Identifying novel molecular targets, signaling pathways and mechanisms underlying fentanyl overdose-induced severe respiratory depression and lethality in rats using TMT phosphoproteomics/proteomics

点击翻译按钮获取中文摘要

Project Summary/Abstract Fentanyl is a potent synthetic, lipophilic phenylpiperidine analgesic that primarily acts through the Oprm1 Exon 1 (E1)-associated full-length 7TM mu opioid receptors (MORs). Fentanyl has its unique pharmacology that distinguishes from opium-derived mu opioids such as morphine and heroin, which includes its faster onset and higher potency. Our preliminary studies showed that a lethal dose of fentanyl (10 mg/kg, i.v.) had no effect on persistent apnea, muscle rigidity and lethality, but induced modest respiratory depression in our Oprm1 E1-KO (rE1d/d) rats, suggesting that fentanyl overdose-induced severe respiratory depression (FOISRD), persistent apnea, muscle rigidity and lethality are primarily mediated through MORs with additional non-MOR mechanisms, particularly of the respiratory depression, in our rat models. Respiratory regulation in mammalians is very complex and dynamic and involves coordination among multiple brain regions and several peripheral tissues such as the lung and carotid body at the molecular, cellular, and network levels. MORs are widely expressed at these sites. Increasing evidence has indicated that MORs play an essential role in mediating opioid-induced respiratory depression (OIRD). However, the molecular mechanisms and related signaling pathways underlying FOISRD and other toxicities via MORs or non-MOR systems remain largely unknown. Reversible protein phosphorylation by protein kinases and phosphatases is one of the most important post-translational modifications that plays a fundamental role in regulating almost all physiological and pathological processes. Mu opioid-induced phosphorylation occurs very rapidly, commonly within seconds or minutes, dynamically shaping the signaling. However, we know little about in vivo phosphorylation induced by mu opioids, particularly by fentanyl overdose. Our preliminary data using tandem mass tags-based phosphoproteomics/proteomics (TMT PP/P-omics) showed that fentanyl overdose can rapidly modify phosphorylation status of many signaling molecules, some of which likely contribute to FOISRD, persistent apnea, muscle rigidity and death. These data strongly support our overarching hypothesis that fentanyl overdose induces differential phosphorylation of crucial signaling molecules in multiple brain regions and peripheral tissues via MORs or non-MOR system and subsequently alters their related signaling pathways, leading to severe respiratory depression, persistent apnea, muscle rigidity and lethality. To test the hypothesis, we proposed the following Specific Aims: (1) Examine fentanyl overdose-induced phosphoproteins/proteins' changes in multiple brain regions and peripheral tissues of our rat Oprm1 gene targeting models using TMT PP/P-omics; (2) Identify molecular targets and signaling pathways that are responsible for FOISRD from selected candidate phosphoproteins from TMT PP/P-omics study. The proposed studies well fall into the goal of this specific RFA and promise to identify novel molecular mechanisms underlying FOISRD, persistent apnea, muscle rigidity and lethality and may identify the potential molecular targets for developing novel therapeutic strategies to combat FOISRD and fentanyl overdose death.