Proc. Nati. Acad. Sci. USAVol. 91, pp. 4766-4770, May 1994Neurobiology

Advanced glycation end products contribute to amyloidosis inAlzheimer disease

(I-amyloid peptide/aggregation/nucleation-dependent kinetics/seed structure and function)

MICHAEL P. VITEK*t, KESHAB BHATTACHARYA*, J. MICHAEL GLENDENING*, EDWARD STOPAt,HELEN VLASSARA*, RICHARD BUCALA*, KIRK MANOGUE*, AND ANTHONY CERAMI**The Picower Institute for Medical Research, Manhasset, NY 11030; and *Department of Pathology, Division of Neuropathology, Brown University School ofMedicine/Rhode Island Hospital, Providence, RI 02903

Contributed by Anthony Cerami, January 7, 1994

ABSTRACT zheimer disease (AD) is characterized bydeposits of an aggregated 42-amino-acid P-amyloid peptide(I3AP) in the brain and cerebrovasculature. After a concen-tration-dependent lag period during in vitro incubations, sol-uble preparations of synthetic .8AP slowly form fibrillar ag-gregates that resemble natural amyloid and are measurable bysedimentation and thioflavin T-based fluorescence. Aggrega-tion of soluble flAP in these in vitro assays is enhanced byaddition ofsmall amounts ofpre-aggregated gamyloid "seed"material. We also have prepared these seeds by using anaturally occurring reaction between glucose and proteinamino groups resulting in the formation of advanced "glyco-sylation" end products (AGEs) which chemically crosslinkproteins. AGE-modified flAP-nucleation seeds further accel-erated aggregation of soluble flAP compared to non-modiflied"seed" material. Over time, nonenzymatic advanced glycationalso results in the gradual accumulation of a set of posttrans-lational covalent adducts on long-lived proteins in vivo. In astandardized competitive ELISA, plaque fractions of ADbrains were found to contain about 3-fold more AGE adductsper mg ofprotein than preparations from healthy, age-matchedcontrols. These results suggest that the in vivo half-life offi-amyloid is prolonged in AD, resulting in greater accumula-tion of AGE modifications which in turn may act to promoteaccumulation of additional amyloid.

Alzheimer disease (AD) is the major cause of dementia andthe fourth leading cause of death in the United States,affecting 10%o of the population age 65 and older. In combi-nation with gradual cognitive decline, AD is characterized bythe progressive accumulation of neurofibrillary tangles, amy-loid plaques, and cerebrovascular amyloid deposits in thebrain. Typically, 5- to 10-fold greater numbers of amyloidplaques accumulate in the brains of AD patients than inage-matched healthy controls. These amyloid plaques mainlycomprise aggregated copies of a 42-amino-acid (-amyloidpeptide (PAP; ref. 1), which derives by cleavage from afamily of larger amyloid peptide precursor proteins (2, 3).Although the amount of aggregated PAP or 3-amyloid maydirectly relate to the increased release of soluble 8lAP in somerare genetically linked forms of familial AD (4-6), the levelsof soluble SlAP in cerebrospinal fluid of sporadic AD casesare similar to those in age-matched healthy counterparts (7,8). Ifincreased levels of soluble PAP monomers generally failto account for the increased numbers ofamyloid plaques seenin the majority ofAD patients, then the increased amyloid inAD must instead reflect an increase in the rate of aggregationand amyloid formation, a decreased rate of amyloid degra-dation, or some combination of both.

To better understand the factors which contribute to amy-loid formation and stability, we have studied the in vitroaggregation of soluble synthetically prepared PAP mono-mers, a process which displays nucleation-dependent kinet-ics, especially at physiological (nanomolar) concentrations ofmonomer (refs. 9 and 10; this report). Although millimolarconcentrations of (3AP exhibit extensive aggregation withinminutes, micromolar and lower concentrations of monomerdisplay a concentration-dependent lag period during whichlittle or no measurable aggregate is formed, followed by a"growth" phase of more rapid aggregation (9-11). This lagperiod can be eliminated by adding trace amounts of pre-formed aggregate as "seed" material which serves to imme-diately induce aggregation (9). Seeding apparently eliminatesthe need for de novo formation of aggregation nuclei, a muchslower process than the subsequent growth of nucleatedaggregates. Thus, amyloid seeds can be seen to representcritical protein masses with special structural features capa-ble of nucleating the formation of larger insoluble aggregatesof amyloid from pools of soluble P3AP. The nature of thesestructural characteristics is unknown, but the net result ofseeding is significant acceleration in the rate of aggregationcompared with unseeded incubations of soluble PAP. Byextrapolation from this simple in vitro model to the similarnanomolar concentrations of soluble fAP found in cerebro-spinal fluid in vivo, it might be expected that the depositionand accumulation of PAP as amyloid plaques would reflect,in part, the amount or availability of seed material able tonucleate aggregation. Some cases of AD might correspond-ingly arise as a consequence of increased availability ofnucleation seeds in the disease-prone brain relative to normalcounterparts not destined to suffer AD at a similar chrono-logical age.

In the present communication, we explore the possibilitythat the formation or stability of amyloid structures whichseed PAP aggregation may be enhanced by covalentcrosslinks that chemically polymerize components of theaggregate. Under physiological conditions, long-lived pro-teins become markedly modified and extensively crosslinkedby advanced "glycosylation" end products (AGEs). AGEsresult from the spontaneous, nonenzymatic glycation reac-tions between proteins and reducing sugars (12), including forinstance the ubiquitous monosaccharide glucose, which is theprimary energy source used by the brain. Initially in theprocess of advanced glycation, condensation reactions be-tween reducing sugars and protein amino groups form apopulation of readily reversible Schiff bases, some of whichrearrange over days into more stable Amadori products. Over

Abbreviations: AD, Alzheimer disease; MAP, (-amyloid peptide;AGE, advanced "glycosylation" end products.tTo whom reprint requests should be addressed at: The PicowerInstitute for Medical Research, 350 Community Drive, Manhasset,NY 11030.

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Proc. Natl. Acad. Sci. USA 91 (1994) 4767

weeks and months, these early products chemically evolveby additional rearrangement, dehydration, cleavage, andaddition reactions into a widely heterogeneous set of cova-lently bound adducts known collectively as AGEs. Theaccumulation of such AGEs in vivo mainly depends on thebioavailability of susceptible primary amino groups, theambient concentration of reducing sugars, and the half-life ofthe modified protein. Accordingly, AGE modifications arestrikingly evident on very long-lived proteins such as lenscrystallins, nerve myelin, and matrix collagen; are markedlyenhanced in diabetic patients (13-15); and might well beexpected to accumulate on the aggregated proteins ofAD andother amyloidoses.By accumulating slowly over time, the AGE modifications

that appear on proteins in vivo not only serve as an index ofprotein half-life but also can initiate a variety of biologicaleffects mediated both by cellular receptor systems for AGE-modified ligands (16) and by processes which depend on theinherent chemical reactivity of both nascent and late AGEmoieties (17). As a first step in understanding the potentialconsequences ofAGE accumulation on f3-amyloid in AD, weinvestigated the effects of advanced glycation on the aggre-gation of P3AP monomers in vitro. Our data indicate thatadvanced glycation ofH3AP accelerates aggregation of solublePAP and that small amounts of preformed AGE-modified(3AP seeds more efficiently nucleate the further merging of(3AP peptides into insoluble aggregates than does nongly-cated seed material. We also report that purified plaquefractions from AD brains contain more AGE adducts per mgof protein than do parallel fractions prepared from healthyage-matched controls, further underlining the potential phys-iological and pathogenic importance of AGE-regulated SAPaggregation and amyloid accumulation.

MATERIALS AND METHODSAggregation and Seeding Reactions. Synthetic, HPLC-

purified peptides representing the first 28 [(3AP-(1-28)] andthe first 40 [38AP-(1-40)] amino acids of the 42-amino-acid,3AP were obtained from Bachem. Aggregation of soluble(3AP-(1-28) or 3AP-(1-40) at different concentrations wasinitiated by addition of 0.1 M sodium acetate and continuedfor the indicated times. Quantitative aggregate formation athigh micromolar PAP concentrations was detected by theprocedure of LeVine (18). Briefly, fluorescence of aggregatesadded to 10 ,uM thioflavin T (Aldrich) in 50 mM potassiumphosphate buffer at pH 6.0 was measured upon excitation at450 ± 5 nm, with emission detected at 482 ± 10 nm on aPerkin-Elmer LS-50B spectrofluorimeter. Where indicated,small amounts of preformed aggregates ("nucleation seeds")were added to the monomeric PAP and 0.1 M sodium acetate.Lysines at positions 16 and 28 of 8AP contain primary aminogroups which may react with reducing sugars to generateAGEs. Monomeric I3AP-(1-40) (250 uM) and 0.2 M sodiumphosphate buffer (pH 7.5) were incubated with or without 1M glucose at 37TC for 4 months to generate preformedaggregates of glycation-modified PAP, referred to as "AGE-P3AP seed" or "PAP seed," respectively. After incubation,protein concentrations of seed preparations were measuredand adjusted with buffer to 150 uM final concentration. Bycompetitive ELISA (19), AGE-I3AP seed contained 50 AGEunits/mg of protein and HAP seed contained <0.5 AGEunit/mg of protein. Indicated amounts of glucose and/oraminoguanidine, a potent inhibitor of advanced glycation andcrosslink formation (20), were also added to solutions of 3APbefore sodium acetate in some experiments.

Aggregation of physiological concentrations of SAP wasquantitated by the method of Burdick et aL (11). Syntheticpreparations of IAP-(1-40) were labeled with 125I (NEN) andchloramine-T (Sigma) for 1 min before the reaction was

quenched with 10mM tyrosine/50 mM sodium metabisulfite.Unincorporated label was removed by filtration through aSephadex G-10 column equilibrated with 0.5x phosphatebuffered saline (PBS) at pH 7.4. The 125I-labeled 3AP (spe-cific activity, -3 x 106 cpm/pg) was immediately diluted to5 nM final concentration in the presence of various seeds,glucose, and/or aminoguanidine at the indicated concentra-tions. After various incubation periods at 370C, aggregationreaction mixtures were underlaid with 20%6 sucrose/0.1 Msodium acetate at the same pH as the incubation, centrifugedfor 30 min at 50,000 x g, and frozen in liquid nitrogen. Eachmicrocentrifuge tube was cut and the bottom 5-mm section,representing the aggregated, sedimentable fraction which hadpelleted through the sucrose cushion, was analyzed forradioactivity in a 'y counter. The remainder of the tube andliquid were also analyzed. The amount of aggregate formedwas calculated as a percentage: (pellet cpm/total cpm) X 100.Measurement of AGEs by Competitive ELISA. Aliquots of

frozen samples of prefrontal cortex (Broadman areas 9 and10) from patients with or without behaviorally and neuro-pathologically confirmed AD were suspended in 10 volumes(per wet weight) of 2% sodium dodecyl sulfate/0.1 M 2-mer-captoethanol and Dounce homogenized. The homogenateswere boiled for 10 min and then centrifuged at 10,000 X g for10 min. Supernatants were aspirated and the resulting pelletswere washed and recovered three times with PBS at 10,000x g for 10 min. In some experiments, this crude plaquefraction was further washed twice with 4 M urea and twicemore with PBS before protease digestion. PBS-washed pel-lets were resuspended in 1/10th the original homogenatevolume of PBS with 0.1% proteinase K (Boehringer Mann-heim) and digested overnight at 37rC. Quadruplicate 5-, 10-,and 20-I1 aliquots of the digested plaque-containing pelletfractions were assayed for AGE content by competitiveELISA (19) against standardized preparations of AGE-modified bovine serum albumin (AGE-BSA). Only values inthe linear range of the standard curve were included in theanalyses. Protein amounts were quantified with a micro-BCAkit (Pierce) and with fluorescamine (21). AGE units pervolume of sample were interpolated from a standard dilutioncurve of AGE-BSA and divided by the sample proteinconcentration to give AGE units per mg of protein. Statisticalanalysis using Student's t test was performed with theSTATWORKS program for MacIntosh.

RESULTSPAP Aggregation Displays Nucleation-Dependent Kinetics.

Aggregation of synthetic 3AP was analyzed in vitro, wherethe aggregation rate of soluble PAP was found to dependmainly upon pH and the initial concentration of monomer.We empirically determined kinetics of aggregation and foundthat at 600 pkM, B3AP-(1-28) spontaneously and rapidlyformed thioflavin T fluorescent aggregates within minutes atpH 7.2 (Fig. 1). At concentrations below 300 uM, however,,SAP aggregated very slowly with a considerable lag beforefluorescent aggregates were measurable.At low concentrations of I3AP, the lag period preceding

measurable aggregation is reminiscent of crystallization re-actions where solutions of protein very slowly assume asingle conformation and then aggregate in a well-defined,molecular packing arrangement (9, 10, 22). The kinetics ofsuch aggregation in many cases can be significantly acceler-ated by the addition of a preformed aggregate or "seed"material as nucleation centers for aggregate formation. Weprepared stable SAP seed material by incubating 250 pM(3AP-(1-40) for 4 months at 370C. Compared with the smallamounts of fluorescent aggregate measured when 300 pAMP3AP monomer or 75 juM PAP seed was separately incubatedin control preparations, incubation of monomer plus PAP

Neurobiology: Vitek et A

Proc. Natl. Acad. Sci. USA 91 (1994)

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0 50-0

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40 50

FIG. 1. Fluorescent aggregates formed with synthetic PAP de-pend on initial concentration of monomer and time. (3AP wasincubated with 0.1 M sodium acetate at pH 7.2, aliquots wereremoved at various times, and fluorescence in the presence ofthioflavin T was measured. The mean (± SE) of three measurementsis plotted for 600 AM (>), 300 p.M (o), and 150 p.M (n) BAP.

seed resulted in the accumulation of much larger amounts offluorescent aggregates (Fig. 2). We also prepared AGE-modified PAP seed by incubating PAP monomer with 1 Mglucose in pH 7.5 phosphate buffer at 37TC for 4 months(AGE-P3AP seed). AGE formation was confirmed by com-petitive ELISA, where SAP seed contained <0.5 AGEunit/mg of protein and AGE-pAP seed contained 50 AGEunits/mg of protein, which is comparable, within an order ofmagnitude, to the AGE content of plaques from AD brains.When 75 FLM AGE-(AP seed was incubated with 300 A.MPAP monomer, much more fluorescent aggregate was de-tected than in parallel incubations with unmodified PAP seedand monomer (Fig. 2). As with PAP seed, separate incubationof AGE-pAP seed alone did not lead to a change in the smallamount of fluorescent aggregate with time.To test whether this seeding phenomenon occurred at

concentrations of PAP typically found in vivo, a sedimenta-tion assay was employed to measure aggregation. Aggrega-

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FIG. 2. Fluorescent-aggregate formation displays nucleation-dependent kinetics. Stable aggregates of ,AP were formed in thepresence or absence of glucose to generate AGE-PAP seed and 3APseed. Seeds were added to 300 puM NAP monomer and incubated forvarious times. The mean (+ SE) of triplicate thioflavin T fluores-cence measurements is plotted as a function of time for AGE-PAPseed plus PAP (o) and 3AP seed plus SAP (>). Control incubationsincluded AGE-fAP seed alone (A), PAP seed alone (r), and PAPmonomer without seeding (+).

tion of 10 nM 125I_3AP-(1-40) monomer in 0.1 M sodiumacetate at pH 7.0 increased slowly over a 2-day incubation(Fig. 3). When 10 nM labeled monomer was incubated with200 nM unlabeled SAP seed material, the amount of sedi-mentable label was similar to the no-seed, monomer-onlycurve. In contrast, incubation of 10 nM monomer with 200nM AGE-,SAP seed increased the amounts of sedimentablelabel compared with the pAP-seed and no-seed experimentalpoints. Thus, the amount of labeled SAP associated withsedimentable aggregates observed in incubations of PAPmonomer with AGE-pAP seed was greater than that seen inincubations with PAP seed or no seed under conditions inwhich pH and soluble 8AP concentrations were physiologi-cal.

Glucose Modifies Kinetics of Aggregation. In the process ofadvanced glycation, formation of a Schiff base betweenglucose and a protein amino group precedes subsequentmaturation of Amadori products into AGE-modified PAP.Aminoguanidine is a compound which prevents AGE forma-tion following initial glucose reactions with protein amines(20). Incubation of 400 uM pAP-(1-28) in 0.1 M sodiumacetate (pH 7.0) with 100 mM glucose stimulated fluorescent-aggregate formation when compared with parallel incuba-tions of monomer, monomer plus aminoguanidine, or mono-mer plus aminoguanidine plus glucose (Fig. 4). Separateincubations of glucose or aminoguanidine failed to generateany detectable fluorescent signals.AGE Content of Brain Fractions. On average, plaque-

enriched fractions isolated from samples of frontal cortex of10 AD brains contained significantly more AGE modifica-tions than did corresponding preparations from 7 healthycontrols (8.9 ± 1.4 versus 2.7 ± 0.5 AGE units/mg ofprotein,P = 0.002; see Fig. 5). The average chronological age ofboththe AD and control groups was 77 years. The presence ofAGEs in similar fractions of parietal cortex from AD andnormal brains has also been observed (data not shown).Supernatant fractions of sodium dodecyl sulfate-soluble pro-teins from AD and control brains routinely contained <0.1AGE unit/mg of protein.

DISCUSSIONAlthough advanced glycation adducts form spontaneously invivo, their accumulation is slow and becomes most notablewith increasing time on long-lived tissue components. Alongwith time and the availability of susceptible protein amino

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FIG. 3. AGE-P3AP seeds nucleate more aggregation at physio-logical concentrations of fAP than fAP seeds. 125I-.lAP-(1-40) (10nM) was mixed with no seed (o), PAP seed (>) or AGE-PAP seed(o) and incubated for the indicated times at 370C. The mean (± SE)of quadruplicate measurements is plotted.

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4768 Neurobiology: Vitek et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 4769

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FIG. 4. Glucose modifies the kinetics of fluorescent-aggregateformation. Soluble SAP-(1-28) (400 pM) with 0.1 M sodium acetatein 0.1 M phosphate buffer at pH 7.0 (o) was mixed with 0.1 M glucose(>), 0.05 M aminoguanidine (A), or glucose and aminoguanidine (o).The mean (± SE) of triplicate thioflavin T fluorescence measure-ments is plotted.

groups, ambient glucose is the other major determinant ofAGE formation. Thus, quantitative analysis of the degree ofAGE modification of a single protein species under normo-glycemic conditions yields an index of protein half-life invivo. We found that plaque-enriched fractions isolated fromAD brain samples contained about 3-fold more AGE modi-fications than did comparable fractions prepared from age-matched control brains, suggesting that PAP half-life isprolonged in AD. That amyloid components exhibit a pro-longed half-life in AD is also supported by studies thatdemonstrated the time-dependent, nonenzymatic isomeriza-tion of aspartic residues occurring at positions 1 and 7 ofPAPisolated from AD brain (23); unfortunately, comparable BAPfractions from normal brain were not tested in parallel. Towhat degree this alteration in 8AP turnover accounts for theaccumulation of fAP as aggregated amyloid in AD remains tobe established. Likewise, it is of interest to determine thenormal mechanism of BlAP removal during turnover of amy-loid components and how this process is slowed in AD.AD is characterized by progressive dementia and increased

numbers of amyloid plaques relative to healthy age-matched

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FIG. 5. Amyloid plaque-enriched fractions of AD frontal cortexcontain more AGE adducts per mg of protein than equivalentlyprepared fractions of age-matched, nondemented controls. Eachcontrol patient is represented by a circle and AD patients bytriangles. Each symbol represents the average of at least fourindependent measurements of immunoreactive AGE adducts foreach sample patient, with the mean of each patient group marked bya cross.

controls. While a causal relationship between increaseddementia and plaque numbers has not been proven, thegradual onset ofsymptoms appears to parallel the progressivedeposition of f3amyloid. From in vitro studies, it is clear thatmillimolar concentrations of monomeric SAP will spontane-ously aggregate into fibrillar amyloid structures by followinga nucleation-dependent mechanism. At lower concentra-tions, the requirement for nucleus formation introduces asubstantial lag period during which a solution of 3AP that stillrequires most of this time to form aggregation nuclei isindistinguishable from one on the verge of rapid aggregationand growth into a "one-dimensional crystal" (10). The effectof this concentration-dependent nucleus formation is ex-treme as illustrated by published calculations showing that amutation of amyloid precursor protein in a Swedish form offamilial AD which raises soluble 3AP concentrations 6-fold(4, 5) should reduce the lag time before aggregate growthoccurs from 100 years to about 3 hr (9). Since the cerebro-spinal fluid concentration of soluble flAP in AD patients is thesame as in age-matched controls (7, 8), the rate of concen-tration-dependent self-formation of nuclei as reflected by theamounts of amyloid formed and deposited might also beexpected to be the same. As this is not the case andAD brainsform substantially more aggregated deposits of PAP thantheir nondiseased counterparts, then one explanation for theincreased amount of amyloid present in afflicted brain tissueis that aggregation is more efficiently nucleated than inhealthy brain parenchyma.Although the physical characteristics of such aggregation

nuclei are presently unknown, we operationally define nu-cleation seeds as structures of SAP possessing a specificConformation that promotes the rapid accretion of additionalsoluble 8AP resulting in the growth of insoluble 8lAP aggre-gates. Since the spontaneous formation of nuclei is thermo-dynamically unfavorable (9), we investigated the possibilitythat processes known to chemically crosslink proteins in vivomight stabilize specific conformations offlAP with nucleatingcharacteristics. Advanced glycation is a naturally occurringprocess ofcovalent posttranslational modification ofproteinsthat readily occurs extracellularly. AGEs in other contextsare well known as protein-protein crosslinking agents in vivo,and AGE accumulation on matrix proteins is associated withincreased resistance to proteolysis (24). At physiologicalconcentration and pH in vitro, fAP monomers aggregateslowly. Of note, the addition of preformed aggregates ofAGE-modified PAP stimulated markedly more rapid aggre-gation of nanomolar solutions of soluble BAP than didpreformed aggregates of unmodified BAP in 2-day incuba-tions. That this acceleration occurred at normal pH, atphysiological concentrations of PAP monomer, and withseeds containing amounts ofAGE modifications comparableto those found in AD plaque fractions suggests that a similarprocess could occur in vivo. Glycation adducts comprise astructurally heterogeneous family of products that slowlyevolve chemically by a variety of rearrangement, condensa-tion, and elimination reactions. The particular AGE speciesthat enhance nucleation remain unknown, but these may berelatively early glycation products, as evidenced by the timecourse over which glucose accelerated the formation offluorescent PAP aggregate compared with aggregation in thepresence of glucose and aminoguanidine, a specific inhibitorof AGE formation. Given that plaque numbers increase inassociation with neuronal degeneration and cognitive declinein AD, and that aggregated but not monomeric SlAP isactively neurotoxic (25, 26), interference with the processesby which AGE formation enhances 8AP aggregation mayprovide new therapeutic opportunities to reduce the patho-physiological changes associated with AD.

We thank Yoonsun Lynda Yi and Victoria LeBlanc for their expert

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Proc. Natl. Acad. Sci. USA 91 (1994)

assistance. This work was generously supported by grants fromAlteon, Inc., and the Alzheimer's Association.

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