Modulation of α-synuclein in vitro aggregation kinetics by its alternative splice isoforms

Significance The existence of proteoforms—distinct versions of a protein originating from the same gene via mechanisms such as genetic variation, RNA transcript alternative splicing, and post-translational modifications—considerably enriches the range of possible behaviors of that protein. Here, we investigated the impact of alternative splice isoforms of α-synuclein on its propensity to aggregate, a phenomenon closely associated with Parkinson’s disease and related synucleinopathies. Our observations reveal that even a relatively small amount of an isoform that is more prone to aggregation can hasten the overall aggregation of α-synuclein. These results illustrate the importance to factor in proteoforms when investigating protein behavior and their implications on disease pathology.


FTIR
FTIR measurements were performed on a Vertex 70 FTIR spectrometer (Bruker) using a DiamondATR unit and a deuterated lanthanum α-alanine-doped triglycine sulphate detector (DOI: dx.doi.org/10.17504/protocols.io.dm6gp3xqpvzp/v1).αSyn aggregation reactions were performed as described for kinetic experiments, whereas no ThT was used in these samples.Fibrils were recovered after 6 days of incubation by centrifugation (20 min, 21100 g, RT), the pellet was washed once in MQ water, centrifuged again and resuspended in a final volume of 20 µL MQ water.5 μL of protein solution were deposited on the prism and spectra were acquired over the spectral range of 4000-900 cm -1 .The spectra were then smoothened (25 points) and normalized.Due to the clumpy nature and therefore scattering effects of αSyn-112 and αSyn-98 aggregates, no FTIR spectra could be obtained at first.For comparison of the isoforms alone (Figure S5A), aggregates were therefore sonicated in an Ultrawave Ultra BT U100 water bath sonicator for 40 min, which did not affect the FTIR spectra comparing sonicated (Figure S5A) and unsonicated αSyn-140 fibrils (Figure S5B,C) but allowed the acquisition of FTIR spectra of αSyn-112 and αSyn-98.

Figure S1 .
Figure S1.Predicted solubility profiles of the four αSyn splice isoforms analyzed in this work.(A-D) Overall solubility scores and residue-specific solubility profiles of αSyn-140 (A), αSyn-126 (B), αSyn-112 (C) and αSyn-98 (D) at pH 7.4; high values (blue) correspond to soluble regions, and low values (red) to insoluble regions.Due to the lack of the highly soluble region of residues 103-130, which is encoded by exon 5, the overall solubility scores are lower for αSyn-112 and αSyn-98 compared to αSyn-140 and αSyn-126.

Figure S2 .
Figure S2.Quality assessment of purified αSyn isoforms.(A) Purity of the final protein product was assessed by SDS-PAGE followed by Coomassie stain.(B) The molecular mass of the produced αSyn isoforms was validated by LC-MS, as shown in the deconvoluted mass spectra.

Figure S3 .
Figure S3.Misfits of αSyn isoform aggregation kinetics.Normalized traces (left) were fitted on the AmyloFit platform to the (A) Nucleation Elongation and (B) Saturating Elongation models, shown as the solid lines.These additional models are not in line with the scaling exponent, show a marked deviation from the data and are therefore less suitable than the Saturating Elongation and Fragmentation model chosen in Figure 1.

Figure S6 .
Figure S6.Monomers of αSyn isoforms are not cytotoxic to SH-SY5Y cells.Cell viability of SH-SY5Y cells was determined using an MTT assay after treatment with monomers of αSyn-140, αSyn-126, αSyn-112 and αSyn-98 at 50 µM.Data are expressed as percentage of medium control and shown as mean ± SEM of independent cell treatments (n = 3).One-way ANOVA with Dunnett's post-hoc test, ns = non-significant.