Structure-neurotoxicity relationships of amyloid beta-protein oligomers - PubMed (original) (raw)

Structure-neurotoxicity relationships of amyloid beta-protein oligomers

Kenjiro Ono et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Amyloid beta-protein (Abeta) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). "Oligomer" is an ill-defined term because many kinds have been reported and they often exist in rapid equilibrium with monomers and higher-order assemblies. We report here results of studies in which specific oligomers have been stabilized structurally, fractionated in pure form, and then studied by using a combination of CD spectroscopy, Thioflavin T fluorescence, EM, atomic force microscopy (AFM), and neurotoxicity assays. Abeta monomers were largely unstructured, but oligomers exhibited order-dependent increases in beta-sheet content. EM and AFM data suggest that dimerization and subsequent monomer addition are processes in which significant and asymmetric monomer conformational changes occur. Oligomer secondary structure and order correlated directly with fibril nucleation activity. Neurotoxic activity increased disproportionately (order dependence >1) with oligomer order. The structure-activity correlations reported here significantly extend our understanding of the conformational dynamics, structure, and relative toxicity of pure Abeta oligomers of specific order.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Stability of purified oligomers. Aβ samples were subjected to PICUP and SDS/PAGE. Individual gel bands were stained with Coomassie blue, excised, and then extracted under alkaline conditions. The extracts then were reconstituted in F12K medium and analyzed by SDS/PAGE and silver staining. Lane 1, cross-linked Aβ immediately after PICUP (cross-linking control). Lane 2, cross-linked Aβ subjected to the entire protocol, but with all bands pooled together (control for unfractionated Aβ subjected to alkaline extraction). Lane 3, monomer band. Lane 4, dimer band. Lane 5, trimer band. Lane 6, tetramer band. Lane 7, a “band-equivalent”-sized piece of gel (“no protein” control). The data are representative of results from each of three independent experiments.

Fig. 2.

Fig. 2.

Secondary structure dynamics of Aβ assemblies. Uncross-linked (UnXL), cross-linked (XL), or fibrillar (Fib) Aβ (A) or isolated oligomers [monomer (Mo), dimer (Di), trimer (Tr), and tetramer (Te)] (B) were prepared in 10 mM PBS, pH 7.4, and then monitored immediately by CD. Data are representative of three independent experiments.

Fig. 3.

Fig. 3.

Nucleation of Aβ assembly. The nucleation activity of different Aβ preparations was assessed by addition of each preparation to uncross-linked Aβ, which then was incubated for 7 d at 37 °C in 10 mM PBS, pH 7.4. Aliquots were assayed periodically by using ThT. The preparations were uncross-linked Aβ (○, UnXL), 10% (vol/vol) cross-linked Aβ (●, XL), or 10% (vol/vol) sonicated Aβ fibrils (△, Fib) (A) or 10% (vol/vol) Aβ monomer (○, Mo), dimer (●, Di), trimer (△, Tr), or tetramer (▲, Te) (B). Binding is expressed as mean fluorescence [in arbitrary fluorescence units (FU)] ± SE. Data were obtained in three independent experiments. Arrows indicate times at which half maximal ThT binding was observed.

Fig. 4.

Fig. 4.

LDH activity. Uncross-linked (UnXL), cross-linked (XL), fibrillar, monomeric, and purified oligomeric (dimers, trimers, tetramers) Aβ samples were added at final nominal concentrations of 25 μM to differentiated PC12 cells. LDH activity in the supernatant fluid then was measured after 48 h. Data are representative of that obtained in three independent experiments. Each column represents means ± SE. The statistical significance of the toxicity differences among samples was determined by one-way fractional ANOVA and multiple comparison tests. *, P < 0.01; **, P < 0.001. NS, not significant.

Fig. 5.

Fig. 5.

Aβ assembly. The data are consistent with an initial oligomerization process in which dimerization involves the self-association of two monomers, each of which exists in a folded (F) state in the dimer. It is not clear when folding from the unfolded (U) state occurs, e.g., before dimerization, contemporaneous with dimerization, or through conformational rearrangement within the dimer. Once the dimer forms, subsequent addition of a monomer to form the trimer involves accommodation (dotted arrow) of the incoming monomer into the dimer structure. The structure of this third monomer within trimers (F*) is different from that of free monomer or of monomers comprising a dimer because the size of the trimer is not thrice that of the monomer or 150% that of the dimer. Each monomer addition past the dimer stage produces the same size increase, and this increase is larger than that observed in dimerization; thus each of these stages would involve monomer accommodation, i.e., F*. It should be noted that every step of peptide oligomerization or fibril formation may not involve simple monomer addition. Other pathways are possible and likely occur (e.g., tetramer formation by dimer association). The scheme presented here illustrates one pathway that is both reasonable and consistent with the experimental data.

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References

    1. Blennow K, de Leon MJ, Zetterberg H. Alzheimer's disease. Lancet. 2006;368:387–403. - PubMed
    1. Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer's amyloid β-peptide. Nat Rev Mol Cell Biol. 2007;8:101–112. - PubMed
    1. Kirkitadze MD, Bitan G, Teplow DB. Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: The emerging role of oligomeric assemblies. J Neurosci Res. 2002;69:567–577. - PubMed
    1. Walsh DM, Selkoe DJ. Aβ oligomers: A decade of discovery. J Neurochem. 2007;101:1172–1184. - PubMed
    1. Shankar GM, et al. Amyloid-β protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med. 2008;14:837–842. - PMC - PubMed

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