Dynamic Effects Of AΒ Oligomers On Autophagy In Neuronal Cells And Exploration Of Regulation Of Autophagy Via PrP~C

Alzheimer Disease(AD)is the most common cause of dementia in the central nervous system(CNS)resulting in significant problems with learning,memory,thinking and behavior.The neurodegenerative disorder disease’s typical pathological feature is the deposition of senile plaques(SPs)formed by extracellular β-amyloid(Aβ)and neurofibrillary tangles(NFTs)formed by intracellular hyper-phosphorylation Tau in AD brains.Amyloid-β(Aβ)generated through the sequential cleavage of amyloid precursor protein(APP)by β-and γ-secretases is the major component of SPs,therefore considered to be a central trigger of AD pathogenesis.Autophagy is a lysosomal mediated cellular programmed self-processing system,which is closely related to the clearance of abnormal accumulation proteins associated with AD.Autophagy plays a "double-edged sword" role in AD.There is increasing evidence that a large number of autophagosomes and autolysosome were found in the brains of AD patients with autophagic dysfunction during AD development.Autophagy is considered a potential treatment for AD.In this study,by adding Aβ oligomers to SK-N-SH cells at different concentrations and culturing different days,we found that the level of LC3 B continued to increase in a short time,and LC3 B began to decrease at 7 days and was negatively correlated with the dose with Aβ.The level of p62 level is significantly increased.Furthermore,the SK-N-SH cells infected by AAV containing the m RFP-GFP-LC3 B fluorescent protein were cultured with different concentrations of Aβ for 3 days.The results of detecting autophagy showed that number of red fluorescence spots in 0.5μM group was significantly higher than those in the control group.The number of yellow spots increased significantly in the 1μM and 2μM groups.Western Blot and Confocal results indicated that autophagy was activated in a short time;long-term Aβ incubation led to blocking of autophagic flow suggesting autophagosome and lysosome fusion were blocked and autophagy was inhibited by feedback.Because of the close relationship between autophagy and ubiquitin,we detected ubiquitin-related proteins by Western Blot and found that MYSM1 was decreased significantly in 7d group relative to 1d and 3d groups,but the levels of XIAP were significantly higher than the control group in 1,3 and 7 days.It is suggested that Aβ oligomers can accelerate the ubiquitin protease dysfunction protein,and the stimulation intensity of oxidative stress caused by Aβ oligomers entering cells can be alleviated by the continuous activation of deubiquitinating enzymes,but with the extension of time,deubiquitination decreased and the ubiquitin system was disordered.We used SA-β-Gal kit to detect cell senescence,and further used Western Blot to detect the levels of p53 and Rb proteins.The results showed that Aβ oligomers can cause cell senescence.We used immunofluorescence to observe the effect of Aβon endogenous Aβ production in cells.We can see that Aβ production in cells increased in 3 days.We inferred that cells increase the intracellular Aβ production by up-regulating β-secretase by producing ROS.The results from Western Blot showed that Aβ oligomers can be increased in intracellular tau protein levels.Then we used nuclear and cytoplasmic extraction reagents to extraction nuclear and cytoplasmic protein.The result of Western Blot showed a decrease in Beclin 1 in the cytoplasm and an increase in Beclin 1 in the nucleus.It is suggested that part of the Beclin1 in cytoplasm was translocated into the nucleus.Finally,we used 2-(2-furan)quinoline-4-carboxylic acid to interfer the process,and the results showed that 2-(2-furan)quinoline-4-carboxylic acid can significantly reduce the level of p62 and the level of Tau,suggesting that Aβ inhibition of autophagy by cells is relieved by 2-(2-furan)quinoline-4-carboxylic acid.In conclusion,short-term incubation of Aβ oligomers activates autophagy in neuronal cells and increases abnormal protein clearance by ubiquitin system;under prolonged conditions,Aβ oligomers cause autophagosome and lysosomal fusion to be blocked,autophagy inhibition,ubiquitin system disorder,increased intracellular Tau protein levels,and cellular senescence.2-(2-furan)quinoline-4-carboxylic acid blocks the pathological pathways caused by Aβ.In conclusion,we established a model of Alzheimer’s disease cells model in vitro,studied the effect of Aβ on the process of autophagy and ubiquitin scavenging abnormal proteins,and found that Beclin 1translocated into the nucleus of SK-N-SH cells.It was also demonstrated that small molecule prion compounds could alleviate the inhibition of autophagy and the increased level of Tau induced by Aβ oligomer.