Exercise promotes lipophagy and mitigates cognitive impairment in Alzheimer’s disease through downregulating microglial TMEM9

Background Autophagy-lysosomal dysfunction contributes to lipid droplet (LD) accumulation, which drives pathological progression in Alzheimer's disease (AD). Exercise is reported to restore autophagy-lysosomal function and attenuate cognitive impairment in AD, but its regulation of LD deposition remains unknown. Methods 5×FAD transgenic mice underwent a three-month exercise intervention. Cognitive and emotional behaviors were assessed using the Morris water maze, novel object recognition, and open field test. LD deposition was evaluated by oil red O and BODIPY staining. Proteomic sequencing was performed to identify proteins affected by exercise. Pathological changes were examined by immunofluorescence, western blotting, and transmission electron microscopy. Intracerebroventricular injection of AAV was used to overexpress or knock down microglial TMEM9 to investigate the mechanisms by which physical exercise affects behavior and pathology in AD. In vitro, BV2 cells were cultured and incubated with oligomeric amyloid-β (oAβ), with or without oleic acid to induce LD formation. Chloroquine and rapamycin, which regulate autophagy-lysosomal function, were used to examine the effects of LDs on mitochondrial integrity under glucose deprivation conditions. Lentiviral or plasmid transfection was used to explore the key role of TMEM9 in regulating lipophagy, and the interaction between TMEM9 and ATP6AP1 was detected by co-immunoprecipitation. Clinically, plasma TMEM9 and ATP6AP1 levels in AD patients were measured by ELISA, and receiver operating characteristic (ROC) analysis was performed to evaluate diagnostic performance. Results Exercise mitigated cerebral LD deposition through enhanced lipophagy, preserving mitochondrial integrity and attenuating cognitive decline. Mechanistically, exercise suppressed microglial TMEM9 expression, restoring LD degradation. Tmem9 downregulation facilitated LKB1 lysosomal translocation, activating AMPK signaling. In vitro, oleic acid-induced LDs preserved mitochondrial membrane potential and increased ATP production in BV2 microglia under glucose deprivation and oAβ exposure. Tmem9 overexpression exacerbated lipophagy impairment and mitochondrial dysfunction, whereas Tmem9 knockdown enhanced lipophagy and cytoprotection. Aβ clearance was negatively regulated by TMEM9. Clinically, AD patients exhibited characteristic Aβ deposition and glucose hypometabolism, with plasma TMEM9 levels strongly correlating with p-tau217 progression. Plasma TMEM9 and the TMEM9/ATP6AP1 ratio emerged as potential AD diagnostic biomarkers (AUC=0.791 and 0.853, respectively). Conclusion These findings establish exercise-induced TMEM9 inhibition as a novel therapeutic strategy targeting lipidostasis to combat AD progression.