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

Cohen SI, Arosio P, Presto J, Kurudenkandy FR, Biverstål H, Dolfe L, Dunning C, Yang X, Frohm B, Vendruscolo M, Johansson J, Dobson CM, Fisahn A, Knowles TP, Linse S - Nat. Struct. Mol. Biol. (2015)

Bottom Line: Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.

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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Inhibition by Brichos of Aβ42 surface-catalysed secondary nucleation of oligomers(a-d) Kinetics of aggregation when pre-formed Aβ42 fibrils, grown in the absence (a, c) or presence (b, d) of Brichos, were added to monomeric Aβ42 with (c, d) or without (a, b) Brichos in solution. Fibrils grown (b) in the presence of Brichos do not accelerate the reaction to the same extent as (a) fibrils that have never been exposed to Brichos, showing that Brichos binds to fibrils. When Brichos was added in to solutions in which aggregation was underway (c, d), it arrested the reaction and prevented the acceleration due to added fibrils even with fibrils grown in the absence of chaperone. The dashed lines show predictions19 for the reaction profiles with the secondary nucleation rate constant set to (a) the value measured previously in the absence of the chaperone19, (b) 60% of this value, and (c, d) zero. The solution concentrations of Aβ42 and Brichos were 3 μM; the concentration of pre-formed fibrils was 6 nM. All data show four technical replicas overlaid.
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Figure 2: Inhibition by Brichos of Aβ42 surface-catalysed secondary nucleation of oligomers(a-d) Kinetics of aggregation when pre-formed Aβ42 fibrils, grown in the absence (a, c) or presence (b, d) of Brichos, were added to monomeric Aβ42 with (c, d) or without (a, b) Brichos in solution. Fibrils grown (b) in the presence of Brichos do not accelerate the reaction to the same extent as (a) fibrils that have never been exposed to Brichos, showing that Brichos binds to fibrils. When Brichos was added in to solutions in which aggregation was underway (c, d), it arrested the reaction and prevented the acceleration due to added fibrils even with fibrils grown in the absence of chaperone. The dashed lines show predictions19 for the reaction profiles with the secondary nucleation rate constant set to (a) the value measured previously in the absence of the chaperone19, (b) 60% of this value, and (c, d) zero. The solution concentrations of Aβ42 and Brichos were 3 μM; the concentration of pre-formed fibrils was 6 nM. All data show four technical replicas overlaid.

Mentions: Inhibition of secondary nucleation requires perturbation of the interactions between soluble monomers and amyloid fibrils, and in principle either species could be targeted by Brichos. To identify the molecular species on which this molecular chaperone acts, therefore, we performed experiments in which newly formed Aβ42 fibrils, produced in the presence or absence of Brichos, were diluted and added to freshly prepared monomer solutions and then also incubated in the presence and absence of Brichos (Fig. 2). We observed that pristine fibrils that had not been exposed to the chaperone at any stage enhance the process of secondary nucleation and increase the rate of aggregation19 (Fig. 2a). By contrast, we have found in the present work that pre-formed fibrils that had been generated in the presence of Brichos (Fig. 2b) accelerate aggregation, even in the absence of Brichos in solution, to a significantly smaller extent than do fibrils formed in the absence of chaperone (Fig. 2a). Indeed, the kinetic analysis indicates a reduction of approximately 40% in the secondary nucleation rate in this latter case (Fig. 2b), showing that a significant fraction of the chaperones must have remained bound to the fibrils during the time course of the aggregation reaction. Furthermore, the surface catalytic activity of fibrils was observed to be entirely abolished when pre-formed aggregates generated in the absence of molecular chaperones were brought into contact with solutions containing both Brichos and monomeric Aβ42 at equal stoichiometry (Fig. 2c), demonstrating the ability of Brichos to arrest even ongoing reactions. Identical results were obtained when fibrils formed in the presence of Brichos were added to solutions containing both monomeric Aβ42 and Brichos (Fig. 2d). These results are consistent with observations that Brichos is able to arrest ongoing Aβ42 and Aβ40 aggregation reactions when added at various time-points.31


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

Cohen SI, Arosio P, Presto J, Kurudenkandy FR, Biverstål H, Dolfe L, Dunning C, Yang X, Frohm B, Vendruscolo M, Johansson J, Dobson CM, Fisahn A, Knowles TP, Linse S - Nat. Struct. Mol. Biol. (2015)

Inhibition by Brichos of Aβ42 surface-catalysed secondary nucleation of oligomers(a-d) Kinetics of aggregation when pre-formed Aβ42 fibrils, grown in the absence (a, c) or presence (b, d) of Brichos, were added to monomeric Aβ42 with (c, d) or without (a, b) Brichos in solution. Fibrils grown (b) in the presence of Brichos do not accelerate the reaction to the same extent as (a) fibrils that have never been exposed to Brichos, showing that Brichos binds to fibrils. When Brichos was added in to solutions in which aggregation was underway (c, d), it arrested the reaction and prevented the acceleration due to added fibrils even with fibrils grown in the absence of chaperone. The dashed lines show predictions19 for the reaction profiles with the secondary nucleation rate constant set to (a) the value measured previously in the absence of the chaperone19, (b) 60% of this value, and (c, d) zero. The solution concentrations of Aβ42 and Brichos were 3 μM; the concentration of pre-formed fibrils was 6 nM. All data show four technical replicas overlaid.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4595974&req=5

Figure 2: Inhibition by Brichos of Aβ42 surface-catalysed secondary nucleation of oligomers(a-d) Kinetics of aggregation when pre-formed Aβ42 fibrils, grown in the absence (a, c) or presence (b, d) of Brichos, were added to monomeric Aβ42 with (c, d) or without (a, b) Brichos in solution. Fibrils grown (b) in the presence of Brichos do not accelerate the reaction to the same extent as (a) fibrils that have never been exposed to Brichos, showing that Brichos binds to fibrils. When Brichos was added in to solutions in which aggregation was underway (c, d), it arrested the reaction and prevented the acceleration due to added fibrils even with fibrils grown in the absence of chaperone. The dashed lines show predictions19 for the reaction profiles with the secondary nucleation rate constant set to (a) the value measured previously in the absence of the chaperone19, (b) 60% of this value, and (c, d) zero. The solution concentrations of Aβ42 and Brichos were 3 μM; the concentration of pre-formed fibrils was 6 nM. All data show four technical replicas overlaid.
Mentions: Inhibition of secondary nucleation requires perturbation of the interactions between soluble monomers and amyloid fibrils, and in principle either species could be targeted by Brichos. To identify the molecular species on which this molecular chaperone acts, therefore, we performed experiments in which newly formed Aβ42 fibrils, produced in the presence or absence of Brichos, were diluted and added to freshly prepared monomer solutions and then also incubated in the presence and absence of Brichos (Fig. 2). We observed that pristine fibrils that had not been exposed to the chaperone at any stage enhance the process of secondary nucleation and increase the rate of aggregation19 (Fig. 2a). By contrast, we have found in the present work that pre-formed fibrils that had been generated in the presence of Brichos (Fig. 2b) accelerate aggregation, even in the absence of Brichos in solution, to a significantly smaller extent than do fibrils formed in the absence of chaperone (Fig. 2a). Indeed, the kinetic analysis indicates a reduction of approximately 40% in the secondary nucleation rate in this latter case (Fig. 2b), showing that a significant fraction of the chaperones must have remained bound to the fibrils during the time course of the aggregation reaction. Furthermore, the surface catalytic activity of fibrils was observed to be entirely abolished when pre-formed aggregates generated in the absence of molecular chaperones were brought into contact with solutions containing both Brichos and monomeric Aβ42 at equal stoichiometry (Fig. 2c), demonstrating the ability of Brichos to arrest even ongoing reactions. Identical results were obtained when fibrils formed in the presence of Brichos were added to solutions containing both monomeric Aβ42 and Brichos (Fig. 2d). These results are consistent with observations that Brichos is able to arrest ongoing Aβ42 and Aβ40 aggregation reactions when added at various time-points.31

Bottom Line: Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.

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.

Show MeSH
Related in: MedlinePlus