IRE1α-XBP1 Affects The Mitochondrial Function Of Aβ25-35-treated SH-SY5Y Cells By Regulating Mitochondria-associated Endoplasmic Reticulum Membranes

BackgroundAlzheimer’s Disease(AD)is a degenerative disease of the central nervous system that severely impairs the cognitive and memory functions of patients.The neurotoxicity induced by amyloid-β(Aβ)is one of the most important pathological mechanisms of Alzheimer’s disease(AD),but its mechanism of action is unknown.Studies have shown that Aβ accumulates in the mitochondria,activates mitochondrial fission and increases mitochondrial calcium levels,impairing mitochondrial function.Mitochondrial dysfunction can promote the cleavage of APP to produce Aβ,leading to abnormal accumulation of Aβ,forming a vicious circle and promoting the occurrence and development of AD.Therefore,exploring mitochondrial dysfunction at the molecular level may provide new clues for understanding Aβ-induced neuronal damage and developing effective treatments for AD.The lipid raft-like region where the endoplasmic reticulum physically contacts the mitochondria is called the mitochondrial-associated endoplasmic reticulum membrane(MAM).Recently,the potential role of MAM disorders in the pathogenesis and pathological process of AD has received increasing attention.MAM behavior changes may be related to various dysfunctions in the AD phenotype,such as changes in Aβ;mitochondrial damage.IP3R-Grp75-VDAC1 is a tethering protein on MAM,which structurally connects the endoplasmic reticulum and mitochondria and enables calcium transfer from the ER to mitochondria,affecting mitochondrial function.Inositol-requiring enzyme 1α(IRE 1α)is a transmembrane protein with serine/threonine protein kinase and endonuclease activity.Once activated,IRE1αexcises a 26-base intron from the x-box binding protein 1(XBP1)mRNA to produce an active transcription factor(XBP1s).A growing number of studies have demonstrated that IRE1α-XBP1 is activated in AD and triggered by Aβ peptide,and the activation of IRE1α exacerbates the histopathological progression of AD.It was recently discovered that IRE1α interacts with IP3R to regulate calcium transfer from the endoplasmic reticulum to the mitochondria.In addition,IRE1α-XBP1 inhibits mitochondrial respiration of ovarian cancer T cells.However,the relationship between mitochondrial function,IRE1α-XBP1 and MAM in the pathogenesis of AD is still unclear.ObjectiveThis study conducted a cellular research to investigate the effects of IRE1α-XBP1 on Aβ-induced cytotoxicity and mitochondrial function,and clarify whether IRE1α-XBP1 affects the mitochondrial function of Aβ-treated cells through regulation of MAM,thereby contributing to find prevention and treatment methods for the cause of AD.Methods1.SH-SY5Y cells were treated with AP25-35 for different time,and the expression of IRE1α,pIRE1α and XBP1s was detected by RT-PCR and Western Bolt experiment.2.SH-SY5Y cells were divided into control group,4μ8c(small molecule inhibitor of IRE1α),Aβ group,and Aβ+4μ8c group.Four groups of cells were tested by the MTT experiment to detect the effect of inhibiting IRE1α-XBP1 on the cytotoxicity of Aβ,and the effect of inhibiting IRE1α-XBP1 on the functional state of Aβ treated cells was tested by calcium ion imaging technology.3.ATP production was detected by fluorescence method,mitochondrial membrane potential was detected by TMRM staining,and mitochondrial reactive oxygen species was detected by MitoSOX Red staining.4.The number and length of MAM of each group of cells were observed by electron microscope5.The calcium ion level in the cytoplasm and endoplasmic reticulum was detected by calcium imaging technology,and the calcium ion level in the cytoplasm,mitochondria and endoplasmic reticulum was further detected by immunofluorescence technology.6.The expression of MAM tether protein IP3R-Grp75-VDAC1 was detected by western blot,and the interaction of IP3R-Grp75-VDAC1 was detected by CO-IP.Results1.Aβ activates the IRE1α-XBP1 pathway.2.Inhibition of IRE1α-XBP1 reduces the cytotoxicity of Aβ.3.Inhibition of IRE1α-XBP1 enhances ATP content,restores mitochondrial membrane potential,reduces mitochondrial reactive oxygen levels,and reduces Aβ-induced mitochondrial dysfunction.4.Inhibition of IRE1v-XBP1 reduces the increase in the number and length of MAM caused by Aβ.5.Inhibition of IRE1α-XBP1 reduces calcium transfer between endoplasmic reticulum and mitochondria caused by Aβ.6.Inhibition of IRE1α-XBP1 reduces the expression and interaction of IP3R-Grp75-VDAC1 induced by Aβ.ConclusionAβ activates the IRE1α-XBP1 pathway,and inhibiting the IRE1β-XBP1 pathway can alleviate the Aβ-induced mitochondrial dysfunction.This may be achieved by reducing the expression and interaction of the MAM tether protein IP3R-Grp75-VDAC1,further reducing the formation of MAMs and reducing the endoplasmic reticulum Calcium transfer to the mitochondria.