C-Terminal Maturation Fragments of Presenilin 1 and 2 Control Secretion of APPα and Aβ by Human Cells and Are Degraded by Proteasome (2025)

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Molecular Medicine 5: 160-168, 1999

Molecular Medicine

C)1999 The Picower Institute Press

C-Terminal Maturation

Fragments

Presenilin

and 2 Control Secretion

of APPa and

A13

Human Cells and Are

Degraded by

Proteasome

Cristine Alves

Costa,

Karine

Ancolio,

and Frederic

Checler

Institut de

Pharmacologie

Moleculaire et Cellulaire du

CNRS,

Valbonne, France

CommunicatedbyP.Chambon.Accepted February 15, 1999.

Abstract

Background: Most early-onset forms of Alzheimer'sdisease are due to missense mutations located on two

homologousproteinsnamedpresenilin1and2(PS1 andPS2).Several lines of evidence indicate thatPS 1andPS2undergo various post-transcriptional events including

endoproteolytic cleavages, giving rise to 28-30 kD

N-terminal (NTF) and 18-20 kD C-terminal (CTF)

frag-mentsthat accumulate invivo. Whether the biologicalactivity of presenilins is borne by the processed frag-ments ortheirholoprotein precursor remains in

ques-tion. We have examined the putative control of I3APPmaturationbyCTF-PS1/PS2andthe catabolic process ofthe latter proteinsbythemulticatalytic complex,

protea-some.

Materials and Methods: We transiently and stablytransfected HEK293 cells with CTF-PS1 or CTF-PS2

cDNA. We examined these transfectants for their pro-duction ofA,B40,Af342,andAPPaby

immunoprecipita-tion using specific polyclonals. The effect of a series ofproteases inhibitors on the immunoreactivity of

CTF-PS1/PS2 was examined by Western blot. Finally, theinfluence of proteasome inhibitorsonthe generation off3APP fragmentsby CTF-expressing cells was assessed bycombined immunoprecipitation anddensitometricanal-yses.

Results:We showedthat transient and stable transfec-tion of CTF-PS1 and CTF-PS2 cDNAs in human cellsleadstoincreased secretion ofAPPaand A,B, the

matu-rationproductsof j3APP.Furthermore,wedemonstratedthattwoproteasomeinhibitors, lactacystinand Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs.Accordingly, we established that proteasome inhibitorsdrastically potentiatethe phenotypic increased

produc-tionofAPPaand A,B elicitedby CTF-PS1/PS2.

Conclusion: Our data establish that the C-terminalproducts of PS1 and PS2 maturation exhibit biologicalactivity andinparticular control 13APP maturation

up-streamtoa-and13/y-secretase cleavages.This function isdirectlycontrolledbythe proteasome that modulates theintracellularconcentrationof CTFs.

Introduction

Familial forms ofAlzheimer's disease are dueto

inherited mutations on genes, the loci of which

have been identified on chromosomes 21, 14,

and 1 (forreview seeref. 1). The gene products

ofchromosomes 1 and 14havebeen showntoberesponsible formostearly-onset formsof

Alzhei-Addresscorrespondenceandreprintrequeststo:Dr. F.Checler, Institut dePharmacologie Moleculaire etCellulaireduCNRS,UPR411, 660 Route desLucioles, SophiaAnti-polis,06560Valbonne, France.Phone: (33) 4 93 95 7760;Fax:(33) 4 93 95 7704/08; E-mail:[emailprotected]

mer'sdisease and have been identified as prese-nilins 2 and 1 (PS2 andPS1), respectively (2-4).

PS1 and PS2 are highlyhomologous transmem-braneproteins (5,6) locatedmainlyinthe endo-plasmic reticulum (7-11) and Golgi apparatus

(10,11). These proteins undergo various post-transcriptional modifications (for reviews see

refs. 12,13) and are particularly susceptible toendoproteolytic cleavages givingrise to

N-termi-nal (NTF) and C-terminal (CTF) fragments (14-17). This cleavage by an as-yet unknown

(2)

holoproteinsarepoorlydetectable in the brain of

transgenic mice and affected patients and the

twoproteolytic fragments appear to accumulate

witha 1 / 1 stochiometry (15). Interestingly, NTF

anf CTF undergophosphorylation events (9,18),behaveastargetsofcaspase-mediated proteolysis(19-21), and can physically interact (12,13).Whether thesepost-transcriptionaleventsreflect

regulatory mechanisms aimedatmodulating

pu-tative NTFand CTFbiological activityremains to

be established.

We have examined whether the expressionof CTF-PS1/PS2 modulates the processing of

,BAPPin HEK293 cells.We show that both

CTF-PSI and CTF-PS2 expression leads to increasedsecretion of

AP3

and APPa. Furthermore, we

es-tablish that both PS maturated fragments are

catabolizedby the proteasome. Accordingly, two

proteasome inhibitors drastically potentiate theCTF-mediated increase in the recovery of both

,3APP

maturation

products. Altogether,

our data

indicate that CTF-PS1 and CTF-PS2 are

biologi-callyactive andinparticular, control ,APP

mat-uration. This function appears to be directlymodulated by the proteolytic catabolism elicitedby the proteasome.

Materials and Methods

Design ofCTF-PS1 and CTF-PS2 cDNAs

The cDNA encoding CTF-PS1

(ct-i)

was engi-neered by introducing the Kosak sequence ofPS1 upstream of the ATG codon encoding thePSI methionine in position 292 (oligo: 5'-CAT-AGG-ATC-CGT-TGC -TCC

-AAT-GGT-GTG-GTT-GGT-GAA-TAT-GGC-AGA-A-3'). An additional

BamHl restriction site was also added, adjacent

tothe Kosak sequence for further subcloning ofthe construction in pcDNA3

(ct-i).

The cDNAconstruction coding for CTF-PS2 (ct-2) was en-gineered similarly, with the Kozak sequence of

PS2 upstream of the ATG codon encoding themethionine in position 298 and an additionalKpn restriction site adjacent to the Kozaksequence (oligo: 5'

-ATC-TGG-TAC-CGG-CAG-GGC

-TAT-GGT-GTG-GAC-GGT-TGG-CAT-GGC-GAA-G-3').

Stable Transfections ofHEK293 Cells

HEK293 cells were grown as previously de-scribed (22). Stable transfectants were obtainedby calcium phosphate precipitation with 1 ,ug ofempty pcDNA3 vector,

ct-i,

or ct-2 and called

Mock, CPS1, and CPS2, respectively. CPS1 and

CPS2 wereidentified after Western blotanalysis

of electrophoresed proteins by means of the

aPSlLoop or aPS2Loop antibodies (15) as de-scribed previously (23,24). HEK293 cells

ex-pressing

wild-type-f3APP751

(referred to as WT)

were obtained as described previously (22).

Transient Transfections of HEK293 Cells

Mock or WTstable transfectants weretransientlytransfected with 2

jig

of ct-i, ct-2, or emptyvector bymeans of DAC30 according to

manu-facturer recommendations (Eurogentec).

Trans-fection efficiency was checked by Western blot

withaPSILooporaPS2Loopantibodies.Analysis

of ,BAPPmaturation productswas performed 48

to 72 hr after transfection.

Immunoprecipitation and Detection of APPa and

Transiently or stably transfected cellsweremain-tained in the above F12/DMEM-supplemented

medium then washed, and secretion of APPaand

AP3

was initiated for 5 hr at 370C in theabsence orwith presence of proteasome

inhibi-tors. Aliquots of media were recovered, diluted

in an equal volume of RIPA buffer, and

incu-batedovernightwith a 3000-fold dilution of207

antibody (APPa) or with a 350-fold dilution ofFCA18 (total Af3) as previously described (25).Samples were centrifuged, and the pellets were

washed three times with RIPA 1X containing

NaCl (350 mM), rinsed with TBS buffer, then

resuspended in the loading buffer, electropho-resed on 8% SDS-PAGE (APPa) or Tris-tricinegels (Af3), andWesternblotted for 1-3 hr. Nitro-cellulosesheetswerecapped with skim milk (5%in TBS buffer) and exposed overnight to a

200-fold dilutionofmAblOD5C (APPa) or 1,g/mlof

W02 (A,3) antibodies. The nitrocellulose sheets

wererinsedwith TBSbuffer then incubated withadequate anti-IgGs, revealed, and quantified aspreviously described (25).

Western BlotAnalysis of ,BAPP and CTFs

Immunoreactivity

Stably transfected cells were treated as abovethenlysedin50 mMTris-HCl,pH 7.5,containing150mM of NaCl andprotease inhibitors (5 mMEDTA, 1 mM leupeptin, 1 ,uM pepstatin and 1mM AEBSF). Analysis for ,3APP and CTF-PS1/CTF-PS2 contents was performed by means of

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162 Molecular Medicine,Volume5, Number3, March 1999

300

200

ct-1

ct-2

ct-I

mlwtmlwt

_CL

- 150

'Pa

-I

100

II I I

CPS1

CPS2

Mock

CPS1

CPS2

Mock

CPS1-6

CPS2-8

Mock 1 (6) (8)

(6)

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Fig. 2. Influence of CTF-PS1 and CTF-PS2

overexpressiononAPPa secretion. Stably

trans-fected HEK293 cells overexpressing CTF-PS1

(CPS1-6 andCPS1-20), CTF-PS2 (CPS2-1 andCPS2-8), oremptypcDNA3 (Mock)were examined forAPPasecretion from endogenous,BAPP (A, B) or

aftertransient transfection (C, D) with f3APP751

(wt) orpcDNA3 (m). APPawasdetected after

im-munoprecipitation with 207antibody andwestern

blot with mAB OD5C asdescribed in Materials and

and CTF-PS2 increased the recovery oftotal

se-creted

AP3

(Fig. IA, left panel) by about 200 to300% (Fig. LB) overcontrol (Mock/m) cells,

in-dicating that transient transfection of ct-I andct-2 modulates the

AP3

production derived from

endogenous ,APP. The same phenotypic in-crease in A,B secretion was observed (Fig. IA,

rightpanel) with stably transfectedwt-f3APP751

-expressing cells (WT) transiently transfectedwith ct-I and ct-2 (about 200% over controlWT/m).

To further document the influence of theoverexpression of CTF-PS 1/2 onthe f3/y-derived

productof,BAPP maturation,clonesstablymock-transfected (Mock) or overexpressing CTF-PS1

(CPS1) and CTF-PS2 (CPS2) were transiently

transfected with empty pcDNA3 (m) (Fig. 1C).

Both CTF-expressing clones secreted higheramounts of

AP3

than Mock/m control cells

(Fig. 1D). Transient transfection ofMock, CPS1,and CPS2 cloneswithwt-4APP751 cDNA(wt)led

to increased

A,8

secretion, the production ofwhich was higher in CPS1 and CPS2 than inMock-transfected cells (Fig. 1C, D).

Methods. BarsinB andDrepresenttheAPPadensi-tometricanalysis ofexperiments A (normalized forCTF-PS1/PS2 expressions) and C (normalized forwtexpression), respectively. Valuesare expressedas thepercent of controlAPPasecretionobtained withMock cells (B) orMock-transfected cells (D) tran-sientlytransfectedwith emptyvector (m) or

wt-13APP751 (wt).Values arethemeans ± SEM of inde-pendent experiments.

Weexaminedthe putative influence ofCTF-PSI and CTF-PS2 expression onthe secretion of

the a-secretase-derived physiological product of

,3APP maturation, APPa. Two independent

clones of CPS 1 and CPS2 produced higheramounts of APPa than Mock-transfected cells

(Fig. 2A, B). Transient transfection of Mock,CPS1, and CPS2 (Fig. 2C) with empty pcDNA3

(m) or wt-f3APP751 cDNA (wt) confirmed that

the CTF-PSs-expressing cells secrete more APPathan the Mock-transfected cells (Fig. 2D).

The effect of the proteasome inhibitorZ-IE(Ot-Bu)A-Leucinal on CTF-PS1 and

CTF-PS2immunoreactivityinstabletransfectantswas

also examined. Figure 3 indicatesthat

immuno-reactivityofboth CTF-PS1 (Fig. 3A) and CTF-PS2

(Fig. 3B)is highly enhanced by Z-IE(Ot-Bu)A-Leucinal. The involvement of theproteasome inCTF degradation is further demonstrated by theenhancement of CTF immunoreactivity upon

treatment with lactacystin, a very potent andselective proteasomeblocker (Fig. 3C). Itshouldbe noted that a smir of high-molecular-weight

proteins is detectable upon proteasome

inhibi-A

APPa

1% %

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164 Molecular Medicine, Volume 5,Number3, March 1999

CPS1-6

C-PSI-17

ZIE

|1- +I|

-14

-_14

B CPS2-1

ZIE m-+

CPS2-8

CTF-PS2

-0

0sU

COLL

_ w

r J

4I)0L

Fig. 3. Effect ofvarious protease in-hibitorsonCTF-PS1 and CTF-PS2immunoreactivityin HEK293 trans-fected cells. Stably transfectedHEK293

cells expressingCTF-PS1 (A, C) or CTF-PS2 (B, C) wereincubated for 5 hrin

thepresence of theproteasome

inhibi-tors Z-IE(Ot-Bu)A-leucinal (ZIE, 2.5 XCTF-PSI 10-5M) and lactacystin (5 X 10-6M) or

with

trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64, 10-4M),

4-(2-amido-ethyl) benzenesulfonylfluo-ride (AEBSF, 10-4M), phosphoramidon(10-5M),pepstatinA (10-5M),andZ-L-Leucinal (ZL, 2.5 X 10-5M). Cells4CTF-PS2 were lysed, then immunoreactivity of

CTF-PS wasrevealedwithaPSILoop andaPS2Loopantibodiesas describedin Ma-terialsand Methods.

tion (seeFig. 3B), which could reflectprotectionof ubiquitinated forms of CTF as was shown forpresenilin 1 (23) andpresenilin 2 (29).

Other protease blockers unable to inhibit theproteasome activity do not modify CTF-PS1/2

re-covery (Fig. 3C). Thus, Z-L-Leucinal (calpain andcathepsin B inhibitor), E64 (thiol and serine

pro-teaseinhibitor),AEBSF (serine proteaseinhibitor),

phosphoramidon (endopeptidase 3.4.24.11 inhibi-tor), and pepstatin (acid protease inhibitor) donot

protectCTF-PS 1 and CTF-PS2 from degradationin

HEK293 cells (Fig. 3C).

IfCTF-PS 1/2 degradation by the proteasome

is physiologically relevant, one would expect topotentiate their stimulatory effect on A,B and

APPa secretion upon proteasome inhibition. Z-IE(Ot-Bu)A-Leucinal augments the APPa

secre-tion triggered by CPS1 and CPS2 transfectants(Fig. 4A, B). This inhibitor also potentiates the

secretionof

AP3

wt-,3APP751

-expressing

trans-fectants (WT) transiently transfected with ct-I

andct-2 constructions (Fig. 4C, D). Therefore, it

can be concluded that inhibition of the

protea-somepotentiates the effectof CTF-PS 1 and CTF-PS2 on botha- and1/,y-secretase-derived

prod-uctsof ,3APP maturationin HEK293 cells.

Discussion

We have previously established that the

protea-some contributes to the maturation of

endoge-nous ,3APP in human cells. Thus, proteasomeinhibitors increase the recovery of both

AP3

and

APPa in naiveHEK293 cells (22). This led us to

hypothetize that a cellular intermediate locatedupstream tothe a- and 3/-y-secretases cleavagesand behaving as substrate of the proteasomeplaysanimportantroleinIAPPmaturation. Thesearch for suchaneffector ledus to suggestthat

PS1 and PS2 could fulfill such a role. First, we

showed that overexpression ofPSI (26) andPS2

(24) elicitanincreasedsecretionofboth AB and

APPa. Second, we demonstrated that PSI and

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CPS1-6

ZIE

-CPS1-17

CPS2-1

CPS2-8

I.-

APPa

ct-i

ct-2

1 +1 1_ +1

200

0-vL

300

_O20

200

-g

100

-PS1-

CPSI-6

CPS1-17

CPS2-1

CPS2-8

Fig. 4. Effect ofZ-IE(Ot-Bu)A-leucinalonAf3and APPa secretionby stably transfectedHEK293 expressing CTF-PS1 and CTF-PS2.

Sta-ble transfectants overexpressing CTF-PS1 (CPS1-6and CPS1-17) and CTF-PS2 (CPS2-1 andCPS2-8)

were grownas describedinMaterials andMethodsthenAPPasecretion wasinitiated intheabsence (-)

orpresence (+) ofZ-IE(Ot-Bu)A-leucinal. APPa wasimmunoprecipitated after 5 hrbymeans of 207 anti-body andrevealed with mAblOD5C (A). Stabletransfectants overexpressing wt-f3APP751 (WT) were

PS2 behave as excellentsubstrates of the

protea-some (23,24), whichis inagreement with otherstudies (29,30). Third, we established that theproteasome inhibitors exacerbate the PS1/PS2-induced increased recovery of both

f3APP

matu-ration products (23,24). Interestingly,

protea-some inhibitors also exacerbate the phenotypicalterations of ,BAPP maturation triggered byFAD-linked PS mutations (23,24).Therefore, we

suggestedthat PS1 and PS2 control

P3APP

matu-rationupstreamtosecretases, and that this

func-tion is directly modulated by catabolic events

triggered by theproteasome.

Presenilins undergo several post-transcrip-tional modifications that include

phosphoryla-tion andproteolytic cleavages byunknown

pro-teases as well as by caspase 3 (12,13). Whether

Inl

ct-i

ct-2

transiently transfected with CTF-PS1 (ct-i) or

CTF-PS2 (ct-2) cDNAs. Secretedtotal

AP3

wasobtainedafter immunoprecipitation withFCA18 andrevealedwith mAbWO2 as described in Materials andMeth-ods (C). Densitometric analysesofAPPa (B) or

AP3

(D) [normalizedforCTF-PS1/2 (A) or ct-1/2 (C)]secretions areexpressed asthepercentofcontrolcorrespondingto secretion obtained inabsence ofinhibitor.Values are the mean of independent ex-periments carriedout ontheindicatedindependentclones.

the degradation products of PSI /PS2 correspond

to intermediate or final catabolites en route tofinal clearance or, alternatively, to maturatedfragments bearing biological activity is not yetestablished. However, the fact that these prod-uctsaccumulateinvivo inhuman and transgenicmice brains (1 5,31) and are able to interact

phys-ically raised the possibility that N- and C-termi-nal PS fragments could display a physiologicalfunction. In line with this hypothesis was theobservation that the overexpression of a C-ter-minal fragment of PS2 that can be

physiologi-cally generated by proteolysis and alternativetranscriptionwas able to rescue HeLa cellsfrom

Fas- andTNF-induced apoptosis (32). Therefore,it wasstillquestionable whether PS-related

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166 Molecular Medicine, Volume5,Number 3,March 1999

maturated fragments, or both, and particularly,

whether CTF-PS1 or CTF-PS2couldplayaputa-tive role inthe control of,BAPP maturation.

We showhere that the expression of CTF-PSI and CTF-PS2 mimicks the phenotype of

overexpression of their precursor holoproteins

PSI and PS2 in HEK293 cells. Thus, both C-terminal fragments augment the secretion of

AP3

and APPa derived from endogenous or

overexpressed

wt-f3APP751.

Furthermore, bothCTF-PSI andCTF-PS2 are susceptible toprote-olysis by the proteasome. Accordingly, treat-ment of transfected cells overexpressing

CTF-PSs with proteasome inhibitors leads to the

exacerbation of the CTF-dependent augmenta-tionof

AP3

andAPPa.The fact that the

produc-tion of botha and f3/y-secretase-derived f3APPfragments areaugmented by the CTFsrulesout

thepossibility thatCTFbehavesas one of theseproteolytic activities.

Ithas been documented that CTF-PS1 andits

N-terminal counterpart (NTF)interact toform

sta-ble heterodimers witha 1:1 stochiometry (15). Ifthis also holds for the CTF-PS2 and NTF-PS2 de-rivatives,it isquestionable whether thephenotype

observed when overexpressing CTF-PS2 mimicksthe physiological function of endogenously pro-duced CTF-PS2, since the 1:1 stochiometry of

en-dogenous fragments theoretically preclude CTFs

from interacting withendogenous NTFs-this frag-mentwouldnotbeavailable as a free entity. How-ever, it should be noted that several studies haveindicated that transient or stable transfection of PS2led to apoptosis and that transfection of CTF-PS2

cDNA antagonized this effect in various cell sys-tems, suggesting that overexpression of this

frag-ment elicits a physiologically relevant phenotype(32).

Interestingly, Tomita and colleagues alsorecently examined the effect of overexpressionof CTF-PS2 in stably transfected cells and con-cluded that this didnot affect ,BAPP maturation(33). These discrepancies with our presentworkcanlikely be explained by several factors.The CTF constructions used in the Tomita

study had different N-termini from ours. Also,the effect ofCTF expression was monitored on

x-40/42 species that likely include a majorcontribution of N-terminally truncated

frag-ments derived from either pathogenic or alsophysiologic cleavages (33), whereas our anti-bodies were able to monitor total but genuineA,B. Part of the datainTomita's studyconcernsthe effect ofCTF on x-40/42fragments derivedfrom the C100 ,BAPPconstruction, the

process-ing/routing of which could be different from

full-length I3APP. Finally, it can be noted that

the respective cells systems are totally distinct

[COS and N2a (33) versus HEK293 cells

(present study)].

Our study documents the fact that CTF-PS1andCTF-PS2 exhibitbiological activity and

par-ticularly, control

P3APP

maturation. This does not

preclude the possibilitythat intact remaining PS

holoproteins also playarolein,BAPPprocessing.The site of action of CTF is likely located up-stream to a- and 3/,y-secretases cell

compart-ments since both Af3 and APPa secretions are

similarly affected. This is in agreement with the

fact that PS and their fragments have been

mainly identified in the endoplasmic reticulum(7-1 1). It isinteresting toemphasize that sucha

compartmenthas been showntobe accessibleto

the proteasome, therefore reinforcing the

likeli-hood of involvement of the proteasome in themodulation off3APP maturationthroughcontrolof the intracellularconcentration ofCTF-PS1 andCTF-PS2.

Acknowledgments

We thank Dr. D. Schenk (Athena

Neuro-science, San Francisco, CA) for providing us

with 1OD5C antibodies. We sincerely thank

Dr. G. Thinakaran (Johns Hopkins University,Baltimore, MD) for supplying us with the

PS2 cDNA and aPS2Loop antibody. We aregrateful to Dr. K. Beyreuther (Heidelberg, Ger-many) for providing us with W02. We thank

Drs. B. Greenberg and M. Savage for their kindsupply of the 207 antibody. We thank J.Kervella for secretarial assistance. A.d.C. is re-cipient of a grant from the

Fundaqao

deAmparo a Pesquisa do Estado de Sao Paulo(FAPESP, Brazil). This work was supported bythe Centre National de la Recherche

Scienti-fiqueand theInstitutNational de la Sante et dela Recherche Medicale.

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