N-terminal Domain of Amyloid-β Impacts Fibrillation and Neurotoxicity

Abstract

Alzheimer's disease is characterized by the presence of distinct amyloid-β peptide (Aβ) assemblies with diverse sizes, shapes, and toxicity. However, the primary determinants of Aβ aggregation and neurotoxicity remain unknown. Here, the N-terminal amino acid residues of Aβ42 that distinguished between humans and rats were substituted. The effects of these modifications on the ability of Aβ to aggregate and its neurotoxicity were investigated using biochemical, biophysical, and cellular techniques. The Aβ-derived diffusible ligand, protofibrils, and fibrils formed by the N-terminal mutational peptides, including Aβ42(R5G), Aβ42(Y10F), and rat Aβ42, were indistinguishable by conventional techniques such as size-exclusion chromatography, negative-staining transmission electron microscopy and silver staining, whereas the amyloid fibrillation detected by thioflavin T assay was greatly inhibited in vitro. Using circular dichroism spectroscopy, we discovered that both Aβ42 and Aβ42(Y10F) generated protofibrils and fibrils with a high proportion of parallel β-sheet structures. Furthermore, protofibrils formed by other mutant Aβ peptides and N-terminally shortened peptides were incapable of inducing neuronal death, with the exception of Aβ42 and Aβ42(Y10F). Our findings indicate that the N-terminus of Aβ is important for its fibrillation and neurotoxicity.

Figure 1

Comparison of human and rat Aβ amino acid sequences and aggregation states. (A) The Aβ peptide variants were used in this study. Note the differences between the human and rat Aβ sequences at positions 5, 10, and 13. (B) SEC analysis of the aggregation states of Aβ monomers and protofibrils in solution using a Superdex 75 column. (C) TEM examination of the morphology of Aβ monomers, ADDL, protofibrils and fibrils using negative staining techniques.

Figure 2

Human Aβ42 N-terminal mutations alter amyloid fibrillation. (A) Silver staining of Aβ fibril preparations at the time intervals indicated. (B) Semi-quantitative analysis of aggregates and oligomers from Aβ fibril preparations in (A). Each measurement was performed in three independent biological replicates. (C) ThT assays for measuring Aβ fibrillation. The raw data was fitted with the mathematical model of Boltzmann’s sigmoidal equation. Each measurement was performed in three independent biological replicates. (D) Representative TEM images of Aβ fibrils in (C).

Figure 3

The N-terminal region of human Aβ42 modulates the secondary structural composition of Aβ monomers, protofibrils and fibrils. (A,B) CD spectroscopy was used to determine the secondary structure composition of the various Aβ aggregates (200 μM) (A), which was afterwards interpreted using the BeStSel online tools (http://bestsel.elte.hu/index.php) (B).

Figure 4

The R5G mutation of human Aβ42 reduces neuronal death caused by protofibrils. (A) Representative images of primary hippocampal neurons treated with the corresponding Aβ protofibrils. MAP2 expression was used to identify the surviving neurons. (B) Survival rates of neurons treated with the indicated Aβ protofibrils were calculated. Each measurement was performed in three independent biological replicates. Statistical significance was assessed by the unpaired Student’s t-test; *: P < 0.01; **: P < 0.001.