Engineering fluorescent probes for tracking lysosomal pH in β-amyloid-induced microglial activation and phagocytosis

Alzheimer's disease (AD) is primarily associated with the aggregation of amyloid-β (Aβ) due to insufficient clearance of Aβ peptides. This leads to deposition of fibrillar Aβ (fAβ), contributing to AD progression. Microglia, the brain's resident immune cells, are central to the phagocytotic-fusion of fAβ. Notably, fAβ itself can activate microglia via toll-like receptor signaling, triggering a phagocytic response. Previous studies have shown that activated microglia exhibit efficient phagocytotic-fusion of fAβ, primarily through lysosomal acidification compared to resting microglia. Therefore, distinguishing microglial activation states is vital for understanding and potentially modulating Aβ clearance mechanisms in AD. Herein, we systematically modified the structure to develop fluorescent probes (FPs), PS-Mor and PM-DMor based on morpholine-conjugated pyrylium and pyridinium derivatives of indigenous "IndiFluors". These probes exhibit strong fluorescence enhancement in lysosomal pH windows by modulating photoinduced electron transfer (PET). The turn-on behavior of the probes was further supported by TD-DFT/PCM theoretical calculations. Confocal imaging revealed that PM-DMor selectively localizes to lysosomes, while PS-Mor targets mitochondria in activated human microglia. PM-DMor effectively monitors intracellular pH changes (ΔpHi) during drug-induced apoptosis and discriminates activated from resting microglial using both fluorescence microscopy and flow cytometry. Importantly, PM-DMor also tracks Aβ-induced microglial activation and subsequent phagocytosis of Aβ. Overall, PM-DMor offers a valuable tool for probing lysosomal dynamics in microglia and holds promise for early-stage therapeutic strategies targeting Aβ clearance in Alzheimer's disease.