Étude de la toxicité du peptide amyloïde beta Aß42 dans la levure Saccharomyces cerevisiae

La maladie d Alzheimer est la maladie neurodégénérative la plus fréquente chez l Homme et constitue un enjeu économique et de santé publique majeur. Les cerveaux de patients atteints présentent une atrophie corticale associée à deux types de lésions : les dégénérescences neurofibrillaires, constituées de protéines Tau agrégées, et les plaques amyloïdes, composées majoritairement de peptides amyloïde beta Aß agrégés. Les peptides Aß et leur agrégation seraient à l origine de la pathogenèse. Afin d éclaircir les mécanismes moléculaires à la base de la toxicité d Aß, nous avons construit un modèle de toxicité d Aß dans la levure Saccharomyces cerevisiae. Ce modèle a permis d établir que la toxicité d Aß dans la levure est intimement liée à sa sécrétion et au trafic vésiculaire. Ce modèle nous a également permis de réaliser une étude structure-toxicité du peptide et de mettre en évidence des éléments en cis importants pour la toxicité d Aß. Une nouvelle voie d agrégation des peptides toxiques en structure riche en feuillet ß anti-parallèle a pu ainsi être mise en évidence. Le modèle de toxicité d Aß et l existence de variants très toxiques d Aß dans la levure nous a permis de réaliser des cribles génétiques afin de rechercher les éléments modulant la toxicité d Aß in vivo. Le trafic vésiculaire, en particulier l endocytose via le remodelage du cytosquelette d actine, un complexe responsable de la formation de vésicules intraluminales appelé ESCRT, forment autant de pistes à étudier pour améliorer notre compréhension de la toxicité d Aß.Alzheimer s disease is the most common neurodegenerative disease. This pathology is caused by aggregation of Aß peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer s disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. In order to clarify the molecular mechanisms underlying the toxicity of Aß, we generated a new model to study Aß toxicity in yeast Saccharomyces cerevisiae. In our model, Aß toxicity is closely related to its secretion and its intracellular traffic. Indeed, when Aß is targeted to the secretory pathway, it is able to produce toxic species. Interestingly, we demonstrated also that even if Aß is addressed to the secretory pathway, it is still able to form cytoplasmic aggregates. Moreover, with this model, we generated new highly toxic mutants of Aß by random mutagenesis. In order to correlate structural conformation signature to Aß toxicity, we performed a structure-toxicity study of these new variants. In vitro, we demonstrated that a new anti-parallel aggregation pathway is associated with highly toxic mutants of Aß. Then, using our Aß yeast model and also these harmful variants, we performed genetic screens in order to identify candidate genes able to modulate Aß toxicity in vivo. Given these different screens, we found that vesicular trafficking, endocytosis via actin cytoskeleton remodeling, and ESCRT-III (Endosomal Sorting Complex Required for Tansport) open new avenues to improve our understanding of Aß toxicity.BORDEAUX2-Bib. électronique (335229905) / SudocBORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Traffic

Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organization and secretion. Here, we use a genetic approach to characterize the effect on late steps of the secretory pathway. The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid-containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with Cdc42p, Rho1p and Rho3p, because of disruption of the GTP/GDP ratio of at least Rho1p and Rho3p GTPases, thereby preventing activation of the exocyst

The impact of ESCRT on Aβ1-42 induced membrane lesions in a yeast model for Alzheimer’s disease

Aβ metabolism plays a pivotal role in Alzheimer’s disease. Here, we used a yeast model to monitor Aβ42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aβ42 toxic potential. Consistent with previously reported data, our data suggests that Aβ42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aβ42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aβ42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aβ42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aβ42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aβ42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders

Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species

Guidelines and Recommendations on Yeast Cell Death Nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research

Guidelines and recommendations on yeast cell death nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research

Etude de cytochromes P-450 exprimes dans la levure S. cerevisae

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78998 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Approches génétiques et biochimiques visant à caractériser le prion de levure [URE3]

Le phénotype prion [URE3]chez la levure S. cerevisiae est associé à la protéine Ure2p. Ure2p est agrégée dans les cellules [URE3] et forme des fibres amyloides in vitro. Il a donc été proposé qu'[URE3] soit le résultat d'un processus amyloide in vivo. Au cours de mon travail de thèse, j'ai en premier lieu étudié la stabilité du prion [URE3]. J'ai montré l'existence de deux voies d'élimination d'[URE3] : la première est l'inhibition de la réplication du prion (par certains composés chimiques) et la deuxième est la formation d'agrégats de haut poids moléculaire (avec certains allèles d'URE2). J'ai ensuite testé si les agrégats [URE3] étaient d'une nature comparable aux amyloides produites in vitro. La comparaison des propriétés de solubilisation et de protéolyse ménagée de ces deux types d'agrégats nous a permis de mettre en évidence qu'ils étaient différents. La nature biochimique des agrégats [URE3] reste énigmatique.[URE3] in yeast is the prion phenotype associated to Ure2p. Ure2p is aggregated in [URE3] cells and this protein form amyloids in vitro. Therefore, it has been proposed that [URE3] could be the result of an amyloid process. During my PhD, I first investigated the stability of [URE3]. I have demonstrated the existence of two ways of curing : the first isthe inhibition of prion replication (ie with guanidine-HC1) and the second is the large aggregation of Ure2p (ie with Ure2-gfp overproduction). These results emphasize the importance of intermediate species in the aggregation process for prion propagation. I then tested the possibility for [URE3] in vivo aggregates to be the result of an amyloid process. Comparison of solubilization properties and limited proteolysis patterns of these two types of aggregates demonstrated that they are different. The biochemical nature of in vivo aggregates is still enigmatic.BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

ETUDE DU PRION URE3 DE LA LEVURE SACCHAROMYCES CEREVISIAE

Centre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Developpement d'un modèle d'étude de la toxicité du peptide amyloïde Ab chez la levure saccharomyces cerevisiae

La Maladie d Alzheimer (MA) est un des challenges sanitaires les plus importants du XXIème siècle. En effet, elle touche 35,6 millions de personnes en 2010, et en atteindra probablement quatre fois plus en 2050.Cependant, peu d éléments sont à ce jour connus concernant les mécanismes de toxicité de cette maladie. Il semble néanmoins acquis que l agrégation du peptide amyloïde Ab est l élément déclenchant une cascade d événements cellulaires aboutissant à trois types de lésions : les dégénérescences neurofibrillaires, les plaques amyloïdes et une atrophie corticale, révélatrice d une importante mort neuronale.Différents modèles transgéniques d étude de la toxicité du peptide Ab ont été créés au cours des vingt dernières années. Cependant, aucun modèle de levure n a encore vu le jour, alors que cet organisme est utilisé depuis de nombreuses années pour l étude des protéines amyloïdes.J ai donc cherché à créer ce modèle de toxicité au cours de ma thèse. L adressage d une protéine de fusion Ab-GFP à la voie de sécrétion permet de rendre son expression toxique chez la levure. J ai démontré que le trafic intracellulaire était un élément important pour la génération d espèces toxiques. Les orthologues de PICALM, facteur de prédisposition à la MA, sont impliqués dans la toxicité, montrant une conservation des mécanismes de toxicité avec l Homme. Les constructions semblent avoir la capacité de traverser les membranes afin d atteindre des cibles cellulaires comme la mitochondrie.Le modèle ainsi construit nous permettra de mettre en place une étude de la relation entre la structure et la toxicité du peptide Ab et mieux comprendre les mécanismes cellulaires régissant la MA.Alzheimer s Disease (AD) is one of the most important sanitary challenges of the XXIst century. Indeed, 35.6 million people are affected in 2010, and there will be probably four times more in 2050. However, little is known about the mechanisms of toxicity of this disease. Nevertheless, it seems that aggregation of the Ab peptide is the triggering factor of a cascade of cellular events that leads to three characteristic lesions: neurofibrillary tangles, amyloid plaques and a cortical atrophy, revealing an important neuronal death. Different transgenic models for the study of the Ab peptide toxicity have been created during the past twenty years. However, no yeast model has yet seen the light of day, whereas this organism is used for several years to study amyloid proteins.Therefore I worked to create this model during my thesis. Addressing an Ab-GFP fusion to the secretory pathway enable this construction to become toxic in yeast. I proved intracellular pathways are important for generation of toxic species. PICALM orthologs, an AD predisposing factor, are involved in toxicity, showing conservation in the mechanisms of toxicity between yeast and man. The constructions seem to be able to cross membranes and reach cytoplasmic targets as mitochondria.Thus, this model will allow us to set a study of the relationship between structure and toxicity of the Ab peptide and better understand the cellular mechanisms governing ADBORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF