PMC

AFM analysis of Aβ40 or Aβ42 assemblies. AFM was performed on 25 μm un-cross-linked (A, C, E, G, I, and K) and cross-linked (B, D, F, H, J, and L) Aβ40 (A–F) and Aβ42 (G–L) of WT (A, B, G, and H), H6R (C, D, I, and J), or D7N (E, F, K, and L) peptides. Scale bars are 100 nm.

EM analysis of Aβ40 or Aβ42 assemblies. EM was performed on 25 μm un-cross-linked (A, C, E, G, I, and K) and cross-linked (B, D, F, H, J, and L) Aβ40 (A-F) and Aβ42 (G-L) of wild type (WT) (A, B, G, and H), H6R (C, D, I, and J), or D7N (E, F, K, and L) peptides. Scale bars are 100 nm.

Secondary structure of Aβ oligomers. CD spectroscopy was performed after cross-linking of 25 μm Aβ40 (A) or Aβ42 (B) of wild type (WT) (○), H6R (▾), or D7N (□). Each figure is representative of data obtained in each of three independent experiments.

MTT metabolism. MTT assays were performed on differentiated PC12 cells incubated for 24 h with un-cross-linked (UnXL) wild type (WT) (○), H6R (▿), D7N (□) or cross-linked (XL) WT (●), H6R (▾), D7N (■) of Aβ40 (A) and Aβ42 (B). Each figure is representative of results obtained in each of three independent experiments. Data are expressed as mean percent toxicity ± S.E.

LDH activity. LDH release was measured after a 48-h incubation of differentiated PC12 cells with un-cross-linked (UnXL) or cross-linked (XL) Aβ40 (A) and Aβ42 (B) of WT, H6R, or D7N at final nominal concentration of 25 μm. Each figure is representative of data obtained in each of three independent experiments. Bars are mean LDH activity ± S.E. Significance was determined using one-way fractional analysis of variance and multiple comparison tests (*, p < 0.05; **, p < 0.01).

Nucleation of Aβ fibril assembly. Zero % (v/v) (●) or 10% (v/v) cross-linked (XL) WT (○), H6R (▾), or D7N (□) oligomers of Aβ40 (A) or Aβ42 (B) were added to un-cross-linked (UnXL) WT Aβ40 and Aβ42, which then were incubated for 3 or 7 days at 37 °C in 10 mm phosphate-buffered saline, pH 7.4. Aliquots were assayed periodically using ThT. Binding is expressed as mean fluorescence (in arbitrary fluorescence units (FU)) ± S.E. Each figure comprises data obtained in three independent experiments.

Aβ oligomerization. PICUP, followed by SDS-PAGE and silver staining, was used to study oligomerization of 25 μm Aβ40 (A, C, and E) or Aβ42 (B, D, and F) of wild type (WT) (A and B), H6R (C and D), or D7N (E and F). Lanes C, un-cross-linked Aβ; lanes 0, Aβ cross-linked at the beginning of incubation at 37 °C; lanes 2, Aβ cross-linked after 2 h; lanes 4, Aβ cross-linked after 4 h; lanes 6, Aβ cross-linked after 6 h. Oligomer order is indicated by white numerals over the respective gel bands. The gel is representative of each of three independent experiments.

Densitometric analysis of Aβ oligomerization. To produce intensity profiles and calculate the relative amounts of each oligomer type of Aβ40 (A) or Aβ42 (B), One-Dscan software (version 2.2.2; BD Biosciences Bioimaging, Rockville, MD) was used. The metric of oligomer/monomer ratio = (total lane intensity-monomer intensity)/monomer intensity of wild type (WT) (○), H6R (▾), or D7N (□) is expressed as mean ratio ± S.E. Statistical significance of oligomer/monomer differences between each mutant peptide and wild type peptide is indicated by: *, p < 0.05; or **, p < 0.01. Each figure comprises data obtained in three independent experiments.

Aβ secondary structure dynamics. 25 μm Aβ40 (A, C, and E) or Aβ42 (B, D, and F) of wild type (WT) (A and B), H6R (C and D), or D7N (E and F) were incubated at 37 °C for 7 days in 10 mm phosphate, pH 7.4. Spectra were acquired immediately at the start of the incubation period, day 0 (○), and after days 0.5 (●), 1 (▾), 2 (□), 3 (■), 4 (◇), 5 (▴), and 6 (▿). The spectra presented at each time are representative of those obtained during each of three independent experiments. G and H, molar ellipticity [θ]₁₉₈ versus time for Aβ40 (G) or Aβ42 (H).