Colocalization of α-syn species and selected intracellular targets in donor cells before transfer.

<p>Monomeric, oligomeric or fibrillar Cy3-labeled (red) α-syn was added to RA-differentiated SH-SY5Y cells and incubated for 3 h. After 24 h, the cells were fixed and then subjected to immunocytochemistry using Alexa Fluor 488-conjugated secondary antibody (green) showing subcellular targets. (A) Confocal microscopy images showing α-syn (red) colocalization (yellow) with LAMP2, VAMP2, KDEL, SV2 or TSG101 (green). Graphs show quantification of the total proportions of the intracellular α-syn monomers (B), oligomers (C), and fibrils (D) that are colocalized with the respective subcellular targets. The results are presented as the mean percentage of colocalization ± SEM. ** p<0.01, *** p<0.001, one-way ANOVA with Bonferroni correction. (Scale bar = 10 μm).</p

Colocalization of α-syn species and intracellular selected targets in acceptor cells after transfer.

<p>(A) Confocal images of donor and acceptor cells after 24 h co-culture show colocalization (magenta) of Cy3-labeled α-syn (red) with LAMP2, VAMP2, KDEL, SV2 or TSG101 using the Alexa Fluor 405-conjugated secondary antibody (blue) in GFP-expressing acceptor cells (green). (B-D) Quantification of the total proportions of the intracellular α-syn monomers (B), oligomers (C), and fibrils (D) that are colocalized with the respective subcellular targets. The results are presented as the mean percentage of colocalization ± SEM. * p<0.05, one-way ANOVA with Bonferroni correction. (Scale bar = 10 μm).</p

Uptake of α-synuclein in differentiated SH-SY5Y donor cells.

<p>SH-SY5Y cells differentiated for 7 d with 10 μM retinoic acid were treated for 3 h with 1 μM monomeric, oligomeric or fibrillar Cy3-labeled α-synuclein and reseeded onto cover slips. After 24 h or 48 h of incubation, the cells were fixed and mounted onto slides with ProLong antifade reagent containing DAPI. (A) Confocal images showing cellular uptake of monomeric, oligomeric and fibrillar species of α-synuclein (red) and DAPI (blue) staining for the nuclei. (B) The Cy3 fluorescence intensity at different concentrations of α-syn species. (C) Quantification of α-syn uptake. Data are presented from an average of 1855 counted donor cells from an average of 67 pictures per treatment and time point. The results are presented as the mean percentage of positive transfer ± SEM. n.s. not significant, *** p<0.001, Chi-test with Bonferroni correction (Scale bar = 10 μm).</p

Transfer of Cy3-labeled α-syn between neuronal cells.

<p>Co-culture of neuronally differentiated donor cells containing Cy3-labeled α-syn (red) and acceptor cells transfected with EGFP-tagged endosomal Rab5a (green). (A) Confocal images show transfer (black and white arrows) of monomeric, oligomeric and fibrillar α-syn from donor cells to acceptor cells after 24 h of co-culture. (B) Quantification of acceptor cells with successfully transferred α-syn isoforms. Data are presented from an average of 460 counted acceptor cells from an average of 83 pictures per treatment. The results are presented as the mean percentage of positive transfer ± SEM. *** p<0.001, Chi-test with Bonferroni correction (Scale bar = 20 μm).</p