Thalamic PDK-PDHA1 Signaling is Associated with Opioid-Induced Loss-of-Righting-Reflex Sensitivity: Evidence from Phosphoproteomic Profiling and PDK Pathway Intervention.

Opioids are widely used in clinical sedation and anesthesia, but the downstream phosphorylation events associated with opioid-induced reductions in arousal remain incompletely understood. Here, we investigated whether thalamic phosphoproteomic remodeling and the pyruvate dehydrogenase kinase (PDK)-pyruvate dehydrogenase E1 alpha subunit (PDHA1) pathway are associated with opioid-induced loss of righting reflex (LORR) in rats. Phosphoproteomic profiling of rat thalamic tissues after treatment with fentanyl, FENT-001 (a synthetic fentanyl analog), and other sedative and anesthetic agents revealed broad changes in thalamic phosphopeptide abundance.Differentially phosphorylated proteins were enriched in neuronal projections, synapses, cytoskeletal organization and synaptic transmission. PDHA1 was identified as a significantly differentially phosphorylated protein and was further examined by Western blotting. Fentanyl and 3-methylfentanyl altered phosphorylation at multiple PDHA1 sites. Pharmacological inhibition of PDKs with dichloroacetate reduced PDHA1 Ser293 phosphorylation in cultured cortical neurons and produced numerical increases in the estimated ED50 values for fentanyl- and 3-methylfentanyl-induced LORR. These changes were consistent with a trend toward reduced LORR sensitivity. Conversely, CM-targeted overexpression of PDK2 or PDK4 shifted the fitted fentanyl-induced LORR dose-response curves to the left relative to the Vector control, with a larger numerical reduction in ED50 following PDK2 overexpression.These findings suggest that opioid treatment remodels the thalamic phosphoproteome and that the PDK-PDHA1 phosphorylation pathway is functionally associated with opioid-induced LORR sensitivity.