Báo cáo khoa học: b-Amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH docx

b-Amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH Genevieve M. J. A. Klug 1 , Dusan Losic 2,3 , Supundi S. Subasinghe 1,2 , Marie-Isabel Aguilar 2 , Lisandra L. Martin 3 and David H. Small 2 1 Department of Pathology, University of Melbourne, Victoria, Australia; 2 Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia; 3 Department of Chemistry, Flinders University, Adelaide, Australia Amyloid protein (Ab1–40) aggregation and conformation was examined using native and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis, and the results com- pared with those obtained by atomic force microscopy, and with Congo red binding, sedimentation and turbidity assays. The amount of Ab aggregation measured was different, depending upon the method used. Incubation for 15 min at pH 5.0 or in the presence of Fe 2+ , Cu 2+ or Zn 2+ did not alter the level of Ab oligomers observed on SDS and native gels. However, the slow aggregation of Ab to form high molecular mass species over 5 days was inhibited. In contrast, when Ab aggregation was monit- ored using a Congo red binding assay or sedimentation assay, a rapid increase in Ab aggregation was observed after incubation for 15 min at pH 5.0, or in the presence of Fe 2+ , Cu 2+ or Zn 2+ .ThelowpH-,Zn 2+ -orCu 2+ - induced Ab aggregation measured in a turbidity assay was reversible. In contrast, a considerable proportion of the Ab aggregation measured by native and SDS/PAGE was stable. Atomic force microscopy studies showed that Ab aged at pH 5.0 or in the presence of Zn 2+ produced larger looser rod-shaped aggregates than at pH 7.4. Ab that had been aged at pH 7.4 was more cytotoxic than Ab aged at pH 5.0. Taken together, the results suggest that Ab oligomerizes via two mutually exclusive mechanisms to form two different types of aggregates, which differ in their cytotoxic properties. 1 Keywords: Alzheimer’s disease; amyloid; Ab aggregation; toxicity; fibril. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by the accumulation of amyloid in the brain in the form of amyloid plaques and cerebral amyloid angiopathy. The major component of the amyloid plaques, the amyloid-b protein (Ab), is a polypeptide of 39–43 amino-acid residues, which is derived from a larger amyloid-b protein precursor (APP) [1–4]. Ab can poly- merize via a nucleation-dependent process [5,6] generating insoluble fibrillar aggregates which form amyloid plaques. Analysis of plaque amyloid has revealed that these aggre- gates adopt a b-sheet arrangement [7,8]. Aggregation of Ab in vivo may also lead to the formation of ill-formed, nonfibrillar amorphous aggregates known as the diffuse or ÔfleecyÕ plaques [9]. There is strong evidence that Ab has a causative role in the development of AD. The neurotoxicity of Ab has been demonstrated in neuronal cultures [10–12] and aggregation of Ab, which can be generated by ÔagingÕ (i.e. incubation of the peptide for several days), is required for this effect [10,11]. Recent studies have shown that low molecular mass oligomeric species are also neurotoxic [13–15]. In contrast, diffuse, amorphous aggregates of Ab do not appear to possess the neurotoxic properties of the fibrillar forms [16]. The mechanism by which monomeric Ab is converted to high molecular mass species in vivo is unknown. The influence of metal ions on aggregation in vitro has been investigated extensively. Zn 2+ and Cu 2+ have been shown to promote aggregation [17–20] and it has been suggested that the toxicity of Ab involves free radical-induced oxidative damage through the involvement of Cu 2+ [20,21]. Several studies have demonstrated that the aggre- gation of Ab can occur under acid pH conditions [22,23], such as those which occur in intracellular vesicular com- partments. Thus, some Ab aggregation could also occur intracellularly, prior to secretion. Not all studies have yielded similar conclusions about Ab aggregation. For example, it has been reported that at pH 5.0, Ab rapidly aggregates to form fibrils [24]. However, a more recent study suggested that aggregated species generated at low pH are nonfibrillar and are unable to be converted into fibrils or to seed fibril formation [25]. The aim of the present study was to examine the effect of pH and metal ions on the aggregation and conformation of Correspondence to D. H. Small, Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3800, Australia. Fax: + 61 3 9905 3726, Tel.: + 61 3 9905 1563, E-mail: david.small@med.monash.edu.au Abbreviations:Ab, b-amyloid protein; AD, Alzheimer’s disease; AFM, atomic force microscopy; APP, amyloid precursor protein; CR, Congo red; EDTA, ethylenediaminetetraacetic acid; HOPG, highly oriented pyrolytic graphite; MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium; VSMC, vascular smooth muscle cell. (Received 17 June 2003, revised 19 July 2003, accepted 2 September 2003) Eur. J. Biochem. 270, 4282–4293 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03815.x Ab and to relate this to the peptide’s toxic effects. Most techniques for studying Ab aggregation do not easily discriminate between different oligomeric species. Some studies have shown that Ab can form SDS-resistant oligomers, which can be measured by SDS-gel electrophor- esis [11,24,26]. However, it is unclear whether the formation of SDS-resistant species accurately reflects the overall state of Ab aggregation. For this reason, in the present study, we examined Ab aggregation using both SDS and non-SDS (native) PAGE. We also examined Ab aggregation using atomic force microscopy (AFM), Congo red (CR) binding, sedimentation and turbidity assays. Our results show that some oligomeric Ab species are sufficiently stable to allow their measurement by gel electrophoresis. However, we show that not all forms of oligomeric Ab are observed by PAGE and that different patterns of Ab aggregation are observed, depending upon the method by which aggrega- tion is measured. Taken together these results suggest that Ab aggregates by two separate pathways. One pathway, which is inhibited at pH 5.0 or by metal ions, slowly generates stable species that can be measured by PAGE. The other pathway generates unstable species that rapidly disaggregate and therefore cannot be measured by PAGE. Furthermore, the results of toxicity studies suggest that the slow aggregation at pH 7.4 can produce more toxic forms of Ab than the rapid aggregation at pH 5.0. Materials and methods Materials Electrophoretic molecular mass markers and reagents for enhanced chemiluminescence (ECL) were purchased from Amersham Pharmacia Biotech, Sydney, NSW, Australia. Electrophoretic reagents and Trans-Blot nitrocellullose membranes were obtained from Bio-Rad Laboratories, North Ryde, NSW, Australia. A mouse monoclonal antibody (mAb) WO2, which recognizes the N-terminal region (residues 1–5) of Ab, has been described previously [27]. Congo red was obtained from Sigma Chemical Co. (St Louis, MO, USA). Highly oriented pyrolytic graphite (HOPG) was purchased from Group Scientific Pty. Ltd. (Adelaide, Australia). Synthesis and solubilization of Ab1–40 Ab1–40 was synthesized utilizing manual solid-phase N-tert-butoxycarbonyl (Boc) amino-acid chemistry as des- cribed by He and Barrow [23]. Briefly, peptides were synthesized using manual solid-phase Boc amino-acid chemistry with in situ neutralization. Peptide purification was achieved using an acetonitrile/water (0.01% trifluoro- acetic acid) gradient on a reverse-phase preparative Zorbax HPLC column heated to 60 °C. Peak fractions were lyophilized and the purity (‡ 95%) and identity of the peptide were analysed by analytical HPLC, electrospray mass spectroscopy and amino-acid analysis. Preliminary studies using PAGE demonstrated that Ab peptides dissolved and stored at )80 °C in dimethylsulfoxide were less aggregated than when dissolved and stored in water. Furthermore the results of PAGE experiments were found to be more reproducible when the stock Ab was made up in dimethylsulfoxide. Therefore, routinely Ab1–40 was dis- solved in 100% dimethylsulfoxide at a concentration of 2m M and sonicated for 20 min. Sonication was used to help dissolve the peptide and was not found to have any effects on the final outcome of the experiment as those experiments performed in the absence of sonication showed similar results. Peptide solutions were then filtered using 0.22 lm centrifuge tube filters (Costar) for 3 min at 10 000 g to remove particulate matter. Filtration did not cause any significant loss of Ab as there was no significant change in the concentration of UV-absorbing material following filtration. The peptide was stored at )80 °C. Under these conditions, Ab was stable and no aggregation was observed during storage. Furthermore, no significant differences were observed in the ability of different batches of Ab1–40 stored for different periods of time to aggregate. Just prior to use, all peptide solutions were diluted to 1–2.5% (v/v) dimethylsulfoxide with deionized water or 20 m M sodium phosphate buffer, pH 7.4 that had been prefiltered using 0.45 lm filter units (Millipore, Bedford, MA, USA). Electrophoresis and Western blotting Samples were analysed on 15% native [28] or SDS/ polyacrylamide gels using a Tris/tricine buffer system over 1.5 h [29]. The duration of electrophoresis was 1.5 h in the presence of SDS or 2 h for the ÔnativeÕ gels (in the absence of SDS). After electrophoresis, Ab was detected by Western blotting, which yielded similar results to silver staining, but was much more sensitive for the detection of higher molecular mass complexes. Protein was electrophoretically transferred from the gels onto nitrocellulose at a constant current of 300 mA overnight. Membranes were then pre- blocked with 0.5% (w/v) casein in NaCl/Pi, pH 7.4 with gentle agitation for 1 h at room temperature. The blocking solution was replaced with primary monoclonal mouse antibody, WO2 (1 : 50 dilution in blocking solution) and incubated with gentle agitation for 2 h at room temperature. Blots were then probed with a secondary polyclonal rabbit anti-(mouse IgG) Ig conjugated to horseradish peroxidase (1 : 5000 dilution in blocking solution) (Amersham Phar- macia Biotech, Sydney, NSW, Australia) with gentle agita- tion for 1 h and then developed by the ECL detection system. SDS and non-SDS/PAGE in two dimensions Stock solutions of 2 m M Ab1–40 were thawed and diluted into 20 m M sodium phosphate buffer to a final concentra- tion of 10 l M . Samples were incubated at 37 °Cfor15min and then loaded (2 lg per lane) onto 1 mm thick, 15% Tris/ tricine gels prepared with or without 0.1% SDS and separated in the first dimension. After electrophoresis, gels were removed and single lanes excised, bathed in freshly prepared stacking gel (for SDS/PAGE slices) or separating gel (for non-SDS/PAGE slices) and then loaded horizon- tally onto a second gel with or without 0.1% SDS. The buffer for electrophoresis was the same in the second dimension as in the first. Proteins were separated in the second dimension, after which slab gels were electroblotted onto nitrocellulose and then analysed by Western blotting with the mAb WO2. Ó FEBS 2003 Mechanisms of Ab aggregation (Eur. J. Biochem. 270) 4283 Congo red binding assay Ab1–40 was diluted into NaCl/P i (pH 7.4) in the presence or absence of 1 m M MgSO 4 , CaSO 4 , CuSO 4 , FeSO 4 or ZnSO 4 to give a final peptide concentration of 10 l M .CR (100 l M stockinNaCl/P i , pH 7.4) was then added to the peptide solution to give a final concentration of 10 l M CR and 9.09 l M Ab1–40. This ratio of CR to Ab was required for maximum saturation of all CR binding sites on Ab1–40 aggregates [30]. Solutions of 10 l M CR lacking Ab were also prepared. Solutions were vortexed briefly and then incuba- ted at room temperature for 15 min. Absorbance values at 403 and 541 nm were recorded for samples and CR alone preparations using a Bio-Rad SmartSpec 3000 spectro- photometer in a cuvette with a 1-cm path cuvette length. Background absorbance values of buffer (with or without metal ion) alone were subtracted from the values obtained for each sample. The concentration of aggregated Ab in each preparation was determined as described by Klunk et al.[30]usingtheformula Aggregated AbðlgÁmL À1 Þ¼ð 541nm Abs=4780Þ Àð 403nm Abs=6830Þ Àð 403nm Abs CR alone =8620Þ The amount of aggregated Ab monomer was then calculated assuming a molecular mass for Ab1–40 of 4330. All preparations were prepared in triplicate and the assay was conducted independently three times with similar results in each experiment. Sedimentation assay of Ab aggregation Ab aggregation was essentially measured using a sedimen- tation assay as described by Atwood et al.[19].Ab1–40 (100 l M ) was diluted to a final concentration of 10 l M in 20 m M sodium phosphate buffer (pH 7.4 or 5.0) containing 1m M ZnSO 4 , FeSO 4 , CuSO 4 , MgSO 4 , CaSO 4 or no metal. After incubation for 15 min or 120 h at 37 °C, the samples were centrifuged at 12 000 g in a Z160M microcentrifuge (Hermle Labortechnik, Wehingen, Germany) for 10 min. After centrifugation, the supernatant fractions were removed and the pellets were resuspended in sample buffer (100 lL) containing 0.5 M Tris/HCl, pH 6.8, 5% (v/v) glycerol, 0.005% (w/v) bromophenol blue, 2% (w/v) SDS and 5% 2-mercaptoethanol. Samples were boiled for 5 min, centri- fuged and then analysed by 15% SDS/PAGE. Ab was blotted electrophoretically onto nitrocellulose sheets and Ab immunoreactivity was visualized by ECL. The total immu- noreactivity in each lane was then quantified by densitometry using SCION IMAGE Software (Scion Corporation, Frederick, MD, USA). Mean values of total lane immunoreactivity were then determined from the analyses of the triplicate samples (3 lanes). The percentage increase in immunoreac- tivity in the pellet fraction compared with control incubation (no metal, pH 7.4, 00.25 h) pellet fraction was then calculated. Atomic force microscopy Ab1–40 (2 m M in dimethylsulfoxide) was diluted to 10 l M with 20 m M sodium phosphate buffer, pH 5.0, pH 7.4 or pH 7.4 with 1 m M of Zn 2+ (as ZnSO 4 ). Solutions were incubated at room temperature for 15 min and 120 h without agitation. Immediately prior to AFM imaging, the solutions were diluted 50–100 times using same buffer solution. Five lL of the prepared solution was applied to the substrate (HOPG), left for one minute, andthenrinsedwith100lL of water twice. This sample was dried with stream of nitrogen for one min and used for imaging immediately. Aged solutions were prepared in the same manner following incubation for 120 h. Some samples were left to age while on the substrate, in air for 120 h. AFM imaging was performed using a MultiMode microscope in conjunction with a Nanoscope IV system (Digital Instruments, Santa Barbara, CA). Tapping mode in air was used for the experiments reported in this work, but contact mode was also used to obtain higher resolution images of fibrils. Silicon cantilevers (Digital Instruments, Santa Barbara, CA, model TESP), which operate at frequencies of the 300–400 kHz were used. Height and phase data were simultaneously collected at a scan rate between 1 and 3 Hz. Typical images were acquired from several regions on the substrate. Data processing (particle size measurement) and cross section analysis of Ab oligo- mers was performed using NANOSCOPE III software (Veeco Instruments Inc., Santa Barbara, CA, USA) 2 . Turbidity assay of Ab aggregation Ab aggregation was measured using a turbidity assay as described by Huang et al. [31]. To examine the effect of metal ions, solutions of 50 l M FeSO 4 , Zn SO 4 and CuSO 4 in 40 m M sodium phosphate buffer, pH 7.4 were pre- pared. Ab1–40 (2 m M in dimethylsulfoxide) was diluted with H 2 O to a 50-l M concentration. Metal and Ab solutions were combined to give a final ratio of 25 l M Ab1–40–25 l M metal ion in 20 m M sodium phosphate buffer. Solutions (200 lL) were immediately added to flat- bottomed microtitre plate wells (Nunclon, Nunc, Den- mark) in triplicate. Plates were incubated at room temperature. The absorbance at 405 nm was monitored at 1-min intervals using a Wallac 1420 Multilabel counter and 1420 software 2.0, release 8 (Perkin Elmer Life Sciences, Turku, Finland). Plates were agitated by orbital shaking every 30 s between measurements to resuspend peptide aggregates. After 4 min, 20 lL aliquots of either 10 m M ethylenediaminetetraacetic acid (EDTA), 10 m M metal ion or H 2 Owereaddedtoeachwell.Aftereach addition of metal or chelator, samples were equilibrated for 2 min at room temperature with agitation every 30 s (equilibration period) and then absorbance measurements were recorded. To assess the stability of Ab1–40 oligomers formed at low pH, the turbidity of an Ab1–40 solution was examined at pH 5.5 and after conversion to neutral pH. Ab1–40 (2 m M in dimethylsulfoxide), was diluted to 25 l M with H 2 O. Samples (200 lL) were added to microtitre plate wells (in triplicate) and absorbance measured at 405 nm at 1-min intervals. The pH was adjusted as appropriate by addition of 10 lL of 100 m M sodium acetate buffer, pH 5.5, H 2 Oor40lL of 500 m M sodium phosphate buffer, pH 7.8. 4284 G. M. J. A. Klug et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Cell viability (MTS) assay Cell lines of Wistar-Kyoto (WKY) rat aortic VSMC were obtained from G. Dusting (Howard Florey Institute of Experimental Physiology, Melbourne, Australia). Cells were grown in 96-well titre plates in DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin until 80% confluent before treatment with Ab40 preparations. Ab (100 l M ) was aged for 15 min or 120 h in 20 m M NaPO 4 buffer at pH 5.0 or 7.4. Aged Ab wasthendilutedinculture medium and added to VSMC cultures at 10 l M for 24 h. To determine cell viability after treatment, a 10-lL aliquot of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTS) was added to 100 lL cell culture medium per well. Culture plates were covered in foil and then allowed to incubate for 2 h at 37 °C. Absorbance values were then determined using a Wallac 1420 Workstation at a wavelength of 560 nm. Results Analysis of Ab by PAGE Previous studies [26] have shown that Ab oligomers are stable enough to be analysed by SDS/PAGE. Therefore, initially, we examined the state of aggregation of freshly prepared Ab by SDS/PAGE. A stock solution of Ab1–40 (2 m M in dimethylsulfoxide) was diluted into 20 m M sodium phosphate buffer, pH 7.4 to achieve a final concentration of 100 l M .Ab was incubated for 15 min at 37 °Candthen analysed by electrophoresis (2 lg per lane) for 1.5 h on a 15% Tris/tricine polyacrylamide gel in the presence of 0.1% SDS. After electrophoresis, proteins were blotted onto nitrocellulose, which was stained for Ab immunoreactivity. Ab-immunoreactive bands with apparent molecular masses of 4-, 8-, and 12- kDa were observed (Fig. 1A). Trace amounts of higher molecular mass species (> 50 kDa) were also observed. To determine the stability of the oligomeric Ab species observed upon SDS/PAGE, a two dimensional gel electro- phoresis approach was used. Ab was incubated for 15 min as before, separated by Tris/tricine SDS/PAGE and then sub- jected to an identical electrophoresis step in the second dimension over 1.5 h. The time taken between the two electrophoresis steps was 30 min. After electrophoresis in the second dimension, the gel was electroeluted onto nitrocellu- lose and stained for Ab immunoreactivity (Fig. 1B). Analysis showed that a proportion of the Ab immuno- reactivity migrated at the same relative molecular mass in the second dimension as that seen in the first dimension, indicated by the presence of a diagonal band of Ab immunoreactivity running across the gel (Fig. 1B). Some immunoreactivity was spread in a horizontal staining pattern, indicating that little oligomeric Ab had dissociated to lower molecular mass forms. From this experiment, it was evident that a proportion of SDS-resistant Ab oligomeric species are relatively stable over the time course required to perform the two electrophoretic steps (a total of 3.5 h). Ab was also analysed by ÔnativeÕ PAGE in the absence of SDS (Fig. 1C). In contrast to SDS/PAGE, the majority of the Ab immunoreactivity was seen in the higher molecular mass region of the gel. To assess the stability of Ab oligomers seen by native PAGE, PAGE was again performed using the same two-dimensional approach as that for SDS/PAGE, except that SDS was omitted from the electrophoresis buffer in both dimensions (Fig. 1D). The total time for electrophoresis on non-SDS/PAGE in each dimension was 2 h and the time taken between each electrophoretic step was 30 min. Similar to the SDS/PAGE, the relative mobility of the Ab bands on native PAGE performed in two dimensions remained constant over the period of electrophoresis (4.5 h). A small amount of dissociation was also observed in the second dimension, indicated by horizontal bands directly below the higher molecular mass species. However, most of the Ab migrated at the same relative mobility, producing a diagonal staining pattern. This finding showed that a proportion of the Ab oligomers seen on non-SDS/PAGE systems were stable over at least a 4.5-h period. Time course of Ab oligomerization Previous studies have shown that when Ab is incubated for several days, the peptide aggregates [10,16,24,32]. To examine the time course of oligomerization of Ab using PAGE, Ab1–40 (10 l M ) was dissolved in 20 m M sodium phosphate buffer, pH 7.4 and incubated at 37 °C for up to 120 h. After 0.25, 24, 48 and 120 h of incubation at 37 °C, aliquots (15 lL) were collected and analysed by SDS- and non-SDS/PAGE. After electrophoresis, the gels were elec- troblotted onto nitrocellulose and stained using the mAb WO2. The time-dependent aggregation of Ab was observed on both native and SDS gel systems (Fig. 2). On SDS/PAGE, Fig. 1. Western blot analysis of Ab1–40 analysed by SDS and native PAGE. Ab1–40 (100 l M )wasincubatedfor15minin20m M sodium phosphate buffer at 37 °C and then aliquots (2 lg) were loaded per lane onto the SDS (A) or native (C) gels. After electrophoresis in one dimension, the gels were blotted onto nitrocellulose and stained for Ab immunoreactivity with the Ab-specific mAb, WO2. In a second experiment, after electrophoresis in the first dimension, single lanes were excised and loaded horizontally onto a second gel of the same type, either a SDS (B) or a native gel (D). After separation in the second dimension, protein was transferred to nitrocellulose and then probed with the mAb WO2. m, molecular mass in kDa. R f (relative mobility with respect to the bromophenol dye front), distance from the origin/distance migrated by the dye front. Ó FEBS 2003 Mechanisms of Ab aggregation (Eur. J. Biochem. 270) 4285 Ab-immunoreactive bands with apparent molecular masses of 4-, 8-, 12- and 95-kDa were observed in samples incubated for 15 min (Fig. 2). After 24 h incubation, an increase in the 12- and 95-kDa species and the appearance of a 16-kDa species was observed. These changes were accompanied by a decrease in the 4-kDa species. Between 24 and 120 h a reduction in the 4-, 8- and 12-kDa bands and an increase in high molecular mass species was observed, suggesting that the majority of low molecular mass species had been converted to higher molecular mass aggregates. Loss of the low molecular mass species was not due to proteolysis by contaminating proteases, because inclusion of a cocktail of broad spectrum protease inhibitors did not block the disappearance of low molecular mass Ab species (data not shown). In contrast to the results for SDS/PAGE, both high and low molecular mass oligomeric species were observed on native PAGE, even after 15 min of incubation (Fig. 2). After 24 h, little of the lower molecular mass species was observed. An immunoreactive band at the top of the gel was also diminished over the time course. This apparent loss of high molecular mass immunoreactivity was due to Ab aggrega- ting to such an extent that it was unable to enter the gel. Taken together, the results from both gel systems demonstrated that initially, most of the Ab1–40 was present in an aggregated form, although these aggregates were not stable in the presence of SDS. However, with increasing time of incubation over several days, the proportion of SDS-stable aggregates increased. Effect of pH and metal ions – PAGE analysis The effect of pH on Ab1–40 aggregation was examined by PAGE. Despite several reports [22,23] demonstrating that Ab aggregation is promoted at pH 5.0, analysis of Ab aggregation at pH 5.0 revealed that the slow aggregation over 5 days of Ab to higher molecular mass species that can be seen by PAGE was less than at pH 7.4 (Fig. 2). Initially, after 15 min of incubation, there was little difference between the pH 7.4 and pH 5.0 incubation. However, at later time points, there was a greater proportion of lower molecular mass forms in the pH 5.0 incubation than in the pH 7.4 incubation. A small increase in a 12-kDa Ab oligomer on SDS/PAGE was seen at pH 5.0. However, no increase was seen in the level of other oligomeric species at pH 5.0, and the loss of the 4 kDa Ab monomer was less at pH 5.0 than at 7.4. A similar result was obtained by non-SDS/PAGE, as the rapid conversion of low to high molecular mass species seen at pH 7.4 was not observed at pH 5.0. Previous studies have shown that metal ions, notably Zn 2+ and Cu 2+ can stimulate Ab aggregation [17–19]. However, similar to the results obtained at acid pH, metal ions were found to inhibit Ab aggregation analysed by PAGE (Fig. 3). Once again, little difference was seen in the extent of oligomerization in the presence of metal ions after 15 min of incubation. However, on SDS/PAGE, the slow aggregation of low molecular mass species to larger, SDS- stable oligomeric species was found to be strongly inhibited by Zn 2+ , Cu 2+ , and slightly less so by Ca 2+ and Fe 2+ . Mg 2+ appeared to have little effect on the aggregation of Ab to SDS-stable species. Similarly, the slower aggregation of Ab over several days observed on non-SDS/PAGE was strongly inhibited by Zn 2+ and Cu 2+ , weakly inhibited by Ca 2+ and Fe 2+ and largely unaffected by Mg 2+ . Effect of pH and metal ions – CR binding and sedimentation analysis Because of the apparent discrepancy between our results using PAGE, which showed that metal ions and acid pH inhibited aggregation, and previous studies which reported increased aggregation [17–20,22–25], we compared the results obtained by PAGE with those obtained using CR binding and sedimentation assays of Ab aggregation. Ab1– 40 was incubated in the presence or absence of 1 m M CuSO 4 , FeSO 4 , CaSO 4 , ZnSO 4 , or MgSO 4 at pH 7.4 for 15 min and then the amount of fibrillar Ab was measured using a CR binding assay (Fig. 4). In agreement with the previous studies, and in contrast to our PAGE results (Figs 2 and 3) a significantly greater concentration of CR- binding material was observed in the presence of all metals than in incubations lacking metal ions. Preparations with Zn 2+ , Cu 2+ and Fe 2+ showed the highest concentra- tions of Ab aggregates (P < 0.05) compared with the control preparation. It was not possible to measure the effect of pH using the CR binding assay, because lowering the pH to 5.0 altered the absorbance spectrum of CR (data not shown). Fig. 2. Western blot analysis of the time course of Ab1–40 aggregation at pH 7.4 and pH 5.0. Ab1–40 (10 l M )wasincubatedin20m M sodium phosphate buffer, pH 5.0 or 7.4 at 37 °C for 120 h. Aliquots (15 lL) were removed at 0.25, 24, 48 and 120 h, added to an equivalent volume of 2 · tricine sample buffer and analysed by 15% Tris/tricine SDS- and native PAGE. Proteins were electrophoretically transferred to nitrocellulose and Ab-immunoreactivity was detected using the mAb WO2. All incubations were performed in triplicate. m, molecular mass in kDa. R f (relative mobility with respect to the bromophenol dye front), distance from the origin/distance migrated by the dye front. 4286 G. M. J. A. Klug et al.(Eur. J. Biochem. 270) Ó FEBS 2003 A sedimentation assay was also employed to examine the effect of low pH and metal ions on Ab aggregation. Ab1–40 (10 l M )wasfreshlypreparedin20m M sodium phosphate buffer (pH 7.4 or 5.0) containing 1 m M ZnSO 4 , FeSO 4 , CuSO 4 , MgSO 4 and CaSO 4 or no metal. Samples were incubated for 15 min or 120 h and then centrifuged at 12 000 · g (10 min) to separate aggregated from soluble material. After centrifugation, the supernatant fractions containing soluble Ab were removed and the pellets containing aggregated Ab species were resuspended in sample buffer and analysed by SDS/PAGE and Western blotting. The results obtained with the sedimentation assay method were similar to those from the CR assay (Fig. 5). In the presence of Ca 2+ , Fe 2+ , Zn 2+ , Cu 2+ and at pH 5.0, significantly more Ab was precipitated than at pH 7.4 and in the absence of metal ions at the initial time point. No significant increase in the amount of precipitated Ab was observed in the presence of Mg 2+ . After 120 h of incuba- tion, a significant increase in sedimentable material was observed at pH 7.4 in the absence of metal ions. In the presence of all metal ions and at pH 5.0, a significant increase in sedimentable material was also observed. Analysis of Ab oligomerization by AFM AFM has proven to be a valuable technique for the study of Ab aggregation [33–38]. We therefore used the morpholo- gical information obtained by AFM to probe the aggrega- tion process over time at pH 7.4 and pH 5.0. Topographic AFM images of Aß1–40 in phosphate buffer at pH 7.4 were taken (Fig. 6). Fresh Ab samples (incubated 00.25 h) showed spherical, globular structures of 15–20 nm in diameter evenly dispersed across the substrate (panel A). The height of these globules was less than 5 nm, suggesting that the Ab collapses on the surface and interestingly, many Fig. 3. Western blot analysis demonstrating the effect of divalent cations on Ab1–40 aggregation. Ab1–40 (10 ll)wasincubatedin20m M sodium phosphate buffer, pH 7.4 in the presence or absence of 1 m M MgSO 4 , CaSO 4 , FeSO 4 , ZnSO 4 , or CuSO 4 for 0.25, 24, 48 or 120 h at 37 °C. Aliquots (15 lL)wereremovedateachtimepointandanalysedbySDSandnativePAGE.Ab immunoreactivity was detected by Western blotting using the mAb WO2. All experiments were performed in triplicate. m, molecular mass in kDa. R f (relative mobility with respect to the bromophenol dye front), distance from the origin/distance migrated by the dye front. Fig. 4. Congo red (CR) spectrophotometric analysis of Ab1–40 aggre- gation in the presence or absence of 1 m M MgSO 4 ,CaSO 4 ,FeSO 4 , ZnSO 4 ,orCuSO 4 at pH 7.4. CR (20 lL) was added to the peptide solution to give a final concentration of 10 l M CR and 9.09 l M Ab1– 40. Solutions were allowed to incubate for 15 min at room tempera- ture. Incubations of CR alone were also prepared. Absorbance values were then read at 403 and 541 nm. The concentration of aggregated Ab was calculated from the equation, Ab (lgÆmL )1 ) ¼ ( 541nm Abs/ 4780) ) ( 403nm Abs/6830) ) ( 403nm Abs CR alone /8620) from Klunk et al. [30]. The amount of aggregated Ab monomer was then calculated assuming a molecular mass of 4330.9. **Significantly different (P < 0.001) from control incubations with no added metal ion. *Significantly different (P < 0.05) from control incubations with no added metal ion (two-tailed Student’s t-test). Ó FEBS 2003 Mechanisms of Ab aggregation (Eur. J. Biochem. 270) 4287 doughnut-shaped annuli were observed. Images of samples aged for 5 days (120 h) showed considerable aggregation in solution. The aggregates observed after aging were larger than in the fresh solutions (panel B). The addition of Zn 2+ ions to the solution during ageing at pH 7.4 resulted in some alignment of the Ab with branched fibril-like structures (panel C). However, although the branched fibril-like arrangements were visible, these structures were funda- mentally small aggregates of Ab, which were somewhat aligned in an organized manner. Solutions of Ab, aged 120 h at pH 5.0, showed medium-sized spherical structures with some similarities to those at pH 7.4, although the spheres were much better defined and reproducible (panel D). However at pH 5.0 the tendency to form linear fibers was clearly apparent and these fibers resembled those observed with Zn 2+ ions (pH 7.4). Distinct mature fibrils comprised of spherical aggregates apparently ÔirreversiblyÕ attached to each other were also seen (panel E). Reversibility of Ab aggregation – turbidity assay As low pH, Zn 2+ or Cu 2+ rapidly stimulated Ab aggregation in the CR binding and sedimentation assays but inhibited aggregation on PAGE, we examined the possibility that the aggregated Ab measured by the sedimentation assay and CR binding assay may be unstable and consequently not detectable by PAGE. The reversibility of Ab aggregation was examined using a turbidity assay. Ab1–40 (25 l M ) was incubated in the presence of 25 l M metal ions in 200 lLof20m M sodium phosphate buffer in microtitre plate wells. Absorbance was monitored at Fig. 6. AFM images of aggregates and fibrils of Ab1–40 on HOPG substrate after incubation at 15 min and 120 h in phosphate buffer at pH 7.4, or at pH 5.0 for 120 h or at pH 7.4 with Zn 2+ ions. (A) pH 7.4 incubated for 15 min, inset figure of observed Ab structure with characteristic ÔdoughnutÕ shape. (B) pH 7.4 incubated for 120 h, inset figure of typical Ab aggregates. (C) pH 7.4 with Zn 2+ ions incubated for 120 h, showing formation of long linear aggregates, inset figure of typical small branched Ab fibrils (arrows). (D) and (E) pH 5.0 incubated for 120 h, assemblies of Ab aggregates are seen. Panel E shows shows a mature fibril. All large topographic images are 2 lm · 2 lm in size and with a height range from 5 nm to 10 nm. Inset image in (A) is 100 nm · 100 nm in size while inset images in (B), (C) and (D) are 200 nm · 200 nm in size. Image in (E) is 100 nm · 500 nm with size. Fig. 5. Analysis of the effect of pH 5.0 and divalent cations on Ab1–40 aggregation using a sedimentation assay. The percentage increase of total immunoreactivity of Ab aggregation on SDS/PAGE is shown. Ab1–40 (10 l M )wasincubatedin20m M sodium phosphate buffer, pH 7.4 containing 1 m M MgSO 4 , CaSO 4 , FeSO 4 , ZnSO 4 , or CuSO 4 or in 20 mm sodium phosphate buffer, pH 5.0 for 15 min (0.25 h) or 120 h at 37 °C. Samples were then centrifuged at 12 000 g for 10 min after which time supernatants were removed. The peptide pellet was resuspended in sample buffer and analysed by 15% Tris/tricine SDS/ PAGE. Total immunoreactivity in each lane was determined using SCION IMAGE software. Percentage increase from the control prepar- ation (20 m M sodium phosphate buffer, pH 7.4, 15 min) was calcula- ted for each incubation type. Bars show the mean of three determinants ± SEM. **Significantly different (P < 0.001) from control incubations with no added metal ion. *Significantly different (P < 0.05) from control incubations with no added metal ion (two- tailed Student’s t-test). 4288 G. M. J. A. Klug et al.(Eur. J. Biochem. 270) Ó FEBS 2003 405 nm. Initially there was slightly more aggregated Ab in the presence of metal ions than in their absence (Fig. 7A). EDTA (20 lL, 10 m M )wasthenaddedtoeachwellbut caused no change in the turbidity of the preparations. Subsequent addition of 20 lLof10m M Zn 2+ , or Cu 2+ caused a sharp increase in the amount of Ab aggregation. In comparison, addition of H 2 O had little effect on the aggregation of Ab. After a second addition of 10 m M EDTA (20 lL), the turbidity of the solutions containing Zn 2+ and Cu 2+ decreased rapidly indicating that the induced aggregates were unstable and dissociated after chelation of the metal ions. A further addition of 10 m M Zn 2+ and Cu 2+ (20 lL) increased Ab aggregation, which was rapidly reversible with further addition of EDTA. To examine the stability of Ab oligomers induced at low pH, Ab1–40 (25 l M ) was dissolved in distilled water. After an initial absorbance measurement was recorded, a 10-lL aliquotof100 m M sodium acetate buffer, pH 5.5 was added to reduce the pH. The effect of this addition was a marked and steady increase in turbidity over a 20-min period, suggesting a rapid promotion of aggregation at low pH (Fig. 7B). The subsequent addition of water (10 lL) had little effect on Ab aggregation. The turbidity of the solution remained stable over a 25-min period. After this, an aliquot of 500 m M sodium phosphate buffer, pH 7.8 (40 lL) was added to each well to raise the pH. The absorbance sharply declined after the pH change and remained lower over a 15-min period. This result showed that low pH promoted aggregation but that the aggregation was readily reversible at higher pH. Cytotoxicity of oligomeric Ab We also examined which forms of oligomeric Ab are toxic to vascular smooth muscle cells (VSMC). Preliminary experiments demonstrated that metal ions (Cu 2+ , Zn 2+ ) were very toxic to VSMC (data not shown). Therefore we did not determine the effect of metal-ion pretreatment on Ab cytotoxicity. However, we were able to examine the effect of pH on the generation of cytotoxic Ab species. VSMC were treated with Ab incubated (aged) for 15 min or 5 days at pH 5.0 or 7.4. After treatment, the pH of the Ab solution was adjusted to 7.4 as appropriate and the effect of the peptide solution on VSMC viability was measured. Using the MTS assay, cell viability was reduced 20% and 40% when treated with Ab aged at pH 7.4 for 15 min and 5 days, respectively (Fig. 8). There was a significant increase in cytotoxicity after aging for 5 days. In contrast, Ab was significantly less toxic when aged at pH 5.0 at both time points. These results indicate that Ab1–40 oligomers generated at pH 7.4 are more toxic to VSMCs than those generated at pH 5.0. Discussion This study demonstrates that some Ab oligomers are sufficiently stable to enable measurement by SDS- or non- SDS/PAGE systems. Our experiments suggest that, like SDS/PAGE, non-SDS PAGE can also be used for the analysis of Ab aggregation. Differences between the amounts of aggregated Ab in the presence or absence of SDS are probably a reflection, at least in part, of differences Fig. 7. Analysis of the effect of divalent metal ions and low pH on Ab1– 40 aggregation by turbidity assay. (A) Ab1–40 (50 l M ), prepared in H 2 Owasdilutedin50l M ZnSO 4 , or CuSO 4 and no metal in 40 m M sodium phosphate buffer, giving a starting ratio of Ab1–40:metal ion of 25 l M :25 l M . Solutions (200 lL)wereaddedtomicrotitreplate wells and absorbance at 405 nm was measured at four 1-min intervals. After the initial reading, a 20-lL aliquot of 10 m M EDTA was added per well and allowed to incubate at room temperature for 2 min before absorbance measurement. Following measurement, a 20-lL aliquot of 10 m M metal ion (M 2+ ) or water (control) was added and the absorbance was recorded. This sequence was repeated as indicated to determine the effect of repeated metal/chelator doses. The data rep- resent the mean difference ± SEM (n ¼ 3). (B) Solutions (200 lL) of Ab1–40 (25 l M ) prepared in H 2 O, were added to microtitre plates. After an initial absorbance was measured, a 10-lL aliquot of 100 m M acetate buffer, pH 5.5 was added to each well to reduce the sample pH. Following an equilibration period, three absorbance measurements were made. A 10-lLaliquotofH 2 O was then added to each well and absorbance was recorded as for the acetate addition. To raise the pH to neutral, 500 m M sodium phosphate buffer, pH 7.8 (40 lL) was then added to each well and two final absorbance measurements were recorded. During both assays, plates were agitated every 30 s to resuspend aggregated Ab and each absorbance measurement of four 1-min intervals (except initial) was preceded by a 2-min equilibration period. The experiment in panel B was repeated three times with similar results in each experiment. Ó FEBS 2003 Mechanisms of Ab aggregation (Eur. J. Biochem. 270) 4289 in the sensitivity of specific Ab oligomers to disassembly by SDS. Interestingly, the results of the PAGE experiments suggested that some of the Ab was aggregated, even after 15 min of incubation in aqueous solution. However, some of this Ab aggregation may have occurred during the electrophoresis procedure as well. Using PAGE, Zn 2+ , Cu 2+ , Fe 2+ or low pH were observed to have little effect on Ab aggregation initially, although the slow production of oligomeric Ab species was inhibited. In contrast, Zn 2+ , Cu 2+ , Fe 2+ or low pH rapidly promoted Ab aggregation observed in CR binding, sedi- mentation and turbidity assays. This discrepancy between the different assay methods is explained by the fact that Ab can oligomerize via at least two distinct and mutually exclusive mechanisms to form two different types of aggregates (Fig. 9). The first mechanism is rapid, generates unstable aggregates, is stimulated at pH 5.0 or by Cu 2+ and Zn 2+ . The second mechanism is slow (occurs over several days), generates stable aggregates and is inhibited by low pH, Cu 2+ or Zn 2+ . Therefore, our results suggest that some caution is needed in the interpretation of Ab aggregation data. Different proportions of Ab aggregation may be measured using different techniques. In addition to the differences between the PAGE assays and the other assays, we also found discrepancies between the amount of Ab aggregation measured in a CR binding assay and that obtained with a sedimentation assay. In a CR binding assay, Fe 2+ , Zn 2+ and Cu 2+ were approximately equipotent in stimulating Ab aggregation, whereas in a sedimentation assay, Cu 2+ was more potent than Zn 2+ or Fe 2+ in stimulating aggregation. One possible interpretation of this finding is that the two assays do not measure exactly the same thing. It is likely that not all of the Ab aggregates measured in the sedimentation assay bind CR. Furthermore, it would be expected that the sedimentation assay would favour the measurement of high molecular mass (more readily sedimentable) aggregates, whereas CR might bind less readily to higher molecular mass forms of Ab due to steric hindrance. The results showed that although a proportion of the aggregated Ab measured by PAGE was stable over the time course of the PAGE experiment, most of the Ab aggregation (measured in a turbidity assay) induced by metal ions or by low pH could be easily reversed. Once again, this indicated that the two assay methods are measuring different forms of aggregated Ab. Of course it was not always possible to exactly match the conditions of incubation in each experi- ment. For example, the metal: peptide ratio in the turbidity experiments differed from that used in the other experiments of the study because it was not possible to easily chelate the metal ion with EDTA at the concentration (1 m M )usedin the other studies. Therefore a much lower concentration was used. Similarly the buffer conditions could not be exactly reproduced in the turbidity experiment looking at the reversibility of pH because of the need to alter the pH during the course of the experiment. It was not possible to maintain the same buffer and salt conditions and change the pH. Nevertheless, the results of this experiment explain the discrepancies between the other experiments. The conclusion that Ab1–40 can aggregate via distinct mechanisms was supported by the AFM results, which show that aggregates formed slowly at pH 7.4 are distinct in appearance from those formed in the presence of Zn 2+ or at pH 5. At pH 7.4, fresh solutions (00.25 h) clearly demon- strated the presence of small aggregates. Based on their dimensions on both substrates, it can be estimated that these Fig. 9. A hypothetical model of Ab aggregation and toxicity. Ab aggregates via two pathways. The first pathway occurs slowly and at neutral pH leading to the generation of stable toxic aggregates [Ab**] n thatcanbeobservedonPAGE.Thesecondpathwayisreversibleand leads to a rapid oligomerization of Ab forming unstable nontoxic aggregates [nAb*] 3 that are not observed when analysed by PAGE. This pathway is stimulated in the presence of Cu 2+ and Zn 2+ and at pH 5.0. The stimulation of this pathway under these conditions leads to an inhibition of the generation of stable aggregates as the peptide starting product is redirected toward the generation of an unstable oligomeric species. Fig. 8. Percentage decrease in cell viability of VSMC in the presence of fresh and 120 h aged Ab40 at pH 7.4 and 5.0. Ab40 (100 l M )wasaged at 37 °Cfor0or120hin20m M NaPO 4 buffer at pH 7.4 or 5.0. Ab preparations were diluted in VSMC culture medium to 10 l M which was then applied to confluent VSMC and incubated for 24 h at 37 °C. Cell viability was then determined using the MTS assay. The decrease in cell viability was calculated as a percentage of the pH 7.4 or 5.0 buffer control. Graph shows the mean of three independent experi- ments ± SEM. *Significant difference (P < 0.05) between the pH 7.4 and the corresponding pH 5.0 time point as calculated by the Student’s two-tailed t-test. 4290 G. M. J. A. Klug et al.(Eur. J. Biochem. 270) Ó FEBS 2003 aggregates contained up to 4–8 units. Aging of the Ab1–40 solution at pH 7.4 caused an increase in aggregation of Ab in solution. Changes in the oligomeric structure of Ab were evident upon addition of Zn 2+ ions at pH 7.4 or low pH (pH 5.0). The presence of Zn 2+ ions increased aggrega- tion at pH 7.4 and these aggregates were organized into proto-fibrils (Fig. 6C,D). The proto-fibrils were more regular in appearance in the low pH solution. The data are consistent with a reversible mechanism functioning at pH 7.4 in the presence of Zn 2+ ions or at low pH, in which a conformational change in Ab occurs which leads to the formation of fibrils. The mechanism by which metal ions or low pH stimulate aggregation is not yet clear. Low pH would alter the positive charge density at the N-terminus of Ab, in the region of the histidines (residues 6, 13 and 14). Furthermore, several studies have shown that histidine 13 and 14 are involved in metal-ion binding [19,39,40]. Therefore, one possibility is that the binding of metal ions or protonation of histidines may induce rapid Ab aggregation by altering the positive charge density at the N-terminus of the Ab polypeptide chain. This increase in charge density may, in turn, increase the proportion of b-structure. At pH 7.4, Ab1–40 would be predicted to possess a charge of between )2and)3. Most of this negative charge density would be located in the N-terminal region. While it must remain only as specula- tion, this negative charge might decrease intermolecular interactions needed for promotion of a b-sheet configur- ation. If this is the case, then protonation or binding of a metal ion could reduce this charge-charge repulsion and thereby allow for a b-sheet structure supporting aggrega- tion. Indeed, the circular dichroism studies of He and Barrow [23] support this view. Several studies [17–20,40–42] suggest that metal ions bind and promote Ab aggregation and subsequently induce toxicity via the generation of reactive oxygen species. Bush and coworkers [17] have suggested that Zn 2+ -promoted aggregation of Ab may be a key step in the generation of toxic Ab species. However, the role of metal ions in toxicity is unclear [43] and Mok et al. [44] have demonstrated that the generation of Ab toxicity to VSCMs cannot be blocked by the antioxidant catalase. Acid pH conditions may also contribute to Ab aggregation, as Ab is first secreted into the lumen of the endoplasmic reticulum, from which it is trafficked into the Golgi apparatus, where it is exposed to the acid pH environment [45]. Our study clearly demonstrates that Ab aggregation induced by metal ions (or by low pH) occurs via a different pathway from that which involves the slow aggregation of stable Ab species. Furthermore, cell culture studies suggest that Ab toxicity can be increased through a process of ÔagingÕ in which higher molecular mass aggregated forms are produced [10,11,32]. We consistently found that Ab aged at pH 7.4 over 5 days was more toxic to VSMCs in culture than Ab that had been incubated for 5 days at pH 5.0. Interestingly, significant toxicity was observed in fresh Ab solutions (incubated for 15 min at pH 7.4). The lack of large aggregates in the solution, as observed by AFM, would suggest that low molecular mass oligomeric forms of Ab are also toxic, which would be consistent with pre- vious studies showing that low molecular mass (diffusible) oligomeric Ab is toxic [13–15,37,46,47]. Indeed, studies by Lambert et al. [13], Stine et al. [37] and Bitan et al.[48] suggest that low molecular mass Ab species may be the most toxic form of Ab. In summary, the major conclusion of this study is that Ab can aggregate to form different types of oligomeric complexes and that these complexes may have different toxicities. Not all of the Ab in the brain may be toxic, and the mechanism by which Ab aggregates in vivo is likely to be very important in understanding its toxicity. So far, very little is known about this mechanism or how toxic species are generated in vivo.Ab forms at least two types of plaques in the brain [49]. Amorphous plaques appear to have no associated neurotoxicity, yet fine fibrillar material has been detected in these deposits [50,51]. In contrast, neurodegen- eration is more commonly associated with compact amyloid deposits. However, even here, not all amyloid plaques may be toxic, as neuritic pathology is not an invariant feature of all amyloid plaques [49]. The results presented here raise an important issue relating to the development of new therapies for AD. 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Amyloid b -protein (Ab) assembly: Ab40 and Ab42 oligomerize through distinct pathways Proc Natl Acad Sci USA 100, 330–335 49 Probst, A., Langui, P & Urlich, J (1991) Alzheimer’s disease: a description of the structural lesions Brain Path 1, 229–239 50 Yamaguchi, H., Hirai, S., Morimatsu, M., Shoji, M & Harigaya, Y (1988) Diffuse type of senile plaques in the brains of Alzheimer-type dementia Acta Neuropath . b-Amyloid protein oligomers induced by metal ions and acid pH are distinct from those generated by slow spontaneous ageing at neutral pH Genevieve M. J. A. Klug 1 ,. aged at pH 5.0 at both time points. These results indicate that Ab1–40 oligomers generated at pH 7.4 are more toxic to VSMCs than those generated at pH 5.0. Discussion This study demonstrates that. that Ab aggregates by two separate pathways. One pathway, which is inhibited at pH 5.0 or by metal ions, slowly generates stable species that can be measured by PAGE. The other pathway generates unstable