Asparagine Endopeptidase Cleaves α-Secretases And Amphiphysin1,Mediating The Pathogenesis Of Alzheimer’s Disease

Objective1.To investigate the degradation pathway of α-secretase(ADAM10 and ADAM17),and whether asparagine endopeptidase(AEP)is involved in the degradation.2.To explore whether amphiphysin1(Amph1)is a substrate of AEP in the brain of Alzheimer’s disease(AD).3.To explore the effects of AEP-generated Amph1 fragments in synaptic dysfunction and tau phosphorylation,and whether blocking AEP-mediated cleavage of Amph1 can alleviate the above pathological changes.Methods1.To investigate the degradation pathway of ADAM10/17,inhibitors of lysosome and proteasome were used to explore the role of lysosome and protease in the degradation of ADAM10/17.Meanwhile,we tested the half-life of ADAM10/17 when AEP was overexpressed.Immunofluorescence was used to detect the co-localization of ADAM10/17 and AEP in mouse brain slices overexpressing AEP.The pulse/chase experiment was applied to observe the effect of AEP on the endocytic traffic and degradation of ADAM10/17.2.To investigate the cleavage of ADAM10/17 and Amph1 by AEP in vitro,Western blot was used to detect the time-dependent cleavage of Amph1 or ADAM10/17 by AEP,the cleavage of purified Amph1 or ADAM10/17 by AEP,and the cleavage of Amph1 or ADAM10/17 in cell lysates co-transfected with myc-AEP or myc-AEP C189 S and Amph1 or ADAM10/17.Western blot was used to detect the effect of AEP inhibitor AENK on the cleavage of Amph1 by AEP.3.To investigate AEP-mediated cleavage of Amph1 in AD,mass spectrometry was used to analyze the cleavage site of Amph1 by AEP.Mutation of Amph1 which blocks its cleavage by AEP was used to confirm the cleavage site.An antibody that specifically recognizes the N terminal of Amph1 cleaved by AEP was generated.Western blot and immunohistochemistry were used to detect the level of Amph1 fragment in tau P301 S mice,normal human,and AD patients.4.To investigate the effect of AEP-generated Amph1 fragments on synaptic function and tau phosphorylation,we overexpressed Amph1 full-length and its fragments in cells,and performed transferrin uptake test,FM 4-64 dye,and f EPSPs recording.Western blot and immunofluorescence were used to observe the effect of Amph1 on tau phosphorylation,and the activity of tau phosphorylation kinase CDK5.5.To confirm the effect of AEP-generated Amph1 fragments on AD-related pathology and behavioral impairments in vivo,we overexpressed Amph1 full-length,fragments,and mutations that blocking its cleavage by AEP in tau P301 S mice brain.Electron microscopy was used to observe the synapse density.Golgi staining was used to detect dendritic spine density.Water maze and Y maze were used to observe learning and memory function.The electrophysiological test was used to detect LTP of the brain.Results1.Inhibiting the lysosomal degradation pathway increases the protein level of ADAM10/17,while inhibiting the proteasomal pathway did not significantly change the protein levels of ADAM10/17,indicating that ADAM10/17 are maily degraded via the lysosomal pathway.Half-life experiments and pause/chase experiments found that AEP can accelerate the degradation of ADAM10/17.AEP was co-localized with endocytosed ADAM10/17,and AEP cleaved ADAM10/17 in vitro.Knockout of AEP increased the protein level of ADAM10/17.2.AEP cleaved Amph1 in vitro.The major cleavage site was N278.N278 A mutant blocked the cleavage of Amph1 by AEP.The AEP-generated Amph1 N-terminal fragment existed in tau P301 S mice and increased with aging.Furthermore,the Amph1(1-278)fragment was more abundant in tau P301 S mice than wild-type mice,as well as in AD patients than the normal human.3.Amph1(1-278)fragment and(279-695)fragments generated by AEP partially lost the ability to combine other endocytic proteins,which induced the impairment of clathrin-mediated endocytosis and synaptic dysfunction.4.Amph1(1-278)fragment promoted the tau phosphorylation and enhanced CDK5 activity.CDK5 inhibitor reserved tau hyperphosphorylation induced by Amph1(1-278)fragment.Amph1(1-278)fragment promoted the translocation of p35 to the cell membrane,and facilitated its activation.5.Tau P301 S mice overexpressing Amph1 fragments exhibited a decrease of synaptic density and dendritic spine density,synaptic dysfunction,and impaired learning and memory ability.However,overexpression of Amph1 N278 A that blocks the AEP cleavage alleviated the AD-like pathological and behavioral defects.ConclusionAEP is activated in an age-dependent manner in the brain.Active AEP mediates the degradation of ADAM10/17 and the cleavage of Amph1.On one hand,the AEPmediated degradation of ADAM10/17 induces the dysregulation of α-secretase.On the other,AEP-mediated cleavage of Amph1 generates Amph1 fragments that causes synaptic dysfunction and tau hyperphosphorylation.Thus AEP may be a novel target for the treatment of AD.