The properties of α-synuclein secondary nuclei are dominated by the solution conditions rather than the seed fibril strain

Alessia Peduzzo, Sara Linse, Alexander K. Buell

Preprint posted on 4 September 2019

Think again when copying amyloid fibrils – the properties of α-synuclein fibrils change when amplification occurs through secondary nucleation in altered buffer conditions

Selected by Tessa Sinnige

Categories: biochemistry, biophysics


Amyloid fibrils are a hallmark of various neurodegenerative diseases. In Parkinson’s disease, fibrils formed by the protein α-synuclein are found in the characteristic Lewy Bodies in dopaminergic neurons. Recent evidence suggests that α-synuclein pathology may spread through the brain in a prion-like fashion, whereby existing fibrils serve as templates for the formation of new fibrils. Different mechanisms can lead to an increase in fibril mass: 1) primary nucleation, the first step for monomeric protein molecules to convert to a fibrillar structure; 2) fibril elongation, in which monomers are added to the ends of the growing filament; 3) secondary nucleation, which entails the formation of new nuclei catalysed at the surface of previously formed fibrils. Whereas it is well established that the structural features of fibrils are preserved upon elongation of short fibril seeds, the properties of fibrils generated by secondary nucleation with respect to the ‘parent’ fibrils have not yet been investigated.


Results of the preprint

The authors of this preprint – recently posted on ChemRxiv – make use of the fact that α-synuclein adopts fibrils with different properties depending on the buffer conditions in vitro, most notably pH and ionic strength. They first characterise Thioflavin T aggregation kinetics and morphology under three different buffer conditions, leading to fibrils with a ribbon-like appearance, twisted fibrils, and short needle-like fibrils, respectively. They also show that the needle-like fibrils are more sensitive to proteinase K digestion than the other two polymorphs, providing a straightforward read-out to distinguish this fibril type.


The three α-synuclein fibril types as seen by AFM, reproduced from Figure 1 of the preprint under a CC BY-NC-ND 4.0 international licence.


The authors then tweak the conditions of their aggregation assay to favour amplification of the fibril mass by elongation versus secondary nucleation. When a high concentration of seeds is provided, elongation dominates, and as expected the authors find that the fibril types are preserved even if they switch to a buffer condition that would lead to the formation of a different fibril type de novo. However, when they perform the assay with only a small amount of seeds and at lower pH, favouring secondary nucleation, they find that adding seeds of the ribbon and twisted fibrils leads to the formation of the needle type that would spontaneously form at this pH. Thus, secondary nucleation generates fibrils that have the morphology dictated by the buffer conditions, as if they were formed by primary nucleation.


Why I chose this preprint

The spreading of α-synuclein pathology in a prion-like fashion throughout the brain, and even starting from the gut (1), has been studied extensively in recent years (see (2-4) for reviews). Many questions remain about the molecular processes that underlie this phenomenon, and in particular about the propagation of different α-synuclein fibril types, or ‘strains’ analogously to prions (5).

This preprint is the first to show that the properties of α-synuclein fibrils are not retained if buffer conditions are changed and secondary nucleation is the dominant mechanism of fibril amplification. These findings are important for the interpretation of α-synuclein spreading experiments. Furthermore, they reveal fundamental insights into the process of secondary nucleation, which also occurs for other disease-associated proteins such as Alzheimer’s amyloid-β peptide, and is thought to be a key mechanism that generates toxic oligomeric species.



If the structural features of the fibrils are not preserved during secondary nucleation, do they matter at all? Can fibrils formed by different proteins promote each other’s aggregation by secondary nucleation? Could any other surface with the right charge, hydrophobicity etc. perform this role?

Do these results suggest that secondary nucleation plays a minor role in the spread of α-synuclein pathology in vivo, compared to fibril fragmentation followed by elongation? Would you expect α-synuclein fibrils to encounter different solution conditions while travelling from cell to cell?



  1. Kim, S. et al. Transneuronal Propagation of Pathologic α-Synuclein from the Gut to the Brain Models Parkinson’s Disease. Neuron 103, 627-641.e7 (2019).
  2. Dehay, B., Vila, M., Bezard, E., Brundin, P. & Kordower, J. H. Alpha-synuclein propagation: New insights from animal models. Mov. Disord. 31, 161–168 (2016).
  3. Goedert, M., Masuda-Suzukake, M. & Falcon, B. Like prions: the propagation of aggregated tau and α-synuclein in neurodegeneration. Brain 140, 266–278 (2016).
  4. Karpowicz, R. J., Trojanowski, J. Q. & Lee, V. M.-Y. Transmission of α-synuclein seeds in neurodegenerative disease: recent developments. Lab. Investig. 99, 971–981 (2019).
  5. Peelaerts, W. et al. α-Synuclein strains cause distinct synucleinopathies after local and systemic administration. Nature 522, 340 (2015).

Tags: amyloid fibrils, secondary nucleation, α-synuclein

Posted on: 20 September 2019 , updated on: 17 April 2020


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Author's response

Alessia Peduzzo and Alexander K. Buell shared

If the structural features of the fibrils are not preserved during secondary nucleation, do they matter at all?

One of the intriguing features of amyloid fibrils is the fact that a given amino acid sequence can adopt very different folds. This probably reflects that the amyloid state has not been subjected to the same degree of evolutionary pressure as functional protein folds. There is increasing evidence from cryo-EM analysis of ex vivo fibrils, in particular in the case of the protein tau, that different diseases (tauopathies) are associated with different fibril structures of the same protein. Hence the fibril structure seems to matter a great deal. Whether or not this also applies to alpha-synuclein is not yet clear at this stage. However, in vitro a range of different fibril structures have been generated and there is evidence that these may have different toxicities and spreading properties in cellular and animal models of the disease.

Can fibrils formed by different proteins promote each other’s aggregation by secondary nucleation?

Yes, this has been shown in a few cases. When fibrils of one protein accelerate the nucleation of fibrils of another protein, we would not call that secondary nucleation (a term reserved to autocatalytic amplification of a given type of fibril), but heterogeneous primary nucleation. This phenomenon may partly explain why in late stages of neurodegenerative protein misfolding diseases, various types of proteins are found to aggregate.

Could any other surface with the right charge, hydrophobicity etc. perform this role?

It is very likely that in many cases the nucleation of protein aggregates, in vitro as well as in vivo, is a heterogeneous process. Given the ubiquitous nature and diversity of surfaces in living organisms, in particular lipid bilayers, there is ample opportunity for different proteins to interact with a suitable surface that can facilitate nucleation. If this happens on the surface of another fibril, this may just be a special case of heterogeneous primary nucleation.

Do these results suggest that secondary nucleation plays a minor role in the spread of α-synuclein pathology in vivo, compared to fibril fragmentation followed by elongation?

Even though our results seem to suggest that, it is probably too early to rule out that secondary nucleation is responsible for the spread of pathology in synucleinopathies. In our experiments, we made the fibrils at pH 7, but amplified them at mildly acidic pH, where secondary nucleation has a non-negligible rate. We cannot rule out that the propagation of structural information was overcome by the lack of stability of the secondary nuclei at this different pH. Further experiments with multiple strains formed and stable at acidic pH would help to address this point in more detail. What is also needed is data that shows whether or not the amplification processes active in vivo are able to propagate the structural properties of alpha-synuclein fibrils.

Would you expect α-synuclein fibrils to encounter different solution conditions while travelling from cell to cell?

Yes, alpha-synuclein experiences different pH environments in the cytosol, in endosomes and lysosomes. In fact, there is a strong association between PD and lysosomal storage disorders, such as Gaucher disease. So the pH range where α-synuclein aggregation features secondary nucleation in in vitro experiments is physiologically relevant. Furthermore, we have shown recently that C-terminal truncation of α-synuclein is able to shift the pH-window of secondary nucleation into the neutral range (van der Wateren et al., Chemical Science 2018).

In your view, what were the most important things you improved in your study as a result of peer review?

This turned out to be a particularly useful case of peer review. One of the referees asked us to perform a more detailed and quantitative analysis of our data, in particular of the AFM images and the SDS-PAGE data. We did that and where thus able to fully convince the referee, who had been somewhat critical at first, of the validity of our findings. The lesson to be learned from that is that it is worth to go the extra mile to be even more convincing, even though it is already totally obvious to oneself that the data and message are correct. While one should try to be one’s own hardest critic, this is often easier said than done. That is why peer review is indispensable.

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