Yeast as a Model System to Study Tau Biology

Hyperphosphorylated and aggregated human protein tau constitutes a hallmark of a multitude of neurodegenerative diseases called tauopathies, exemplified by Alzheimer's disease. In spite of an enormous amount of research performed on tau biology, several crucial questions concerning the mechanisms of tau toxicity remain unanswered. In this paper we will highlight some of the processes involved in tau biology and pathology, focusing on tau phosphorylation and the interplay with oxidative stress. In addition, we will introduce the development of a human tau-expressing yeast model, and discuss some crucial results obtained in this model, highlighting its potential in the elucidation of cellular processes leading to tau toxicity

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The evolutionary conserved TOR complex 1 controls growth in response to the quality and quantity of nutrients such as carbon and amino acids. The protein kinase Sch9 is the main TORC1 effector in yeast. However, only few of its direct targets are known. In this study, we performed a genome-wide screening looking for mutants which require Sch9 function for their survival and growth. In this way, we identified multiple components of the highly conserved vacuolar proton pump (V-ATPase) which mediates the luminal acidification of multiple biosynthetic and endocytic organelles. Besides a genetic interaction, we found Sch9 also physically interacts with the V- ATPase to regulate its assembly state in response to glucose availability and TORC1 activity. Moreover, the interaction with the V-ATPase has consequences for ageing as it allowed Sch9 to control vacuolar pH and thereby trigger either lifespan extension or lifespan shortening. Hence, our results provide insights into the signaling mechanism coupling glucose availability, TORC1 signaling, pH homeostasis and longevity. As both Sch9 and the V-ATPase are highly conserved and implicated in various pathologies, these results offer fertile ground for further research in higher eukaryotes

Recent insights on Alzheimer’s disease originating from yeast models

In this review article, yeast model-based research advances regarding the role of Amyloid-? (A?), Tau and frameshift Ubiquitin UBB+1 in Alzheimer’s disease (AD) are discussed. Despite having limitations with regard to intercellular and cognitive AD aspects, these models have clearly shown their added value as a complementary model for the study of the molecular aspects of these proteins, including their interplay with AD related cellular processes such as mitochondrial dysfunction and altered proteostasis. Moreover, these yeast models have also shown their importance in translational research, e.g. in compound screenings and for AD diagnostics development. In addition to well-established Saccharomyces cerevisiae models, new upcoming Schizosaccharomyces pombe, Candida glabrata and Kluyveromyces lactis yeast models for A? and Tau are briefly described. Finally, traditional and more innovative research methodologies, e.g. for studying protein oligomerization/aggregation, are highlighted

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

Study of two types of immune responses in insects: nodulation in the flesh fly, Neobellieria bullata, and prophenoloxidase activation in the desert locust, Schistocerca gregaria.

In deze thesis werden twee aspecten van de aangeboren immuniteit bij insecten bestudeerd, nl. nodulatie en activering van profenoloxidase. Aangezien Diptera in voorgaande studies omtrent nodulatie over het hoofd werden gezien, ontwikkelden we een in vivo assay om de vorming van nodules te bestuderen bij een vertegenwoordiger van deze belangrijke insectenorde. Het injecteren van laminarine in het derde larvestadium van N. bullata induceerde op een dosis-afhankelijke wijze nodulatie rondom de krop. De laminarine-geïnduceerde respons kon geblokkeerd worden door behandeling van de larven met dexamethasone en naproxen, twee inhibitoren van de eicosanoïd-biosynthese. Deze resultaten tonen aan dat de PLA2- en cyclooxygenase-activiteiten, noodzakelijk voor prostaglandine-biosynthese, een belangrijke rol spelen in de regulatie van de nodulatiereactie. Naast eicosanoïden bestudeerden we eveneens de invloed van de hormonen 20-hydroxyecdyson (20E) en juveniel hormoon (JH) op de laminarine-geïnduceerde nodulatiereactie. Behandeling van de larven met 20E resulteerde in een dosis-afhankelijke stijging van het aantal gevormde nodules in respons op laminarine. Tegengesteld aan dit effect, stelden we bovendien vast dat een behandeling van de larven met JH leidde tot een reductie van het aantal gevormde nodules in respons op laminarine. Deze resultaten suggereren alvast dat componenten van het endocrien systeem betrokken zijn bij de ontwikkeling en regulatie van de laminarine-geïnduceerde nodulatiereactie bij deze tweevleugelige (Diptera). Alhoewel meerdere studies aantoonden dat eicosanoïden betrokken zijn bij de regulatie van nodulatiereacties, is het nog onduidelijk volgens welke mechanismen deze regulatoren hun biologische werking uitoefenen bij arthropoda. Daarom onderzochten we de invloed van diverse prostaglandines op de concentraties van secundaire boodschappers in hemocyt-afgeleide cellijnen van Drosophila. Bovendien identificeerden we een vermoedelijke prostaglandine receptor in het Drosophila genoom. Het cDNA van deze receptor werd gecloneerd, tot expressie gebracht en bestudeerd. Vermits geen van de geteste eicosanoïden deze receptor echter bleek te activeren bij fysiologische concentraties, blijft de moleculaire identiteit van de natuurlijke agonist ervan nog steeds onbekend. In een tweede deel van deze thesis bestudeerden we het profenoloxidase activerend systeem van de woestijnsprinkhaan Schistocerca gregaria. Met behulp van een in vitro assay toonden we aan dat zowel laminarine als LPS de PO-activiteit bij S. gregaria induceerde. Daarnaast onderzochten we de invloed van de S. gregaria serine protease inhibitoren SGPI-1 en SGPI-2 op de laminarine-geïnduceerde PO-activiteit. Onder de geteste experimentele condities hadden deze inhibitoren echter geen invloed op de laminarine-geïnduceerde PO-activiteit. Niettemin werd met behulp van real-time RT-PCR aangetoond dat de mRNA-gehaltes voor de precursoren SGPP-1 en SGPP-2 opgereguleerd waren in het vetlichaam van sprinkhanen, 20 u na laminarine injectie. Deze resultaten suggereren dat SGPI-1, SGPI-2 en SGPI-3 mogelijkerwijze toch een rol vervullen in de immuunrespons bij de woestijnsprinkhaan.status: publishe

A Mitochondria-Associated Oxidative Stress Perspective on Huntington's Disease

Huntington's disease (HD) is genetically caused by mutation of the Huntingtin (HTT) gene. At present, the mechanisms underlying the defect of HTT and the development of HD remain largely unclear. However, increasing evidence shows the presence of enhanced oxidative stress in HD patients. In this review article, we focus on the role of oxidative stress in the pathogenesis of HD and discuss mediators and potential mechanisms involved in mutant HTT-mediated oxidative stress generation and progression. Furthermore, we emphasize the role of the unicellular organism Saccharomyces cerevisiae in investigating mutant HTT-induced oxidative stress. Overall, this review article provides an overview of the latest findings regarding oxidative stress in HD and potential therapeutic targets for HD.status: publishe

Yeasts as Complementary Model Systems for the Study of the Pathological Repercussions of Enhanced Synphilin-1 Glycation and Oxidation

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases

Development and validation of a humanized yeast model for Abeta42-induced cytotoxicity

Alzheimer’s Disease (AD) is the most common neurodegenerative disorder. Today, more than 25 million people are suffering from AD, a number that has been estimated to double every 20 years. Brains from AD patients have two major hallmarks. They contain intracellular deposits of hyperphosphorylated protein tau, known as neurofibrillary tangles, as well as extracellular deposits of Aβ peptides, known as amyloid plaques. The latter are derived from cleavage of the amyloid precursor protein by the β- and gamma-secretases. Despite enormous efforts, the exact molecular mechanisms underlying the pathology and neuronal loss in AD brain are still not known. According to a current hypothesis, the accumulation of intracellular oligomeric complexes of Aβ-peptides induces cytotoxicity and cellular stress, which in turn activates stress-sensitive kinases that hyper-phosphorylate and induce aggregation of protein tau. In our project, we aim to develop a humanized yeast model to investigate Aβ-induced toxicity and its causal effect on the behavior of protein tau in the unicellular eukaryote Saccharomyces cerevisiae or baker’s yeast, a strategy already successfully applied to decipher the toxicity mechanisms for other proteins triggering neurodegeneration in e.g. Parkinson’s and Huntington’s disease. Preliminary data shows that the proteasome plays an important role in clearing Aβ42, as severe growth defects are observed in proteasomally-impaired strains.status: publishe