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Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's β-amyloid

Nature Structural & Molecular Biology volume 14, pages 1157–1164 (2007)Cite this article

Abstract

Diffusible subfibrillar aggregates of amyloid proteins are potent neurotoxins and primary suspects in amyloid diseases including Alzheimer's disease. Despite widespread interest, the molecular structures of the amyloid intermediates and the conformational conversions in amyloid misfolding are poorly understood. Here we present a molecular-level examination of sequence-specific secondary structures and supramolecular structures of a neurotoxic amyloid intermediate of the 40-residue β-amyloid (Aβ) peptide involved in Alzheimer's disease. Using solid-state NMR and electron microscopy, we show that, before fibrillization, natively unstructured monomeric Aβ is subject to large conformational changes into a spherical amyloid intermediate of 15–35 nm diameter, which has predominantly parallel β-sheet structures. Structural comparison with Aβ fibrils demonstrates that formation of this β-sheet intermediate (Iβ) largely defines conformational transitions in amyloid misfolding. Neurotoxicity assays on PC12 cells show that Iβ shows higher toxicity than the fibril, indicating that the β-sheet formation may trigger neurotoxicity.

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Figure 1: Morphological properties of Aβ1–40 aggregates after different incubation periods (t).
Figure 2: Toxicity of Aβ1–40 monomers, intermediates and fibrils assessed by MTT assay.
Figure 3: Comparison of SSNMR data for the amyloid intermediate and the fibril.
Figure 4: Conformation and 13C chemical shift analyses of Iβ and fibril by SSNMR.
Figure 5: Kinetic properties of Iβ formation assessed by ThT fluorescence and Iβ seeding experiments.

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References

  1. Selkoe, D.J. Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases. Nat. Cell Biol. 6, 1054–1061 (2004).

    Article  CAS  Google Scholar 

  2. Caughey, B. & Lansbury, P.T. Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 26, 267–298 (2003).

    Article  CAS  Google Scholar 

  3. Sunde, M. & Blake, C.C.F. From the globular to the fibrous state: protein structure and structural conversion in amyloid formation. Q. Rev. Biophys. 31, 1–39 (1998).

    Article  CAS  Google Scholar 

  4. Dobson, C.M. Protein folding and misfolding. Nature 426, 884–890 (2003).

    Article  CAS  Google Scholar 

  5. Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507–511 (2002).

    Article  CAS  Google Scholar 

  6. Lorenzo, A. & Yankner, B.A. β-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc. Natl. Acad. Sci. USA 91, 12243–12247 (1994).

    Article  CAS  Google Scholar 

  7. Walsh, D.M., Lomakin, A., Benedek, G.B., Condron, M.M. & Teplow, D.B. Amyloid β-protein fibrillogenesis—detection of a protofibrillar intermediate. J. Biol. Chem. 272, 22364–22372 (1997).

    Article  CAS  Google Scholar 

  8. Lambert, M.P. et al. Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. USA 95, 6448–6453 (1998).

    Article  CAS  Google Scholar 

  9. Hoshi, M. et al. Spherical aggregates of (β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β. Proc. Natl. Acad. Sci. USA 100, 6370–6375 (2003).

    Article  CAS  Google Scholar 

  10. Nilsberth, C. et al. The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Aβ protofibril formation. Nat. Neurosci. 4, 887–893 (2001).

    Article  CAS  Google Scholar 

  11. Lashuel, H.A. et al. Mixtures of wild-type and a pathogenic (E22G) form of Aβ40 in vitro accumulate protofibrils, including amyloid pores. J. Mol. Biol. 332, 795–808 (2003).

    Article  CAS  Google Scholar 

  12. Conway, K.A. et al. Acceleration of oligomerization, not fibrillization, is a shared property of both α-synuclein mutations linked to early-onset Parkinson's disease: Implications for pathogenesis and therapy. Proc. Natl. Acad. Sci. USA 97, 571–576 (2000).

    Article  CAS  Google Scholar 

  13. McLean, C.A. et al. Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann. Neurol. 46, 860–866 (1999).

    Article  CAS  Google Scholar 

  14. Lue, L.F. et al. Soluble amyloid β peptide concentration as a predictor of synaptic change in Alzheimer's disease. Am. J. Pathol. 155, 853–862 (1999).

    Article  CAS  Google Scholar 

  15. Hsia, A.Y. et al. Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models. Proc. Natl. Acad. Sci. USA 96, 3228–3233 (1999).

    Article  CAS  Google Scholar 

  16. Walsh, D.M. et al. Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535–539 (2002).

    Article  CAS  Google Scholar 

  17. Lesne, S. et al. A specific amyloid-β protein assembly in the brain impairs memory. Nature 440, 352–357 (2006).

    Article  CAS  Google Scholar 

  18. Kirkitadze, M.D., Condron, M.M. & Teplow, D.B. Identification and characterization of key kinetic intermediate in amyloid β-protein fibrillogenesis. J. Mol. Biol. 312, 1103–1119 (2001).

    Article  CAS  Google Scholar 

  19. Krishnan, R. & Lindquist, S.L. Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature 435, 765–772 (2005).

    Article  CAS  Google Scholar 

  20. Liu, K., Cho, H.S., Lashuel, H.A., Kelly, J.W. & Wemmer, D.E. A glimpse of a possible amyloidogenic intermediate of transthyretin. Nat. Struct. Biol. 7, 754–757 (2000).

    Article  CAS  Google Scholar 

  21. Jahn, T.R., Parker, M.J., Homans, S.W. & Radford, S.E. Amyloid formation under physiological conditions proceeds via a native-like folding intermediate. Nat. Struct. Mol. Biol. 13, 195–201 (2006).

    Article  CAS  Google Scholar 

  22. Eakin, C.M., Berman, A.J. & Miranker, A.D. A native to amyloidogenic transition regulated by a backbone trigger. Nat. Struct. Mol. Biol. 13, 202–208 (2006).

    Article  CAS  Google Scholar 

  23. Dobson, C.M. An accidental breach of a protein's natural defenses. Nat. Struct. Mol. Biol. 13, 295–297 (2006).

    Article  CAS  Google Scholar 

  24. Chromy, B.A. et al. Self-assembly of Aβ(1–42) into globular neurotoxins. Biochemistry 42, 12749–12760 (2003).

    Article  CAS  Google Scholar 

  25. Chimon, S. & Ishii, Y. Capturing intermediate structures of Alzheimer's β-amyloid, Aβ(1–40), by solid-state NMR spectroscopy. J. Am. Chem. Soc. 127, 13472–13473 (2005).

    Article  CAS  Google Scholar 

  26. Shearman, M.S. Toxicity of protein aggregates in PC12 cells: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Methods Enzymol. 309, 716–723 (1999).

    Article  CAS  Google Scholar 

  27. Kayed, R. et al. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489 (2003).

    Article  CAS  Google Scholar 

  28. Lansbury, P.T. et al. Structural model for the β-amyloid fibril based on interstrand alignment of an antiparellel-sheet comprising a c-terminal peptide. Nat. Struct. Biol. 2, 990–998 (1995).

    Article  CAS  Google Scholar 

  29. Petkova, A.T. et al. A structural model for Alzheimer's β-amyloid peptide fibrils based on experimental constraints from solid-state NMR spectroscopy. Proc. Natl. Acad. Sci. USA 99, 16742–16747 (2002).

    Article  CAS  Google Scholar 

  30. Jaroniec, C.P., MacPhee, C.E., Astrof, N.S., Dobson, C.M. & Griffin, R.G. Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril. Proc. Natl. Acad. Sci. USA 99, 16748–16753 (2002).

    Article  CAS  Google Scholar 

  31. Heise, H. et al. Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR. Proc. Natl. Acad. Sci. USA 102, 15871–15876 (2005).

    Article  CAS  Google Scholar 

  32. Ritter, C. et al. Correlation of structural elements and infectivity of the HET-s prion. Nature 435, 844–848 (2005).

    Article  CAS  Google Scholar 

  33. Benzinger, T.L.S. et al. Propagating structure of Alzheimer's β-amyloid(10–35) is parallel β-sheet with residues in exact register. Proc. Natl. Acad. Sci. USA 95, 13407–13412 (1998).

    Article  CAS  Google Scholar 

  34. Weliky, D.P. et al. Solid-state NMR evidence for an antibody-dependent conformation of the V3 loop of HIV-1 gp120. Nat. Struct. Biol. 6, 141–145 (1999).

    Article  CAS  Google Scholar 

  35. Igumenova, T.I. et al. Assignments of carbon NMR resonances for microcrystalline ubiquitin. J. Am. Chem. Soc. 126, 6720–6727 (2004).

    Article  CAS  Google Scholar 

  36. Castellani, F. et al. Structure of a protein determined by solid-state magic-angle- spinning NMR spectroscopy. Nature 420, 98–102 (2002).

    Article  CAS  Google Scholar 

  37. Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).

    Article  CAS  Google Scholar 

  38. Studelska, D.R., McDowell, L.M., Espe, M.P., Klug, C.A. & Schaefer, J. Slowed enzymatic turnover allows characterization of intermediates by solid-state NMR. Biochemistry 36, 15555–15560 (1997).

    Article  CAS  Google Scholar 

  39. Saito, H. Conformation-dependent 13C chemical shifts: a new means of conformational characterization as obtained by high-resolution solid-state NMR. Magn. Reson. Chem. 24, 835–852 (1986).

    Article  CAS  Google Scholar 

  40. Spera, S. & Bax, A. Empirical correlation between protein backbone conformation and C-α and C-β C-13 nuclear-magnetic-resonance chemical shifts. J. Am. Chem. Soc. 113, 5490–5492 (1991).

    Article  CAS  Google Scholar 

  41. Ishii, Y. 13C–13C dipolar recoupling under very fast magic angle spinning in solid-state NMR: Applications to distance measurements, spectral assignments, and high-throughput secondary-structure elucidation. J. Chem. Phys. 114, 8473–8483 (2001).

    Article  CAS  Google Scholar 

  42. Petkova, A.T. et al. Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science 307, 262–265 (2005).

    Article  CAS  Google Scholar 

  43. Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. & Sykes, B.D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J. Biomol. NMR 5, 67–81 (1995).

    Article  CAS  Google Scholar 

  44. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  Google Scholar 

  45. Petkova, A.T., Yau, W.M. & Tycko, R. Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils. Biochemistry 45, 498–512 (2006).

    Article  CAS  Google Scholar 

  46. Levine, H., III Quantification of β-sheet amyloid fibril structures with thioflavin T. Methods Enzymol. 309, 274–284 (1999).

    Article  CAS  Google Scholar 

  47. Naiki, H., Gejyo, F. & Nakakuki, K. Concentration-dependent inhibitory effects of apolipoprotein E on Alzheimer's β-amyloid fibril formation in vitro. Biochemistry 36, 6243–6250 (1997).

    Article  CAS  Google Scholar 

  48. Reches, M. & Gazit, E. Casting metal nanowires within discrete self-assembled peptide nanotubes. Science 300, 625–627 (2003).

    Article  CAS  Google Scholar 

  49. Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 21, 1171–1178 (2003).

    Article  CAS  Google Scholar 

  50. Lambert, M.P. et al. Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies. J. Neurochem. 79, 595–605 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Rasenick and J.-Z. Yu (University of Illinois at Chicago) for the PC-12 cells; L. Juarez and B. Lee for assistance with TEM studies and peptide synthesis, respectively; C. Bhardwaj, W. Cho and members of his group, and G. Fenteany for assistance with the neurotoxicity assay; R. Tycko for the structural model of Aβ1–40 used in Fig. 4b and C. Jameson, T. Keiderling and W. Klein for suggestions. This work was supported in part by the Alzheimer's Association (NIRG 035123), the Dreyfus Foundation Teacher-Scholar Award program, the US National Science Foundation CAREER program (CHE 449952), and the National Institutes of Health RO1 program (AG028490).

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  1. Department of Chemistry, University of Illinois at Chicago, Chicago, 60607, Illinois, USA

    Sandra Chimon, Medhat A Shaibat, Christopher R Jones, Diana C Calero, Buzulagu Aizezi & Yoshitaka Ishii

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Correspondence to Yoshitaka Ishii.

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Chimon, S., Shaibat, M., Jones, C. et al. Evidence of fibril-like β-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's β-amyloid. Nat Struct Mol Biol 14, 1157–1164 (2007). https://doi.org/10.1038/nsmb1345

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