Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Les encéphalopathies spongiformes transmissibles (EST) sont des maladies neurodégénératives infectieuses qui résultent de l'agrégation de formes anormales de la protéine prion cellulaire (PrP[indice supérieur C]). La maladie de Creutzfeldt-Jakob est la plus répandue chez les humains alors que chez les animaux, la maladie de la vache folle est celle qui a l'impact économique le plus important et est la seule EST animale clairement transmissible à l'homme. Des études suggèrent que des agrégats cytoplasmiques de la protéine prion (CyPrP) pourraient être responsables de la neurodégénérescence observée lors des EST, toutefois, le mécanisme de toxicité de ces agrégats est encore inconnu. Nous avons émis l'hypothèse que la production d'agrégats cytoplasmiques de la protéine prion entraîne un stress considérable et possiblement létal pour les cellules neuronales. Nous avons analysé la capacité de ces agrégats d'activer ou d'inhiber certaines composantes de la réponse au stress intégrée. Cette réponse a pour but de limiter les dommages cellulaires en conditions de stress et consiste en l'arrêt de la synthèse protéique, l'assemblage de granules de stress et l'induction de chaperonnes moléculaires. Nos résultats démontrent que les agrégats cytoplasmiques de la protéine prion induisent un stress pour la cellule qui résulte en l'activation de la kinase du stress PKR, la phosphorylation du facteur d'initiation de la traduction eIF2[alpha] et une diminution d'environ 80% de la synthèse protéique. De façon surprenante, la formation des granules de stress est inhibée dans les cellules qui produisent des agrégats cytoplasmiques de PrP. L'hybridation in situ et la chromatographie d'affinité sur résine de cellulose-oligo(dT) nous ont permis de démontrer que les ARNm étaient séquestrés en grande partie au sein des agrégats de CyPrP. Nous avons aussi démontré que l'induction de Hsp70 était inhibée suite à un stress dans les cellules qui produisent des agrégats et que ces cellules sont beaucoup plus sensibles à un stress oxydatif. Nous proposons que l'activation d'une réponse au stress inadéquate par les agrégats cytoplasmiques de la protéine prion altère considérablement la capacité des neurones de résister à de nombreux stress physiologiques. Nous suggérons que ces événements pourraient contribuer à la toxicité et à la neurodégénérescence observée au cours des maladies à prions.

No description available.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative diseases affecting humans and animal species. Prion diseases are characterized by the conversion of the host encoded prion protein (PrPC) into a disease-associated isoform (PrPSc), which (according to the prion hypothesis) is thought to be the main component of the infectious agent. PrPSc has been traditionally distinguished from PrPC by its biochemical properties, such as partial resistance to proteolysis and detergent-insolubility. In the absence of a foreign nucleic acid genome associated with prion diseases, efforts to provide a molecular basis for the biological diversity of prions have focused on biochemical characterization of PrPSc. In Creutzfeldt-Jakob disease (CJD) and other forms of human prion disease, the biochemical characterization of PrPSc has been largely restricted to the analysis of PK-resistant fragments of PrPSc (PrPres) by Western blot. However, given recent findings on the complexity of PrPSc identified in laboratory prion strains, PrPres analysis alone may not provide a complete description of PrPSc present in CJD brains. For a more complete characterization of PrPSc in human prion diseases, this study investigated biochemical properties of PrPSc in different forms of CJD by employing approaches that differ in principle from conventional Western blot analysis of PrPres. The novel biochemical approaches used in this study have identified further complexity of PrPSc accumulated in CJD brains, not only between different forms of CJD but also within single cases of individual disease entities. In this study, the two biochemical criteria most frequently used to define PrPSc (3F4 epitope accessibility versus resistance to limited proteolysis) did not always correlate, indicating probable non-uniform distribution of PK-sensitive isoform of PrPSc within the same CJD brains. In variant CJD (vCJD) brains, the thalamic region, which is characterized by distinct neuropathological features, could also be distinguished from frontal cortex and cerebellum by the sedimentation profiles of PrPC and PrPSc on sucrose step gradients. Moreover, the conformational stability of PrPSc was found not to be uniform among human prion diseases and did not correlate with PrPres type or prion protein genotype. Taken together, the results from this study provide a more complete description of PrPSc species occurring in CJD brains and contribute to a fuller understanding of the agents and the disease processes involved in humans.

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Memoria para optar al Título Profesional de Médico Veterinario / El scrapie ovino pertenece al grupo de las encefalopatías espongiformes transmisibles (EET), las que corresponden a enfermedades degenerativas y progresivas del sistema nervioso central (SNC), que se caracterizan por presentar un largo período de incubación y que culminan con la muerte del animal afectado. Datos históricos indican que el scrapie es la enfermedad más antigua del grupo de las EET, siendo descrita hace más de 250 años en Europa. El agente etiológico de la enfermedad corresponde a un prion (proteinaceus infectious particle), que es la isoforma anormal de una proteína celular, que aunque carece de ADN tiene la capacidad de replicarse sin la presencia de genes. Se caracteriza por una gran resistencia a la acción de agentes químicos y físicos y por lo mismo es muy difícil de erradicar de los lugares infectados. Se sabe que el scrapie es una enfermedad del ganado ovino, que se reconoce por su signología clínica, sin embargo para un diagnóstico específico se requiere del examen histopatológico del encéfalo de los animales sospechosos, los que en el caso de ser positivos a la enfermedad, se caracterizan por presentar cambios espongiformes bilaterales y simétricos, tanto en el pericarion como en el neuropilo. La detección de formas anormales de una proteína de membrana del tejido nervioso del huésped llamada “proteína prion”, es útil como criterio adicional para confirmar el diagnóstico de la enfermedad, sin embargo en nuestro país el principal método de confirmación de la enfermedad es hasta ahora el estudio histopatológico del encéfalo. La importancia a nivel mundial de esta enfermedad, es la de haber participado en la diseminación masiva de la enfermedad a otras especies animales, como lo es el ganado bovino Europeo en la denominada Encefalopatía Espongiforme Bovina (EEB). Este proceso se cree que se produce por medio del consumo de harinas de carne y hueso de origen ovino. Además existe la potencialidad de constituir una zoonosis, por el consumo de carne o subproductos de ovinos que cursan con la enfermedad. Este hecho concuerda con la aparición de una nueva variante de la enfermedad de Creutzfeldt - Jakob (nv CJD) en los humanos. El presente trabajo constituye una parte de un estudio poblacional a nivel nacional que realiza el Servicio Agrícola y Ganadero (SAG), para detectar la eventual presencia del scrapie en Chile y de este modo, ayudar a conocer la situación epidemiológica de esta enfermedad en nuestro país. Esta investigación se realizó en base al estudio histológico de 135 cerebros de ovinos mayores de 5 años, provenientes de mataderos de la XI región de Chile, en los que se utilizaron métodos, procedimientos y criterios histoanatomopatológicos reconocidos internacionalmente. De esta forma el análisis de los resultados del presente estudio nos permite indicar que las principales alteraciones encontradas se agruparon en : lesiones vacuolares tanto en el pericarion neuronal como en el neuropilo (de presentación unilateral y en ningún caso bilaterales) y alteraciones que involucran cambios de tipo inflamatorio (manguitos perivasculares), depósitos pigmentarios, hallazgos parasitarios y lesiones traumático hemorrágicas. Sin embargo, se puede concluir que los cambios observados en las muestras de los encéfalos examinados al microscopio, de acuerdo a sus características histopatológicas, no corresponden a lesiones atribuibles a scrapie ovino, sino que simplemente constituyen hallazgos histopatológicos, por ser lesiones aisladas y no presentar un patrón específico de ubicación en el SNC. La importancia de este estudio radica en contribuir al conocimiento de la situación epidemiológica del scrapie ovino en nuestro país, así como también el de servir de base para futuras investigaciones relacionadas con el tema. / Proyecto financiado por el Servicio Agrícola y Ganadero (SAG)

Thesis advisor: Jianmin Gao / Protein aggregation can be highly detrimental to organisms, and has been associated with diseases including Alzheimer's, Huntington's, type II diabetes, and transmissible spongiform encephalopathies such as Mad Cow disease. There is no single amino acid sequence responsible for aggregation into amyloid-like structures, but rather a large range of amyloidogenic peptides have been discovered. A fragment of the yeast Sup35 prion, GNNQQNY, has been found to aggregate using a "dry, steric zipper" structure. This study looks at mutants of GNNQQNY in order to elucidate the exact contributions of various amino acids to the aggregation process. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

Les synucléionopathies représentent un groupe de maladies neuro-dégénératives incurables du système nerveux central. Elles regroupent entre autres la maladie de Parkinson, l’atrophie multi-systématisée et la maladie à corps de Lewy. Toutes ces maladies se caractérisent par un déclin progressif des fonctions motrices, cognitives, comportementales et autonomiques. La mal-conformation et l’agrégation de la protéine α-synuclein qui forme des inclusions intraneuronales sont des éléments communs à toutes les synucleinopathies. Ces inclusions portent le nom de corps de Lewy et se forment dans des neurones ou cellules gliales appartenant à des régions cérébrales spécifiques. Elles sont vraisemblablement à l’origine de la perte progressive de neurones dans certaines parties du cerveau. Dans le cas de la maladie de Parkinson et dans d’autres maladies neuro-dégénératives, il a été démontré que la pathologie se propage anatomiquement d’une manière spécifique et prévisible au niveau cérébrale. Ceci suggère donc que la progression de la maladie est étroitement liée au transfert des agrégats d’α-synucléine. Ce procédé est très similaire à celui impliqué dans la maladie du prion qui elle en revanche est infectieuse. Par ailleurs, des inclusions neuronales d’α-synucléine ont été identifiées dans des neurones dopaminergiques d’origine fœtaux qui avaient été transplanté dans des cerveaux de patients parkinsoniens. Cette étude a permis d’envisager pour la première fois la possibilité de la transmission d’inclusions d’α-synucléine entre les neurones. Bien que de nombreuses études aient démontré la propagation d’α-synucléine in vitro et in vivo, le mécanisme permettant ce transfert n’est pas clairement établi. Par conséquent, ma thèse s’attache à étudier le mécanisme de transfert d’assemblages d’α-synucléine (i.e., oligomères et fibrilles). Dans un premier temps, j’ai apporté la preuve que les assemblages d’α-synucléine transfèrent de manière efficace entre les cellules neuronales via les Tunneling nanotubes (TNT). Les TNT sont définis comme étant des ponts membranaires riches en F-actine et permettant de connecter physiquement le cytoplasme de cellules éloignées. Au niveau subcellulaire, j’ai démontré que les assemblages d’α-synucléine qui transfèrent se trouvent dans des lysosomes. En revanche, après le transfert, ces assemblages se retrouvent libres dans le cytoplasme. J’ai également mis en évidence qu’à la suite du transfert, permis par les TNT, les fibrilles d’α-synucléine sont capables de recruter et d’induire l’agrégation de l’α-synucléine soluble afin de perpétuer le processus d’agrégation à l’infinie. Ces résultats indiquent que les TNT peuvent représenter un moyen efficace permettant le transfert d’assemblages d’α-synucléine. Cette découverte offre de nouvelles opportunités pour le développement de nouveaux agents neuro-protectifs contre la propagation des synucléinopathies. / Synucleinopathies are a group of fatal neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, characterized by a chronic and progressive decline in motor, cognitive, behavioral, and autonomic functions. The hallmark of these diseases is the misfolding and aggregation of α-synuclein protein accumulating into intracellular inclusions Lewy bodies in neurons and glial cells which leads to the loss of neurons in specific brain regions. In the case of Parkinson’s disease and other neurodegenerative diseases, the pathology was shown to progress throughout the brain in a specific and predictable manner suggesting that the progression of the diseases is linked to the transfer of aggregated α-synuclein that is reminiscent of prion diseases that are infectious. Importantly, upon transplantation of fetal dopaminergic neurons in the brain of Parkinson’s patients, neuronal inclusions were found in the grafted neurons strongly suggesting that α-synuclein inclusions could transmit between neurons. While several studies showed α-synuclein propagation in vitro and in vivo the mechanism of intercellular transfer remains elusive. The aim of my thesis was to study the mechanism of transfer of α-synuclein assemblies (i.e., oligomers and fibrils) involved in Parkinson’s pathogenesis. I evidenced that α-synuclein assemblies transferred efficiently via tunneling nanotubes (TNT), F-actin based membranous bridges connecting the cytoplasm of remote cells. I demonstrated that, at the sub-cellular level, the transferred α-synuclein assemblies were specifically confined in lysosomes and that upon transfer a large amount of α-synuclein was found free in the cytosol of acceptor cells. Finally, I showed that after TNT-mediated transfer α-synuclein fibrils recruited and seeded the aggregation of the soluble α-synuclein protein in order to perpetuate aggregation. The identification of TNT as an efficient means of α-synuclein transfer opens new avenues to the development of novel therapies targeting the spreading into the brain of amyloidogenic proteins involved in neurodegenerative diseases.