Poly-Q expanded exon 1 of huntingtin (Q103) fused to GFP is toxic to yeast cells containing endogenous yeast prions, [PIN+] ([RNQ+]) and/or [PSI+], which presumably serve as aggregation nuclei. Propagation of yeast prions is modulated by the chaperones of Hsp100/70/40 complex. While some chaperones were reported to influence poly-Q aggregation in yeast, it was not clear whether they do it directly or via affecting yeast prions. Our data show that while dominant negative Hsp104 mutants antagonize poly-Q aggregation and toxicity by eliminating endogenous yeast prions, some mutant alleles of Hsp104 decreases size and ameliorate toxicity of poly-Q aggregates without affecting prion propagation. Elevated levels of the yeast Hsp40 proteins, Ydj1 and Sis1, exhibit opposite effects on poly-Q aggregation and toxicity without influencing prion propagation. Among the yeast Hsp70s, only overproduction of Ssa4 antagonized poly-Q toxicity. We have also isolated dominant Anti-poly-Q-toxicity (AQT) mutants counteracting poly-Q toxicity only in the absence of the major ubiquitin-conjugating enzyme Ubc4. Prion forming potential of other Q-rich proteins and influence of Q and P-rich regions on prion propagation were also studied. Our data connects poly-Q aggregation and toxicity to the stress defense pathway in yeast. As many stress-defense proteins are conserved between yeast and mammals, our data shed light on possible mechanisms modulating poly-Q aggregation and toxicity in mammalian cells.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Chernoff, Yury; Committee Member: Bommarius, Andreas; Committee Member: Doyle, Donald; Committee Member: Lobachev, Kirill; Committee Member: Yi, Soojin. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2006. / Jung Choi, Committee Member ; Yury Chernoff, Committee Chair ; kirill.lobachev@biology.gatech.edu, Committee Member.

No

錯誤折疊並聚集的促澱粉樣變蛋白和多肽分子通常以β折疊含量豐富的纖維狀澱粉態存在，這種纖維狀澱粉態被認為與多種神經退行性疾病的發病有關，例如老年癡呆症，多聚穀氨醯胺症以及傳染性海綿狀腦病。澱粉態沉積物作為多種神經退行性疾病的顯著標誌，促澱粉樣變蛋白和多肽發生錯誤折疊並聚集進而導致神經毒性的機理仍未被闡明。在當前的研究中，我們選擇酵母感染性蛋白Sup35作為探索促澱粉樣變蛋白聚集機理的模型。Sup35是一種存在於釀酒酵母細胞中的感染性蛋白，作為一種翻譯終止因子，它可以通過改變自身構象，進而形成不溶的纖維狀澱粉態沉澱。根據位置和功能的不同，Sup35可被劃分為3個結構域，即N，M和C。作為控制其感染性的區域，Sup35-NM被廣泛接受為一種用於研究促澱粉樣變蛋白的模型。研究人員已經針對Sup35的聚集機理開展了很多研究，其中最為廣泛接受的是Lindquist 等人提出的β螺旋模型。在這個模型中，相鄰的氨基酸片段形成了一種頭對頭和尾對尾的構象。我們的研究目的就是要探究這種聚集機理模型是否正確。如果不正確，我們將對聚集機理提出一種新的假設。 / 作為探索促澱粉樣變蛋白聚集過程的重要前提，研究人員必須首先製備出只含有單獨的蛋白單體的樣品溶液。否則，相關的動力學過程研究將被干擾。我們通過動態激光光散射研究發現，使用現有的多種用於溶解促澱粉樣變蛋白和多肽的實驗方法並不能製備出真正的蛋白溶液，得到的樣品中總含有微量的、尺寸大約為10-10² nm的聚集體。這些聚集體會極大地影響聚集的動力學過程。這也可以在一定程度上解釋為什麼在不同的文獻報導中，同一種蛋白在相同的環境中卻表現出差異巨大的動力學過程。在當前的研究中，我們將傳統方法與我們實驗室新進開發的超濾法相結合，發展出了一套可以用於製備真正的、不含有聚集體的促澱粉樣變蛋白或多肽溶液的方法。製備出的溶液可以保持其中的蛋白或多肽處於單體狀態至少一個星期，這為研究在生理條件下蛋白的聚集過程提供了重要的基礎。 / 為了研究Sup35不同亞基之間的相互作用，我們分別在其N結構域的頭，腰和尾做了半胱氨酸點突變，並用兩種相互獨立的方法研究亞基之間的相互作用。第一種方法是在突變位點引入空間位阻，從而減弱所謂的頭對頭尾對尾的相互作用。我們的想法很直接，如果Lindquist等人提出的機理是正確的，那麼突變後的蛋白將無法形成纖維狀澱粉態沉澱。第二種方法是通過形成二硫鍵在不同蛋白的半胱氨酸突變位點之間引入連接分子，共有兩種連接分子，一種長約2 Å，另一種長約11 Å。選擇這兩種連接分子的原因是，聚集體中兩條Sup35蛋白鏈之間的距離通常約4.7 Å，連接分子長於或短于這個距離應會對聚集產生不同影響，從而反映出聚集體的結構資訊。 / 在這篇博士論文中，首先，我將介紹促澱粉樣變蛋白研究的背景和激光光散射測量的原理以及研究中用到的主要實驗方法。然後，我將闡述如何將傳統方法和我們實驗室新進發展出的超濾法相結合，從而製備出真正的、不含聚集體的蛋白溶液。接下來，我還將證明通過動態和靜態激光光散射相結合，我們可以得到更多關於促澱粉樣變蛋白的微觀參數，包括分子量，蛋白單體和聚集體的流體力學半徑等。最後，我將針對不同Sup35突變體的聚集動力學過程來研究其亞基之間的相互作用並提出Sup35的聚集模型。 / Misfolding and aggregation of amyloidogenic protein/peptide are frequently found in a β-sheet-rich fibrillar protein conformation known as amyloids, which are related to the onset of neurodegenerative diseases, ranging from Alzheimer and polyglutamine diseases to transmissible spongiform encephalopathies. While amyloid deposits are hallmarks of many neurodegenerative diseases, the mechanism by which these proteins/peptides gain their neurotoxic function upon misfolding and aggregation remains unclear. In the current study, we choose the yeast prion Sup35 as a model system to investigate the aggregation mechanism of amyloidogenic protein. The Sup35 protein is a yeast (Saccharomyces cerevisiae) prion protein, a translation termination factor that can convert into insoluble amyloid fibril. The structure of Sup35 protein can be divided into three regions; namely, N, M, and C based on their positions and different functions. Being the prion-determining region, Sup35-NM has been widely accepted as a model to study the amyloidogenic proteins. Many studies have been focused on the aggregation mechanism. The β-helix model proposed by Lindquist and her coworkers is mostly accepted. In such a model, Sup35-NM is folded to form a “head and a “tail region in the N region and different Sup35-NM chains aggregate together via a cooperative Head-to-Head and Tail-to-Tail stacking. The aim of our current study is to check whether this proposed mechanism is valid. / To gain insight into the mechanism of aggregation process, one must start with a solution that contains only individual (monomeric) protein chains. Otherwise, the kinetic study would be compromised. Our dynamic laser light scattering (LLS) study shows that the existing protocols of dissolving amyloidogenic protein/peptide do not result in a true solution; namely, there always exist a trace amount of interchain aggregates with an average size of ~10-10² nm, which greatly affect the association kinetics, partially explaining why different kinetics were reported even for a solution with identical protein and solvent. In this thesis study, by using a combination of the conventional dissolution procedure and our newly developed ultra-filtration method, we have developed a novel protocol to prepare a true solution of amyloidogenic protein/peptide without any interchain aggregates. The resultant solutions remain in their monomeric state for more than one week, which is vitally important for further study of the interchain association under the physiological conditions / To investigate the inter-subunit relationship, cysteine variants mutated at “Head, Waist, Tail" of the N region have been constructed. Two independent assessments have been proposed to study the inter-subunit interaction. One is to provide steric hindrance to the mutated sites so that the so called “Head-to-Head and Tail-to-Tail" interaction will be attenuated. Our strategy is quite straightforward, if the mechanism proposed by Lindquist and her coworkers is valid, the modified protein should lose its ability to form amyloid fibrils. The other strategy is to introduce disulfide cross-linkage between different mutation sites. Two types of disulfide cross-linkage have been chosen, one with a bond length of ~2 Å and the other, ~11 Å. The reason for such choices is that Sup35-NM has a characteristic inter-strand distance of ~4.7 Å. The disulfide bond length shorter or longer than this distance is supposed to play a different role in the protein aggregation, shedding light on the structural information. / In this Ph.D. thesis, we first introduce the background of amyloidogenic protein research and present the principle and instrumentation of laser light scattering, the main technique applied in our study. Next we show how to obtain a true solution of amyloidogenic protein with no oligomeric aggregates by combining a conventional dissolution procedure and our newly developed ultra-filtration method. We also show how to combine static and dynamic laser light-scattering measurements in the study of protein solutions, which leads to more microscopic parameters, such as the molar mass and the hydrodynamic sizes of individual protein chains and their aggregates. Our focus is on the aggregation kinetics of modified Sup35-NM variants and on the investigation of the inter-subunit interaction. Finally, we propose a model for the aggregation of Sup35-NM prion protein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Diao, Shu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 99-101). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT (in Chinese) --- p.i / ABSTRACT --- p.iii / Table of content --- p.v / Acknowledgement --- p.viii / Chapter Chapter 1 --- Introduction and background --- p.1 / Chapter 1.1 --- The biological role of amyloidogenic protein --- p.1 / Chapter 1.1.1 --- The role of amyloidogenic protein in human disease --- p.1 / Chapter 1.1.2 --- The functional role of amyloidogenic protein in living system --- p.2 / Chapter 1.1.3 --- The role of amyloidogenic protein asnonchromosomal genetic elements --- p.3 / Chapter 1.2 --- The structure of amyloid fibrils --- p.4 / Chapter 1.2.1 --- Macromolecular structure of amyloid fibrils --- p.5 / Chapter 1.2.2 --- Structure models for protofilament --- p.6 / Chapter 1.2.3 --- The polymorphism of amyloid fibrils --- p.9 / Chapter 1.3 --- Aggregation mechanism of amyloidogenic protein --- p.10 / Chapter 1.3.1 --- The nucleated polymerization mechanism --- p.11 / Chapter 1.3.2 --- Multiple conformations adopted by amyloidogenic protein chains --- p.13 / Chapter 1.3.3 --- Sequence effect on amyloid formation --- p.15 / Chapter 1.4 --- The pathogenesis of amyloid diseases --- p.16 / Chapter 1.4.1 --- Prefibrillar aggregates may be the real culprits --- p.16 / Chapter 1.4.2 --- Strategies to prevent amyloid diseases --- p.17 / Chapter 1.5 --- References and Notes --- p.19 / Chapter Chapter 2 --- Principle of Laser Light Scattering and Instrumentation --- p.27 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Static Laser Light Scattering --- p.28 / Chapter 2.2.1 --- Scattering by a small particle --- p.28 / Chapter 2.2.2 --- Scattering by many small-particle system --- p.30 / Chapter 2.2.3 --- Scattering by real systems --- p.31 / Chapter 2.3 --- Dynamic Laser Light Scattering --- p.37 / Chapter 2.3.1 --- Power spectrum of scattered light --- p.37 / Chapter 2.3.2 --- Siegert relation --- p.39 / Chapter 2.3.3 --- Translational diffusions --- p.40 / Chapter 2.3.4 --- Analysis of the correlation function profile --- p.42 / Chapter 2.4 --- Combination of Static and Dynamic Light Scattering --- p.44 / Chapter 2.5 --- Practice of Laser Light Scattering --- p.45 / Chapter 2.5.1 --- Light source --- p.45 / Chapter 2.5.2 --- Optics and cell design --- p.46 / Chapter 2.5.3 --- Detector --- p.47 / Chapter 2.5.4 --- Sample Preparation --- p.47 / Chapter 2.5.5 --- Differential refractometer --- p.48 / Chapter 2.6 --- References and Notes --- p.49 / Chapter Chapter 3 --- How to obtain a true solution of amyloidogenic protein/peptide with no oligomeric aggregates --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Experimental section --- p.53 / Chapter 3.3 --- Results and discussion --- p.59 / Chapter 3.4 --- Conclusion --- p.68 / Chapter 3.5 --- References and Notes --- p.71 / Chapter Chapter 4 --- Aggregation mechanism investigation of the Yeast prion protein Sup35-NM --- p.73 / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Experimental section --- p.75 / Chapter 4.3 --- Results and discussion --- p.82 / Chapter 4.3.1 --- Aggregation kinetics of Sup35-NM protein initiated from monomeric state --- p.82 / Chapter 4.3.2 --- Does Sup35-NM protein aggregate in a head-to-head and tail-to-tail fashion? --- p.87 / Chapter 4.3.2.1 --- The effect of dimerization on Sup35-NM aggregation --- p.88 / Chapter 4.3.2.2 --- Inter-subunit investigation by Pyrene excimer fluorescence assay --- p.92 / Chapter 4.3.2.3 --- The effect of PEGylation on Sup35-NM aggregation --- p.94 / Chapter 4.4 --- Conclusion --- p.98 / Chapter 4.5 --- References --- p.100 / Publications --- p.102

La conversion de la protéine prion cellulaire (PrPC) en protéine prion scrapie (PrPSc) est à l’origine des encéphalopathies spongiformes transmissibles (EST). La toxicité de la PrPSc est restreinte aux neurones et implique la déviation de la (des) fonction(s) de la PrPC. Une action neurospécifique de la PrPC est le recrutement de la src kinase Fyn. Dans ce travail, nous montrons que la PrPC est capable de mobiliser une autre src kinase, Lyn, via la cavéoline (cav) dans les neurones. Une cible du couplage PrPC-cav-Lyn, restreint aux corps cellulaires, est la kinase GSK3ß. L’inactivation de GSK3ß par la PrPC intervient dans la régulation des fonctions neuronales, en limitant l’activité du récepteur sérotoninergique 1B. D’autre part, nous montrons que dans les cellules infectées par les prions, l’accumulation de PrPSc induit le recrutement constitutif de la plateforme PrPC-cav-Fyn, à l’origine d’un stress oxydant. Ce gain de fonction entraine notamment un défaut de clivage par la metalloprotéase MMP-9, dont une des conséquences est l’accumulation du peptide Aß. Un autre impact de l’infection est la suractivation de la kinase PDK1, qui génère une perte de fonction de la metalloprotéase TACE. Dans des neurones « Alzheimer », on observe également cette cascade délétère, qui dépend de la PrPC. L’inhibition de PDK1 dans des modèles d’infection par les prions ou de maladie d’Alzheimer permet de rétablir l’activité d’alpha-clivage de TACE vis-à-vis de ses substrats, la PrPC, APP et le récepteur au TNF-alpha. L’effet bénéfique observé in vivo permet de définir PDK1 comme une cible thérapeutique potentielle pour les EST et la maladie d’Alzheimer. En résumé, ce travail illustre comment une meilleure connaissance de la fonction de signalisation de la PrPC peut permettre de progresser dans la compréhension des mécanismes de neurodégénérescence associés non seulement aux maladies à prions, mais également à la maladie d’Alzheimer. / Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of the cellular prion protein (PrPC) into its scrapie isoform (PrPSc). PrPSc-mediated toxicity is restricted to neurons and results from the subversion of PrPC function(s). Some neuronal specificity of PrPC signaling relates to its coupling to the Fyn src kinase. In this work, we report that PrPC has the capacity to mobilize another src kinase, Lyn, via caveolin in neurons. A downstream effector of the PrPC-cav-Lyn signaling complex, which is spatially restricted to cell bodies, is the GSK3ß kinase. The inactivation of GSK3ß by PrPC takes part to the control of neuronal functions by negatively regulating the activity of the serotonin 1B receptor. Furthermore, we show that in prion-infected cells, PrPSc constitutively activates the PrPC-cav-Fyn platform, leading to oxidative-stress conditions. This toxic gain of PrPC function induces a defect in metalloproteinase MMP-9 activity, leading to increased Aß levels. Another impact of prion infection is the overactivation of the PDK1 kinase, which results in the loss of function of the TACE metalloproteinase. PDK1 overactivation is also observed in neurons from Alzheimer’s disease model mice, and is shown to be PrPC dependent. PDK1 inhibition in models of prion infection or Alzheimer’s disease restores TACE-mediated alpha-cleavage of PrPC, APP and TNF-alphareceptor. Since positive effects of PDK1 inhibition are observed in vivo, our data posit PDK1 as a putative therapeutic target to combat TSE disorders and Alzheimer’s disease. In summary, achieving a better knowledge of PrPC signaling function may help to improve our understanding of the mechanisms sustaining neurodegeneration in prion and Alzheimer’s diseases.

Les Encéphalopathies Spongiformes transmissibles (EST) sont des maladies neurodégénératives fatales caractérisées par l’accumulation d’un conformère anormal (PrPSc) d’une protéine de l’hôte (PrP). Chez l’homme, plusieurs centaines de cas de transmissions iatrogènes de la maladie de Creutzfeldt Jakob (MCJ) ont été identifiées, notamment chez des patients ayant fait l’objetd’ une greffe de dure-mère, de cornée ou des injections d’hormones de croissance extractives. Plus récemment, plusieurs cas du variant de la maladie de Creutzfeldt Jakob (vMCJ) ont été observés chez des patients transfusés avec des produits sanguins issus de donneurs en incubation de la maladie. D’un point de vue sanitaire, l’évaluation du risque de contamination interindividuelle par des tissus ou des fluides biologiques issus de patients atteints représente un enjeu important en matière de santé publique. La première partie de notre travail a consisté à comparer la sensibilité relative de modèles de souris transgéniques sur-exprimant la PrP à celle de l’hôte conventionnel exprimant la même séquence. Les résultats obtenus ont validé le concept d’une absence d’impact du niveau d’expression de la PrP ou du fond génétique de l’hôte sur la sensibilité finale du modèle à l’infection. A l’aide de ces modèles de souris transgéniques, nous avons alors mesuré les niveaux d’infectiosité dans le sang de patients atteints de différentes formes d’’EST. Chez un patient atteint de vMCJ, nous avons mis en évidence de faibles niveaux d’infectiosité dans les concentrés de globules rouges, les leucocytes et le plasma. Nous avons également pu détecter de l’infectiosité dans le plasma issu de 2 patients atteints de sMCJ sur 4 testés. Parallèlement à ces expériences, nous avons démontré dans un modèle expérimental d’infection chez le mouton, que l’administration de 104 à 105 leucocytes suffisent à transmettre par voire transfusionnelle la maladie. Ces résultats soulignent l’intérêt et les limites de la leuco-déplétion appliquée de manière standard en médicine transfusionnelle, pour limiter les risques de transmission du vMCJ. Enfin, nous avons testé la capacité de différents outils in vitro à détecter la présence des Prions dans le sang. / Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders occurring in a wide spectrum of animals. They are characterized by accumulation of abnormally folded conformers (PrPSc) derived from normal cellular PrP protein (PrP) of the host. In human, many iatrogenic transmissions of Creutzfeldt Jakob disease (CJD) have been reported after dura mater graft, corneal graft or extractive growth hormone injections, prepared from affected donors. More recently, several cases of vCJD transmissions were reported in individuals that were transfused with blood from asymptomatic donors that subsequently developed vCJD. Risk assessment of interindividual transmission with contaminated tissues or body fluids remains a major public health issue. In a first part, we validated the final pertinence of infectious titers as measured in mice overexpressing PrP to the risk of transmitting the disease in the natural host species. In a second time, we used this model to evaluate the presence of infectivity in blood from TSE affected patients. We were able to detect the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with vCJD and in the plasma of 2 out of 4 persons whose tests were positive for sporadic CJD. We then demonstrated in a sheep TSE model, that intravenous administration of 104 to 105 leucocytes was sufficient to cause disease in recipient sheep, underlying the efficacy and potential limits of leuko-reduction processes that are currently applied in transfusion medicine to mitigate the TSE transmission risk. Finally, using the same model, we tested different in vitro methods to detect prions in blood.

Les protéines prions sont associées à une classe de maladies neurodégénératives, dont l'encéphalopathie spongiforme transmissible (EST) est la mieux connue. La protéine prion Sup35p représente un tel modèle car elle est non associée à une maladie. Sup35p se compose de trois domaines : un domaine N-terminal qui est responsable de la formation de prion, d'un domaine de milieu (M) qui affiche un degré élevé de flexibilité, et un domaine C-terminal fonctionnel et globulaire. Le fragment Sup35pNM est souvent utilisé comme modèle pour documenter l'assemblage et les propriétés infectieuses de Sup35p. Les études de Sup35p et Sup35pNM par RMN du solide ont révélé d'étonnantes différences structurelles entre les deux cœurs amyloïdes de Sup35p et Sup35pNM. Nos résultats sur Sup35p apportent un nouvel éclairage sur le monde étonnamment diversifié des prions où la variabilité conformationnelle joue un rôle énorme et perturbant. Ils reflètent l'image émergente que les prions sont des unités structurelles complexes. En effet, même s'il affiche une structure très définie, un domaine donné peut adopter des conformations différentes selon les circonstances (en isolation, dans le contexte d'un fragment ou la protéine entière) ou de l'environnement (conditions de tampon, présence de chaperonnes). Nos résultats donnent une explication au niveau moléculaire pour la contractante propension à l'assemblage et l'infectiosité de Sup35pNM et Sup35p, et soulignent l'importance primordiale d'une caractérisation structurale au niveau moléculaire des agrégats utilisés dans des études fonctionnelles / Prion proteins are associated with a class of neurodegenerative diseases, including transmissible spongiform encephalopathy (TSE) which is the best known. The prion protein Sup35p displays a model system because it is not associated with disease. Sup35p consists of three domains: an N-terminal domain which is responsible for the prion formation, a middle domain (M) that displays a high degree of flexibility, and a functional C-terminal domain. Sup35pNM the fragment is often used as a model to document for the assembly and infectious properties of Sup35p. Solid-state NMR studies of Sup35p and Sup35pNM fibrils showed amazing structural differences between the two amyloid cores. Our results shed new light on the surprisingly diverse world of prions where conformational variability plays a huge role. They reflect the emerging picture that prions are complex structural units. Even if it displays a very defined structure, a given field may adopt different conformations depending on the circumstances (in isolation, in the context of the whole protein or fragment) or the environment (buffer conditions, presence of chaperones). Our results provide an explanation at the molecular level for the contrasting propensity assembly and infectivity Sup35pNM and Sup35p, and emphasize the central importance of a structural characterization at the molecular level

Les Encéphalopathies Spongiformes Subaigües Transmissibles (ESST) sont des maladies neurodégénératives caractérisées par une longue période d'incubation asymptomatique à l'issue fatale. Elles sont induites par l'accumulation, au niveau du système nerveux central (SNC), de l'isoforme pathogène de la protéine du prion (PrPSc) entraînant une dégénération des cellules neuronales ainsi qu'une astrogliogénèse. La PrPSc, qui joue un rôle central dans la transmission de la maladie, est produite par conversion de la forme physiologique de la protéine du prion (PrPC). Les mécanismes de conversion de la PrPC et de propagation de la PrPSc sont incertains ainsi que les mécanismes moléculaires à la base des maladies à prions. Dans le cadre de la création et l'amélioration de modèles de culture cellulaire, il a été montré que les Cellules Souches Neurales (CSN) issues du SNC permettent la conversion in vitro de la PrPC en PrPSc. Dans cette étude, nous avons utilisé les CSN pour optimiser et caractériser les conditions d'infection des cellules et émis l'hypothèse que la modification des conditions de culture pouvait moduler la production de PrPSc dans les CSN. Pour cela, nous avons ajouté des facteurs influençant l'identité cellulaire dans nos cultures et avons montré qu'ils étaient capables d'augmenter la propagation du prion. Ces modèles nous permettent l'étude des mécanismes moléculaires pouvant être à l'origine de l'infection. En parallèle, nous avons montré que l'ajout d'HEPES dans nos cultures inhibe la production de PrPSc dans les CSN de façon dose-dépendante. Par ailleurs, à ce jour il n'existe aucune thérapie capable de stopper la progression de la maladie chez l'homme. Ainsi, nous avons utilisé les CSN dans le but d'élaborer une approche thérapeutique permettant de distribuer des anticorps au sein du SNC pour stopper la réplication du prion. Ces cellules permettront, de plus, de réparer les zones endommagées du cerveau combinant ainsi thérapie cellulaire et génique. / Transmissible Spongiform Encephalopathies (TSE) are neurodegenerative disorders with long asymptomatic incubation periods and fatal issue. They are induced by accumulation of the pathogen isoform of the prion protein (PrPSc) in the central nervous system (CNS) resulting in neuronal degeneration and astrogliosis. PrPSc, produced by the conversion of the physiological form of the prion protein (PrPC), plays a key role in the disease transmission. The mechanisms underlying the conversion of PrPC and the propagation of PrPSc are uncertain just as the molecular mechanisms giving rise to prion diseases. In the aim of creating or improving cell culture models, it has been shown that CNS Neural Stem Cells (NSC) could support PrPC conversion into PrPSc in vitro. In this project, we used NSC to improve and characterize cellular infection and hypothesized that modification of culture conditions could modulate PrPSc production in NSC. Hence, we used factors known to influence cellular identity in our culture model and showed that higher amount of prions were produced. These models also allow molecular mechanisms studies that could be at infection origin. During the course of this study, we also demonstrated that HEPES added to our culture medium could stop prion propagation in a dose-dependant manner. Moreover, to date no therapy aimed at stopping disease progression has been established in humans. We therefore used NSC with the ultimate goal to elaborate a therapeutic strategy based on the delivery of antibodies into the CNS to block prion replication. These cells will also able to repair damaged brain area thus combining cell and gene therapy.