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

Microglia, which are the brain's resident macrophages, have received considerable attention as the leading player in the progression of neurodegenerative disease. However, there are currently no tools available for the tracking of biomolecules within microglia in diverse brain environments. In this study, we developed a multipotent microglial fluorescent probe called CDr20-CO1 that can be regarded as a toolbox for fluorescent labeling of live microglia’s intracellular biomolecules, from in vitro to in vivo. The compound CDr20-CO1 has a dual role namely as a binder for bioconjugation to an azido compound and as a microglia labeler by its specific fluorescent substrate activity for the UGT1A7C enzyme. It visualizes microglial lysosome, choline, or newly synthesized protein in the live heterogeneous brain cell culture condition by sequentially treating CDr20-CO1 following the addition of a binding partner such as morpholine-azide, choline-azide, or L-azidohomoalanine. This allows observing the low responsiveness of lysosomal fusion and choline metabolism of microglia in contrast to neuroinflammatory stimuli comparing coexistence with other glial cells against isolated microglia to see how they are different. Eventually, the labeling system works even in intact live mouse embryos, suggesting the application extends up to the microglia's organelles/biomolecules in vivo. A new approach for tracking microglial organelles/biomolecules using CDr20-CO1 will be a potent tool for deciphering microglia’s pathophysiological role in real-time.