Cargando…

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-oc...

Descripción completa

Detalles Bibliográficos
Autores principales: Lashuel, Hilal A., Mahul-Mellier, Anne-Laure, Novello, Salvatore, Hegde, Ramanath Narayana, Jasiqi, Yllza, Altay, Melek Firat, Donzelli, Sonia, DeGuire, Sean M., Burai, Ritwik, Magalhães, Pedro, Chiki, Anass, Ricci, Jonathan, Boussouf, Manel, Sadek, Ahmed, Stoops, Erik, Iseli, Christian, Guex, Nicolas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9584898/
https://www.ncbi.nlm.nih.gov/pubmed/36266318
http://dx.doi.org/10.1038/s41531-022-00388-7
_version_ 1784813377409777664
author Lashuel, Hilal A.
Mahul-Mellier, Anne-Laure
Novello, Salvatore
Hegde, Ramanath Narayana
Jasiqi, Yllza
Altay, Melek Firat
Donzelli, Sonia
DeGuire, Sean M.
Burai, Ritwik
Magalhães, Pedro
Chiki, Anass
Ricci, Jonathan
Boussouf, Manel
Sadek, Ahmed
Stoops, Erik
Iseli, Christian
Guex, Nicolas
author_facet Lashuel, Hilal A.
Mahul-Mellier, Anne-Laure
Novello, Salvatore
Hegde, Ramanath Narayana
Jasiqi, Yllza
Altay, Melek Firat
Donzelli, Sonia
DeGuire, Sean M.
Burai, Ritwik
Magalhães, Pedro
Chiki, Anass
Ricci, Jonathan
Boussouf, Manel
Sadek, Ahmed
Stoops, Erik
Iseli, Christian
Guex, Nicolas
author_sort Lashuel, Hilal A.
collection PubMed
description Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies.
format Online
Article
Text
id pubmed-9584898
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-95848982022-10-22 Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity Lashuel, Hilal A. Mahul-Mellier, Anne-Laure Novello, Salvatore Hegde, Ramanath Narayana Jasiqi, Yllza Altay, Melek Firat Donzelli, Sonia DeGuire, Sean M. Burai, Ritwik Magalhães, Pedro Chiki, Anass Ricci, Jonathan Boussouf, Manel Sadek, Ahmed Stoops, Erik Iseli, Christian Guex, Nicolas NPJ Parkinsons Dis Article Antibodies against phosphorylated alpha-synuclein (aSyn) at S129 have emerged as the primary tools to investigate, monitor, and quantify aSyn pathology in the brain and peripheral tissues of patients with Parkinson’s disease and other neurodegenerative diseases. Herein, we demonstrate that the co-occurrence of multiple pathology-associated C-terminal post-translational modifications (PTMs) (e.g., phosphorylation at Tyrosine 125 or truncation at residue 133 or 135) differentially influences the detection of pS129-aSyn species by pS129-aSyn antibodies. These observations prompted us to systematically reassess the specificity of the most commonly used pS129 antibodies against monomeric and aggregated forms of pS129-aSyn in mouse brain slices, primary neurons, mammalian cells and seeding models of aSyn pathology formation. We identified two antibodies that are insensitive to pS129 neighboring PTMs. Although most pS129 antibodies showed good performance in detecting aSyn aggregates in cells, neurons and mouse brain tissue containing abundant aSyn pathology, they also showed cross-reactivity towards other proteins and often detected non-specific low and high molecular weight bands in aSyn knock-out samples that could be easily mistaken for monomeric or high molecular weight aSyn species. Our observations suggest that not all pS129 antibodies capture the biochemical and morphological diversity of aSyn pathology, and all should be used with the appropriate protein standards and controls when investigating aSyn under physiological conditions. Finally, our work underscores the need for more pS129 antibodies that are not sensitive to neighboring PTMs and more thorough characterization and validation of existing and new antibodies. Nature Publishing Group UK 2022-10-20 /pmc/articles/PMC9584898/ /pubmed/36266318 http://dx.doi.org/10.1038/s41531-022-00388-7 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Lashuel, Hilal A.
Mahul-Mellier, Anne-Laure
Novello, Salvatore
Hegde, Ramanath Narayana
Jasiqi, Yllza
Altay, Melek Firat
Donzelli, Sonia
DeGuire, Sean M.
Burai, Ritwik
Magalhães, Pedro
Chiki, Anass
Ricci, Jonathan
Boussouf, Manel
Sadek, Ahmed
Stoops, Erik
Iseli, Christian
Guex, Nicolas
Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title_full Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title_fullStr Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title_full_unstemmed Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title_short Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity
title_sort revisiting the specificity and ability of phospho-s129 antibodies to capture alpha-synuclein biochemical and pathological diversity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9584898/
https://www.ncbi.nlm.nih.gov/pubmed/36266318
http://dx.doi.org/10.1038/s41531-022-00388-7
work_keys_str_mv AT lashuelhilala revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT mahulmellierannelaure revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT novellosalvatore revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT hegderamanathnarayana revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT jasiqiyllza revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT altaymelekfirat revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT donzellisonia revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT deguireseanm revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT burairitwik revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT magalhaespedro revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT chikianass revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT riccijonathan revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT boussoufmanel revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT sadekahmed revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT stoopserik revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT iselichristian revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity
AT guexnicolas revisitingthespecificityandabilityofphosphos129antibodiestocapturealphasynucleinbiochemicalandpathologicaldiversity