TY - JOUR
T1 - The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson’s disease brain as revealed by multicolor STED microscopy
AU - Moors, Tim E.
AU - Maat, Christina A.
AU - Niedieker, Daniel
AU - Mona, Daniel
AU - Petersen, Dennis
AU - Timmermans-Huisman, Evelien
AU - Kole, Jeroen
AU - El-Mashtoly, Samir F.
AU - Spycher, Liz
AU - Zago, Wagner
AU - Barbour, Robin
AU - Mundigl, Olaf
AU - Kaluza, Klaus
AU - Huber, Sylwia
AU - Hug, Melanie N.
AU - Kremer, Thomas
AU - Ritter, Mirko
AU - Dziadek, Sebastian
AU - Geurts, Jeroen J. G.
AU - Gerwert, Klaus
AU - Britschgi, Markus
AU - van de Berg, Wilma D. J.
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. We thank the Advanced Optical Microscopy Core O|2 (www.ao2m.amsterdam ) and John Bol, Allert Jonker and Bram van der Gaag for support with immunostainings and confocal imaging. Further, we thank Lidia Janota for performing immunofluorescent stainings of the tissue sections used for CARS microscopy. This work was supported by Roche. 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. We thank the Advanced Optical Microscopy Core O|2 ( www.ao2m.amsterdam ) and John Bol, Allert Jonker and Bram van der Gaag for support with immunostainings and confocal imaging. Further, we thank Lidia Janota for performing immunofluorescent stainings of the tissue sections used for CARS microscopy. This work was supported by Roche. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/9
Y1 - 2021/9
N2 - Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)—including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn—accumulate in Lewy bodies (LBs) in different regions of the Parkinson’s disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.
AB - Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)—including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn—accumulate in Lewy bodies (LBs) in different regions of the Parkinson’s disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.
KW - Alpha-synuclein
KW - Lewy bodies
KW - Parkinson's disease
KW - Post-mortem human brain
KW - Post-translational modifications
KW - Super-resolution microscopy
UR - http://www.scopus.com/inward/record.url?scp=85107802927&partnerID=8YFLogxK
U2 - https://doi.org/10.1007/s00401-021-02329-9
DO - https://doi.org/10.1007/s00401-021-02329-9
M3 - Article
C2 - 34115198
SN - 0001-6322
VL - 142
SP - 423
EP - 448
JO - Acta Neuropathologica
JF - Acta Neuropathologica
IS - 3
ER -