NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002 1263

ARTICLES

The amyloid β-peptide (Aβ) has a central role in initiating neurode-generation in Alzheimer disease (AD)1. Transgenic (Tg) CRND8mice, which carry a mutant human Alzheimer precursor protein(APP)695 transgene under the control of the prion protein promoter2,develop an AD-like phenotype characterized by progressive cogni-tive impairment accompanied by the accumulation of Aβ42 peptideand neuritic amyloid plaques in the cerebral cortex3. Immunizationof these and other murine models of AD with Aβ42 significantly re-duces both the density of cerebral amyloid plaques and the degreeof cognitive impairment3–5. However, inflammatory reactions thatdeveloped in a portion of patients after immunization with Aβ42 re-sulted in cancellation of this trial6. In order to identify the immunemechanisms in mice that lead to beneficial effects without inducinga cellular inflammatory response, we have investigated both the hu-moral and cellular immune responses generated by vaccinatingTgCRND8 mice with protofibrillar/oligomeric assemblies of Aβ42.We report that therapeutically effective immunization that reducescerebral Aβ deposits and cognitive impairments in TgCRND8 micecan be achieved by induction of immunoglobulin G2b (IgG2b) sub-class antibodies against residues 4–10 of Aβ. These antibodies caninhibit Aβ fibril assembly and toxicity without activating microglialor other cellular inflammatory responses.

Therapeutically active anti-sera recognize Aβ4–10

TgCRND8 mice and their non-transgenic littermates (n=18 or 16, re-spectively) were immunized with protofibrillar aggregates of either

Aβ42 or an amyloid peptide not expressed in the central nervous sys-tem (islet-associated polypeptide (IAPP); 10 TgCRND8 and 7 non-Tgmice)3. Antibody responses were robust (titers: Aβ42,1:5,000–1:50,000; IAPP, 1:5,000–1:30,000). Western-blot analysesshowed that Aβ42-immune sera specifically recognized Aβ42

monomers, tetramers, hexamers and large oligomers of mass greaterthan 98 kD (data not shown). Western blots also demonstrated thatsera stained mature Αβ plaques, but not diffuse Aβ deposits, in his-tological brain sections from non-immunized TgCRND8 mice. Incontrast, IAPP-immune and non-immune sera were non-reactive inthese assays3.

To identify both the linear sequences and the conformationalepitopes recognized by these therapeutically active antibodies, weused selective proteolytic cleavage of intact, immobilized immunecomplexes followed by high-resolution Fourier-transform ion cy-clotron resonance mass spectrometry (FT-ICR-MS)7–13. Briefly, IgGfrom Aβ42-immunized TgCRND8 mice (n = 6, randomly chosen),were immobilized in microcapillaries, and exposed to Aβ42 aggre-gates, synthetic Aβ42 or isolated amyloid plaques from human ADand TgCRND8 brain tissue. The resultant antigen–antibody com-plexes identified Aβ4–10 (FRHDSGY) as the minimal peptide bound tothe antibody (Fig. 1). As a control, no peptides were detected fromrat Aβ42 (which has a different amino-acid sequence), nor from anti-bodies from IAPP-immunized mice (n = 4). Our results were inde-pendently confirmed by experiments in which proteolyticfragments of Aβ42 were presented to each antibody, and by classical

Therapeutically effective antibodies against amyloid-βpeptide target amyloid-β residues 4–10 and inhibit

cytotoxicity and fibrillogenesis

J. MCLAURIN1,2, R. CECAL3, M.E. KIERSTEAD1,2, X. TIAN3, A.L. PHINNEY1, M. MANEA3, J.E. FRENCH1, M.H.L. LAMBERMON1, A.A. DARABIE1, M.E. BROWN1, C. JANUS1, M.A. CHISHTI1, P. HORNE1, D. WESTAWAY1,2, P.E. FRASER1,4, H.T.J. MOUNT1,5,

M. PRZYBYLSKI3 & P. ST GEORGE-HYSLOP1,5

1Centre for Research in Neurodegenerative Diseases, 2Departments of Laboratory Medicine and Pathobiology, and4Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

3Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, Germany5Department of Medicine, University of Toronto, and University Health Network/Toronto Western Hospital,

Toronto, Ontario, CanadaCorrespondence should be addressed to J.M. (email: j.mclaurin@utoronto.ca) or

M.P. (email: Michael.Przybylski@uni-konstanz.de)

Published online 15 October 2002; doi:10.1038/nm790

Immunization of transgenic mouse models of Alzheimer disease using amyloid-β peptide (Aβ)reduces both the Alzheimer disease–like neuropathology and the spatial memory impairmentsof these mice. However, a therapeutic trial of immunization with Aβ42 in humans was discontin-ued because a few patients developed significant meningo-encephalitic cellular inflammatoryreactions. Here we show that beneficial effects in mice arise from antibodies selectively directedagainst residues 4–10 of Aβ42, and that these antibodies inhibit both Aβ fibrillogenesis and cyto-toxicity without eliciting an inflammatory response. These findings provide the basis for im-proved immunization antigens as well as attempts to design small-molecule mimics asalternative therapies.

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

1264 NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002

ARTICLES

peptide mapping using competitive ELISA and a series of nested Aβpeptides (1–42, 1–40, 1–11, 4–10, 10–20 and 17–40; data notshown). Both of these independent methods demonstrated thatAβ4–10 was the dominant peptide that anti-Aβ42 IgGs specifically rec-ognized with high affinity.

Anti-Aβ4–10 sera inhibit fibrillogenesis and cytotoxicityAβ fibrillogenesis—especially the formation of small oligomers andprotofibrils—is closely related to the cytotoxic effects of Aβ (ref. 14).To assess whether these sera might protect against Aβ-induced celldeath, PC12 cells were incubated with sera and with Aβ42 prepara-tions, similar to those used for the initial immunization and fibril-assembly studies, that contained numerous assembled oligomers,including amyloid-derived diffusible ligands (ADDLs) and protofib-rils15,16 (electron microscopy data not shown). Non-immune andIAPP-immune sera did not inhibit Aβ-toxicity (Fig. 2a and Table 1).The efficacy of sera towards cytotoxicity and fibrillogenesis are cor-related (Fig. 2b). Moreover, Αβ42-immune sera prevented Αβ42 cyto-toxicity in two independent assays (n = 22, P < 0.01) (i.e. Fig. 2a).The activity of these sera on Aβ cytotoxicity was concentration-de-pendent and occurred at low antibody-to-Aβ ratios (50% effector

concentration (EC50) for inhibition of Aβ cytotoxicity was 1:234 ±39; mean ± s.d.) (Fig. 2c).

In order to investigate potential mechanisms for inhibiting cyto-toxicity, we examined the effect of the Aβ42- and IAPP-immunizedsera upon the assembly of Aβ42 fibrils. Sera were incubated withnanomolar concentrations of Aβ42 at 37 °C, and subsequent assem-bly of fibers was monitored by negative-stain electron microscopyat days 1, 3, 7, 10 and 14 (Fig. 3). Typical Aβ42 fibers (∼ 7,500 Å longand 50–70 Å in diameter)14 were formed when Aβ42 was incubatedby itself or with pre-immune sera (n = 18/18 samples) or with IAPP-immune sera (n = 13/17 samples) (Fig. 3a–c). Of the 17 IAPP-im-mune sera, 4 slightly reduced Aβ fibril assembly. However, this islikely to be a non-specific consequence of upregulation of acute-phase protein, which binds Aβ,because none of the IAPP-sera recog-nized Aβ. In contrast, fiber assembly was completely blocked byΑβ42-immunized mouse sera (n = 27/34 sera; Fig. 3d–f). This in-hibitory effect was equivalent in Aβ-immunized sera fromTgCRND8 mice and from non-transgenic littermates, indicatingthat the antibody repertoire was dependent on the immunizingantigen rather than on either the amount or sequence of endoge-nously produced Aβ42.

To explore whether anti-Aβ sera might disrupt pre-existing Αβ42

fibers, we incubated preformed fibrils with these sera for up to 30days. Preformed Aβ fibrils were stable when incubated with serum-free buffer (Fig. 4a), non-immune sera or IAPP-immune sera (Fig.4b). In contrast, the preformed Αβ42 fibers disassembled into amor-

LysC

(1-16)

1400 1600 1800 2000 2200 2400 2600 m/z

1 3 1610

DAE FRHDSGY EV HHQKLV

(4-10) (1-10)

Aminopeptidase M

1000 1200 1400 m/z

a b

c

Fig. 1 Mass spectrometric identification of the antisera epitope of Aβ42.a, Lys-C protease fragments eluted after epitope excision from plaques re-vealed a single fragment (1–16), 1954.8806. b, ESI and MALDI spectraeluted upon epitope extraction with α-chymotrypsin followed byaminopeptidase M digestion revealed fragments (1–10) 1195.4968 Da and(4–10) 880.3827 Da. c, Sequence of Aβ42, residues 1–18. Shaded sequenceindicates the epitope, residues 4–10 (FRHDSGY). Proteolytic cleavagesyielding fragments identified upon epitope excision are indicated by solidarrows (Lys-C (K16), Glu-C (E11), α-chymotrypsin (Y10) and aminopepti-dase M (D1, A2, E3)). Residues shielded from proteolytic digestion are indi-cated by broken arrows.

30

40

50

60

70

80

90

2 10 12 14 16

Cel

l sur

viva

l (%

)

Disaggregation of fibers (d)4 6 8

60

70

80

90

100

5 10 15 20 25 30

Cel

l sur

viva

l (%

con

trol

)

Concentration of Aβ ( µg/ml)0

Fig. 2 Anti-Aβ4-10 sera inhibit Aβ-induced cytoxicity. a, Aβ-immunizedsera-mediated inhibition of Aβ42-induced cytotoxicity is concentration-de-pendent, with non-immunized and IAPP-immunized sera having no activ-ity. Two different Aβ42-immunized sera are shown. Values are mean ±s.e.m. of 3 separate experiments. b, Correlation between cell survival andextent of fibril disaggregation was plotted for individual sera (�). c, Aβdose-survival curves demonstrate that the inhibition of toxicity was di-rectly related to the presence of the sera. Curves represent Aβ42 alone (�)and in the presence of Aβ42-immunized sera (�). Statistical analyses for allgraphs using one-way ANOVA with Fischer’s PLSD. *, P < 0.01; , P <0.001.

a b

c

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

phous aggregates or into short (∼ 30–100 Å) fibers when incubatedwith Αβ42-immune sera (n = 26/34) (Fig. 4c and d). The rate of fibrildisaggregation was dependent on the titer of antibody used (Fig.4e), and occurred at low molar ratios of antibody-to-Aβ (1:20–1:50).Furthermore, the sera that most efficiently inhibited Aβ cytotoxicityalso most efficiently inhibited Aβ fibrillogenesis and promoted Aβfibril disassembly. Together, the results of these three experimentssuggest that the induced antibodies against Aβ target only a smallsubset of toxic Aβ species, such as protofibrillar oligomers. It re-mains to be determined whether the activity of these sera in vitro iscorrelated with their activity in suppressing Aβ-like neuropathologyand cognitive impairment in vivo. If such a correlation can be deter-mined, the ability of individual sera to modulate Aβ fibrillogenesisand/or toxicity might be used to predict clinical response to Aβ vac-cination.

Anti-Aβ4–10 IgG2b are active componentsTo confirm that the effects of Aβ-immunization were specificallydue to Aβ-induced antibodies as op-posed to changes in the expression ofother serum proteins, we repeated theseexperiments using purified IgG fractionsfrom Aβ42-immune sera. These experi-ments revealed that the purified IgG andcrude sera induced equivalent degrees ofAβ-fiber disaggregation, inhibition of Aβfibrillogenesis and inhibition of toxicity;these results are in agreement with pre-vious passive immunization experi-ments in murine models of AD (refs.17,18) (Fig. 5a).

Immunoglobulins have several iso-types, and expression of a particular iso-type in response to antigen exposuredetermines its functional effect. Analysisof the distribution of the isotypes in ran-domly chosen, non-immunized (n = 4),IAPP-immunized (n = 4) and Aβ42-immu-nized (n = 7) sera revealed that the non-immunized sera displayed the full rangeof IgG isotypes, whereas IAPP-immu-nized sera predominantly expressedIgG1, IgG2b and IgM. In contrast, all ofthe Aβ42-immunized sera expressed IgG2band very little IgG3. The predominant ex-pression of IgG2b in Aβ42-immunized sera

NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002 1265

ARTICLES

may explain the lack of inflammation detected in these animals, be-cause IgG2b is the least effective activator of the complement systemand binds with the lowest affinity to cell-associated Fcγreceptors19.

Cumulatively, these experiments show that IgG2b antibodies tar-geted to the N-terminal Aβ4–10 epitope are likely to be therapeuticallyactive. To provide independent, biological validation of this conclu-sion, we repeated the fibrillogenesis and toxicity studies using com-mercial monoclonal antibodies to specific epitopes of Aβ.Monoclonal antibodies specific for residues 17–24 of Aβ (4G8) or toresidues 11–17 (6E10) did not inhibit fibrillogenesis, although theydid slightly decrease the number of fibers (probably by binding to asmall portion of free Aβ peptide in solution and sequestering it fromfiber formation) (Fig. 5b and c). In contrast, Bam10, which recog-nizes a sequence within Aβ1–10, inhibited fiber formation (Fig. 5d).This conclusion is also consistent with a previous report that mono-clonal antibodies to the N-terminal region of Αβ42 can inhibit bothin vitro Αβ fibrillogenesis and the cytotoxic effects of Aβ on PC-12cells, whereas antibodies to the C terminus were ineffective20–22.Together, the results of these experiments strongly suggest that an-tibodies to residues 4–10 of Aβ are effective in vivo inhibitors of thespatial memory disturbances and amyloid plaque formation in theTgCRND8 mouse model of AD, and probably act by inhibiting Aβ-fibrillogenesis and toxicity.

Anti-Aβ4–10 antibodies recognize Aβ in AD brain tissueSome previous studies have suggested that human AD brain has apreponderance of N-terminally truncated and modified pep-tides23,24. Most of these N-terminal truncations either involve thefirst 5 amino acids (generating Aβ peptides that would be detectedby antibodies to Aβ4–10), or extend beyond residue 14 (generating Aβpeptides that would be insoluble and possibly less toxic24,25). If cor-rect, this might raise a concern about the therapeutic utility of anti-bodies to Αβ4–10 in human AD. However, when the anti-Aβ4–10

antibodies described here were used to probe amyloid plaques from

Table 1 Summary of effects of non-immune, Aβ42-immune and IAPP-immune sera on fibril formation, fibril disassembly and cytotoxicity

Inhibition studies

Sera (n) Aggregation Disaggregation Toxicity

Non-immunized sera(18) 0 / 18 0 / 18 0 / 18

Aβ42-immunized sera(34) 27 / 34 26 / 34 22 / 30

IAPP-immunized sera(17) 4 / 17 1 / 17 2 / 11

Data represent the number of sera, isolated from all animals used in the study, thatwere effective at prevention of Aβ aggregation and cytotoxicity, and able to disaggre-gate preformed Aβ42 fibers.

Fig. 3 Aβ42-immunized mouse sera inhibit Aβ42 fiber formation. a, Fibers are formed at low Aβ42 concen-trations over the 14-day incubation and have a characteristic diameter of 50–70 Å. b, No difference in thestructure of the fibers could be detected when incubated in the presence of non-immunized mouse sera.c, Sera immunized with IAPP decreased the extent of fiber formation but fibers that did form were similarto Aβ42 alone. d–f, Aβ42-immunized sera inhibited fibrillogenesis completely (d and e) or slightly decreasedfiber density (f). Magnification, ×60,000.

a b c

d e f

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

1266 NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002

ARTICLES

human AD brain, mass spectrometric epitope-excision analysis ofthe peptide generated showed the presence of abundant Aβ speciescontaining the Aβ4–10 epitope. These results indicate that antibodiesto N-terminal Aβ do in fact react to relevant Aβ species in vivo and,therefore, might have therapeutic utility in humans.

Anti-Aβ4–10 response need not be inflammatoryImmunization of humans with full-length Aβ1–42 has resulted in sev-eral instances of severe meningo-encephalitic cellular inflammatoryreactions6. To determine whether induction of anti-Aβ4–10 was neces-sarily accompanied by a similar cellular inflammatory response inthe TgCRND8 mouse, we investigated both the cellular immuneand the brain microglial responses that accompanied the inductionof these antibodies. Immunization with antigen usually stimulatesthe adaptive immune system to produce either T-helper cell 1 (Th1)or Th2 responses. Classically, Th1 responses stimulate pro-inflam-matory reactions, whereas Th2 reactions aid B cells. To differentiatethese responses, we examined in vitro T-cell activation and subse-quent cytokine production in splenic T cells isolated from Aβ42- orIAPP-immunized mice. After restimulation with Aβ42 or Aβ4–10,splenic T cells from Aβ42-immunized TgCRND8 mice produced Th2cytokines interleukin-4 (IL-4) (32.5 ± 5.4 pg/ml) and IL-5 (33 ± 10.8pg/ml). IAPP-immunized mice did not respond to Aβ peptides butdemonstrated a Th2 response to IAPP (IL-4, 21.7 ± 10.5 pg/ml; IL-5,30.1 ± 6.9 pg/ml). In contrast, the positive control, Concavalin A,stimulated both Th1 and Th2 cytokines, IL-2 (314.6 ± 42.6 pg/ml),interferon-γ (IFN-γ) (127.8 ± 32 pg/ml), IL-4 (7.0 ± 1.0 pg/ml) and IL-

5 (26.9 ± 9.6 pg/ml). The extent of cytokine production was low,and is consistent with a low number of activated T cells present inthe spleen of Aβ42-immunized mice (a classic characteristic of pep-tide challenge of a Th2 response26). Unstimulated splenic T cells iso-lated from both Aβ42- and IAPP-immunized mice did notspontaneously produce cytokines, suggesting that the Th2 responseis peptide-specific.

The over-expression of Aβ and subsequent deposition of plaquesare accompanied by development of microgliosis in TgCRND8mice2. Induction of therapeutically active anti-Aβ4–10 could eitherdecrease this microglial response due to decreased plaque load3, orenhance it due to increased microglial activation upon phagocyto-sis of IgG–Aβ complexes. However, using the immunohistochemi-cal density of CD68 staining as a marker of activated microglia, wefound that the decrease in plaque load after immunization withAβ42 was in fact accompanied by a decrease in microgliosis; the per-centage of hippocampus covered by microgliosis in Aβ-immunizedmice was 0.11 ± 0.04% (n = 4) compared with 0.31 ± 0.01% in IAPP-immunized and nonimmunized TgCRND8 mice (n = 4).

Finally, to determine whether the clearance of plaques by Aβ4–10

IgG2b antibodies is accompanied by markers of T-cell activation andinflammation in the brain, we measured the expression of the cy-tokines tumor necrosis factor-α (TNF-α) and IFN-γ within the cere-brospinal fluid of immunized mice. TNF-α expression was equivalentin Aβ42-immunized (n = 3), IAPP-immunized (n = 3) and non-immu-nized mice (n = 3) (relative density in pixels: 179.33 ± 27.5, 162.26 ±21.52 and 199.69 ± 30.81, respectively). IFN-γ was not detected inAβ42-, IAPP- or non-immunized TgCRND8 mice, indicating that Tcells were not present in the cerebrospinal fluid (data not shown).

DiscussionOur results must be interpreted with the qualification that theywere generated from a single animal model and that both the ge-netic background and specific immunization protocol employedcan profoundly alter the immune response to a vaccine.Nevertheless, taken together, our results lead to the conclusion thatimmunization with protofibrillar forms of Aβ42 can induce thera-peutically effective IgG2b antibodies that recognize Aβ4–10. These an-tibodies can reduce both AD-like cognitive impairments andneuropathology, probably by inhibiting Aβ-protofibrillar aggrega-tion and toxicity. Aβ4–10 contains a predicted B cell–activating epi-tope in both humans and mice, but no predicted T cell–activatingepitopes (identified using B- and T-cell epitope-prediction algo-rithms available commercially). In contrast, the C terminus of Aβ42,which is present in the AN1792 vaccine used in human trials, con-tains several epitopes that are predicted to activate T-cell responsesin a proportion of both mice and humans (identified using B- and T-cell epitope-prediction algorithms available commercially).Although immunization with full-length Aβ42 caused significantand adverse cellular inflammatory responses in some patients, ourdata also show that it is possible to obtain a more restricted, but po-tentially therapeutically effective immune response; that is, IgG2bsubclass anti-Aβ4–10, a Th2 response, reduced microgliosis and no

a b

c d

Fig. 4 Disaggregation of preformed Aβ42 fibers by immunized sera. aand b, Preformed Aβ42 fibers in the absence (a) and presence (b) ofIAPP-immunized sera were stable for up to 30 d in vitro. c and d, In con-trast, Aβ42-immunized sera disaggregated preformed fibers over thesame time frame. e, The time required for fiber disassembly was directlycorrelated with the extent of antibody production. Magnification,×60,000. Error bars indicate means ± s.e.m.

0

2

4

6

8

10

12

14

5,000 7,500 10,000 25,000 50,000

Day

s

Titer (half maximal binding)

e

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002 1267

ARTICLES

Th1 activation either in splenic T cells or T-cell activation in thecentral nervous system.

Our data also help explain another conundrum. Only smallamounts of anti-Aβ will gain access to the brain across theblood–brain barrier (∼ 0.1% of serum levels)17. How then do suchsmall amounts of anti-Aβ reduce amyloid plaque load and neuro-toxicity? How can this be accomplished without necessarily alter-ing total Aβ peptide levels? Why are the effects of immunotherapyon brain Aβ levels different in different mouse models? The sim-plest answers to these questions lie in the fact that the anti-Aβ4–10

induced by our immunization regime seem to target only a specificsubset of Aβ species (that is, protofibrillar species or other solubleprecursors). Although protofibrillar Aβ species are a low-abun-dance species, they are the critical intermediaries of plaque forma-tion and are important causes of neuronal dysfunction and,ultimately, neuronal death both in human AD (ref. 27) and in ro-dent models14–16,28. Although we cannot demonstrate directly thatthe antibodies generated in our mice specifically targeted protofib-rillar forms of Aβ42 in vivo, two lines of evidence strongly supportthis conclusion. First, protofibrillar Aβ42 was the immunizing anti-gen. Second, the resultant sera detected Aβ oligomers on westernblots, and recognized fibrillar but not diffuse Aβ deposits in histo-logical sections of brain. Finally, total brain Aβ levels were not de-creased in the CNS of TgCRND8 mice immunized using ourprotocol3. In contrast, PDAPP mice show decreased levels of Aβ inthe CNS when immunized with AN1792 Aβ42 vaccine, which alsoactivated cell-mediated events, such as Fc-receptor mediated mi-croglial clearance4,28. PDAPP mice also show decreased Aβ whenpassively immunized with the m266 monoclonal anti-Aβ anti-body. This antibody, which recognizes Aβ13–28, enhances plasmaclearance and may exhibit disparate binding specificity towards Aβoligomers, protofibrils and plaques, and have differential access tothe CNS17,18. These differential effects of vaccination on CNS Aβlevels probably arise from variation in the predominant immuneepitope presented by the specific immunization protocol, as well asfrom differences in the immunogenetic backgrounds of the twomurine models. Nevertheless, induction of IgG2b antibodies toAβ4–10 seems to have both a therapeutic effect and low propensityfor the genesis of unwanted and deleterious cellular immune re-sponses.

A final conclusion that derives from this work is that in additionto developing better immunological reagents it provides a novel tar-get for treating AD. Knowledge of the three-dimensional structureof this antibody–antigen interaction may permit the developmentof small-molecule mimics that could circumvent the problems ofimmunotherapy.

MethodsMice. The TgCRND8 mice have been described2. The mice maintained in anoutbred C3H/C57BL6 (82%/18%) background overexpress the βAPPSwedish

and βAPPV717F mutations in cis on the βAPP695 transcript under control of theSyrian hamster prion gene promoter. All experimental groups were sex- andweight-matched. All experiments were performed according to the CanadianCouncil on Animal Care guidelines.

Immunization protocol and sera isolation. Synthetic Aβ42 and IAPP pep-tides were isolated by reverse-phase high pressure liquid chromatography(HPLC). The immunization protocol and schedule have been described4.Antibody titers were assayed in serum (200 µl blood) collected at 13 and 25wk of age. Prior to use, the complement was deactivated by incubation at 56 °C for 30 min. Immunoglobulin fractions were isolated using protein G(Sigma).

Antibody immobilization. A solution of 100 µg IgG in 0.2 M NaHCO3, 0.5 M NaCl (pH 8.3) was added to n-hydroxysuccinimidyl (NHS)-activated6-aminohexanoic acid–coupled sepharose (Sigma), for 60 min at 20 °C be-fore transferring onto a 100-µm microcapillary8,12. The column was washedalternately with blocking (ethanolamine/NaCl) and washing buffers(NaAc/NaCl)8.

Epitope excision and extraction. Epitope excision was performed by appli-cation of 2–5 µg antigen to the antibody microcolumn for 60 min at 20 °C.After washing, digestion was performed on the column for 2 h at 37 °C with0.2 µg protease in 200 µl PBS. Unbound peptides were removed and the epi-tope dissociated using 500 µl 0.1% trifluoroacetic acid (TFA). After incubationfor 15 min at 20 °C, the epitope eluate was lyophilized and reconstituted in10 µl 0.1% TFA for analysis. Exopeptidase digestion was performed using 0.1µg aminopeptidase M or carboxypeptidase Y for 30 min, as described.Epitope extraction was performed similarly, with the proteolytic digest ap-plied directly to the antibody column.

Mass spectrometry. FT-ICR-MS was performed with a Bruker (BrukerDaltonik, Bremen, FRG) Apex II FT-ICR spectrometer equipped with a 7T su-perconducting magnet and ICR analyzer cell29,30. The matrix-assisted laser

a b c

d

Fig. 5 Targeting Aβ4-10 necessary for activity of sera. a, Purified IgG fraction replicated the beneficial ef-fect of sera suggesting that IgG and no other serum components was the active component for inhibitionof cell death. Two different Aβ42-immunized sera are shown. Statistical analyses were performed usingone-way ANOVA with Fischer’s PLSD; *, P < 0.01. Values are mean ± s.e.m. of 3 separate experiments. b–d,Control antibodies 6E10 (b) and 4G8 (c) did not inhibit Aβ42 fiber formation, whereas Bam10 (d) was ef-fective, suggesting that the 4–10 epitope is necessary for the inhibition. Magnification, ×60,000.

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

1268 NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002

ARTICLES

desorption/ionization (MALDI)-FT-ICR source with pulsed collision gas31,Apollo-nano-ESI-source, instrumental conditions and mass calibration havebeen described32. Mass determination accuracies were ∼ 1 part per million(p.p.m.) (MALDI) and typically, 0.5–1 p.p.m. (ESI) at a mass resolution of ∼ 2× 105. 2,5-Di-hydroxybenzoic acid (DHB) was the matrix for MALDI-MS sam-ple preparation. Electrospray ionization (ESI)-MS was performed with aque-ous 0.01% TFA solutions.

Epitope confirmation. Antigen–antibody affinities were assessed as de-scribed8–13. Syntheses of peptides (Aβ42, Aβ40, Aβ30 and Aβ4–10) were performedwith an ABIMED EPS-221 (Foster City, California) synthesizer. Peptides werepurified by HPLC and characterized by MALDI-MS and ESI-MS. Biotinylationof peptides was performed using sulfo-LC-PEO4-biotin (Pierce)33 and charac-terized by ELISA (Promega, Madison, Wisconsin)34. Aβ peptides, 1–11, 10–20and 17–40 were purchased from American Peptide Inc. (Sunnyvale,California). Reactivity of sera isolated from Aβ42-immunized mice were seriallydiluted and analyzed using ELISA. Antibody reactivity is presented as half-maximal binding.

Aβ-induced toxicity. PC-12 cells were plated at 500 cells per 96 well and sus-pended in 30 ng/ml NGF (Alamone Labs, Jerusalem, Israel) diluted inN2/DMEM (Gibco/BRL, Rockville, Maryland). Cells were differentiated over5–7 d. Aβ was aged 3 d before addition to cultures at a final concentration of0.1 µg/µl, and incubated for 24 h at 37 °C. Toxicity was assayed using theLive/Dead assay (Molecular Probes, Eugene, Oregon) and Alamar Blue assay(Biosource Inc, Camarillo, California).

Electron microscopy. Aβ42 was used directly after solubilization in water at aconcentration of 10 mg/ml, or after assembly into mature amyloid fibers. Aβ42

was incubated in the presence or absence of sera at a final peptide concentra-tion of 100 µg/ml. Serial dilutions of sera were added to Aβ42 and incubated atroom temperature for up to 2 wk. For negative-stain electron microscopy,carbon-coated pioloform grids were prepared and visualized as described35.

Antibody isotyping. Sera and purified immunoglobulin were isotyped usingthe Serotec (Oxford, UK) mouse monoclonal antibody isotyping kit.

T-cell stimulation assays. Splenic T cells were isolated by negative-selectioncolumn chromatography (Cedarlane, Hornby, Ontario). Purity of the T cellsdetermined by flow cytometry and found to be at least 90% pure. Resting T cells (1 × 106) were cultured with irradiated allogenic antigen presentingcells (1 × 105) plus 1 µM Aβ4–10, Aβ1–42, IAPP, concavalin A and alone. Following3 d stimulation, supernatants were collected and frozen at –70 °C. For the last6 h, proliferation was monitored using the Alamar blue assay.

Cytokine detection. Mouse Th1/Th2 cytokines in culture media were ana-lyzed using the Pharmingen Cytometric Bead Array kit (Mississauga, Ontario)that simultaneously measures TNF-α, IL-2, IFN-γ, IL-4 and IL-5 by flow cytom-etry. Western-blot analysis was used to examine the presence of TNF-α andIFN-γ (BD Biosciences, San Jose, California) in cerebrospinal fluid.

Microgliosis quantification. Five randomly selected, evenly spaced, frozensagittal sections were collected from one paraformaldehyde-fixed hemi-sphere of all animals. Sections were immunolabeled for microglia cells onfree-floating sections36. In brief, sections were incubated overnight at 4 °Cwith anti-rat CD68 IgG2a (Serotec; diluted 1:50). Digital images were cap-tured using a Coolsnap digital camera (Photometrics, Tuscon, Arizona)mounted to a Zeiss, Axioscope 2 Plus microscope. Images were analyzedusing Openlab 3.08 imaging software (Improvision, Lexington,Massachusetts).

Acknowledgments:We thank N. Wang for the synthesis of all peptides used in this study, N.Youhnovski and E. Damoc for expert assistance with the FT-ICR mass spectrome-try, and the Electron Microscopy Suite at the University of Toronto for use ofHitachi 7000 electron microscope. This study was supported by the OntarioAlzheimer’s Society (to P.H., P.E.F., D.W., H.M. & J.M.), Canadian Institutes ofHealth Research (to P.H., P.E.F., D.W., H.M. & J.M.), the Natural Sciences and

Engineering Research Council of Canada (to J.M.), Howard Hughes Foundation(to P.H.), Canadian Genetic Diseases Network (to P.H.), the Scottish RiteCharitable Foundation (to P.E.F. & J.M.), the Deutsche Forschungsgemeinschaft(M.P.), Research & Arts Ministery Baden-Württemberg (M.P.) and theAlexander-von-Humboldt Foundation through a fellowship (X.T.).

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 25 JUNE; ACCEPTED 1 OCTOBER 2002

1. Small, D.H., Mok, S.S. & Bornstein, J.C. Alzheimer’s disease and Aβ-toxicity: Fromtop to bottom. Nature Rev. Neurosci. 2, 595–598 (2001).

2. Chishti, M.A. et al. Early-onset amyloid deposition and cognitive deficits in trans-genic mice expressing a double mutant form of amyloid precursor protein 695. J.Biol. Chem. 276, 21562–21570 (2001).

3. Janus, C. et al. Aβ peptide immunization reduces behavioural impairment andplaques in a model of Alzheimer’s disease. Nature 408, 979–982 (2000).

4. Schenk, D. et al. Immunization with amyloid-β attenuates Alzheimer-disease-likepathology in the PDAPP mouse. Nature 400, 173–177 (1999).

5. Morgan, D. et al. Vaccination with Aβ peptide prevents memory deficits in an ani-mal model of Alzheimer’s disease. Nature 408, 982–985 (2000).

6. Check, E. Nerve inflammation halts trial for Alzheimer’s drug. Nature 415, 462(2002).

7. Marshall AG, Hendrickson, CL, Jackson, GS. Fourier transform ion cyclotron reso-nance mass spectrometry: A primer. Mass Spectrom. Rev. 17, 1–35 (1998).

8. Macht, M., Fiedler, W., Kürzinger, K. & Przybylski, M. Mass spectrometric mappingof protein epitope structures of the myocardial infarct markers myoglobin and tro-ponin T. Biochemistry 35, 156333–15639 (1996).

9. Suckau, D. et al. Molecular epitope identification by limited proteolysis of an im-mobilized antigen-antibody complex and mass spectrometric peptide mapping.Proc. Natl. Acad. Sci. USA 87, 9848–9852 (1990).

10. Przybylski, M. et al. Approaches to the characterisation of tertiary and supramolec-ular protein structures by combination of protein chemistry and mass spectrome-try. in New Methods for the Study of Biomolecular Complexes, 17–43 (Kluwer Acad.Publ., Amsterdam, 1998).

11. Papace, C.I., Hoyes, J. & Tomer, K.B. Direct analysis of affinity-bound analytes byMALDI/TOF-MS. Anal. Chem. 199, 2609–2613 (1994).

12. Kohlmann, M. et al. Epitope identification of the carboxy-terminal cytosolic do-main of the Alzheimer’s amyloid precursor protein (APP) with a monoclonal mouseanti-APP antibody by high resolution mass spectrometry. J. Pept. Sci. (in the press).

13. Hochleitner, E.O., Borchers, C., Parker, C., Bienstock, R.J. & Tomer, K.B.Characterisation of a discontinuous epitope of the human immunodeficiency virus(HIV) core protein p24 by epitope excision and differential chemical modificationfollowed by mass spectrometric peptide mapping analysis. Protein Sci. 9, 487–496(2000).

14. Yang, D.S. et al. Assembly of Alzheimer’s amyloid-β fibrils and approaches for ther-apeutic intervention. Amyloid 8, 10–19 (2001).

15. Walsh, D.M. et al. Amyloid β-protein fibrillogenesis. Structure and biological activ-ity of protofibrillar intermediates. J. Biol. Chem. 274, 25945–25952 (1999).

16. Hartley, D.M. et al. Protofibrillar intermediates of amyloid β-protein induce acuteelectrophysiological changes and progressive neurotoxicity in cortical neurons. J.Neurosci. 19, 8876–8884 (1999).

17. Bard, F. et al. Peripherally administered antibodies against amyloid β-peptide enterthe central nervous system and reduce pathology in a mouse model of Alzheimerdisease. Nature Med. 6, 916–919 (2000).

18. DeMattos, R.B. et al. Peripheral anti-Aβ antibody alters CNS and plasma Aβ clear-ance and decreases brain Aβ burden in a mouse model of Alzheimer’s disease. Proc.Natl. Acad. Sci. USA 98, 8850–8855 (2001).

19. Fridman, W.H. & Sautes, C. Cell-mediated effects of immunoglobulins. in MolecularBiology Intelligence Unit (Springer, New York, 1997).

20. Solomon, B., Koppel, R., Hanan, E. & Katzav, T. Monoclonal antibodies inhibit invitro fibrillar aggregation of the Alzheimer β-amyloid peptide. Proc. Natl. Acad. Sci.USA 93, 452–455 (1996).

21. Solomon, B., Koppel, R., Frankel, D. & Hanan-Aharon, E. Disaggregation ofAlzheimer β-amyloid by site-directed mAb. Proc. Natl. Acad. Sci. USA 94,4109–4112 (1997).

22. Frenkel, D., Solomon, B. & Benhar, I. Modulation of Alzheimer’s β-amyloid neuro-toxicity by site-directed single-chain antibody. J. Neuroimmunol. 106, 23–31(2000).

23. Kuo, Y.-M. et al. Comparative analysis of amyloid-β chemical structure and amyloidplaque morphology of transgenic mouse and Alzheimer’s Disease brains. J. Biol.Chem. 276, 12991–12998 (2001).

24. Kalback, W. et al. APP Transgenic mice Tg2576 accumulate Aβ peptides that aredistinct from the chemically modified and insoluble peptides deposited inAlzheimer’s disease senile plaques. Biochemistry 41, 922–928 (2002).

25. Gowing, E. et al. Chemical characterization of Aβ 17-42 peptide, a component ofdiffuse amyloid deposits of Alzheimer disease J. Biol. Chem. 269, 10987–10990(1994).

26. Delgado, M. & Ganea, D. VIP and PACAP enhance the in vivo generation of mem-ory TH2 Cells by inhibiting peripheral deletion of antigen-specific effectors. ArchPhysiol Biochem. 109, 372–376 (2001).

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine

NATURE MEDICINE • VOLUME 8 • NUMBER 11 • NOVEMBER 2002 1269

ARTICLES

27. McLean, C.A. et al. Soluble pool of Aβ amyloid as a determinant of severity of neu-rodegeneration in Alzheimer’s disease Ann. Neurol. 46, 860–866 (1999).

28. Walsh, D.M. et al. Naturally secreted oligomers of amyloid β protein potently in-hibit hippocampal long-term potentiation in vivo. Nature 416, 535–539 (2002).

29. Youhnovski, N. et al. Thin chip microspray systems coupled to fourier transform-ICR mass spectrometry. Angew. Chem. Int. Ed. Engl. (in the press).

30. Fligge, T.A., Reinhard, C., Harter, C., Wieland, F.T. & Przybylski, M.Oligomerisation peptides analogous to the cytoplasmic domains of coatamer re-ceptors revealed by mass spectrometry. Biochemistry 39, 8491–8496 (2000).

31. Bauer, S.H., Wiechers, M.F., Bruns, K., Przybylski, M. & Stuermer, C.A.O. Isolationand identification of the plasma membrane-associated intracellular protein reggie-2 from goldfish brain by chromatography and fourier transform-ion cyclotron reso-nance mass spectrometry. Anal. Biochem. 298, 25–31 (2001).

32. Mayer-Fligge, P. et al. Synthesis and structural characterisation of human-identicallung surfactant protein SP-C. J. Pept. Sci. 4, 355–363 (1998).

33. Altin, J.G. et al. A one-step procedure for biotinylation and chemical crosslinking oflymphocyte surface intracellular membrane-associated molecules. Anal. Biochem.224, 382–389 (1995).

34. Craig, D.B., Wong, J.C.Y. & Dovici, N.J. Detection of attomolar concentrations ofalkaline phosphatase by capillary electrophoresis using laser-induced fluorescencedetection. Anal. Chem. 68, 697–701 (1996).

35. McLaurin, J., Golomb, R., Jurewicz, A., Antel, J.P. & Fraser, P.E. Inositol stereoiso-mers stabilize an oligomeric aggregate of Alzheimer amyloid β peptide and inhibitaβ-induced toxicity. J. Biol. Chem. 275, 18495–18502 (2000).

36. Jucker, M. et al. Age-related deposition of glia-associated fibrillar material in brainsof C57BL/6 mice. Neuroscience 60, 875–889 (1994).

©20

02 N

atu

re P

ub

lish

ing

Gro

up

h

ttp

://w

ww

.nat

ure

.co

m/n

atu

rem

edic

ine