Activity-Based Multimodal Probes Uncover the AChE-ADGRB Axis in Cellular and Murine Models of Depression

Acetylcholinesterase (AChE) is a cholinergic enzyme that hydrolyzes the neurotransmitter acetylcholine (ACh). Dysregulated AChE activity is closely linked to neurodegenerative diseases and cancer. Current chemical probes for AChE detection mostly rely on single-modality fluorescence readouts, limiting their utility. Herein, we developed a set of activity-based multimodal probes, termed AFP1, AFP2, AFP3, and AFPP, which combine the complementary strengths of near-infrared fluorescence (NIRF) imaging, photoacoustic (PA) detection, and proteome profiling. These probes offer a comprehensive toolset for deciphering the AChE code in complex biological systems. AFP1/2/3 enable continuous monitoring of AChE activity via NIRF/PA detection, while AFPP represents the first trimodal probe for simultaneous NIRF/PA imaging and proteome profiling in live systems. Notably, the usage of AFP2 and AFPP (1) highlighted the superior capability of PA imaging for deep-tissue studies in live animals, (2) revealed significantly elevated AChE activity in microglia compared to astrocytes in the depressive brain, and (3) identified a functional link between AChE and adhesion G protein-coupled receptor B2 and B3 (ADGRB2 and ADGRB3) in both cellular and murine models of depression. Our study not only provides powerful molecular tools for studying cholinergic systems but also reveals novel therapeutic targets for depression intervention.

乙酰胆碱酯酶（Acetylcholinesterase, AChE）是一种可水解神经递质乙酰胆碱（acetylcholine, ACh）的胆碱能酶。AChE活性失调与神经退行性疾病及癌症密切相关。当前用于AChE检测的化学探针大多依赖单模态荧光读出，限制了其应用范围。本文中，我们开发了一套基于活性的多模态探针，命名为AFP1、AFP2、AFP3与AFPP，这些探针整合了近红外荧光（near-infrared fluorescence, NIRF）成像、光声（photoacoustic, PA）检测与蛋白质组谱分析的互补优势，可为复杂生物系统中AChE的功能解析提供一套全面的工具集。AFP1/2/3可通过NIRF/PA检测实现AChE活性的连续监测，而AFPP则是首款可在活体系统中同时实现NIRF/PA成像与蛋白质组谱分析的三模态探针。值得关注的是，借助AFP2与AFPP的应用，我们取得了三项重要发现：(1) 证实了PA成像在活体动物深部组织研究中的优异性能；(2) 揭示出抑郁模型大脑中小胶质细胞的AChE活性显著高于星形胶质细胞；(3) 在抑郁症的细胞模型与小鼠模型中，明确了AChE与黏附型G蛋白偶联受体B2、B3（ADGRB2、ADGRB3）之间的功能性关联。本研究不仅为胆碱能系统的研究提供了强有力的分子工具，还为抑郁症干预发掘了全新的治疗靶点。