Fig. 1: Comprehensive exploration of aSyn heterogeneity - pathology diversity, fibril polymorphism, aSyn PTMs in synucleinopathy brains and antibody generation/ validation steps.

a Diversity of aSyn pathology in synucleinopathies with aa granular/punctate cytoplasmic inclusions in the neurons; ab classical LBs in the neuronal soma; ac LNs in the neuronal processes; ad astrocytic aSyn accumulations; ae oligodendroglial cytoplasmic inclusions. These pathological structures show differences in their positivity to aggregation markers, including ubiquitin (Ub) and p62. Schematic created with BioRender.com (agreement no: QW23G6FJ76). b Cryo-EM three-dimensional reconstructions of the recombinant full-length aSyn PFFs to show the polymorphism of aSyn fibrils generated in vitro^38,39. Four distinct polymorphs were identified based on the protofilament fold and inter-protofilament interfaces: Polymorph 1a ‘rod’ (PDB-6CU7, EMD-7618); polymorph 1b ‘twister’ (PDB-6CU8, EMD-7619); polymorph 2a (PDB-6SSX, EMD-10307); and polymorph 2b (PDB-6SST, EMD-10305). c aSyn PTMs identified in synucleinopathy brain tissues, which include acetylation^29,30,81,82, ubiquitination^{29,30,32,52,72,73,74,75,76,77,78}, phosphorylation^{7,29,30,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71}, nitration^79,80, and truncation^{1,29,49,74,76,78,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96} across the whole sequence of the protein. d A schematic representation of the steps followed for the generation, characterization, validation and application of aSyn antibodies. These involved da antibody design via the selection of immunogens comprising of aSyn recombinant proteins and peptides; db immunization of the mice followed by lymphocyte–myeloma fusion; dc screening of the hybridomas via ELISA, DB and WB, isotyping and subcloning, and dd acquisition of purified antibodies. The antibodies were then de characterized using a library of aSyn recombinant proteins for their epitopes, conformational selectivity, sensitivity, specificity and reactivity via DB and WB. The antibody specificity was further validated on df aSyn KO mouse primary hippocampal and cortical neurons, and in aSyn KO mouse tissue of amygdala. dg The antibodies were validated on human brain tissues of different LB diseases. dh The mouse aSyn-reactive antibodies were applied to neuronal seeding model and PFF-injected mouse brain tissues to profile the newly formed aggregates. Schematic created with BioRender.com (agreement no: FN23G6E1SR). aSyn alpha-synuclein, DB dot/slot blot, cryo-EM cryogenic electron microscopy, ELISA enzyme-linked immunoassay, KO knockout, LB Lewy body, LN Lewy neurite, PFFs pre-formed fibrils, PTM post-translational modification, Ub ubiquitin, WB Western blot.