Simultaneous profiling of cholinesterases and NAD(P)H in murine hippocampus via a dual-responsive indicator to investigate cholinergic impairment in Alzheimer's disease

The cholinergic hypothesis constitutes a pivotal theoretical framework for elucidating the pathological mechanisms underpinning Alzheimer's disease (AD). Quantitative assessment of cholinesterases (ChEs) and NAD(P)H levels in neuronal cells, particularly within the septo-hippocampal pathway, is imperative for the comprehensive study of the cholinergic system. In this study, we designed and synthesized a dual-channel, dual-enzyme-responsive fluorescent indicator that was chemically optimized for detecting levels of ChEs and NAD(P)H in neuronal cells. Biological experiments demonstrated that this indicator can sensitively and reliably visualize the dynamics of intracellular ChEs and NAD(P)H following stimulation. The corresponding ChEs activities and NAD(P)H concentrations demonstrated a linear correlation with fluorescence intensity, reaching equilibrium within a few minutes. This innovative and precise technique for visualizing ChEs and NAD(P)H dynamics with high spatial resolution during the progression of AD is expected to serve as a robust tool for investigating alterations in the cholinergic nervous system, including changes in acetylcholine neurotransmission and neuronal energy metabolism.