An <i>oxa1</i> deletion does not influence the strains growth rate.

<p>(A) Growth curves of an <i>oxa1</i> deletion strain and the corresponding wild-type strain. Experiment was performed in triplicate. The y-axis is scaled logarithmically. (B) Size of isolated colonies of the indicated strains grown for 3 and 5 days on SCGlu plates, respectively. Diameters were evaluated by processing the photos with Metamorph Imaging (mean±SEM, <i>n</i> = 2).</p

Abrogation of respiration suppresses apoptosis and ROS production in a maximal proliferating solid cell population.

<p>(A) Isolated colonies were grown from single cells, manipulated onto agar plates. Clonogenic assay of cells removed from colonies of the wild-type strain, rho⁰ (no mitochondrial DNA) and two single gene-deletion strains (<i>mgm1</i> and <i>oxa1</i>), grown on SCGlu. After 3 days 500 cells of whole colonies, after 5 days 500 cells of the central colony-region were analysed (mean±SEM, <i>n</i> = 2). Statistical significance of p<0.006 compares colony forming units (cfu) of mutant strains to wild-type strain at respective time-points. (B) Cells from the whole colony (3 days) as well as from the central region (5 days) were stained for reactive oxygen species (ROS) with dihydroethidium and analysed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 2; **p<0.01, ***p<0.001 compared to deletion strains at respective time-points). (C) Approximately 50 (2 days) and 10 (3 days) colonies grown on SCGlu plates were washed off and clonogenic assays were performed with the collected cells (mean±SEM, <i>n</i> = 5; **p<0.01, ***p<0.001). (D) Approximately 50 colonies grown on SCGlu plates for 2 days were stained for phosphatidylserine exposition (AnnV) and DNA breakage (TUNEL) and analyzed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 3; **p<0.01, ***p<0.001). (E) Fluorescence microscopy from central and outer region of 5 days old wild-type colony cells, harbouring plasmid dsRED-MLS.</p

Data_Sheet_1_The Enzymatic Core of the Parkinson’s Disease-Associated Protein LRRK2 Impairs Mitochondrial Biogenesis in Aging Yeast.pdf

<p>Mitochondrial dysfunction is a prominent trait of cellular decline during aging and intimately linked to neuronal degeneration during Parkinson’s disease (PD). Various proteins associated with PD have been shown to differentially impact mitochondrial dynamics, quality control and function, including the leucine-rich repeat kinase 2 (LRRK2). Here, we demonstrate that high levels of the enzymatic core of human LRRK2, harboring GTPase as well as kinase activity, decreases mitochondrial mass via an impairment of mitochondrial biogenesis in aging yeast. We link mitochondrial depletion to a global downregulation of mitochondria-related gene transcripts and show that this catalytic core of LRRK2 localizes to mitochondria and selectively compromises respiratory chain complex IV formation. With progressing cellular age, this culminates in dissipation of mitochondrial transmembrane potential, decreased respiratory capacity, ATP depletion and generation of reactive oxygen species. Ultimately, the collapse of the mitochondrial network results in cell death. A point mutation in LRRK2 that increases the intrinsic GTPase activity diminishes mitochondrial impairment and consequently provides cytoprotection. In sum, we report that a downregulation of mitochondrial biogenesis rather than excessive degradation of mitochondria underlies the reduction of mitochondrial abundance induced by the enzymatic core of LRRK2 in aging yeast cells. Thus, our data provide a novel perspective for deciphering the causative mechanisms of LRRK2-associated PD pathology.</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

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

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

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

MetR specifically regulates induction of autophagy.

<p>MET⁺, Δ<i>met15</i> and Δ<i>met2</i> strains from chronological aging experiments were analyzed for vacuolar ALP activity (with a fluorescent plate reader) (A) (n = 6), and GFP-Atg8p processing (by Western-blot analysis) (B). (C) GFP-Atg8p localization was determined by using fluorescent microscopy (white arrows indicate vacuolar localization or autophagosome formation) and statistical analysis thereof (330–600 cells of each GFP-Atg8p expressing strain were evaluated from two independent samples) (D). (E) <i>MET2</i> deletion strain (Δ<i>met2</i>) was grown to stationary phase in SCD (supplemented with all aa) and shifted to SCD media with given methionine concentrations. Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) (n = 6). (F) ALP assays of chronological aging of MET⁺ strain, in SCD media supplemented with all aa except for methionine which was added at given concentrations (n = 2). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s003" target="_blank">Figure S3</a>.</p