Báo cáo khoa học: Co-incorporation of Ab40 and Ab42 to form mixed pre-fibrillar aggregates ppt

Co-incorporation of Ab40 and Ab42 to form mixed pre-fibrillar aggregates David Frost 1 , Paul M. Gorman 1 , Christopher M. Yip 2 and Avijit Chakrabartty 1 1 Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, and 2 Department 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 proximal cause 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 with neurodegeneration, consist mainly of unstructured Ab42. We have investigated the interaction between Ab40 and Ab42 through an assay, which involves labeling both vari- ants with an environment-sensitive fluorophore. We have monitored association of Ab without fibrillar seeds, which allows investigation of molecular species preceding fibrils. Immediately upon mixture, Ab40 and Ab42 associate into mixed aggregates, in which the peptides are unstructured and relatively 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 show that in vitro the two Ab variants coassemble early in the fibrillogenesis pathway. The ease of formation for mixed and homogeneous aggregates is similar. A change in the local Ab variant ratio can therefore have a significant impact on Ab aggregation; indeed such a change has been reported in some types of familial AD. Keywords: Alzheimer’s disease; amyloid; fibril; peptide; fluorescence. Alzheimer’s disease (AD) is a significant and increasing health concern. Neurohistological studies have uncovered several hallmarks that distinguish the AD brain from its normal counterpart. Chief among these are neurofibrillary tangles (NFTs) andsenile plaques (SPs). NFTs are composed of paired helical filaments of the (normally) microtuble- associated Tau protein, while senile plaques are primarily comprised of the 40- and 42-residue forms of the Ab peptide [1,2]. The interaction between Ab40 and Ab42, the two major variants of the Ab peptide, is the subject of this study. The Ab family of peptides is enzymatically cleaved from the amyloid precursor protein (APP), a 563–770 residue membrane protein that is expressed in both neuronal and non-neuronal tissue [3,4]. Ab40, and to a lesser extent Ab42, are normal constituents of cerebrospinal fluid [5–8]. Both forms are capable of assembling into 60–100 A ˚ diameter b-sheet fibrils, which form the core of the aforementioned senile plaques. An impressive body of evidence points to Ab depo- sition in senile plaques as the causal event in AD pathology. Upon postmortem examination of AD brains, senile plaques are invariably found in the limbic and association cortices, surrounded by dead or dying neurons, as well as activated microglia and reactive astrocytes [1]. In several forms of familial AD, mutations in the APP gene have been identified [9,10]. Also, mutations of the presenilin genes have been linked to familial AD, and appear to lead to an increase in the ratio of Ab42 to Ab40 [11]. Transgenic mice over expressing a mutant form of APP develop neurohistolo- gical characteristics similar to those of AD patients [12–14]. Perhaps most convincingly, Down’s syndrome patients, who receive a triple dose of the genes present on chromosome 21, including the APP gene, often show senile plaque deposition and classical AD neurohistology in their late 20s or early 30s, followed by progressive cognitive and behavioral dysfunction in their mid 30s [15]. Unlike senile plaques, diffuse plaques are more loosely packed depositions of mostly unstructured Ab42 [1]. Diffuse plaques are not associated with dead or dying neurons, and have been found upon post mortem exami- nation of the brains of elderly people who had not exhibited AD symptoms [16–20]. Diffuse plaques are also referred to as Ôpreamyloid plaquesÕ because of several lines of evidence that point to them as precursors to senile plaques. 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 also develop diffuse Ab42 plaques before fibrillar plaques surrounded 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: chakrab@uhnres.utoronto.ca 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) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03415.x The observation that Ab42 diffuse plaques lead to senile plaques, consisting of both Ab variants, and the apparent importance of the Ab40/Ab42ratioinAD[22]haveledus to investigate the interaction between these two peptides. Studies by Hasegawa et al.[23]havedemonstratedthe ability of preformed Ab42 fibrils to seed the fibrillogenesis of Ab40, as well as the ability of Ab40 to seed Ab42 fibrillogenesis. However, with little conclusive evidence that Ab fibrils are neurotoxic, more attention is being paid to prefibrillar Ab species in the search for a clear culprit in AD pathology [24]. Recent work by our group [25] as well as others [26–28], has shown a complex series of reactions, which precedes the formation of mature fibrils. To our knowledge, no study has been undertaken to determine at which point in the fibrillogenesis pathway Ab40 and Ab42 can form mixed molecular species, and the relative ease of formation of mixed vs. homogenous species. We believe that the approach of studying fibrillogenesis in the context of Ab40 and Ab42 mixtures is advantageous. Studies are usually of either Ab40 or Ab42, while it is known that in vivo, both Ab40 and Ab42 are present, and their interaction may play a key role in the transition between relatively innocuous diffuse plaques and possibly neurotoxic senile plaques. Furthermore, many in vitro studies examine fibril formation without rigorously removing all fibril seeds, thereby making it impossible to characterize all species preceding fibrils. In the present study, we ensure a homo- geneous starting solution of monomeric Ab peptides, thereby permitting an examination of the interaction between Ab40 and Ab42 throughout the fibrillogenic pathway. Materials and methods Peptide synthesis A PerSeptive Biosystems 9050 Plus peptide synthesizer was used to separately prepare both Ab40 and Ab42 by solid phase peptide synthesis. An active ester coupling procedure, employing O-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate of 9-fluorenyl- methoxycarbonyl amino acids was used. The magnitude of the syntheses was 0.05 mmol, and a three times excess of reagents was used. The peptides were cleaved from the resin with 95 : 5 trifluoroacetic acid and anisol mixture. The cleavage mixture was incubated at room temperature for 30 min, and the resin removed by filtration. Bromo- trimethylsilane was added to a final concentration of 12.5% (v/v). The peptides were then precipitated and washed five times in cold ether. The peptides were removed from the ether and dissolved in 6 M guanidine hydrochloride, 0.15% NH 4 OH (pH 10) and purified by HPLC. The purified peptide was chromatographed on a Sephadex G-75 column (Amersham Pharmacia Biotech) and the fractions corresponding to the correct monomer molecular mass collected. Electrospray mass spectrometry confirmed the presence of the correct molecular mass, and purity was determined by six cycles of PTH peptide sequencing by the Edman degradation reaction (Porton gas-phase Microsequencer, model 2090), which revealed that the purified peptides had the correct sequence. Sequencing proceeds from the N- to the C-terminus, while automated synthesis proceeds in the opposite direction. By confirming that the main peptide present has the correct N-terminal sequence, purity is established, as any errors in synthesis usually result in a truncated N-terminus. Fluorescent labeling A glycine residue was added to the N-terminus of both Ab40 and Ab42 prior to addition of the fluorophore. This acts as a flexible linker to prevent the fluorophore from interfering 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 sequencing revealed the major peptide present in each synthesis to be the correct labeled Ab, with a minor contaminant of unlabeled Ab. Preparation of stock peptide solutions without fibril seeds After chromatographic separation as described above, both the labeled and unlabeled peptides were separately stored at pH 10 at 4 °C until use. This method of stock storage has previously been used by us [25] and others [29] to successfully prevent the formation of fibril seeds. Peptide concentration determination For unlabeled Ab peptides, tyrosine absorbance of UV light (275 nm) was used to determine concentration in 0.15% NH 4 OH by Beer–Lambert law (e ¼ 1390 cm )1 Æ M )1 [30]). Each concentration obtained was multi- plied by the appropriate dilution factor to obtain stock concentrations. The EDANS-labeled peptide stock con- centration was determined by EDANS absorbance at 338 nm (e ¼ 6500 cm )1 Æ M )1 [31]). This method of con- centration determination was used because it ensures the correct concentration of labeled peptide is obtained, and is not affected by the minor unlabeled Ab contaminant described above. All absorbance measurements were made on a Milton-Roy Spectronic 3000 spectrophoto- meter. Sample preparation All samples were measured at pH 7 with 40 m M phos- phate buffer. Into Eppendorf tubes, first the amount of unlabeled Ab stock (stored at pH 10, 4 °C) appropriate for each concentration tested was added. Stock peptide concentrations were 0.212 m M for Ab40 and 0.165 m M for Ab42. As the final solution volume was 500 lL, each increment of 10 l M unlabeled Ab40 required the addition of 23.6 lL of stock. Similarly, each 10 l M increment of unlabeled Ab42 required 30.3 lL of stock. Next, the labeled Ab was added to the solution. Stock concentra- tions were 8.65 l M for EDANS-Ab40 and 31.9 l M for EDANS-Ab42, both stored at pH 10, 4 °C. All solutions tested that included labeled Ab had a concentration of 0.1 l M EDANS-Ab. Therefore, for the solutions with Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 655 EDANS-Ab40, 5.8 lL of stock was added. Similarly, for solutions with EDANS-Ab42, 1.6 lL of stock was added. Deionized water was then added to each sample, such that the final volume was 300 lL. A 100-m M phosphate- buffered solution was prepared, and separated into aliquots, with pHs ranging from 6.8 to 7.0 in increments of 0.05 pH units. Two hundred microliters of the buffer with appropriate pH to make the final pH near 7.0 (i.e. compensate for the addition of pH 10 Ab stocks) was added, and 0.1 m M NaOH or HCl was added to make the pH exactly 7.0, just prior to measurement. In most cases the final adjustment involved no more than 1–2 lL of acid or base, and therefore had negligible effect on the final volume. This pH adjustment just prior to measure- ment ensures that the fibrillogenesis process does not start before measurements are taken. Controls (i.e. unlabeled Ab alone and EDANS-Ab + hen lysozyme) were pre- pared exactly as above, with the exception that the hen lysozyme (Sigma Chemical) stock was kept at pH 7, at 1.57 m M ,4°C. As the hen lysozyme stock was kept at pH 7, a phosphate buffer of the same pH was added for all samples. Sample incubation All samples were incubated for approximately 3 months at room temperature. They were kept in a dark area to prevent photobleaching of EDANS. Fluorescence spectroscopy Fluorescence assays were carried out at room temperature using a Photon Technology International QM-1 fluores- cence spectrophotometer equipped with excitation intensity correction and a magnetic stirrer. All samples were scanned in a quartz cuvette with 2 mm path length in the excitation direction and 1 cm path length in the emission direction. Total sample volume was 0.5 mL. All constituents of the samples (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 EDANS fluorescence. To measure EDANS fluorescence, emission spectra were collected from 360 to 600 nm (k ex ¼ 350 nm; step size ¼ 1nm; 2sÆnm )1 ; bandpass ¼ 2nm). After obtaining the spectra, the control of unlabeled Ab alone (of appropriate concentration) was subtracted in order to correct for the effect of light scattering by large aggregates. The resultant spectrum was then integrated over the wavelengths of 400–550 nm. In order to correct for daily variation in the UV lamp and slight variations in bandpass, as well as the minor unlabeled contaminant of EDANS-Ab stocks described above, the fluorescence of EDANS-Ab alone was subtracted from all other measurements, giving a normalized measure of the fluorescence of EDANS-Ab at different concentrations over time. All fluorescence experi- ments were conducted in three different trials on different days. Circular dichroism spectroscopy CD spectra were recorded on an Aviv Circular Dichroism Spectrometer 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 images were acquired using a combination contact/tapping mode liquid cell fitted to a Digital Instruments Nanoscope IIIA MultiMode scanning probe (Digital Instruments, Santa Barbara, CA, USA). The AFM images were acquired using the E scanning head, which has a maximum lateral scan area of 14.6 · 14.6 lm. Samples were made by diluting the appropriate Ab stocks with 100 m M phosphate buffer (pH 7). Five microliters of the mixed sample solution were transferred onto a freshly cleaved mica 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 the shareware image analysis program NIH - IMAGE (version 1.62). Results Monitoring Ab association We have employed a variation on the strategy used by our group to monitor Ab40 fibrillogenesis [32]. First, the Ab40 and Ab42 peptides were synthesized separately. EDANS, an environment-sensitive fluorophore, was added to the N-terminus of aliquots of both Ab40 and Ab42, separated from the rest of the sequence by a glycine linker. Samples of 0.1 l M EDANS-labeled Ab40 (AED-Ab40) and 0, 10, 20 and 30 l M unlabeled Ab40 or Ab42 were separately prepared. Similarly, samples of 0.1 l M AED-Ab42 were prepared with 0, 10, 20 and 30 l M unlabeled Ab40 or Ab42. Thus, every combination 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 l M [27], 0, 10, 20 and 30 l M Ab are ideal concentrations to monitor prefibrillar species. To start the fibrillogenesis process, the pH of the solution is lowered from 10 to 7 by addition of phosphate buffer. The AEDANS fluorophore absorbs at approximately 350 nm, and emits at approximately 480 nm. In samples with only AED-Ab, fluorescence at 480 nm is relatively low due to fluorescence quenching by water (Fig. 1). As the label is sequestered by unlabeled Ab, fluorescence increases. In order to control for light scattering by the peptides as an explanation for increased fluorescence readings, we also scanned 10, 20 and 30 l M unlabeled Ab over the same wavelengths, and subtracted these spectra from the corresponding ones with EDANS-Ab. In Fig. 3B, we show the unsubtracted fluorescence for EDANS-Ab40 incorporating into unlabeled Ab40 at the early time period, as well as the subtracted fluorescence and the difference, over the three concentrations tested. As a second control, we prepared samples of EDANS-Ab40 or EDANS-Ab42 with 0, 10, 20 and 30 l M hen lysozyme. This is to ensure that any observed association is Ab-specific, and not simply due 656 D. Frost et al. (Eur. J. Biochem. 270) Ó 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 the various aggregate species. d, EDANS-labeled Ab; s,unlabeledAb.AstheEDANS-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. Thesampleconsistsof30l M Ab40 in 40 m M 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. Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 657 to hydrophobic interactions. Hen lysozyme is another peptide that forms amyloid deposits [33]. Atomic force microscopy (AFM) at early time period Immediately upon inducing fibrillogenesis by lowering pH, small Ab aggregates, approximately 5–10 nm in height, are visible across the freshly cleaved mica surface (Fig. 2). Both mixed and homogeneous Ab solutions form these aggregates, and both Ab variants form early aggregates of similar morphology and size. These early aggregates are similar to those identified in previous studies 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 lowering pH (Fig. 3). Addition of labeled Ab40 to increasing concentrations of both unlabeled Ab40 or unlabeled Ab42 resulted in an increase in fluorescence intensity indicating that labeled Ab40 incorporates into both aggregates of unlabeled Ab40 and Ab42. Similarly, labeled Ab42 was found to incorporate into both aggregates 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 yielded these results consistently. The observed incorporation is not due to light scattering from increasing protein concentrations, as spectra of unlabeled peptide alone were subtracted from their counterparts with EDANS- labeled Ab to generate the data shown in Fig. 3. All EDANS peaks in early spectra (i.e. upon mixture) occur around the known maximum of approximately 480 nm (Fig. 4). CD spectroscopy at early time period Immediately upon mixing, at the time point when association between Ab species occurs and aggregates are small and amorphous, CD shows a spectrum of a random coil or unstructured conformation (data not shown). The spectrum shows a minimum at approxi- mately 190 nm. The presence of small aggregates in these samples can confound the interpretation of CD spectra. However, we are confident that light-scattering effects have not adversely influenced the results because the spectrum so closely resembles that of a typical random coil. AFM after extended incubation After 3 months of incubation, spherical prefibrillar aggre- gates (approximately 15 nm in height) have replaced the unstructured aggregates observed initially (Fig. 5A). These aggregates form in all samples examined, including samples with mixed Ab40 and Ab42. AFM on the control EDANS- Ab40 mixed with unlabeled hen lysozyme shows large aggregates (Fig. 5B). Fluorescence assay for association after extended incubation AsshowninFig.6,whenEDANS-Ab40 is allowed to incorporate into unlabeled Ab40 or Ab42 for extended time periods, it exhibits greater incorporation into Ab40, although incorporation into Ab42 also occurs. However, EDANS-Ab42 incorporates to a similar extent into either unlabeled Ab40 or Ab42. These results suggest that Ab40 late aggregate formation displays a slight preference for homogeneous vs. mixed aggregation, while Ab42 late 0 50000 100000 150000 200000 250000 300000 0 5 10 15 20 25 30 3 5 0 50000 100000 150000 200000 250000 300000 350000 0 5 10 15 20 25 30 35 [Unlabeled peptide] (µM) [Unlabeled peptide] (µM) Fluorescence (400-550nm) Fluorescence (400-550nm) 0 50000 100000 150000 200000 250000 300000 350000 400000 10 20 30 Fluorescence (400-550nm) [Unlabeled peptide] ( M) A B C Fig. 3. Fluorescence of EDANS-Ab40 with unlabeled Ab40, Ab42 or hen lysozyme immediately after mixing. (A) Fluorescence of 0.1 l M EDANS-Ab40 with 0, 10, 20 and 30 l M unlabeled Ab40 (d),Ab42 (s) or hen lysozyme (j) immediately after mixing. (B) Fluorescence of 0.1 l M EDANS-Ab40 with 10, 20 and 30 l M 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 l M EDANS-Ab42 with 0, 10, 20 and 30 l M unlabeled Ab40 (d),Ab42 (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 l M unlabeled peptide alone over the same wavelengths were subtracted from the EDANS spectra obtained. 658 D. Frost et al. (Eur. J. Biochem. 270) Ó FEBS 2003 aggregate formation does not display such a preference. In all cases, incorporation into the lysozyme control remains negligible even after extended incubation. The lysozyme aggregates observed by AFM (Fig. 5B) there- fore do not include the labeled Ab that was present in the solution. Significantly, the EDANS peak of approximately 480 nm shifts to approximately 420 nm, concomitant with late aggregate formation. In addition, the magnitude of EDANS fluorescence is approximately five- to 10-fold higher with late aggregates relative to early aggregates, indicating that the fluorophore is more sequestered in the late aggregate. We have consistently observed that structured aggregate formation is accompanied by a blue shift and increased intensity in the EDANS spectrum. Figure 7 demonstrates that a biphasic distribution of EDANS fluorescence exists at certain Ab concentrations, indicating a mixture of unstructured and structured Ab aggregates in the solution. All fluorescence scans of EDANS-Ab alone show maxima at 480 nm, indicating no structured aggregate formation in these samples, as expected by the trace concentration of labeled Ab (i.e. 0.1 l M ). The unshifted spectrum of AEDANS-Ab alone after extended incubation (Fig. 7) also eliminates the possibility that the behavior of the EDANS fluorophore changes due to the incubation itself rather than a change in the aggregate species. As mentioned above, three trials were conducted over separate days, and yielded similar results. 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 CD data, but the spectrum obtained shows definite secondary structure. The spectrum is somewhat similar to the typical b-sheet spectrum, with a positive band around 200 nm and a negative band around 218 nm (data not shown). Although not fully b-sheet, these blue-shifted, large late aggregates clearly show secondary structure in the CD spectrum, in stark contrast to the early aggregates which are fully unstructured, both morphologically, shown by AFM, and spectroscopically, shown by CD. Discussion With growing interest in the process preceding fibril forma- tion in identifying a conclusively neurotoxic species, our approach avoids the problem associated with nucleation- extension studies, namely that only addition to a pre-existing fibril is studied. By starting the fibrillogenesis pathway and monitoring association of Ab as soon the conditions permit association (i.e. lower pH from 10 to 7), we examine the interaction between Ab40 and Ab42 throughout the entire pathway. We have demonstrated that early aggregates form in vitro at pH 7, and Ab40 and Ab42 prefer to incorporate into Ab40 at this stage. Our assay allows us to distinguish unstructured aggregate from structured aggregate formation Fig. 4. Fluorescence spectrum of EDANS- Ab40 with unlabeled Ab40 immediately upon mixing. (A) Fluorescence spectrum of 0.1 l M EDANS-Ab40 with 0 (h),10(j),20(s) and 30 (d) l M unlabeled Ab40 immediately upon mixing. Spectra of 0, 10, 20 and 30 l M unlabeled Ab40 alone subtracted. Peaks occur at normal EDANS fluorescence maximum of approximately 480 nm. (B) Fluorescence spectrum of 0.1 l M EDANS-Ab40 with 0 (h), 10 (j),20(s) and 30 (d) l M unlabeled Ab42 immediately upon mixing. Spectra of 0, 10, 20 and 30 l M unlabeled Ab42 alone subtracted. Peaks occur at normal EDANS fluorescence maximum of approximately 480 nm. Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 659 because the EDANS spectrum shifts to a 420-nm peak when structured aggregates are present. Hence, we are able to detect the formation of structured aggregates after an extended incubation period (3 months). There is evidence to suggest that the aggregates formed immediately upon lowering the pH are similar to the diffuse plaques observed in vivo. The aggregates are morphologically unstructured, and form a diffuse lawn A B Fig. 5. AFM images of Ab40 (A) and hen lysozyme (B) after extended incubation. Samples consist of 30 l M Ab40 or hen lyso- zyme in 40 m M 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 Ab formed large aggregates (note that the scale of the hen lysozyme image is five times larger than that of the Ab40 image). 660 D. Frost et al. (Eur. J. Biochem. 270) Ó FEBS 2003 on the mica AFM surface. They are accessible to water relative to the aggregates formed later (recall the 10-fold increase in EDANS fluorescence after extended incuba- tion). The fluorescence spectrum of the EDANS-labeled Ab incorporated into them shows a distinct peak from that which occurs after extended incubation, suggesting a difference in the fluorescent behavior of the fluorophore in each of the aggregates. Finally, CD spectroscopy shows these early aggregates to be random coil (i.e. without secondary structure). Similarly, evidence can link the late aggregates to senile plaques. AFM shows a large, well-defined spherical structure, whose height is consistent with the diameter of typical Ab amyloid fibrils. Fluorescence shows that the peptides are highly sequestered from water, indicating tighter packing. The fluorescence spectrum shifts to 420 nm from 480 nm, indicating a significant change in fluorophore behavior. Finally, CD shows the aggregates to be structured, with spectra similar to those of b-sheet (Ab fibrils found in senile plaques also have b-sheet secondary structure). After 3 months of incubation, fibrils were not detected by EM or AFM in any of the 0, 10, 20 and 30 l M Ab samples tested. Because we undertook this study to examine the early aggregation events, not the fibrils per se,wehavechosenAb concentrations near or below the known threshold for Ab40 fibril formation under the conditions tested. It is therefore not surprising that fibrils have not formed in these samples. It is also important to note that fibril formation is quite difficult to achieve de novo. As described in the Materials and methods section, we have employed a rigorous proce- dure to prevent the formation of fibrillar seeds in our stock Ab solutions. This allows us to examine prefibril- lar structures. We are confident therefore that by examining concentrations at or near threshold for fibril formation, prefibrillar structures are the major species present. After sufficient time for structured aggregates to form, we find that both homogeneous and mixed aggregates have formed, but that Ab40 shows a slight bias towards associating with Ab40 to form spherical aggregates. Significantly, Ab42 associates equivalently with itself or Ab40 in these aggregates. Given that diffuse plaques consist mostly of Ab42, and senile plaques of both variants, the addition of monomer Ab42 to local Ab40 appears to favor the production of structured aggregates, which could lead to senile plaques. It appears less likely for these structured aggregates to form when only Ab42 is present. In vivo,sucha transition could conceivably be caused by an increase in overall cerebral Ab, which would probably be mostly an increase in Ab40, as this variant is generally more abundant. Mixed aggregates occur in vitro, and the association between the Ab variants begins before fibrils form. The transition between unstructured and structured aggregate could be vital in the progression from diffuse to senile plaque; this transition is unlikely to be direct, as solid-to- solid transitions are rare, and usually require rather extreme conditions (e.g. graphite to diamond). More likely is that unstructured and structured aggregates are alternate aggregation products of soluble Ab. This study shows that the local Ab42/Ab40 ratio can significantly influence the ease of formation of structured aggregates, as in some cases mixed aggregates form more easily than homogeneous ones; indeed such a change in Ab42/Ab40 ratio has been identified in some forms of familial AD [35]. The easier formation of mixed aggregates in some cases tested in vitro may also help explain the difference in Ab variant content in diffuse vs. senile plaques. This work has demonstrated the possibility for Ab to form both mixed early unstructured aggregates (similar to diffuse plaques) and late structured aggregates (possibly an intermediate in the transition to senile plaques), and has shown that, in vitro, Ab40 and Ab42 associate early in the fibrillogenesis pathway. We have also demonstra- ted an interesting property of the EDANS fluorophore, namely that its fluorescence spectrum shifts concomitant with structured aggregate formation. This could be quite useful in other fibrillogenesis studies. More work is needed to elucidate not only the aggregation and fibrillogenesis pathway of Ab40, which is an area of much active research, but also the role that Ab42/Ab40 interaction plays in the formation of senile plaques. This study provides a starting point for further investigation in this 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 l M unlabeled peptide alone over the same wave- lengths were subtracted from the EDANS spectra obtained. (A) Fluorescence of 0.1 l M EDANS-Ab40 with 0, 10, 20 and 30 l M unlabeled Ab40 (d),Ab42 (s) or hen lysozyme (j) after incubation for approximately 3 months at pH 7. (B) Fluorescence of 0.1 l M EDANS-Ab42 with 0, 10, 20 and 30 l M unlabeled Ab40 (d),Ab42 (s) or hen lysozyme (j) after incubation for approximately 3 months at pH 7. Ó FEBS 2003 Pre-fibrillary association between Ab40 and Ab42 (Eur. J. Biochem. 270) 661 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. 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Gorman 1 , Christopher M. Yip 2 and Avijit Chakrabartty 1 1 Division