TY - JOUR
T1 - Multi-platform quantitation of alpha-synuclein human brain proteoforms suggests disease-specific biochemical profiles of synucleinopathies
AU - Moors, Tim E.
AU - Mona, Daniel
AU - Luehe, Stefan
AU - Duran-Pacheco, Gonzalo
AU - Spycher, Liz
AU - Mundigl, Olaf
AU - Kaluza, Klaus
AU - Huber, Sylwia
AU - Hug, Melanie N.
AU - Kremer, Thomas
AU - Ritter, Mirko
AU - Dziadek, Sebastian
AU - Dernick, Gregor
AU - van de Berg, Wilma D. J.
AU - Britschgi, Markus
N1 - Funding Information: We are grateful to all individuals that donated their brain to the Netherlands Brain Bank (NBB; www.brainbank.nl). We thank the team of the NBB, in particular Michiel Kooreman, for their cooperation and their help in the selection of brain tissue. Further, we would like to acknowledge the support of Tim Moors by a fellowship of the Roche internships for Scientific Exchange (RiSE) and support of Wilma van de Berg by a fellowship of the Dutch Parkinson association. We also thank Gaby Walker for initial support and critical input to tissue processing, and Robin Barbour and Wagner Zago for kindly providing several antibodies from Prothena Biosciences Inc. Lastly, we would like to thank Kayhan Binazir, Matthias E. Lauer, Henk Berendse and Jeroen Geurts for critically reading and helping to optimize the manuscript over the course of its development. Funding Information: We are grateful to all individuals that donated their brain to the Netherlands Brain Bank (NBB; www.brainbank.nl ). We thank the team of the NBB, in particular Michiel Kooreman, for their cooperation and their help in the selection of brain tissue. Further, we would like to acknowledge the support of Tim Moors by a fellowship of the Roche internships for Scientific Exchange (RiSE) and support of Wilma van de Berg by a fellowship of the Dutch Parkinson association. We also thank Gaby Walker for initial support and critical input to tissue processing, and Robin Barbour and Wagner Zago for kindly providing several antibodies from Prothena Biosciences Inc. Lastly, we would like to thank Kayhan Binazir, Matthias E. Lauer, Henk Berendse and Jeroen Geurts for critically reading and helping to optimize the manuscript over the course of its development. Funding Information: The work was funded by Roche. Publisher Copyright: © 2022, The Author(s).
PY - 2022/6/3
Y1 - 2022/6/3
N2 - Based on immunostainings and biochemical analyses, certain post-translationally modified alpha-synuclein (aSyn) variants, including C-terminally truncated (CTT) and Serine-129 phosphorylated (pSer129) aSyn, are proposed to be involved in the pathogenesis of synucleinopathies such as Parkinson's disease with (PDD) and without dementia (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, quantitative information about aSyn proteoforms in the human brain in physiological and different pathological conditions is still limited. To address this, we generated sequential biochemical extracts of the substantia nigra, putamen and hippocampus from 28 donors diagnosed and neuropathologically-confirmed with different synucleinopathies (PD/PDD/DLB/MSA), as well as Alzheimer's disease, progressive supranuclear palsy, and aged normal subjects. The tissue extracts were used to build a reverse phase array including 65 aSyn antibodies for detection. In this multiplex approach, we observed increased immunoreactivity in donors with synucleinopathies compared to controls in detergent-insoluble fractions, mainly for antibodies against CT aSyn and pSer129 aSyn. In addition, despite of the restricted sample size, clustering analysis suggested disease-specific immunoreactivity signatures in patient groups with different synucleinopathies. We aimed to validate and quantify these findings using newly developed immunoassays towards total, 119 and 122 CTT, and pSer129 aSyn. In line with previous studies, we found that synucleinopathies shared an enrichment of post-translationally modified aSyn in detergent-insoluble fractions compared to the other analyzed groups. Our measurements allowed for a quantitative separation of PDD/DLB patients from other synucleinopathies based on higher detergent-insoluble pSer129 aSyn concentrations in the hippocampus. In addition, we found that MSA stood out due to enrichment of CTT and pSer129 aSyn also in the detergent-soluble fraction of the SN and putamen. Together, our results achieved by multiplexed and quantitative immunoassay-based approaches in human brain extracts of a limited sample set point to disease-specific biochemical aSyn proteoform profiles in distinct neurodegenerative disorders.
AB - Based on immunostainings and biochemical analyses, certain post-translationally modified alpha-synuclein (aSyn) variants, including C-terminally truncated (CTT) and Serine-129 phosphorylated (pSer129) aSyn, are proposed to be involved in the pathogenesis of synucleinopathies such as Parkinson's disease with (PDD) and without dementia (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, quantitative information about aSyn proteoforms in the human brain in physiological and different pathological conditions is still limited. To address this, we generated sequential biochemical extracts of the substantia nigra, putamen and hippocampus from 28 donors diagnosed and neuropathologically-confirmed with different synucleinopathies (PD/PDD/DLB/MSA), as well as Alzheimer's disease, progressive supranuclear palsy, and aged normal subjects. The tissue extracts were used to build a reverse phase array including 65 aSyn antibodies for detection. In this multiplex approach, we observed increased immunoreactivity in donors with synucleinopathies compared to controls in detergent-insoluble fractions, mainly for antibodies against CT aSyn and pSer129 aSyn. In addition, despite of the restricted sample size, clustering analysis suggested disease-specific immunoreactivity signatures in patient groups with different synucleinopathies. We aimed to validate and quantify these findings using newly developed immunoassays towards total, 119 and 122 CTT, and pSer129 aSyn. In line with previous studies, we found that synucleinopathies shared an enrichment of post-translationally modified aSyn in detergent-insoluble fractions compared to the other analyzed groups. Our measurements allowed for a quantitative separation of PDD/DLB patients from other synucleinopathies based on higher detergent-insoluble pSer129 aSyn concentrations in the hippocampus. In addition, we found that MSA stood out due to enrichment of CTT and pSer129 aSyn also in the detergent-soluble fraction of the SN and putamen. Together, our results achieved by multiplexed and quantitative immunoassay-based approaches in human brain extracts of a limited sample set point to disease-specific biochemical aSyn proteoform profiles in distinct neurodegenerative disorders.
UR - http://www.scopus.com/inward/record.url?scp=85131631303&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s40478-022-01382-z
DO - https://doi.org/10.1186/s40478-022-01382-z
M3 - Article
C2 - 35659116
SN - 2051-5960
VL - 10
JO - Acta Neuropathologica Communications
JF - Acta Neuropathologica Communications
IS - 1
M1 - 82
ER -