Regulation Of Autophagic Flux By Aβ And The Underlying Molecular Mechanism

Objective:Alzheimer’s disease (AD) is the most common form of age-related neurodegenerative disease in old people, and the main clinical features are cognitive disorders. AD has an insidious onset of symptoms and the mechanisms are not well understood. Recently, the abmornal state in protein modification and degeneration caused by autophagy dysfunction in AD has been the popular topics. A large number of autophagosomes are present in the AD brain before the formation of pathological features, which indicates that autophagy should be activated during the early stage of AD. In present study, we established the stable cell line expressing the molecular marker of autophagy, microtubule-associated protein1light chain3(LC3), so as to investigate the effects of Aβ-induced autophagy flux, corresponding cell fate changes and the underlying mechamism.Methods:(i) The establishment of EGFP-LC3-HEK293stable cell line. Six types of cells were considered as the candidates, and the cell type was determined according to the transfection efficiency and LC3protein expression level. The EGFP-LC3stable cells were developed via geneticin (G418) stress screen,(ii) The development of AP-induced autophagy cell model. EGFP-LC3puncta were detected in the pcDNA3.1(-)-AP transfected cells or the AP peptide-treated cells via fluorescence microscope, and AP dose-dependant, time-dependent, and secondary structure-dependent assays were performed. The ultrastructures and morphology were studied via transmission electron microscope (TEM).(iii) The mechanisms of AP-induced autophagy. The cells were incubated with10μmol/L of AP or the truncated peptides with various percentage of hydrophobic amino acids (Aβ40, Aβ42, Aβ1-11, Aβ12-24, Aβ25-35, and AP35-42) for24h. EGFP-LC3puncta were detected by fluorescence microscopy, the changes of LC3BII/I, p62and HSP70/90were detected via Western blot, cell viability was measured via MTT assays.Results:(i) An EGFP-LC3-HEK293stable cell line has been established under the stress of700μg/mL of G418in6weeks,(ii) The cell model of AP induced-autophagy has been established. EGFP-LC3puncta can be induced by transfectd AP or peptides in dose, time and secondary structure-dependant manner. The best conditions in this study for AP-induced autophagy were determined as:10μmol/L of AP oligomers assembled in24h were used to treat cells for24h. Ultrastructures of autophagosomes were detected in Aβ-treated cells via TEM.(iii) The underlying mechanism in AP-induced autophagy. First, quantitative fluorescence microscope data showed that EGFP-LC3puncta were induced in both AP-transfected and AP peptide-treatment groups. All of the AP isoforms used, particularly Aβ25-35, Aβ40and Aβ42induced EGFP-LC3puncta in this stable cell line. No relationship between autophagy and the amount of hydrophobic amino acids had been found. Second, Western blot results showed that LC3BII and LC3BII/LC3BI ratio increased in both AP42and positive control groups compared with the cell control group. The LC3BI/II shifting indicated LC3lipidation. At the same time, p62expression level decreased indicating its degradation with autophagy activation. If autophagesome-lysosome pathway was blocked by bafilomycin A1(Baf A1), LC3BII and LC3BII/LC3BI ratio increased significantly (p<0.01), and no reduction of p62was detected. Compared with Aβ42group, LC3BII level, LC3BII/LC3BI ratio (p<0.01) and p62level (p<0.05) increased significantly in AP42+Baf A1group. Similarly, compared with serum-free group, LC3BII level, LC3BII/LC3BI ratio (p<0.05) and p62level (p0.01) increased significantly in serum-free+Baf A1group. As for the heat shock protein (HSP) reaction, HSP90increased whereas HSP70decreased in AP42group or serum-free group, compared with control group. Last, much more cytotoxicity (p<0.05) were detected in AP42group than in Aβ40group via MTT assays.Conclusion:EGFP-LC3-HEK293cell line was established in our group and the effects and underlying mechanism of AP-induced autophagy were investigated. The findings also indicated that Aβ-induced autophagy may contribute to the cytotoxicity in AD. Our data laid the foundation for further mechanism by which autophagy regulates AD pathogenesis, prophylaxis and treatment.