Study On Mechanism Of GPR50Regulating Amyloid-βProduction

Alzheimer’s disease (AD) is a neurodegenerative disease which is characterizedclinically by progressive impairments of memory and cognition. The importantpathological features of Alzheimer’s disease are the presence of neurofibrillary tanglesand extracellular amyloid plaques in the brain. Senile Amyloid plaques are consisted ofβ--amyloid (Aβ), which is derived from the sequential cleavage of amyloid-β precursorprotein (APP) by β-secretase and γ-secretase. Aβ, especially the oligomeric form,causes losses in spines, suppression of synaptic plasticity, neuroinflammation, cellapoptosis, eventually causes cognitive deficits. Therefore, inhibition of the generation ofAβ is one of the prime strategies of AD therapy.β-secretase activity is believed to be the rate limiting step in the amyloidogenicpathway. BACE1(β-site APP cleaving enzyme1) is the major rate limiting enzyme. Themajor form of the enzyme responsible for Aβ production, BACE1, is highly expressedin brain. Several studies indicate that BACE1level and activity are elevatedsignificantly in brain. Hence, BACE1is a major drug target for Alzheimer’s diseasebecause BACE1-mediated cleavage of APP is the first step in the generation ofamyloid-β peptides.G protein coupled receptors (GPCRs) have been suggested recently to playessential roles in the pathogenesis of AD. G protein coupled receptor50(GPR50) is anorphan GPCR. GPR50interacts with Nogo-A, a member of reticulon family, whichplays essential roles in neurodegenerative diseases including AD. These studies suggestthat GPR50may have a role in AD pathology. However, the precise function of GPR50in the nervous system remains unknown. In this study, we demonstrated that GPR50inhibited β-secretase cleavage of APP and generation of Aβ, which was a novel role ofGPR50in the pathogenesis of AD.Contents1. To investigate the function and the mechanisms of GPR50on the generation of Aβ.2. To investigate the function and the mechanisms of GPR50on regulation ofBACE1protein.Methods:1. Cell viability was measured by MTT assay．2. Immunofluorescence staining was performed to investigate the location ofGPR50and BACE1.3. RT-PCR and Western blot were used to detect the mRNA levels of and theprotein levels of GPR50, BACE1, respectively.4. Levels of Aβ40and Aβ42were measured by ELISA．5. performed to investigate the interaction between GPR50and either BACE1orAPP.Results:1. GPR50on the regulation of the generation of Aβ1.1GPR50inhibited the generation of Aβ and myloid-β precursor proteinmetabolism.1.2Overexpression of GPR50in HEK-APP cells (HEK293cells stably expressingAPP) inhibited the generation of Aβ in does-dependent manner.1.3Knocking-down of GPR50by siRNA increaseed the generation of Aβ incultured primary neurons.1.4GPR50supressed the cleavage of APP by β-secretase. Overexpression ofGPR50reduceed the levels of β-CTF, but not that of α-CTF.2. GPR50on the regulation of BACE12.1BACE1and GPR50colocalized and interacts with each other.2.2BACE1activity and protein expression were significantly reduced byoverexpression of GPR50in HEK-293cells. Overexpression of GPR50did not affectthe levels of BACE1mRNA in cultured neurons and HEK-293cells.2.3GPR50accelerated the degradation of BACE1, which could be blocked bypretreatment the transfected cells with lysosomal inhibitors.2.4Overexpression of or knocking-down of GPR50, enhanced or decreased the colocalization ratio of BACE1and LAMP1, respetively.Conclusion:Interaction of GPR50and BACE1inhibited the generation of Aβ. GPR50accelerated BACE1degradation by facilitating the translocation of BACE1tolysosomes. GPR50will play a role in the pathogenesis of AD through regulating thelevel of BACE1.Hence, GPR50may be a new drug target for the trerapy of AD.