Fibrillar oligomers nucleate the oligomerization of monomeric amyloid beta but do not seed fibril formation

Abstract

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid beta (Abeta) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 x g, rich in beta-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Abeta FOs do not seed the formation of thioflavin T-positive fibrils from Abeta monomers but instead seed the formation of FOs from Abeta monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

FIGURE 1.

Kinetics and solubility of Aβ42 FO assembly. Aβ42 was dissolved in HFIP and incubated in double distilled H₂O, pH 7.4, with stirring (70 μm final concentration). At the time indicated, aliquots were removed and analyzed by dot blot and ultracentrifugation. A, aliquots of Aβ42 were spotted onto nitrocellulose membrane and probed with OC, A11, and 6E10 antibodies. FO-specific immunoreactivity formed within 1 day (d) of aggregation. Under this condition, the FOs formed are homogenous and did not react with the anti-PFO antibody A11. B, aggregation assay and fibrils were also examined by ultracentrifugation at 100,000 × g for 1 h. The supernatant (Sup) and pellet fractions were separated and resuspended in equal volumes of desired buffers. Aβ FOs were soluble in contrast to mature fibrils that sedimented at high speed.

FIGURE 2.

FTIR spectroscopy reveals structural similarity between fibrils and FOs. FTIR spectra of Aβ42 fibrils (dashed line) and FOs (solid line) indicate similar β-sheet-rich secondary structures.

FIGURE 3.

FOs are distinct from fibrils by thioflavin T binding. Aliquots of Aβ42 and Aβ40 were added to 3 μm ThT in 10 mm sodium phosphate, pH 7.4. ThT fluorescence was measured as described under “Experimental Procedures.” Significant dosage-dependent increase in ThT binding is only observed in fibrils but not in FOs or monomers.

FIGURE 4.

Comparison of EPR spectra for Aβ labeled at position 14 in different oligomeric or fibrillar forms. A shows the EPR spectrum of the fibrils, which was reported previously and is reproduced here for comparison. The EPR spectra for FOs (B, solid line) are of much lower amplitude than those of PFOs (B, dashed line). The reduction in amplitude coincides with significant spectral broadening beyond 70 G indicating the presence of strong spin-spin interaction. The presence of spin-spin interaction is illustrated in C, which overlays the spectrum of FOs labeled at 100% (solid line) with the spectrum of FOs generated from 10% of spin-labeled peptide and 90% of wild-type peptide (dashed line). In contrast to the FOs, the spectra of the fibrils contain a clear component of spin exchange, which is characterized by single line EPR spectra (D) (32). The spectral component was obtained by subtraction of the hyperfine structure from the spectrum in A. Similar spectral subtractions did not yield any evidence for single line, exchange narrowed EPR spectra for FOs. All spectra were obtained at the X-band, normalized to the same number of spins, and are shown at a scan width of 150 G.

FIGURE 5.

Morphological analysis of Aβ42 PFOs, Fos, and fibrils. A–C, AFM shows that Aβ42 PFOs, FOs, and fibrils have different distinct morphologies. FOs are small spherical aggregates that are 5–10 nm in diameter. Mature fibrils are filaments that are 6–10 nm in diameter and 1–3 μm long (scale bars, 200 nm). D, height distribution of Aβ42 FOs (solid) and fibrils (dashed) as analyzed by AFM. The data were collected by an AFM operating in air using tapping mode by measuring the heights in cross-section of >100 oligomers or fibrils.

FIGURE 6.

Aβ40 FOs do not serve as nuclei for fibril formation. Aβ40 fibrils (A) and FOs (B) were sonicated for 5 min in a water bath and incubated at 25 °C with 45 μm Aβ40 monomers (Mon). The addition of fibril seeds to monomers accelerated fibril formation (A). In contrast, when Aβ40 monomers were seeded with FOs, the lag phase of fibril formation increased slightly in a seed percentage-dependent manner (B). Experiments were performed in triplicate. ThT data were normalized by subtracting ThT fluorescence values of fibril seeds and FO seeds incubated alone and buffer, respectively. AU, arbitrary units.

FIGURE 7.

Aβ40 FOs seed the formation of FOs from monomers. Aβ40 fibrils and FOs were sonicated and incubated at 25 °C with 45 μm Aβ40 monomers. At the time indicated, aliquots were spotted onto nitrocellulose membrane and probed with the fibril- and FO-specific OC-like antibody LOC. A, Aβ40 monomers seeded with 2 and 5% of FOs formed LOC-immunoreactive aggregates within 1.5 h. In contrast, Aβ40 monomers in the absence of seeds developed LOC immunoreactivity after 1.5 h. Monomers seeded with fibrils developed LOC reactivity at 1.5 and 2 h. B, Aβ40 FO and fibril seeds control. Immunoreactivity of seeds diluted to 2% is below the limit of LOC detection. C, quantification of LOC immunoreactivity of Aβ40 monomers alone and monomers seeded with 2% FOs. The amount of LOC reactivity represents the amount of FOs or fibrils in each reaction. AU, arbitrary units.

FIGURE 8.

TEM analysis of seeded assembly reactions. Aβ40 monomers alone, monomers seeded with FOs, and monomers seeded with fibrils were analyzed by TEM at times indicated. FOs seeded formation of more FOs from monomers after 1 h of incubation. At the same time point, monomers alone did not form oligomers. The fibril-seeded reaction formed fibrils after 2 h. Scale bar = 100 nm.

FIGURE 9.

Size distribution of FO seeds (gray) and the resulting seeded FOs (black). Data represent measurements from two EM images and >150 oligomers.

FIGURE 10.

Schematic diagram of amyloid aggregation pathways. Misfolded or random coil monomers can assemble along two divergent pathways into two distinct types of oligomeric intermediates as follows: prefibrillar oligomers and fibrillar oligomers. Although the structure of PFOs is unknown, we propose that FOs are pieces of single fibril protofilament. Fibrils, on the other hand, contain two or three protofilaments composed of Aβ organized in parallel in-register β-sheets. Only the end of the aggregates are shown, but they contain multiple intermolecularly hydrogen-bonded strands. Once FOs or fibrils have formed, monomer can add on to the ends, resulting in elongation of fibrils and replication of FOs that appear to reach a size at which they become unstable and break into smaller units. It is not yet clear whether FOs coalesce via stacking of the two carboxyl-terminal sheets or whether they dissociate into monomer prior to fibril formation.