Amyloid formation of alternatively spliced variants of α‐synuclein

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are disorders characterized by the presence of cytosolic α-synuclein (SNCA) amyloids. The gene SNCA is alternatively spliced, generating three variants of SNCA, missing exon 3 (SNCAΔ3) or 5 (SNCAΔ5), or both exons (SNCAΔ3Δ5). Despite purported upregulation in disease states, their pathological relevance is ill-defined. Here, we investigated the amyloid formation of alternatively spliced variants under physiological conditions. Aggregation kinetics, secondary structure, and fibril morphology of N-terminally acetylated SNCAΔ3, SNCAΔ5, and SNCAΔ3Δ5 were assessed by thioflavin-T fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. Compared to SNCA, both SNCAΔ5 and SNCAΔ3Δ5 aggregate faster and adopt a more twisted fibril morphology, whereas SNCAΔ3 is more sensitive to solution conditions, exhibiting similar or modestly faster aggregation kinetics compared to SNCA. Cross-seeding experiments using spliced-variant fibrils and soluble SNCA showed that despite fibril morphological differences, SNCAΔ5 were competent seeds for SNCA, which is explained by their similar protease-K resistant regions. Contrastingly, neither SNCAΔ3 nor SNCAΔ3Δ5 fibrils cross-seed SNCA, indicating exon 3 (residues 41-54) is essential in modulating fibril structure. Notably, SNCA aggregation is stimulated by sub-stoichiometric amounts of soluble SNCAΔ5 and SNCAΔ3Δ5, but not SNCAΔ3, suggesting that exon 5 (residues 103-130) is more important in modulating aggregation kinetics. Taken together, we propose that alternatively spliced variants are pathogenic by exacerbating aggregation of the main SNCA isoform.