Synphilin-1 Enhances α-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner

© 2010 Büttner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Parkinson’s disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive. Methodology/Principal Findings: We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation. Conclusions/Significance: Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.This work was supported by grants from IWT-Vlaanderen (SBO NEURO-TARGET), the K.U.Leuven Research Fund (K.U.Leuven BOF-IOF) and K.U.Leuven R&D to JW, a Tournesol grant from Egide (Partenariat Hubert Curien) in France in collaboration with the Flemish Ministry of Education and the Fund of Scientific Research of Flanders (FWO) in Belgium to JW, MCG and LB, a shared PhD fellowship of the EU-Marie Curie PhD Graduate School NEURAD to JW, MCG and LB, grants of the Austrian Science Fund FWF (Austria) to FM and DR (S-9304-B05), to FM and SB (LIPOTOX), and to SB (T-414-B09; Hertha-Firnberg Fellowship) and an EMBO Installation Grant, a Marie Curie IRG, and a grant of the Fundação para a Ciência e Tecnologia (PTDC/SAU-NEU/105215/2008) to TFO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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

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

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

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