Genetic Characterization and Exploration of Mu-Opioid Receptor Signaling in C. elegans

PROJECT SUMMARY/ABSTRACT Opioids are the most effective pharmacological treatment for pain but feature severe negative side effects including tolerance and abuse liability, highlighting the need for strategies to make opioid drugs safer. The key molecular target modulating opioid analgesia and problematic side effects is the mu-opioid receptor (MOR), a G protein-coupled receptor (GPCR) found in the nervous system. To investigate MOR signaling, our lab pioneered a translationally relevant, cross-species transgenic model where mammalian MOR is expressed pan-neuronally in C. elegans (tgMOR). This yields opioid-induced behavior as a primary readout where addition of opioid drugs induces behavioral paralysis in a dose-dependent manner. Utilizing this innovative platform, my proposal aims to identify and characterize genetic regulators of opioid drug action in the context of behavioral sensitivity and tolerance. This project directly addresses NIDA’s Priority Scientific Area 1 that aims to further understand the genetic, neuropharmacological, and behavioral changes induced by opioid use. At present, we do not fully understand how tgMOR utilizes endogenous signaling components to generate opioid-induced behavioral effects. To address this, Aim 1 will investigate how G-protein signaling influences behavioral outcomes in tgMOR C. elegans. Aim 1 will use traditional genetics and CRISPR engineering to insert stop cassettes in genes of four selected mediators of MOR signaling. These four tgMOR mutants will undergo automated behavioral assays to evaluate changes in opioid sensitivity. Additional experiments will evaluate behavioral responses in a transgenic rescue model using Mos1-mediated single copy insertion (MosSCI) to express human and C. elegans genetic constructs of two selected MOR mediators in tgMOR mutants. Aim 2 will test whether genetic regulators of MOR signaling alter behavioral tolerance responses. The speed and genetic tractability of tgMOR C. elegans will allow comprehensive testing of two selected tgMOR mutants as well as multiple opioid drugs. Experiments will utilize automated behavioral assays to evaluate opioid sensitivity and tolerance in two tgMOR mutants. These results will be confirmed by performing transgenic rescue experiments with human and C. elegans constructs of two regulators of MOR signaling using MosSCI technology. Thus, our tgMOR C. elegans model provides an innovative, unbiased behavioral platform to genetically investigate MOR signaling with the potential to open up new avenues to manage opioid substance use disorder.