α-Syn and synphilin-1 equally enhance cell death in aged yeast cells.

<p>A: Quantification of ROS accumulation using DHE staining at different times during growth of yeast strains transformed with an empty plasmid (□, Ctrl.) or constructs allowing for expression of α-Syn (▪), SY^WT (Δ), SY^R621C (▴),α-Syn and SY^WT (○) or α-Syn and SY^R621C (•). B: Quantification of the number of cells that display phosphatidylserine externalization or loss of membrane integrity using annexinV/propidium iodide (PI) co-staining at 36 h of growth in the strains used in A. C: Quantification of viable cells present in the strains used in A at 36 h of growth as determined by their ability to form colonies. D: Fluorescence microscopic visualization of cells expressing combinations of α-Syn, SY^WT or SY^R621C as indicated and stained with DHE (upper panels) or co-stained with annexinV and PI (lower panels) after 36 h of growth. E and F: Quantification of viable cells (E) and cells producing ROS (F) in the strains used in A when kept in culture for two weeks. All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Co-expression of synphilin-1 increases α-Syn S129-phosphorylation.

<p>A: Phosphorylation of α-Syn at S129 in the BY4741 wild-type strain when the expression of α-Syn was combined with an empty plasmid or constructs allowing for co-expression of SY^WT or SY^R621C as indicated. The panel on the left represents the average S129-phosphorylation as determined by immunodetection using a P-S129 specific monoclonal antibody, shown in the right panel, and quantified relative to intensity obtained for immunodetection with a polyclonal α-Syn antibody. B: The left panel shows the average number of H4 neuroglioma cells containing inclusions formed by α-Syn–EGFP when expressed alone or in combination with SY^WT as determined by fluorescence microscopic visualization, for which a representative picture is shown in the right panel. C: Phosphorylation of α-Syn at S129 in H4 neuroglioma cells as detected by immunodetection using a P-S129 specific monoclonal antibody and quantified relative to the intensity obtained for immunodetection with a polyclonal α-Syn antibody. The panel on the left represents the relative average phosphorylation, the panel on the right a corresponding Western blot analysis. All data represent the mean ± SEM of at least three independent experiments. Significance was assayed using a 1-way ANOVA (A) or t-test (B and C)(* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Sir2 mediates synphilin-1 toxicity in yeast.

<p>A: Relative quantification of viable cells as determined by their ability to form colonies at different times after inoculation of the wild-type strain or the isogenic <i>sir2Δ</i> mutant transformed with empty plasmids or constructs allowing for expression of α-Syn or SY^WT, either alone or in combination as indicated. The number of viable cells in samples taken after 24 h of growth of the two strains transformed with the empty plasmids was set at 100%. B and C: Quantification of viable cells (B) and cells producing ROS (C) during chronological ageing of the wild-type strain transformed with an empty plasmid (□) or expressing SY^WT (▪) and the isogenic <i>sir2Δ</i> mutant transformed with an empty plasmid (○) or expressing SY^WT (•). All data represent mean ± SEM of six independent transformants. Significance of the data was determined by t-tests (* = p<0.05; ** = p<0.01; *** = p<0.001).</p

Transport of synphilin-1 inclusions along actin cables.

<p>A: Fluorescence microscopy images of late exponential <i>sir2Δ</i> cells expressing dsRed-SY^WT and α-Syn-EGFP showing that daughter cells inherit cytosolic synphilin-1 inclusions and plasma membrane associated α-Syn. B: Fluorescence microscopy images of late exponential wild-type cells expressing dsRed-SY^WT stained with Alexa Fluor 488 phalloidin to visualize actin patches and actin fibers and with Calcofluor to visualize the cell wall. Shown are the pictures obtained with the fluorescent proteins or dyes as well as the corresponding merges. C: Assessment of growth of wild-type cells with or without expression of native SY^WT or SY^R621C when plated on media supplemented with either Latranculin-B, Benomyl or the solvent DMSO.</p

Synphilin-1 induces inclusion formation of α-Syn in yeast.

<p>A: Fluorescence microscopic visualization and intracellular localization of the α-Syn–EGFP (upper panels), dsRed-SY^WT (middle panels) or dsRed-SY^R621C (lower panels) fusion proteins expressed separately in the BY4741 wild-type yeast strain. The panels display cells without (left) or with (right) aggregates. The percentages refer to the number of cells with or without inclusions in an exponential culture. Cells expressing native EGFP or dsRed served as controls and showed a dispersed cytoplasmic localization. B: Fluorescence microscopic visualization and intracellular localization of α-Syn–EGFP and dsRed-SY^WT upon combined expression in the BY4741 wild-type yeast strain. The upper panels display cells where both fusion proteins co-localize, the middle panels cells with intracellular inclusions of synphilin-1 and plasma membrane localized α-Syn, and the lower panels cells with peripheral inclusions of α-Syn and a dispersed cytoplasmic distribution of synphilin-1. C: Western blot analysis of strains transformed with an empty plasmid (Ctrl.) or a construct allowing the expression of dsRed-SY^WT or dsRed-SY^R621C. Immunodetection was performed using primary antibodies directed against synphilin-1 or Adh2 as indicated on the left. Molecular weight markers are indicated on the right. D and E: The percentage of cells containing inclusions of wild-type or mutant synphilin-1 (D) or α-Syn (E) in exponential cultures. Data represent the combined results of at least three independent experiments. Error bars represent the variation between different counts. Significance was assayed on the total amount of cells counted using a t-test (*** = p<0.001).</p

Synphilin-1 forms aggresomes in cells approaching stationary phase.

<p>A: Fluorescence microscopy pictures of post-diauxic wild-type and <i>sir2Δ</i> cells with large inclusions formed by dsRed-SY^WT and stained with DAPI to visualize the nucleus. B: Fluorescence microscopy pictures of post-diauxic wild-type and <i>sir2Δ</i> cells expressing either EGFP or EGFP-SY^WT, as indicated, and stained with DHE to visualize ROS producing cells (left panels) or with PI to discriminate death cells (right panels).</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

SIRT2 interacts with aSyn and deacetylates lysine 6 and 10.

<p><b>(A)</b> Detection of aSyn acetylation on lysine 6 (K6) and lysine 10 (K10) by peptide mass fingerprinting analysis in total mouse brain lysates. Spectrum shows acetylated peptides in red and aSyn peptides in blue. The corresponding peptide sequences are shown (red residues are acetylated and green residues are oxidated). <b>(B)</b> Human HEK293T cells expressing the indicated proteins were lysed and immunoprecipitated (IP) with anti-aSyn (left panels) or anti-SIRT2 polyclonal antibodies (right panel). The whole-cell lysates (WCL) and immunoprecipitation samples were probed for GFP (left panels) or aSyn (right panels). <b>(C)</b> Mouse whole-brain lysates (WBL) were lysed and IP with anti-aSyn polyclonal antibody. The IP sample was probed with anti-SIRT2 and anti-aSyn. Rabbit IgG was used as a negative control for the IP sample. <b>(D)</b> Immunoprecipitations were probed with an anti–acetyl-lysine antibody in cells expressing either SIRT2 or the SIRT2-H187Y inactive mutant. <b>(E)</b> Immunoblot of thermoenriched aSyn from mouse-brain lysate probed for acetyl-lysine and aSyn. <b>(F)</b> Brain protein extracts from WT and SIRT2 knockout (T2.KO) mice were separated by SDS-PAGE and immunoblotted with antibodies against acetyl-lysine and aSyn (<i>n</i> = 3). The ratio of acetyl-lysine to aSyn is presented. *<i>p</i> < 0.05, unpaired <i>t</i> test with equal standard deviation (SD). <b>(G)</b> Overlay of the deconvoluted intact protein mass spectra obtained from chemically acetylated aSyn (theoretical mass 14,460 Da) in buffer (green) and treated with SIRT2 (purple). The observed masses of the different species correlate with the presence of multiple acetyl modifications (+42 Da), ranging from 2 to 8 before treatment with SIRT2 and from 0 to 6 after deacetylation with SIRT2. <b>(H)</b> Mass spectrometry fragmentation analysis of a peptide from aSyn carrying acetylations at K6 and K10. Red peaks correspond to y-ion series, green peaks to b-ion series, and purple peaks to a-ion series. Corresponding amino acids to mass intervals of y-ion and b-ion series are represented (red and green, respectively). <b>(I)</b> Semisynthetic aSyn acetylated at K6 and K10 were incubated with increasing amounts of recombinant SIRT2 in the presence or absence of NAD at 37°C for 3 h. Proteins were probed for acetyl-lysine residues, aSyn, and SIRT2. All images are representative out of three independent experiments. 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

aSyn acetylation-resistant mutant induces nigral dopaminergic neuronal loss in vivo.

<p><b>(A)</b> AAV6-mediated delivery of EGFP and mutant aSyn (KQ or KR) into the SN of the rat brain. TH and GFP or aSyn expression was examined in brain sections 3 wk after injection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. <b>(B)</b> Stereological counting of the number of TH-positive neurons in the SN. The contralateral SN of the different groups of animals was used as a control (intact). Data in panels are average ± SD. <b>(C)</b> Brain sections stained for aSyn (green), pS129 aSyn (red), and DAPI (Blue). Representative sections are shown. Dashed square boxes delineate the magnification presented on the right. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm and 50 μm. *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001, one-way ANOVA with Bonferroni correction used for statistical calculations. In (B), GFP was used as a control; <i>n</i> = 6–7 animals per condition; five sections from a one-in-six series were analyzed per brain. 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

aSyn acetylation mimic is neuroprotective in primary cortical neurons.

<p><b>(A)</b> Cultured primary neurons were transduced with AAVs encoding for EGFP, WT aSyn, KQ, or KR-mutant aSyn at day in vitro 3 (DIV3). Whole-cell lysates were analyzed by immunoblotting 7 d postinfection with antibodies against aSyn and β-actin (<i>n</i> = 3). <b>(B)</b> Primary neuronal cells were coinfected with aSyn and EGFP at DIV3 and monitored over time. 16 images per condition were acquired, and the EGFP fluorescence signal was recorded in living neurons at 7, 10, 15, 18, and 21 d posttransduction (<i>n</i> = 3). Representative images are shown. Scale bar 20 μm. <b>(C)</b> Total number of EGFP positive cells normalized to the number of neurons on WT 7 d posttransduction is presented (<i>n</i> = 6). Data in all panels are average ± SD, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, two-way ANOVA with Bonferroni post-test. <b>(D)</b> Cortical neuronal cells 15 d posttransduction were processed for ICC (aSyn, red; microtubule-associated protein 2 [MAP2], green; nuclei, Hoechst, blue). Representative images are presented. Scale bar 200 μm. 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