Alzheimers Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes 英文参考文献.docVIP

Alzheimer’sDisease-LinkedMutationsinPresenilin-1 ResultinaDrasticLossofActivityinPurifiedc-Secretase Complexes MatthiasCacquevel,Lore`neAeschbach,JemilaHouacine,PatrickC.Fraering* E′colePolytechniqueFe′de′raledeLausanne,BrainMindInstitute,LaboratoryofMolecularandCellularBiologyofAlzheimer’sDisease,Lausanne,Switzerland Abstract Background: Mutations linked to early onset, familial forms of Alzheimer’s disease (FAD) are found most frequently in PSEN1,thegeneencodingpresenilin-1(PS1).Togetherwithnicastrin(NCT),anteriorpharynx-defectiveprotein1(APH1),and presenilinenhancer2(PEN2),thecatalyticsubunitPS1constitutesthecoreofthec-secretasecomplexandcontributesto the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Ab) peptides. Although there is a growing consensusthatFAD-linkedPS1mutationsaffectAbproductionbyenhancingtheAb1–42/Ab1–40ratio,itremainsunclear whetherandhowtheyaffectthegenerationofAPPintracellulardomain(AICD).Moreover,controversyexistsastohowPS1 mutationsexerttheireffectsindifferentexperimentalsystems,byeitherincreasingAb1–42production,decreasingAb1–40 production,orboth.Becauseitcouldbeexplainedbytheheterogeneityinthecompositionofc-secretase,wepurifiedto homogeneitycomplexesmadeofhumanNCT,APH1aL,PEN2,andthepathogenicPS1mutantsL166P,DE9,orP436Q. Methodology/Principal Findings: We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generatedifferentstablecelllinesoverexpressinghumanc-secretasecomplexeswithdifferentFAD-linkedPS1mutations.A multi-step affinity purification procedure was used to isolate semi-purified or highly purified c-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of c-secretase activityphenotype,intermsofAb1–40,Ab1–42andAPPintracellulardomainproductionsinvitro. Conclusion/Significance: Our data support the view that PS1 mutations lead to a strong c-secretase loss-of-function phenotypeandanincreasedAb1–42/Ab1–40rati