. How specific are the conformation-specific α-synuclein antibodies? Characterization and validation of 16 α-synuclein conformation-specific antibodies using well-characterized preparations of α-synuclein monomers, fibrils and oligomers with distinct struct. Neurobiol Dis. 2020 Sep 22;146:105086. PubMed.

Recommends

Please login to recommend the paper.

Comments

  1. This is an extremely useful analysis of the current α-Syn antibodies used in the field, and quite an ‘eye-opener’.

    The study clearly shows that all the antibodies that have claimed to be ‘oligomer/conformation-specific’ also detect fibrils, i.e. the antibodies cannot distinguish between oligomeric and fibrillar forms. Although they may have preferential binding toward aggregated forms, they still do pick up also monomeric forms in a concentration-dependent manner. This is an extremely important point to raise, as it continues to cause confusion in the field even among the experts and misinterpretation of some previous studies.

    The pathological heterogeneity of α-Syn-positive structures in the human brain when using different antibodies is well-recognized in the neuropathology field (Beach 2018, Croisier 2006). However, it has not been studied in detail due to lack of well-characterized antibodies (to the level done here), as well as lack of AI-based digital imaging tools which are only now emerging.

    Our own preliminary studies have shown that this heterogeneity can be more profound than previously accepted, e.g. α-Syn aggregation in astroglial cells is only detected with α-Syn antibodies that have their epitope within the central NAC region. This suggests that the aggregates in different cell types are most likely formed of different α-Syn proteoforms, and that we really need to use several better-characterized antibodies to capture the diversity of a-Syn species in the brain.

    References:

    . Evaluation of alpha-synuclein immunohistochemical methods used by invited experts. Acta Neuropathol. 2008 Sep;116(3):277-88. Epub 2008 Jul 15 PubMed.

    . Comparative study of commercially available anti-alpha-synuclein antibodies. Neuropathol Appl Neurobiol. 2006 Jun;32(3):351-6. PubMed.

    View all comments by Laura Parkkinen
  2. I think most thoughtful people in the field have wished for better antibodies for some time. This work will make it clear how urgent that is. The question is whether it’s possible to get them.

    Structural biologists have often been suspicious of ‘conformation-specific’ antibodies in the absence of direct data about what exactly is being detected. Reproducibility of biological experiments has been a topic of much discussion in the past few years, and many of the people involved have come to the conclusion that a lot of irreproducibility is due to, for lack of a better word, crappy antibodies.

    In the neurodegenerative disease field in particular, given the use of antibodies as therapeutic agents in clinical trials, these results should sound a loud alarm bell. The authors were able to identify a few antibodies that were somewhat more specific than most, which should be somewhat encouraging, but even those had their issues.

    Let me end with a word of praise and thanks to the authors. The painstaking quality control study that you see here might well be almost impossible to do in an academic lab in the United States these days, given the current funding situation and the drive to publish hot new findings. It’s probably not surprising that, even in Europe, this study had participation by industry as well as academia. The authors have done the field a service. Let’s hope the field responds by finding better ways to tackle this problem.

    View all comments by Gregory Petsko
  3. In this important study, Kumar and colleagues conducted an impressive amount of rigorous work on the study of different forms and assemblies of alpha-synuclein, a central protein in synucleinopathies. The study is systematic, carefully executed, and highlights the need for uniformization in terms of protocols and language used in the field.

    Recent evidence from numerous studies in cell and animal models of disease suggest that oligomeric species of different proteins might be more toxic that the larger, fibrillar forms. This idea is backed up by a lack of correlation between the presence of the typical pathological hallmark inclusions and disease. E.g. at autopsy, amyloid-beta plaques and Lewy bodies are often found in the brains of people who had no overt signs of Alzheimer’s or Parkinson’s disease, respectively.

    The idea that oligomeric forms of proteins are formed “on pathway” to the formation of the mature amyloid-like fibrils is also supported by numerous studies. However, the field suffers from major limitations, including the ambiguity associated with the term “oligomer,” as this is often used to refer to a variety of protein species that is difficult to compare between different laboratories.

    The present study assessed the behavior of a panel of 18 antibodies in the context of in vitro-prepared alpha-synuclein species: monomers, oligomers, and fibrils. The findings may appear surprising but, in fact, I believe they were to be expected. The antibodies were developed using alpha-synuclein species produced in different laboratories and, therefore, they may not be directly comparable to those used in this study. In addition, and as the authors point out, a major limitation is to know how the alpha-synuclein species used as reference in the study relate to those accumulating in the human brain. One may expect them to be very different in fact, due to the absence of the posttranslational modifications that take place in any biological context.

    Nevertheless, the study has great merit as it highlights the fact that we need to be cautious when assuming the specificity of the numerous antibodies used in the field. The study also highlights the need for more precise guidelines and standardization in the field. This is the only way we might move forward and rationally develop tools and strategies for advancing our understanding of these devastating diseases.

    View all comments by Tiago Outeiro
  4. Kumar and colleagues set out in their study to characterize a variety of available ‘conformation- specific antibodies’ for α-Syn to test the antibodies’ specificity toward monomeric, oligomeric and fibrillar forms of α-Syn. The results largely agree with what is known in the field, i.e. most conformation-specific antibodies detect oligomeric and fibrillar α-Syn with higher sensitivity compared to monomers, but are ultimately not completely selective.

    The study also finds that there does not seem any distinction between fibrillar and smaller oligomeric material, even though this might be due to the authors’ preparation method of oligomeric samples.

    This study will be a helpful guide for people looking to use the described antibodies in their research, and it emphasizes the importance of being mindful of the relative concentrations of monomers in your preparations. The results do imply that most of these antibodies have higher sensitivity toward oligomers and fibrils than monomer. Given that, using these antibodies mostly in comparative studies, in which total α-Syn protein concentration (i.e. monomer + multimers/oligomers + fibrils), independent of conformation, is similar between samples will still deliver meaningful results.

    View all comments by Tim Bartels
  5. Few questions have led to more confusion than that of the relative importance of oligomers and filaments. Part of the problem stems from the fact that filaments are easily observed in human brain, whereas oligomers are largely invisible. It would therefore be valuable to have reagents that are specific for protein oligomers.

    One option is to produce antibodies that can distinguish between monomers, oligomers, and filaments. It has been reported that such antibodies exist for α-synuclein. Kumar and colleagues have now studied these and other antibodies using a wide variety of techniques, including single molecule assays, surface plasmon resonance, and immunoblotting. They report that none of the 18 anti-α-synuclein antibodies analysed were specific for oligomers of recombinant α-synuclein.

    Although some antibodies did not visualize monomeric alpha-synuclein, they recognised both oligomers and filaments. We do not know how the structures of recombinant α-synuclein oligomers relate to those of oligomers from human brain. However, we do know that the structures of alpha-synuclein filaments from multiple system atrophy (MSA) differ from those of recombinant assembled protein (Schweighauser et al., 2020).

    Distinct conformers of assembled alpha-synuclein are probably present in Parkinson’s disease, dementia with Lewy bodies and MSA (Spillantini et al., 1998a, Spillantini et al., 1998b; Crowther et al., 2000; Schweighauser et al., 2020). We are looking forward to the production of antibodies that are specific for the conformational states of assembled alpha-synuclein from human brain.

    References:

    . Characterisation of isolated alpha-synuclein filaments from substantia nigra of Parkinson's disease brain. Neurosci Lett. 2000 Oct 6;292(2):128-30. PubMed.

    . Structures of α-synuclein filaments from multiple system atrophy. Nature. 2020 Sep;585(7825):464-469. Epub 2020 May 27 PubMed.

    . alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6469-73. PubMed.

    . Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies. Neurosci Lett. 1998 Jul 31;251(3):205-8. PubMed.

    View all comments by Michel Goedert

Make a Comment

To make a comment you must login or register.

This paper appears in the following:

News

  1. Blunt Instruments: α-Synuclein Antibodies Poorly Distinguish Forms