Thursday, November 21, 2024
11/21/2024
Project Summary The current pain killer crisis, fueled by the overuse of opioids to manage chronic inflammatory pain, urges the development of non-addictive pain relief medications. Local injection of drugs, at the site of injury, is one way to circumvent adverse drug reactions, including diversion potential. For example, injection of corticosteroids into arthritic joints is one of the mainstay therapeutic approaches to treat arthritic pain, circumventing opioid use. However, recent studies have put into question their efficacy and point to potential long-term deleterious effects of steroid therapy for arthritis. Thus, we require further identification of analgesic targets specifically at nociceptive peripheral terminals, i.e. local targets. We have recently identified the adaptin 2 alpha 2 subunit Ap2A2 and nociceptor endocytosis as promising targets for pain reduction in recoverable acute and chronic animal pain models. We showed that AP2A2 binds to KNa channels to cause their internalization and precipitate dorsal root ganglion (DRG) neuronal hyperexcitability after protein kinase A stimulation. The AP2A2 subunit localized to CGRP+/IB4- nociceptors. Genetically knocking down AP2A2 or locally inhibiting endocytosis with a lipidated decoy peptide provided prolonged reductions in pain behaviors in mouse and rats during inflammatory pain. We showed that nociceptor endocytosis was required for both the development and maintenance of inflammatory pain. Our exciting new preliminary data indicates that the AP2A2 subunit localizes to large dense core vesicles and are transported down axons where the AP2A2 subunit eventually finds residence at peripheral terminals. The first Aim of this study is to further understand the subcellular localization of AP2A2 and other members of the AP2 complex in peptidergic nociceptors. We will also study their expression during chronic pain. In the second Aim, we will test the contribution of AP2A2 and nociceptor endocytosis to pain in a non-recoverable inflammatory pain model: the monoiodoacetate-induced osteoarthritic pain model. We will use a genetic knockdown approach and intraarticular injection of our lipidated AP2 inhibitor peptide to study the consequences of nociceptor endocytosis inhibition on pain behaviors. Maintenance of inflammatory pain has been shown to be dependent upon protein kinase C (PKC) signaling. We had previously shown that Slack KNa channels constitutively associate with the 14-3-3 family of proteins in DRG neurons. Our preliminary data indicates that 14-3-3 zeta causes PKC-dependent Slack KNa channel internalization. In Aim 3 we will test the hypothesis that 14-3-3 proteins confer additional endocytotic motifs to KNa channels important for inflammatory signaling. We will use a novel lipidated 14-3-3 uncoupler peptide to study KNa channel trafficking in DRG neurons and pain behavior. Positive outcomes should establish the central role of nociceptor endocytosis to inflammatory pain and reveal druggable targets.
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