Simultaneous Sensing of H2S and ATP with a Two-Photon Fluorescent Probe in Alzheimer’s Disease: toward Understanding Why H2S Regulates Glutamate-Induced ATP Dysregulation

Energy deprivation and reduced levels of hydrogen sulfide (H₂S) in the brain is closely associated with Alzheimer's disease (AD). However, there is currently no fluorescent probe for precise exploration of both H₂S and adenosine triphosphate (ATP) to directly demonstrate their relationship and their dynamic pattern changes. Herein, we developed a two-photon fluorescent probe, named AD-3, to simultaneously image endogenous H₂S and ATP from two emission channels of fluorescent signals in live rat brains with AD. The probe achieved excellent selectivity and good detection linearity for H₂S in the 0-100 μM concentration range and ATP in the 2-5 mM concentration range, respectively, with a detection limit of 0.19 μM for H₂S and 0.01 mM for ATP. Fluorescence imaging in live cells reveals that such probe could successfully apply for simultaneous imaging and accurate quantification of H₂S and ATP in neuronal cells. Further using real-time quantitative polymerase chain reaction and Western blots, we confirmed that H₂S regulates ATP synthesis by acting on cytochrome C, cytochrome oxidase subunit 3 of complex IV, and protein 6 of complex I in the mitochondrial respiratory chain. Subsequently, we constructed a high-throughput screening platform based on AD-3 probe to rapidly screen the potential anti-AD drugs to control glutamate-stimulated oxidative stress associated with abnormal H₂S and ATP levels. Significantly, AD-3 probe was found capable of imaging of H₂S and ATP in APP/PS1 mice, and the concentration of H₂S and ATP in the AD mouse brain was found to be lower than that in wild-type mice.