Prion diseases are formed by a conformational change of prion-like protein (PrPC) with alfa-helix structure to the pathological isoform - prion (PrPSc) which acquires beta-sheet structure. PrPC physiological properties in the brain are insufficiently described but there is an assumption of its affinity to metal ions. Another protein with metal-binding ability is metallothionein (MT). Brain specific isoform of MT is called MT-III and it is assumed to participate in maintenance of metal ions concentration in the brain. Aim of this study was to prepare recombinant human PrPC in E. coli. Furthermore, this protein was used to detect interactions between metal ions (Cu, Zn), MT and PrPC by differential pulse voltammetry method. The final part was devoted to the MT-III determination in different genotypes of prion-infected and non-infectious mouse brain tissues.

Orientador: Prof. Dr. Luis Paulo Barbour Scott / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2015. / Mecanismos biologicos envolvidos nas fibras amiloides e sua agregacao ainda sao um dos topicos mais investigados cientificamente. As prions estao entre estas proteinas que agregam-se sob certas condicoes. Estas proteinas adotam duas diferentes formas: i) a forma celular (PrPC) e ii) a forma infecciosa denominada scrapie (PrPSc) que possui propensao a agregacao. Na forma infecciosa, essa proteina pode causar diversas doencas tais como a encefalopatia espongi-forme bovina, doenca de Creutzfeldt-Jakob e doenca de Gerstmann-Straussler-Scheinker. As PrPC e PrPSc sao diferentes com relacao as estruturas secundarias e terciarias. A PrPC contem um numero menor de folha-¿À quando comparada a PrPSc. Nenhuma estrutura da PrPSc foi resolvida ate o presente momento e, alem disso, o mecanismo molecular da transicao entre a PrPC e PrPSc e o processo de agregacao da forma scrapie nao sao bem compreendidos apesar dos numerosos estudos realizados nesta area. Neste trabalho, foi aplicada uma nova metodologia de simulacao recentemente desenvolvida que permite promover grandes mudancas estruturais, nomeada como MDeNM (Molecular Dynamics with excited Normal Modes), com o objetivo de investigar e compreender a transicao conformacional entre a PrPC e PrPSc. Este metodo combina modos normais e dinamica molecular, a partir de movimentos coletivos ativados cineticamente que devem contribuir para um novo rearranjo estrutural dificilmente alcancado por simulacoes de dinamica molecular tradicional. Alem disso, estudos termodinamicos foram realizados com o modelo baseado em estrutura (SBM), que caracteriza as transicoes energeticas. Os resultados do SBM mostraram que as estruturas geradas por MDeNM podem corresponder a via de transicao entre a conformacao nao-infecciosa e a desnovelada. A combinacao de MDeNM e SBM mostrou-se util para extrair informacoes estruturais e energeticas relacionadas a conversao da prion para as formas infecciosas. Os resultados mostraram um aumento significativo na formacao da folha-¿À sob condicoes de baixo pH e considerando a PrP na forma trimerica e tambem em agregados. Estas simulacoes permitiram a caracterizacao de estados intermediarios na transicao da PrPC para a PrPSc, e a proposicao um mecanismo de conversao da PrPC para PrPSc. Estudos iniciais relacionados aos mutantes da prion e o modelo Coarse-Grained tambem foram realizados neste trabalho. / Biological mechanisms involved in the amyloid fibrils and aggregation belong yet to very hot topics of scientific investigation. Prions are among proteins that aggregate under certain conditions. They adopt two different forms: i) the cellular form (PrPC) and ii) infectious form called scrapie (PrPSc) having the propensity to aggregate. In the infectious form, the prion may cause many diseases such as bovine spongiform encephalopathy (commonly known as mad cow disease), Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker disease. The PrPSc and PrPC are widely different regarding secondary and tertiary structures. PrPC contains a much smaller number of ¿À-strands compared to PrPSc. No structures of PrPSc have been solved until nowadays, and furthermore the molecular mechanism of the transition between PrPC and PrPSc, and the aggregation process of the scrapie form are not well understood besides numerous studies achieved in this field. In this work, we applied a recently developed simulation method allowing to promote large structural changes, namely the MDeNM (Molecular Dynamics with excited Normal Modes) in order to better investigate and understand the conformational transition between PrPC and PrPSc. This method makes a combined use of normal modes and molecular dynamics, consisting to kinetically activate collective motions that might contribute to new structural rearrangements difficult to achieve by simple standard MD simulations. Furthermore, a thermodynamical study was achieved with the well know ¿¿-carbon structure-based model (SBM), which characterizes the energetic transitions. SBM results showed that the PrPSc structures that were generated with MDeNM must correspond to the transition pathways between the non-infectious native PrPC and the fully unfolded conformations. The combination of MDeNM and SBM was useful to extract structural to energetical information related to the prion conversion to the frustrated infections forms. The results showed a significant increase in ¿À-sheet formation under low pH condition and when PrPC was in trimeric form, and also when larger assemblies. These simulations allowed us to characterize intermediate states in the transition from the cellular prion to PrPSc, and a model of conversion from PrPC to PrPSc. Also, studies with mutants of prion and Coarse-Grained were performed in this work.

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Ein Ziel dieser Arbeit war die Entwicklung eines differentialdiagnostischen Tests (FLI-Test), der die Abgrenzung einer BSE- von einer Scrapieinfektion durch die direkte Untersuchung des Hirnstammmaterials ermöglicht. Bei einem Teil der dabei untersuchten deutschen klassi-schen Scrapiefälle wurde diese Charakterisierung zusätzlich im bis dahin zur Differenzierung verwendeten klassischen Mausbioassay durchgeführt, um die Ergebnisse aus dem FLI-Test zu verifizieren und um die vorhandenen Scrapieisolate weitergehend zu charakterisieren. Im zweiten Teil dieser Arbeit wurden die biochemischen Eigenschaften atypischer deutscher Scrapieisolate analysiert und ihre Infektiosität anhand von Übertragungsversuchen auf drei Wildtypmauslinien und eine transgene Mauslinie beurteilt. Darüber hinaus wurden diese Isolate dem klassischen BSE-Isolat gegenüber gestellt.

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Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources.

La Protéine Cellulaire du Prion (PrPc), isoforme non pathogène de la Protéine Scrapie, est une protéine ubiquitaire qui a été impliquée dans de nombreux processus cellulaires tels que la prolifération, la migration, l’adhésion, la différenciation et l’apoptose, par des mécanismes qui restent en grande partie à élucider. L’épithélium intestinal est en constant renouvellement et son homéostasie repose sur une régulation fine et coordonnée de l’ensemble de ces processus. Notre équipe s’est intéressée au rôle de la PrPc dans l’épithélium intestinal et a mis en évidence son expression dans le type cellulaire majoritaire de cet épithélium, les entérocytes, et sa double localisation selon leur état de différenciation. En effet, dans les cellules différenciées, la PrPc est majoritairement présente au niveau des desmosomes, alors que dans les cellules prolifératives, elle est principalement nucléaire. Nous mettons en évidence que la PrPc desmosomale est impliquée dans le maintien et l’intégrité de l’ensemble des jonctions intercellulaires (jonctions serrées, adhérentes et desmosomes) et contribue à la fonction de barrière de l’épithélium intestinal. La PrPc nucléaire, quant à elle, interagit avec plusieurs effecteurs de la voie de signalisation Wnt : la -caténine, la -caténine et le facteur de transcription TCF7L2. Dans ce contexte, nous révélons la capacité de la PrPc nucléaire à moduler l’expression de gènes cibles de la voie Wnt canonique. L’ensemble de ces travaux permet de révéler la PrPc comme un nouvel élément clé de l’homéostasie de l’épithélium intestinal – du maintien de la fonction de barrière jusqu’à la régulation de l’expression de gènes – et de définir la PrPc comme un nouveau membre de la famille des protéines NACos. / The cellular Prion Protein (PrPc), the normal conformer of the Scrapie protein, is a ubiquitous protein, which has been involved in several cellular processes such as proliferation, migration, adhesion, differentiation and apoptosis, through mechanisms that are not fully characterized. Intestinal epithelium is renewing constantly and its homeostasis requires a fine and coordinated regulation of all these processes. Our team has focused on PrPc functions in this tissue and has demonstrated that it is expressed in enterocytes, the major cell type in the intestinal epithelium, with a dual localization depending on the differentiation state of the cells. Indeed, in differentiated cells PrPc is localized in desmosomes while being mostly in the nucleus in proliferative cells. We demonstrated the involvement of desmosomal PrPc in the maintenance and integrity of all the intercellular junctions (tight, adherens junctions and desmosomes) and its requirement for the intestinal barrier function. PrPc in the nucleus interacts with key effectors of the Wnt pathway: -catenin, -catenin and the transcription factor TCF4/TCF7L2. In this context, we revealed the ability of nuclear PrPc to modulate the expression of a subset of Wnt target genes. Altogether, this work highlights the role of PrPc as a new key element of the intestinal epithelial homeostasis – from the barrier function to gene regulation – and allows considering PrPc as a new member of the NACos family proteins (proteins associated with the Nucleus and Adhesion Complexes).

The host encoded cellular prion protein (PrPC) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrPC can undergo conversion into a conformationally-altered isoform (PrPSc) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Thus, tissues expressing PrPC are potential sites for conversion of PrPSc during TSE pathogenesis. Although much is known about the role of PrPSc in prion diseases, the normal function of PrPC is poorly understood. Lines of mice and cattle in which PrPC has been ablated by gene knockout show no major phenotypical alterations other than resistance to TSE infection. However, recent reports using Prnp-null mouse models have suggested the participation of PrPC in neural stem/progenitor cell proliferation and differentiation. The first objective in our study was to map the expression of PrPC in twenty six somatic and reproductive tissues in ruminants. Our second objective was to characterize the ontogeny of PrPC expression during bovine embryonic and early fetal development. Finally, we used a mouse embryonic stem cell (mESC) model to study the potential role of PrPC during neurogenesis. In adult tissues, intense expression of PrPC was detected in the central nervous system (CNS), thymus and testes, whereas the liver, striated muscle and female reproductive tissues showed the lowest expression. We observed that PrPC was associated with tissues undergoing cellular differentiation including spermatogenesis, lymphocyte activation and hair follicle regeneration. Analyses in bovine embryos and fetuses indicated peaks in expression of PrPC at days 4 and 18 post-fertilization, stages associated with the maternal-zygote transition and the maternal recognition of pregnancy and initiation of placental attachment, respectively. Later in development, PrPC was expressed in the CNS where it was localized in mature neurons of the neuroepithelium and emerging neural trunks. Based on these observations, we hypothesized that PrPC was involved in neurogenesis. We tested this hypothesis in a murine embryonic stem cell model (mESC). mESC were induced to form embryoid bodies (EBs) by placing them in suspension culture under differentiating conditions and allowed to differentiate in vitro for 20 days. We detected increasing levels of PrPC starting on day 12 (8.21- fold higher vs. day 0; P < 0.05) and continuing until day 20 (20.77-fold higher vs. day 0; P < 0.05). PrPC expression was negatively correlated with pluripotency marker Oct-4 (r= -0.85) confirming that mESC had indeed differentiated. To provide a more robust system for assessing the role of PrPC in neural differentiation, mESC were cultured with or without retinoic acid (RA) to encourage differentiation into neural lineages. Induction of EBs with retinoic acid (RA) resulted in an earlier up-regulation of PrPC and nestin (day 12 vs. day 16; P < 0.05). In addition, immunofluorescence studies indicated co-expression of PrPC and nestin in the same cells. The results of these experiments suggested a temporal link between PrPC expression and expression of nestin, a marker of neural progenitor cells. We next tested whether PrPC was involved in RA-enhanced neural differentiation from mESC using a PrPC knockdown model. Plasmid vectors designed to express either a PrP-targeted shRNA or scrambled, control shRNA were transfected into mESC. Stable transfectants were selected under G418 and cloned. PrP-targeted and control shRNA clones, as well as wild-type mESC, were differentiated in presence of RA and sampled as above. PrPC expression was knocked down in PrP-targeted shRNA cultures between days 12 and 20 (62.2 % average reduction vs. scrambled shRNA controls). Nestin expression was reduced at days 16 and 20 in PrPC knockdown cells (61.3% and 70.7%, respectively vs. scrambled shRNA controls). These results provide evidence that PrPC plays a role in the neural differentiation at a point up-stream from the stages at which nestin is expressed. In conclusion, the widely distributed expression of PrPC in ruminant tissues suggests an important biological role for this protein. In the present work we have provided evidence for the participation of PrPC in the differentiation of mESC along the neurogenic pathway. / Ph. D.

Die genaue physiologische Funktion des zellulären Prion-Proteins (PrPC) ist noch immer nicht vollständig verstanden. Eine mögliche Funktion des PrPC auf das neuronale Überleben nach einem hypoxischen oder ischämischen Insult wird diskutiert. In einem Vorversuch zeigten sich nach zerebraler Ischämie deutlich größere Infarktvolumina in den Gehirnen von Prion-Knock-Out-Mäusen im Vergleich zu denen der Wild-Typ-Mäuse. Das Identifizieren der molekularen Mechanismen der PrPC-vermittelten Neuroprotektion ist daher von großem Interesse und machte die Etablierung eines Zell-Modells erforderlich. Neuere Studien konnten einen Einfluss des zellulären Prion-Proteins auf die Glykolyse nachweisen. Unter Sauerstoffmangelbedingungen kommt es zu einer vermehrten Bildung von Laktat durch das Enzym Laktat-Dehydrogenase (LDH). Neurone benötigen unter hypoxischen oder ischämischen Bedingungen dieses Laktat als Energiesubstrat. Je mehr Laktat den Neuronen zur Verfügung steht, umso höher ist das neuronale Überleben. In dieser Arbeit konnte die Beteiligung der Laktat-Dehydrogenase an der durch das zelluläre Prion-Protein vermittelten Neuroprotektion nach Hypoxie nachgewiesen werden. Das Ziel dieser Arbeit bestand darin, mögliche Unterschiede der LDH-Expression in WT-Zellen, Prnp0/0-Zellen und HEK-293-Zellen unter normalen und hypoxischen Bedingungen in vitro zu untersuchen. Die Expression der LDH war unter hypoxischen Bedingungen in den WT-Zellen im Vergleich zu den Prnp0/0-Zellen deutlich höher. Dies konnte auch in PrPC-überexprimierenden HEK-293-Zellen nach Hypoxie gezeigt werden. Ebenso konnte nachgewiesen werden, dass Hypoxie zu einem größeren Schaden des Tubulinzytoskelettes in Prnp0/0-Zellen führt als in WT-Zellen, was eine neuroprotektive Wirkung von PrPC vermuten lässt. Eine direkte oder indirekte Interaktion von LDH-A und PrPC konnte durch eine Co-Immunpräzipitation in HEK-293-Zellen nachgewiesen werden. Die genauen Mechanismen über die PrPC möglicherweise zu einer vermehrten Laktat-Produktion führt, sind noch nicht eindeutig identifiziert und müssen noch näher untersucht werden. Zusammengefasst kann gesagt werden, dass die erhobenen Daten die Vermutung verstärken, dass das Enzym LDH und sein Produkt Laktat in die durch das zelluläre Prion-Protein vermittelte Neuroprotektion nach Hypoxie involviert sind. Es ist das erste Mal, dass gezeigt wurde, durch welchen Mechanismus PrPC zur Neuroprotektion beiträgt.