Lighting Up Arginine Metabolism Dynamics in Neural Differentiation through Ratiometric Fluorescence Imaging

Arginine (Arg) is a critically important amino acid in the central nervous system and plays a pivotal role in the differentiation of neural stem cells (NSCs). However, specifically and quantitatively tracking Arg dynamics in situ presents a significant challenge. Herein, we developed a series of Arg probes (Rap1-9) based on the nucleophilic substitution reaction between a phenyl-activated ester and a primary amine. Among these, the optimized NIR-emission ratiometric fluorescent probe Rap-9 demonstrated the most pronounced electrophilicity at its reactive site, enabling rapid and specific detection of Arg both in vitro and in vivo. Notably, Rap-9 was successfully utilized for real-time imaging of Arg dynamics during O₂^·--stimulated NSC differentiation. Further studies revealed that Arg-mediated activation of the mTORC1 pathway could coordinate qNSC reactivation and neuronal differentiation by regulating mitochondrial energy metabolism from glycolysis to mitochondrial oxidative phosphorylation. These findings not only enhanced our understanding of the metabolic pathways governing NSC differentiation but also offered new perspectives for neuroscience research and potential therapeutic interventions.