Proc. Natl. Acad. Sci. USAVol. 90, pp. 9195-9198, October 1993Neurobiology

Protein phosphorylation inhibits production of Alzheimer amyloid,B/A4 peptideJOSEPH D. BUXBAUM*t, EDWARD H. Koot, AND PAUL GREENGARD**Laboratory of Molecular and Cellular Neuroscience, The Rockefelier University, New York, NY 10021; and *Department of Pathology, Brigham andWomen's Hospital and Harvard Medical School, Boston, MA 02115

Contributed by Paul Greengard, June 30, 1993

ABSTRACT The major component of amyloid plaquecores and cerebrovascular amylold deposits found in Alzheimerdisease Is the P/A4 peptide, which is derived from the AIz-heimer amylold protein precursor (APP). Recent evidencesugests that abnormalities in f/A4 peptide production or,B/A4 peptide agregation may underlie cerebral amyloidosis.In the present study, treatment of cells with phorbol dibu-tyrate, which activates protein kinase C, and/or okadaic acid,which inhibits protein phosphatases 1 and 2A, reduced P/A4peptide production by 50-80%. These effects were observedwith APP6. and APP75s expressed in stablY btrasfected CHOcells, as well as with endogenous APP in human glioma (Hs 683)cells. Phorbol dibutyrate also decreased P/A4 peptide pro-duction in cells expressing various mutant forms of APPassociated with familial Alzheimer disease, one of which wasreported tom est greatly incrased P/A4 peptide produc-tion in cultured cells. Mastoparan and mastoparan X, com-pounds which can activate phospholipase C and hence proteinkhin C, also decreased f/A4 peptide production inCHO cellsstably transfected with APP695. A model is presented in whichdecreases in 3/A4 peptide production can be achieved byaccelerating the metabolism of APP through a nonamyloid-genic secretory pathway.

Alzheimer disease is characterized by distinct neuropatho-logical lesions, including intracellular neurofibrillary tangles,and extracellular parenchymal and cerebrovascular amyloiddeposits. The principal component of parenchymal amyloidplaque cores and cerebrovascular amyloid is the P3/A4 pep-tide, which is derived from cleavage of a large transmem-brane protein, the Alzheimer amyloid protein precursor(APP) (1-7). Several families with early-onset, autosomaldominant forms of Alzheimer disease have mutations in APP(8-13) which cosegregate with the disease, implicating ab-normalities in APP metabolism or ,B/A4 peptide metabolismin the symptomatology of Alzheimer disease. It is thereforeof considerable importance to identify ways of reducing theproduction of ,B/A4 peptide. Agents that lead to increasedphosphorylation, including phorbol esters (which activateprotein kinase C) and okadaic acid (which inhibits proteinphosphatases 1 and 2A), accelerate processing and secretionof APP (14-16). In the present study we have examined theeffects of these compounds, and of toxins known to activatephospholipase C, on the production of,/A4 peptide.

MATERIALS AND METHODSCell culture conditions and the sources of analytical reagentshave been described (14-17). Monoclonal antibodies 4G8 (18)and 6E10 (37) and polyclonal antibodies SP40 (19), SGY1234(20), and R1280 (21) have been described.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Pulse-chase labeling of cells was carried out on confluentcell monolayers in six-well culture dishes (Corning) with 1 mlof methionine-free Dulbecco's modified Eagle's medium(DMEM) supplemented with 1 mCi (37 MBq) of [35S]methio-nine (EXPRE35S35S, NEN). Metabolic labeling was carriedout for 2 hr, followed by a chase period of2 hr. The chase wasinitiated by replacing the labeling medium with DMEMcontaining excess unlabeled methionine. Two minutes afterthe start of the chase, the indicated test compounds wereadded. This protocol maximized the probability that anyobserved effects were attributable to changes in APP metab-olism rather than APP transcription. After incubation, con-ditioned medium was centrifuged for 5 min at 10,000 x g toremove cellular debris. 3/A4 peptide and p3 were immuno-precipitated with an anti-p/A4 antibody (4G8 except whereindicated), and secreted APP (APPs) was immunoprecipi-tated with an anti-APP NH2-terminal antibody (22C11). Im-munoprecipitated APP fragments were subjected to SDS/PAGE in either 10-20% gels with Tris/tricine buffer (forf/A4 peptide and p3) or 6% gels with Tris/glycine buffer (forAPPs), autoradiographed, and quantified with a Phosphor-Imager (Molecular Dynamics). Analysis of variance followedby Fisher's post-hoc analysis was used to determine thesignificance of observed differences.

RESULTSWhen the medium of metabolically labeled CHO cells stablytransfected with cDNA encoding the 695-amino-acid isoformofAPP (APP695) was subjected to immunoprecipitation withan antibody raised against a synthetic peptide correspondingto ,/A4 peptide, two small peptide bands of apparent mo-lecular mass 3 and 2 kDa were observed (Fig. 1). Thesepeptides had migration rates similar to those of two peptidesidentified previously as f/A4 peptide and p3 (20-23). p3 wasshown to be a //A4 peptide fragment deleted of the first16-17 amino acids (23). On the basis of the similarities inmigration rate and immunochemical properties shown inFigs. 1 and 2, we conclude that the two small peptide bandsof 3 and 2 kDa represent f/A4 peptide and p3, respectively.The levels of 3/A4 peptide decreased dramatically when

the cells were incubated with either PBt2, okadaic acid, or thetwo compounds together. The decreased recovery of ,B/A4peptide in the presence of PBt2 and/or okadaic acid wasobserved with each of several antibodies raised against (/A4peptide sequences, including the monoclonal antibodies 4G8(Fig. 1) and 6E10 (data not shown), as well as the polyclonalantibodies R1280 (Fig. 2), SP40 (Fig. 2), and SGY2134 (datanot shown). Under the same conditions, the levels ofp3 roseby 30-50% (Figs. 1 and 2); quantitation of p3 was difficultbecause it was poorly resolved from a nonspecific band (Fig.2).

Abbreviations: APP, amyloid protein precursor; APPs, secretedAPP; PBt2, phorbol 12,13-dibutyrate.tTo whom reprint requests should be addressed.

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/3/A4 peptide - -

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FIG. 1. Production of 8/A4 peptide is regulated by proteinphosphorylation. 8/A4 peptide was immunoprecipitated with anti-body 4G8 from the medium ofmetabolically labeled CHO cells stablyexpressing APP695. (Upper) Representative autoradiogram. (Lower)Quantitation of ,/A4 peptide. Control, no additions; PBt2, 1 uMphorbol 12,13-dibutyrate; OKA, 2.5 pM okadaic acid. Results are themeans ± SEM of three to six experiments performed in duplicate ortriplicate. *, Different from control (P < 0.0001); **, different fromcontrol (P < 0.0001), from PBt2 alone (P < 0.0005), and from OKAalone (P < 0.0005).

It has recently been demonstrated that chloroquine andammonium chloride, two agents that inhibit acid-dependenthydrolases by neutralizing acidic organelles, decrease thelevels of /A4 peptide recovered from conditioned mediumof cultured cells (20, 23). Incubation of labeled cells for 2 hrin the presence ofchloroquine (50 ug/ml) alone decreased thelevels of ,B/A4 peptide recovered in cell culture supematants(Fig. 3), consistent with those previous reports (20, 23).Incubating cells with chloroquine together with either PBt2 orokadaic acid led to decreases in f/A4 peptide levels whichwere significantly greater than the decreases observed in thepresence of chloroquine alone (Fig. 3) or of PBt2 or okadaicacid alone (data not shown).When cells were incubated in the absence or presence of

PBt2, okadaic acid, or chloroquine, alone or in combination,

B/A4peptide- 4p3 -

1 2 3 4 5 6

FIG. 2. Identification of the 3- and 2-kDa peptide bands as ,B/A4peptide and p3, respectively. P/A4 peptide and p3 were immunopre-cipitated from the medium of metabolically labeled CHO cells stablyexpressing APP695. Lanes: 1, 125I-labeled ,/A4-(1-40); 2, immuno-precipitation with R1280 of medium from untreated cells; 3, immu-noprecipitation with R1280 of medium from cells treated with 1 pMPBt2 plus 2.5 .M okadaic acid; 4, immunoprecipitation with R1280, inthe presence of synthetic ,B/A4-(1-40) at 10 JAg/ml, of medium fromuntreated cells; 5, immunoprecipitation with SP40 of medium fromunbrated cells; 6, immunoprecipitation with SP40 ofmedium from cellstreated with 1 pM PBt2 plus 2.5 pM okadaic acid. (3/A4 peptidecomigrted with radioiodinated synthetic ./A4-(1--40) (compare lanes1 and 2). Several antibodies were able to immunoprecipitate .8/A4peptide, including R1280 (lanes 2 and 3), two other anti-fi/A4-(140)antibodies (SP40, lanes 5 and 6, and SGY2134, data not shown), and amonoclonal antibody raised against P/A4-(1-27) (4G8) (Fig. 1). p3 couldbe immunoprecipitated with R1280 (lanes 2 and 3), as well as with 4G8(Fig. 1). The monoclonal antibody 6E10, which recognizes an epitopearound amino acid 11 of the P/A4 peptide (37), immunoprecipitated,B/A4 peptide but not p3 (data not shown). Neither (3/A4 peptide norp3was recovered when immunoprecipitation was carried out in thepresence of /A4-(140) (lane 4). A minor band migratingjust above p3in R1280 immunoprecipitates proved to be nonspecific (compare lanes2 and 3 with lane 4).

C C00au+<0 O ED 0O

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FIG. 3. Production of P/A4 peptide is regulated by proteinphosphorylation in the presence of chloroquine. 8/A4 peptide wasimmunoprecipitated from the medium of metabolically labeled CHOcells stably expressing APP695. (Upper) Representative autoradio-gram. (Lower) Quantitation of ,B/A4 peptide. Control, no additions;CQ, chloroquine (50 Hg/ml); PBt2, 1 ,uM PBt2; OKA, 2.5 .tM okadaicacid. Results are the means ± SEM of three or four experimentsperformed in duplicate or triplicate. *, Different from control (P <0.0001); **, different from control (P < 0.0001), and from CQ alone(p < 0.05).

and the cell lysates were examined for P/A4 peptide, nonewas detected (data not shown). These results indicate that, inthese cells, 8/A4 peptide either is formed extracellularly oris secreted very efficiently upon its intracellular formation. Ineither case the data indicate that protein phosphorylationregulates .3/A4 peptide production, rather than secretion.We have also characterized the effects ofPBt2 and okadaic

acid on ,B/A4 peptide production from endogenous APP in ahuman glioma cell line, Hs 683. Incubation of Hs 683 cellswith PBt2 or okadaic acid decreased the production of 13/A4peptide (Table 1) in a manner similar to that observed fortransfected CHO cells, supporting the physiological signifi-cance of the observations with the transfected cells.

It was recently reported (24, 25) that a mutant form ofAPP(APP[K595 -* N; M596 - LI) (13) was associated withincreased 8/A4 peptide production. When CHO cells stablytransfected with cDNA encoding either wild-type APP751 ormutant APP751 (APP751[K595-- N; M596-+ LI) were incubatedwith 1 AM PBt2, there was a significant decrease in the levelsof f3/A4 peptide recovered from the medium (Table 2). Theseresults indicate that protein phosphorylation regulates P/A4peptide production from APP751 as well as from APP695 andthat even the elevated level of ,B/A4 peptide productionreportedly associated with APP[K595-- N; M596- L] (24, 25)may be sensitive to regulation by protein phosphorylation.PDBu also inhibited B/A4 peptide production from cellsexpressing any of three other mutations in APP751(APP751[E618- Q], APP751[V642- I], and APP751[V'2-- PI),Table 1. Production of 8/A4 peptide is regulated by proteinphosphorylation in Hs 683 cells

,8/A4 peptide,Treatment relative amount

Control 1.00 ± 0.029PBt2 (1 AM) 0.50 ± 0.083*Okadaic acid (2.5 ,tM) 0.55 ± 0.052*

,B/A4 peptide was immunoprecipitated from the medium of met-abolically labeled Hs 683 cells. Results are the means ± SEM ofthreeexperiments performed in duplicate or triplicate. *, Different fromcontrol (P < 0.05).

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Table 2. Production of f3/A4 peptide is regulated by proteinphosphorylation in CHO cells stably expressing wild-typeor mutant APP751

,3/A4 peptide, relative amount

APP7s1 Control PBt2

Wild type 1.00 ± 0.075 0.53 ± 0.033*K595 - N; M596 - L 1.00 ± 0.046 0.37 ± 0.079*E618 Q 1.00 ± 0.086 0.32 ± 0.073*V642 I 1.00 ± 0.143 0.35 ± 0.051*V642 p 1.00 ± 0.061 0.30 ± 0.070*

,B/A4 peptide was immunoprecipitated from the medium of met-abolically labeled CHO cells stably expressing either wild-type ormutant APP751. Control, no additions; PBt2 was added at 1 ,uM.Results are the means ± SEM of three experiments performed intriplicate. *, Different from control (P < 0.001).

each of which is associated clinically with abnormal cerebral,8/A4 peptide deposition (8-11, 26, 27) (Table 2).The effects of PBt2 and okadaic acid on the production of

APPs, the secreted form of APP (28, 29), were opposite tothose observed for ,/A4 peptide. For example, when CHOcells, stably expressing APP695 were treated with PBt2,okadaic acid, or both compounds together, production ofAPPs increased significantly (Fig. 4) while P/A4 peptideproduction decreased (Figs. 1 and 2). A similar stimulatoryeffect of PBt2 and okadaic acid on APPs production was seenin previous studies of other cell types (15, 16). Under thesame conditions, the levels of p3 increased in the medium(Figs. 1 and 2), consistent with the idea that p3, like APPs, isderived from a secretory pathway.The results obtained with PBt2 and okadaic acid indicate

that compounds which stimulate APPs formation could pro-vide a way to inhibit ,B/A4 peptide production (see below).Experiments with mastoparan and mastoparan X, toxinswhich activate the phospholipase C/protein kinase C cas-cade, apparently through activation of a G protein, furthersupport this idea. Thus, when CHO cells stably expressingAPP695 were incubated with mastoparan or mastoparan X,,B/A4 peptide production decreased, with a concomitantincrease in APPS production (Fig. 5).

APPs

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FIG. 4. Production of APPs is regulated by protein phosphory-lation. APPs was immunoprecipitated from the medium of metabol-ically labeled CHO cells stably expressing APP69s. (Upper) Repre-sentative autoradiogram. (Lower) Quantitation of APPS. Control, no

additions; PBt2, 1 AM PBt2; OKA, 2.5 AM okadaic acid. Results are

the means + SEM of three experiments performed in duplicate or

triplicate. *, Different from control (P < 0.005).

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FIG. 5. Production of f3/A4 peptide is regulated by mastoparanand mastoparan X. 13/A4 peptide and APPs were immunoprecipi-tated from the medium of metabolically labeled CHO cells stablyexpressing APP695. (Left) Quantitation of 8/A4 peptide. (Right)Quantitation of APPs. Control, no additions; mastoparan and mas-toparan X were added at 50 1.M. Results are the means ± SEM ofthree to four experiments performed in duplicate or triplicate. *,Different from control (P < 0.005).

DISCUSSIONThe various results presented here suggest that compoundswhich, directly or indirectly, increase protein kinase C ac-tivity or decrease protein phosphatase 1 or 2A activity mightconceivably prove useful in efforts to slow the developmentof Alzheimer disease. Examples of such compounds includea variety of neurotransmitters and hormones known to actthrough the phospholipase C/protein kinase C cascade. Mus-carinic cholinergic agonists and interleukin 1, representativesof this class of substances, have, in fact, been shown toincrease APPs production (16, 30).APP is processed by at least two pathways: (a) a nonamy-

loidogenic a-secretory (31) pathway, in which the extracel-lular portion ofAPP (APPs) is released into the extracellularspace (28, 29, 32, 33), and (b) an alternative pathway whichgenerates ,B/A4 peptide (Fig. 6). The reciprocal effects ofPBt2, okadaic acid, mastoparan, and mastoparan X on pro-duction of APPs and ,B/A4 peptide are consistent with amodel in which APPs and (3/A4 peptide are derived fromcompeting pathways ofAPP metabolism. Thus, according tothis model, the amount of APP available for processingrepresents the limiting step in the formation of f3/A4 peptide.The ability of phorbol esters, okadaic acid, mastoparan, andmastoparan X to reduce the formation of ,B/A4 peptide maytherefore be attributable to an ability of phosphorylation to

protein phosphorylationstimulates

\ APPS

Mature APP

1,.I%R/A4 peptide

FIG. 6. Schematic diagram ofAPP processing. Scheme illustratestwo known pathways, the a-secretory (nonamyloidogenic) pathwayand an alternative (amyloidogenic) pathway, for metabolic process-ing of APP. It is hypothesized that increased protein phosphoryla-tion, by accelerating the removal of mature APP via the a-secretorypathway, diminishes the amount of substrate available for thealternative pathway which generates ,B/A4 peptide.

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divert APP from the alternative pathway to the a-secretorypathway.Lysosomotropic agents, including chloroquine and ammo-

nium chloride, can inhibit 83/A4 peptide production (20, 23).It is possible that the alternative pathway leading to theformation of 8/A4 peptide involves acidic intracellular com-partments (34-36). The fact that PBt2 (or okadaic acid) pluschloroquine gave a larger inhibition of ,/A4 peptide produc-tion than either compound alone is consistent with thehypothesis that these compounds act at different sites off3/A4 peptide production.The molecular mechanism by which protein phosphoryla-

tion diverts APP metabolism from the amyloidogenic to thenonamyloidogenic pathway remains to be determined. Mu-tation ofthe putative phosphorylation sites in the cytoplasmicdomain of APP to nonphosphorylatable residues had noeffect on the stimulation of the secretion of APPs by proteinphosphorylation (0. B. da Cruz e Silva, K. Iverfeldt, T.Oltersdorf, S. Sinha, T. V. Ramabhadran, T. Suzuki, S.Sisodia, S. Gandy, and P.G., unpublished work). These dataimplicate one or more components of the APP processingpathway as a target for the stimulation of secretion by proteinphosphorylation.

Note Added in Proof. Another group has recently reported (38) dataimplicating protein kinase C in the regulation of f8/A4 peptideproduction.

We thank Dr. S. S. Sisodia for providing the CHO cells transfectedwith APP695, Drs. K. S. Kim and H. M. Wisniewski for providingascites 6E10 and 4G8, Dr. D. Selkoe for providing antiserum R1280,Drs. J. Ghiso and B. Frangione for providing antiserum SP40, Drs.S. Estus and S. Younkin for providing antiserum SGY1234, Drs. T.Oltersdorf and M. Citron for providing APP cDNAs, and Dr. M.Citron for providing 1251-labeled synthetic f3/A4 peptide used as astandard. This work was supported by United States Public HealthService Grants AG07911 (to E.H.K.), AG09464 (to P.G.), andAG10491 (to P.G.). J.D.B. is the recipient of Alzheimer's Associa-tion Faculty Scholar Award, and E.H.K. is the recipient of Alzhei-mer's Association/George F. Berlinger Memorial Faculty ScholarAward.

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