Abstract
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.
Highlights
The presence of neuronal inclusions—termed Lewy Bodies (LBs) and Lewy Neurites (LNs)—in predilected brain regions pathologically defines Parkinson’s disease (PD) and dementia with Lewy bodies (DLB)
The initial characterization and validation of these antibodies has been described in literature [3, 23, 24] Syn105 is an antibody raised against an immunogenic peptide corresponding with residues 118–122 of aSyn, which displays a high affinity for 122CTT aSyn fragments compared to full-length aSyn [23], while 11A5 is directed against aSyn phosphorylated at Ser129 [3]
The specificity of these novel antibodies was confirmed using enzyme-linked immunosorbence assays (ELISAs) and surface plasmon resonance (SPR) on aSyn peptides as well as by Western blots (WB) on recombinant protein
Summary
The presence of neuronal inclusions—termed Lewy Bodies (LBs) and Lewy Neurites (LNs)—in predilected brain regions pathologically defines Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). LBs are described as eosinophilic inclusion bodies with different morphologies, typically dependent on brain region (brainstem, limbic or cortical) [40, 70]. The mechanisms determining their formation and morphology remain elusive. An important role has been proposed for the lipophilic N-terminus (NT) and non-amyloid-β component domain (NAC domain) in the interaction of aSyn with lipid membranes [7, 18], while the residues 96–140 encompass the negatively charged, acidic C-terminus (CT) of aSyn for which important regulatory roles have been proposed in the interaction of aSyn with other proteins or metal ions [17]. The CT further harbors many sites where aSyn can be post-translationally modified (PTM) [57]
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