Journal: PLoS Genetics

Article Title: shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity

doi: 10.1371/journal.pgen.1005995

Figure Lengend Snippet: A. Stable H4 cells silenced for selected hits or with a scrambled shRNA were co-transfected with aSynT and Synphilin-1. Cells were fixed 48 h post-transfection and subjected to immunocytochemistry for aSyn (green) and for Synphilin-1 (red) followed by fluorescence microscopy. DAPI was used as a nuclear counterstain. White arrowheads point to aSyn inclusions. Scale bars: 10 μm. B. Percentage of cells with no inclusions (gray), less than 10 inclusions (light green) or more than 10 inclusions (dark green). C. Cytotoxicity (measured by LDH release in the media) from stable cells subjected to hits silencing and normalized to control (cells transduced with scrambled shRNA). The ratio represented refers to cells with aSyn inclusions versus cells with no aSyn. Bars represent mean±95% CI (*: 0.05

0.01; **: 0.01

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. kd, knockdown.

Article Snippet: Then, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT, blocked for 1 h at RT with 1% normal goat serum in 0.1% Triton X-100 in PBS, incubated with primary antibody against aSyn (mouse anti-aSyn 1:1000; BD Biosciences) and Synphilin-1-V5 (only for loss of function assays; mouse anti-V5, 1:1000, Cell Signaling) at 4°C overnight followed by secondary antibody incubation (1:1000, goat anti-mouse IgG-Alexa488 for aSynT (or igG-Alexa 568 for aSynT when co-transfected with GFP-SYTL5) and goat anti-mouse IgG-Alexa568 (Life Technologies) for Synphilin-1-V5 (only for loss of function assays), for 2 h at RT.

Techniques: shRNA, Transfection, Immunocytochemistry, Fluorescence, Microscopy, Transduction

Journal: PLoS Genetics

Article Title: shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity

doi: 10.1371/journal.pgen.1005995

Figure Lengend Snippet: A. 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 , , and Figs. Scale bars: 10 μm. 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. C. Quantification of alexa-647 transferrin intensity normalized to the control condition. D. 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 E. 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

0.01; **: 0.01

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.

Article Snippet: Then, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT, blocked for 1 h at RT with 1% normal goat serum in 0.1% Triton X-100 in PBS, incubated with primary antibody against aSyn (mouse anti-aSyn 1:1000; BD Biosciences) and Synphilin-1-V5 (only for loss of function assays; mouse anti-V5, 1:1000, Cell Signaling) at 4°C overnight followed by secondary antibody incubation (1:1000, goat anti-mouse IgG-Alexa488 for aSynT (or igG-Alexa 568 for aSynT when co-transfected with GFP-SYTL5) and goat anti-mouse IgG-Alexa568 (Life Technologies) for Synphilin-1-V5 (only for loss of function assays), for 2 h at RT.

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Incubation, Immunocytochemistry, Confocal Microscopy, Transferring, Imaging, Lactate Dehydrogenase Assay, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Posttranslational modification and mutation of histidine 50 trigger alpha synuclein aggregation and toxicity

doi: 10.1186/s13024-015-0004-0

Figure Lengend Snippet: Intracellular aggregation of WT and H50 mutant aSyn. H4 neuroglioma cells were transfected with WT and H50Q/R mutant aSyn. SDGC analysis of lysates from H4 cells transfected with WT and H50Q/R mutant aSyn was performed. A shift of aSyn positive fractions from lower density fractions to higher density fractions was observed for the aSyn H50Q mutant. The quantification of aSyn immunointensity in fractions 11 - 17 in 3 independent experiments is shown in the lower panel. To allow comparison, all fractions collected from WT and H50Q/R mutants in each experiment were loaded on the same dot blot membrane. The relative aSyn intensity was generated by normalizing the intensity of each fraction to the intensity of the fraction 17 from WT aSyn in each experiment.

Article Snippet: After blocking the nitrocellulose membranes, aSyn was visualized using a mouse anti-aSyn primary antibody (Syn-1, 1:2000, BD Transduction Laboratories), and a HRP-conjugated goat anti-mouse secondary antibody (1:10000, Dianova).

Techniques: Mutagenesis, Transfection, Dot Blot, Generated

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: Scheme representing the structure of human ASYN with the three distinct domains (N-terminal, NAC and C-terminal). Amino acid residues are indicated in the bottom. Brown bars inside protein domains represent the imperfect hexameric KTKEGV repeats. Arrows indicate the sites of phosphorylation and the broken lines show the mutated sites.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Phospho-proteomics

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC). ASYN BiFC constructs in anti-parallel orientation. B. Representative pictures of ASYN oligomerization. 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. C. Oligomerization efficiency. 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. D. Intracellular distribution of oligomeric ASYN. 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. E-F. Levels of ASYN. E . Representative immunoblot showing the expression levels of ASYN. F . Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Bimolecular Fluorescence Complementation Assay, Construct, Fluorescence, Expressing, Transfection, Western Blot

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Constructs used in the aggregation model. This model consists of co-expressing SynT together with synphilin-1. B. Inclusion pattern in H4 cells. Different SynT mutants resulted in the formation of distinct inclusion formation in human H4 cells. Scale bar: 10 µm. C. Inclusion quantification. >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. D-E. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Construct, Expressing, Western Blot

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: WT and ASYN mutants were subjected to a variety of assays as depicted in the schematic.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques:

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A . Yeast cells expressing GAL1-driven ASYN-GFP variants from 2 µ plasmids were spotted in 10-fold dilutions on selection plates containing 2% glucose (control) or 2% galactose. After incubation for 3 days at 30°C the plates were photographed. Expression of GFP from the same promoter was used as a control. B. Live-cell fluorescence microscopy of yeast cells expressing ASYN-GFP. Yeast cells, pre-grown to mid-log phase, were induced in galactose-containing medium and examined for aggregates at 6 hours of induction. GFP-expressing cells were used as control. Scale bar: 1 µM. C. Aggregate quantification of yeast cells, expressing ASYN-GFP. For each strain, the number of cells displaying cytoplasmic foci is presented as percent of the total number of cells counted. For quantification of aggregation at least 300 cells were counted per strain and per experiment. GFP-expressing cells were used as a control. Student's t test (*p<0.05, **p<0.01, ***p<0.001). D. Protein levels of ASYN-GFP variants. Expression of ASYN-GFP variants was induced for 6 hours in galactose-containing medium. Equal amounts of crude protein extracts were used for Western analysis with anti-ASYN antibody and anti-cdc28 antibody as a loading control. n = 2.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Expressing, Selection, Control, Incubation, Fluorescence, Microscopy, Western Blot

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Native Gels. 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. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. 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. D-E . Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Western Blot, Clear Native PAGE, Transfection, Construct, Microscopy, Staining, Expressing, Incubation, Solubility

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C . 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 ) and extracellular ( C ) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p<0.05, **p<0.01, ***p<0.001) D . Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's t test (*p<0.05, **p<0.01, ***p<0.001) E-F. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Construct, Luciferase, Transfection, Activity Assay, Western Blot, Expressing

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Secretion of ASYN. B. Toxicity measurements. 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. C. Correlation between Secretion and Toxicity. The graph shows the inverse trend in secretion and toxicity.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. 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.

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Immunocytochemistry, Expressing, Marker

Journal: PLoS Genetics

Article Title: Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

doi: 10.1371/journal.pgen.1004741

Figure Lengend Snippet: 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).

Article Snippet: Immunoblotting was performed following standard procedures using anti-ASYN monoclonal antibody (AnaSpec, CA, USA) or Cdc28 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) as a loading control.

Techniques: Selection