The aggregation of β-amyloid (Aβ) peptide in Alzheimer's disease (AD) is characterized by mitochondrial dysfunction and mitophagy impairment. Mitophagy is a homeostatic mechanism by which autophagy selectively eliminates damaged mitochondria. Valinomycin is a respiratory chain inhibitor that activates mitophagy via the PINK1/Parkin signaling pathway. However, the mechanism underlying the association between mitophagy and valinomycin in Aβ formation has not been explored. Here, we demonstrate that genetically modified (N2a/APP695swe) cells overexpressing a mutant amyloid precursor protein (APP) serve as an in vitro model of AD for studying mitophagy and ATP-related metabolomics. Our results prove that valinomycin induced a time-dependent increase in the mitophagy activation of N2a/APP695swe cells as indicated by increased levels of PINK1, Parkin, and LC3II as well as increased the colocalization of Parkin–Tom20 and fewer mitochondria (indicated by decreased Tom20 levels). Valinomycin significantly decreased Aβ_1–42 and Aβ_1–40 levels after 3 h of treatment. ATP levels and ATP-related metabolites were significantly increased at this time. Our findings suggest that the elimination of impaired mitochondria via valinomycin-induced mitophagy ameliorates AD by decreasing Aβ and improving ATP levels.

β-淀粉样蛋白（Aβ）肽在阿尔茨海默病（AD）中的聚集以线粒体功能障碍和线粒体损害为特征。线粒体吞噬是一种体内平衡机制，通过这种机制，自噬选择性地消除了受损的线粒体。Valinomycin是一种呼吸链抑制剂，可通过PINK1 / Parkin信号通路激活线粒体。然而，尚未探索线粒体和缬氨霉素之间在Aβ形成中的关联的机制。在这里，我们证明过表达突变淀粉样前体蛋白（APP）的基因修饰（N2a / APP695swe）细胞充当AD的体外模型，用于研究线粒体和ATP相关的代谢组学。我们的结果证明，如PINK1水平升高所示，缬氨霉素诱导N2a / APP695swe细胞线粒体激活的时间依赖性增加，Parkin和LC3II以及增加Parkin–Tom20的共定位作用和减少线粒体（Tom20水平降低表明）。缬霉素明显降低Aβ治疗3小时后，其_1–42和_Aβ1–40的水平。ATP水平和与ATP相关的代谢产物此时显着增加。我们的研究结果表明，通过缬氨霉素诱导的线粒体消除受损的线粒体可通过降低Aβ和改善ATP水平来改善AD。