Major Carboxyl Terminal FragmentsGenerated by g-Secretase Processing of theAlzheimer Amyloid Precursor Are 50 and 51

Amino Acids Long

Inga Pinnix, Ph.D., Jorge A. Ghiso, Ph.D., Miguel A. Pappolla, M.D., Ph.D.,Kumar Sambamurti, Ph.D.

Received MarchDepartment of Pof Medicine, UniSend correspondSC 29425. e-mail

� 2013 Amehttp://dx.d

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Objectives: To understand the cleavage of the amyloid b protein (Ab) precursor

(APP) by g-secretase and to determine its changes in a representative familial

Alzheimer disease (FAD) mutation. Methods: Transfected cells expressing wild-type

and FAD mutant APP were analyzed for changes in the levels of the major secreted

Ab species and of the corresponding intracellular C-terminal APP fragments (APP

intracellular domain, AICD) generated by g-secretase, whereas radio-sequencing was

used to precisely identify the resulting cleavage site(s). Results: The AICD fragment(s)

generated by g-secretase cleavage comigrated in gels with a 50-residue synthetic

peptide used as control, which is smaller than the 59 and 57 residues predicted from

Ab ending at positions 40 (Ab40) and 42 (Ab42), respectively. In agreement with

previous findings, an FAD mutant form of presenilin 1 (PS1-M139V) significantly

increased the longer Ab42 while showing trends toward reducing Ab40. AICD levels

were reduced by the mutation, suggesting that g-secretase activity may be actually

impaired by the mutation. Radiosequence analysis in cells expressing wild-type PS1

detected g-secretase cleavage sites at the Ab peptide bond L49-V

50to generate a 50-

amino acid (aa) AICD fragment (AICD50) and the Ab peptide bond T48-L49, gener-

ating an AICD of 51 aa (AICD51). No other cleavage sites were reliably detected.

Conclusions: Based on findings that the FAD mutation that increases Ab42 also

reduces AICD, we propose that g-secretase activity is impaired by FAD mutations and

predict that physiologic and environmental agents that inhibit g-secretase will actually

induce AD pathogenesis rather that prevent it. Furthermore, we propose that the

cleavage site to generate AICD is naturally ragged and occurs predominantly at two

sites 48 and 49 aa from the start of the Ab sequence. Thus, end specific antibodies to

these two sites will need to be generated to study the quantitative relationships between

these two cleavages in sporadic AD and FAD. (Am J Geriatr Psychiatry 2013;21:474e483)

14, 2012; revised February 4, 2013; accepted February 15, 2013. From Sandalwood High School (IP), Jacksonville, FL;athology and Psychiatry, New York University School of Medicine (JAG), New York, NY; Department of Neurology, Schoolversity of Texas Medical Branch (MAP), Galveston, TX; and the Medical University of South Carolina (KS), Charleston, SC.ence and reprint request to Kumar Sambamurti, Ph.D., Professor of Neuroscience, 173 Ashley Avenue, BSB 403, Charleston,: sambak@musc.edurican Association for Geriatric Psychiatryoi.org/10.1016/j.jagp.2013.02.009

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Pinnix et al.

Key Words: AICD, Carboxy-terminal fragment (CTF)-g, amyloid, Alzheimer disease,g-secretase

INTRODUCTION

Amyloid b (Ab) protein precursor (APP) is a type Iintegral membrane protein with a short half-life dueto its cleavage in the juxtamembrane region by anenzyme named a-secretase.1e3 This cleavage yieldsa secreted derivative, sAPPa (595e670 amino acid[aa]), which includes the first 16 aa Ab sequence anda membrane-bound Carboxy-terminal fragment(CTF) of 83 aa e CTFa, which is further cleaved byin the intramembrane protease, g-secretase, to yielda 3-kDa fragment (P3 or Aa) starting at Ab position17.4 In contrast, Beta Site APP Cleaving Enzyme(BACE1) cleaves APP on the N-terminal side ofAb (b site) to generate the secreted derivative,sAPPb (579e654 aa) and a membrane-bound frag-ment, CTFb (99 aa), which is cleaved to Ab of 4 kDaby g-secretase.4 Although most (>90%) secreted Ab is40 aa long, the accumulated deposit in the Alzheimerdisease (AD) brain is mostly 42e43 aa long, and theselonger forms selectively increase in familial AD (FAD)mutations on APP or presenilin 1 (PS1) or 2 (PS2).

A large body of literature has demonstrated thatPS1 and PS2 are active subunits of g-secretase, whichis a complex of four integral membrane proteins,PS1/2, anterior pharynx 1, presenilin enhance 2, andNicastrin, that can be coexpressed in yeast togenerate the active enzyme.5,6 Ab42 tends to formmultimeric complexes more readily than Ab40, andsome of the oligomeric intermediates are neurotoxicin culture, supporting a hypothesis that a neurode-generative cascade triggered by Ab aggregates(amyloid hypothesis) causes AD.7,8 The longer Ab42forms increase in FAD and provide the strongestsupport for the amyloid hypothesis.

Approximately 8 years after the discovery of Ab,one group isolated the APP intracellular fragment,AICD, frombrain.9 At the same time,we developed anassay to show that g-secretase cleavage yieldssubstantial quantities of a cytoplasmic APP-CTF andnamed it CTFg (a.k.a. AICD, Cg, CTFε, AID) that islost in cells due to rapid degradation.10,11 Our study

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triggered several follow-up studies that identified theAICD cleavage site as the junction of Ab49e50 and thefragment size as 50 aa and called it the ε site.12e14 Onegroup studied the membrane Ab fragment moreextensively and demonstrated the presence ofsubstantial quantities of Ab46 and called it the z site.15

Other studies described a novel serial cleavagepathwaywhere the initialg-secretase cleavage atAb49generates additional cleavages in 3-aa intervals togenerate Ab of 38e43 aa.15,16 Detection of an alterna-tive minor cleavage site at Ab48 led to the hypothesisthat Ab48 generates Ab45 and Ab42, whereas Ab49 isprocessed to Ab46, Ab43, and Ab40, suggesting themisprocessing occurs early in the sequential g-secre-tase cleavage.17,18 However, these studies failed todetect any Ab49 and suggested that Ab49 is rapidlyconverted to Ab46 but Ab48 is more stable and giverise to lower levels of Ab45 and Ab42.17e19 Moreover,it was proposed that Ab46 could not give rise to Ab42,based on inhibitor and kinetic analyses.17 However,these studies are contradicted by site-directed muta-genesis studies providing strong evidence that Ab46can generate Ab40, Ab42 and Ab38.16,20

Mass spectroscopy studies showed that ε cleavageby g-secretases generates mostly AICD50 and smallquantities of AICD51.21 Because matrix effects anddifferential ability to quantitatively detect fragmentsare known to affect the interpretation of mass spec-troscopy data, we chose to detect the exact cleavagesite of AICD by radio-sequencing.22,23 Our dataconfirm that primary cleavage of APP by g-secretaseto generate AICD is predominantly at residues Ab48and Ab49, providing independent evidence that thereis nohint of cleavage atAb40orAb42.Wealsodescribea second cleavage that generates AICD51 and provideevidence that these are the only two major cleavagesites for AICD production. Further, we showed thatreduction of g-secretase using antisense PS1 RNAincreases Ab42, which provides evidence supportingthe idea that FAD mutations impair g-secretaseactivity.24 These studies support a hypothesis thatinhibition of g-secretase may be a cause of AD,

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a problem that needs to be evaluated in typical late-onset AD as proposed independently by othergroups25 and by us previously.26

METHODS

Cells and Reagents

Chinese hamster ovary (CHO) cells transfectedwith the 695-aa neuronal variant of wild-type APP(2B7) was described previously.27 Transient trans-fections of wild-type and mutant PS1 were carriedout using HEK293H (Invitrogen, Carlsbad, CA),which is very efficiently transfected by lipofectamine2000 (Invitrogen) as described previously.28,29

Radioactive 35S-methionine was purchased from Per-kin Elmer-New England Nuclear (Waltham, MA).Antibodies O443, BAN50, BCO5, and BA27 againstthe C-terminal 20 aa of APP, 1e16 aa of Ab, Ab42 end,and Ab40 end were described previously.11,28 Peptidestandards were from rPeptide (Augusta, GA) for Ab

and Calbiochem (EMD, San Diego, CA) for AICDfragments of 50 and 57 aa. Mutant APP and PS1constructs were described previously.24,28 Precast Bis-Tris gels and related reagents were obtained fromInvitrogen. Other reagents and cell culture media andassay kits were obtained from either VWR (Radnor,PA) or Fisher Scientific (Waltham, MA).

Western Blot Analysis for AICD

CHO-2B7 cells were cultured in 100-mm dishes inOpti-MEM (Invitrogen) containing 10% fetal bovineserum to about 70% saturation. The cells from twoplates were scraped with a rubber policeman andresuspended in ice-cold 50 mM HEPES, 0.15 M NaCl,pH 7.0, containing the Roche protease inhibitorcocktail and differentially centrifuged at 2,500 rpm(600g) for 10 minutes, and the supernatant (S1) wascentrifuged at 10,000 rpm (12,000g) for 15 minutes tocollect the pellet (P2). The pellet was resuspended inice-cold Roche protease inhibitor cocktail, washed,and resuspended in the same buffer. The washed P2was incubated at 37�C for 2 hours and centrifugedonce again at 14,000 rpm (20,000g) for 15 minutes toremove the membrane and recover the supernatantcontaining AICD as described previously.11

Supernatants were separated on precast 10%Bis-Tris gels and electrophoresed using the

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2-(N-morpholino)ethanesulfonic acid (MES) buffersystem to ensure separation of low-molecular-weightfragments in the 4- to 6-kDa range (Invitrogen). Thegels were transferred onto nitrocellulose membranes(VWR) in a transfer buffer consisting of 25 mM Tris,191 mM glycine, and 20% methanol using a wettransfer apparatus (Bio-Rad, Hercules, CA) for 90minutes at 90 V. The blots were dried overnight,washed extensively in distilled water for 15 minutes,soaked in boiling phosphate-buffered saline (FisherScientific) for 10 minutes, rinsed in distilled water,and blocked for 1e2 hours in 10% newborn bovineserum in 50 mM Tris, pH 8.0, 0.15 M Nacl con-taining 0.05% Tween-20 (TBST). We find that thisstep improves signal of nearly all Western blots andtherefore extends beyond antigen retrieval.

The blocked membranes were washed extensivelywith TBST (6 � 10 minutes) before incubation withO443 (10,000 fold dilution) in 50 mM Tris, pH 8.0, 0.15MNacl containing 2% newborn bovine serum for 1e2hours. After an extensive wash in TBST as describedearlier, the blots were probed with horseradish per-oxidaseelabeled goat anti-rabbit antibody (1:7,500;Jackson Immunoresearch Philadelphia, PA) for 1 hourandwashed extensively again in TBST. The blots werethen incubated in super signal west pico chemilumi-nescent substrate (Fisher Scientific) and exposed to x-ray film to capture the signal. The blots were scannedusing a Umax technologies (Fremont, CA) scannerwith transparency attachment and quantified by theMolecularDynamics (Piscataway,NJ) Storm software-image quant. The procedure detects as little as 0.1fmole of synthetic AICD-C50 andAICD-C57 peptides.

Radiolabeling and Enzyme Assay

Ten dishes of 100 mm were seeded with CHO-2B7cultures and grown to 70% confluence. Cells werewashed once with Hank’s balanced salt solution(Invitrogen), equilibrated in methionine-deficientmedium (Invitrogen) for 30 minutes, and subjectedto metabolic labeling for 16 hours in methionine-deficient medium containing 1 mCi of 35S-methio-nine (Perkin Elmer-New England Nuclear), as previ-ously reported.22 AICDs were generated as describedabove and immunoprecipitated with the O443 anti-body against the 20 C-terminal residues of APP,separated on 10% Bis-Tris gels with MES buffer andtransferred to Immobilon P (Millipore Billerica, MA)

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FIGURE 1. AICD derived from CHO-2B7 cells migrate with the APP-C50 synthetic peptide. CHO-2B7 cells were cultured, membranesisolated for an in vitro g-secretase assay, separated on Tris-Tricine 10%e20% gels (Invitrogen) along with synthetic APP-C50 and APP-C57 standards, and analyzed by Western blotting with the O443 antibody as described in Methods andreported previously.11 MG132 was used to inhibit g-secretase cleavage as described previously. Fifteen- and 10-kDamarkers are indicated and AICD migrates at about 7 kDa.

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using a Western blotting apparatus (Bio-Rad), asdescribed by the manufacturer. The membrane wasexposed to x-ray film overnight, and the radioactiveband was identified, marked, and excised along withsurrounding regions for Edman degradation usinga protein sequencer (Applied Biosystems, Foster City,CA). The released amino acid from each sequencingcycle was recovered and 35S-methionine radioactivityassessed in a liquid scintillation counter (modelLS-250; Beckman, Brea, CA).

Statistical Analysis

Ab and AICD from wild-type and mutant PS1-expressing cells were compared by paired two-tailedStudent’s t test using GraphPad (La Jolla, CA) Prismversion 6.

RESULTS

Most AICD Released Comigrates with C50 Ratherthan C57

CHO-2B7 cell membranes were fractionated toobtain the P2 fragment and incubated in vitro for

Am J Geriatr Psychiatry 21:5, May 2013

g-secretase to release the cytoplasmic C-terminaldomain, AICD or CTFg. Western blot analysis of thesupernatants from the in vitro assay was carriedout as described in Methods. It shows that most ofthe C-terminal fragment obtained in the in vitrog-secretase assay comigrates with C50, suggestingit is the major peptide generated by g-secretasecleavage (Figure 1). A high level of MG132 (50and 200 nM), a proteasome inhibitor that also inhibitsg-secretase,29 reduces production of the AICD frag-ment (Figure 1). These studies indicate that little, ifany, AICD of 57 or 59 residues that complementsAb40 or Ab42 is generated by g-secretase cleavage.

Radiosequencing Identifies Two Cleavage Sites forAICD Generation

Although the Western blot data in Figure 1 aredefinitive in not showing larger AICD fragment,a problem with this approach is that the methodhas a limited resolution in its ability to identifythe exact cleavage sites and sizing on gel canbe affected by conformation and post-translationalmodification. Therefore, we used Edman degrada-tion to radio-sequence the AICD fragment generated

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FIGURE 2. Distribution of 35S-methionine by cycles of Edman degradation. CHO-2B7 cells were cultured and radio-labeled andAICD generated from the membranes were immunoprecipitated with O443 as described in Methods. The quantity of35S-methionine released in each cycle after Edman degradation was determined by scintillation counting and thepercentage of counts per minute versus total were plotted as described in Methods.

TABLE 1. Yield of 35S-Methionine from AICD Radio-Sequencing Indicates Cleavage Occurs After Ab48and Ab49

Cycle Percent Yield Predicted AICD Predicted Ab

1 3.4 C49 AB502 55.0 C50 AB493 37.8 C51 AB484 0.0 C52 AB475 3.4 C53 AB466 0.0 C54 AB457 �0.7 C55 AB448 �0.7 C56 AB439 1.7 C57 AB4210 0.0 C58 AB4111 0.0 C59 AB4012 0.0 C60 AB39

g-Secretase Processing of the Alzheimer Amyloid Precursor

from 35S-methionineelabeled 2B7 cells. The productsof Edman degradation were analyzed by scintillationcounting; the number of cycles necessary to detect theradioactive methionine indicates the length of thepeptide that extends before the N-terminus of theradioactive residue.

Figure 2 and Table 1 show the distribution of theradioactive amino acid peaks obtained in the first12 cycles of Edman degradation analysis in a repre-sentative experiment. Two radioactive peaks weredetected at cycles 2 and 3, whereas the rest of thecycles consistently rendered background levelsof radioactivity, indicating the presence of twofragments—one containing methionine at position 2and the other at position 3—identifying the cleavagesites at Ab peptide bonds L49-V50 and T48-L49 and thegeneration of AICD50 and AICD51 (Figure 3). Nofragments complementing Ab40 (AICD59) or Ab42(AICD57) were detected.

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These studies confirm previous mass spectroscopyfindings regarding g-secretase cleavage generatingAICD fragments of 50 and 51 aa.18 The study detectsno AICD fragment other than the 50 and 51 aa forms,

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FIGURE 3. Target sequence for g-secretase cleavage showing the major cleavage sites that generate AICD. The sequence of theC-terminal APP 101 residues starting 2 aa before Ab to Ab99 is shown. The known cleavage sites are indicated by “j” andmajor cleavage sites identified by Edman degradation analysis are shown below the target sequence and labeled AICD50and AICD51. The reference radioactive methionine detected by radio-sequencing is indicated in bold by an asterisk M*.

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suggesting that we need to study the biologic effectsof these two forms instead of using AICD57 andAICD59 predicted from the Ab40 and Ab42 cleavagesites.

FAD Mutant PS1 Reduces AICD Levels

PS1 M139V is an FAD mutation that increases thelevels of Ab42 and represents an example of themajor hypothesis in the AD field that toxic amyloidoligomers are responsible for the characteristic neu-rodegeneration and dementia.30 To determinewhether AICD is affected by this FAD mutation,HEK293H cells were transiently transfected witheither APPwt þ PAG3 vector or APPwt andPS1-M139V in PAG3. Both these transiently trans-fected cells showed detectable Ab40 and Ab42 levelsby a standard ELISA assays (Figure 4). The data(Figure 4, left panel) show that the relative Ab42levels increase in cells transfected with the FADmutant PS1 as reported by several groups previ-ously.31 In addition, the studies show a significantdrop in the levels of AICD levels in the cellsexpressing mutant PS1 (Figure 4, right panel).However, there was no significant change in Ab40levels. These results suggest that FAD mutations maylead to partial inhibition of g-secretase rather thanmodify its specificity and thereby act as dominant-negative mutations.

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DISCUSSION

The finding that FAD mutations subtly alter APPprocessing to generate increased levels of Ab42 hasbeen a major foundation of the amyloid hypothesisand has been a widely repeated finding.7 However,only a few studies have examined the effects of PS1mutations on AICD with varying results.21,32 In thisstudy, we show that an FAD mutant form of PS1increases Ab42 and reduces AICD levels. The drop issignificant (Figure 4) and suggests that FAD muta-tions can reduce g-secretase activity, at least withrespect to AICD generation. We have previouslyshown that antisense RNA against PS1 increasesAb42 like some inhibitors of g-secretase, which alsoleads to the same conclusion.24 Furthermore, wehave previously shown that overexpression of allg-secretase subunits together in HEK293H cellsincreases Ab and AICD but actually reduces theAb42/Ab40 ratio conversely, showing that increasingg-secretase reduces processing to the longer Ab42form and therefore reinforces the conclusion that theFAD-associated increase in Ab42 may be due toreduction of g-secretase.33

However, it is important to note that AICDproduction may be independent of Ab given thatg-secretase has two activities, the first, namedε cleavage, cuts CTFb to Ab45e49 and the second,g cleavage, generates secreted Ab of 39e43 residues

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FIGURE 4. FAD mutant PS1 M139V increases Ab42 and reduces AICD levels. APP and either wild-type or mutant PS1 were transiently transfected into HEK293H cells orCHO cells and the secreted Ab40 (pM) and Ab42 (pM) detected by a standard sandwich ELISA assay with means and standard deviations plotted [Left].Membranes from the cells were isolated and assayed for changes in AICD levels by Western blotting quantified by densitometry showing means and standarddeviations [Right]. Unpaired two-tailed Student’s t test for Ab42 (t [ 3.496, df [ 6, p [ 0.0129*) and AICD (t [ 2.789, df [ 4, p [ 0.0494*) showed significantdifferences between wild-type and mutant cells, but Ab40 (t [ 1.748, df [ 6, p [ 0.131) did not show statistically significant difference.

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FIGURE 5. Model showing APP processing by g-secretase at the g and ε sites. APP is cleaved to CTFa and CTFb, which are cleaved byg-secretase at the ε site to release AICD of the indicated sizes with relative levels. The most common cleavage siteconfirmed in this study based on AICD length (see Table 1) should give rise to Ab49, but this fragment has not beenreported, whereas considerable amounts of Ab48 are efficiently detected.19 This study detects substantial amounts ofAICD of 51 aa consistent with the reports showing Ab48. The final step consists of g-site cleavages, which occur atresidues 42 and 43. Although Ab43 is readily detected in lysates, unlike Ab42, it is not detected in media.19 In contrast,a second g-site cleavage at residues 38e40 release secreted Ab primarily of 40 residues. However, it is possible that mostof these g site cleavages are independent of each other, even though they may be dependent on the ε site cleavage.

Pinnix et al.

(Figure 5).19,21 In addition to Ab48/49, one studyidentified a critical intermediate, Ab46, in Ab40and Ab42 generation and named this z cleavagewithout attempts to detect the correspondingAICD.34 The current study suggests that there is noAICD corresponding in size to forms other thanAb48/49, supporting the view that intermediatesare derived from further processing of these longerAb forms.16,19,20,34e36 Given a sequential cleavagepattern from Ab48/49 to Ab45/46 to Ab42/43 toAb39/40, a slow or impaired g-secretase will likelyrelease the longer Ab42/43 forms into the mediarather than convert it to the shorter 39/40 forms.We have also proposed that accumulation of theimmediate substrate, CTFb, may lead to increasedrelease of Ab in spurts, which may facilitateaggregation.3 However, if the proximal substrate forsecreted Ab42 is Ab45e49, the phenomenon mayfostered by accumulating these intermediates ratherthan CTFb. Indeed, we find that partial g-secretaseinhibition can yield very high amounts of Ab40 andAb42, presumably by accumulation CTFb and

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longer Ab intermediates (Barnwell et al, unpub-lished data).

The model that inhibition of g-secretase to explainthe generation of differential amounts of Ab40 andAb42 by simple enzyme kinetics rather than byinvoking a change in enzyme specificity is moreparsimonious and provides a simple kinetic mecha-nism to explain the three observed phenomena:(1) multiple FAD mutations spanning the entiresequence of PS1 increase Ab42/Ab40,31 (2) treatmentwith antisense RNA against PS1 or g-secretaseinhibitors increases Ab42/Ab40 ratios,24 and (3)overexpression of all g-secretase subunits reducesAb42/Ab40 ratios.28

Another implication of our model is that g-secretaseinhibition may lead to increases in secreted Ab levelssimply by accumulating substrate (CTFb and longerAb forms) and releasing them in spurts. This typeof accumulationerelease mechanism should resultin a high local Ab concentration and facilitateAb aggregation to its toxic oligomers.37 Thus, evenif Ab oligomers are the only cause of AD-related

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g-Secretase Processing of the Alzheimer Amyloid Precursor

neurodegeneration, one of the major triggeringevents may be the inhibition of g-secretase. Becauseg-secretase is an essential enzyme, in the wholeorganism complete inhibition may lead to rapid death.Even complete inhibition limited to some cell pop-ulations may lead to accumulation of high levels ofmembrane-bound fragments: CTFa and CTFb, whichmay trigger inflammatory pathways to clear thedysfunctional cells as recently noted in the g-secretasedeficiency associated with acne inverse.25,38 Althoughpartial g-secretase inhibition may be less damaging, itcan clearly affect critical processes such as endothelialtight junction barrier by facilitating vascular endo-thelial growth factor.39,40 Thus, partial g-secretaseinhibition may increase Ab and may be damagingby multiple mechanisms that include impair-ment of signaling by other regulatory substrates,inflammation in response to accumulation of mis-folded proteins in the membrane, and impairedmembrane function due to congestion in the proteintrafficking pathways caused by accumulatedpeptides.26,41

Ab is certainly one of the major accumulatedpeptides and could be the major toxin in initiating thedisease process. However, Ab40 and Ab42 representmetabolites that are generated by normal processingof APP that is highly conserved in higher vertebrates.Under normal conditions, it is well tolerated anddoes not accumulate. The finding that FAD

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mutations inhibit g-secretase but increase Ab42suggests that partial inhibition of g-secretase can bethe cause of AD pathogenesis.3,26 Prevention of ADmust therefore involve preservation of g-secretaserather than its inhibition.

CONCLUSION

Due to extensive efforts spanning behavior,imaging, neuropathology, genetics, and toxicology,Ab accumulation is recognized as an importanttrigger in dementia of the Alzheimer type. This studypresents a slightly different twist to the hypothesis bypresenting evidence that FAD mutations may actu-ally impair g-secretase, the final step in Ab biogen-esis, suggesting that even if Ab toxicity is the triggerin AD, g-secretase inhibition may be an incorrectapproach for its treatment. In addition, the studypresents details on intracellular g-secretase cleavageproducts of APP and identifies fragments whosefunction and toxicity need to be evaluated withrespect to their role in neurodegeneration anddementia.

We thank Todd Golde for kindly providing CHO-2B7and the PS1-M139V construct. Supported by Alzheimer’sAssociation IIRG 10-173180 and NIH RO1AG023055(to KS) and NIH-RO1AG030539 (to JG).

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