GPR50 Promotes Degradation Of BACE1 Via Induction Of Autophagy

Objective:GPR50 is a member of the rhodopsin-like subclass of GPCRs(G protein coupled receptor).To investigate the functionof GPR50 in the pathogenesis of Alzheimer’s disease, we explore the function of GPR50 on degradation of BACE1(β-site APP cleaving enzyme 1)and the underlying mechanisms.Methods:1) Analysis of the levels of BACE1 by Western Blotting in HEK-293 T cellscotransfected with BACE-HA and different amount of GPR50-FLAG plasmids.2) To investigate whether BACE1 is degraded throught autophagy by treatingHEK-293 T or MEF cells with autophagy inhibitors(3-MA) or autophagyinducer(Trehalose and Rapamycin).3) To investigate whether GPR50 promotes degradation of BACE1 throughautophagy by transfection of GPR50 in wild type MEF cells, ATG5 deficientMEF cells and CHO cells pretreated with 3-MA.4) To investigate whether GPR50 induces autophagy by analysis of levels ofLC3II and the density of LC3 positive puncta in cells overexpressing GPR50 orknocking-down of GPR50.5) To investigate the molecular mechanisms underlying the induction ofautophagy by GPR50 by investigating the levels of phosphorylated mTOR,phosphorylated CREB, ATG5, ATG7 and BACE1 in cells transfected withGPR50 and in the brains of GPR50 deficient mice using western blotting andqPCR.Results:1. BACE1 can be degradated by autophagy.2. GPR50 promotes degradation of BACE1 through autophagy.3. GPR50 induces autophagy through activation of CREB, which mediates transcription of ATG5 and ATG7.Conclusion:Experimental results demonstrate that GPR50 can activate autophagy through activating P-CREB. In addition, GPR50 plays a critical role in degradating BACE1 by autophagy pathway, which promotes a new drug target for the treatment of Alzheimer’s disease.