Co-incorporation of Aβ40 and Aβ42 to form mixed pre-fibrillar aggregates

Co-incorporation of Ab40 and Ab42 to form mixed pre-fibrillaraggregates

David Frost1, Paul M. Gorman1, Christopher M. Yip2 and Avijit Chakrabartty1

1Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, and2Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering,

Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

Senile plaques, the invariable hallmark and likely proximalcause of Alzheimer’s disease (AD), are structured deposi-tions of the 40- and 42-residue forms of the Ab peptide.Conversely, diffuse plaques, which are not associated withneurodegeneration, consist mainly of unstructured Ab42.We have investigated the interaction between Ab40 andAb42 through an assay, which involves labeling both vari-ants with an environment-sensitive fluorophore. We havemonitored association of Ab without fibrillar seeds, whichallows investigation of molecular species preceding fibrils.Immediately upon mixture, Ab40 and Ab42 associate intomixedaggregates, inwhich thepeptides areunstructuredandrelatively accessible to water. When left to incubate for an

extended period, larger, more tightly packed aggregates,which show secondary structure, replace the small,unstructured aggregates formed earlier. Our results showthat in vitro the two Ab variants coassemble early in thefibrillogenesis pathway. The ease of formation formixed andhomogeneous aggregates is similar. A change in the local Abvariant ratio can therefore have a significant impact on Abaggregation; indeed such a change has been reported in sometypes of familial AD.

Keywords: Alzheimer’s disease; amyloid; fibril; peptide;fluorescence.

Alzheimer’s disease (AD) is a significant and increasinghealth concern. Neurohistological studies have uncoveredseveral hallmarks that distinguish the AD brain from itsnormal counterpart. Chief among these are neurofibrillarytangles (NFTs)andsenileplaques (SPs).NFTsarecomposedof paired helical filaments of the (normally) microtuble-associated Tau protein, while senile plaques are primarilycomprised of the 40- and 42-residue forms of the Ab peptide[1,2].The interactionbetweenAb40andAb42, the twomajorvariants of the Ab peptide, is the subject of this study.

The Ab family of peptides is enzymatically cleaved fromthe amyloid precursor protein (APP), a 563–770 residuemembrane protein that is expressed in both neuronal andnon-neuronal tissue [3,4]. Ab40, and to a lesser extentAb42, are normal constituents of cerebrospinal fluid[5–8]. Both forms are capable of assembling into60–100 A diameter b-sheet fibrils, which form the core ofthe aforementioned senile plaques.

An impressive body of evidence points to Ab depo-sition in senile plaques as the causal event in ADpathology. Upon postmortem examination of AD brains,senile plaques are invariably found in the limbic andassociation cortices, surrounded by dead or dyingneurons, as well as activated microglia and reactiveastrocytes [1]. In several forms of familial AD, mutationsin the APP gene have been identified [9,10]. Also,mutations of the presenilin genes have been linked tofamilial AD, and appear to lead to an increase in theratio of Ab42 to Ab40 [11]. Transgenic mice overexpressing a mutant form of APP develop neurohistolo-gical characteristics similar to those of AD patients[12–14]. Perhaps most convincingly, Down’s syndromepatients, who receive a triple dose of the genes present onchromosome 21, including the APP gene, often showsenile plaque deposition and classical AD neurohistologyin their late 20s or early 30s, followed by progressivecognitive and behavioral dysfunction in their mid 30s [15].

Unlike senile plaques, diffuse plaques are more looselypacked depositions of mostly unstructured Ab42 [1].Diffuse plaques are not associated with dead or dyingneurons, and have been found upon post mortem exami-nation of the brains of elderly people who had notexhibited AD symptoms [16–20]. Diffuse plaques are alsoreferred to as �preamyloid plaques� because of several linesof evidence that point to them as precursors to senileplaques. In the Down’s syndrome patients discussed earlier,diffuse plaques are observed as early as age 12 years [21].Similarly, mice transgenic for mutant human APP alsodevelop diffuse Ab42 plaques before fibrillar plaquessurrounded by dead and dying neurons [12–14].

Correspondence to A. Chakrabartty, Division of Molecular and

Structural Biology, Ontario Cancer Institute and Department of

Medical Biophysics, University of Toronto, 610 University Avenue,

Toronto, Ontario, Canada, M5G 2M9.

Fax: + 416 9466529, Tel.: + 416 9464501 ext. 4910,

E-mail: [email protected]

Abbreviations: Ab, Alzheimer beta amyloid; AD, Alzheimer’s disease;

AFM, atomic force microscopy; APP, amyloid precursor protein;

EDANS, ethyldiaminonaphthalene-1-sulfonic acid;

NFT, neurofibrillary tangle; SP, senile plaque.

(Received 13 September 2002, revised 27 November 2002,

accepted 5 December 2002)

Eur. J. Biochem. 270, 654–663 (2003) � FEBS 2003 doi:10.1046/j.1432-1033.2003.03415.x

The observation that Ab42 diffuse plaques lead to senileplaques, consisting of both Ab variants, and the apparentimportance of the Ab40/Ab42 ratio in AD [22] have led usto investigate the interaction between these two peptides.Studies by Hasegawa et al. [23] have demonstrated theability of preformed Ab42 fibrils to seed the fibrillogenesis ofAb40, as well as the ability of Ab40 to seed Ab42fibrillogenesis. However, with little conclusive evidencethat Ab fibrils are neurotoxic, more attention is beingpaid to prefibrillar Ab species in the search for a clearculprit in AD pathology [24]. Recent work by our group[25] as well as others [26–28], has shown a complex seriesof reactions, which precedes the formation of maturefibrils. To our knowledge, no study has been undertakento determine at which point in the fibrillogenesis pathwayAb40 and Ab42 can form mixed molecular species, andthe relative ease of formation of mixed vs. homogenousspecies.

We believe that the approach of studying fibrillogenesis inthe context of Ab40 and Ab42 mixtures is advantageous.Studies are usually of either Ab40 or Ab42, while it is knownthat in vivo, both Ab40 and Ab42 are present, and theirinteraction may play a key role in the transition betweenrelatively innocuous diffuse plaques and possibly neurotoxicsenile plaques. Furthermore, many in vitro studies examinefibril formation without rigorously removing all fibril seeds,thereby making it impossible to characterize all speciespreceding fibrils. In the present study, we ensure a homo-geneous starting solution of monomeric Ab peptides,thereby permitting an examination of the interactionbetween Ab40 and Ab42 throughout the fibrillogenicpathway.

Materials and methods

Peptide synthesis

A PerSeptive Biosystems 9050 Plus peptide synthesizerwas used to separately prepare both Ab40 and Ab42 bysolid phase peptide synthesis. An active ester couplingprocedure, employing O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate of 9-fluorenyl-methoxycarbonyl amino acids was used. The magnitudeof the syntheses was 0.05 mmol, and a three times excessof reagents was used. The peptides were cleaved from theresin with 95 : 5 trifluoroacetic acid and anisol mixture.The cleavage mixture was incubated at room temperaturefor 30 min, and the resin removed by filtration. Bromo-trimethylsilane was added to a final concentration of12.5% (v/v). The peptides were then precipitated andwashed five times in cold ether. The peptides wereremoved from the ether and dissolved in 6 M guanidinehydrochloride, 0.15% NH4OH (pH 10) and purified byHPLC. The purified peptide was chromatographed on aSephadex G-75 column (Amersham Pharmacia Biotech)and the fractions corresponding to the correct monomermolecular mass collected. Electrospray mass spectrometryconfirmed the presence of the correct molecular mass, andpurity was determined by six cycles of PTH peptidesequencing by the Edman degradation reaction (Portongas-phase Microsequencer, model 2090), which revealedthat the purified peptides had the correct sequence.

Sequencing proceeds from the N- to the C-terminus,while automated synthesis proceeds in the oppositedirection. By confirming that the main peptide presenthas the correct N-terminal sequence, purity is established,as any errors in synthesis usually result in a truncatedN-terminus.

Fluorescent labeling

A glycine residue was added to the N-terminus of bothAb40 and Ab42 prior to addition of the fluorophore. Thisacts as a flexible linker to prevent the fluorophore frominterfering with the normal behavior of the peptides.Ethyldiaminonaphthalene-1-sulfonic acid (EDANS; Mole-cular Probes) was then coupled to the glycine linker.Purification was performed as above, and sequencingrevealed the major peptide present in each synthesis to bethe correct labeled Ab, with a minor contaminant ofunlabeled Ab.

Preparation of stock peptide solutions without fibrilseeds

After chromatographic separation as described above, boththe labeled and unlabeled peptides were separately storedat pH 10 at 4 �C until use. This method of stock storagehas previously been used by us [25] and others [29] tosuccessfully prevent the formation of fibril seeds.

Peptide concentration determination

For unlabeled Ab peptides, tyrosine absorbance of UVlight (275 nm) was used to determine concentrationin 0.15% NH4OH by Beer–Lambert law (e ¼ 1390cm)1ÆM)1 [30]). Each concentration obtained was multi-plied by the appropriate dilution factor to obtain stockconcentrations. The EDANS-labeled peptide stock con-centration was determined by EDANS absorbance at338 nm (e ¼ 6500 cm)1ÆM)1 [31]). This method of con-centration determination was used because it ensures thecorrect concentration of labeled peptide is obtained, andis not affected by the minor unlabeled Ab contaminantdescribed above. All absorbance measurements weremade on a Milton-Roy Spectronic 3000 spectrophoto-meter.

Sample preparation

All samples were measured at pH 7 with 40 mM phos-phate buffer. Into Eppendorf tubes, first the amount ofunlabeled Ab stock (stored at pH 10, 4 �C) appropriatefor each concentration tested was added. Stock peptideconcentrations were 0.212 mM for Ab40 and 0.165 mM

for Ab42. As the final solution volume was 500 lL, eachincrement of 10 lM unlabeled Ab40 required the additionof 23.6 lL of stock. Similarly, each 10 lM increment ofunlabeled Ab42 required 30.3 lL of stock. Next, thelabeled Ab was added to the solution. Stock concentra-tions were 8.65 lM for EDANS-Ab40 and 31.9 lM forEDANS-Ab42, both stored at pH 10, 4 �C. All solutionstested that included labeled Ab had a concentration of0.1 lM EDANS-Ab. Therefore, for the solutions with

� FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 655

EDANS-Ab40, 5.8 lL of stock was added. Similarly, forsolutions with EDANS-Ab42, 1.6 lL of stock was added.Deionized water was then added to each sample, suchthat the final volume was 300 lL. A 100-mM phosphate-buffered solution was prepared, and separated intoaliquots, with pHs ranging from 6.8 to 7.0 in incrementsof 0.05 pH units. Two hundred microliters of the bufferwith appropriate pH to make the final pH near 7.0 (i.e.compensate for the addition of pH 10 Ab stocks) wasadded, and 0.1 mM NaOH or HCl was added to makethe pH exactly 7.0, just prior to measurement. In mostcases the final adjustment involved no more than 1–2 lLof acid or base, and therefore had negligible effect on thefinal volume. This pH adjustment just prior to measure-ment ensures that the fibrillogenesis process does not startbefore measurements are taken. Controls (i.e. unlabeledAb alone and EDANS-Ab + hen lysozyme) were pre-pared exactly as above, with the exception that the henlysozyme (Sigma Chemical) stock was kept at pH 7, at1.57 mM, 4 �C. As the hen lysozyme stock was kept atpH 7, a phosphate buffer of the same pH was added forall samples.

Sample incubation

All samples were incubated for approximately 3 monthsat room temperature. They were kept in a dark area toprevent photobleaching of EDANS.

Fluorescence spectroscopy

Fluorescence assays were carried out at room temperatureusing a Photon Technology International QM-1 fluores-cence spectrophotometer equipped with excitation intensitycorrection and a magnetic stirrer. All samples were scannedin a quartz cuvette with 2 mm path length in the excitationdirection and 1 cm path length in the emission direction.Total sample volume was 0.5 mL. All constituents of thesamples (i.e. buffer, water, unlabeled Ab and labeled Ab)were first screened for the presence of fluorescent contami-nants, and only the labeled Ab stocks exhibited EDANSfluorescence. To measure EDANS fluorescence, emissionspectra were collected from 360 to 600 nm (kex ¼ 350 nm;step size ¼ 1 nm; 2 sÆnm)1; bandpass ¼ 2 nm). Afterobtaining the spectra, the control of unlabeled Ab alone (ofappropriate concentration) was subtracted in order tocorrect for the effect of light scattering by large aggregates.The resultant spectrum was then integrated over thewavelengths of 400–550 nm. In order to correct for dailyvariation in the UV lamp and slight variations in bandpass,as well as the minor unlabeled contaminant of EDANS-Abstocks described above, the fluorescence of EDANS-Abalone was subtracted from all other measurements, giving anormalized measure of the fluorescence of EDANS-Ab atdifferent concentrations over time. All fluorescence experi-ments were conducted in three different trials on differentdays.

Circular dichroism spectroscopy

CD spectra were recorded on an Aviv Circular DichroismSpectrometer model 62DS at 25 �C. Spectra were obtained

from 190 to 260 nm (1 mm path length, 1 nm step size,1 nm bandwidth).

Atomic force microscopy

All solution tapping atomic force microscopy imageswere acquired using a combination contact/tapping modeliquid cell fitted to a Digital Instruments Nanoscope IIIAMultiMode scanning probe (Digital Instruments, SantaBarbara, CA, USA). The AFM images were acquiredusing the E scanning head, which has a maximum lateralscan area of 14.6 · 14.6 lm. Samples were made bydiluting the appropriate Ab stocks with 100 mM

phosphate buffer (pH 7). Five microliters of the mixedsample solution were transferred onto a freshly cleavedmica surface, and the sample was sealed in the liquid cell.Sizes and volumes were calculated using Digital Instru-ments’ NANOSCOPE software (version 4.21) and theshareware image analysis program NIH-IMAGE (version1.62).

Results

Monitoring Ab association

We have employed a variation on the strategy used byour group to monitor Ab40 fibrillogenesis [32]. First, theAb40 and Ab42 peptides were synthesized separately.EDANS, an environment-sensitive fluorophore, wasadded to the N-terminus of aliquots of both Ab40 andAb42, separated from the rest of the sequence by aglycine linker. Samples of 0.1 lM EDANS-labeledAb40 (AED-Ab40) and 0, 10, 20 and 30 lM unlabeledAb40 or Ab42 were separately prepared. Similarly,samples of 0.1 lM AED-Ab42 were prepared with 0,10, 20 and 30 lM unlabeled Ab40 or Ab42. Thus, everycombination of Ab40 and Ab42 heterogeneous associ-ation, as well as homogeneous association was examined.Given that the threshold concentration for fibril forma-tion of Ab40 at neutral pH is between 10 and 40 lM [27],0, 10, 20 and 30 lM Ab are ideal concentrations tomonitor prefibrillar species.

To start the fibrillogenesis process, the pH of thesolution is lowered from 10 to 7 by addition ofphosphate buffer. The AEDANS fluorophore absorbsat approximately 350 nm, and emits at approximately480 nm. In samples with only AED-Ab, fluorescence at480 nm is relatively low due to fluorescence quenchingby water (Fig. 1). As the label is sequestered byunlabeled Ab, fluorescence increases. In order to controlfor light scattering by the peptides as an explanation forincreased fluorescence readings, we also scanned 10, 20and 30 lM unlabeled Ab over the same wavelengths, andsubtracted these spectra from the corresponding oneswith EDANS-Ab. In Fig. 3B, we show the unsubtractedfluorescence for EDANS-Ab40 incorporating intounlabeled Ab40 at the early time period, as well as thesubtracted fluorescence and the difference, over the threeconcentrations tested. As a second control, we preparedsamples of EDANS-Ab40 or EDANS-Ab42 with 0, 10,20 and 30 lM hen lysozyme. This is to ensure that anyobserved association is Ab-specific, and not simply due

656 D. Frost et al. (Eur. J. Biochem. 270) � FEBS 2003

Fig. 1. Assay for Ab association through the use of Ab labeled with an environment sensitive fluorophore (EDANS) and the characteristic spectra of thevarious aggregate species.d, EDANS-labeled Ab; s, unlabeled Ab. As the EDANS-Ab peptides reorganize into the structure late aggregates, the

emission peak wavelength shifts to 420 nm from the characteristic 480 nm.

Fig. 2. AFM image of Ab40 at early time period. The sample consists of 30 lM Ab40 in 40 mM phosphate, pH 7. The image was acquired

immediately after sample preparation. Homogeneous and heterogeneous mixtures of Ab40 and Ab42 formed these early aggregates of similar

morphology and size.


to hydrophobic interactions. Hen lysozyme is anotherpeptide that forms amyloid deposits [33].

Atomic force microscopy (AFM) at early time period

Immediately upon inducing fibrillogenesis by loweringpH, small Ab aggregates, approximately 5–10 nm inheight, are visible across the freshly cleaved mica surface(Fig. 2). Both mixed and homogeneous Ab solutionsform these aggregates, and both Ab variants form earlyaggregates of similar morphology and size. These early

aggregates are similar to those identified in previousstudies of Ab40.

Fluorescence assay for association at early time period

Immediate incorporation of EDANS-Ab40 and EDANS-Ab42 into unlabeled Ab40 or Ab42 occurs upon loweringpH (Fig. 3). Addition of labeled Ab40 to increasingconcentrations of both unlabeled Ab40 or unlabeledAb42 resulted in an increase in fluorescence intensityindicating that labeled Ab40 incorporates into bothaggregates of unlabeled Ab40 and Ab42. Similarly,labeled Ab42 was found to incorporate into bothaggregates of unlabeled Ab40 and Ab42 (Fig. 3C).Significantly, the observed incorporation is Ab-specific;controls of EDANS-Ab mixed with the same concentra-tions of hen lysozyme showed negligible incorporation.Three trials were conducted on separate days, and yieldedthese results consistently. The observed incorporation isnot due to light scattering from increasing proteinconcentrations, as spectra of unlabeled peptide alonewere subtracted from their counterparts with EDANS-labeled Ab to generate the data shown in Fig. 3. AllEDANS peaks in early spectra (i.e. upon mixture) occuraround the known maximum of approximately 480 nm(Fig. 4).

CD spectroscopy at early time period

Immediately upon mixing, at the time point whenassociation between Ab species occurs and aggregatesare small and amorphous, CD shows a spectrum of arandom coil or unstructured conformation (data notshown). The spectrum shows a minimum at approxi-mately 190 nm. The presence of small aggregates in thesesamples can confound the interpretation of CD spectra.However, we are confident that light-scattering effectshave not adversely influenced the results because thespectrum so closely resembles that of a typical randomcoil.

AFM after extended incubation

After 3 months of incubation, spherical prefibrillar aggre-gates (approximately 15 nm in height) have replaced theunstructured aggregates observed initially (Fig. 5A). Theseaggregates form in all samples examined, including sampleswith mixed Ab40 and Ab42. AFM on the control EDANS-Ab40 mixed with unlabeled hen lysozyme shows largeaggregates (Fig. 5B).

Fluorescence assay for association after extendedincubation

As shown in Fig. 6, when EDANS-Ab40 is allowed toincorporate into unlabeled Ab40 or Ab42 for extendedtime periods, it exhibits greater incorporation into Ab40,although incorporation into Ab42 also occurs. However,EDANS-Ab42 incorporates to a similar extent into eitherunlabeled Ab40 or Ab42. These results suggest that Ab40late aggregate formation displays a slight preference forhomogeneous vs. mixed aggregation, while Ab42 late

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Fig. 3. Fluorescence of EDANS-Ab40 with unlabeled Ab40, Ab42 orhen lysozyme immediately after mixing. (A) Fluorescence of 0.1 lM

EDANS-Ab40 with 0, 10, 20 and 30 lM unlabeled Ab40 (d), Ab42

(s) or hen lysozyme (j) immediately after mixing. (B) Fluorescence of

0.1 lM EDANS-Ab40 with 10, 20 and 30 lM unlabeled Ab40 imme-

diately after mixing. Data are shown before subtracting scattering

control of unlabeled Ab40 alone (h), after subtracting control of Ab40

alone (j) and the difference, which is signal due solely to scattering by

unlabeled Ab40 (filled grey square). (C) Fluorescence of 0.1 lM


(s) or hen lysozyme (j) immediately after mixing. Samples were

scanned after initiating reaction by dropping pH from 10 to 7. Samples

were excited at 350 nm and scanned from 360 to 600 nm. The resultant

spectra were integrated over 400–550 nm. Scans of 0, 10, 20 and 30 lM

unlabeled peptide alone over the same wavelengths were subtracted

from the EDANS spectra obtained.


aggregate formation does not display such a preference.In all cases, incorporation into the lysozyme controlremains negligible even after extended incubation. Thelysozyme aggregates observed by AFM (Fig. 5B) there-fore do not include the labeled Ab that was present inthe solution.

Significantly, the EDANS peak of approximately480 nm shifts to approximately 420 nm, concomitantwith late aggregate formation. In addition, the magnitudeof EDANS fluorescence is approximately five- to 10-foldhigher with late aggregates relative to early aggregates,indicating that the fluorophore is more sequestered in thelate aggregate. We have consistently observed thatstructured aggregate formation is accompanied by a blueshift and increased intensity in the EDANS spectrum.Figure 7 demonstrates that a biphasic distribution ofEDANS fluorescence exists at certain Ab concentrations,indicating a mixture of unstructured and structured Abaggregates in the solution. All fluorescence scans ofEDANS-Ab alone show maxima at 480 nm, indicatingno structured aggregate formation in these samples, asexpected by the trace concentration of labeled Ab (i.e.0.1 lM). The unshifted spectrum of AEDANS-Ab aloneafter extended incubation (Fig. 7) also eliminates thepossibility that the behavior of the EDANS fluorophorechanges due to the incubation itself rather than a changein the aggregate species. As mentioned above, three trialswere conducted over separate days, and yielded similarresults.

CD spectroscopy after extended incubation

CD spectra of samples showing large, structured aggre-gates and blue-shifted EDANS fluorescence were taken.As mentioned above, large aggregates can confound CDdata, but the spectrum obtained shows definite secondarystructure. The spectrum is somewhat similar to the typicalb-sheet spectrum, with a positive band around 200 nmand a negative band around 218 nm (data not shown).Although not fully b-sheet, these blue-shifted, large lateaggregates clearly show secondary structure in the CDspectrum, in stark contrast to the early aggregates whichare fully unstructured, both morphologically, shownby AFM, and spectroscopically, shown by CD.

Discussion

With growing interest in the process preceding fibril forma-tion in identifying a conclusively neurotoxic species, ourapproach avoids the problem associated with nucleation-extension studies, namely that only addition to a pre-existingfibril is studied. By starting the fibrillogenesis pathway andmonitoring association of Ab as soon the conditions permitassociation (i.e. lower pH from 10 to 7), we examine theinteraction between Ab40 and Ab42 throughout the entirepathway. We have demonstrated that early aggregates formin vitro at pH 7, and Ab40 and Ab42 prefer to incorporateinto Ab40 at this stage. Our assay allows us to distinguishunstructured aggregate from structured aggregate formation

Fig. 4. Fluorescence spectrum of EDANS-

Ab40 with unlabeled Ab40 immediately uponmixing. (A) Fluorescence spectrum of 0.1 lM

EDANS-Ab40 with 0 (h), 10 (j), 20 (s)

and 30 (d) lM unlabeled Ab40 immediately

upon mixing. Spectra of 0, 10, 20 and 30 lM

unlabeled Ab40 alone subtracted. Peaks occur

at normal EDANS fluorescence maximum

of approximately 480 nm. (B) Fluorescence

spectrum of 0.1 lM EDANS-Ab40 with 0 (h),

10 (j), 20 (s) and 30 (d) lM unlabeled

Ab42 immediately upon mixing. Spectra of 0,

10, 20 and 30 lM unlabeled Ab42 alone

subtracted. Peaks occur at normal EDANS

fluorescence maximum of approximately

480 nm.


because the EDANS spectrum shifts to a 420-nm peak whenstructured aggregates are present. Hence, we are able todetect the formation of structured aggregates after anextended incubation period (3 months).

There is evidence to suggest that the aggregates formedimmediately upon lowering the pH are similar to thediffuse plaques observed in vivo. The aggregates aremorphologically unstructured, and form a diffuse lawn

A

B

Fig. 5. AFM images of Ab40 (A) and henlysozyme (B) after extended incubation.

Samples consist of 30 lM Ab40 or hen lyso-

zyme in 40 mM phosphate, pH 7. Images were

acquired 3 months after sample preparation.

Homogeneous and heterogeneous mixtures of

Ab40 and Ab42 formed these spherical prefi-

brillar aggregates with a uniform distribution

of morphology and size. CD spectroscopy

indicates that they contain secondary struc-

ture. Hen lysozyme mixed with labeled Abformed large aggregates (note that the scale of

the hen lysozyme image is five times larger

than that of the Ab40 image).


on the mica AFM surface. They are accessible to waterrelative to the aggregates formed later (recall the 10-foldincrease in EDANS fluorescence after extended incuba-tion). The fluorescence spectrum of the EDANS-labeledAb incorporated into them shows a distinct peak fromthat which occurs after extended incubation, suggesting adifference in the fluorescent behavior of the fluorophorein each of the aggregates. Finally, CD spectroscopyshows these early aggregates to be random coil (i.e.without secondary structure).

Similarly, evidence can link the late aggregates to senileplaques. AFM shows a large, well-defined sphericalstructure, whose height is consistent with the diameterof typical Ab amyloid fibrils. Fluorescence shows that thepeptides are highly sequestered from water, indicatingtighter packing. The fluorescence spectrum shifts to420 nm from 480 nm, indicating a significant change influorophore behavior. Finally, CD shows the aggregatesto be structured, with spectra similar to those of b-sheet(Ab fibrils found in senile plaques also have b-sheetsecondary structure).

After 3 months of incubation, fibrils were not detectedby EM or AFM in any of the 0, 10, 20 and 30 lM Absamples tested. Because we undertook this study toexamine the early aggregation events, not the fibrils perse, we have chosen Ab concentrations near or below the

known threshold for Ab40 fibril formation under theconditions tested. It is therefore not surprising thatfibrils have not formed in these samples. It is alsoimportant to note that fibril formation is quite difficultto achieve de novo. As described in the Materials andmethods section, we have employed a rigorous proce-dure to prevent the formation of fibrillar seeds in ourstock Ab solutions. This allows us to examine prefibril-lar structures. We are confident therefore that byexamining concentrations at or near threshold for fibrilformation, prefibrillar structures are the major speciespresent.

After sufficient time for structured aggregates to form,we find that both homogeneous and mixed aggregateshave formed, but that Ab40 shows a slight bias towardsassociating with Ab40 to form spherical aggregates.Significantly, Ab42 associates equivalently with itself orAb40 in these aggregates. Given that diffuse plaquesconsist mostly of Ab42, and senile plaques of bothvariants, the addition of monomer Ab42 to localAb40 appears to favor the production of structuredaggregates, which could lead to senile plaques. Itappears less likely for these structured aggregates toform when only Ab42 is present. In vivo, such atransition could conceivably be caused by an increase inoverall cerebral Ab, which would probably be mostly anincrease in Ab40, as this variant is generally moreabundant.

Mixed aggregates occur in vitro, and the associationbetween the Ab variants begins before fibrils form. Thetransition between unstructured and structured aggregatecould be vital in the progression from diffuse to senileplaque; this transition is unlikely to be direct, as solid-to-solid transitions are rare, and usually require ratherextreme conditions (e.g. graphite to diamond). Morelikely is that unstructured and structured aggregates arealternate aggregation products of soluble Ab. This studyshows that the local Ab42/Ab40 ratio can significantlyinfluence the ease of formation of structured aggregates,as in some cases mixed aggregates form more easily thanhomogeneous ones; indeed such a change in Ab42/Ab40ratio has been identified in some forms of familialAD [35]. The easier formation of mixed aggregates insome cases tested in vitro may also help explain thedifference in Ab variant content in diffuse vs. senileplaques.

This work has demonstrated the possibility for Ab toform both mixed early unstructured aggregates (similar todiffuse plaques) and late structured aggregates (possiblyan intermediate in the transition to senile plaques), andhas shown that, in vitro, Ab40 and Ab42 associate earlyin the fibrillogenesis pathway. We have also demonstra-ted an interesting property of the EDANS fluorophore,namely that its fluorescence spectrum shifts concomitantwith structured aggregate formation. This could be quiteuseful in other fibrillogenesis studies. More work isneeded to elucidate not only the aggregation andfibrillogenesis pathway of Ab40, which is an area ofmuch active research, but also the role that Ab42/Ab40interaction plays in the formation of senile plaques. Thisstudy provides a starting point for further investigation inthis regard.

Fig. 6. Spectra from samples excited at 350 nm and scanned from 360 to

600 nm. Samples were excited at 350 nm and scanned from 360 to

600 nm. The resultant spectra were integrated over 400–550 nm. Scans

of 0, 10, 20 and 30 lM unlabeled peptide alone over the same wave-

lengths were subtracted from the EDANS spectra obtained. (A)

Fluorescence of 0.1 lM EDANS-Ab40 with 0, 10, 20 and 30 lM

unlabeled Ab40 (d), Ab42 (s) or hen lysozyme (j) after incubation

for approximately 3 months at pH 7. (B) Fluorescence of 0.1 lM


(s) or hen lysozyme (j) after incubation for approximately 3 months

at pH 7.


Acknowledgments

This work was supported by a grant to A.C. from the Canadian

Institutes for Health Research (CIHR) and by a grant to CMY from

CIHR. PMG acknowledges support from the Ontario Student

Opportunity Transfer Fund, a Scace Graduate Fellowship in Alzhei-

mer’s Research, and a Gamble Grant Graduate Fellowship. DF

acknowledges support from a Natural Science and Engineering

Research Council summer studentship.

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0

5000

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20000

350 400 450 500 550 600

Flu

ore

scence

(co

unts

/s)

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0

2000

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6000

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A

B

Fig. 7. Fluorescence spectra of EDANS-Ab40with unlabeled Ab40 after incubation forapproximately 3 months. (A) Fluorescence

spectrum of 0.1 lM EDANS-Ab40 with 0 (h),

10 (j), 20 (s) and 30 (d) lM unlabeled Ab40

after incubation for approximately 3 months.

Spectra of similarly incubated 0, 10, 20 and

30 lM unlabeled Ab40 alone subtracted. Peak

occurs at shifted EDANS fluorescence

maximum of approximately 420 nm, which

corresponds to structured aggregate forma-

tion. A somewhat biphasic distribution of

maxima (420 and 480 nm) is visible in the

10 lM spectrum, corresponding to a mixed

population of structured and unstructured

aggregates. (B) Fluorescence spectrum of

0.1 lM EDANS-Ab40 with 0 (h), 10 (j), 20

(s) and 30 (d) lM unlabeled Ab42 after

incubation for 3 months. Spectra of similarly

incubated 0, 10, 20 and 30 lM Ab42 subtrac-

ted. Biphasic distribution of EDANS peaks

corresponds to a mixed population of struc-

tured and unstructured aggregates.


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Co-incorporation of Aβ40 and Aβ42 to form mixed pre-fibrillar aggregates