An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity
Fig 5
Istradefylline, but not CGS 21680 HCl, attenuates the interaction between A2AR and PS1 and influences the internal conformation of PS1.
(A, B) HEK293 cells co-transfected with CFP-PS1 and A2AR-YFP were treated with Istradefylline (30 nM) for 0–60 min followed by the determination of FRET efficiency using acceptor photobleaching FRET (A) or FRET SE technique (B). Both the FRET efficiency and the attribute units of YFP or CFP intensity are presented in B. N ≥ 20 cells per condition. a.u., arbitrary units. Alternatively, the transfected cells were treated with Istradefylline (C) or CGS21680 HCl (D) at indicated concentrations for 30 min followed by acceptor photobleaching FRET assay. (E) Representative image showing Istradefylline reduces the co-IP of PS1 with Flag-A2AR. HEK293T cells were transfected with Flag-A2AR alone, PS1 alone or co-transfected with Flag-A2AR and PS1. Cells were treated without or with 30 nM of Istradefylline and then lysed with 1% of TritonX-100 IP buffer, followed by immunoprecipitation and western blotting. (F) PS1 conformation revealed by the internal FRET efficiency of CFP-PS1-YFP. HEK293 cells were transfected with CFP-PS1-YFP (C-PS1-Y) followed by the treatment without or with 30 nM of Istradefylline. As a control, cells were co-transfected with CFP-PS1-YFP (C-PS1-Y) and pMLink-Pen2-NCT-APH1aL (PNA). N ≥ 30 cells per condition for FRET assays. Data are mean +/± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.