Genetic Variation of Ultra-Potent Synthetic Opioid Sensitivity in Mice - PROJECT SUMMARY/ABSTRACT The weaponization of Ultrapotent synthetic opioids (UPS) has made finding a novel reversal agent a priority. The current opioid response agent, Naloxone, is not as effective against UPS opioids and does not reverse one of its known effects, wooden chest syndrome. Our long-term goal is to define the biological basis of opioid overdose risk and promote the discovery of safe and effective agents that reverse fentanyl lethality. A key objective is determining the molecular mechanisms underlying individual variability to fentanyl toxicity using genetically diverse mice. In aim 1 we plan to identify genes and variants that modify the influence of Mcoln1 on acute UPS opioid toxicity. Mcoln1 was identified in a GWAS study of overdose risk, and preliminary data support a genetic knockout of Mcoln1 resulting in death more rapidly from morphine or fentanyl. We will create an additional CRISPR knockout of Mcoln1 on a more sensitive genetic background, NOD/ShiLtJ and compare it with the C57BL/6 knockout we have for response to the UPS opioid fentanyl. The LD50 will be determined for these strains using our piezoelectric respiratory depression detection system. We will also study respiratory mechanics and pulmonary and chest wall impedance in response to three doses of fentanyl. In another cohort of mice, they will be tested by plethysmography to acquire respiratory metrics such as tidal volume and minute ventilation. We will also collect arterial blood to measure blood gases of oxygen and carbon dioxide during the plethysmography session to monitor the response to fentanyl at that level. Finally, another cohort of naïve and fentanyl-treated mice will be dissected for brain stems. The pre-bötzinger complex will be identified and analyzed by single-nucleus RNA-Seq, comparing the cellular populations and differential gene expression across genotypes, sexes and treatments. In aim 2 we plan to identify the physiological, neural, and molecular mechanisms of variable fentanyl-induced toxicity and lethality among eight inbred mouse strains. These eight strains, which served as the foundation for the advanced mouse populations of the Collaborative Cross and Diversity Outbred mice, contain approximately 45 million SNPs segregating between them. We have determined that the LD50 for fentanyl varies > 150-fold across both sexes of the eight strains. As in aim 1, in aim 2 we will phenotype cohorts of mice to detect the diverse phenomena associated with UPS opioids, including Opioid- Induced Respiratory Depression (OIRD), Opioid-Induced Persistent Apnea (OIPA), Wooden Chest Syndrome (WCS), closure/collapse of the upper and cardiovascular/hemodynamic disturbances. This phenotyping will be coupled to identifying the cellular populations, through single nucleus RNA-Seq, within the brainstem pre- bötzinger region that varies across naïve and fentanyl-treated strains of both sexes of mice. The differentially expressed genes that define these populations will help us identify targets for therapeutic development associated with the different fentanyl lethality phenotypes.
