Structural Basis for Increased Toxicity of Pathological Aβ₄₂:Aβ₄₀ Ratios in Alzheimer Disease

Background: Amyloid β peptide plays a role in Alzheimer disease.

Results: Interaction of amyloid β peptides with 40 and 42 amino acids has consequences for oligomer formation.

Conclusion: Increased production of amyloid β peptide with 42 amino acids affects the behavior of the entire amyloid β peptide pool.

Significance: This might explain the synaptotoxic effect observed with a shift in amyloid β peptide production.

Abstract

The β-amyloid peptide (Aβ) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aβ₄₂:Aβ₄₀ ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aβ₄₂ in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aβ₄₀ and Aβ₄₂ individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aβ₄₂:Aβ₄₀ ratios by several techniques. We show that Aβ₄₀ and Aβ₄₂ directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aβ₄₀ and Aβ₄₂ mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aβ₄₀ and Aβ₄₂ influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aβ₄₂:Aβ₄₀ in synaptotoxicity assays and in AD patients.

Introduction

Alzheimer disease (AD)⁶ is a multifactorial neurodegenerative disease that mainly affects the growing population of the elderly. The primary agents of AD, the β-amyloid peptides (Aβ), are produced from the amyloid precursor protein by sequential endoproteolytic cleavages. The severity of dementia correlates with soluble assemblies of Aβ peptides rather than with the final fibrillar Aβ deposits observed in the brain (1) and a plethora of different toxic oligomers have been identified (2–5).

Imprecise cleavage of the amyloid precursor protein substrate by γ-secretase or altered catabolism of the Aβ peptides affect the relative amounts of Aβ₄₂ and Aβ₄₀, the two main Aβ fragments (6–8). An increased Aβ₄₂:Aβ₄₀ ratio seems to coincide with more aggressive forms of the disease compared with cases of sporadic AD (9) and affects synaptic activity, viability of neuronal cells, and memory formation in animals (7, 8, 10–12). Recently, minor shifts in the Aβ₄₂:Aβ₄₀ ratio have been reported to drastically influence the formation of neurotoxic oligomers (13, 14). Despite the very similar chemical nature of the two peptides, they seem to have quite different structural and biophysical properties. Aβ₄₂ is known to be highly fibrillogenic and more prone than Aβ₄₀ to form neurotoxic assemblies (13, 15–17). Different architectures of in vitro-generated amyloid fibrils from pure Aβ₄₀ and Aβ₄₂ peptides have been revealed by nuclear magnetic resonance (NMR) (18), electron microscopy (EM) (19), and x-ray fiber diffraction methods (20–22). A limited number of studies have demonstrated that Aβ₄₀ and Aβ₄₂ each affect the aggregation rates of the other, and it is generally reported that Aβ₄₀ inhibits the aggregation of Aβ₄₂ (12, 14, 23–27).

To date, most structural and biophysical studies have been performed using Aβ₄₀ or Aβ₄₂ in isolation. However, the aberrant behavior of neurotoxic Aβ peptides directed by the Aβ₄₂:Aβ₄₀ ratio requires the need to simultaneously investigate Aβ₄₀ and Aβ₄₂. In the present study, we address how Aβ₄₀ and Aβ₄₂ influence and modulate assembly and consider how structural aspects of intermediates along the aggregation pathway can direct the cytotoxic response of Aβ₄₂:Aβ₄₀ ratios. By combining transmission electron microscopy (TEM), x-ray fiber diffraction, surface plasmon resonance (SPR), solution NMR, and high molecular weight mass spectrometry, we have characterized the start and end states of different relevant Aβ₄₂:Aβ₄₀ ratios. Using the unique combination of ¹⁵N-edited and ¹⁵N-filtered NMR experiments, we have been able to isolate the specific behavior of either Aβ₄₀ or Aβ₄₂ in mixtures. We show that Aβ₄₀ and Aβ₄₂ can interact and that they mutually influence their aggregation behavior. Interestingly, cross-seeding and mass spectrometry (MS) experiments reveal differences in the prefibrillar stage of aggregation, which are reflected by different aggregation kinetics.

EXPERIMENTAL PROCEDURES

Preparation of Aβ Peptide Ratios

The Aβ₄₀ and Aβ₄₂ peptides and their uniformly ¹⁵N-labeled variants were purchased from rPeptide (rPeptide catalogue no. A-1153-1, A-1163-1, A-1101-2, and A-1102-2). The Aβ₄₀ and Aβ₄₂ peptides were combined in monomeric form in the desired ratios as described in detail elsewhere (28). In brief, Aβ peptides were dissolved in 1,1,1,3,3,3-hexafluor-2-propanol, Aβ₄₂ and Aβ₄₀ were then mixed in molar ratios of 1:9 and 3:7 together with pure Aβ₄₂ and Aβ₄₀ samples, and after evaporation of 1,1,1,3,3,3-hexafluor-2-propanol, they were redissolved in dimethyl sulfoxide (DMSO). The peptide was passed through a desalting column and eluted in a 50 mm Tris, 1 mm EDTA buffer, pH 7.5. Peptide concentrations were measured by the Bradford assay or by UV absorbance at 280 nm (ϵ₂₈₀ = 1490 m⁻¹cm⁻¹). The samples were kept on ice until required, with a maximum lag time of 30 min.

SPR Analysis

N-terminally labeled biotin-linker chain Aβ₄₀ (biotin-Aβ₄₀) and biotin-Aβ₄₂ (rPeptide catalogue no. A-1112-1 and A-1118-1, respectively) and Aβ₄₀, Aβ₄₂, and a non-assembling peptide with the sequence KAAEAAAKKFFE (29) were treated as described above except using a 10 mm HEPES, 100 mm NaCl, 1 mm EDTA, and 0.05 mm NaN₃, pH 7.4 buffer, and the peptide was eluted using a 2-ml Zeba spin column for buffer exchange. SPR measurements were carried out on a Biacore® 2000 system (GE Healthcare) using carboxymethylated dextran preimmobilized with streptavidin sensor chips (GE Healthcare). A volume of 150 μl of biotin-Aβ₄₀ or biotin-Aβ₄₂ was immobilized to the sensor surface at a concentration of 10 μm at a flow rate of 30 μl/min. Concentrations of 10 μm of Aβ₄₀, Aβ₄₂ or KAAEAAAKKFFE were injected at 3 μl/min. Measurements were done in triplicate and analyzed with the built-in BIAevaluation software. Curve fitting relied on the Marquardt-Levenberg algorithm, and the change in response was fitted to the binding isotherm R_eq = R_max[A]/((k_off/k_on)+[A]), where R_eq is the equilibrium response, R_max is the maximum signal response, [A] is the analyte concentration, k_off is the dissociation rate constant, and k_on is the association rate constant.

Fiber Diffraction

Samples of mature fibers were aligned by suspending a droplet of solution at 4 mg/ml between two wax-tipped capillaries positioned end-to-end. Fibers were mounted on a goniometer head, and x-ray diffraction data were collected using a Rigaku CuKα rotating anode with a wavelength of 1.5419 Å and RAxis IV++ detector. Specimen to detector distances were 160 and 250 mm with an exposure time of 10 min. Diffraction patterns were examined in CLEARER (30). For additional inspection, meridional, and equatorial axes signals were sampled through an angular search width of 60°, exported as a function of distance (pixels), and plotted using Braggs Law.

TEM Analysis

Aliquots of 4 μl Aβ were adsorbed for 30 s onto freshly prepared carbon-coated and glow-discharged copper grids, washed briefly with milli-Q water, and subsequently stained with 1% (w/v) uranyl acetate for 30 s. Samples were examined with a JEOL 1200 transmission electron microscope operating at 100 KV.

Cross-seeding Monitored with ThT

Aliquots (100 μl) of each Aβ ratio at 50 μm were incubated at 25 °C in 50 mm Tris, 1 mm EDTA, pH 7.5. After 24 h incubation these samples were sonicated at 4 °C for 10 min at maximum power and mixed with freshly and simultaneously prepared Aβ ratios to induce (cross-)seeding of Aβ aggregation. Final concentrations in these mixtures were 0.5 μm of sonicated Aβ and 25 μm of monomeric Aβ. The seed preparations were examined by TEM.

ThT Fluorescence

Aβ peptide samples of 25 μm were incubated with 12 μm thioflavin T (ThT) in a total volume of 150 μl in a Greiner 96-well plate. Fibrillization kinetics were followed using a Fluostar OPTIMA fluorescence plate reader using 440 nm excitation wavelength and an emission wavelength of 480 nm. Readings were recorded in triplicate every 10 min for a period of 24 h.

High Molecular Weight MS

High mass measurements were performed at CovalX AG (Schlieren, Switzerland) using an ABI 4800 MALDI TOF mass spectrometer retrofitted with CovalX HM2 TUVO high mass system. A phosphate-buffered saline buffer was used to prepare the Aβ ratios, which were subjected to cross-linking with gluteraldehyde at specific time points. Each sample was mixed with sinapinic acid matrix (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA 0.1% and spotted on the MALDI plate (SCOUT 384, AchorChip). High-mass MALDI TOF MS analysis was performed using standard nitrogen laser and focusing on different mass ranges from 8 to 1000 kDa in linear and positive mode and at a gain voltage of 3.14 kV and an acceleration voltage of 20 kV for HM2 High-Mass detection. The instrument was calibrated using insulin, BSA, and IgG. The analysis was repeated in triplicate.

Solution NMR

Aβ samples for NMR studies varied between 20 and 200 μm (monomer concentration) in 50 mm Tris-HCl, 1 mm EDTA at pH 7.5, supplemented with 10% (v/v) D₂O (>99.96%, Sigma Aldrich). The experiments were performed at 25 °C either on a Bruker Avance (equipped with cryoprobe) or on a Varian Inova spectrometer both operating at 14.1 Teslas (600 MHz). ¹⁵N sofast heteronuclear single quantum coherence (HSQC) spectra were each collected over 30 min to monitor aggregation. ¹⁵N NOESY-HSQC and ¹H,¹H TOCSY experiments were recorded at 5 °C to obtain sequence specific ¹H_N,¹⁵N assignments to identify the HSQC peaks. A combination of ¹⁵N-edited and ¹⁵N-filtered experiments (31) acquired on samples containing uniformly ¹⁵N-labeled and unlabeled Aβ peptides at different ratios was used to selectively monitor Aβ₄₂ and Aβ₄₀ in solution.

Protection factors of mature Aβ fibers were measured by comparing the amide peak intensities obtained for Aβ samples incubated in H₂O or in D₂O after for a period of 672 h to allow amide exchange. The fibers were collected by centrifugation, washed, and incubated in D₂O at 25 °C for 48 h, flash-frozen to quench the hydrogen-deuterium exchange (HDX), lyophilized, and redissolved in 100% DMSO-d₆ (99.9%, Cambridge Isotope Laboratories) acidified with 0.1% (v/v) trifluoroacetic acid (Fluka) for 30 s, followed by a 10-fold dilution with perdeuterated DMSO (32). Amide exchange was measured by collecting two-dimensional ¹⁵N-¹H HSQC spectra in comparison to control samples that were incubated in H₂O. The ¹⁵N-¹H HSQC cross-peak assignment was confirmed with a ¹⁵N-resolved NOESY experiment.

Cross-seeding Monitored via NMR

Seeds were prepared of pure Aβ₄₀ or pure Aβ₄₂ as described above. An aliquot of 30 μl seeds (50 μm equivalent monomeric concentration) was mixed with 330 μl of the corresponding Aβ samples that were preincubated in an NMR tube (Shigemi), whereas the non-seeded signals were monitored. ¹⁵N-edited and ¹⁵N-filtered spectra (31) were acquired as a function of time to simultaneously monitor both Aβ alloforms in the 1:9 and 3:7 ratios, whereby Aβ₄₂ was ¹⁵N-labeled and Aβ₄₀ was unlabeled. Only one-dimensional proton spectra were recorded as a function of time for the pure Aβ₄₀ or pure Aβ₄₂ unlabeled samples.

RESULTS

Direct Interactions between Aβ₄₀ and Aβ₄₂

SPR was used to explore whether Aβ₄₀ and Aβ₄₂ are able to directly associate. Either biotinylated Aβ₄₂ or Aβ₄₀ were tethered to a chip and measurements of interactions between Aβ₄₂-Aβ₄₂ and Aβ₄₀-Aβ₄₀ adsorption resulted in initial fast adsorption of the Aβ peptides, followed by a slower kinetics phase of peptide binding, characteristic for high affinity binding (Fig. 1). A similar binding profile has been reported for the aggregation and fibrillization of isolated Aβ₄₂, where a high incidence of specific binding is observed between 11-mercaptoundecanoic acid tethered Aβ₄₂ and monomeric Aβ₄₂ in bulk solution (33). The interaction between tethered Aβ₄₂ with Aβ₄₀ monomers showed a similar binding profile but slightly weaker binding. Binding between the same Aβ alloform resulted in greater mass adsorption to the sensor surface compared with mixed Aβ oligomeric interactions. These data show that the strongest binding occurs between the same alloform such as Aβ₄₂-Aβ₄₂ or Aβ₄₀-Aβ₄₀. However, strong specific binding was also observed between Aβ₄₂-Aβ₄₀.

FIGURE 1.

Aβ₄₀ and Aβ₄₂ interact directly. 10 μm covalently tethered biotinylated Aβ₄₀ or Aβ₄₂ to streptavidin-coated SA chips show binding between 10 μm Aβ variants. The sensorgram presents the interaction between Aβ₄₀-Aβ₄₂ (orange), Aβ₄₂-Aβ₄₂ (black), Aβ₄₀-Aβ₄₀ (blue), and the negative control nonspecific binding between Aβ₄₂-KAAEAAAKKFFE (gray).

Different Molar Aβ₄₂:Aβ₄₀ Ratios Are Structurally Similar at Beginning of Aggregation Process

Because Aβ₄₂ and Aβ₄₀ were found to interact directly, we used NMR to explore whether the interactions could influence the conformation of Aβ at different Aβ₄₂:Aβ₄₀ ratios immediately following their preparation. The peptides were treated according to a new protocol designed to yield completely solubilized samples without solvent contamination (13). ¹⁵N-labeling of one Aβ alloform at a time allowed us to monitor the individual structural behavior of each alloform within the context of different molar ratios. The structural fingerprints of pure Aβ₄₂ and Aβ₄₀ peptides by the ¹⁵N-¹H_N HSQC spectra are in excellent agreement with data shown in the literature (Fig. 2) (27, 34). The spectra of ¹⁵N-labeled Aβ₄₂ at different molar ratios do not present chemical shift variations, not even for the resonances of the C terminus, which should be the most sensitive to even a small change of environment (Fig. 2B). This indicates that the co-presence of the two alloforms has no influence on the structure at an atomic level, as expected for the monomeric state. The same is observed for Aβ₄₀ (Fig. 2A). We conclude that samples with different Aβ ratios are structurally equivalent to samples of pure individual peptides prior to aggregation.

FIGURE 2.

The monomeric structures of Aβ₄₂:Aβ₄₀ ratios are identical at atomic level. Shown is the overlay of the ¹H-¹⁵N HSQC spectra immediately after sample preparation of a, ¹⁵N-labeled Aβ₄₀ in pure Aβ₄₀ sample (black), ratio 1:9_{(15 N)} (green) and ratio 3:7_{(15 N)} (red). b, ¹⁵N-labeled Aβ₄₂ in pure Aβ₄₂ sample (black), ratio 1_{(15 N)}:9 (red), and ratio 3_{(15 N)}:7 (green). For representation purposes and clarity, we have artificially introduced a systematic shift of the spectra of the 1:9 and 3:7 Aβ₄₂:Aβ₄₀ ratios.

Fibers Formed by Different Aβ Ratios Have Similar Morphology and Cross-β Structure

Negative stain TEM was used for a morphological characterization of the end states of the Aβ aggregation reactions. Upon long term incubation, all Aβ ratios display a similar morphology with long, unbranched amyloid fibrils with detectable helicity and uniform diameters of 5.4, 10.2, and 16 nm, depending on the number of laterally associated protofilaments (Fig. 3A). At a qualitative level, the fibrillar structure of the 3:7 ratio of Aβ₄₂:Aβ₄₀ appeared to be slightly more polymorphic compared with the other ratios.

FIGURE 3.

Morphology and detailed structure analysis of fibrils of Aβ₄₂:Aβ₄₀ ratios reveals similar structural characteristics. a, TEM images of Aβ ratios obtained upon aggregation for 2 weeks at 25 °C without agitation. Top left panel, pure Aβ₄₀; top right panel, ratio 1:9; bottom left panel, ratio 3:7; bottom right panel, pure Aβ₄₂. Bar, 200 nm. b, fiber diffraction patterns of Aβ ratios obtained upon aggregation for 4 weeks at 25 °C without agitation. Top left panel, pure Aβ₄₀; top right panel, ratio 1:9; bottom left panel, ratio 3:7; bottom right panel, pure Aβ₄₂. c, overlay showing the normalized x-ray scattering intensity function of D-spacing plotted from b.

X-ray fiber diffraction showed that the samples of pure Aβ and mixed ratios all exhibit the classic cross-β fiber diffraction patterns described in the literature (38, 39), showing a strong meridional reflection at 4.7 Å and a major equatorial reflection at ∼10 Å consistent with a cross-β architecture (Fig. 3B). The patterns arising from Aβ₄₂ and Aβ₄₀ fibrils both share the same 9.7–9.8 Å major equatorial reflection, which we attribute to the β-sheet spacing perpendicular to the fiber axis. The fiber diffraction pattern obtained from Aβ₄₂ fibrils was distinguishable from Aβ₄₀ fibrils only by the sharper signals likely to arise from a higher degree of order in the Aβ₄₂ fibers, whereas the mixtures of the two peptides give rise to patterns that are virtually indistinguishable from that of Aβ₄₀. This could be due to the large amount of Aβ₄₀ relative to Aβ₄₂ such that the signal from Aβ₄₀ dominates the pattern. Although subtle differences in the fiber diffraction patterns likely arise from differing degrees of order and composition of the samples, the equatorial signal positions and relative intensities of signals are largely comparable for all samples (Fig. 3C).

To confirm structural similarity of fiber architecture of various Aβ ratios at a higher resolution, we measured the protection factors of the Aβ peptides by acquiring ¹⁵N-¹H_N HSQC spectra for monomeric Aβ₄₀ and Aβ₄₂ after resolubilizing amyloid fibers that were subjected to HDX (supplemental Fig. S1). Amide protection factors were measured by comparing the amide peak intensities obtained for the sample in H₂O and the sample in D₂O after the exchange period. The backbone amide chemical shift data are consistent with those previously reported in acidified DMSO-d₆ (32), although we observed (partial) overlaps in the cross-peaks of residues 11/23, 12/18, 13/27, 19/40, and 32/41 for Aβ₄₂ (supplemental data). Only residues 11/23 and 13/27 have overlapping cross-peaks in the Aβ₄₀ spectrum. The HDX pattern of ¹⁵N-labeled Aβ₄₀ for the 1:9_{(15 N)} and 3:7_{(15 N)} ratios and for pure Aβ₄₀ fibers shows that the stretches comprising residues 18–22 and 30–34 are more protected from solvent exchange than the N terminus (supplemental Fig. S2). The C-terminal residues 37–40 also appear more accessible to solvent exchange. This agrees with earlier observations and proposed models for Aβ₄₀ fibrils (18, 40–42). The HDX patterns of ¹⁵N-labeled Aβ₄₂, Aβ₄₀, and the ratios showed only small differences. Extensive exchange times (up to 672 h) (supplemental Fig. S2) of Aβ₄₂ showed no noticeable effects in agreement with the suggestion that Aβ₄₂ fibrils are highly resistant to solvent exchange (32, 43). The pattern for pure Aβ₄₂ and the 3_{(15 N)}:7 and 1_{(15 N)}:9 ratios is less distinct but indicates a clear distinction between the N- and the C-terminal halves with higher protection of the C-terminal half.

We conclude that the architecture of Aβ fibers in pure or mixed form is overall indistinguishable both at a macromolecular and high resolution level, although small differences at atomic level may be present. As these mature fibrils have been shown to have weak toxicity, we will focus on differences in the oligomeric regime.

Aβ₄₀ and Aβ₄₂ Affect Aggregation Kinetics of the Other

To address whether aggregation kinetics are affected by different ratios, we monitored the intensity of NMR spectra as a function of time, exploiting the disappearance of the resonances due to the increased molecular weight, typical of events in a slow exchange regime. We exploited again the possibility of ¹⁵N-isotope labeling only one of the Aβ alloforms, in combination with ¹⁵N-edited filter experiments to monitor the aggregation of both the ¹⁵N-labeled and unlabeled peptides simultaneously. This experimental setup offers the advantage that the individual Aβ alloforms are selectively observed in parallel and within the same sample preparation, thereby circumventing any uncertainty that might arise from different sample preparations or peptide batches. In all cases, we observed the concomitant disappearance of all peaks according to a cooperative behavior along the whole peptide or at least the region of it visible in the NMR spectrum. No sufficiently populated lower molecular weight assemblies were observed, indicating that Aβ aggregates directly into NMR invisible assemblies under these experimental conditions. Due to the long apparent lag phase, we did not detect a sigmoidal transition for Aβ₄₀ and the 1:9 ratio over a time scale of 180 h (Fig. 4, A, E, and F). In contrast, pure Aβ₄₂ aggregates very rapidly with a sigmoidal signal disappearance (Fig. 4B). Interestingly, the Aβ₄₂ component of the 1:9 ratio remains in solution significantly longer in the presence of Aβ₄₀. The kinetics recorded for Aβ₄₀ and Aβ₄₂ within the 3:7 ratios with either ¹⁵N-labeled Aβ₄₂ or ¹⁵N-labeled Aβ₄₀ (named 3_{(15 N)}:7 and 3:7_{(15 N)}, respectively) indicate that Aβ₄₀ remains longer in solution compared with Aβ₄₂, which aggregates more rapidly (Fig. 4, C and D). However, the complete loss of signal for Aβ₄₂ in the presence of Aβ₄₀ is delayed in comparison with pure Aβ₄₂, suggesting that the shorter Aβ₄₀ alloform reduces the aggregation propensity of the longer Aβ₄₂ alloform. To make sure that these observations could not be explained as the average of populations containing only the same alloforms, we analyzed a 5:5 ratio where ¹⁵N-labeled Aβ₄₂ or Aβ₄₀ are present in equimolar amounts of the unlabeled alloform (Fig. 4, G and H). We observed a nearly simultaneous disappearance of the signals of Aβ₄₂ and Aβ₄₀, which strongly suggests co-aggregation of both peptides into mixed fibers. We conclude that Aβ₄₀ and Aβ₄₂ mutually influence the aggregation kinetics of the other.

FIGURE 4.

Aβ₄₀ and Aβ₄₂ show different aggregation behavior in different Aβ₄₂:Aβ₄₀ ratios. a, pure Aβ₄₀ at a concentration of 180 μm does not aggregate within the timeframe of data collection. b, pure Aβ₄₂ at a concentration of 20 μm displays a lag phase and a sigmoidal transition from monomeric species into NMR invisible aggregates. c, 3:7_{(15 N)} ratio, whereby the Aβ sample is composed of 70% ¹⁵N-labeled Aβ₄₀, which is monitored via HSQC (140 μm Aβ₄₀ monomer concentration) and 30% unlabeled Aβ₄₂ (at a monomer concentration of 60 μm), which is simultaneously monitored via the amide region ¹⁵N-filtered one-dimensional NMR spectrum. d, the 3_{(15 N)}:7 ratio whereby the Aβ sample is composed of 30% ¹⁵N-labeled Aβ₄₂ and 70% unlabeled Aβ₄₀. e, the 1:9_{(15 N)} ratio with 10% unlabeled Aβ₄₂ (20 μm) and 90% ¹⁵N-labeled Aβ₄₀ (180 μm). f, the 1_{(15 N)}:9 ratio with 10% ¹⁵N-labeled Aβ₄₂ and 90% unlabeled Aβ₄₀. g, the 5_{(15 N)}:5 ratio whereby the ¹⁵N-labeled Aβ₄₂ and unlabeled Aβ₄₀ are present in equimolar amounts (60 μm of each alloform). h, the 5:5_{(15 N)} ratio whereby equimolar amounts (60 μm) of unlabeled Aβ₄₂ and ¹⁵N-labeled Aβ₄₀ are present. The blue symbols represent Aβ₄₀, and the black symbols correspond to Aβ₄₂.

Aβ₄₀ and Aβ₄₂ Ratios Both Form Complex but Different Ensembles of Oligomers

To investigate whether the observed alloform influence on the aggregation arises from an impact on the formation of intermediates along the aggregation pathway, we followed aggregation of the Aβ mixtures using high molecular weight MS, a technique that uses high voltages to enable detection of high molecular weight species. Because non-covalent complexes disassemble at these voltages, we incubated our samples prior to analysis with glutaraldehyde as cross-linking agent. The resulting masses reveal various interesting features (Fig. 5 and supplemental Table S1). First, the masses of Aβ₄₂ and of the two mixtures are consistently larger than those of Aβ₄₀, in support of the hypothesis that there are appreciable populations of oligomers that contain both alloforms. Second, early aggregation proceeds through a monomer addition process during which oligomers gradually grow by the addition of one monomer at a time. Third, in all cases we observed that assemblies accumulate during aggregation, the maximum size of which depends on the Aβ₄₂:Aβ₄₀ ratio. At an incubation time of 1 h, Aβ₄₀ samples contain oligomers with a range of sizes from dimers up to 13-mers. As the process continues, larger oligomers are formed and after 6 h of incubation, 25-mer assemblies are detected together with larger oligomers at apparent molecular weights of 186 up to 852 kDa. For 1:9 ratios, we observe formation of much smaller oligomers with a maximum of 8-mers and accumulation of larger sized oligomers at apparent molecular masses of 171 up to 515 kDa. The 3:7 ratios aggregate in a similar manner but share features closer to the pattern observed for Aβ₄₂. No very large molecular weight oligomers are observed after 6 h of incubation, presumably because they become so large that either they cannot become mobile or are not efficiently cross-linked. These results reveal clear differences in the pattern of small oligomeric species formed under different ratio conditions, indicating a potential basis for the difference in toxic effect (13).

FIGURE 5.

Oligomer formation by Aβ₄₂:Aβ₄₀ ratios shows a monomer addition process and a dynamic distribution of oligomeric species. Mass spectra of the different ratios with the high molecular weight detection spectra as insets whereby the blue trace is t = 1 h, the black trace is t = 3 h, and the red trace is t = 6 h. a, pure Aβ₄₀; b, 1:9 ratio; c, 3:7 ratio; d, pure Aβ₄₂. These aggregation patterns for the different ratios are also presented in supplemental Table S1.

Differences between Aβ₄₂:Aβ₄₀ Ratios Reside along Aggregation Pathway

The influence of the Aβ₄₀ and Aβ₄₂ ratios on aggregation kinetics is also evident from cross-seeding experiments where sonicated protofibrils were added to monomeric solutions of different Aβ₄₂:Aβ₄₀ ratios. Seed preparations were verified by TEM (Fig. 6A) and added to freshly prepared monomeric Aβ solutions. The aggregation kinetics were followed by in situ ThT fluorescence (Fig. 6, B–E). Aβ₄₀ aggregation was efficiently seeded by Aβ₄₀ seeds and the 1:9 ratio seeds leading to elimination of the lag phase (Fig. 6B). The initial parts of the two curves overlap, indicating that the properties of Aβ₄₀ are predominant, also in the 1:9 mixture. Addition of 3:7 seeds also induces aggregation, but some lag phase is retained. Addition of pure Aβ₄₂ not only does not seed aggregation, but even lengthens the lag phase. Seeding of Aβ₄₀ therefore appears to proceed in a highly specific manner with preference for the same alloform seeds. The 1:9 ratio is seeded by any seed but, as for pure Aβ₄₀, Aβ₄₀, and 1:9 seeds have a similar strong seeding effect, whereas Aβ₄₂ and 3:7 seeds have a milder effect, which, however, still preferentially selects the same alloform (Fig. 6C). In contrast, pure Aβ₄₂ and the 3:7 ratio were equally effectively seeded by any Aβ seed, regardless of whether they were formed from Aβ₄₀ or Aβ₄₂ or a mixture (Fig. 6, D and E). Therefore, it appears that Aβ₄₂ monomers have a higher degree of plasticity so that they may use a less specific surface as a template, whereas Aβ₄₀ oligomers have higher selectivity.

FIGURE 6.

Cross-seeding reveals that Aβ₄₂ oligomers show plasticity, whereas Aβ₄₀ oligomers display a higher selectivity. a, TEM of seed preparations. Seeds were prepared by incubation of 50 μm Aβ ratios for 24 h followed by sonication at maximum power for 10 min. From left to right: pure Aβ₄₀, ratio 1:9; ratio 3:7, pure Aβ₄₂. Bar, 0.2 μm. Freshly prepared seeds were added to monomeric solutions of Aβ₄₂:Aβ₄₀ ratios at final concentrations of 0.5 μm and 25 μm, respectively. b, ThT of Aβ₄₀ monomers seeded with pure Aβ₄₀ seeds (blue dotted trace); with seeds from ratio 1:9 (green dotted trace), with seeds from ratio 3:7 (red dotted trace), and with seeds from pure Aβ₄₂ (black dotted trace). The blue dashed line represents the unseeded Aβ₄₀ control. c, ThT of ratio 1:9 monomers seeded with Aβ ratios as compared with the non-seeded aggregation curve. The colors are as described in b. d, ThT of ratio 3:7 monomers seeded with Aβ ratios as compared with the non-seeded aggregation curve (red dashed line). Colors are as described in b. e, ThT of Aβ₄₂ monomers seeded with Aβ ratios in comparison with the non-seeded sample (black dashed line). Colors are as described in b.

The effect of cross-seeding on the disappearance of the NMR signals of the different Aβ alloforms was also studied. In these experiments, we limited ourselves to the addition of seeds of pure Aβ₄₀ or pure Aβ₄₂ to the preincubated samples of which the NMR signals were monitored. In all cases, adding seeds resulted in appreciable aggregation irrespectively of the time point at which the addition was made. The control experiments were performed without any addition and this ensured that the effect is the direct consequence of the addition. Pure Aβ₄₂ monomers could be easily seeded by both peptides (Figs. 7, C and D). Induction of aggregation of Aβ₄₀ with Aβ₄₀ seeds was also highly efficient (Fig. 7B), whereas Aβ₄₂ seeds induced some initial signal disappearance but with a delayed aggregation (Fig. 7A). In the 1:9 and 3:7 ratios, Aβ₄₀ seeds could efficiently induce aggregation of the two Aβ alloforms in the mixtures, while the Aβ₄₂ seeds efficiently seeded the Aβ₄₂ alloform, whereas the Aβ₄₀ alloform lags behind in the aggregation. This implies that the presence of Aβ₄₂ monomers in the ratios influences the behavior of the Aβ₄₀ alloform. Overall, it is observed that the Aβ₄₀ seeds can efficiently induce aggregation of Aβ samples, whereas the Aβ₄₀ alloform responds less efficiently to the Aβ₄₂ seeds (Fig. 8 and Table 1). These data show a genuine difference between the two peptides at the level of the oligomeric state. They reveal that even a relatively small increase in Aβ₄₂ in the mixture confers aggregation properties to Aβ₄₀ that are markedly more similar to pure Aβ₄₂.

FIGURE 7.

Cross-seeding was monitored by NMR by recording one-dimensional proton spectra as a function of time with unlabeled pure Aβ₄₀ (a and b) and pure Aβ₄₂ (c and d) monomers using preformed Aβ₄₀ seeds (b and d) and Aβ₄₂ seeds (a and c). Pure Aβ₄₀ samples (blue) were prepared at a concentration of 180 μm, whereas pure Aβ₄₂ samples (black) were at a concentration of 20 μm. The addition of 10% (v/v) of a 50 μm (monomeric equivalent) seed preparation was added at the time points indicated by the arrow.

FIGURE 8.

Cross-seeding was monitored by ¹⁵N-filtered and ¹⁵N-edited NMR experiments with Aβ samples composed of the Aβ₄₂:Aβ₄₀ ratio 1_{(15 N)}:9 (a and b), whereby 20 μm¹⁵N-labeled Aβ₄₂ is present with 180 μm unlabeled Aβ₄₀ and the Aβ₄₂:Aβ₄₀ ratio 3_{(15 N)}:7 (c and d) with 60 μm¹⁵N-labeled Aβ₄₂ and 140 μm unlabeled Aβ₄₀. The addition of 10% (v/v) of a 50 μm (monomeric equivalent) preparation of preformed Aβ₄₀ seeds (b and d) and Aβ₄₂ seeds (a and c) is indicated by the arrows.

TABLE 1.

Efficiency of cross-seeding on Aβ alloforms as monitored by isotope-labeled NMR

Monomeric sample	Seeds	Efficiency of seeding monitored by NMR
		Aβ42	Aβ40
Pure Aβ42	Aβ40	+++	NAa
	Aβ42	+++	NA
Aβ42:Aβ40 ratio 3:7	Aβ40	+++	+++
	Aβ42	+++	+
Aβ42:Aβ40 ratio 1:9	Aβ40	+++	+++
	Aβ42	+++	+
Pure Aβ40	Aβ40	NA	+++
	Aβ42	NA	+

^a NA, not applicable.

⁺, seeding induces aggregation, +++, seeding induces aggregation very efficiently.

DISCUSSION

Although previous structural studies have mostly focused on pure Aβ alloforms and the identification of a single oligomeric species, the present work aims to understand the determinants of the toxicity of different Aβ₄₂:Aβ₄₀ ratios. We demonstrate by independent evidence from MS, NMR, and SPR that the two peptides interact, although recognition between the same alloforms is preferred over interactions between different ones. We therefore expect that the populations of the two peptides in the aggregates will be mixed. This explains and expands previous data (23, 25, 27) that indicate that Aβ₄₀ and Aβ₄₂ influence their respective aggregation properties.

To understand which step along the aggregation pathway is responsible for this effect, we compared the structures and morphologies of all the species formed. We show that the initial monomeric and final fibrillar states do not differ to a large extent. NMR analysis of freshly prepared samples at different Aβ₄₂:Aβ₄₀ ratios conclusively reveals the presence of predominant monomeric species that lack a regular and well defined structure. Therefore, at this stage, the peptides are not affected by the presence of the other alloform. Likewise, we do not observe appreciable differences between the mature fibrillar states by TEM, HDX, and fiber diffraction: fibers formed during long incubation times are virtually identical. The absence of significant differences in the start and end points of Aβ fibrillation directed our focus to the formation of transient oligomeric intermediates. Previous data had indicated differences in the protofibrillar morphologies and Fourier transform infrared spectroscopy data following short term incubation, which, together with the present data, underline the importance of the aggregation pathway and of the dynamics of the oligomeric state (13, 44, 45).

We observed subtle but clear differences between the different Aβ ratios along the aggregation pathway. NMR experiments visualizing the spontaneous aggregation (Fig. 4) showed that the presence of monomeric Aβ₄₀ slows down the aggregation kinetics of Aβ₄₂, increasing the time frame that soluble forms are found in solution for any peptide ratio. Vice versa Aβ₄₂ stimulates Aβ₄₀ aggregation as revealed by comparing the 3:7 and 1:9 ratios with pure Aβ₄₀. This is compatible with the view that Aβ₄₂ drives aggregation and acts as a template by lowering the kinetic barriers that prevent Aβ₄₀ from aggregating (15, 25, 46). Aβ₄₀ potentially delays Aβ₄₂ aggregation through “non-productive” interactions. Although these conclusions are in agreement with previous reports (25, 27, 46), our cross-seeding data suggest that Aβ₄₀ monomers specifically require Aβ₄₀ oligomers to induce growth of mature fibrils, whereas Aβ₄₂ monomers are less selective and are stimulated by all types of seeds.

It might be argued that there is an apparent discrepancy between the progressive Aβ aggregation as monitored by NMR (Fig. 4) and the cross-seeding data (Figs. 6–8). By NMR, we observe that Aβ₄₂ stimulates Aβ₄₀ to aggregate while Aβ₄₀ simultaneously delays Aβ₄₂ aggregation. Vice versa, the cross-seeding data reveal that monomeric Aβ₄₀ is not efficiently seeded by sonicated Aβ₄₂ protofibrils. It is reasonable to explain this discrepancy by assuming that the oligomers formed during the aggregation process have features that are distinct from the sonicated protofibrillar species (seeds) that may have undergone advanced structural maturation. For pure Aβ₄₂, these seeds would not be the optimal templates to directly incorporate Aβ₄₀ monomers and perhaps even entail a conformational restructuring to lead to productive aggregation. We cannot rule out the possibility that monomeric Aβ₄₀ could be able to resolubilize Aβ₄₂ seeds, thereby bringing Aβ₄₂ into solution and promoting in this way productive aggregation, as hinted by Yan and Wang (27). Because our observations with high molecular weight MS underline that the aggregation process proceeds through a monomer addition mechanism, the dynamic interplay (productive and non-productive) of monomeric Aβ with soluble Aβ assemblies seems appropriate to explain toxicity of the Aβ₄₂:Aβ₄₀ ratios. This relevance of monomer addition processes for neurotoxicity was recently described by Jan and colleagues for pure Aβ₄₂ aggregation (44). Thus, the modulation of the Aβ oligomer formation by the Aβ₄₂:Aβ₄₀ ratio adds to the cause of neurotoxicity and the Alzheimer disease pathology.

In conclusion, our work indicates that the Aβ₄₂:Aβ₄₀ ratio behavior cannot be simply interpreted by stating that Aβ₄₂ can induce Aβ₄₀ aggregation while at the same time, Aβ₄₀ can prevent or delay Aβ₄₂ aggregation. Rather than the morphology of the amyloid fibrils, the Aβ₄₂:Aβ₄₀ ratio modulates the Aβ oligomer formation. Our data indicate that neurotoxicity is more likely to be explained by the dynamic nature of the ongoing Aβ aggregation rather than by the prevailing view that Aβ toxicity is associated with a distinct assembly. A change in the Aβ₄₂:Aβ₄₀ ratio induces differences in conformational plasticity of the oligomeric peptide mixtures and the pattern of detectable oligomeric species. That the oligomer formation along the amyloid assembly pathway is affected by the different Aβ ratios emphasizes the necessity to further expand our understanding of the exact compositional, temporal, and structural properties of the homo- and hetero-oligomers. The implications of this finding for AD therapy are fundamental: the results imply that it is less important to focus on lowering the total amyloid burden in patients, although it appears crucial to affect the relative ratios of the peptides.