Expression of the cellular prion protein (PrPC) from the PRNP gene is crucial for the development of a group of fatal neurodegenerative disorders called prion diseases. During prion infection a misfolded protein homologue of PrPC, PrPSc causes further misfolding on interaction with native PrPC molecules. PrPSc is highly resistant to proteinase K and aggregation of this protein is considered a hallmark of infection. Sheep are considered a model of natural infection and susceptibility to scrapie in sheep is defined by polymorphisms in the PRNP gene. It is still not fully understood how these polymorphisms regulate the conversion process or which other co-factors are involved. One such factor may be the truncation of PrPC via proteolytic processing in the form of two main cleavage events, known as α- and β-cleavage. In sheep α-cleavage cuts at amino acid 115, creating two truncated proteins C1 and N1 and represents the main cleavage event in healthy brain. β-Cleavage creates a longer C-terminal fragment, C2 and corresponding N-terminal fragment N2, cutting around amino acid 92 in sheep. Truncated forms of PrPC have been shown to represent around 50 % of total residual PrP in brain and may be an important determinant of disease through both decreasing the amount of full length PrPC available for conversion and through functions associated with the truncated fragments. The research presented has shown that increased production of an α-cleavage fragment C1 in brain is associated with TSE resistant genotype ARR/ARR, while the presence of C2 fragment is affiliated with scrapie susceptible PRNP genotypes in brain. There was no difference in the levels of full length PrPC in these genotypes suggesting that PrP expression does not directly correlate to susceptibility in this model. To assess if PrPC fragments could affect the conversion during disease in-vitro fibrillisation assays were performed using novel truncated recombinant proteins. These truncated proteins, although not thought to convert to PK resistant PrPSc during disease, can form amyloid fibrils. However, these fibrils appear to be less neurotoxic when compared to fibrils produced by full length PrPC. Only the truncated fragments derived from the ARR allele inhibit in-vitro fibrillisation of other allelic PrPC variants. Furthermore, treatment of infected cells in culture with recombinant C1ARR led to a decrease in the formation of disease associated PrPSc. In conclusion, genetic variations in levels of PrP truncated fragments may add to the complexity of genetic determinants of prion disease. In parallel with polymorphism-dependant conversion abilities, varying α-cleavage of ovine PrPC may help to explain genetic resistance in sheep. The inhibitory effects of C1, illustrated in-vitro may represent a therapeutic avenue in the treatment of prion disease.

The aim of my thesis was to develop and characterise PrP transgenic Drosophila melanogaster of various genotypes to study the process of prion-induced neurodegeneration in this model. Prion diseases are caused by the occurrence of an abnormally-folded form of PrP (PrPSc) protein that arises either from the environment as an acquired disease, from mutation in the PrP-coding gene as a genetic disease or sporadically from causes unknown. The PrPSc then recruits PrPC, the normal form of PrP, that is ubiquitously present in the mammalian CNS and triggers neurotoxicity and neurodegeneration that is transmissible between individuals of the same or even different species. All prion diseases are currently incurable, fatal and the mechanism of prion-induced neurodegeneration remains to be discovered. In this thesis, Drosophila transgenic for ovine (chromosome 3 and dual PrP transgenic flies), hamster, humanised murine, human and cervid PrP were characterised for expression and biochemical properties. The ultimate goal of my thesis was investigation of cell-to-cell spread of misfolded PrP in Drosophila CNS. To achieve this, a mutant form of PrP that is thought to misfold was co-expressed with the normal form PrPC that served as a substrate in the same dual PrP-transgenic fly. The process was modelled using hamster, humanised murine or ovine PrP transgenes that carry human mutations associated with the spontaneous onset of transmissible neurodegeneration in the natural host. Various approaches towards independent spatial expression of PrP in Drosophila were exploited here in both single and dual PrP expressing flies. Moreover, the ability to initiate misfolding and the impact of this on the fly phenotype was investigated. Both apparent misfolding and phenotypic changes were seen in different fly models suggesting the models were successful. To this extent, PrP transgenic Drosophila were developed to allow for relatively rapid modelling of mammalian prion disease in this invertebrate organism.

L’objectif de cette thèse est d’étudier, sous divers aspects, le processus de formation d’amyloïde à partir de la polymérisation de protéines. Ces phénomènes, aussi bien in vitro que in vivo, posent des questions de modélisation mathématique. Il s’agit ensuite de conduire une analyse des modèles obtenus. Dans la première partie nous présentons des travaux effectués en collaboration avec une équipe de biologistes. Deux modèles sont introduits, basés sur la théorie en vigueur du phénomène Prions, que nous ajustons aux conditions expérimentales. Ces modèles nous permettent d’analyser les données obtenues à partir d’expériences conduites en laboratoire. Cependant celles-ci soulèvent certains phénomènes encore inexpliqués par la théorie actuelle. Nous proposons donc un autre modèle qui corrobore les données et donne une nouvelle approche de la formation d’amyloïde dans le cas du Prion. Nous terminons cette partie par l’analyse mathématique de ce système compose d’une infinité d’équations différentielles. Ce dernier consiste en un couplage entre un système de type Becker-Doring et un système de polymérisation-fragmentation discrète. La seconde partie s’attache à l’analyse d’un nouveau modèle pour la polymérisation de protéines dont la fragmentation est sujette aux variations du fluide environnant. L’idée est de décrire au plus près les conditions expérimentales mais aussi d’introduire de nouvelles quantités macroscopiques mesurables pour l’étude de la polymérisation. Le premier chapitre de cette partie présente une description stochastique du problème. On y établit les équations du mouvement des polymères et des monomères (de type Langevin) ainsi que le formalisme pour l’étude du problème limite en grand nombre. Le deuxième chapitre pose le cadre fonctionnel et l’existence de solutions pour l équation de Fokker-Planck- Smoluchowski décrivant la densité de configuration des polymères, elle-même couplée a une équation de diffusion pour les monomères. Le dernier chapitre propose une méthode numérique pour traiter ce problème. On s’intéresse dans la dernière partie à la modélisation de la maladie d’Alzheimer. On construit un modèle qui décrit d’une part la formation de plaque amyloïde in vivo, et d’autre part les interactions entre les oligomères d’Aβet la protéine prion qui induiraient la perte de mémoire. On mène l’analyse mathématique de ce modèle dans un cas particulier puis dans un cas plus général ou le taux de polymérisation est une loi de puissance / The aim of this thesis is to study, under several aspects, the formation of amyloids from proteins polymerization. The mathematical modelling of these phenomena in the case of in vitro or in vivo polymerisation remains questioned. We then propose here several models, which are also investigated from theoritical and numerical point of view. In the first part we present works done in collaboration with biologists. We propose two models based on the current theory on Prion phenomena that are designed for specific experimental conditions. These models allow us to analyse the experimental data obtained in laboratory and raise phenomena that remain unexplained by the theory. Then, from these results and biophysical considerations, we introduce a model which corroborates with data and provides a new approach on the amyloid formation in the particular case of Prion. This part is ended by the mathematical analysis of the model consisting of an infinite set of differentials equations. The system analysed is a Becker-Doring system coupled to a discrete growth-fragmentation system. The second part is dedicated to the analysis of a new model for polymerization of proteins with fragmentation subject to the surrounding variations of the fluid. Thus, we propose a model which is close to the experimental conditions and introduce new measurable macroscopic quantities to study the polymerization. The first introductory chapter states the stochastic description of the problem. We give the equations of motion for each polymers and monomers as well as a general formalism to study the limit in large number. Next, we give the mathematical framework and prove the existence of solutions to the Fokker-Planck-Smoluchowski equation for the configurational density of polymers coupled to the diffusion equation for monomers. The last chapter provides a numerical method adapted to this problem with numerical simulations In the last part, we are interested in modelling Alzheimer’s disease. We introduce a model that describes the formation of amyloids plaques in the brain and the interactions between Aβ-oligomers and Prion proteins which might be responsible of the memory impairment. We carry out the mathematical analysis of the model. Namely, for a constant polymerization rate, we provide existence and uniqueness together with stability of the equilibrium. Finally we study the existence in a more general and biological relevant case, that is when the polymerization depends on the size of the amyloid

La Protéine Prion cellulaire (PrPc) est une protéine ubiquitaire mais majoritairement présente dans le système nerveux central. Elle est plus particulièrement connue pour sa conversion conformationnelle en PrPSc dans les maladies à Prions qui sont des Protéinopathies comme la maladie d’Alzheimer (MA). La MA est en partie associée à des dépôts de peptides beta-amyloïdes (Aβ) agrégés de façon extracellulaire et issus des clivages successifs par la β- puis la γ-sécrétase de la protéine précurseur amyloïde (APP) exprimée dans les neurones. La PrPc et l’APP partagent des fonctions et des voies protéolytiques communes (α- ou β-sécrétase) les impliquant dans la prolifération, la différenciation, la synaptogenèse et la survie cellulaire. La PrPc est impliquée dans la régulation de la prolifération et la différenciation de différentes cellules souches : neurales adultes (NSC), hématopoïétiques (HSC), embryonnaires humaines (hESC). Si la PrP et l’APP partagent des fonctions communes, plusieurs publications montrent que la PrPc régule négativement le clivage de l'APP en Aβ et positivement le clivage de l’APP en sAPPα suggérant ainsi un rôle anti-amyloïdogénique de la PrPc. La PrP agirait également comme récepteur des Aβ à la surface neuronale induisant notamment l’inhibition des LTP et l’altération synaptique.Dans ce contexte, les objectifs spécifiques de la thèse sont :- L’étude de l’expression de la PrP, de l’APP et ses résidus de clivage au cours de l’induction neurale des hESC en NSC et de la différenciation neuronale- L’impact de la modulation de l’expression de la PrP sur le clivage de l’APP ainsi que sur les propriétés des cellules souches (survie, prolifération, différenciation).1. Induction neurale des hESC en NSC Pour ce projet nous avons utilisé des Cellules Souches Embryonnaires Humaines (hESC) pour lesquelles le laboratoire dispose d’une autorisation de l’Agence de la Biomédecine.Pour l’induction neurale, nous avons testé deux protocoles : l’un permet d’obtenir des neurosphères en suspension puis des «rosettes» constituées de NSC, l’autre protocole en monocouche mime quant à lui la corticogenèse. Une optimisation de ces protocoles a été nécessaire (densité de départ, méthodes de fixation des cellules pour améliorer la détection de la PrP) ainsi que la détermination des conditions d’analyse de l’expression de PrP, d’APP et ses résidus clivés (Aβ, sAPPα/β). 2. Différenciations à partir des NSC Les NSC obtenues ont ensuite été amplifiées puis différenciées en neurones et/ou astrocytes. Les cellules ont été caractérisées notamment par immunofluorescence et RT-qPCR pour l’expression des principaux marqueurs astrocytaires (GFAP) et neuronaux (BIII-tubuline, Doublecortine, Synaptophysine) et la disparition progressive des marqueurs de NSC. Là encore nous avons établi des conditions précises de densité cellulaire ainsi que les points des analyses cinétiques de nos différents paramètres.3. Modulation de l’expression de la PrPc Nous avons utilisés des vecteurs lentiviraux permettant l’expression ou l’inhibition de la PrPc humaine pour transduire des hESC au moment d’initier l’induction neurale et des NSC. Pour cela nous avons également dû réaliser des optimisations de différents paramètres : densité cellulaire, taille des supports d’ensemencement ou MOI de lentivirus afin d’avoir une transduction efficace tout en limitant la cytotoxicité. De même, les échantillons récoltés nous ont permis d’évaluer l’impact de la modulation de la PrPc sur le clivage de l’APP ainsi que sur la biologie des cellules souches (survie, prolifération, différenciation). / The cellular Prion Protein (PrPc) is a ubiquitary protein mainly expressed in the central nervous system. It is particularly known for its conformational conversion in PrPSc in Prion diseases, which are proteinopathies such as Alzheimer’s disease (AD). AD is associated with extracellular deposits of aggregated beta-amyloid peptides (Aβ) derived from successive β- and the γ-secretase cleavages of the amyloid precursor protein (APP) expressed by neurons. PrPc and APP share some common functions and proteolytic pathways (α- or β-secretase), involving them in proliferation, differentiation, synaptogenesis and cellular survival. PrPc is involved in the regulation of proliferation and differentiation of many stem cells: adult neural (NSC), hematopoietic (HSC) and human embryonic (hESC). Several publications also show that PrP downregulates the cleavage of APP in Aβ and positively regulates the cleavage of APP in sAPPα suggesting an anti-amyloïdogenic role of PrPc. PrP could also act as a receptor of Aβ at the neuronal surface inducing LTP inhibition and synaptic alteration. In this context, the specific objectives of my thesis were:- Study of the expression of PrP, APP and its cleavage residues during neural induction of hESC in NSC and neuronal differentiation.- Impact of the modulation of PrP expression on APP cleavages as well as on stem cells properties (survival, proliferation, differentiation). 1. Neural induction of hESC in NSCFor this project, we have used Human Embryonic Stem Cells (hESC) for which the laboratory has an authorization from the “Agence de la Biomédecine”.For the neural induction, we have tested two protocols, the first one allows the obtention of neurospheres in suspension and then figures of “rosettes” composed of NSC, and a “monolayer” protocol that mimics the beginning of corticogenesis. An optimization of these protocols has been necessary (starting cell density, cell fixation methods to improve PrP detection). We have also determined the best conditions to analyze the expression of PrP, APP and its derived peptides (Aß, sAPPα/β). 2. Differentiation of NSCNSC derived from hESC were amplified and differentiated into neurons and/or astrocytes. Cells were characterized in particular by immunofluorescence and RT-qPCR for the expression of the major astrocytic (GFAP) and neuronal markers (BIII-tubulin, doublecortin, synaptophysin) and the progressive decrease of NSC markers. Again we have determined the best conditions for cell density and kinetic time points for our analysis.3. Modulation of PrPC expression We have used lentiviral vectors allowing the expression of an anti-PrP shRNA, human PrP and respective controls. To achieve this task, lentiviral transductions of hESC and NSC were optimized: cell density, size of the seeding culture wells or MOI of lentivirus. Finaly, samples collected allowed us to evaluate the impact of PrPc modulation on the APP cleavages as well as on stem cells properties (survival, proliferation, differentiation).

No description available.

Proteinaceous infectious particles, termed 'prions' are self-perpetuating protein isoforms that transmit neurodegenerative diseases in mammals and phenotypic traits in yeast. Each conformational variant of a prion protein is faithfully propagated to a homologous protein in the same cell environment. However, a reduction in the efficiency of prion transmission between different species is often observed and is termed "species barrier". Prion transmission to a heterologous protein may, in some cases, permanently change the structure of the prion variant, and divergent proteins may interfere with prion propagation in a species-specific manner. To identify the importance of both protein sequence and the cell environment on prion interference and cross-species transmission, we employed heterologous Sup35 proteins from three Saccharomyces sensu stricto species: Saccharomyces cerevisiae (Sc), Saccharomyces paradoxus (Sp), and Saccharomyces bayanus (Sb). We performed our experiments in two different cell environments (Sc and Sp). Our data show that Sup35 from one species can form a prion in another, and we employed a transfection procedure to perform cross-species transfer of the prion. Using a shuffle procedure, we demonstrate that the specificity of prion transmission is determined by the protein itself rather than the cell environment. Interestingly, we noted that variant-specific prion patterns can be altered irreversibly during cross-species transmission through S. bayanus module II. We further show that prion interference does not always correlate with cross-species prion transmission, and the identity of particular regions or even a specific amino acid, rather than the overall level of PrD homology is crucial for determining cross-species transmission and interference. Lastly we provide evidence to suggest that prion interference is specific to the cell environment.

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Chronic neurodegenerative diseases, such as Alzheimer’s disease, prion diseases and many others are unified by the aberrant folding of a host encoded protein to a disease-associated isoform and the predictable cell-to-cell spread of disease-associated misfolded proteins via a putative prion-like mechanism. Prion diseases, for example, are associated with the aberrant folding of host encoded prion protein (PrPC) to a disease-associated isoform, which acts as a seed for the further conversion of PrPC to misfolded protein species. The role of misfolded prion protein in neurodegeneration remains unclear. Accumulation and spread of misfolded prion protein is typically slow and progressive, correlating with neurodegeneration. A number of studies show that mice are susceptible to prion disease with characteristic hallmarks of prion pathology but in the presence of little detectable misfolded prion protein (e.g. the GSS/101LL model). In this thesis I test the hypothesis that detectable species of misfolded prion protein correlate with neurodegeneration and spreads in a predictable, progressive fashion from one anatomically distinct brain region to the next. Using the GSS/101LL model, misfolded prion protein was detected as mostly PK-sensitive isoforms (PrPsen). The progression and pathological presentation is comparable to other prion diseases with larger quantities of PK resistant prion isoforms. A highly sensitive in vitro assay system (the QuIC assay) was subsequently used to establish the extent that misfolded protein was present within the brain. Amyloidogenic prion seeds were found to be widespread throughout the brain from an early stage and spread rapidly throughout the brain. Absence of neurodegeneration in certain brain regions is not due to differing quantities of prion seeds between regions or time exposed to prion seeds, as unaffected regions are exposed to comparative quantities of prion seeds for the same time-period as regions of the brain which eventually succumb to neurodegeneration. These results indicate a clear dissociation between prion seeds and neurotoxicity. They highlight the need to understand regional host responses to prion seeds that may evoke neurodegeneration in some but resilience in others. To test this, transcriptomic analysis was carried out on brain samples from regions undergoing neurodegeneration and unaffected regions. A gene profile signature of hybrid pro-and anti-inflammatory response was observed in regions undergoing neurodegeneration. However, large cohorts of genes were down-regulated across all regions tested, including pro-inflammatory genes and a large proportion of genes involved within transcriptional and translational regulation and function. These results highlight the possible molecular pathways in response to the presence of misfolded protein. In summary, misfolded prion protein accumulates rapidly across the CNS but only specific brain regions undergo neurodegeneration. In the presence of the misfolded protein, the host elicits a robust molecular response. The additional activation of glial cells within regions undergoing neurodegeneration highlights their importance in disease. It is therefore proposed that misfolded prion protein, alone, is not sufficient to trigger neurodegeneration. This gives rise to a “multi-hit” hypothesis whereby two or more factors, for example the accumulation of misfolded protein and glial cell response, are required to trigger neurodegeneration.

No description available.

No description available.