Valinomycin Reduces A? And Improves ATP-related Metabolomics In N2a/APP695swe Cells By Activating Mitophagy

Objectives: amyloid-?(A?)is one of the characteristic pathological changes of Alzheimer's disease(AD).The compromised mitophagy will lead to the accumulation of damaged mitochondria,which is an important factor for the formation of A?.Mitophagy is a selective autophagy mechanism,which maintains the homeostasis of the body by clearing damaged mitochondria.Valinomycin can activate the PINK1/Parkin mediated mitophagy pathway by inducing the decrease of mitochondrial membrane potential.However,the effect of valinomycin-induced mitophagy on A? production needs to be further identified.Here,we demonstrate that genetically modified(N2a / APP695swe)cells that overexpressing a mutant amyloid precursor protein(APP)serve as an in vitro model of AD for studying.We first observed the changes of mitochondria using electron microscope,then detected the experssion of mitochondria and mitophagy-related proteins in N2 a / APP695 swe cells.Then dectected the changes of mitochondria and mitophagy-related proteins activated by valinomycin in N2 a / APP695 swe cells,and further dectected the effect of valinomycin-induced mitophagy on the production of A? and ATP.Finally,the relationship between the changes of metabolites and ATP level in N2 a / APP695 swe cells was analyzed by gas chromatography-mass spectrometry(GC-MS).The purpose of this study is to explore the mechanism of Valinomycin-induced mitophagy to reduce A? levels and improve ATP level,and to provide more theoretical basis for finding ATP-related metabolic targets for the treatment of AD.Methods: First of all,the cells were divided into 2 groups: WT group(N2a / WT cells)and APP group(N2a / APP695 swe cells);Immunofluorescence and Western Blot was used to detect the expression of APP in each group.The morphological difference of mitochondria between the two groups was observed by transmission electron microscope.Western blot was used to detect the expression of mitochondrial outer membrane protein Tom20,mitophagy-related proteins PINK1 and Parkin,as well autophagy-related protein LC3 II in each group.Elisa assay was performed to analyze the expression of A?1-40 and A?1-42 in each group of cell culture mediums.The optimal concentration of Valinomycin was selected by CCK8(Cell Counting Kit-8),after treating N2 a / APP695 swe cells with Valinomycin at fixed concentration(1 ? mol / l),the cells were divided into five groups according to different treating time: APP group(N2a / APP695 swe cells),DMSO group(N2a / APP695 swe cells treated with DMSO),APP-Va3 h group(N2a / APP695 swe cells treated with Valinomycin for 3 h),APP-Va6 h group(N2a / APP695 swe cells treated with Valinomycin for 6 h),APP-Va12 h group(N2a / APP695 swe cells treated with Valinomycin for 12 h).Western blot was employed to detect the expression of the expression of PINK1,Parkin,LC3 II and Tom20.Then the co-localized expression of LC3 and Tom20 in each group was detected by immunofluorescence microscope.Photochemical analysis was used to detect the changes of ATP production in each group.Elisa assay was used to detect the expression of A ? 1-40 and A ? 1-42 in each group of cells culture medium.The changes of energy metabolites were detected by gas chromatography-mass spectrometry(GC-MS).Results: In N2a/APP695 swe cells,mitochondria are damaged and mitophagy is impaired.After activating mitophagy in N2 a / APP695 swe cells by Valinomycin,the levels of A?1-40 and A?1-42 decreased,and the levels of ATP as well the levels of ATP-relating metabolites increased.This study provides more evidence for ameliorating AD and finding metabolic targets for the treatment of AD by activating mitophagy via Valinomycin.However,the activation of mitophagy should be at an appropriate level,which was obtained in N2 a / APP695 swe cells after treating with valinomycin for 3 h,otherwise excessive mitophagy activation may lead to negative effects,such as the increase of A?.