Amyloid formation of alternatively spliced variants of α-synuclein

Authors

• DQ SanGiovanni
• RP McGlinchey
• JC Lee

Abstract

Intracellular accumulation of α-synuclein (SNCA) amyloid fibrils is the pathological hallmark of synucleinopathies including Parkinson’s disease, Lewy body dementia, and multiple system atrophy. The gene that encodes for SNCA is alternatively spliced, generating three additional isoforms (SNCAΔ3, SNCAΔ5, and SNCAΔ3Δ5) through the deletion of exon 3, 5, or both. Vast amount of research has been dedicated to SNCA, overlooking these other isoforms. Despite recent documented upregulation of alternatively spliced variants in disease states, their impact on SNCA aggregation is unknown. To address this deficiency, recombinant expression and purification of all three isoforms were achieved, and their aggregation behaviors were characterized. All three spliced variants exhibited faster aggregation kinetics compared to that of the full-length protein as assessed by thioflavin-T fluorescence (SNCAΔ3Δ5 ≈ SNCAΔ5 > SNCAΔ3 > SNCA). Fibrils were characterized by circular dichroism, Fourier-transform infrared and Raman spectroscopies which revealed distinctive spectral features for SNCAΔ5 and SNCAΔ3Δ5, suggesting different fibril structures, which was supported by morphological variations by negatively stained transmission electron microscopy. Finally, protease-resistant cores were determined by liquid-chromatography mass spectrometry using a broad-spectrum protease, proteinase-K, and lysosomal cathepsins. Interestingly, co-mixing SNCA with SNCAΔ5 as well as with SNCAΔ3Δ5 showed an enhancement of aggregation of SNCA, whereas co-mixing with SNCAΔ3 had no observable effect, highlighting a potential pathogenic connection for SNCAΔ5 and SNCAΔ3Δ5. In future work, we are conducting cross-seeding experiments to test the hypothesis of heterologous propagation of the isoforms, addressing the question whether fibril structural differences could contribute to disease phenotypes.

Scientific Focus Area: Biomedical Engineering and Biophysics

This page was last updated on Tuesday, August 6, 2024