The exquisite specificity of antibodies powers neurodegenerative research, enabling reliable assays, precise diagnostics, and targeted therapeutics. Unless … some antibodies are not as specific as has been thought. In the September 22 Neurobiology of Disease, researchers led by Hilal Lashuel at École Polytechnique Fédérale de Lausanne, Switzerland, present a provocative study of 16 α-synuclein antibodies reported to be selective for oligomeric or fibrillar forms of the protein. In rigorously controlled experiments on recombinant α-synuclein, Lashuel and colleagues found that nearly all the antibodies reacted equally strongly with oligomers and fibrils, and most bound weakly to monomers as well. In other words, none were truly conformation specific. The findings suggest the need to verify previous claims about the role of oligomers in Parkinson’s disease that were based solely on antibody binding, Lashuel told Alzforum.
• Many α-synuclein antibodies are reported to be specific for oligomers or fibrils
• But a survey of 16 such antibodies found they bound all forms of α-synuclein
• The findings raise questions about the accuracy of data obtained with conformation-specific antibodies
Other scientists deemed this study valuable. “This is an extremely useful analysis of the current α-synuclein antibodies used in the field, and quite an eye-opener,” Laura Parkkinen at the University of Oxford, U.K., wrote to Alzforum. Gregory Petsko at Weill Cornell Medical College in New York City noted, “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,” (see full comments below).
Varied Shapes. Electron microscopy reveals a mix of structures among α-synuclein oligomers (upper left), contrasting with amorphous blobs in dopamine-incubated oligomers (bottom left), long chains in HNE-incubated oligomers (bottom right), and filaments in fibril preparations (upper right). [Courtesy of Kumar et al., Neurobiology of Disease.]
As with Aβ in Alzheimer’s disease, α-synuclein oligomers are believed to be more toxic than larger deposits (Dec 2007 conference news; Oct 2010 news). Researchers have tried to develop antibodies selective for both types of aggregate. Of the handful of α-synuclein antibodies in clinical trials, AbbVie’s ABBV-0805 is purported to be oligomer-specific, while Biogen’s cinpanemab and Roche’s prasinezumab recognize any aggregated forms. However, few data are available on how these clinical antibodies, or others used for research, were characterized.
To fill in this knowledge gap, Lashuel and colleagues undertook a survey of 16 α-synuclein antibodies used for research. None are in clinical trials. They tested six antibodies that are reputed to be fibril-specific: three from Biolegend (SYNO2, SYNO3, and SYNO4), two from the University of Pennsylvania (7015 and 9029), and one from Abcam (MJFR-14). Another six were raised against aggregated forms of α-synuclein: four from Biolegend (A17183A, A17183B, A17183E, and A17183G), one from Kovacs (5G4), and one from Agrisera (ASyO5). Finally, with the support of the Michael J. Fox Foundation, the authors generated four antibodies directed against oligomers (24H6, 26B10, 12C6, and 26F1).
Joint first authors Senthil Kumar and Somanath Jagannath prepared standardized synthetic α-synuclein samples against which to test these 16 antibodies. After incubating the protein at high temperature to allow aggregation, they separated different-size molecules via filtration and size exclusion chromatography. This produced pure solutions of monomers, oligomers, and fibrils. The authors created two additional oligomeric mixtures by adding reagents to the monomers during incubation. In one case they added dopamine, which tends to generate disordered oligomers; in the other, the lipid peroxidation product 4-hydroxy-2-nonenal (HNE), which tends to induce β-sheet containing oligomers.
Circular dichroism spectroscopy revealed that the original oligomeric preparations assumed a range of structures, including spheres, tubes, and rings, that all contained β-sheets. The authors did not further characterize potential structural differences among the species in this oligomer solution. On the other hand, when oligomers formed in the presence of dopamine, they clumped into disordered globs that lacked β-sheet structure. In HNE solutions, oligomers assembled into longer chains of β-sheets (see images).
Test Material. Five different preparations of α-synuclein help test antibody specificity. [Courtesy of Kumar et al., Neurobiology of Disease.]
Kumar and colleagues dotted these five α-synuclein preparations onto nitrocellulose membrane and added each antibody to assess binding. In this slot blot paradigm, all antibodies bound with nearly equal affinity to all aggregated forms. Two exceptions were 26F1 and 5G4, which barely reacted with monomers or dopamine-incubated oligomers, suggesting they might be specific for β-sheet structure. Most of the other antibodies bound strongly to all aggregates and weakly to monomers, but the signal increased at high monomer concentration. Some bound all forms of α-synuclein equally.
In a sandwich ELISA assay, which uses low protein concentrations, few of the antibodies bound monomers. However, again, most of the antibodies bound oligomers and fibrils with equal strength. 5G4, A17183E, 24H6, and 26B10 were exceptions, binding to fibrils much better than to oligomers.
Lashuel believes this relative lack of selectivity for putative conformation-specific antibodies points to the need to better define the properties of these reagents. “We need to devote more time, effort, and resources to characterizing and validating the tools and assays that we use to build the foundation of our research and drug discovery programs,” he said.
Tiago Outeiro, University Medical Center Göttingen in Germany, agreed on the need for more precise guidelines and standardization in the field. At the same time, Outeiro noted that the recombinant α-synuclein preparations used here likely do not match the post-translationally modified forms of the protein found in the brain, and so are not the ideal material for this task. Michel Goedert, MRC Laboratory of Molecular Biology, Cambridge, U.K., points out that conformers of α-synuclein aggregates differ between the different synucleinopathies (see full comments below). And Lashuel agrees with that. He plans to isolate α-synuclein oligomers and fibrils from brain tissue and use this material to characterize antibodies in the future.
Tim Bartels at the U.K. Dementia Research Institute at University College London said Lashuel’s study will be a helpful guide for researchers in selecting α-synuclein antibodies. Despite the relative lack of specificity shown here, he believes the antibodies are still useful research tools, since most do bind aggregates more strongly than monomers. Bartels also speculated that the oligomer preparations used in this study might contain some small diffusible fibrils as well, which would further blur the issue of antibody specificity. CD spectroscopy would not distinguish those structural details, Bartels said (see comment below).
In the Alzheimer’s field, antibodies with imperfect selectivity, such as BAN2401 and aducanumab, have made it into late-stage trials and even onto the FDA’s desk for marketing approval.—Madolyn Bowman Rogers
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- 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 Sep 22;146:105086. PubMed.