Inhibitors of  -secretase stabilize the complex and differentially affect processing of amyloid...

The FASEB Journal • Research Communication

Inhibitors of �-secretase stabilize the complex anddifferentially affect processing of amyloid precursorprotein and other substrates

Gael Barthet, Junichi Shioi, Zhiping Shao, Yimin Ren, Anastasios Georgakopoulos,and Nikolaos K. Robakis1

Center of Molecular Biology and Genetics of Neurodegeneration, Departments of Psychiatry andNeuroscience, Mount Sinai School of Medicine, New York University, New York, New York, USA

ABSTRACT �-Secretase inhibitors (GSIs) are drugsused in research to inhibit production of A� and inclinical trials to treat Alzheimer’s disease (AD). Theyinhibit proteolytic activities of �-secretase noncompeti-tively by unknown mechanisms. Here, we used corticalneuronal cultures expressing endogenous levels of en-zymes and substrates to study the effects of GSIs on thestructure and function of �-secretase. We show thatGSIs stabilize the interactions between the C-terminalfragment of presenilin (PS-CTF), the central componentof the �-secretase complex, and its partners the APH-1/nicastrin and PS1-NTF/PEN-2 subcomplexes. This stabi-lization dose-dependently correlates with inhibition ofN-cadherin cleavage, a process limited by enzyme avail-ability. In contrast, production of amyloid precursor pro-tein (APP) intracellular domain (AICD) is insensitive tolow concentrations of GSIs and is limited by substrateavailability. Interestingly, APP is processed by both PS1-and PS2-containing �-secretase complexes, while N-cad-herin and ephrinB1 are processed only by PS1-containingcomplexes. Paradoxically, low concentrations of GSIsspecifically increased the levels of A� without affecting itscatabolism, indicating increased A� production. Our datareveal a mechanism of �-secretase inhibition by GSIs andprovide evidence that distinct �-secretase complexes pro-cess specific substrates. Furthermore, our observationshave implications for GSIs as therapeutics because pro-cessing of functionally important substrates may be inhib-ited at lower concentrations than A�.—Barthet, G., Shioi,J., Shao, Z., Ren, Y., Georgakopoulos, A., Robakis, N. K.Inhibitors of �-secretase stabilize the complex anddifferentially affect processing of amyloid precursorprotein and other substrates. FASEB J. 25, 2937–2946(2011). www.fasebj.org

Key Words: Alzheimer’s disease � A� � presenilin � N-cadherin

Presenilin (PS) is the catalytic component of the�-secretase complex that processes the amyloid precur-sor protein (APP), producing the A� peptides, thestructural components of the amyloid depositions ofAlzheimer’s disease (AD). This complex also promotesthe cleavage of a large number of cell surface proteins,

including APP, Notch1, cadherins, and ephrinB1, pro-ducing peptides that have been shown to regulatesignal transduction and gene expression (1, 2). Inaddition to PS, the �-secretase complex contains at least3 other partners, including the anterior pharynx-defec-tive 1 (APH-1), nicastrin (NCT), and presenilin en-hancer 2 (PEN-2). The latter protein has been reportedto stimulate the endoproteolysis of full-length PS zymo-gen into catalytically active fragments PS-NTF andPS-CTF (3). Genetic studies showed that both PS and itssubstrate APP play causative roles in the developmentof the forms of familial AD (FAD). In addition, brainamyloid depositions of A� are used in the diagnosis ofAD and have also been proposed to play causative rolesin its development. Thus, �-secretase inhibitors (GSIs)have been used to lower the in vivo levels of A� andtreat the disease (4). A number of groups however,reported that prolonged treatment of mice or humanswith micromolar concentrations of GSIs resulted, afteran initial decrease, in levels of A� exceeding thestarting levels (4–6). Furthermore, low (nanomolar)concentrations of GSIs increased the in vivo levels of A�without an initial inhibitory effect (4, 7), although itwas unclear whether this effect resulted from increasedproduction or decreased degradation of A�. The inhib-itory mechanisms of GSIs are under investigation, andrecent data indicate that they inhibit catalysis noncom-petitively, consistent with a model where substratesbind a docking site before migrating to the catalytic site(8–10). To examine whether GSIs modify the confor-mation of the �-secretase, we studied their effects onthe interactions between components of the �-secretasecomplex and on substrate proteolysis. Our data showthat GSIs increase the interactions between PS1-CTFand its binding partners, APH-1/NCT and PS1-NTF/PEN-2 heterodimers, and differentially affect process-

1 Correspondence: Departments of Psychiatry and Neuro-science, Mt. Sinai School of Medicine, New York University,One Gustave L. Levy Pl., Box 1229, New York, NY 10029, USA.E-mail: nikos.robakis@mssm.edu

doi: 10.1096/fj.11-183806This article includes supplemental data. Please visit http://

www.fasebj.org to obtain this information.

29370892-6638/11/0025-2937 © FASEB

ing of substrates. In addition, we obtained evidencesupporting an increased production of A�42 at lowconcentrations of GSIs.

MATERIALS AND METHODS

Materials and antibodies

Mouse monoclonal antibody 33B10 against residues 331–350of PS1, polyclonal antibody R222 against PS1 N-terminalfragment, and R57 antibody against C-terminal domain ofAPP were described previously (11). Mouse anti-N-cadherin(cat. no. 610920) was from Becton Dickenson TransductionLaboratory (Franklin Lakes, NJ, USA). Anti-APH-1 specific ofAPH-1aL isoform (PA1–2010) was from Affinity BioReagents(Golden, CO, USA); anti-NCT (N1660) was from Sigma (St.Louis, MO, USA). Anti-PS2-NTF (7861) and ephrinB1-Cter(c18) were from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Anti-PEN-2 (NE1008), anti-PS2-CTF (PC235), and GSIsL665,458 and DAPT were from Calbiochem (San Diego, CA,USA).

Primary neuronal cultures

Cortices from embryonic day 17 (rat) or 15 (mouse) embryoswere dissected and dissociated in trypsin. Neuronal progeni-tors were plated in serum-free Neurobasal � B27 medium.Cultures were maintained at 37°C in a humidified atmo-sphere in 5% CO2 (106 cells/well in 6-well plate). All exper-iments were performed with neurons cultured for 8 days invitro (DIV).

Analysis of �-secretase complexes

Neuronal cultures were treated or not with inhibitors beforelysis in a dodecylmaltoside-based lysis buffer (50 mM HEPES,pH 7.4; 100 mM NaCl; 10% glycerol; and 0.5% DDM).Samples were immunoprecipitated (IPed) with APH-1, NCT,or PS1-NTF antibodies. Obtained proteins were separated byWBs using Tris-tricine gels.

In vitro �-secretase activity assay

Cortical neurons of 8 DIV were treated or not overnight(O/N) with DAPT or L685,458, and then scraped in hypo-tonic buffer (10 mM MOPS and 10 mM KCl). Membranespurified from postnuclear fraction were either incubated at37°C in a citrate buffer (150 mM, pH 6.4) to allow �-secretaseenzymatic activity or kept at 4°C. In some experiments, DAPTor L685,458 was added to the membrane suspension. After16 h of incubation, the reactions were stopped by theaddition of Laemmli buffer, and proteins in samples wereseparated by Western blot (WB) analysis using 10–20%gradient Tris-tricine gels. Membranes were probed for theanalysis of APP with R1 antiserum specific to cytoplasmic APP(12). N-cadherin full-length and C-terminal fragments weredetected with anti-N-cadherin monoclonal antibodies (BDTransduction Laboratories). In experiments reported in Fig.4A, PS1/2 double-knockout (PS1/2 dKO) mouse embryonicfibroblasts (MEFs) were lysed in hypotonic buffer (10 mMMOPS; 10 mM KCl) and membrane purified by ultracentrif-ugation. Membrane proteins were extracted with CHAPSO1% lysis buffer and used as a source of APP-CTFs (whichaccumulate in fibroblasts lacking PS).

Surface biotinylation

Neuronal cultures were incubated with 1 mg/ml sulfo-NHS-SS-biotin in PBS at 4°C for 1 h, washed with 0.1 M glycine, andlysed in a dodecylmaltoside-based lysis buffer (see above).The cell extracts were incubated with streptavidin-agarosebeads O/N. The bound proteins were washed 3 times withPBS and eluted with Laemmli.

Medium preparation and A� analysis using sandwich ELISAassay

Growth medium from neuronal culture of 8 DIV was changedto fresh Neurobasal medium without B27 (to eliminateinsulin that may interfere with both A� production anddegradation), and cultures were treated with GSIs at variousconcentrations, as indicated in figures, for 16 h. Medium wasthen collected, supplemented with 1 mM pefablock, andcentrifuged at 14,000 g for 15 min to remove any membranecontaminants. In experiments reported in Fig. 5C, mediumfrom a wild-type neuronal culture was transferred to anAPP�/� neuronal culture. Aliquots were analyzed after 16 hof incubation to access A�42 degradation. Collected mediumwas analyzed using the Covance A�42 murine kit (Covance,Madison, WI, USA) following the manufacturer’s instruc-tions.

Study of A� degradation

Medium from 8-DIV neuronal cultures was changed to freshmedium containing S35-labeled methionine/cysteine. AfterO/N incubation, medium was purified and concentrated bydialysis and added to sister neuronal cultures grown in theabsence of labeled methionine/cysteine. DAPT or insulin wasadded for 16 h to evaluate their effects on A� catalysis.Medium was then IPed with anti-p3/A� antibodies, andobtained proteins were separated using 10–20% Tris-Tricinegels. Gels were dried and used for autoradiography.

Statistical analysis

Four independent experiments were performed for statisticalanalysis of A� secreted by neuronal cultures and measured byELISA, as described earlier. Likewise, 3 independent experi-ments were performed for the in vitro �-secretase assay withneuronal membranes. To evaluate statistical significance ofthe pharmacological treatments, paired t tests were per-formed against the value of the untreated basal condition.Values of P � 0.05 were considered significant.

RESULTS

�-Secretase inhibitors enhance interactions betweenPS1-CTF and PS1-NTF and stabilize the �-secretasecomplex

The mechanism by which GSIs block substrate cleavageis under intense investigation, and reports indicate thatboth transition- and nontransition-state analogs arenoncompetitive inhibitors (8, 9). Functional �-secretasecomplexes contain at least 4 subunits, including NCT,APH-1, PS-CTF/PS-NTF heterodimer, and PEN-2.APH-1 and NCT form a subcomplex that binds thePS-CTF component of the PS heterodimer, while PEN-2

2938 Vol. 25 September 2011 BARTHET ET AL.The FASEB Journal � www.fasebj.org

binds PS-NTF, in an arrangement that places PS-CTF inthe center of the proteolytic complex (13–15). Wepostulated that GSIs affect catalysis by changing theinteractions between �-secretase complex subunits andtested this hypothesis using coimmunoprecipitationexperiments of detergent-extracted �-secretase compo-nents. O/N treatment of primary neuronal cultureswith DAPT, a potent GSI (16), increased the amountsof PS1-CTF and PEN-2 associated with either APH-1 orNCT, while the ratio of APH-1 to NCT remainedconstant (Fig. 1A), in agreement with reports of anAPH-1/NCT subcomplex. Interestingly, the amounts ofPS1-CTF and PEN-2 associated with APH-1/NCT wereincreased by 2- and 6-fold, respectively. Since PEN-2 bindsPS-NTF, this observation suggests that in addition to theinteraction between APH-1/NCT and PS1-CTF, DAPT

may also increase the interaction between PS-CTF andPS-NTF and possibly between PS-NTF and PEN-2. To testthese possibilities, we IPed PS1-NTF and observed thatDAPT increases the interaction between PS1-NTF andPS1-CTF (Fig. 1B), but not the interaction between PS1-NTF and PEN-2, which remained constant (Fig. 1B).

We then evaluated the sensitivity of the interactionbetween �-secretase subunits to DAPT concentration.Figure 1C shows that 5 nM DAPT is sufficient toincrease the interactions between NCT and PEN-2. Amaximal effect was observed at 100 nM DAPT, with anEC50 of �30 nM. A comparable sensitivity of theinteractions between PS1-CTF and the other �-secretasecomponents to DAPT was seen when PS1-CTF was IPed(Fig. 1D). In this case, all complexes between PS1-CTFand other subunits increased, in agreement with re-

Figure 1. GSIs increase inter-actions between PS1-CTF andother components of �-secre-tase. A) DAPT treatment in-creases interactions betweenAPH-1/NCTheterodimerandPEN-2. Extract in dodecyl-maltoside-based lysis bufferwas prepared from rat corticalneurons treated O/N with 2�M DAPT, and samples wereIPed with either anti-APH-1or anti-NCT antibodies (Abs).IPed proteins were analyzedon WBs using 10–20% gradi-ent Tris-tricine gels. Experi-ments were repeated 4 times.B) DAPT increases the inter-action between PS1-NTF andPS1-CTF but not between

PS1-NTF and PEN-2. Samples were IPed with anti-PS1-NTF Abs, and obtained proteins were analyzed on WBs as above. Duplicates areshown. C) DAPT stabilizes NCT to PEN-2 interactions in a concentration-dependent manner. Rat neurons were treated O/N withincreasing concentrations of DAPT (molar), and prepared extract was IPed with anti-NCT Abs. Top panel: precipitates were blotted forNCT and PEN-2. Bottom panel: quantification of NCT to PEN-2 interactions, expressed as a percentage of interaction at the highestconcentration of inhibitor. EC50 of DAPT effect is �30 nM. D) DAPT increases interactions between PS1-CTF and other �-secretasesubunits in a concentration-dependent manner. Samples prepared as in C were IPed with anti-PS1-CTF Abs. E) Transition-state analogL685,458 also increases interaction between NCT and other �-secretase components. Rat neuronal cultures were treated O/N with 2 �MDAPT or 1 �M L685,458 (L685), and extract was IPed with anti-NCT Abs. F) Model of �-secretase complex assembly and inhibition. Leftand middle panels depict the �-secretase assembly, with the PS1-CTF fragment of PS1 heterodimer binding the APH-1/nicastrinsubcomplex, while the PS1-NTF fragment of PS1 heterodimer binds PEN-2, as described previously (13–15). The two catalytic aspartateson TMs VI and VII of PS are depicted as yellow-red sparks. Right panel illustrates proposed effects of GSIs on structure of the complex. Byenhancing component interactions (see text), GSIs induce a closed conformation of the enzyme complex, characteristic of inhibitedenzymes (inactive aspartates shown in black).

2939GSIs REVEAL SUBSTRATE-SPECIFIC �-SECRETASE

ports that DAPT acts on PS-CTF (17), the centralcomponent of the �-secretase complex. Since DAPT isroutinely used in inhibition assays at concentrations �100 nM, our data indicate that at these concentrations,the drug acts as an effective stabilizer of the �-secretasecomplex, increasing the interactions between PS1-CTFand its partners. Moreover, this stabilization is notassociated with a defect in �-secretase trafficking, be-cause DAPT does not affect cell surface expression ofthe �-secretase subunits (Supplemental Fig. S1). Toexamine whether this stabilization effect was restrictedto nontransition-state analog inhibitors like DAPT, weanalyzed the effect of �-secretase inhibitor L685,458, atransition-state analog. As shown in Fig. 1E, L685,458also increases the interaction between NCT/APH-1 andother components of the �-secretase complex. To-gether, our results show that GSIs increase the interac-tions between PS1-CTF and its partners, including theAPH-1/NCT and the PS1-NTF/PEN-2 subcomplexes.Furthermore, our data conform with enzymology par-adigms predicting that enzymes exhibit a tensed orclosed conformation when inhibited and a relaxed oropened conformation when active (18). We propose thatGSIs inactivate �-secretase by closing the PS1-CTF/NTFinterface where the catalytic site forms and by stabilizingthe complex in a tensed conformation (Fig. 1F).

GSIs stabilize both PS1- and PS2-containing�-secretase complexes independently of APP

We then asked whether the stabilization of �-secretasecomplexes by GSIs is specific to PS1-containing com-plexes or also occurs with PS2-containing complexes.To avoid interference from PS1, we analyzed the effectof DAPT on PS2-containing complexes in PS1-KO(PS1�/�) mouse cortical neurons. Consistent with dataobtained in cell lines (19), absence of PS1 results indecreased levels of both NCT and PEN-2 but increasedlevels of neuronal PS2 (Fig. 2A, left panels). IP ofAPH-1 revealed increased amounts of PS2 associatedwith APH-1 in PS1�/� neurons compared to WT neu-rons (Fig. 2A, right panels, lanes 1, 5), while IP ofPS2-CTFs revealed increased amounts of both APH-1and NCT bound to PS2, even though total levels ofNCT decreased in these neurons (Fig. 2B). Further-more, DAPT stabilized the interaction of APH-1 withboth PEN-2 and PS2-CTF (Fig. 2A, right panel), as wellas the interaction between the NTF and CTF fragmentsof PS2 (Fig. 2C), indicating that similar to PS1-contain-ing complexes, DAPT also stabilizes interactions be-tween components of the PS2-containing �-secretasecomplexes. To examine whether the GSI-induced sta-bilization of �-secretase complexes involves APP-CTFsubstrates that accumulate during enzyme inhibition,

Figure 2. GSIs stabilize both PS1- and PS2-containing �-secretase complexes independently of APP. A) GSIs also affectPS2-containing �-secretase complexes. Cortical neurons from PS1�/�, PS1�/�, and PS1�/� mouse embryos were treated O/Nwith 2 �M DAPT. Protein samples were IPed with anti-APH-1 Abs, and precipitates were analyzed on WBs as in Fig. 1A.B) PS2-containing �-secretase complexes increase in absence of PS1. Samples as in A were IPed with anti-PS2-CTF Abs. C) DAPTincreases the interaction between PS2-NTF and PS2-CTF. Neuronal cultures were treated or not with 2 �M DAPT, and extractswere IPed with anti-PS2-NTF or anti-PS2-CTF Abs. D) DAPT effect on �-secretase complex is independent of APP. Neurons fromAPP�/� or APP�/� cultures were lysed, and extracts were IPed with anti-APH-1 or anti-NCT Abs.

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we measured inhibitor effects on �-secretase complexesin the presence or absence of APP. Figure 2D showsthat absence of APP has no effect on the DAPT-inducedstabilization of these complexes. These data supportthe conclusion that stabilization of �-secretase com-plexes by GSIs is due to PS binding of these drugs.

GSIs differentially affect the �-cleavage of APP andN-cadherin

We then asked whether modulation of the interactionsof �-secretase components induced by GSIs correlateswith the inhibitory activity of the drugs. Figure 1Dindicates that at 10�7 M, DAPT inhibitor has a signifi-cant effect on the interaction between PS1-CTF andPS1-NTF. To examine whether �-secretase activity issignificantly changed at this DAPT concentration, weused an in vitro assay based on cleavage of endogenoussubstrates that copurify with neuronal membranes(20). We first tested N-cadherin, an abundant neuronalprotein that is processed by �-secretase at the epsilon site toyield C-terminal fragment N-Cad/CTF2 (20). Figure 3A(top panels) shows that incubation of neuronal mem-branes resulted in the production of N-Cad/CTF2 (lanes1, 2), and this process was inhibited by increasing DAPT

concentrations, with an IC50 of �30 nM. At 100 nMDAPT, �-secretase processing of N-cadherin was mostlyinhibited. Surprisingly, however, APP processing was lesssensitive to DAPT inhibition. Indeed, in vitro productionof AICD was insensitive to DAPT concentrations as high as75 nM, but decreased at concentrations above 100 nM(Fig. 3A, bottom panel; B). These differences in dose-response inhibition of N-Cad/CTF2 and AICD suggestthat processing of APP-CTF/� substrates is much lesssensitive to GSIs than processing of N-Cad/CTF1 sub-strate.

The lower sensitivity of APP processing to GSIs isfurther illustrated by experiments involving O/N treat-ment of primary neuronal cultures with GSIs. Produc-tion of N-Cad/CTF2, but not of AICD, was inhibited inmembranes prepared from such cultures incubatedO/N with inhibitor L685,458 (Fig. 3C, lanes 1–4).Since AICD production is still mediated by �-secretase(Fig. 3C, lane 5), the most probable explanation for thisdifferential effect is that drug copurified with themembranes inhibits the �-secretase cleavage of N-Cad/CTF1 but not cleavage of APP-CTF/�, in agreementwith the lower sensitivity of the APP cleavage to GSIscompared to cadherin cleavage. Interestingly, eph-rinB1, another substrate of �-secretase (2) behaves like

Dose-response inhibition ofAICD and Ncad/CTF2 production

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Figure 3. ε-Cleavage of APP is less sensitive to inhibitors compared to other substrates. A, B) In vitro dose-response of �-secretaseinhibition. Rat cortical neurons were lysed in hypotonic buffer. Purified membranes were either incubated at 37°C in citratebuffer to follow �-secretase activity (run) or directly suspended in Laemmli buffer (start; starting material). Under runconditions, membranes were incubated with indicated concentrations of DAPT for 16 h, and reactions were stopped withLaemmli buffer. Proteins were analyzed on WBs as described in Materials and Methods. Membranes were probed for �-secretaseprocessing of APP and N-cadherin by following AICD and N-cad/CTF2. A) Results from a representative experiment.B) Quantifications of 3 experiments. C) O/N treatment of neurons with GSIs induces a rebound effect on APP but notN-cadherin cleavages. Neuronal cultures were either left untreated (Ctrl) or treated O/N (L685,458) with 1 �M L685,458, andthen lysed in hypotonic buffer. Purified membranes from both samples were then incubated at 37°C in citrate buffer (run) ordirectly suspended in Laemmli buffer (start). A duplicate of run in 1 �M L685,458 was included to assess in vitro inhibition of�-secretase, and samples were analyzed on WBs. D) APP-CTF/� substrates of �-secretase do not inhibit N-cadherin cleavage.WT or APP�/� mouse cortical neurons were treated O/N or not with 2 �M DAPT; �-secretase activity was analyzed by incubatingmembranes at 37°C in citrate buffer, and obtained samples were analyzed on WBs.

2941GSIs REVEAL SUBSTRATE-SPECIFIC �-SECRETASE

N-cadherins (Supplemental Fig. S2). This differentialsensitivity of N-cadherin and APP cleavage to GSIs isconsistent with the hypothesis that distinct �-secretasecomplexes cleave N-cadherin and APP, a hypothesisstrongly supported by reports that distinct dockingproteins recruit cadherins and APP to �-secretase com-plexes. Specifically, p120ctn recruits cadherins (11),and GSAP (�-secretase activating protein) recruits APP-CTFs (21) to �-secretase. To further test this hypothesis,we used neuronal cultures from WT and APP�/�-transgenic mice to ask whether there is substrate com-petition for �-secretase enzymes. Figure 3D shows thatcadherin processing and production of N-Cad/CTF2 isindependent of APP (compare lanes 1, 2 to 5, 6).Furthermore, DAPT inhibition of N-Cad/CTF2 doesnot depend on accumulated APP-CTFs (Fig. 3D; com-pare lanes 3, 4 to 7, 8). Together, these observationsindicate the existence of distinct �-secretase complexesthat cleave N-cadherin or APP and suggest that distinctcellular �-secretase complexes may process specificsubstrates.

Substrate quantity limits AICD production, whereasenzyme quantity limits production of N-Cad/CTF2and ephrinB1/CTF2

Since enzymatic reactions can be limited by substrate orenzyme availability, we asked whether APP substrate isthe limiting factor in our assays by examining whetherincreasing levels of APP-CTF/� would increase pro-duction of AICD. To this end, we used MEFs derivedfrom PS1/2 dKO mice to prepare an extract with highconcentrations of APP substrates. The addition of in-creased amounts of this extract as a source of APP-CTFsubstrates to membranes prepared from APP�/� neu-rons resulted in a proportional increase in AICD pro-duction (Fig. 4A). Moreover, production of AICD wasnot reduced by a 50% decrease of PS1 in membranesprepared from PS1�/� neurons (Fig. 4B), indicatingthat enzyme availability is not limiting production ofneuronal AICD. A substantial decrease in the produc-

tion of AICD was only observed in the total absence ofPS1 (Fig. 4B). Although remaining production of AICDin the absence of PS1 is attributed to PS2, our datasuggest that this PS cannot fully support AICD produc-tion in the absence of PS1. In contrast to AICD, whichderives from APP, production of Ncad/CTF2 and eph-rinB1/CTF2, the products of the �-secretase processingof N-cadherin and ephrinB1, respectively, decreased by�50% in membranes from PS1�/� neurons (Fig. 4C),suggesting that cleavage of these substrates is limited byenzyme availability. Interestingly, despite the presenceof PS2 (Fig. 4C, bottom panel), no cadherin or eph-rinB1 cleavage products were detected in the absenceof PS1, indicating that only PS1-containing �-secretasecomplexes process cadherins and ephrinB1, as op-posed to APP, which can be processed by both PS1- andPS2-containing complexes, although at different effi-ciencies. Thus, �-secretase processing of N-cadherinand ephrinB1 is limited by the quantity of �-secretaseenzymatic sites and cannot be performed by PS2-containing �-secretase.

Low concentrations of GSIs stimulate production ofA�42

Paradoxically, an increase, rather than a decrease, ofA�42 levels has been reported in cell lines and animalmodels treated with low concentrations of GSIs (4, 7).We verified these data in primary neuronal culturestreated with either DAPT (Fig. 5A) or L685,458 (Fig. 5B).Both drugs at concentrations � 50 nM increased theamounts of A�42. Since DAPT is a protease inhibitor, weexamined whether it stabilizes A�42 by inhibiting itsdegradation. Figure 5C, D shows that at these concentra-tions, DAPT has no effect on the degradation of thispeptide, suggesting that low DAPT concentrations stimu-late production of A�42. AICD and A� are thought toderive from different substrates, as the former is mostlyproduced from APP-CTF (22), while the latter derivesfrom APP-CTF�. Combined with reports that treatment ofcell cultures with low DAPT concentrations also fails to

Figure 4. Production of AICD is limited by APP-CTF substrates, while production of Ncad- andephrinB1-CTF2 is limited by enzyme availability. A)Production of AICD is proportional to the amountof substrate. Membrane proteins from PS1/2 dKOMEFs were used as a source of APP-CTFs and added to membranes from APP�/� cortical neuronal cultures. Neuronalmembranes were then incubated at 37°C in a citrate buffer (run) or directly suspended in Laemmli buffer (start). Reactionswere stopped by Laemmli buffer, and proteins were analyzed by WBs. B) Fifty percent reduction of PS1 in PS1�/� has noeffect on AICD production. Membranes from mouse cortical neurons of PS1�/�, PS1�/�, and PS1�/� embryos wereprepared as above, and �-secretase activities were analyzed by incubating at 37°C. C) Reduction of PS1 proportionallydecreases production of N-cad/CTF2 and ephrinB1/CTF2. Experiment was performed as in B, except membranes wereprobed for N-cadherin and ephrinB1.

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change the levels of p3 peptides, another product ofAPP-CTF (7), our data suggest that low DAPT concen-trations have different effects on the �-secretase process-ing of different APP substrates.

DISCUSSION

A� peptides, the structural subunits of the amyloiddepositions of AD, have been proposed to play crucialroles in the development of the disease, and recenttherapeutic strategies use inhibitors of �-secretase toblock their production. The mechanism of GSIs is stillunder investigation, but it was reported that transition-state analogs predicted to bind the catalytic site of�-secretase display noncompetitive inhibition towardsubstrate binding (9), suggesting that substrates first

bind a docking site before they migrate to the catalyticsite (10). To understand the mechanisms by which GSIsregulate �-secretase activity, we asked whether thesedrugs affect the interactions between components ofthe �-secretase complex. Because brain A� is mostlyproduced by neurons, we used primary neuronal cul-tures expressing endogenous levels of enzymes andsubstrates to avoid problems associated with overex-pression systems, such as changes in the enzyme tosubstrate ratios. We discovered that both transition- andnontransition-state analogs of GSIs stabilize interac-tions between PS-CTF, the central component of the�-secretase proteolytic complex, and its partners, theAPH-1/nicastrin and PS1-NTF/PEN-2 subcomplexes.These stabilization effects suggest a mechanism bywhich these drugs inhibit substrate processing. Indeed,a common paradigm of enzymology is that enzymes

Figure 5. Low concentrations of GSIs increase A� without affecting its degradation. A, B) GSIs increase A�42 level at nanomolarconcentrations in neuronal culture. Rat cortical neurons were treated with different concentrations of DAPT for 16 h in freshmedium. A�42 levels in the medium were determined using an ELISA assay. B) Same as in A but using GSI L685,458.C) Degradation of exogenous A�42 in APP�/� is inhibited by insulin but not by GSIs. WT mouse neuronal culture medium wasreplaced with fresh Neurobasal and then incubated for 16 h. This 16-h-conditioned medium was used as a source of A�42 andtransferred to APP�/� neuronal cultures treated either with DAPT or insulin, which inhibits A� degradation by insulin-degrading enzyme. A�42 in medium was measured as above. D) Degradation of A� and p3 peptides is inhibited by insulin butnot DAPT. WT mouse cortical neurons were radio-labeled with S35-methionine/cysteine. Medium from these cultures was usedas a source of exogenous A� and added to sister cultures in the presence or absence of DAPT or insulin (100 �g/ml). Cultureswere incubated O/N, and supernatants were then IPed with anti-A� and anti-p3 Abs. Proteins were analyzed on 10–20%gradient Tris-tricine gels and by autoradiography. E) Proposed model of allosteric effects of GSIs containing two �-secretasecomplexes associated in trans. PS1-CTF fragment of one complex associates with the NTF fragment of the adjacent �-secretasecomplex. To help visualize the enzymatic sites, APH-1 and NCT of one complex have been omitted in the right middle panel.The two catalytic aspartates on TM domains VI and VII of each PS are depicted as yellow-red sparks. Right panel illustrates apositive allosteric effect on A�42 production at low concentrations of inhibitors when only one half of active sites are occupied.The two aspartates of the PS on the left are blocked by inhibitor and are inactive (shown in black); in the countercomplex, thetwo aspartates of PS are more active (shown in brighter yellow).

2943GSIs REVEAL SUBSTRATE-SPECIFIC �-SECRETASE

exhibit a relaxed conformation when active and arestrained or “tensed” conformation when inhibited bydrugs (18). We propose that both transition- andnontransition-state analogs of GSIs inactivate �-secre-tase by closing the PS CTF/NTF interface that formsthe active site of the enzyme. This mechanism is alsoconsistent with observations that GSIs inhibit substrateproteolysis without preventing substrate binding (8, 9).

The stabilizing effect of GSIs on �-secretase com-plexes correlates with the inhibitory effects of thesedrugs on N-cadherin processing (Fig. 3A, B). However,higher concentrations of GSIs are required to inhibitproduction of AICD compared to N-Cad/CTF2, sug-gesting that �-secretase complexes processing APP-CTF/� are less sensitive to inhibitors than complexesprocessing N-Cad/CTF1. This lower sensitivity of APPprocessing to GSIs is also supported by our data show-ing that, in contrast to N-cadherin, APP processing isnot inhibited in membranes prepared from neuronalcultures pretreated with GSI (Fig. 3C). Although themolecular basis of this differential sensitivity of sub-strates to GSIs needs further examination, recent evi-dence suggests that the composition of �-secretasecomplexes may be tailored to the processing of specificsubstrates. Indeed, distinct cellular factors, termed�-secretase docking proteins (GSDPs), recruit N-cad-herin or APP to �-secretase complexes (11, 21). PS1binds p120ctn, which links cadherin substrates to�-secretase (11), and recent reports show that APP isrecruited to �-secretase by the GSAP (21). We previ-ously reported that by recruiting �-secretase to cad-herin substrates, p120ctn promotes cleavage of cad-herins, while it suppresses cleavage of APP, indicatingthat p120 recruits �-secretase complexes to cadherinprocessing, thus reducing the number of complexesavailable for APP processing (11). The existence ofdifferent pools of �-secretase complexes specific todistinct substrates is also supported by our data showingthat APP and N-cadherin substrates do not compete for�-secretase (Fig. 3D). Furthermore, we obtained evi-dence that while APP substrates are processed by bothPS1- and PS2-containing �-secretase complexes, N-cad-herin is processed only by PS1-containing complexes, aresult in agreement with reports that sequence 330–360 of PS1-CTF is critical for N-cadherin recruitment(11). Indeed, this sequence is very different in PS2,suggesting that PS2-containing �-secretase complexesdo not bind p120ctn and are thus unable to recruitcadherin substrates. Thus, GSDPs may control access of�-secretase to substrates. Further support for the exis-tence of distinct �-secretase complexes comes from ourdata indicating that neuronal �-secretase complexescontain mostly the low-molecular-weight NCT, in con-trast to cell line �-secretase complexes that incorporatemostly the high-molecular-weight NCT (23). Becausethe two NCT forms differ in their degree of glycosyla-tion (24, 25), our observation suggests that NCT glyco-sylation is not required for �-secretase activity, in agree-ment with previous reports (24). Together, our dataprovide strong evidence that distinct �-secretase com-

plexes process specific substrates and suggest a dynamicsteady-state model, according to which �-secretase com-ponents assemble into functional complexes in re-sponse to cellular needs for specific substrate process-ing.

Our data (Fig. 4C) show that in neuronal systems,enzyme availability limits production of N-cad/CTF2and ephrinB1/CTF2. In contrast, production of AICDis limited by the quantity of �-secretase substratesAPP-CTF/�. We observed that these substrates accu-mulate dramatically in membranes treated with inhibi-tory concentrations of GSIs (Fig. 3C), in contrast toN-Cad/CTF1, which accumulates much less, suggestinga differential removal of these substrates by alternativeproteolytic pathways. This accumulation of APP-CTF/� substrates explains the “A� rebound effect”reported in patients treated with inhibitory concen-trations of GSI (4 – 6, 26). As the in vivo concentra-tions of GSI decrease below inhibitory levels, accu-mulated APP-CTF� substrates are processed by theenzyme, leading to increased amounts of A�, oftenhigher than the initial levels.

However, recent publications report that treatmentof animal models or cell cultures with low concentra-tions of GSIs increases the in vivo levels of A� peptides(4, 7). This increase could result from increased pro-duction or decreased degradation of A�. Our datashowing that A� degradation is unaffected by GSIs (Fig.5C, D) suggest that low concentrations of GSIs stimulateproduction of A�. Indeed, the hyperbolic shape of thedose-response curve of A� production (Fig. 5A, B), isconsistent with a positive allosteric effect. SinceL-685,458 is a transition-state analog, this biphasicresponse is incompatible with a model that incorpo-rates 1 PS molecule/enzymatic unit. Instead, ourdata are consistent with a 2-site model, in which atlow inhibitor concentrations, targeting of one siteresults in increasing activity of the other site througha positive allosteric effect induced by conformationalchanges (Fig. 5E). This explanation is consistent withrecent evidence (27–29), including our own datapresented here (Supplemental Fig. S3) that PS formsdimers. However, the increased level of A�42 se-creted by neuronal cultures contrasts with the un-changed level of AICD produced by neuronal mem-branes treated with low concentrations of GSIs. Thelack of effect on AICD is consistent with a recentreport that low concentrations of GSIs do not in-crease the level of p3 peptide, which, like AICD, isalso derived predominantly from the �-secretasecleavage of APP-CTF (7). Together, these observa-tions suggest that the mechanism involved in theincrease of A�42 may be specific to the processing ofAPP-CTF�.

The finding that processing of APP substrates isless sensitive to GSIs than processing of cadherinsubstrates has important implications for the use ofthese inhibitors as therapeutic agents, because pro-cessing of substrates other than APP may be inhibitedbefore A�. Thus, it would be interesting to determine

2944 Vol. 25 September 2011 BARTHET ET AL.The FASEB Journal � www.fasebj.org

the GSI sensitivity of other �-secretase substrates,including Notch1, ErbB4, and EphB receptors (30 –32). Nonetheless, our results raise the possibility thatcurrently available inhibitors interfere with impor-tant cellular functions before production of A� isinhibited. This observation may explain reportedclinical side effects in patients treated with GSIs (33)and indicates that a different pharmacological strat-egy is required to decrease A�. Indeed, recent effortsconcentrate on the development of selective alloste-ric �-secretase modulators that target PEN-2 andstimulate degradation of A� to shorter peptides (34).Alternatively, it may be possible to selectively targetthe GSDPs that recruit specific APP substrates to�-secretase complexes (21).

This work was supported by U.S. National Institutes ofHealth grants AG-017926, NS047229, and AG-008200.

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Received for publication February 22, 2011.Accepted for publication May 5, 2011.

2946 Vol. 25 September 2011 BARTHET ET AL.The FASEB Journal � www.fasebj.org