Mechanistic Studies Of Frataxin-mediated Ferroptosis In A Cellular Model Of Alzheimer’s Disease

Objective To clarify the expression of FXN in an Alzheimer’s disease（AD）cell model constructed from L-Glutamaic acid（L-Glu）induced human neuroblastoma cells（SH-SY5Y）and to investigate whether ferroptosis is involved in the cell death process.The aim is to further investigate the effects of FXN overexpression on ferroptosis and mitochondrial function-related mechanisms in AD cell models,and to provide a reference laboratory basis for AD therapeutic mechanisms based on the inhibition of ferroptosis.Methods 1.SH-SY5Y cells were used as the target of the study,and the cells were induced with different concentrations of L-Glu for 24 h.The cell viability was detected by CCK-8 method,and the optimal L-Glu concentration was screened to construct an in AD cell model.The cells were divided into:Control（normal control group）,L-Glu（L-glutamate group）,Lentiviral Vector（normal control group+FXN lentivirus null group）,L-Glu+OE-FXN（L-glutamate+FXN overexpression group）and L-Glu+Fer-1（L-glutamate+ferroptosis inhibitor Fer-1 group）.2.In terms of mitochondrial function:mitochondrial membrane potential（MMP）changes were detected by fluorescent probe TMRE method and cellular mitochondrial morphological changes were observed by transmission electron microscopy to verify the effect of FXN overexpression on mitochondrial function in AD cell models.3.Ferroptosis levels:intracellular reactive oxygen species（ROS）changes were detected by fluorescent probe DHE method and kits to detect malondialdehyde（MDA）,The changes in the concentrations of reduced glutathione（GSH）,total iron ions and divalent iron ions were measured by fluorescent probe DHE and kits to determine whether L-Glu could induce ferroptosis in SH-SY5Y cells,and whether overexpression of FXN and Fer-1,an ferroptosis inhibitor,could reverse the above indicators.4.Western blot and cellular immunofluorescence assays were used to detect the expression of xCT and GPX4 proteins in the System Xc-/GPX4 pathway,and Western blot assays were used to detect changes in the expression of ACSL4 and Tf R1,which are important regulatory proteins of ferroptosis,to further explore the possible pathogenesis of ferroptosis in a model of AD cells mediated by overexpression of FXN.Results 1.Different concentrations of L-Glu significantly reduced the survival rate of SH-SY5Y cells in a concentration-dependent manner,and finally the survival rate of SH-SY5Y cells induced by 20 mmol/L L-Glu was 54.6%after 24 h.The cell morphology was significantly changed under ordinary light microscope,with shorter or absent synapses and smaller and rounder cells.Therefore,20 mmol/L L-Glu was set as the best concentration for the subsequent experiments in this study to prepare AD cell model.2.The expression of FXN was reduced in the AD cell model,and the expression of FXN was successfully up-regulated by using lentiviral transfection technique.3.There was no statistical significance in the Control group compared with the Lentiviral Vector group for each detection index,considering that the lentiviral empty vector there was no significant functional effect on SH-SY5Y cells.Compared with the Control group,MMP was reduced in the L-Glu group,and mitochondria were observed to shrink and cristae were deformed,reduced or disappeared by transmission electron microscopy,and after overexpression of FXN,MMP was increased and mitochondrial morphology was significantly improved,suggesting that mitochondrial function was impaired in the AD cell model,and overexpression of FXN could improve the manifestation of mitochondrial function impairment.4.In the L-Glu group,ROS,MDA,iron ion concentration and Fe²⁺levels were increased and GSH was decreased,while GSH was increased and ROS,MDA,iron ion concentration and Fe²⁺levels were decreased after overexpression of FXN.Elevated expression of ACSL4 and Tf R1,important regulatory proteins of ferroptosis,was detected by Western blot in the L-Glu group,and the expression of xCT and GXP4 in the System Xc^-/GPX4 pathway was reduced by Western blot and cellular immunofluorescence.Overexpression of FXN and Fer-1（10μmol/L）reversed the expression of these proteins after intervention.Conclusion 1.FXN expression is reduced in an AD cell model constructed from L-Glu-induced SH-SY5Y cells.2.Overexpression of FXN ameliorates mitochondrial functional impairment in an AD cell model.3.Overexpression of FXN may enhance antioxidant capacity by regulating the System Xc^-/GPX4 pathway and increasing the expression of xCT and GPX4,inhibit lipid peroxidation by suppressing the expression of ACSL4,reduce Fe²⁺accumulation by suppressing the expression of Tf R1,thereby inhibiting ferroptosis and subsequently having a protective effect on AD cell models.