α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

TY - JOUR

T1 - α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity

AU - Ghanem, Simona S.

AU - Majbour, Nour K.

AU - Vaikath, Nishant N.

AU - Ardah, Mustafa T.

AU - Erskine, Daniel

AU - Jensen, Nanna Møller

AU - Fayyad, Muneera

AU - Sudhakaran, Indulekha P.

AU - Vasili, Eftychia

AU - Melachroinou, Katerina

AU - Abdi, Ilham Y.

AU - Poggiolini, Ilaria

AU - Santos, Patricia

AU - Dorn, Anton

AU - Carloni, Paolo

AU - Vekrellis, Kostas

AU - Attems, Johannes

AU - McKeith, Ian

AU - Outeiro, Tiago F.

AU - Jensen, Poul Henning

AU - El-Agnaf, Omar M.A.

N1 - Funding Information: ACKNOWLEDGMENTS. We thank Prof. Michael Schlossmacher (University of Ottawa, Canada) for providing SNCA transgenic and SNCA-null mouse brain tissues, and Dr. Giulia Rosseti (Forschungszentrum J€ulich, Germany) and Prof. Claudio Fernandez (Universidad Nacional de Rosario, Argentina) for their contributions to the modelling of the effect of S129 phosphorylation. The work conducted by the O.M.A.E.-A. laboratory was supported by the Qatar Biomedical Research Institute under Internal Grant VR98. D.E. is funded by an Alzheimer’s Research UK Fellowship (ARUK-RF2018C-005). Human brain tissue was provided by Newcastle Brain Tissue Resource, which is funded in part by a grant from the UK Medical Research Council (G0400074), by National Institute for Health Research Newcastle Biomedical Research Centre awarded to the Newcastle upon Tyne NHS Foundation Trust and Newcastle University, and by a grant from the Alzheimer’s Society and Alzheimer’s Research UK as part of the Brains for Dementia Research Project. P.H.J. is funded by Lundbeck Foundation Grants R223-2015-4222 and R248-2016-2518 for the Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic–European Molecular Biology Laboratory Partnership for Molecular Medicine, Aarhus University, Denmark. Funding Information: We thank Prof. Michael Schlossmacher (University of Ottawa, Canada) for providing SNCA transgenic and SNCA-null mouse brain tissues, and Dr. Giulia Rosseti (Forschungszentrum J?ulich, Germany) and Prof. Claudio Fernandez (Universidad Nacional de Rosario, Argentina) for their contributions to the modelling of the effect of S129 phosphorylation. The work conducted by the O.M.A.E.-A. laboratory was supported by the Qatar Biomedical Research Institute under Internal Grant VR98. D.E. is funded by an Alzheimer?s Research UK Fellowship (ARUK-RF2018C-005). Human brain tissue was provided by Newcastle Brain Tissue Resource, which is funded in part by a grant from the UK Medical Research Council (G0400074), by National Institute for Health Research Newcastle Biomedical Research Centre awarded to the Newcastle upon Tyne NHS Foundation Trust and Newcastle University, and by a grant from the Alzheimer?s Society and Alzheimer?s Research UK as part of the Brains for Dementia Research Project. P.H.J. is funded by Lundbeck Foundation Grants R223-2015-4222 and R248-2016-2518 for the Danish Research Institute of Translational Neuroscience (DANDRITE), Nordic?European Molecular Biology Laboratory Partnership for Molecular Medicine, Aarhus University, Denmark. Publisher Copyright: Copyright © 2022 the Author(s).

PY - 2022/4

Y1 - 2022/4

N2 - α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

AB - α-Synuclein (α-syn) phosphorylation at serine 129 (pS129–α-syn) is substantially increased in Lewy body disease, such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129–α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129–α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129–α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129–α-syn (WT–α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129–α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129–α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129–α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129–α-syn as a measure of efficacy in clinical trials.

KW - Parkinson’s disease

KW - phosphorylation

KW - α-synuclein

KW - Amyloid/metabolism

KW - Protein Aggregates

KW - Phosphorylation

KW - Protein Aggregation, Pathological/genetics

KW - alpha-Synuclein/genetics

KW - Humans

KW - Serine/metabolism

KW - Lewy Body Disease/genetics

KW - Parkinson Disease/genetics

UR - http://www.scopus.com/inward/record.url?scp=85129016122&partnerID=8YFLogxK

U2 - 10.1073/pnas.2109617119

DO - 10.1073/pnas.2109617119

M3 - Journal article

C2 - 35353605

AN - SCOPUS:85129016122

SN - 0027-8424

VL - 119

JO - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

IS - 15

M1 - e2109617119

ER -