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Direct measurement of lipid membrane disruption connects kinetics and toxicity of Aβ42 aggregation

Nature Structural & Molecular Biology volume 27, pages 886–891 (2020)Cite this article

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Abstract

The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer’s disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aβ42 peptide implicated in Alzheimer’s disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.

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Fig. 1: Link between aggregation of Aβ42 and lipid bilayer permeability.
Fig. 2: Role of oligomer cooperativity and structural reorganization.
Fig. 3: Molecular chaperones modulate lipid bilayer permeation induced by the aggregation of Aβ42.
Fig. 4: Common role of secondary Aβ42 oligomers in generating aggregation-associated damage in different systems.

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The authors confirm that all data generated and analyzed during this study are included in this published article and its Supplementary Information. Source data are provided with this paper.

Code availability

All simulation and data analysis codes are included in this article and its Supplementary Information. Codes are available from the corresponding authors on request.

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Acknowledgements

This study has been supported by the Boehringer Ingelheim Fonds (P.F.), the German National Merit Foundation (P.F.), a Marie-Curie Individual Fellowship (S.D.), Peterhouse, Cambridge (T.C.T.M.), the Swiss National Science Foundation (T.C.T.M.), the Wellcome Trust (T.P.J.K., C.M.D., M.V.), the Swedish Research Council (S.L.), the Royal Society (D.K.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Biotechnology and Biological Sciences Research Council (T.P.J.K.) and the Cambridge Centre for Misfolding Diseases (P.F., T.P.J.K., M.V. and C.M.D.).

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Author notes

  1. These authors contributed equally: Patrick Flagmeier, Suman De, Thomas C. T. Michaels.

Authors and Affiliations

  1. Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge, Cambridge, UK

    Patrick Flagmeier, Suman De, Thomas C. T. Michaels, Xiaoting Yang, Alexander J. Dear, Michele Vendruscolo, Tuomas P. J. Knowles & Christopher M. Dobson

  2. Paulson School of Engineering and Applied Science, Harvard University, Cambridge, MA, USA

    Thomas C. T. Michaels & Alexander J. Dear

  3. Department of Chemistry, Division for Biochemistry and Structural Biology, Lund University, Lund, Sweden

    Cecilia Emanuelsson & Sara Linse

  4. Department of Chemistry, University of Cambridge, Cambridge, UK

    David Klenerman

  5. UK Dementia Research Institute, University of Cambridge, Cambridge, UK

    David Klenerman

  6. Cavendish Laboratory, University of Cambridge, Cambridge, UK

    Tuomas P. J. Knowles

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Contributions

P.F. and S.D. performed the experiments. T.C.T.M. developed the theoretical model and performed the kinetic analysis. P.F., S.D., T.C.T.M., X.Y., A.J.D., C.E., M.V., S.L., D.K., T.P.J.K. and C.M.D. participated in designing the study, interpreting the results and writing the paper. P.F., S.D. and T.C.T.M. contributed equally to this work.

Corresponding authors

Correspondence to David Klenerman or Tuomas P. J. Knowles.

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The authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Reproducible aggregation kinetics of recombinant Aβ42.

a, Monomeric Aβ42 is incubated in the presence of 20 μM ThT under quiescent conditions at 37 °C (red: 2 μM, orange: 2.5 μM, light green: 3 μM, dark green: 3.5 μM, light blue: 4 μM, dark blue: 4.5 μM, black: 5 μM). b, Global fit to the kinetic traces of the Aβ42 aggregation to Supplementary Note 1, Eq. 2, yielding the parameters of Supplementary Table 1. c, Half-times of the aggregation reaction. d, Drawing of the microscopic events of Aβ42 aggregation. Monomeric protein forms small aggregates (for example oligomers) during primary nucleation and these convert into fibrils and their fibril mass growths via elongation. An autocatalytic process, namely secondary nucleation, that depends on both the monomer and fibril concentration leads to an amplification of the aggregation reaction.

Extended Data Fig. 2 Oligomer concentrations are dominated by secondary oligomers.

Fraction of primary oligomers (over total oligomer population) calculated using the fitting parameters of Fig. 1c of the main text.

Extended Data Fig. 3 The addition of seed fibrils accelerates the aggregation of Aβ42.

a, Monomeric Aβ42 was incubated at a concentration of 2 μM in the presence of 20 μM ThT at 37 °C under quiescent conditions in the presence of no (red), 0.5% (orange), 1% (light green), 2.5% (dark green), 10% (blue) and 20% (black) preformed fibrils. b, Global fits to the kinetic traces of the aggregation reaction. c, The half-times of the aggregation reaction with varying concentrations of seed fibrils. d, Drawing of the microscopic events of Aβ42 aggregation in the presence of the preformed fibrils that accelerate the aggregation. Addition of seed fibrils allows bypassing de novo formation of new aggregates by primary nucleation, favouring secondary nucleation.

Extended Data Fig. 4 Role of cooperativity and toxic conversion.

a, Best global fit of experimental Ca2+-influx data to secondary oligomer kinetic model with reaction order γ = 3 (Supplementary Note 1, Eq. 8). b, Misfit of permeation measurements to a kinetic model that assumes slow toxic conversion of oligomers generated initially through secondary nucleation (Supplementary Note 1, Eq. 9). Theoretical predictions were generated assuming an arbitrary timescale for conversion comparable to that of the aggregation reaction (τ = 0.5 h in this case).

Extended Data Fig. 5 The chaperone DNAJB6 inhibits primary nucleation of Aβ42 aggregation.

a, Monomeric Aβ42 was incubated at a concentration of 2 μM in the presence of 20 μM ThT at 37 °C under quiescent conditions in the absence (black) and presence (blue: 0.1%, green: 0.25%, orange: and 0.5% relative to monomeric Aβ42) of DNAJB6. b, Global fits to the kinetic traces. c, Relative primary nucleation rate kn. d, Drawing of the microscopic events of Aβ42 aggregation in the presence of DNAJB6 that inhibits primary nucleation.

Extended Data Fig. 6 The BRICHOS domain inhibits secondary nucleation of Aβ42 aggregation and the membrane disruption ability of Aβ42 oligomers.

a, Monomeric Aβ42 was incubated at a concentration of 2 μM in the presence of 20 μM ThT at 37 °C under quiescent conditions in the absence (black) and presence (dark blue: 10%, cyan: 35%, magenta: 50%, green: 75% and orange 100% relative to monomeric Aβ42) of the Brichos domain. b, Fits to the kinetic traces of the aggregation reactions in the presence of the BRICHOS domain to determine its influence on secondary nucleation. c, Relative secondary nucleation rate constants k2 of the aggregation of Aβ42 with increasing concentrations of the BRICHOS domain. d, Drawing of the microscopic events of Aβ42 aggregation in the presence of the BRICHOS domain that inhibits secondary nucleation.

Extended Data Fig. 7 The mutant variant S/T18A of DNAJB6 does neither influence the primary nucleation of Aβ42 nor its ability to disrupt the membrane at the same concentrations.

a, Aggregation kinetics of Aβ42 in the presence of the mutant variant S/T18A. b, Effect of S/T18A on the ability of aliquots taken at a time point corresponding to maximal Ca2+ influx.

Extended Data Fig. 8 Analysis of relative secondary nucleation rate with increasing BRICHOS concentration.

Relative secondary nucleation rate constants k2 of the aggregation of Aβ42 with increasing concentrations of the BRICHOS domain (from Extended Data Fig. 6c) fitted to Supplementary Note 1, Eq. 11 (solid line).

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Supplementary Note 1 and Tables 1–3.

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Flagmeier, P., De, S., Michaels, T.C.T. et al. Direct measurement of lipid membrane disruption connects kinetics and toxicity of Aβ42 aggregation. Nat Struct Mol Biol 27, 886–891 (2020). https://doi.org/10.1038/s41594-020-0471-z

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