Dual-Channel Imaging of Amyloid-β Plaques and Peroxynitrite To Illuminate Their Correlations in Alzheimer’s Disease Using a Unimolecular Two-Photon Fluorescent Probe

Alzheimer's disease (AD) involves multiple pathological factors that mutually cooperate and closely contact to form interaction networks for jointly promoting the AD progression. Therefore, the comonitoring of different factors is particularly valuable for elucidating their level dynamics and complex interactions. However, such significant investigations remain a major challenge due to the lack of unimolecular fluorescent probes capable of simultaneous and discriminative visualization of multiple targets. To address this concern, as proof of principle, we rationally designed a unimolecular fluorescent probe to discriminate and simultaneously profile amyloid-β (Aβ) plaques and peroxynitrite (ONOO^-), which are both the pronounced AD pathological factors. Herein, a novel ONOO^- reaction trigger was installed onto an Aβ plaque binding fluorophore to generate a dual functional fluorescent probe, displaying completely separate spectral responses to Aβ plaques and ONOO^- with high selectivity and sensitivity. With this probe, for the first time, we comonitored the distribution and variation of Aβ plaques and ONOO^- through two independent fluorescence channels, demonstrating their close apposition and tight correlation during AD course in live cell and mouse models through two-photon imaging mode. Notably, Aβ aggregates induce the neuronal ONOO^- generation, which conversely facilitates Aβ aggregation. The two critical events, ONOO^- stress and Aβ aggregation, mutually amplify each other through positive feedback mechanisms and jointly promote the AD onset and progression. Furthermore, by coimaging of the level dynamics of Aβ plaques and ONOO^-, we found that the cerebral ONOO^- is a potential biomarker, which emerges earlier than Aβ plaques in transgenic mouse models. Overall, the dual-channel responsive performance renders this probe as a powerful imaging tool to decipher Aβ plaque-ONOO^- interactions, which will facilitate AD-associated molecular pathogenesis elucidation and multitarget drug discovery.