The presenilin hypothesis of Alzheimer’s disease: Evidencefor a loss-of-function pathogenic mechanismJie Shen*†‡ and Raymond J. Kelleher III†‡§

*Center for Neurologic Diseases, Brigham and Women’s Hospital, §Center for Human Genetic Research and Harvard-Partners Centerfor Genetics and Genomics, Massachusetts General Hospital, and †Program in Neuroscience and Department of Neurology, HarvardMedical School, Boston, MA 02115

Edited by Thomas C. Südhof, University of Texas Southwestern Medical Center, Dallas, TX, and approved November 17, 2006 (received for reviewOctober 5, 2006)

Dominantly inherited mutations in the genes encoding presenilins (PS) and the amyloid precursor protein (APP) are the major causesof familial Alzheimer’s disease (AD). The prevailing view of AD pathogenesis posits that accumulation of �-amyloid (A�) peptides,particularly A�42, is the central event triggering neurodegeneration. Emerging evidence, however, suggests that loss of essentialfunctions of PS could better explain dementia and neurodegeneration in AD. First, conditional inactivation of PS in the adult mousebrain causes progressive memory loss and neurodegeneration resembling AD, whereas mouse models based on overproduction ofA� have failed to produce neurodegeneration. Second, whereas pathogenic PS mutations enhance A�42 production, they typicallyreduce A�40 generation and impair other PS-dependent activities. Third, �-secretase inhibitors can enhance the production of A�42while blocking other �-secretase activities, thus mimicking the effects of PS mutations. Finally, PS mutations have been identified infrontotemporal dementia, which lacks amyloid pathology. Based on these and other observations, we propose that partial loss of PSfunction may underlie memory impairment and neurodegeneration in the pathogenesis of AD. We also speculate that A�42 may actprimarily to antagonize PS-dependent functions, possibly by operating as an active site-directed inhibitor of �-secretase.

Alzheimer’s disease (AD) is anage-related neurodegenerativedementia and is the most com-mon cause of both neurodegen-

eration and dementia. Neurodegenerativedementias are characterized clinically byprogressive impairment of cognitive abili-ties, which most prominently affects mem-ory in AD. Neuronal and synaptic loss isthe essential neuropathological featurecommon to different forms of neurode-generative dementias, including AD, fron-totemporal dementia (FTD) and Lewybody dementia (LBD). These diseases aredistinguished neuropathologically by char-acteristic patterns of abnormal proteinaggregation, such as the presence in theAD brain of cerebral cortical amyloidplaques and neurofibrillary tangles(NFTs). Extracellular amyloid plaquesconsist primarily of 40- to 42-residue�-amyloid (A�) peptides (A�40 andA�42) derived from proteolytic processingof the amyloid precursor protein (APP).NFTs are intraneuronal inclusions com-posed of hyperphosphorylated forms ofthe microtubule-associated protein tau.

Research on AD has been greatlystimulated by the identification of caus-ative mutations in the genes encodingAPP and presenilins (PS1 and PS2).Dominantly inherited missense muta-tions in APP increase the production ofA� peptides and account for �10% ofmutations identified in familial AD(FAD). PSs harbor �90% of identifiedFAD mutations, and many of these mu-tations increase the relative productionof A�42 peptides. The prevailing amy-loid hypothesis posits that accumulationof A� peptides, particularly the morehydrophobic and aggregation-prone

A�42, triggers a pathogenic cascade,leading to neurodegeneration in AD (1).However, amyloid accumulation is notan obligatory feature of dementia orneurodegeneration because neurodegen-erative dementias lacking amyloid pa-thology (e.g., FTD) have been well de-scribed. Accordingly, the regionaldistribution of amyloid plaques corre-lates poorly with the pattern and sever-ity of dementia in AD, whereas synapticloss correlates well with these clinicalfeatures (2). More surprisingly, mousemodels overexpressing mutant humanAPP have reproduced overproduction ofA� peptides and progressive amyloiddeposition, but they have largely failedto reproduce neurodegeneration (e.g.,see ref. 3).

The presenilin hypothesis (Fig. 1) wasprompted by our recent studies of con-ditional knockout mice in which PSs areselectively inactivated in the adult cere-bral cortex (4). These mice develop age-related, progressive neurodegenerationcharacterized by hallmarks of AD neu-ropathology, including synaptic loss,neuronal cell death, astrogliosis and tauhyperphosphorylation (Fig. 2). In theseconditional mutant mice, inactivation ofPS expression occurs at 4 weeks of agepostnatally, and neurodegeneration be-comes evident by 4 months of age. Bythe age of 9 months, 24% of corticalneurons and 35% of cortical volume arelost. Neurodegeneration is preceded bymemory loss, synaptic plasticity impair-ments, reductions in NMDA receptor-mediated synaptic responses, and de-creases in cAMP-response element(CRE)-dependent gene expression (e.g.,BDNF, c-fos), suggesting that these mo-

lecular defects mediate the subsequentneurodegeneration. Among mouse mod-els of AD, conditional PS knockoutmice are the only mutant mice derivedfrom genetic manipulation of AD genesthat reproduce the central features ofAD, namely neurodegeneration anddementia.

The fact that loss of PS function inthe mouse brain phenocopies the essen-tial manifestations of AD raised thepossibility that FAD-linked mutations inPS may cause the disease by means ofthe partial loss of essential PS functions.Indeed, substantial experimental evi-dence supports the view that pathogenicPS mutations cause partial impairmentof PS-mediated activities. These findingsprovided the initial impetus to rethinkhow PS and APP may be involved inAD. Below, we will summarize accumu-lating evidence for the presenilin hy-pothesis and discuss how it can explainfamilial and sporadic AD.

FAD-Linked PS Mutations Impair�-Secretase-Dependent and-Independent PS ActivitiesPSs are essential components of �-secre-tase, a multisubunit protease complex

Author contributions: J.S. and R.J.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: AD, Alzheimer’s disease; A�, �-amyloid; FAD,familial AD; FTD, frontotemporal dementia; LBD, Lewy bodydementia; NFT, neurofibrillary tangle; APP, amyloid precursorprotein; PS, presenilin, NICD, Notch intracellular domain;AICD, APP intracellular domain; SEL12, suppressor/enhancerof LIN12; cDKO, conditional double knockout.

‡To whom correspondence may be addressed. E-mail:jshen@rics.bwh.harvard.eduorkelleher@helix.mgh.harvard.edu.

© 2006 by The National Academy of Sciences of the USA

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that catalyzes the intramembranouscleavage of a number of type I trans-membrane proteins, including Notch,APP, and cadherins. Notch is a key

physiological substrate of �-secretase, asevidenced by similar developmental phe-notypes exhibited by PS and Notch mu-tant mice (5), and the dependence ofNotch signaling on the �-secretase-medi-ated release of its intracellular domain(NICD). The APP intracellular domain(AICD), which is similarly released by�-secretase-mediated cleavage, has beenimplicated in transcriptional regulation(6). Cadherins seem to undergo similar�-secretase-dependent cleavages, al-though the physiological significance ofthis cleavage is unclear. One unusualfeature of �-secretase is its relaxed se-quence specificity, as evidenced by lackof strong sequence similarity in its sub-strates and its tendency to cleave somesubstrates at a series of neighboring in-tramembranous residues. PSs also pos-sess �-secretase-independent activities,

such as down-regulation of Wnt signal-ing through destabilization of �-catenin.

The first direct evidence that FAD-linked mutations impair the biologicalactivity of PS came from geneticcomplementation studies in Caenorhabdi-tis elegans (7). The C. elegans PS ho-molog, SEL12 (suppressor/enhancer ofLIN12), was originally identified throughits ability to revert the phenotypecaused by constitutive activation of theNotch homolog LIN12. Loss-of-functionmutations in SEL12, which exhibits�50% sequence identity to PS1 andPS2, reduce LIN12 activity and conferan egg-laying defective phenotype (Egl).Transgenic expression of wild-type hu-man PS1 and PS2 rescued the Egl phe-notype caused by a strongly hypomor-phic SEL12 mutation to a levelcomparable with that of wild-type (wt)

Fig. 1. The presenilin hypothesis. This diagramdepicts the cascade of events leading to neurode-generation and dementia in AD, as proposed bythe presenilin hypothesis. Pathogenic mutations inPS partially impair �-secretase-dependent and-independent activities through a dominant-negative mechanism. Elevated levels of A�, partic-ularly A�42, resulting from pathogenic mutationsin APP or PS, or in association with sporadic AD,may act to inhibit PS function, mimicking the effectof PS mutations. Because production of A�42 isenhanced by partial loss of PS and �-secretase ac-tivity, A�42-mediated inhibition may create a vi-cious cycle leading to progressively greater impair-ment of PS function. Loss of PS activity results insynaptic dysfunction, such as deficits in synapticplasticity, and alterations in molecular signalingevents, including impairment of NMDA receptor-mediated functions and reduction in CRE-depen-dent gene expression. Loss of PS function ulti-mately leads to age-related, progressiveneurodegeneration characterized by loss of syn-apses, dendrites, and neurons; astrogliosis; and tauhyperphosphorylation.

Fig. 2. Loss of PS function in the adult cerebralcortex causes striking neurodegeneration. Coronalsections of control (Left) and PS conditional doubleknockout (cDKO) (Right) brains at 9 months of ageare shown to illustrate the extent of neurodegen-eration in PS cDKO mice. Thin lines mark theboundaries of cortical layers and show the thick-ness of the cerebral cortex. Note the diffusethinning of the cerebral cortex and underlyinghippocampal atrophy. Labels indicate the locationsof the neocortex (NCX) and hippocampus (HI).

Table 1. Pathogenic PS mutations impair �-secretase-dependentactivities

Presenilin A�40 A�42 NICD AICD sel12

PS1A79V 2 7�I83/�M84 2C92S 7 2Y115H 2N135D 2 1I143T 2 1 2M146L 7 1 7 7 2M146V 7 1 2 2H163R 7 1 2L166P 2 1/7 2 2L166R 2 2G206A 7 1 2 2G209V 7 1 2 7I229F 7 2A231V 2 7M233L 7 2M233T 2 1 7 2M233V 7 2F237I 7 2A246E 7 1 2P264L 2 1L286V 7 2�ex9 2 1/7 2 2 2insR352* 2 2 2G384A 2 1 2L392V 7 1 2 2C410Y 2 1 2 2 2

PS2T122P 2 1 2 2N141I 2 1/7 2 2M239V 2 1 7 2M239I 2 1 7 2

This table summarizes the reported effects of FAD-linked PS mutations on the�-secretase-dependent production of A�40, A�42, and the intracellular domainsof APP (AICD) and Notch (NICD), as well as activity in complementing SEL12deficiency in C. elegans. Increases and decreases are indicated by up (1)and down (2 ) arrows, respectively, and results in which the level of � -secretaseproduct was found to be unchanged are indicated by a sideways arrow (7 ). Theasterisk after insR352 indicates its association with familial FTD rather than AD.Consensus data are tabulated from published studies (7, 9–19, 45, 58–60).

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SEL12. In contrast, six different FAD-linked mutations reduced the ability ofPS1 to substitute functionally for SEL12to varying degrees (Table 1). Impor-tantly, the ability of mutant PSs to sup-press the Egl phenotype is dose-depen-dent, with higher levels of expression ofsome mutants resulting in full rescue,but lower levels of expression revealinga significant impairment. More recentstudies in Drosophila have also shownthat FAD-associated mutations reducethe ability of fly PS to complementNotch-like phenotypes exhibited byPS�/� mutants (8).

Although initial investigations ofFAD-linked PS mutations in mammaliansystems focused on enhancement ofA�42 production, it has now becomeclear that a large number of pathogenicmutations cause impairments in otherPS activities (Table 1). Of the majorcleavages of APP mediated by �-secre-tase, PS mutations typically increase theproduction of A�42 (and A�43), butcan impair the normally predominant�-cleavage following A� residue 40 andthe more distal ‘‘� cleavage’’ followingresidue 49, resulting in significantly re-duced generation of A�40 and AICD,respectively. Similarly, impairment ofthe �-secretase-dependent S3 cleavageof Notch and consequent reduction inproduction of NICD have been welldocumented with a variety of PS muta-tions. The first study in a vertebrate sys-tem to demonstrate reduction of PS ac-tivity by pathogenic mutations analyzedthe effects on NICD generation of sixFAD-linked PS1 mutations distributedacross the coding sequence (Y115H,I143T, M146V, G209V, G384A, andC410Y) (9). All six mutations causedreductions in proteolytic release ofNICD ranging from 40% to �90% rela-tive to wild-type PS1. Subsequent stud-ies confirmed the reduction in NICDgeneration conferred by several of thesemutations, and identified a number ofadditional FAD-linked mutations inboth PS1 (V96F, L166P, L166R,G206A, �exon9, and L392V) and PS2(T122P and N141I) that substantiallyimpair NICD production (10–13).

Interestingly, the vast majority of PSmutations that impair NICD productionalso impair AICD production, indicatinga general impairment of �-secretase-dependent function that is not limited toa single substrate (Table 1) (11–15).This correspondence of the effects ofmutations on liberation of NICD andAICD may reflect mechanistic similari-ties between the S3 and � cleavages ofNotch and APP, respectively, which oc-cur at similar intramembranous posi-tions near the cytoplasmic face of theplasma membrane. Conversely, most

mutations that interfere with normalAICD generation also reduce NICDgeneration.

Perhaps more surprisingly, numerousmutations in PS1 (e.g., N135D, L166P,M233T, P264L, G384A, and C410Y)and PS2 (e.g., T122P, N141I, M239V,and M239I) can cause significant reduc-tions in the production of A�40, oftendespite a concomitant increase in theproduction of A�42 (12–14, 16–19). Thedifferential effect of PS mutations onalternative cleavage positions in theAPP transmembrane domain seems notto represent a simple shift in substratepreference or specificity, because thedegree of elevation in A�42 levels withindividual mutations does not correlatewith the degree of depression in A�40or AICD levels (e.g., ref. 13). Thus, aconcerted effect of many FAD-linkedPS mutations on �-secretase activityseems to be enhancement of cleavagefollowing A� residue 42, accompaniedby inhibition of cleavage at other possi-ble positions in the APP transmembranedomain, with the magnitude of the ef-fects varying with the specific mutation.

FAD-linked PS mutations also impair�-secretase-dependent proteolysis ofsubstrates other than APP and Notch.N-cadherin undergoes a PS- and�-secretase-dependent �-cleavage analo-gous to that of APP, and a series ofFAD-linked mutations (Y115H, M146L,A246E, E280A, E280G, G384A, and�exon9) uniformly suppressed thiscleavage (20). In addition, FAD-linkedmutations have been reported to cause avariable but general impairment of ‘‘pre-senilinase’’ activity. One of the first re-ports describing PS1 endoproteolysisnoted inhibition of this activity by twoFAD-linked mutations (M146V andA246E) (21). In a survey of 29 distinctFAD-linked PS1 mutations, PS1 endo-proteolysis was significantly reduced inall cases, with the most severe impair-ments (�80% reduction) observed withthe V96F, E280G, and C410Y mutations(22). Reduced PS1 endoproteolysis wasalso observed in mice bearing a targetedgerm-line P264L missense mutation(23). The functional significance of defi-cient presenilinase activity is unclear, asPS1 mutants (e.g., �exon9) refractory toendoproteolysis still support �-secretaseactivity, and whether PS endoproteolysisis in fact autocatalytic remains unre-solved (24, 25).

PS also possess �-secretase-indepen-dent activities, such as their down-regu-lation of the Wnt signaling pathwaythrough interaction with and destabiliza-tion of �-catenin. Interestingly, FAD-linked mutations (M146L, �exon9,C263R, and P246L) have been found toimpair this �-secretase-independent ac-

tivity as well, resulting in enhanced�-catenin stability and �-catenin-depen-dent signaling (26, 27). Recently, PS ho-loproteins were found to function aspassive ER calcium leak channels inde-pendent of �-secretase activity, and twoFAD-associated mutations (PS1 M146Vand PS2 N141L) impaired this function(28). Collectively, these observationsstrongly suggest that pathogenic muta-tions cause a general impairment of PSfunction affecting both �-secretase-de-pendent and -independent activities.Given the large number of pathogenicmutations that do not localize to a par-ticular domain, the deleterious func-tional impact of pathogenic mutationsmay reflect a general destabilization ofPS structure.

The ability of PS1 bearing the FAD-linked A246E mutation to rescue thephenotypic defects of PS1�/� mice hasbeen taken as evidence that mutant PSpossess normal biological activity, andthat pathogenic PS1 mutations do notact through a loss-of-function mecha-

Fig. 3. �-Secretase inhibitors mimic the effects ofpathogenic PS mutations. The graph depicts sche-matically the effects of increasing concentrationsof �-secretase inhibitors on A�40 and A�42 pro-duction, based on data from published reports(32–41). Similar findings have been reported withinhibitors of different structural classes, assayed ineither cell culture systems or partially purifiedmembrane preparations. Three distinct patterns ofchange in the levels of A�40 and A�42 productionare observed in response to increasing concentra-tions of �-secretase, as indicated above the graph:(i) increased A�42 and unchanged A�40; (ii) in-creased A�42 and decreased A�40; and (iii) un-changed or decreased A�42 and decreased A�40.Pathogenic PS mutations can be classified into sim-ilar patterns based on their effect on A�42 andA�40 (representative mutations are shown foreach pattern), with most mutations correspondingto the intermediate pattern. Thus, the impact of PSmutations on �-secretase activity can be equatedwith the effects of varying concentrations of anactive site-directed �-secretase inhibitor. Note thatthe A�42/A�40 ratio is consistently increased acrossall concentrations of �-secretase inhibitor, suggest-ing that this ratio provides a more reliable index of�-secretase inhibition than the individual levels ofA�42 or A�40.

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nism (21, 29). However, PS1�/� micewith only one functional PS1 allele arephenotypically normal, indicating that50% of the normal PS1 dosage is suffi-cient to rescue the phenotype of PS1�/�mice (30). Therefore, even if the A246Emutation caused a 50% reduction inPS1 activity, expression at a level equalto the normal PS1 expression levelwould yield a phenotypic rescue; greaterlevels of overexpression would compen-sate for greater decrements in activity.Consistent with this view, analysis oftransgene expression levels in embryosin one study revealed that the degree ofphenotypic rescue correlated with theextent of overexpression of mutant PS1(21). Moreover, the A246E mutationexhibits reduced ability to rescue pheno-types caused by PS homologue defi-ciency in C. elegans and Drosophila, fur-ther arguing that this mutation doesindeed compromise the biological activ-ity of PS1 (7, 8).

Thus, studies of pathogenic PS muta-tions have revealed that a large num-ber of mutations confer a partial lossof function (Table 1). Indeed, suchanalyses have shown that impairmentof PS activities by pathogenic muta-tions is the rule rather than the excep-tion. Because these studies typicallyinvolve overexpression of mutant PS,

the observed reductions in PS-depen-dent activities are likely to representunderestimates; in cases in whichPS-dependent activities were not ap-parently impaired by mutations, over-expression may have obscured a partialloss of activity, as observed in the C.elegans studies described above. Com-parison of the effects of pathogenic PSmutations with those of inactivating ornull mutations has confirmed that thereductions in �-secretase-dependentand �-secretase-independent activitiesrepresent a partial loss of function.The sole discrepancy is the relative in-crease in production of A�42 causedby pathogenic mutations, which hasbeen generally interpreted as a gain offunction, because genetic inactivationof PS impairs all �-secretase-mediatedcleavages of the APP transmembranedomain (but see next section). Recentanalysis of a series of FAD-linked PSmutations in cultured human cells andDrosophila has further shown that thedegree of reduction in PS-dependentactivity correlates well with the corre-sponding clinical severity, as indicatedby age of disease onset (8, 18). Theseobservations lend additional supportto the view that impaired PS functionplays an important role in diseasepathogenesis.

�-Secretase Inhibitors Mimic the Effectsof Pathogenic PS MutationsPeptidomimetic compounds of severalstructural classes act as small moleculeinhibitors of �-secretase (reviewed in ref.31). These compounds, many of which arebased on the APP substrate sequence sur-rounding the A�42 cleavage site, occupythe enzyme’s active site and function asanalogs of the transition state intermedi-

Fig. 4. Large numbers of pathogenic PS1 mutations are diffusely distributed throughout the codingsequence. This diagram shows the distribution of the missense, small insertion and deletion mutations inPS1. In addition to the depicted mutations, in-frame deletion of exon 9 has also been reported in FAD.Residues highlighted in red indicate the sites of identified FAD mutations, and the three residueshighlighted in green (L113P, G183V, and insR352) indicate the sites of mutations identified in familial FTD.The two aspartates (D257 and D385) implicated as catalytic residues in the active site of �-secretase arehighlighted in yellow. PS endoproteolysis occurs within the protein sequence derived from exon 9.

Fig. 5. Longer forms of A� could act as compet-itive inhibitors of �-secretase. (A) The diagramshows the APP amino acid sequence surroundingthe sites of intramembranous cleavage by �-secre-tase and the major A� peptides produced. Theintramembranous portion of APP is indicated bythe shaded region. Cleavage sites are indicated byinverted arrowheads, with the size of the arrow-head denoting the relative frequency of eachcleavage site. Under normal circumstances, A�40 isthe predominant species produced by �-secretasecleavage, and A� peptides of 43, 42, and 38 resi-dues in length are produced in lower amounts.FAD-linked mutations in PS and APP typically en-hance the production of A�42 and A�43. APP res-idues at which FAD-linked mutations have beenidentified are highlighted in red. Interestingly, thelonger forms of A� (A�42, A�43) retain the majorcleavage site for generation of A�40. Althoughthey may be capable of interacting with the en-zyme active site, these longer forms of A� areunlikely to be efficient substrates for cleavage ow-ing to the absence of distal residues, raising thepossibility that they may occupy the active sitenonproductively after their generation. Thus,A�42 and A�43 may act as �-secretase inhibitors,and their increased production in familial and spo-radic AD may result in inhibition of �-secretase. (B)The amino acid sequence of APP surrounding themultiple intramembranous �-secretase cleavagesites is diagrammed at top, with the cleavage sitesdesignated by asterisks; the length of the corre-sponding A� peptide is indicated above the se-quence. The structure of a typical substrate-based�-secretase inhibitor and the C-terminal sequencesof A�43 and A�42 are shown below. Substrate-based �-secretase inhibitors are peptide analogstypically derived from the amino acid sequencesurrounding the A�42 cleavage site. Residues im-mediately flanking the cleavage site (A*T) are of-ten modified to incorporate hydroxyethyl isostereor difluoroketone moieties, which mimic the tran-sition state intermediate in aspartic protease catal-ysis. The C-terminal sequences of A�43 and A�42resemble substrate-based peptide inhibitors di-rected against the A�38–A�40 cleavage sites.

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ate in aspartic protease catalysis. An in-triguing property shared by �-secretaseinhibitors is their paradoxical ability toenhance A�42 production while blockingother �-secretase-dependent cleavages,thus mimicking the effects of PS muta-tions (Fig. 3) (32–41). Low to moderatedoses of essentially all �-secretase inhibi-tors used to date increase A�42 produc-tion, whereas higher doses produce theexpected inhibition of A�42 production.In contrast to this biphasic effect onA�42, �-secretase inhibitors cause a pro-gressive inhibition of A�40 production.Enhancement of A�42 production oftenoccurs even at subinhibitory concentra-tions, arguing that the increase in A�42levels is not simply a consequence of in-creased substrate availability owing to re-duced A�40 generation. In addition, ob-servation of this phenomenon in both cellculture systems and partially purifiedmembrane preparations makes it unlikelythat increased A�42 production is a resultof inhibitory effects on other intracellularprocesses (36, 41). Interestingly, the de-gree of enhancement of A�42 productionelicited by �-secretase inhibitors isstrongly correlated with their inhibitorypotency (42), suggesting that increasedA�42 generation is mechanistically relatedto active site-directed inhibition.

The fact that �-secretase inhibitors canmimic the effects of pathogenic PS muta-tions (i.e., increase A�42 production whileinhibiting other �-secretase activities) sup-ports the hypothesis that pathogenic mu-tations cause a partial loss of PS function,equivalent to the effects of low to moder-ate doses of �-secretase inhibitors (Fig. 3).Thus, the elevated A�42 productioncaused by PS mutations may represent asymptom of a ‘‘sick’’ and otherwise im-paired �-secretase. The opposing effect onA�42 production relative to other sub-strate cleavages exerted by �-secretaseinhibitors and PS mutations is not easilyreconciled with a monomeric enzymestructure, and suggests instead a multi-meric enzyme subject to allosteric regula-tion. This view is consistent with evidencefor PS dimerization and dominant-nega-tive effects of PS mutations (13). Interest-ingly, substrate or transition state analogscan have biphasic effects on the activity ofallosteric enzymes, similar to the effect of�-secretase inhibitors on A�42 produc-tion. This phenomenon has been well doc-umented with the classic allosteric enzymeaspartate transcarbamylase: L-aspartateanalogs (i.e., dicarboxylic acids such asmaleate) perform as activators at low con-centrations and inhibitors at higher con-centrations (43, 44).

PS Mutations Can CauseNeurodegenerative Dementia in theAbsence of A� AccumulationThree PS1 mutations (L113P, G183V,and insR352) have been identified in

families with FTD, a common neurode-generative dementia that lacks amyloidpathology (45–47). The absence of A�accumulation in FTD implies that thesePS1 mutations may confer a strongerloss of function than those causing fa-milial AD, equivalent to high doses of�-secretase inhibitor. Indeed, one ofthese FTD-associated PS1 mutations(insR352) strongly impairs �-secretaseactivity in a dominant-negative manner(46). The remaining two mutations oc-cur at exon-intron boundaries, probablygiving rise to both full-length transcriptsencoding PS1 bearing the identified mis-sense mutation as well as aberrantlyspliced transcripts encoding markedlytruncated PS fragments (46, 47). Thus,PS1 mutations can cause neurodegen-eration and dementia in humans withoutincreasing A� production, possibly byimposing a generalized reduction in PSactivity. Moreover, PS mutations alsooccur in families in which early-onsetAD is associated with cortical Lewybodies, which contain �-synuclein aggre-gates (48–50). Collectively, these obser-vations further suggest that the variableprotein aggregates associated with PSmutations do not play an essential rolein the pathogenic mechanism leading toneurodegeneration and dementia.

The Diffuse Distribution of PS MutationsIs Most Compatible with a Lossof FunctionIn contrast to pathogenic mutations inAPP, which cluster around sites of pro-teolytic cleavage, PS mutations are scat-tered throughout the protein’s extracel-lular, cytosolic and transmembranedomains, occurring at �20% of theamino acid residues (Fig. 4). This sug-gests that ‘‘random’’ alterations of singleamino acid residues in PS are sufficientto cause AD, highlighting the impor-tance of normal PS functions in ADpathogenesis. The large number (�150)and diffuse distribution of PS mutationsare most compatible with a loss of pro-tein function, such as might be causedby a general destabilization of thefolded protein structure. The absence ofpathogenic mutations that would resultin a complete loss of functional protein(e.g., non-sense and frame-shift muta-tions) suggests that PS mutations areunlikely to act through a simple loss offunction. Rather, these observations col-lectively suggest that pathogenic PS mu-tations may act through a dominant-negative mechanism, in which mutantPS protein with diminished activityand/or stability interferes with the func-tion of wild-type protein. Such a mecha-nism would be consistent with the domi-nant inheritance of PS mutations, asdiscussed below.

Is a Pathogenic Mechanism Based onLoss of PS Function Compatible with ADGenetics?One might question whether loss of PSfunction can provide a tenable explana-tion for AD pathogenesis, because APPmutations alone are sufficient to causeAD. APP mutations increase the pro-duction of A�, suggesting that A� itselfcan be pathogenic, as proposed by theamyloid hypothesis. How A� causesneurodegeneration, however, is presentlyunclear. We propose that increased lev-els of A� may cause AD by interferingwith PS function and/or expression, re-sulting in a loss of PS-dependent activi-ties. An interesting possibility that hasnot previously been considered is thatelevated A�42 levels may inhibit�-secretase activity through a product-based negative feedback mechanism,effectively mimicking the effects of PSmutations and �-secretase inhibitors.Because of the relaxed substrate speci-ficity of �-secretase, longer forms of A�(i.e., A�42/43) contain potential cleav-age sites for generation of shorter formsof A� (i.e., A�38/40). Such longer formsof A�, whose production is favored inboth familial and sporadic AD, arelikely to be ineffective substrates forcleavage, owing to the absence of down-stream residues important for productiveactive site-substrate interactions. Thus,longer forms of A� may act as �-secre-tase inhibitors by occupying the en-zyme’s active site nonproductively, in amanner similar to peptidomimetic ac-tive-site directed inhibitors (Fig. 5).These putative inhibitory forms of A�could remain kinetically associated withthe �-secretase active site after theirproduction, or they could reassociatewith �-secretase after retention in orreinsertion into the plasma membrane.

Increased A� levels may also inhibit theexpression of normal levels of �-secretase,because A� negatively regulates CRE-dependent gene expression, and expres-sion of the genes encoding both PS1 andthe essential �-secretase subunit Pen-2 isCRE-dependent (51–53). In support of arole for reduced PS expression in ADpathogenesis, PSEN1 promoter polymor-phisms that reduce PS1 expression havebeen reported as risk factors for sporadicAD (54, 55). Alternatively, loss of PSfunction and increased A� productionmay converge at common downstreamsignaling pathways that are required forsynaptic plasticity and neuronal survival.For example, both PS inactivation andincreased A� lead to reductions in synap-tic NMDA receptors and CRE-dependentgene expression (4, 51, 52, 56).

The presenilin hypothesis must alsobe reconciled with the fact that muta-

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tion of a single PS allele is sufficient tocause AD, despite the presence of threeremaining intact PS alleles. In contrast,inactivation of a single PS1 allele inmice, whether germ-line or conditional,is insufficient to cause neurodegenera-tion (30, 57). The most straightforwardsolution to this apparent dilemma is thata single mutant allele may be patho-genic because the resulting mutant pro-tein can act in a dominant-negativemanner to inhibit the activities of nor-mal PS produced from the remainingwild-type PS alleles. In genetic terms,FAD-linked PS mutations are likely tobe antimorphic, causing an intrinsic lossof function as well as a gain of negativefunction, thereby bringing about anoverall loss of PS activity. Such a modelwould reconcile the dominant inheri-tance of PS mutations with the evidencefor a loss-of-function pathogenic mecha-nism outlined above. Indeed, PS appearto form dimers within the �-secretasecomplex (13), suggesting a possible allo-steric mechanism that could allow fordominant-negative effects of PS muta-tions, as well as their capacity to en-hance A�42 production while impairingother �-secretase activities. Consistentwith this view, dominantly inherited mu-tations causing a variety of familial hu-man diseases have been found to actthrough a dominant-negative mecha-nism, causing both intrinsic and overalllosses of protein function.

ConclusionBased on several independent lines of evi-dence, we propose that loss of PS functionmay be a primary event triggering neuro-degeneration in AD, and possibly in otherforms of neurodegenerative dementia,such as FTD. The presenilin hypothesisderives from the following observations:(i) inactivation of PS function in the adultbrain provides the only mouse modelbased on genetic manipulation of PS orAPP that recapitulates dementia andwidespread neurodegeneration; (ii) al-though FAD-linked PS mutations causeincreased production of A�42, a large andincreasing number of FAD-linked PS mu-tations have been shown to inhibit otherPS activities; (iii) �-secretase inhibitorscan mimic the effects of PS mutations,stimulating A�42 production while inhibit-ing other �-secretase activities; (iv) PS

mutations have recently been identified inFTD, indicating that PS mutations cancause dementia and neurodegeneration inthe absence of amyloid accumulation; and(v) the large number, diffuse distributionand missense nature of pathogenic PSmutations is most compatible with muta-tions causing a loss of PS function througha dominant-negativemechanism.

Our model does not discount an impor-tant role for elevated levels of A� pep-tides, particularly A�42, in the pathogene-sis of AD. However, we suggest thatincreased A� levels may cause neurode-generation and dementia primarily by in-terfering with PS-dependent activities,thereby causing an effective loss of PSfunction. As one possible mechanism, wespeculate that A�42 may act as an inhibi-tor of �-secretase through prolonged oc-cupation of the enzyme’s active site. Re-gardless of the specific mechanism, onewould expect that indirect A�-mediatedinhibition would have an inherentlyweaker influence on PS function than themore direct effect of PS mutations. In thisrespect, the presenilin hypothesis mayhelp to explain several perplexing obser-vations: (i) the paucity of pathogenic mu-tations in APP relative to PS; (ii) the fail-ure of mutant mice overproducing A� todevelop neurodegeneration, whereas mu-tant mice with complete loss of PS func-tion exhibit striking neurodegeneration;and (iii) the earlier age of onset and moreaggressive course of PS-linked FAD incomparison to APP-linked FAD, despitethe fact that APP mutations often elicit afar greater elevation in A� levels. Theproposed antagonistic effect of A� onPS-dependent functions could apply toany mechanism that increases the steadystate levels of A�, including familial andsporadic forms of AD, although elevatedA�42/A�43 production by �-secretasemay have the most deleterious impact.

Recent analysis of a PS conditionaldouble knockout (cDKO) mouse has pro-vided insight into the consequences of lossof PS function in the adult mammalianbrain, outlining a putative pathogenic cas-cade in which loss of PS function compro-mises NMDA receptor function, synapticplasticity and CRE-dependent gene ex-pression, ultimately precipitating wide-spread and progressive neuronal atrophyand death (4). Neurodegeneration in the

PS cDKO mouse displays many of thehallmark features of AD neuropathology,including synaptic and neuronal loss, as-trogliosis and tau hyperphosphorylation.Although the PS cDKO mouse lacks theamyloid pathology characteristic of AD,the evidence outlined above suggests thatloss of PS function may be a centralpathogenic mechanism that operatesdownstream of A� accumulation in ADand independent of A� accumulation inFTD. Furthermore, complete PS inactiva-tion models the proposed dominant-nega-tive effects of a single partial loss of func-tion mutant PS allele, which may lead togradual and progressive inhibition of totalPS activity over time, an effect that maybe further exacerbated by overproductionof A�42. Genetic exaggeration of theseeffects through complete PS inactivationpresumably facilitated the development ofneurodegeneration within the short (2-year) life span of mice, whereas similarneuropathology becomes manifest onlyafter 5–6 decades in FAD patients. Suchgenetic exaggeration has thus far beenlargely unsuccessful in producing neurode-generation in a variety of transgenic miceoverexpressing mutant human APP, possi-bly because the pathogenic effects of A�are less direct and/or less potent than PSmutations in causing neurodegeneration.

The presenilin hypothesis of AD po-tentially reconciles important discrepan-cies in our current understanding ofAD, thereby uniting a fragmented set ofobservations. Although this hypothesisderives in part from genetic observa-tions, progressive loss of PS function,whether due to inhibitory effects of A�accumulation or to independent mecha-nisms, could similarly explain the patho-genesis of sporadic, late-onset AD. Ifcorrect, this hypothesis has significanttherapeutic implications. It suggests that�-secretase inhibitors would aggravateinstead of ameliorate neurodegenerationand dementia, and that boosting PS-dependent pathways or inhibiting oppos-ing pathways will offer the most promis-ing therapeutic strategies for AD.

We thank Mike Brown and John Hardy fordiscussions and comments and Mary Wines-Samuelson for preparation of Fig. 2. Re-search in the authors’ laboratories is sup-ported by grants from the National Institutesof Health and the Alzheimer’s Association.R.J.K. is a Pew Scholar in the BiomedicalSciences.

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