ASYN biochemical state.

<p><b>A. Native Gels.</b> Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. <b>B. STED microscopy.</b> Selected mutants were imaged in order to characterize the fine structure of the inclusions. <b>C. Thioflavin S staining.</b> H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. <b>D-E</b>. <b>Triton X-100 solubility assay and quantification.</b> H4 cells show that all mutants form detergent insoluble species. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.</p

ASYN mutation effects in the inclusion formation.

<p><b>A. Constructs used in the aggregation model.</b> This model consists of co-expressing SynT together with synphilin-1. <b>B. Inclusion pattern in H4 cells.</b> Different SynT mutants resulted in the formation of distinct inclusion formation in human H4 cells. Scale bar: 10 µm. <b>C. Inclusion quantification.</b>>50 cells were scored per experiment and classified in different groups according to the pattern of inclusions. Representative cells were drawn to show type of inclusions present in each categories. Lysine mutants (E35K, E57K) increase the percentage of cells with inclusions and the number of inclusions per cell, whereas A30P and proline mutants reduce percentage of cells with inclusions and also the number of inclusions per cell. <b>D-E. Levels of ASYN.</b> Immunoblot analysis of the expression levels of ASYN. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 3.</p

ASYN secretion is inversely correlated with toxicity.

<p><b>A. Secretion of ASYN. B. Toxicity measurements.</b> Medium from H4 cells were collected to determine the secretion and the percentage cytotoxicity for each mutant. To measure the release of ASYN, an ELISA assay was performed. Using the same media we also measured the release of lactate dehydrogenase as a measure of cytotoxicity. We observed that these values were inversely correlated with those obtained in the release/secretion experiments. A decrease trend particularly for TP and Y125F detected in terms of secretion, was higher in toxicity. n = 3. <b>C. Correlation between Secretion and Toxicity.</b> The graph shows the inverse trend in secretion and toxicity.</p

ASYN partially co-localizes with endosomes/lysosomes.

<p><b>A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants.</b> Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. <b>B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1.</b> We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.</p

ASYN bPCA.

<p><b>A. Schematic representation of the ASYN bPCA constructs.</b> Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. <b>B-C</b>. Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular (<b>B</b>) and extracellular (<b>C</b>) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001) <b>D</b>. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001) <b>E-F. Levels of ASYN.</b> Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.</p

A-B morphology analysis of Golgi apparatus.

<p>The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (<b>A</b>). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus <b>(B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E).</b> n = 3. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001).</p

Mutations effect on ASYN oligomerization.

<p><b>A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC).</b> ASYN BiFC constructs in anti-parallel orientation. <b>B. Representative pictures of ASYN oligomerization.</b> HEK-293 cells overexpressing VN-ASYN and ASYN-VC constructs. The green fluorescence results from the reconstitution of the Venus fluorophore, promoted by the interaction of the proteins of interest. Scale bar: 10 µm. <b>C. Oligomerization efficiency.</b> Mean fluorescence intensity of cells expressing different ASYN mutants was assessed 24 hours post-transfection, using a microcapillary system (GuavaeasyCyte HT system). For each sample 25,000 events were counted. <b>D. Intracellular distribution of oligomeric ASYN.</b> Nuclear and cytoplasmic venus fluorescence intensities in HEK-293 cells were quantified using ImageJ. The graph demonstrates an increase in nuclear fluorescence in cells expressing ASYN mutants. For each experiment>25 cells were analysed. <b>E-F. Levels of ASYN.</b><b>E</b>. Representative immunoblot showing the expression levels of ASYN. <b>F</b>. Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 3.</p

Correlation between the effects of ASYN mutations on oligomerization and inclusion formation.

<p>The graph depicts how mutations affect oligomerization and inclusion formation, enabling the selection of mutants with different effects. Values were attributed to ASYN mutations according to the results from the two models (oligomerization and inclusion formation) using WT ASYN as reference (center of the graph).</p

Experimental design.

<p>WT and ASYN mutants were subjected to a variety of assays as depicted in the schematic.</p

SIRT2 regulates aSyn aggregation and toxicity.

<p><b>(A)</b> H4 cells were infected with lentiviruses encoding shRNAs against SIRT2 (T2.KD) or scramble shRNA (Ctrl) and selected with puromycin. Cells were then cotransfected with SynT and synphilin-1 (Synph1). SIRT2, synphilin-1, aSyn, and GAPDH levels were assessed by immunoblot analyses. <b>(B)</b> Ctrl and T2.KD cells transiently expressing SynT and synphilin-1 for 48 h were processed for immunocytochemistry (ICC) (aSyn, green). Data show percentage of cells with aSyn inclusions (<i>n</i> = 3). Scale bar 15 μm. <b>(C)</b> Triton X-100 insoluble and total fractions of cells as in (B) probed for aSyn and GAPDH. <b>(D)</b> Native protein extracts from H4 cells as in (B) were separated on a sucrose gradient. Fractions were immunoblotted and probed for aSyn. <b>(E)</b> Anti-aSyn IP from cells as in (B). Fractions were immunoblotted and probed for acetyl-lysine and aSyn. <b>(F)</b> Toxicity of Ctrl and T2.KD measured by lactate dehydrogenase (LDH) release assay (<i>n</i> = 3). Data in all panels are average ± SD, ** <i>p</i> < 0.01, **** <i>p</i> < 0.0001. For (B) and (F), unpaired, two-tailed <i>t</i> test with equal SD. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p