Autophagy Plays A Significant Role In The Degradation Of C31,the Toxic C-terminal APP Fragments In Alzheimer’s Disease,via Its Interaction With LC3

Objective:Alzheimer’s disease(AD)is characterized by the buildup of amyloid-β(Aβ)aggregates and tau protein,which are studied intensively before.C31,the cytoplasmic tail of the amyloid precursor protein(APP)was found to be toxic and has a critical role in AD.Autophagy,found to be closely related to AD,is one major cellular pathway leading to the removal of aggregated proteins.In this report,we initiated the investigation about the relationship between the autophagy and c31.Specifically,to explore the role of autophagy in the degradation of C31 and the relevant mechanism.Methods:The recombinant eukaryotic constructs,p3xFLAG-CMV-10-mCherry and p3xFLAG-CMV-10-mCherry-C31,were built through molecular cloning methods.Using liposomes to deliver the two constructs into SH-SY5Y and APPsw HEK293 cells respectively.Then,the transfection and expression of the constructs were detected by immunofluorescence and Western blot.The effects of C31 on the viability of SH-SY5Y and APPsw HEK293 cells were observed using methyl thiazolyl tetrazolium(MTT)assay.To investigate the turnover of C31 in either upregulated or downregulated autophagic levels in the two cell lines via Western blot.Finally,the interaction between autophagic marker light chain 3(LC3)and C31 was observed using immunoprecipitation and immunofluorescence methods.Results:In the SH-SY5Y and APPsw HEK293 cell lines,the viability of cells transfected with the C31 plasmid was significantly lower than the vector group(P<0.05).There was no significant difference in the level of autophagy between the C31 group and control in both cell lines.Under conditions of promoting autophagy(rapamycin and starvation),the level of LC3-Ⅱ was slightly increased,but didn’t reach the statistical significance and the C31 levels were not statistically changed.When using autophagy inhibitors,3-methyl adenine(3-MA)on the APPsw HEK293 cells,both LC3-Ⅱ and C31 levels were not statistically changed,but LC3-Ⅱ levels were mildly reduced.While inhibiting the fusion of autophagosomes and lysosomes,either by chloroquine(CQ)and ammonium chloride(NH4C1),both LC3-Ⅱ and C31 levels were statistically increased.In SH-SY5Y cells,there were similar results,but when incubating with the CQ and NH4C1,the C31 levels were not significantly changed.Moreover,C31 could induce higher levels of APP in SH-SY5Y and APPsw HEK293 cells.C31 could be anchored to the autophagosome via its interaction with LC3.Conclusion:C31 could produce significant cytotoxicity in SH-SY5Y and APPsw HEK293 cells.Overexpressing C31 could elevate the level of APP,but didn’t change the level of LC3-Ⅱ.Autophagy mediated the clearance of C31 via its interaction with LC3,indicating that autophagy may play a protective role in Alzheimer’s disease.This study further clarifies the pathogenesis of AD and may provide new insights into the therapeutic implications for AD.