An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity
Fig 4
A2AR colocalizes with γ-secretase complex in endosomes and interacts with PS1.
(A-E) Representative images showing the subcellular distribution of PS1 and A2AR with endocytic markers. HEK293 cells were co-transfected with indicated protein constructs and pMLink-Pen2-NCT-APH1aL and stained for EEA1 or Flag-PS1 or labeled with lysostracker. (A) Subcellular colocalization of CFP-PS1 (C-P), A2AR-YFP (A-Y) and EEA1. (B-D) Subcellular colocalization of Flag-PS1 (F-P), A2AR-CFP (A-C) and Rab5-GFP (Rab5-G, B), Rab7-GFP (Rab7-G, C), Rab11-GFP (Rab11-G, D). (E) Subcellular colocalization of CFP-PS1 (C-P), A2AR-YFP (A-Y) and lysotracker (lyso). Scale bar = 10 μm. White box indicates the enlarged area. N ≥ 10 cells for each group. The number on the right-top corner presents the Mander’s colocalization coefficiency of two channels. (F) Representative image showing co-IP of γ-secretase complex components or BACE1 with Flag-A2AR in HEK293T cells. Cells, transfected with four γ-secretase complex components (PS1, NCT, APH1aL and Pen2) or BACE1 and β-gal or Flag-A2AR, were lysed in the buffer containing 1% of CHAPSO or 1% of TritonX-100 (TX-100) and immunoprecipitated with anti-Flag slurry. The antibodies were previously verified [11]. (G) Flag-A2AR co-immunoprecipitates with HA-C99, but not HA-APPswe. Cells were transfected with HA-APPswe or HA-C99 without or with Flag-A2AR and then lysed, immunoprecipitated and blotted. Representative image is shown. (H) A2AR interacts with PS1 in APP/PS1 mouse brain. Brain membrane fractions were extracted from a 9 month APP/PS1 mouse. PS1 was immunoprecipitated with anti-PS1 antibody (1563 as labeled in the figure) and samples were subjected to Western-blot analysis. The antibody recognizing the endogenous A2AR has been verified [58]. (I) FRET efficiency of A2AR-CFP with YFP-fused other proteins. HEK293 cells were transfected with A2AR-CFP and YFP alone (Y), A2AR-YFP (A2AR-Y), APH1aL-YFP (APH1aL-Y), NCT-YFP (NCT-Y), YFP-PS1 (Y-PS1) or BACE1-YFP (BACE1-Y). Cells were then fixed and subjected to acceptor photobleaching FRET experiment. N ≥ 30 cells per condition. Data are mean + SEM. *, p < 0.05; ***, p < 0.001.