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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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Brichos inhibits the formation of Aβ42 oligomers by suppressing secondary nucleation and redirecting the reaction pathway towards elongation events(a-b) Quantification of low molecular weight oligomers of Aβ42 in the absence (a) and presence (b) of Brichos using size-exclusion chromatography and the 6E10 antibody. The reduced intensities of the fractions corresponding to the oligomers are shown after incubation times of 15, 30, 45 and 60 min, verifying the reduction in the population of oligomers due to the chaperone. The soluble concentrations of Aβ42 and chaperone were 3 μM, and the concentration of pre-formed fibrils was 30 nM. (c-d) Corresponding predictions for the nucleation rate as a function of time with and without Brichos. (e-f) Cryo-TEM images of fibrils formed in the absence (e) and presence (f) of Brichos show that longer fibrils are formed in the presence of Brichos. Quantification of the fibril lengths over multiple images reveals that fibrils formed in the presence of Brichos are on average at least four times longer than those formed in the absence of Brichos. The samples contained 10 μM Aβ42 and 6 μM Aβ42 + 6 μM Brichos respectively, and were taken at the reaction end point (as measured by ThT) in each case.
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Figure 4: Brichos inhibits the formation of Aβ42 oligomers by suppressing secondary nucleation and redirecting the reaction pathway towards elongation events(a-b) Quantification of low molecular weight oligomers of Aβ42 in the absence (a) and presence (b) of Brichos using size-exclusion chromatography and the 6E10 antibody. The reduced intensities of the fractions corresponding to the oligomers are shown after incubation times of 15, 30, 45 and 60 min, verifying the reduction in the population of oligomers due to the chaperone. The soluble concentrations of Aβ42 and chaperone were 3 μM, and the concentration of pre-formed fibrils was 30 nM. (c-d) Corresponding predictions for the nucleation rate as a function of time with and without Brichos. (e-f) Cryo-TEM images of fibrils formed in the absence (e) and presence (f) of Brichos show that longer fibrils are formed in the presence of Brichos. Quantification of the fibril lengths over multiple images reveals that fibrils formed in the presence of Brichos are on average at least four times longer than those formed in the absence of Brichos. The samples contained 10 μM Aβ42 and 6 μM Aβ42 + 6 μM Brichos respectively, and were taken at the reaction end point (as measured by ThT) in each case.

Mentions: The combination of kinetic studies and direct measurements of binding affinity discussed above demonstrates that Brichos is able to bind with high affinity to the surfaces of Aβ42 fibrils. These findings suggest that this molecular chaperone inhibits specifically secondary nucleation events occurring on the surfaces of fibrils. To probe explicitly the degree of inhibition of the production of low molecular weight oligomeric species that are created through this mechanism, we studied their concentrations during Aβ42 aggregation in the absence (Fig. 4a) and presence (Fig. 4b) of Brichos in solution. We monitored the generation of low molecular weight oligomeric species in each sample by removing aliquots at a series of time points and isolating in each case the fraction corresponding to low molecular weight species by means of size exclusion chromatography19. Eluted fractions were pooled in to three categories of samples: monomers, small oligomers (ca. 3-14 mers, equivalent to globular proteins of 14-65 kDA molecular weight) and large oligomers (ca. 15-20 mers, 66-90 kDa).


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)

Brichos inhibits the formation of Aβ42 oligomers by suppressing secondary nucleation and redirecting the reaction pathway towards elongation events(a-b) Quantification of low molecular weight oligomers of Aβ42 in the absence (a) and presence (b) of Brichos using size-exclusion chromatography and the 6E10 antibody. The reduced intensities of the fractions corresponding to the oligomers are shown after incubation times of 15, 30, 45 and 60 min, verifying the reduction in the population of oligomers due to the chaperone. The soluble concentrations of Aβ42 and chaperone were 3 μM, and the concentration of pre-formed fibrils was 30 nM. (c-d) Corresponding predictions for the nucleation rate as a function of time with and without Brichos. (e-f) Cryo-TEM images of fibrils formed in the absence (e) and presence (f) of Brichos show that longer fibrils are formed in the presence of Brichos. Quantification of the fibril lengths over multiple images reveals that fibrils formed in the presence of Brichos are on average at least four times longer than those formed in the absence of Brichos. The samples contained 10 μM Aβ42 and 6 μM Aβ42 + 6 μM Brichos respectively, and were taken at the reaction end point (as measured by ThT) in each case.
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Related In: Results  -  Collection

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Figure 4: Brichos inhibits the formation of Aβ42 oligomers by suppressing secondary nucleation and redirecting the reaction pathway towards elongation events(a-b) Quantification of low molecular weight oligomers of Aβ42 in the absence (a) and presence (b) of Brichos using size-exclusion chromatography and the 6E10 antibody. The reduced intensities of the fractions corresponding to the oligomers are shown after incubation times of 15, 30, 45 and 60 min, verifying the reduction in the population of oligomers due to the chaperone. The soluble concentrations of Aβ42 and chaperone were 3 μM, and the concentration of pre-formed fibrils was 30 nM. (c-d) Corresponding predictions for the nucleation rate as a function of time with and without Brichos. (e-f) Cryo-TEM images of fibrils formed in the absence (e) and presence (f) of Brichos show that longer fibrils are formed in the presence of Brichos. Quantification of the fibril lengths over multiple images reveals that fibrils formed in the presence of Brichos are on average at least four times longer than those formed in the absence of Brichos. The samples contained 10 μM Aβ42 and 6 μM Aβ42 + 6 μM Brichos respectively, and were taken at the reaction end point (as measured by ThT) in each case.
Mentions: The combination of kinetic studies and direct measurements of binding affinity discussed above demonstrates that Brichos is able to bind with high affinity to the surfaces of Aβ42 fibrils. These findings suggest that this molecular chaperone inhibits specifically secondary nucleation events occurring on the surfaces of fibrils. To probe explicitly the degree of inhibition of the production of low molecular weight oligomeric species that are created through this mechanism, we studied their concentrations during Aβ42 aggregation in the absence (Fig. 4a) and presence (Fig. 4b) of Brichos in solution. We monitored the generation of low molecular weight oligomeric species in each sample by removing aliquots at a series of time points and isolating in each case the fraction corresponding to low molecular weight species by means of size exclusion chromatography19. Eluted fractions were pooled in to three categories of samples: monomers, small oligomers (ca. 3-14 mers, equivalent to globular proteins of 14-65 kDA molecular weight) and large oligomers (ca. 15-20 mers, 66-90 kDa).

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