Direct observation of prion protein oligomer formation reveals an aggregation mechanism with multiple conformationally distinct species

Sang, Jason C.; Lee, Ji Eun; Dear, Alexander J.; De, Suman; Meisl, Georg; Thackray, Alana M.; Mathiason, Candace K.; Knowles, Tuomas P. J.; Klenerman, David

doi:10.1039/c8sc05627g

Cited by 24 publications

(22 citation statements)

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“…13 ) and PrP. 52 To highlight that this is a desired property of the definition, consider the case where all fibrils are formed via a specific oligomeric species, yet the fate of most of those oligomers is to dissociate and not to from fibrils; given that this particular species is a necessary intermediate on the path to fibrils it would be assigned a p value of 1.0, and thus correctly identified as on-pathway in our framework. Any alternative definition by which this oligomeric species would be classified as off-pathway would be misleading.…”

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confidence: 99%

Identification of on- and off-pathway oligomers in amyloid fibril formation

et al. 2020

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confidence: 99%

Identification of on- and off-pathway oligomers in amyloid fibril formation

et al. 2020

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“…These oligomers are also relatively abundant, as might be expected. *Prion protein PrP data were taken from ref 44. (proteinase K-sensitive species); abundance was not measured.…”

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Kinetic diversity of amyloid oligomers

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et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer’s and Parkinson’s diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.

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“…Thus, by using only the ThT assay,i ti s challenging to compareb etween weak and potent inhibitors of oligomerization. The small increase in the signal could be associated to the fact that ThT fails to detect all types of oligomers, especially those with low amountso fb sheets, as described recently by Sang et al [12] On the other hand, the fluorescence intensity of BODIPY (Figures 2B and S1 B, red curve) showedapronounced exponential kinetic curve, as characterized by an elongation slope between 0a nd 4h and as teady state between 5a nd 15 h( Figures 2B and S1 B), followed by a slight increase at 24 h( Figure S1 C). The higherdetection sensitivity of BODIPY is correlated not only to its higher receptive- Figure 1.…”

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“…[9,10] However,T hT only exhibits as ubstantial fluorescencei ncreaseu pon binding to b-sheet-rich amyloid protofilaments and fibrils, but has low sensitivity to soluble early-stage oligomers. [11][12][13][14] Molecules that do not show any apparenti nhibition of amyloid aggregation,a ccording to the ThT assay,a re usually not considered for any subsequentt esting. It may be presumed that several lead compounds have been discarded, although they would be capable of preventing the formation of smallc ytotoxic oligomers.…”

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Real‐Time BODIPY‐Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1–42 Peptide

et al. 2020

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Misfolding and aggregation of amyloid b1-42 peptide( A b1-42) play ac entral role in the pathogenesis of Alzheimer's disease (AD). Ta rgeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents ap romising therapeutic strategy to reduce the toxicity associated with Ab1-42. Currently,t he thioflavin T( ThT) assay is the only established spectrofluorometric methodt os creen aggregation inhibitors. The successo ft he ThT assay is that it can detect Ab1-42 aggregates with high b-sheet content,s uch as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately,b yu sing the ThT assay,t he detection of inhibitors of early soluble oligomers that present al ow b-sheet character is challenging. Herein, an ew,f acile, and robust boron-dipyrromethene( BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selectivei nhibitors of the formation of Ab1-42 oligomers, is reported.T hese inhibitors decrease the cellular toxicityo fA b1-42, although they fail in the ThT assay.T he findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is ac onvenient methodt o screen and discover new candidate compoundst hat slow down or stop the pathological earlyo ligomerization process and are active in the cellular assay.T herefore, it is as uitable complementary screening method of the current ThT assay.Alzheimer's disease (AD) is ad evastating neurodegenerative disease that leads to progressive cognitive decline, functional impairment, and loss of independence. [1] The number of people worldwide suffering from AD is expected to reach 75 million by 2030, [2] butn oc ausative treatment exists for AD.The search for small molecules that inhibitt he aggregation of the 42-residue amyloid b protein fragment (Ab1-42; Figure 1A) is still ongoing to find at herapy for AD. Ab1-42 spontaneously self-associates into soluble oligomers and insolublea ggregates, such as protofibrilsa nd fibrils with high b-sheetc ontent.I th as been recognized that small and soluble Ab1-42 oligomers are particularly cytotoxic. [3][4][5][6][7] Thus, therapeutics trategies that intervene in the early oligomerization process, rather than in the later fibril formation step, have recently attracted attention. [8] However,d espite its therapeutic significance,t he screening of potentiali nhibitors of the early oligomerization process is still challenging. The thioflavin T( ThT) fluorescencea ssayi su sed routinelytodetermine the influence of compounds on amyloid aggregation kinetics. [9,10] However,T hT only exhibits as ubstantial fluorescencei ncreaseu pon binding to b-sheet-rich amyloid protofilaments and fibrils, but has low sensitivity to soluble early-stage oligomers. [11][12][13][14] Molecules that do not show any apparenti nhibition of amyloid aggregation,a ccording to the ThT assay,a re usually not considered for any subsequentt est...

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Direct observation of prion protein oligomer formation reveals an aggregation mechanism with multiple conformationally distinct species

Abstract: The aggregation of the prion protein (PrP) plays a key role in the development of prion diseases.

Cited by 24 publications

References 71 publications

Identification of on- and off-pathway oligomers in amyloid fibril formation

Identification of on- and off-pathway oligomers in amyloid fibril formation

Kinetic diversity of amyloid oligomers

Real‐Time BODIPY‐Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1–42 Peptide

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