Simultaneous Visualization of Labile Ferrous Iron Pool and Biothiols Reveals Divergent Pathological Trajectories in Ferroptosis and Alzheimer’s Disease

Abstract Alzheimer’s disease (AD) and ferroptosis share certain pathobiochemical features, yet the spatiotemporal evolution patterns and metabolic trajectories of their core hallmarks, labile ferrous iron pool and biothiols, and their pathophysiological relationship remain elusive. Herein, we developed a single-molecule dual-site fluorescent probe, FCG, for simultaneous discrimination of Fe2+, Cys/Hcy, and GSH via analyte-specific cascade reaction mechanisms. The probe exhibits rapid kinetics (k = 0.16−1.23 min−1), nanomolar sensitivity (detection limits: 38.8−509.2 nM), and well-separated emission signatures (Δλem ≥ 60 nm), facilitating concurrent visualization of these endogenous species dynamics in living systems. Importantly, application to cellular and mouse models revealed divergent metabolic pathways: ferroptosis displays synchronized labile ferrous iron pool accumulation, catastrophic lipid peroxidation, and global biothiols depletion, whereas AD exhibits modest iron elevation, a unique biothiols profile (elevated total Cys and Hcy but depleted GSH), and protein aggregation that is absent in ferroptosis. Comparative proteomic analysis further elucidated the molecular landscape of these dynamically regulated networks, as well as their distinct pathological metabolic trajectories. This study not only clarifies the mechanistic basis for the fundamentally different pathological trajectories underlying ferroptosis and AD but also provides critical evidence and analytical tools for targeted interventions in neurodegenerative diseases.