Abeta42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete Abeta42 species

Abstract

The identification of toxic Aβ species and/or the process of their formation is crucial for understanding the mechanism(s) of Aβ neurotoxicity in Alzheimer disease and also for the development of effective diagnostic and therapeutic interventions. To elucidate the structural basis of Aβ toxicity, we developed different procedures to isolate Aβ species of defined size and morphology distribution, and we investigated their toxicity in different cell lines and primary neurons. We observed that crude Aβ42 preparations, containing a monomeric and heterogeneous mixture of Aβ42 oligomers, were more toxic than purified monomeric, protofibrillar fractions, or fibrils. The toxicity of protofibrils was directly linked to their interactions with monomeric Aβ42 and strongly dependent on their ability to convert into amyloid fibrils. Subfractionation of protofibrils diminished their fibrillization and toxicity, whereas reintroduction of monomeric Aβ42 into purified protofibril fractions restored amyloid formation and enhanced their toxicity. Selective removal of monomeric Aβ42 from these preparations, using insulin-degrading enzyme, reversed the toxicity of Aβ42 protofibrils. Together, our findings demonstrate that Aβ42 toxicity is not linked to specific prefibrillar aggregate(s) but rather to the ability of these species to grow and undergo fibril formation, which depends on the presence of monomeric Aβ42. These findings contribute significantly to the understanding of amyloid formation and toxicity in Alzheimer disease, provide novel insight into mechanisms of Aβ protofibril toxicity, and important implications for designing anti-amyloid therapies.

FIGURE 1.

Fibril formation and toxicity of subfractionated Aβ42 protofibrils. A, subfractionation of Aβ42 protofibrils (F1–F4) and isolation of monomers (F5 and F6) on a Superose 6 SEC column. B, analytical SEC of protofibrillar fractions (F1–F4) on a Superose 6 pc 3.2/30 column. C, cell viability (MTT reduction assay) of cultured PC12 and SHSY5Y cells after 24 h of treatment with 10 μm Aβ42 CR protofibrils, protofibrils fractions (F1–F4), and monomers (F5) (one-way ANOVA, n = 9, *, p < 0.05; **, p < 0.01, mean ± S.D.). D, ThT binding over time by 10 μm Aβ42 CR protofibrils, protofibril fractions (F1–F4), and monomers (F5) in supplemented 10× DMEM (96 h of incubation; 37 °C, mean ± S.D.). E–L, representative TEM images of Aβ42 (CR, 0 and 96 h, respectively) (E and I), F1 (0 and 96 h, respectively) (F and J), F3 (0 and 96 h, respectively) (G and K), and F5 (0 and 96 h, respectively) (H and L) in aliquots of the culture medium (scale bar = 200 nm) are shown. (a.u., arbitrary units; Veh, buffer vehicle.)

FIGURE 2.

Toxicity of isolated Aβ42 monomers, protofibrils, and fibrils toward cultured cells. A, fractionation of Aβ42 CR protofibrils on a Superdex 75 SEC column to separate PF and M. B, analytical SEC of protofibrillar fractions (PF) from A on a Superose 6 pc 3.2/30 column. It is noteworthy that some of the monomers observed in the PF fractions are due to PF dissociation during the SEC separation. C, cell viability (MTT reduction assay) of cultured rat primary neurons, PC12 cells, and SHSY5Y cells after 24 h of treatment with 10 μm Aβ42 CR, PF, M, and F (one-way ANOVA, n = 6 (neurons) and n = 9 (PC12 and SHSY5Y); *, p < 0.05; **, p < 0.01, mean ± S.D.). D, ThT binding over time by 10 μm Aβ42 CR protofibrils, fractionated PF, M, F, and Aβ40 M in supplemented 10× DMEM (24 h of incubation; 37 °C, mean ± S.D.). E–L, representative TEM images of Aβ42 CR (0 and 24 h, respectively) (E and I), Aβ42 PF (0 and 24 h, respectively) (F and J), Aβ42 M (0 and 24 h, respectively) (G and K), and Aβ42 F (0 and 24 h, respectively) (H and L) in aliquot of the culture medium (scale bar = 200 nm) are shown. (a.u., arbitrary units; Veh, buffer vehicle.)

FIGURE 3.

Effect of monomer addition on fibril formation and toxicity of subfractionated protofibrils. A, ThT binding over time by 1:1 molar mixtures of Aβ42 protofibrils fractions (F1–F5) obtained from Superose 6 with Aβ42 monomers in supplemented 10× DMEM (10 μm Aβ42, 96 h of incubation; 37 °C, mean ± S.D.). B and C, cell viability (MTT reduction assay) of cultured PC12 and SHSY5Y cells after 24 h of treatment with 1:1 molar mixture of Aβ42 protofibrils fractions (F1–F5) and Aβ42 monomers (10 μm Aβ; one-way ANOVA, n = 9, *, p < 0.05; **, p < 0.01, mean ± S.D.). C, monomers were pretreated with IDE before addition to the fractions F1–F5 (Aβ:IDE, 20:1, w/w). D, cell viability (MTT reduction assay) of cultured PC12 and SHSY5Y cells 24 h after treatment with mixtures of sonicated Aβ42 fibrils (2 μm) with SEC protofibrils fractions F1–F5 (8 μm) (one-way ANOVA, n = 9; *, p < 0.05; **, p < 0.01, mean ± S.D.) (CR, crude Aβ42 protofibrils; a.u., arbitrary units; Veh, buffer vehicle).

FIGURE 4.

Time course of Aβ42 fibril formation and toxicity. A, ThT binding over time by 10 μm Aβ42 CR protofibrils in DMEM (37 °C; mean ± S.D.). B, PC12 cell viability (MTT reduction assay and quantification of lactate dehydrogenase release in the medium) after treatment with 10 μm Aβ42 CR. For the MTT assay, at the indicated time points, the cultured medium was completely removed and replaced with MTT assay solution. For lactate dehydrogenase release assay, aliquots of the phenol red-free culture medium were removed from each well at the indicated time points. The data are expressed as the percentage of the buffer vehicle (Veh) (mean ± S.D.). C–F, representative TEM images of Aβ42 CR in culture medium aliquots obtained at 3 h (C), 6 h (D), 9 h (E), and 24 h (F) (scale bar in C–F = 200 nm).

FIGURE 5.

Effect of Aβ42 fibril formation on glucose utilization by cultured astrocytes. 2-[³H]DG utilization by cultured (DIV 21) mouse astrocytes after 24 h of treatment with Aβ42 CR protofibrils, F, fractionated PF, and M (10 μm final Aβ concentration). Monomeric Aβ40 (10 μm) was included for comparison (n = 16 from eight independent experiments, ANOVA followed by a Dunnett's post hoc test; ***, p < 0.005, mean ± S.E.; Veh, buffer vehicle).