will be conducted to broaden the user base. Special care has been taken to ensure that the Core capabilities do not duplicate those offered by other COBRE and INBRE supported research cores. Instead, the Core has strategically specialized in unique services such as a specialty in the processing of non-standard samples in a variety of research models. By these efforts, the UNE H&I Core will provide critical research support to its many users and ensure maximum utility to the broader research community in the State of Maine and neighboring IDeA states.

Chronic pain affects more than 50 million Americans per year, resulting in extraordinary personal and societal costs. Adding to the dilemma, deaths involving prescription opiate analgesics have almost quadrupled in the last ten years. The clinical challenge of pain management is underscored by evidence that chronic pain is mechanistically distinct from acute pain, therefore a thorough understanding of the molecular and cellular mechanisms underlying the transition to chronic pain is fundamental to improving and expanding treatment options. Hyperalgesic priming is a compelling model of the transition to chronic pain in which an initial injury resolves, but leaves the animal in a primed state in which a second insult induces a greatly prolonged pain response. While previous studies have explored the development of sustained mechanical hypersensitivity, we have adapted this model to examine sustained hypersensitivity mediated by nociceptors expressing the heat-gated ion channel TRPV1, which can drive pain in a range of inflammatory and neuropathic conditions. Experiments proposed here will test the contribution to heat hyperalgesic priming of adenylyl cyclase isoform AC2, which has not been previously characterized in sensory neurons, and which is insensitive to inhibition by Gi/o-coupled receptors such as opioid receptors. Preliminary analysis indicates that AC2 is highly expressed in TRPV1-expressing neurons and required for the manifestation of heat hyperalgesic priming. Specific Aim 1 will examine the impact of AC2 gene deletion and pharmacological inhibition on behavioral nociceptive thresholds at baseline, in acute hyperalgesia and in the setting of hyperalgesic priming. Specific Aim 2 will determine whether AC2 and the downstream effector Epac are functionally coupled through AKAP family member scaffolding proteins by co-immunoprecipitation-mass spectrometry, and will determine the impact of deleting identified AKAP genes on Epac signaling and nociceptor function in vitro using a CRISPR/Cas9-based approach. Specific Aim 3 will determine the consequences for Epac function of AC2 and AKAP gene deletion through Epac-dependent PKC phospho- substrate profiling and behavioral assessment of heat hyperalgesia. This proposal will use innovative approaches to explore novel mechanisms contributing to the development of persistent hyperalgesia.