Wednesday, February 5, 2025
2/5/2025
SUMMARY Opioids are the most widely used analgesics but are also heavily abused substances. Their adverse actions include peripheral side effects, dependence and tolerance which severely limit utility of opioid analgesics for long term pain management. The µ-opioid receptor (MOR) is the primary target of opioid analgesia, addiction, and withdrawal. Thus, efforts to develop safer opioid treatments as well as manage addiction and withdrawal will require a much deeper understanding of the genetic and molecular mechanisms that regulate MOR signaling and trafficking. Our long-term goal is to use unbiased forward genetics to dissect the MOR signaling network using behavioral responses to opioids as a phenotypic readout. Towards this goal, we deploy a transgenic MOR (tgMOR) model where mammalian MOR is expressed in the C. elegans nervous system. This imbues C. elegans with opioid-sensitive behaviors. Importantly, tgMOR animals exhibit the behavioral hallmarks of opioid responses in mammals including acute depressant effects, desensitization, and tolerance. We previously used forward genetics and the tgMOR C. elegans model to discover two novel regulators of MOR signaling: 1) The GPR139 orphan receptor and 2) the Ptchd1 receptor. Importantly, GPR139 and Ptdhd1 regulate opioid sensitivity and tolerance from C. elegans through rodents. Thus, our strategy has revealed major new players in MOR signaling, and we are uniquely positioned to break further new ground in opioid receptor biology. Our goal for the next funding period is to build on our success by continuing to identify novel regulators of opioid signaling while performing deep mechanistic studies on validated gene candidates. Towards this goal, our first aim focuses on using forward genetics and the tgMOR C. elegans model to identify new genetic players that affect opioid drug responsivity and MOR signaling. We focus on two principal phenotypic categories of tgMOR mutants, opioid hypersensitive and hyposensitive mutants. We plan to pursue and fully validate a subset of mutants from each phenotypic category. To demonstrate the conserved nature of potential candidates, we deploy two complementary approaches: an engineered yeast model of MOR signaling, and mammalian cell-based MOR signaling assays. In the second aim, we pivot to deep mechanistic studies with two players that we identified as regulators of MOR-mediated behavior using forward genetics with C. elegans. We deploy a combination of targeted C. elegans genetics, and a comprehensive battery of cell- based assays to monitor MOR trafficking and various aspects of MOR signaling. The third aim pivots to begin addressing the molecular landscape of how MOR interconnects with other GPCR signaling networks. We aim to build upon our success with the tgMOR C. elegans model by developing new engineered models of GPCR signaling in C. elegans. Moreover, we plan to deploy unbiased, forward genetics to interrogate opioid-related GPCRs for the first time in any system.
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