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. 2010 Jul 23;285(30):23186-97.
doi: 10.1074/jbc.M109.086496. Epub 2010 May 7.

Effects of the English (H6R) and Tottori (D7N) familial Alzheimer disease mutations on amyloid beta-protein assembly and toxicity

Kenjiro Ono  1 , Margaret M Condron, David B Teplow
Affiliations

Effects of the English (H6R) and Tottori (D7N) familial Alzheimer disease mutations on amyloid beta-protein assembly and toxicity

Kenjiro Ono et al. J Biol Chem. .

Abstract

Mutations in the amyloid beta-protein (Abeta) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid beta-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Abeta40 and Abeta42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil --> alpha/beta --> beta and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial beta-strand (H6R) or alpha/beta (D7N) structure, in contrast to the predominately statistical coil structure of wild type Abeta oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Abeta assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.

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Figures

FIGURE 1.
FIGURE 1.
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).
FIGURE 2.
FIGURE 2.
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.
FIGURE 3.
FIGURE 3.
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.
FIGURE 4.
FIGURE 4.
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.
FIGURE 5.
FIGURE 5.
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.
FIGURE 6.
FIGURE 6.
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.
FIGURE 7.
FIGURE 7.
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.
FIGURE 8.
FIGURE 8.
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.
FIGURE 9.
FIGURE 9.
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).
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References

    1. Teplow D. B. (1998) Amyloid 5, 121–142 - PubMed
    1. Selkoe D. J. (2004) Ann. Intern. Med. 140, 627–638 - PubMed
    1. Bertram L., Tanzi R. E. (2008) Nat. Rev. Neurosci. 9, 768–778 - PubMed
    1. Borchelt D. R., Thinakaran G., Eckman C. B., Lee M. K., Davenport F., Ratovitsky T., Prada C. M., Kim G., Seekins S., Yager D., Slunt H. H., Wang R., Seeger M., Levey A. I., Gandy S. E., Copeland N. G., Jenkins N. A., Price D. L., Younkin S. G., Sisodia S. S. (1996) Neuron 17, 1005–1013 - PubMed
    1. Tomita T., Maruyama K., Saido T. C., Kume H., Shinozaki K., Tokuhiro S., Capell A., Walter J., Grünberg J., Haass C., Iwatsubo T., Obata K. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2025–2030 - PMC - PubMed

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