Visualizing biomolecules within living microglia in complex environments using a clickable small fluorogenic compound

Microglia, the brain's resident macrophages, have received considerable attention as a key player in the progression of neurodegenerative diseases. Understanding how organelles and biomolecules within microglia change in response to their state is crucial to advancing treatments for neurodegenerative diseases. However, these mechanisms remain poorly understood due to technical limitations. In this study, we developed a multipotent microglial fluorescent probe called CDr20-CO1 to label intracellular biomolecules of live microglia. This probe has a dual role as a binder for bioconjugation to biomolecule-specific azido compounds and as a microglia labeler through its specific fluorescent substrate activity for the UGT1A7C enzyme. Using these properties, we visualized microglial lysosomes, choline, or newly synthesized proteins in the live heterogeneous brain cell culture and demonstrated that microglia coexisting with other glial cells were less responsive to lysosomal fusion and choline metabolism in response to neuroinflammatory stimuli compared to isolated microglia. Finally, the labeling system works even in intact live mouse embryos, suggesting that the application extends to microglial organelles/biomolecules in vivo. A new approach for tracking microglial organelles/biomolecules using CDr20-CO1 will be a potent tool for deciphering the pathophysiological role of microglia in real time.