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A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers

Abstract

Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

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Figure 1: Kinetics of Aβ42 aggregation in the presence of Brichos.
Figure 2: Inhibition by Brichos of Aβ42 surface-catalyzed secondary nucleation of oligomers.
Figure 3: Brichos interacts with fibrillar but not monomeric Aβ42.
Figure 4: Brichos inhibits the formation of Aβ42 oligomers by suppressing secondary nucleation and redirecting the reaction pathway toward elongation events.
Figure 5: Brichos reduces the toxicity associated with the aggregation of Aβ42 in mouse brain slices.
Figure 6: Brichos inhibits the catalytic cycle that generates toxic Aβ42 oligomers.

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Acknowledgements

We acknowledge financial support from the Schiff Foundation (S.I.A.C.), the Swedish Research Council (S.L., J.J. and J.P.) and its Linneaus Centre Organizing Molecular Matter (S.L.), the Crafoord Foundation (S.L.), Alzheimerfonden (S.L.), the Frances and Augustus Newman Foundation (P.A. and T.P.J.K.), the European Research Council (T.P.J.K. and S.L.), the Biotechnology and Biological Sciences Research Council (T.P.J.K.), the Nanometer Structure Consortium at Lund University (S.L.), the KID PhD studentship grant (F.R.K. and L.D.), the Swedish Medical Association (A.F.), the Brain Fund (A.F.), the Strategic Program in Neurosciences at the Karolinska Institutet (A.F.), the Swiss National Science Foundation (P.A.) and the Wellcome Trust (C.M.D. and T.P.J.K.). We thank T. Weaver (University of Cincinnati) for reagents.

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S.I.A.C., T.P.J.K., A.F. and S.L. designed the study. J.P., F.R.K., A.F., L.D., C.D., X.Y., B.F., H.B. and S. L. performed the experiments. S.I.A.C., P.A., T.P.J.K., F.R.K., C.M.D., A.F. and S.L. analyzed the data. S.I.A.C., P.A., C.M.D., T.P.J.K. and S.L. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to André Fisahn, Tuomas P J Knowles or Sara Linse.

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Integrated supplementary information

Supplementary Figure 1 Saturation of the effect of Brichos at high chaperone concentrations.

Kinetic reaction profiles for the aggregation of Aβ42 are shown from left to right for reactions in the absence of Brichos and with 67%, 100%, 120%, 170%, 250% and 330% Aβ42 monomer equivalents of Brichos. At high chaperone concentrations, secondary nucleation is completed inhibited and additional chaperone has no effect on the kinetics of aggregation. The inhibitory effect reaches this saturation at a critical Brichos concentration which corresponds to approximately 100% of Aβ42 monomer equivalents.

Supplementary Figure 2 Kinetic analysis of the mode of action of Brichos.

(a) Kinetic reaction profiles for the aggregation of Aβ42 are shown from left to right for reactions in the absence of Brichos and with 10%, 15%, 22%, 35%, 50% and 75% Aβ42 monomer equivalents of Brichos. Continuous lines represent fits of the integrated rate for Aβ42 aggregation, Eq. 1, to the data19. (b) Decrease of the effective microscopic rates for secondary nucleation (k2) and fibril elongation (k+) with increasing chaperone concentration evaluated from the fits. The microscopic rate constants are normalised relative to the values in the absence of Brichos. The Brichos concentration is given in Aβ42 monomer equivalents. The analysis indicates that Brichos affects specifically the secondary nucleation pathway. To further quantify the behaviour as a function of chaperone concentration, we also performed a first-principles kinetic analysis (see Supplementary Note for a derivation) that introduces explicitly into the reaction scheme the reversible binding of the chaperone along the fibril surface by considering a Langmuir-type adsorption, resulting in the fits in Fig. 1c (thin dotted lines).

Supplementary Figure 3 Control experiments with other proteins.

Kinetic reaction profiles with 3µM Aβ42 and four control proteins at varying concentration (0.45µM pale blue; 1.5µM cyan; 3µM green). The proteins are calbindin D9k, calmodulin, protein G B1-domain and fatty-acid free human serum albumin (HSA). Calbindin D9k and PGB1 have a more negative net charge and are more hydrophilic than Brichos, and calmodulin is significantly more negatively charged. (a-c) Calbindin, PGB1 and calmodulin have no effect on Aβ42 aggregation in this concentration range. (d) HSA affects the aggregation reaction but does not match the prediction for selective inhibition of secondary nucleation (green dashed line), implying that the mechanism of inhibition is different to that identified for Brichos. Four replicates of each condition are shown.

Supplementary Figure 4 Cell viability and cytotoxicity assays.

(a) Cell viability as measured by the MTS assay shows that Brichos removes toxicity even in reactions containing pre-formed fibrils. (b, c) Cell apoptosis as measured by caspase assay for two cell lines: (b) more sensitive to perturbations of the medium, and (c) less sensitive to perturbations of the medium. For both cell lines, the change in the caspase signal relative to the control that is induced by Aβ42 aggregation is abolished by Brichos. The change in caspase signal induced by Aβ42 toxicity after a fixed time for these two cell lines differs in direction since the measured signal depends on the time of the readout relative to the characteristic time of cell death (see Supplementary Note). In both cases the change in caspase signal indicates increased toxicity, and is suppressed by Brichos. The concentration of monomeric peptide was 1µM in (a) and (b) and 10µM in (c); the concentration of pre-formed fibrils was 10 nM in (a) and (b) and 100 nM in (c). The average and standard deviations shown are over four replicates of each condition in (a) and (b) and six replicates of each condition in (c).

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Cohen, S., Arosio, P., Presto, J. et al. A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers. Nat Struct Mol Biol 22, 207–213 (2015). https://doi.org/10.1038/nsmb.2971

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