The Role of the Nociceptor Endocytosis in Inflammatory Pain

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.

Grant Number: 5R01NS128543-04
NIH Institute/Center: NIH
Principal Investigator: Arindam Bhattacharjee