Effect Study Of Autophagy Induced By ADDLs In SH-SY5Y Cells In Vivo

Autophagy has been existed generally in Alzheimer's disease (AD) before the classic pathologic features. During this stage,βamyloid-peptide (Aβ) derived diffusible ligands (ADDLs) has also been recognized as the key agent. However, it has been poorly undestood about the relationship between ADDLs and autophagy initiating in AD. In order to study the effects of autophagy induced by ADDLs in SH-SY5Y in vivo and decipher the mechanism, we mainly completed three parts of work as follows:1) The effects of autophagy induced by ADDLs in SH-SY5Y cells in vitro. ADDLs were assamblied as before, and which were identified by SDS-PAGE and TEM. The cyctosolic level of autophagy marker, microtubule-associated protein light chain 3, LC3-Ⅰ/Ⅱwas detected by Western blot, after cells were treated with different dosage or incubation time. cAMP was used as the positive control.2) The role of the secondary structure in ADDLs induced autophagy. Cells were treated with ADDLs of different secondary structures. The structures were conformed by circular dichroism (CD). The contents of a-helix andβ-sheet were determined via specific software. LC3-Ⅰ/Ⅱwas detected by Western blot as above.3) The pathway of autophay induced by ADDLs. The mTOR substrates p-p70 and p70 (t-p70) were detected by WB to determine the pathway.As the result, data showed that:1) The activation of SH-SY5Y autophagy induced by ADDLs is in a time and dose dependent manner. When the induction time were 0 h, 12 h, and 24 h, LC3 level induced by ADDLs increased while it declined on 48 h. When the induction doses were 0μmol/L,5μmol/L,10μmol/L,20μmol/L and 40μmol/L, the expression of LC3 accordingly increased. After that, the expression kept a certain level although the dosage increased.2) During the ADDLs assembling the secondary structure changes, which can induce the autophagy of SH-SY5Y cells. When the polymerization time was between 12 and 24 h, a helix content decreased to 4.5%, andβfold increase in the content to 13.9%. During this period, the expression increasing of LC3 can be detected.3) p-p70 and the ratio of p-p70/p70 decreased accompanied the ADDLs assembling. This suggested that the autophagy induced by ADDLs may through the mTOR pathway.In summary, time, dosage and secondary structure of ADDLs may determine the activation of autophagy of SH-SY5Y cells, and it seems that it is in a mTOR-dependent pathway. This study may provide the experimental basis for further study of ADDLs in autophagy activation in AD.