Kumar ST, Jagannath S, Francois C, Vanderstichele H, Stoops E, Lashuel HA. 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 Dec;146:105086. Epub 2020 Sep 22 PubMed.

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  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:

    Beach TG, White CL, Hamilton RL, Duda JE, Iwatsubo T, Dickson DW, Leverenz JB, Roncaroli F, Buttini M, Hladik CL, Sue LI, Noorigian JV, Adler CH. Evaluation of alpha-synuclein immunohistochemical methods used by invited experts. Acta Neuropathol. 2008 Sep;116(3):277-88. Epub 2008 Jul 15 PubMed.

    Croisier E, MRes DE, Deprez M, Goldring K, Dexter DT, Pearce RK, Graeber MB, Roncaroli F. 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:

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

    Schweighauser M, Shi Y, Tarutani A, Kametani F, Murzin AG, Ghetti B, Matsubara T, Tomita T, Ando T, Hasegawa K, Murayama S, Yoshida M, Hasegawa M, Scheres SH, Goedert M. Structures of α-synuclein filaments from multiple system atrophy. Nature. 2020 Sep;585(7825):464-469. Epub 2020 May 27 PubMed.

    Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. 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.

    Spillantini MG, Crowther RA, Jakes R, Cairns NJ, Lantos PL, Goedert M. 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

  6. The study from Kumar and colleagues is an important contribution to the field. It attempts to bring some consensus on the validity and characterization of these commonly used "conformation-specific" antibodies. Characterizing and validating antibodies with different orthogonal assays is a significant undertaking, but it will be important for the field to agree on criteria to validate antibodies (and other tools/assays for that matter), so that companies and labs developing these tools can include comprehensive tool characterization in their data packages.

    The study shows these antibodies' general inability to distinguish oligomers from fibrils. This was somewhat expected as we have a limited understanding of the structural determinants of oligomers and how they compare to fibrils, and there is a lack of consensus on the defining characteristics of an oligomer. Therefore, these antibodies might have been raised against heterogeneous species ranging from small oligomers to soluble sonicated preformed fibrils.

    The ability for most of these antibodies to also bind to monomeric α-Syn suggests that binding preference to aggregated forms of α-Syn is driven by avidity rather than specificity, an important observation that should be taken into consideration in future studies. This will be especially important in studies aimed at measuring synuclein species in human bio-specimens, where aggregated forms of α-Syn are either transient or exist at much lower concentrations than monomeric α-synuclein. There should be an effort to ensure that assay conditions are set up to minimize/avoid binding to α-Syn monomers.

    Finally, while this study may be mostly perceived as a negative outcome for the field, it is nevertheless positive that some of these antibodies are able to capture different types of oligomers and fibrils, suggesting the potential to capture the diversity of α-Syn aggregates in human bio-specimens.

    View all comments by Luis Oliveira

  7. This is a tour de force assessment of different antibodies previously reported to be specific for certain types of α-synuclein aggregate. They are not!

    I commend the authors for this thorough, systematic, honest, and self-critical approach to preparing samples. This is exactly what the field needs.

    This study contains nice details, such as tests using multiple freeze-thaw rounds followed by painstaking EM analyses to check for sample stability, and a robust assessment of the study’s limitations.

    The conclusion is a good starting point for researchers choosing the right antibodies for their studies. This study is a service to the Parkinson’s community, and a reminder to the whole amyloid field to be extra vigilant about the conclusions they draw from antibodies claimed to be specific for certain species or conformations. I thoroughly enjoyed reading it.

    View all comments by Daniel Otzen

  8. The comprehensive comparison of aggregate-selective α-synuclein antibodies by Kumar et al. has been published after the preprint version earlier was published on BioRxiv. The field’s responses are positive—but then what?

    I think the title of this Alzforum piece is misleading. The antibodies are not blunt, and far from those crappy ones that Gregory Petsko mentions have marred the field of neurodegenerative diseases.

    One reason for the confusion is that we have been studying in vitro-formed oligomers and fibrils for more than a decade. However, not until the recent Schweighauser 2020 Nature paper did we actually see the true high-resolution cryoEM structures of aggregates from brains affected by MSA. What did we see? Two different folding strains isolated from one brain and evidence that aggregates from DLB brains are different.

    The two resolved strains were not identical to the cryoEM structures from in vitro-formed filaments, but did share significant similarity. So how much can we ask for in terms of specific antibodies? What if aggregates vary within the brains of individual patients, and between patients with identical clinically perceived diseases? The currently generated α-synuclein aggregates, amplified from patient samples by in vitro RT-QuIC or PMCA techniques, may help us get an idea of the variability between patient aggregates, although they of course may be artifactually shaped by the in vitro amplification protocol.

    Regarding the oligomers we are more in the open. They are hypothesized to exist in patient brains but have not yet shown their coat. We have built our belief on structures generated in vitro, using different α-synuclein aggregation protocols often spiced up with a twist of chemicals like dopamine, lipid aldehydes, etc. Our reason to believe these oligomers resemble what may exist in brain is meager, and our techniques to actually isolate them haven’t yet been blessed by techniques like PMCA or RT-QuIC and still lack reproducible protocols. This will likely remain a controversial field for quite some time.

    Back to the antibodies that were tested by Kumar et al. Most were able to distinguish between monomer and aggregated α-syn species. The SRP-based kinetic analysis in table 3 demonstrates the highest difference in KD for monomer/fibril of >4000 for the MJFR-14. This is pretty good.

    The study actually demonstrates we have antibodies that not just bind α-synuclein but can distinguish between some species. Of course we shall continue to improve our tools, but this will take time and resources, and another paper might be written in five years that states these new tools are blunt.

    I suggest we focus on refining our protocols with the tools available. This may be done by an online assembly of protocols from those who manage to get positive results from the antibodies. Other investigators can then ask questions and challenge their results. This approach should enable us to have a platform representing best practice that, importantly, is constantly curated by researchers in the field. BioRxiv may serve the purpose by allowing us to upload data that can be commented on by readers.

    References:

    Schweighauser M, Shi Y, Tarutani A, Kametani F, Murzin AG, Ghetti B, Matsubara T, Tomita T, Ando T, Hasegawa K, Murayama S, Yoshida M, Hasegawa M, Scheres SH, Goedert M. Structures of α-synuclein filaments from multiple system atrophy. Nature. 2020 Sep;585(7825):464-469. Epub 2020 May 27 PubMed.

    View all comments by Poul Henning Jensen

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