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.

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.

Cellular prion protein (PrPC ) is a membrane bound glycoprotein. The protein is expressed in all vertebrates, mainly in the nervous system, but it is present also in the cells of gastrointestinal tract, bone marrow, germ cells and heart. PrPC is necessary for pathogenesis of prion diseases, which are deadly and without the possibility of therapy. The pathogenic isoform of prion protein is formed by changing of secondary structure of PrPC and it's the main constituent of infectious prion particles. Pathological form of prion protein accumulates in brain of infected patients and this process is associated with neurodegradation. Physiological function of PrPC is poorly understood. Knock-out of the PrPC gene (PRNP) is not connected with any noticeable phenotype. Potential functions of PrPC are dispersed, protein may have antiapoptotic effect, it can be involved in ions metabolism or in protection against oxidative stress. Latest results show, that PrPC can play important role in cell differentiation. During the differentiation PrPC can influence the development of cells and their typing. It could affect cell cycle and have an influence on formation of nervous system. Aim of the present study was to elucidate, whether the down-regulation of PrPC or infection with prions has an impact on differentiation of...

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Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that affect several species, such as scrapie in sheep or goats and CJD in humans. In several species, neurological disease is preceded by TSE agent accumulation in lymphoid tissues prior to neuroinvasion. While oral transmission is considered the most common route for scrapie, transmission can also occur through lesions to the skin or mucosa, for example in the mouth or gastrointestinal tract due to rough feed, or birth associated skin damage. Scrapie has also been experimentally transmitted through skin scarification in mice. Following scrapie infection via skin scarification, PrPSc accumulates in the draining lymph node (LN) before spreading to other organs in the lymphoreticular system. It is not yet known by what means the scrapie agent is transported from the skin to the draining LN. Dendritic cells (DCs) in the skin have been found to transport viruses, such as HIV or Dengue, from the skin, thereby raising the question whether DCs or Langerhans cells (LCs), located within the epidermis, play a role in the uptake and transport of the TSE agent from the skin to the draining LN. CD11c is a cell surface marker traditionally used to identify or isolate DCs from other cell types. Mice and rats are naturally resistant to Diphtheria toxin (DTX). A transgenic mouse line was created where the Diphtheria toxin receptor (DTR) was expressed on CD11c+ cells. The presence of this receptor on CD11c+ cells allowed for the temporary conditional depletion of CD11c+ cells following a single injection of DTX. The cells repopulate the tissues within a time frame specific to the tissues the cells are located in. These mice were used to determine whether the absence of CD11c+ cells at the time of scrapie infection via the skin had an effect on the early accumulation of PrPSc within the lymphoid tissues and on disease progression. Immunohistochemical analysis demonstrated that early PrPSc accumulation in the draining LNs was delayed following depletion of CD11c+ cells, indicating that their potential role in the transport of the scrapie agent from the skin. Scrapie incubation period was not affected by the absence of the CD11c+ cells at the time of infection. Recent findings show that CD11c is not exclusive to DCs and is also expressed on macrophage populations. Following DTX-mediated depletion, DCs repopulate the tissues much faster than CD11c+ macrophages. Scrapie infection was carried out in the skin in DTX treated mice after DCs had repopulated the tissues but before macrophage numbers had returned, to determine whether macrophages rather than DCs played a role in the early accumulation of PrPSc in the draining LNs. No differences in PrPSc accumulation were observed in mice depleted of macrophages compared to controls and there was no effect on disease incubation period. Another transgenic mouse line was used, where DTX expression on langerin+ cells (LCs and langerin+ DCs in the dermis), allowed for their temporary depletion through DTX treatment. Following langerin+ cell depletion, increased PrPSc accumulation was observed in the draining LNs 7 weeks post infection, but did not affect the incubation period of disease. These results indicate that the absence of LCs somehow accelerated PrPSc accumulation, and that LCs might play a preventative role in early stages after infection. Histopathological analysis was used to complement microarray studies aimed to determine what immune responses were associated with scarification and DTXmediated depletion of cells within the skin and whether these responses might be linked to disease transmission. DCs and LCs in the skin appear to play different roles in the early stages following scrapie infection via the skin, but the lack of effect on incubation period does not rule out the involvement of other cell types or cell-free mechanisms of scrapie agent spread from the skin.

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.

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.

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.

Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrP accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.