Systems-Level Phosphoproteomic and RPPA Profiling Reveals Stress and DNA Damage Signalling as Early Drivers of Polymyxin B Neurotoxicity.

Polymyxins remain indispensable last-line antibiotics for multidrug-resistant Gram-negative infections, yet their clinical use in central nervous system (CNS) infections is constrained by poorly understood neurotoxicity. Here, we define the early molecular signalling events underlying polymyxin B-induced CNS toxicity using an integrated phosphoproteomic and Reverse Phase Protein Array (RPPA) approach in rat brain following intracerebroventricular administration. Global phosphoproteomics revealed extensive phosphosite coverage but identified a highly selective set of significantly regulated phosphosites, implicating calcium-dependent signalling, transcriptional stress regulation, synaptic signalling, and cytoskeletal control, while parallel total proteomics showed minimal changes in protein abundance. RPPA profiling independently confirmed coordinated modulation of stress, apoptotic and survival-associated signalling pathways, including p53, CREB, SQSTM1, Bcl-2, and NFκB related nodes. Network and functional enrichment analyses converged on DNA damage signalling, apoptotic regulation and growth factor-mediated pathways as central features of the polymyxin B early neurotoxicity response, while phosphor to total protein analyses demonstrated suppression of proliferative and pro-survival signalling. Together, these data establish phosphorylation-driven signalling reprogramming as a primary early mechanism of polymyxin B-induced neurotoxicity, providing a mechanistic framework that links membrane-active antibiotic exposure to neuronal stress signalling and identifies candidate pathways for toxicity biomarkers and neuroprotective strategies.