Cohen SI, Cukalevski R, Michaels TC, Šarić A, Törnquist M, Vendruscolo M, Dobson CM, Buell AK, Knowles TP, Linse S. Distinct thermodynamic signatures of oligomer generation in the aggregation of the amyloid-β peptide. Nat Chem. 2018 May;10(5):523-531. Epub 2018 Mar 26 PubMed.
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Drexel University
This Nature Chemistry paper by Cohen and collaborators describes a beautiful and thorough study of Aβ-protein fibril formation. The focus is the 42-amino-acids-long alloform Aβ42, which is the most strongly associated with Alzheimer's disease. Aβ42 preparation involved sequential applications of size exclusion chromatography (SEC) and filtration to ensure the initial monomeric state of Aβ42. The initial monomeric Aβ42 is important from the viewpoint of the nucleation theory, which posits formation of a nucleus of a fibril directly from monomers as a rate-limiting step in protein fibrillogenesis. Within the nucleation theory, which is consistent with a typical sigmoidal shape of the Thioflavin T (ThT) fluorescence intensity versus protein incubation time, fibril formation process is split into the lag phase (during which ThT fluorescence intensity is minimal), primary nucleation, elongation, and secondary nucleation phases when ThT fluorescence intensity increases rapidly before it reaches a steady-state value. This study derived the temperature dependence of the kinetics of fibril formation by using ThT fluorescence data at different Aβ42 concentrations (ranging from 1.9–5 μM) and temperatures (ranging from 26–45 C), the fibril elongation rate, as well as the rates of primary and secondary nucleation processes. The results revealed that unlike elongation and primary nucleation, the secondary nucleation process does not strongly depend on temperature and requires significantly lower activation energy than the primary nucleation process. These results are consistent with the interpretation of secondary nucleation as a self-seeding process, whereby the existing fibrils act as templates for nuclei formation via monomer-fibril interactions.
The elephants in the room are the toxic oligomeric species that are linked to AD. The title of the study is intriguing, considering that with the exception of SEC and filtration, no experimental technique used in this study targets specifically Ab42 oligomers. Why then is the term “oligomer” in the title? This is clearly a rhetorical question. After reading the discussion, it appears that the term toxic oligomers refers to the species formed during the secondary nucleation process, i.e., significantly later than the lag phase and primary nucleation phase. The view that toxic oligomers arise from secondary nucleation, which is a self-seeding process, is highly fashionable and fits the pathogenic-protein-spread hypothesis, which draws parallels between prion, Alzheimer’s, and Parkinson’s diseases, even though key details are missing to be able to support or discard this hypothesis (Walsh and Selkoe, 2016).
It is important to establish whether Aβ42 oligomers can form prior to fibril formation both in vitro and in vivo. The presence of oligomers in the lag phase might complicate the application of the nucleation theory (Schreck and Yuan, 2013), which is to some extent fuzzy with respect to microscopic details of protein-protein interactions that lead to formation of the nucleus. In this study by Cohen et al., surface plasmon resonance was used to find dissociation constants for monomer-fibril interactions at various Aβ42 concentrations up to 40 μM. While at low concentrations of 5 uM and below (for which ThT data in the study were acquired) one could argue that Aβ42 may be at least predominantly monomeric. This is certainly not the case for higher Aβ42 concentrations, where a soluble state consists of monomers and oligomers of various sizes, as demonstrated by substantial evidence from many research groups worldwide. Even if you use SEC to isolate monomeric Aβ42, after a while a heterogeneous soluble state, containing monomers as well as oligomers, will re-emerge. It is a quite common misconception that a soluble state is monomeric. For example, insulin at neutral pH does not fibrillize under quiescent conditions, yet we have recently shown that soluble 10 μM insulin at neutral (as well as at acidic) conditions consists of monomers and oligomers of different sizes (Mawhinney et al., 2017). It is unclear whether or not Cohen et al.’s results would be affected if the initial soluble state consisted of monomers as well as oligomers. Regardless, their study offers fresh insights into our understanding of Aβ42 fibril formation and associated Aβ42 toxicity.
References:
Walsh DM, Selkoe DJ. A critical appraisal of the pathogenic protein spread hypothesis of neurodegeneration. Nat Rev Neurosci. 2016 Apr;17(4):251-60. PubMed.
Schreck JS, Yuan JM. A kinetic study of amyloid formation: fibril growth and length distributions. J Phys Chem B. 2013 May 30;117(21):6574-83. Epub 2013 May 20 PubMed.
Mawhinney MT, Williams TL, Hart JL, Taheri ML, Urbanc B. Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers. Proteins. 2017 Nov;85(11):2096-2110. Epub 2017 Aug 28 PubMed.
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