An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity
Fig 3
The increased Aβ generation induced by Istradefylline is not through PKA, PKC or β-arrestins.
(A) A2AR ligands-modulated cAMP response in HEK293/APPswe cells. Cells were transfected with pGloSensorTM-22F cAMP plasmid and then stimulated with CGS 21680 HCl (CGS), ZM 241385 (ZM), Istradefylline (Istrad.) or SCH 442416 (SCH) at indicated concentrations. (B) A2AR antagonists block agonist-induced cAMP increase. Cells were pre-treated with ZM 241385 (ZM, 1 nM), Istradefylline (Istrad., 100 nM) or SCH 442416 (SCH, 1 nM) followed by the stimulation with CGS 21680 HCl (CGS, 1 μM). (C) The effect of A2AR ligands on Aβ production in the absence or presence of H89. Cells were incubated without or with 10 μM of H89, prior to the treatment with 0.1% of DMSO (Ctrl), CGS 21680 HCl (CGS, left) or Istradefylline (Istrad., right). H89 was present in the indicated groups throughout the experiments. (D) The effect of PKC activator or Istradefylline on Aβ production in the absence or presence of GO6983. HEK293/APPswe cells were pre-treated without or with 1 μM of PKC inhibitor GO6983 followed by the treatment with 0.1% of DMSO (Ctrl), 1 μM of PMA (left) or 30 nM of Istradefylline (Istrad., right). (E-G) The effect of β-arrestin1 or 2 on Istradefylline-modulated increase of Aβ generation. (E, F) Quantification of mRNA levels after transfection with scrambled, β-arrestin1 (E) or β-arrestin2 (F) siRNA in HEK293/APPswe cells. (G) After the transfection with scrambled, β-arrestin1 or β-arrestin2 siRNA, HEK293/APPswe cells were subjected to the treatment with 0.1% of DMSO (Ctrl) or Istradefylline (30 nM) followed by the detection of secreted total Aβ levels. (H) Representative images and the accompanied analysis of A2AR endocytosis upon the treatment with 0.1% of DMSO (Ctrl), CGS 21680 HCl (CGS) or Istradefylline (Istrad.) at 1 μM in HEK293 cells transfected with Flag-A2AR. Scale bar = 10 μm. N = 18–24 cells. Data are representative or mean +/± SEM of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. PMA, phorbol 12-myristate 13-acetate.