Cross-talk between α-synuclein post-translational modifications in yeast as model of Parkinson’s disease

Posttranslationale Modifikationen modulieren verschiedene Charakteristika von Proteinen. Sie können die Aktivität, Lokalisierung und Stabilität ihrer Substrate regulieren, verändern aber auch Eigenschaften und Strukturvon Proteinen, die mit Krankheiten assoziiert sind. Ein wichtiges Kennzeichen der Parkinson-Krankheit ist die Akkumulation von Proteinaggregaten (Lewy Körperchen). Dies führt zu neuronalem Zelltod durch verschiedene, bisher oft unbekannte Mechanismen. α-Synuclein, ein präsynaptisches, neuronales Protein, ist der Hauptbestandteil der Lewy-Körperchen und spielt eine wichtige Rolle in der Pathogenese der Parkinson-Krankheit. Es unterliegt verschiedenen posttranslationalen Modifikationen unter pathologischen Bedingungen. Die Zytotoxizität und Aggregation von α-Synuclein kann in Hefe imitiert werden. In dieser Studie werden zwei wichtigen posttranslationalen Modifikationen von α-Synuclein, Sumoylierung und Phosphorylierung von Serin 129 (S129), untersucht. Heterolog exprimertes Wildtyp-α-Synuclein und die A30P Mutante sind in Hefe an den gleichen Resten, Lysin 96 (K96) und Lysin 102 (K102), sumoyliert wie im Menschen. Eine Absenkung des zellulären Pools des Ubiquitin-ähnlichen Proteins SUMO führte zu einer starken Wachstumsreduktion von Zellen, welche α-Synuclein exprimieren. Dies korrelierte mit einer erhöhten Zahl an Zellen, die Einschlüsse bildeten. Dies legt nahe, dass Sumoylierung die Hefen vor α-Synuclein vermittelter Toxizität und Einschlussbildung schützt. Die Expression von sumoylierungsdefizienten α-Synuclein verursachte die gleiche Wachstumsrate, was die protektive Rolle der α-Synuclein Sumoylierung in cis bestätigt. Eine Überexpression der humanen Kinasen GRK5 und PLK2 erhöhten den Anteil an S129 phosphoryierten α-Synuclein. Interessanterweise wurde die α-Synuclein–vermittelte Zytotoxizität in Zusammenhang mit einer beeinträchtigten Sumoylierung durch eine höhere Kinase-abhängige S129 α-Synuclein Phosphorylierungsrate kompensiert. Phosphorylierung reduzierte die Einschlussbildung und verminderte die Wachstumshemmung. Um mehr Einblicke in eine plausible wechselseitige Beeinflussung zwischen α-Synuclein Sumoylierung und S129 Phosphorylierung zu erhalten, wurde die Beseitigung der α-Synuclein Aggregate beobachtet. Promotor „shut-off“ Studien wurden parallel mit chemischer Inhibition der zellulären Abbauwege durchgeführt. In der Abwesenheit von SUMO wurden α-Synuclein-Aggregate hauptsächlich durch das Ubiquitin-Proteasom-System abgebaut. Dies legt nahe, dass Sumoylierung den Abbau der α-Synuclein-Aggregate durch Autophagie unterstützt. In Anwesenheit der humanen Kinasen GRK5 oder PLK2, wurden die sumoylierungsdefizienten α-Synuclein-Aggregate Kinasen abhängig sowohl dem Ubiquitin-Proteasom als auch dem Autophagie-System zugeführt. Dies ging einher mit einem veränderten Ubiquitinierungs-Profil von α-Synuclein. GRK5 war in der Lage den Abbau von sumoylierungsdefizienten α-Synuclein-Aggregaten durch Autophagie partiell zu retten und außerdem das Proteasom-System zu unterstützen. In Abwesenheit von SUMO, wenn PLK2 überexprimiert wird, trugen beide Abbauwege gleich stark zur Beseitigung der α-Synuclein-Aggregate bei. Diese wechselseitige Beeinflussung zwischen α-Synuclein Phosphorylierung und Sumoylierung könnte neue Wege für eine therapeutische Intervention in der Parkinsonkrankheit und anderen Synucleinopathien eröffnen

Systematic comparison of the effects of Alpha-synuclein mutations on its oligomerization and aggregation

Copyright: © 2014 Lázaro 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.Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.This work was supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).info:eu-repo/semantics/publishedVersio

Correlation between the effects of ASYN mutations on oligomerization and inclusion formation.

<p>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).</p

ASYN biochemical state.

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

ASYN mutation effects in the inclusion formation.

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

ASYN bPCA.

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

A-B morphology analysis of Golgi apparatus.

<p>The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (<b>A</b>). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus <b>(B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E).</b> n = 3. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001).</p

Mutations effect on ASYN oligomerization.

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

Experimental design.

<p>WT and ASYN mutants were subjected to a variety of assays as depicted in the schematic.</p

ASYN secretion is inversely correlated with toxicity.

<p><b>A. Secretion of ASYN. B. Toxicity measurements.</b> 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. <b>C. Correlation between Secretion and Toxicity.</b> The graph shows the inverse trend in secretion and toxicity.</p