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The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis

The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different c...

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Autores principales: Leupold, Lukas, Sigutova, Veronika, Gerasimova, Elizaveta, Regensburger, Martin, Zundler, Sebastian, Zunke, Friederike, Xiang, Wei, Winner, Beate, Prots, Iryna
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9334871/
https://www.ncbi.nlm.nih.gov/pubmed/35911883
http://dx.doi.org/10.3389/fneur.2022.869103
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author Leupold, Lukas
Sigutova, Veronika
Gerasimova, Elizaveta
Regensburger, Martin
Zundler, Sebastian
Zunke, Friederike
Xiang, Wei
Winner, Beate
Prots, Iryna
author_facet Leupold, Lukas
Sigutova, Veronika
Gerasimova, Elizaveta
Regensburger, Martin
Zundler, Sebastian
Zunke, Friederike
Xiang, Wei
Winner, Beate
Prots, Iryna
author_sort Leupold, Lukas
collection PubMed
description The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology.
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spelling pubmed-93348712022-07-30 The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis Leupold, Lukas Sigutova, Veronika Gerasimova, Elizaveta Regensburger, Martin Zundler, Sebastian Zunke, Friederike Xiang, Wei Winner, Beate Prots, Iryna Front Neurol Neurology The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology. Frontiers Media S.A. 2022-07-15 /pmc/articles/PMC9334871/ /pubmed/35911883 http://dx.doi.org/10.3389/fneur.2022.869103 Text en Copyright © 2022 Leupold, Sigutova, Gerasimova, Regensburger, Zundler, Zunke, Xiang, Winner and Prots. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neurology
Leupold, Lukas
Sigutova, Veronika
Gerasimova, Elizaveta
Regensburger, Martin
Zundler, Sebastian
Zunke, Friederike
Xiang, Wei
Winner, Beate
Prots, Iryna
The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title_full The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title_fullStr The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title_full_unstemmed The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title_short The Quest for Anti-α-Synuclein Antibody Specificity—Lessons Learnt From Flow Cytometry Analysis
title_sort quest for anti-α-synuclein antibody specificity—lessons learnt from flow cytometry analysis
topic Neurology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9334871/
https://www.ncbi.nlm.nih.gov/pubmed/35911883
http://dx.doi.org/10.3389/fneur.2022.869103
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