Construction And Characterization Of PH-sensitive ?-site APP Cleaving Enzyme 1 Expression Plasmids

Alzheimer's disease(AD)is a chronic neurodegenerative disease that usually starts slowly and gradually worsens over time.At the microscopic level,the characteristic lesions in AD are senile or neuritic plaques and neurofibrillary tangles in the medial temporal lobe structures and cortical areas of the brain,together with a degeneration of the neurons and activation of astrocytes.Several competing hypotheses exist trying to explain the cause of the disease.The central hypothesis for the cause of AD is the amyloid cascade hypothesis,which states that ?-site APP-cleaving enzyme 1(BACE1)initiates amyloid precursor protein(APP)cleavage and ?-amyloid(A?)production,ultimately leading to neuronal degeneration and dementia.It is thus of considerable interest to investigate how BACE1 activity is regulated.Taking the advantage of BACE1 has its maximal activity at acidic pH,we generated a dual-fluorescence-based BACE1 reporter.It is our hope that this pHluorin-BACE1-mCherry fusion protein can be a useful tool to visualize active/inactive BACE1 tracking in cultured cells and in vivo.GFP and its variant—pHluorin—displays pH dependence.GFP and its variant do not emit obvious fluorescence at acidic vesicle pH(4.5-6)but emits green fluorescence at basic pH(7-7.5);mCherry is a pH-insensitive protein that emits red fluorescence at pH 4.5 to 7.5 and is thus useful to indicate fusion protein expression and localization.In light of these characteristics,we generated three tandem fluorescence-tagged BACE1 fusion proteins,named vesicle pH indicator of BACE1(pHluorin-BACE1-mCherry),cytosol pH indicator of BACE1(BACE1-mCherry-pHluorin)and control pH indicator of BACE1(BACE1-mCherry-EGFP)to investigate their expression in vitro and in vivo.Comparing the fluorescence characteristics of these BACE1 fusion proteins in response to intracellular pH changes induced by chloroquine(CQ)or bafilomycin A1(BafA1),we found that vesicle pH indicator of BACE1,pHluorin-BACE1-mCherry,is a better pH sensor for BACE1 because its fluorescence intensity responds to pH changes more dramatically and more quickly in NLT cells(a GnRH neuroblastoma cell line)and cortical neurons.Furthermore,we found that pHluorin-BACE1-mCherry have ?-secretase activity,and such BACE1 activity was abolished in the presence of the BACE1 inhibitor-LY2886721,suggesting its sensitivity to the inhibitor.(pro)renin receptor(PRR),also identified as adenosine triphosphatase(ATPase),H+-transporting,lysosomal accessory protein 2(ATP6AP2)—is a subunit of the V-ATPase,which is critical for maintaining vesicular pH.We found that PRR regulates pHluorin's fluorescence and BACE1 activity in pHluorin-BACE1-mCherry expressing cells.PRR knock-out increases green fluorescence intensity of pHluorin-BACE1-mCherry,and overexpression of PRR decreases green fluorescence intensity of pHluorin-BACE1-mCherry,supporting the view that the PRR/V-ATPase is a positive regulator of BACE1 activation,which is important for A? production.Finally,we found that the expression of Swedish mutant APP(APPswe)suppresses pHluorin fluorescence in pHluorin-BACE1-mCherry expressing cells in culture and in vivo,implicating APPswe not only as a substrate but also as an activator of BACE1.Taken together,these results suggest that the pHluorin-BACE1-mCherry fusion protein may serve as a useful tool for visualizing active/inactive BACE1 in culture and in vivo.The in vitro and in vivo evaluation models established in this study are a simple,rapid and economical tool for screening BACE1 inhibitors,which is important for AD drug development.