APSH-OP-2: INTRACELLULAR TRANSPORTATION OF AMYLOID BETA VIA INTERACTION BETWEEN ANGIOTENSIN II TYPE 1 RECEPTOR AND RECEPTOR OF ADVANCED GLYCATION END PRODUCTS

Abstract

Objective: We recently reported that the binding of the receptor for advanced glycation end-products (RAGE) to AGE activates the G protein-coupled angiotensin II (AII) type 1 receptor (AT1) through membrane complex of the receptors (2021 Sci Rep). It has been reported that the binding of RAGE to amyloid beta is involved in the development of Alzheimer disease (AD) via processes involving intracellular transport of Amyloid beta. Here, we performed cell experiments and utilized animal models to investigate if the activation of AT1 is critically involved in Amyloid beta-RAGE-mediated cellular phenomenon, leading to the AD pathology. Design and Methods: We used CHO-cells stably transfected with either AT1 (CHO-AT1) or RAGE(CHO-RAGE), RAGE and AT1 (CHO-RAGE-AT1) and either mutation of AT1 G protein pathway(CHO-RAGE-AT1mb) or beta-arrestin pathway(CHO-RAGE-AT1 mg). Activation of Galphaq and Galphai were quantified by accumulation of IP1 and redcution of cAMP assay, respectively. Endocytosis of the Amyloid beta-RAGE complex through the AT1- beta-arrestin pathway was demonstrated by real-time imaging of the membrane dynamics of AT1 and RAGE by a spinning disk-type confocal super-resolution microscope, detection of the AT1- betaarrestin2 interaction by BRET, detection of beta-arrestin2-AP2 interaction by TR-FRET. Visualization of intracellular fluorescent Amyloid beta, and detection of intracellular aggregated Amyloid beta by PROTEOSTAT® reagent in several types of human endthelial cells and microlgilal cells. In vivo experiment, wild type and AT1 KO mice were used for analysis. Results: Using genetically modified CHO cells, we found that Amyloid beta activates Galphai, but not the Galphaq pathway of AT1, in the presence of RAGE. Furthermore, in the presence of both RAGE and AT1, we could detect and visualize Amyloid beta indcued beta-arrestin activation and endocytosis of receptors complex. We also found that inhibition of either AT1 or beta-arrestin pathway could attenuate intracellular Amyloid beta upatke and aggregation in several types of human endthelial cells and microlgilal cells. Finally, serum concentration of Amyloid beta 1–40 and Amyloid beta 1–42 was higher in AT1 knockout mice than wild type mice at 15–19 month, while the concentration of the Amyloid beta peptides in cerebrospinal fluid was equivalent between two types of mice. Conclusions: These findings indicate that Amyloid beta triggers selective G protein activation and the beta-arrestin-dependent internalization of AT1 whereby the Amyloid beta-RAGE complex undergo endocytosis. This machinery can be relevant in mice in which the deletion of AT1 led to increase in circulating Amyloid beta, implying the ablation of intracellular transporting system of Amyloid beta.