Study On Processing Of Amyloid Precursor Protein(APP) Mutations And The Formation Of Amyloid-β Peptides

Currently,Alzheimer’s disease has become a health hazard for the elderly in our major diseases, and there is as yet no effective durg treatment. Amyloidβplaques are a defining characteristic of Alzheimer Disease. Aβ is a 38~43 amino acid peptide which is derived from the β-amyloid precursor protein(APP) through sequential cleavage of β- secretases and γ-secretases. Previous studies indicate that compared with other forms(Aβ1-40, 1-38), Aβ(1-42, 3-40) are more amyloidogenic. Lot of studies suggest that pathogenic mutations in APP have previously been linked to familial Alzheimer’s disease, therefore studies with these APP mutations will help us understand more about the pathogenesis of Alzheimer’s disease. So this study selected 5 APP mutations: A673T、A673V、E682K、E693G、E693Q for research. First we use overlap extension PCR to got 5 APP mutations, which were correctly cloned and identified by restriction enzyme digestion and sequencing. Then we co-transfected BACE1 and APP-WT or APP mutations into HEK293 cell line via lipofectamin 2000. The pcDNA4 was kept as a control. Finally, we used Western Blot, ELISA and LC-MS/MS method to analyze the level of related proteins and unique peptide of APP proteolysis pathway. The results indicated that proteolysis of APP of A673V、E682K and E693 Q significantly increased compared with APP-WT, and APP proteolysis of the rest two mutations had no differences with APP-WT; Although compared with APP-WT, C99 levels of E682K、E693G increased, the ratio of C99/C83 in A673 T was lower than APP-WT. ELISA detection revealed that Aβ42 level of A673 T was significantly lower than APP-WT, and Aβ42 levels of the other four mutation were significantly higher than APP-WT; Compared with APP-WT, Aβ40 level of A673 T has no obvious difference, while Aβ40 level of the other four mutations were remarkablely lower; The ratio of Aβ42/Aβ40 of A673 T was significantly lower than APP-WT, but Aβ42/Aβ40 ratio of the other four mutations significantly increased than APP-WT. The LC-MS/MS suggested that both Aβ40 and Aβ42 levels in A673 T decreased compared with APP-WT; both Aβ40 and Aβ42 levels in E682 K increased compared to APP-WT; Aβ40 levels of A673 V and E693 Q decreased, while the Aβ42 levels increased; Aβ40 level of E693 G increased, while the Aβ42 level decreased. Above all, A673 T mutation may play a protective role on Alzheimer’s disease by inhibiting toxic Aβ generation, whereas the other 4 mutations can promote APP cleavage process to produce Aβ42. All the results provided more data support for understanding Alzheimer’s disease pathology mechanism related to APP.