The Ratio of Monomeric to Aggregated Forms of Aβ40 and Aβ42 Is an Important Determinant of Amyloid-β Aggregation, Fibrillogenesis, and Toxicity

Aggregation and fibril formation of amyloid-β (Aβ) peptides Aβ40 and Aβ42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Aβ40 to Aβ42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Aβ. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Aβ40 to Aβ42, we probed the concentration- and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric Aβ40 alters the kinetic stability, solubility, and morphological properties of Aβ42 aggregates and prevents their conversion into mature fibrils. Aβ40, at approximately equimolar ratios (Aβ40/Aβ42 ∼ 0.5–1), inhibits (>50%) fibril formation by monomeric Aβ42, whereas inhibition of protofibrillar Aβ42 fibrillogenesis is achieved at lower, substoichiometric ratios (Aβ40/Aβ42 ∼ 0.1). The inhibitory effect of Aβ40 on Aβ42 fibrillogenesis is reversed by the introduction of excess Aβ42 monomer. Additionally, monomeric Aβ42 and Aβ40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Aβ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Aβ42 protofibrils selectively seed the fibrillogenesis of monomeric Aβ42 but not monomeric Aβ40. Finally, we also show that the amyloidogenic propensities of different individual and mixed Aβ species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of Aβ40 to Aβ42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease. Aggregation and fibril formation of amyloid-β (Aβ) peptides Aβ40 and Aβ42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Aβ40 to Aβ42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Aβ. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Aβ40 to Aβ42, we probed the concentration- and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric Aβ40 alters the kinetic stability, solubility, and morphological properties of Aβ42 aggregates and prevents their conversion into mature fibrils. Aβ40, at approximately equimolar ratios (Aβ40/Aβ42 ∼ 0.5–1), inhibits (>50%) fibril formation by monomeric Aβ42, whereas inhibition of protofibrillar Aβ42 fibrillogenesis is achieved at lower, substoichiometric ratios (Aβ40/Aβ42 ∼ 0.1). The inhibitory effect of Aβ40 on Aβ42 fibrillogenesis is reversed by the introduction of excess Aβ42 monomer. Additionally, monomeric Aβ42 and Aβ40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Aβ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Aβ42 protofibrils selectively seed the fibrillogenesis of monomeric Aβ42 but not monomeric Aβ40. Finally, we also show that the amyloidogenic propensities of different individual and mixed Aβ species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of Aβ40 to Aβ42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease. Alzheimer disease is a progressive neurodegenerative disorder characterized by age-related accumulation of amyloid-β (Aβ) 2The abbreviations used are: Aβ, amyloid-β; AD, Alzheimer disease; FAD, familial Alzheimer disease; APP, amyloid precursor protein; PF, protofibrillar; M, monomeric; F, fibrillar; SF, sonicated fibrils; SEC, size exclusion chromatography; ThT, thioflavin T; TEM, transmission electron microscopy; PBS, phosphate-buffered saline. proteins in the form of diffuse and neuritic plaques in regions of the brain that are affected by the disease (1Glenner G.G. Wong C.W. Quaranta V. 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Biol. 2007; 369: 909-916Crossref PubMed Scopus (82) Google Scholar) used differential NMR isotope labeling to demonstrate that Aβ40 prevents aggregation of monomeric Aβ42 and is capable of being exchanged for Aβ42 monomer in Aβ42 aggregates. In the present study, we determined the preferential effect of Aβ40 on the kinetic stability, solubility, and fibrillogenesis rate of specific aggregation states of Aβ42, including monomers, protofibrils, and fibrils. Additionally, we explored the dynamics of exchange between monomeric Aβ40 and Aβ42 at the end of protofibrils and fibrils formed by each peptide and determined the effect of these interactions on the aggregate growth and morphology in vitro. Finally, we examined the ability of Aβ42 fibrils and protofibrils to seed the aggregation of Aβ40 and vice versa by monitoring the seeding effects of homologous and heterologous sequence on the lag phase, elongation phase, and steady-state phase of fibril formation of monomeric Aβ40 and Aβ42. The specificity of interaction between Aβ40 and different Aβ42 aggregation states was validated by using Aβ40-1 (reverse) as a control peptide. The present work provides novel mechanistic and structural insights into the molecular mechanisms underlying the consequences associated with altered Aβ40/Aβ42 ratio. The implications of these findings for intervention strategies for AD are also discussed. Chemicals and reagents of analytical grade were purchased from Sigma-Aldrich unless indicated otherwise. Best quality distilled water was used for preparation of buffers and solutions, which were filtered through 0.65-μm DVPP membranes (Millipore) before use. Aβ peptides 1-40, 1-42, and 40-1 were synthesized and purified by Dr. James I. Elliott at Yale University (New Haven, CT). Protofibrillar (PF) and monomeric (M) forms of Aβ were prepared according to the protocols described previously (42Lashuel H.A. Hartley D.M. Petre B.M. Wall J.S. Simon M.N. Walz T. Lansbury Jr., P.T. J. Mol. Biol. 2003; 332: 795-808Crossref PubMed Scopus (208) Google Scholar). Briefly, Aβ peptides were dissolved in 100% DMSO and adjusted to 1 mg/ml by adding distilled H2O. The pH of the resultant solutions was adjusted with 2 m Tris base, pH 7.4. After centrifugation (8000 × g at 4 °C for 10 min) the supernatant was injected into a size exclusion chromatography column Superdex 75 HR 10/30 (GE Healthcare) that had been equilibrated previously with 10 mm Tris-HCl, pH 7.4. Peptides were fractionated at a flow rate of 0.5 ml/min and eluted in 1.5-column volumes. Aβ elution was monitored by UVabsorbance at three different wavelengths: 210, 254, and 280 nm. Under these conditions, Aβ42 eluted as two well separated peaks; one corresponding to the void volume of Superdex 75 containing Aβ protofibrils (Aβ42PF) and the second peak corresponding to monomeric Aβ (Aβ42M) (supplemental Fig. 1). Aβ40 and Aβ40-1 elute predominantly as single peaks corresponding to monomeric species (data not shown). The protein concentrations of the various Aβ fractions was estimated by UV absorbance at 280 nm in 10-mm path-length cuvettes using the theoretical molar extinction coefficient at 280 nm (1490 m–1 cm–1) (43Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3523) Google Scholar) and/or using the Micro BCA protein assay (Pierce) when needed. Co-incubation of Monomeric Aβ40 with Protofibrillar or Monomeric Aβ42—Monomeric and protofibrillar preparations of Aβ42 and Aβ40 obtained by size exclusion chromatography (SEC) were adjusted to a final concentration of 10–20 μm in 10 mm Tris-HCl, pH 7.4, and placed in a 37 °C incubator without agitation. For co-incubation studies, protofibrillar and monomeric Aβ42 fractions were mixed with monomeric Aβ40 at molar ratios (Aβ42M:Aβ40M) of 10:1, 10:5, and 10:10 (all concentrations in μm) and allowed to aggregate at 37 °C without agitation. Fibril formation and protein solubility were monitored by thioflavin T (ThT) binding assay, negative staining transmission electron microscopy (TEM), and analytical SEC as described below. In parallel, the specificity of Aβ40 interactions with Aβ42 was validated by co-incubation with the control peptide Aβ40-1. Fibril Elongation Studies—To probe the effect of monomeric Aβ on the elongation and reassociation of Aβ42 fibrils, we first generated Aβ42 fibrils by incubating 10 μm monomeric Aβ42 (Aβ42M) for 96 h. The fibrils were mechanically fragmented into smaller fibrillar structures (100–300 nm long) by ultra-sonication on ice using a Vibra Cell™ instrument (Sonics Inc.) equipped with a 2-mm diameter microtip (20 × 5 s pulses; amplitude, 40%; output, 6 watts). The sonicated fibrils (Aβ42SF) were incubated: (i) in isolation; (ii) with monomeric Aβ42 (10 μm); (iii) with monomeric Aβ40 (10 μm); or (iv) with a 1:1 mixture of monomeric Aβ42:40 (20 μm final Aβ concentration). Fibril elongation and reformation of mature Aβ fibrils was monitored by ThT fluorescence and TEM. Seeding the Fibrillogenesis of Monomeric Aβ (Aβ40 and Aβ42) with Fibrils Derived from Aβ40 and Aβ42—To probe the ability of Aβ42 fibrils to accelerate the fibrillogenesis of monomeric Aβ40 and vice versa, 20 μm monomeric Aβ (Aβ42 or Aβ40) was incubated with the following: (i) 10 μg/ml sonicated Aβ42 fibrils (Aβ42SF, once); (ii) 20 μg/ml sonicated Aβ42 fibrils (Aβ42SF, twice); (iii) 10 μg/ml sonicated Aβ40 fibrils (Aβ40SF, once); and (iv) 20 μg/ml sonicated Aβ40 fibrils (Aβ40SF, twice). The extent of fibril formation was determined by ThT fluorescence and TEM. ThT fluorescence data were normalized by subtracting the contribution from sonicated fibrils. Seeding the Fibrillogenesis of Monomeric Aβ (Aβ40 and Aβ42) with Aβ42 Protofibrils—20 μm monomeric Aβ (Aβ40 or Aβ42) was co-incubated with different molar ratios of protofibrillar Aβ42 (monomeric Aβ:Aβ42PF; 20:1, 20:2 and 20:4; all final concentrations in μm) and fibril formation was monitored by ThT fluorescence after subtracting the contribution from protofibrillar Aβ42. To probe the specificity of interactions between the monomeric and aggregated forms of Aβ42 and Aβ40, we also evaluated the capacity of sonicated fibrils and protofibrillar species of both peptides (Aβ40 and Aβ42) to seed the fibrillogenesis of Aβ40-1 (20 μm) in a similar fashion. ThT binding assay was performed by mixing aliquots of 10–20 μm Aβ with 10–20 μm ThT dye (Aβ:ThT 1:1) and 50 mm glycine-NaOH, pH 8.5, in Nunc 384-well fluorescence plates (Fisher Scientific). ThT fluorescence of each sample was measured in an Analyst AD fluorometer (Molecular Devices) at excitation and emission wavelengths of 450 and 485 nm, respectively. The samples were analyzed in duplicates at selected time points. 5 μl of sample was applied to carbon-coated Formvar 200 mesh grids (Electron Microscopy Sciences) and incubated at room temperature for 60 s. The grids were then washed sequentially by depositing 10-μl droplets of double distilled sterile water (2 times) followed by a 10-μl droplet of fresh 2% (w/v) uranyl acetate, which remained on the grid for 30 s. After each step, the excess solution was blotted with Whatman filter paper, and the grids were vacuum-dried from the edges. The samples were analyzed using a Philips CM-10 TEM microscope operated at 100 kV acceleration voltage. Analytical SEC was performed to quantify the relative amount of soluble (monomeric and protofibrillar) Aβ in solution at selected time points during the aggregation experiments. For this purpose a SEC column Superdex 75 PC 3.2/30 (GE Healthcare) was connected to a Waters Separation Module 2795 equipped with a photo diode array detector (Waters Corp.). Aliquots (150 μl) of the samples were centrifuged (8500 × g at 4 °C for 10 min), and 50 μl of supernatant was injected into the column. Samples were individually analyzed by UV absorbance (wavelengths 210, 254, and 280 nm) at a flow rate of 0.05 ml/min. Primary Cell Cultures—Rat embryonic (E16) cortical cultures were established using a previously described procedure (44Zala D. Benchoua A. Brouillet E. Perrin V. Gaillard M.C. Zurn A.D. Aebischer P. Deglon N. Neurobiol. Dis. 2005; 20: 785-798Crossref PubMed Scopus (71) Google Scholar). Briefly, neurons were plated at a density of 30,000 cells/well in 96-well dishes (Costar™, Corning) previously coated with poly-l-lysine (Mr 30′000–70′000). On in vitro day 4, half of the medium was replaced with freshly prepared Neurobasal™ medium supplemented with 2% B27 (Invitrogen), 1× penicillin-streptomycin (Invitrogen), 0.5 mm l-glutamine, and 15 mm KCl. Subsequently, half of the medium was changed weekly. On in vitro day 23, half of the primary culture medium was replaced with complete Neurobasal medium containing one-fifth or one-tenth volume of amyloid peptide species with varying concentrations of Aβ40M, Aβ42M, Aβ42PF, and Aβ42F and 1:1 molar mixtures thereof. Cells were subsequently incubated with Aβ species for 7 days. All amyloid peptide species were delivered in 140 mm NaCl, 10 mm Tris, pH 7.4. Immunostaining—Cell cultures were washed with PBS and fixed with 4% paraformaldehyde (Fluka) for 15 min at 4 °C. Cultures were subsequently washed with PBS and then incubated for 1 h in a blocking solution of PBS supplemented with 10% normal goat serum (DakoCytomation) and 0.1% Triton X-100 in PBS. The cells were then incubated overnight at 4 °C in blocking solution containing mouse monoclonal anti-NeuN antibody (1/400, Chemicon Inc.). The next day, cells were washed and incubated for 2 h with a fluorescent secondary antibody (Cy3-conjugated goat anti-mouse; 1:1,000; Jackson ImmunoResearch Laboratories) followed by PBS washes. Immunostained cells were then analyzed with a BD Pathway 855 Bioimager (BD Biosciences). Images for quantitative analyses were acquired under nonsaturating exposure conditions, and image analysis was performed with NIH ImageJ software. Isolation and Characterization of Protofibrillar and Monomeric Aβ—TEM images of protofibrillar Aβ42 (Aβ42PF) fractions revealed predominantly curvilinear structures with an average length and diameter of 60–100 and 4–6 nm, respectively (supplemental Fig. 1B). In addition, spherical aggregates of different diameters (6–10 nm) were also observed occasionally. The second elution peak corresponding to monomeric Aβ42 (Aβ42M) did not show the presence of any recognizable aggregates by TEM (supplemental Fig. 1D). 10–20 μm concentrations of protofibrillar and monomeric Aβ42 species converted readily into fibrils upon incubation at 37 °C (supplemental Fig. 1, C and E). After 96 h of incubation, almost all Aβ42M had aggregated into a network of long intertwining fibrils (supplemental Fig. 1E), whereas significant amounts of Aβ42 protofibrils persisted and coexisted with fibrils under identical conditions (supplemental Fig. 1C). Unlike Aβ42, Aβ40 and Aβ40-1 (reverse) eluted predominantly as a single monomeric peak under the same solubilization conditions (data not shown) and did not form fibrils under the conditions used for Aβ42 fibrillogenesis studies (supplemental Fig. 2, A, C, and E). Fibril formation by monomeric Aβ40 (Aβ40M) required gentle agitation (300 rpm) and higher concentrations (supplemental Fig. 2, B, D, and F). Monomeric Aβ40 Inhibits the Fibrillogenesis of Monomeric Aβ42 in a Concentration-dependent Manner—To probe the effects of monomeric Aβ40 interactions on the self-assembly and fibril formation of monomeric Aβ42, we co-incubated SEC-isolated Aβ42M with increasing concentrations of Aβ40M (see “Experimental Procedures”). We found that Aβ40M inhibited the fibrillogenesis of Aβ42M in a concentration-dependent manner. After co-incubation for 96 h, strong (>50%) inhibition of monomeric Aβ42M fibrillogenesis was observed in samples containing both peptides at Aβ40M/Aβ42M ratios ≈ 0.5–1 (Fig. 1A). However, the presence of Aβ40M, even at lower concentrations (Aβ40M:Aβ42M ∼ 0.1) led to a transient population of protofibrillar species (Fig. 1B), which disappeared subsequently. In the absence of Aβ40, Aβ42M aggregated rapidly and formed long intertwining fibrillar networks without the accumulation of protofibrils (supplemental Fig. 1E). In contrast, co-incubation with Aβ40M favored the formation of short, flexible protofibrillar structures (Fig. 1, C and D, and Scheme 1), which did not appear to convert to mature elongated fibrils. Our data suggest that Aβ40M interferes with the ability of Aβ42M to form mature fibrils but does not interfere with its ability to form higher order prefibrillar aggregates. Under no conditions did we observe that mixtures of monomeric Aβ40 and Aβ42 remained as stable monomers or formed stable heterodimers on the time scale of our experiments (24–96 h).SCHEME 1Monomeric Aβ40 inhibits the ability of Aβ42 to form mature amyloid fibrils. The arrows represent the relative concentration of each species.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Kinetic Stabilization of Aβ42 Protofibrils by Monomeric Aβ40—Next, we probed the interactions of monomeric Aβ (Aβ40 and Aβ42) with protofibrillar Aβ42 and assessed the consequences of such interactions on the elongation Aβ42 protofibrils into mature fibrils. For this purpose, we co-incubated Aβ42PF with monomeric Aβ (Aβ40M or Aβ42M) at different molar ratios (AβPF:AβM, 10:1, 10:5, and 10:10; all final concentrations in μm) (Scheme 2). The addition of Aβ42M to Aβ42PF at increasing molar ratios resulted in enhanced ThT binding (Fig. 2A), consistent with the accelerated conversion of Aβ42PF into mature fibrillar structures (Fig. 2E). SEC analysis after 48 h of co-incubation revealed that the majority of monomeric and protofibrillar Aβ42 were converted into insoluble aggregates and were no longer detectable in solution supernatant after centrifugation (Fig. 2C). Interestingly, the effect of Aβ40M co-incubation on inhibition of Aβ42PF fibrillar conversion was independent of ratio. In the presence of Aβ40M, ThT fluorescence of Aβ42PF remained virtually unchanged even after co-incubation for 96 h (Fig. 2B). To determine whether the lack of a rise in ThT fluorescence was due to stabilization of Aβ42PF or formation of low ThT-binding Aβ aggregates, samples containing mixtures of Aβ42PF and Aβ40M were subjected to further analyses by SEC and TEM. When purified Aβ42PF were reinjected into an analytical Superdex 75 PC 3.2/30 SEC column, we consistently observed two peaks corresponding to Aβ42PF and Aβ42M. This may have happened because Aβ42PF are in rapid equilibrium with monomers and/or a population of the Aβ42PF is more susceptible to dissociation upon interaction with the column matrix. After 24 h of co-incubation in the presence of ≥1 μm Aβ40M, we observed a disappearance of the monomeric peak (Fig. 2D, 25 min), whereas the intensity and area of the protofibril peak increased (Fig. 2D, 18 min). Upon further incubation, the intensity and area of the protofibril peak was markedly reduced and exhibited a slight shift to higher molecular weight elution (16 min