α-Asarone attenuates synaptic loss in APP/PS1 mice by restoring microglial phagocytic-lysosomal balance via the SPP1-mTOR-TFEB axis.

BACKGROUND: Synaptic loss in Alzheimer disease (AD) is closely linked to aberrant microglial phagocytosis. Complement-dependent synaptic pruning and microglial lysosomal dysfunction drive this process, yet effective pharmacological strategies targeting this axis are lacking. α-Asarone (ASA) is neuroprotective, but its effects on SPP1-centered microglial synaptic pathology remain unclear. PURPOSE: To determine whether ASA protects against AD-related synaptic injury by modulating SPP1-associated microglial phagocytic-lysosomal dysfunction and define the underlying mechanism. STUDY DESIGN: An integrated in vivo and in vitro pharmacological study was performed using APP/PS1 mice and BV2 microglia. METHODS: Six-month-old APP/PS1 mice were treated with ASA for 100 days and evaluated behaviorally. ASA effects on microglial complement activation, complement-tagged synapses, lysosomal dysfunction, and hippocampal synaptic injury were assessed by transcriptomic analysis, qPCR, western blotting, immunofluorescence, Golgi staining, and transmission electron microscopy. In Aβ-stimulated BV2 cells, the role of SPP1 in phagocytic-lysosomal dysfunction was evaluated using SPP1 knockdown and recombinant SPP1, together with assays of lysosomal acidification, DQ-BSA degradation, and TFEB subcellular localization. Potential ASA-SPP1 engagement was further assessed by molecular docking, molecular dynamics simulation, and cellular thermal shift assay. RESULTS: ASA improved behavioral performance and preserved hippocampal synaptic integrity in APP/PS1 mice. It reduced complement-associated microglial activation, CR3 signal, and complement deposition on PSD95-positive synapses, while restoring lysosomal acidification and proteolysis. SPP1 was upregulated in APP/PS1 mice and suppressed by ASA. ASA also inhibited SPP1-associated mTOR activation, reduced TFEB Ser211 phosphorylation and cytoplasmic retention, and improved lysosomal function. SPP1 silencing or mTOR inhibition mimicked ASA, whereas rSPP1 or mTOR activation attenuated these effects. CONCLUSION: ASA attenuated AD-related synaptic injury by restoring microglial phagocytic-lysosomal balance through the SPP1-mTOR-TFEB pathway, supporting its potential as a phytomedicine candidate for AD intervention.