aSyn forms oligomers in cell lines of different origin.

<p>GN-link-aSyn and aSyn-GC were co-transfected into HEK, CHO, or MES23.5 cells showing complementation can occur in the different cellular environments provided by each cell line (Scale bar, 50 µm).</p

Constructs used in this study and complementation results.

<p>Complementation occurred only with the combinations shown in green. Co-transfection of GN-link-aSyn with aSyn-GC resulted in the strongest fluorescent signal (+++) indicating the most favorable interaction. All negative controls and other combinations tested showed detectable fluorescent signal.</p

aSyn oligomers are stabilized by complementation and show increased cytotoxicity.

<p>A. The half lives of EGFP (1), aSyn-GC (2), GN-link-aSyn (3), and GN-link-aSyn+aSynGC (4) were monitored via immunoblotting of H4 cells transfected with the different constructs and treated with cycloheximide for the indicated period of time prior to cell harvesting. Immunoblots were probed with an antibody against EGFP (Abcam, Cambridge, USA).B. Toxicity assay of cells transfected with EGFP (1), WT aSyn (2), GN-link-aSyn (3), aSyn-GC (4), and GN-link-aSyn+aSynGC (5) showing that stabilization of aSyn oligomers leads to increased toxicity (*t-test, n = 3, p<0.005).</p

Hsp70 reduces aSyn oligomerization and toxicity in living cells.

<p>A. H4 cells were co-transfected with WT aSyn or GN-link-aSyn+aSynGC and either with an empty vector or with Hsp70. Cytotoxicity was reduced by Hsp70 (t-test, n = 3, p<0.001). B. Confocal microscopy analysis showing that overexpression of Hsp70 reduces aSyn oligomerization (Scale bar, 50 µm). C. Quantification of the pixel intensity of the same cells as in B showing a statistically significant reduction in fluorescence in cells overexpressing Hsp70 (t-test, p<0.0001). D. Immunoblot of a native PAGE of cells co-transfected with GN-link-aSyn+aSynGC and either an empty vector of with Hsp70 showing a strong reduction in high molecular weight oligomeric species by Hsp70. E. Immunoblot of an SDS-PAGE of the same samples as in E showing Hsp70 overexpression does not lead to decreased levels of GN-link-aSyn nor aSynGC. F. Quantification of the SDS-PAGE confirms that Hsp70 does not reduce the protein levels of GN-link-aSyn nor aSyn-GC.</p

PD-associated aSyn mutations form oligomeric species.

<p>A. H4 cells were transfected with WT or mutant combinations of GN-link-aSyn+aSynGC and analyzed via flow cytometry to quantify the fluorescence intensity. The fluorescence signal was identical for WT and mutant aSyn. B. Immunoblots of native-PAGE showing oligomeric species formed by GN-link-aSyn+aSynGC (lane 1), WT aSyn (lanes 2 and 3), A53T (lane 4), A30P (lane 5), and E46K (lane 6) and the corresponding expression levels (SDS-PAGE). C. Subcellular distribution, analyzed by confocal microscopy, of WT and mutant aSyn oligomers in H4 cells showing cytoplasmic and nuclear localization (green). Hoescht staining highlight the nuclei in blue (Scale bar, 50 µm).</p

Rab8b, Rab11a, Rab13 and Slp5 are involved in different steps of cellular trafficking and modulate different aggregated species of aSyn.

<p>Rab8b is localized in cell membranes and vesicles and may be involved in polarized vesicular trafficking (endoplasmic reticulum (ER) to plasma membrane) and, specifically, in neurotransmitter release. Rab11a regulates endocytic recycling pathway and participates specifically in transferrin recycling. Rab13 plays a role in regulating membrane trafficking between trans-Golgi network, recycling endosomes (RE) and cell/tight junctions. Slp5 is localized throughout the nucleus and cytosol, and binds to phospholipidic structures. Slp5 is a Rab27a effector protein and plays a role in exocytosis. Rab8b, Rab11a and Rab13 overexpression rescues aSyn-induced toxicity and inhibits its oligomerization and aggregation. Moreover, Rab11a and Rab13 increases aSyn secretion through recycling endocytic route only when aSyn inclusions are present within cells. Although Slp5 also rescues aSyn induced toxicity when oligomerization or aggregation are the readout, it increases aSyn secretion only in a context of aSyn oligomerization. EE, early endosome; V, vesicle.</p

RNAi-based screen for genes that modify aSyn oligomerization in living cells.

<p><b>A.</b> A human shRNA library targeting trafficking and phosphotransferase genes was screened using a stable cell line expressing aSyn-BiFC constructs (1). Genes modifying aSyn oligomerization by at least 50% (2) were identified using fluorescence microscopy analysis and considered for further validation. <b>B.</b> Representative live cell imaging pictures of aSyn-BiFC stable H4 cells silenced for hits that increase (<i>RAB8B</i>, <i>RAB11A</i>, <i>RAB13</i>, <i>RAB39B</i>, <i>CAMK1</i>, <i>DYRK2</i>) or decrease (<i>CC2D1A</i>, <i>CLK4</i>, <i>SYTL5)</i> aSyn oligomerization (green). A scrambled shRNA was used as control. Scale bars: 20 μm. <b>C.</b> Representative immunoblot of aSyn-BiFC cells subjected to silencing of the selected hits <b>D.</b> Relative fluorescence quantification of aSyn oligomerization (green) and quantification of aSyn protein levels (white). The ratio of protein levels and aSyn oligomerization is presented (yellow). <b>E.</b> LDH release in the media from cells with aSyn oligomers versus no aSyn (orange). Bars represent mean±95% CI (*: 0.05</p><p>0.01; **: 0.01</p><p>0.001; ***: p<0.001) and are normalized to the control of at least three independent experiments. Single comparisons between the control and experimental groups were made through Wilcoxon test. Silencing of hits was performed using at least three different shRNAs against the same gene. For simplicity, only one shRNA is shown. Results with additional shRNAs are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s003" target="_blank">S1 Fig</a> kd, knockdown.</p

Overexpression of Rab8b, Rab11a, Rab13 and Slp5 reduces aSyn aggregation and modulates endosomal recycling and secretion.

<p><b>A.</b> H4 cells were triple-transfected with aSynT, Synphilin-1 and constructs expressing Rab8b, Rab11a, Rab13, Slp5 (red) or empty vector. 48 h post-transfection, media with no serum was replaced in cells for 1 h. Cells were incubated with Alexa-647 human transferrin (magenta) for 30 min, prior to fixation and subjected to immunocytochemistry for aSyn (green), and followed by confocal microscopy. DAPI was used as a nuclear counterstain. Control with empty vector is shown. Amplifications within cells were made to show co-localization between aSyn, the hit and transferring within inclusions. For simplicity, only Rab8b-WT is shown. Imaging of the remaining constructs is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s007" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s008" target="_blank">S6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s009" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s010" target="_blank">S8</a> Figs. Scale bars: 10 μm. <b>B.</b> Quantification of the number of aSyn inclusions per cell. The cells displaying aSyn inclusions were divided in: cells with no inclusions (represented in black), cells with less than 10 inclusions (in light gray) and cells with more than 10 inclusions (in dark gray). Only triple transfected cells were considered for the quantifications. <b>C.</b> Quantification of alexa-647 transferrin intensity normalized to the control condition. <b>D.</b> Cytotoxicity was measured by the LDH-release assay. The represented ratios in C and D refers to cells with aSyn inclusions versus cells with no aSyn <b>E.</b> Representative immunoblot of extracellular conditioned media from cells overexpressing the selected genes in the aSyn aggregation model, and respective quantification. In graphs, Rab8b is represented in yellow, Rab11a in orange, Rab13 in green and Slp5 in blue. Bars represent mean±95% CI (*: 0.05</p><p>0.01; **: 0.01</p><p>0.001; ***: p<0.001) and are normalized to the control of at least three independent experiments. Single comparisons between the control and experimental groups were made through Wilcoxon test.</p

Overexpression of selected hits reduces aSyn oligomerization and modulates endocytic recycling and secretion.

<p><b>A.</b> H4 stable cells for aSyn-BiFC (green) or H4 cells with no aSyn were transfected with constructs expressing Rab8b, Rab11a, Rab13, Slp5 (red) or empty vector. 48 h post-transfection, media with no serum was replaced in cells for 1 h. Cells were incubated with Alexa-647 human transferrin (magenta) for 30 min, prior to fixation. DAPI was used as a nuclear counterstain. Cells were subjected to confocal microscopy. For simplicity, because all expressed hits have similar subcellular locations, only wild type form of Rab8b is shown. Imaging of the remaining constructs is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s007" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s008" target="_blank">S6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s009" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005995#pgen.1005995.s010" target="_blank">S8</a> Figs. Squares are regions zoomed in showing transferrin localization within endocytic recycling compartment in cells with no aSyn oligomers or hit, at cells extremities in cells with aSyn oligomers and no hit, and colocalizing the hit in cells with or without aSyn. Scale bars: 10 μm. <b>B.</b> Relative aSyn-BiFC fluorescence upon hits overexpression compared to the control (empty vector transfection). <b>C.</b> Quantification of Alexa-647 transferrin intensity normalized to the control condition. The represented ratio refers to cells with aSyn oligomers versus cells with no aSyn. <b>D.</b> LDH extracellular levels were measured to assess cytotoxicity. The ratio represented refers to cells with aSyn oligomers versus cells with no aSyn. <b>E.</b> Relative quantification of aSyn intracellular total protein (stripe pattern) and extracellular conditioned media (clear pattern) for each condition. A representative immunoblot is shown. In graphs, Rab8b is represented in yellow, Rab11a in orange, Rab13 in green and Slp5 in blue. Bars represent mean±95% CI (*: 0.05</p><p>0.01; **: 0.01</p><p>0.001; ***: p<0.001) and are normalized to the control of at least three independent experiments. Single comparisons between the control and experimental groups were made through Wilcoxon test.</p

Autophagy modulates SNCA/α-synuclein release, thereby generating a hostile microenvironment

<p>SNCA/α-synuclein aggregation plays a crucial role in synucleinopathies such as Parkinson disease and dementia with Lewy bodies. Aggregating and nonaggregating SNCA species are degraded by the autophagy-lysosomal pathway (ALP). Previously, we have shown that the ALP is not only responsible for SNCA degradation but is also involved in the intracellular aggregation process of SNCA. An additional role of extracellular SNCA in the pathology of synucleinopathies substantiating a prion-like propagation hypothesis has been suggested since released SNCA species and spreading of SNCA pathology throughout neural cells have been observed. However, the molecular interplay between intracellular pathways, SNCA aggregation, release, and response of the local microenvironment remains unknown. Here, we attributed SNCA-induced toxicity mainly to secreted species in a cell culture model of SNCA aggregation and in SNCA transgenic mice: We showed that ALP inhibition by bafilomycinA₁ reduced intracellular SNCA aggregation but increased secretion of smaller oligomers that exacerbated microenvironmental response including uptake, inflammation, and cellular damage. Low-aggregated SNCA was predominantly released by exosomes and RAB11A-associated pathways whereas high-aggregated SNCA was secreted by membrane shedding. In summary, our study revealed a novel role of the ALP by linking protein degradation to nonclassical secretion for toxic SNCA species. Thus, impaired ALP in the diseased brain not only limits intracellular degradation of misfolded proteins, but also leads to a detrimental microenvironmental response due to enhanced SNCA secretion. These findings suggest that the major toxic role of SNCA is related to its extracellular species and further supports a protective role of intracellular SNCA aggregation.</p