Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia / Made available in DSpace on 2013-03-04T20:33:26Z (GMT). No. of bitstreams: 1 313812.pdf: 1486669 bytes, checksum: 8cf915721b889545c16ee9700e4ce593 (MD5) / O consumo de drogas com potencial aditivo, como o etanol, induz alterações sinápticas profundas na via mesocorticolímbica, que estão associadas com as alterações comportamentais observadas em indivíduos adictos. Desta maneira, a investigação de sistemas capazes de modular a neurotransmissão dopaminérgica pode revelar novos alvos, com intuito de interferir no processo de adição às drogas. Neste trabalho, demonstramos de maneira inédita a interação entre a proteína prion celular (PrPC) e o sistema dopaminérgico e que esta interação é capaz de modular diversas propriedades aditivas do etanol. Demonstramos a presença da PrPC em neurônios dopaminérgicos e a importância desta proteína na modulação dos níveis de dopamina em diferentes áreas cerebrais, na expressão de receptores dopaminérgicos e do seu mecanismo efetor mais importante, a DARPP-32. A deleção genética ou o bloqueio farmacológico da PrPC também alteraram processos seminais para o desenvolvimento da adição ao etanol, como o desenvolvimento da tolerância rápida, o consumo oral voluntário, e comportamentos motivacionais, assim como propriedades de transmissão basal e de plasticidade utilizando eletrofisiologia extracelular cortico-estriatal. Em conjunto, nossos dados indicam que a PrPC tem um papel fundamental sobre a homeostasia da neurotransmissão dopaminérgica e que a interação PrPC/dopamina modula as propriedades aditivas ao etanol em camundongos

A proteína prion celular (PrPC) consiste em uma glicoproteína de membrana que atua como receptora para diversas moléculas, desencadeando sinais intracelulares. Ao interagir com a co-chaperona STIP1, PrPC promove a autorrenovação e proliferação de células-tronco/progenitoras neurais (NSPCs) durante a fase embrionária. De fato, PrPC tem se destacado por sua participação na neurogênese embrionária e adulta, porém o papel de sua interação com a proteína STIP1 na neurogênese adulta permanece obscuro. Deste modo, o presente trabalho adotou abordagens in vitro para avaliação do complexo PrPC-STIP1 em processos celulares que culminam na neurogênese adulta. Para isso, culturas primárias de NSPCs de camundongos deficientes (Prnp-/-) e tipo-selvagens (Prnp+/+) para PrPC foram realizadas, e a cultura foi devidamente padronizada e caracterizada. Através de ensaios de autorrenovação, proliferação e migração celular sugere-se que PrPC promove estes eventos celulares independentemente de STIP1, e que possivelmente a proteína laminina seja um alvo crítico para migração via PrPC. / Cellular prion protein (PrPC) consists in a membrane glycoprotein that acts as a receptor to several molecules, triggering intracellular signals. By interacting with co-chaperone STIP1, PrPC promotes self-renewal and proliferation of neural stem/progenitor cells (NSPCs) during embryonic stage. Indeed, PrPC has excelled for its participation in embryonic and adult neurogenesis, but the role of its interaction with STIP1 protein in adult neurogenesis remains unclear. Thus, herein it was adopted in vitro approaches in order to evaluate the PrPC-STIP1 complex on cellular processes that culminate in adult neurogenesis. In order to assess that, NSPC primary cultures of PrPC deficient (Prnp-/-) and wild-type (Prnp+/+) mice were performed, and the culture was properly standardized and characterized. Through self-renewal, proliferation and cell migration assays, it was suggested that PrPC promotes these cellular events regardless of STIP1, and possibly the laminin protein is a critical target for migration via PrPC.

A proteína prion celular (PrPC) consiste em uma glicoproteína de membrana que atua como receptora para diversas moléculas, desencadeando sinais intracelulares. Ao interagir com a co-chaperona STIP1, PrPC promove a autorrenovação e proliferação de células-tronco/progenitoras neurais (NSPCs) durante a fase embrionária. De fato, PrPC tem se destacado por sua participação na neurogênese embrionária e adulta, porém o papel de sua interação com a proteína STIP1 na neurogênese adulta permanece obscuro. Deste modo, o presente trabalho adotou abordagens in vitro para avaliação do complexo PrPC-STIP1 em processos celulares que culminam na neurogênese adulta. Para isso, culturas primárias de NSPCs de camundongos deficientes (Prnp-/-) e tipo-selvagens (Prnp+/+) para PrPC foram realizadas, e a cultura foi devidamente padronizada e caracterizada. Através de ensaios de autorrenovação, proliferação e migração celular sugere-se que PrPC promove estes eventos celulares independentemente de STIP1, e que possivelmente a proteína laminina seja um alvo crítico para migração via PrPC. / Cellular prion protein (PrPC) consists in a membrane glycoprotein that acts as a receptor to several molecules, triggering intracellular signals. By interacting with co-chaperone STIP1, PrPC promotes self-renewal and proliferation of neural stem/progenitor cells (NSPCs) during embryonic stage. Indeed, PrPC has excelled for its participation in embryonic and adult neurogenesis, but the role of its interaction with STIP1 protein in adult neurogenesis remains unclear. Thus, herein it was adopted in vitro approaches in order to evaluate the PrPC-STIP1 complex on cellular processes that culminate in adult neurogenesis. In order to assess that, NSPC primary cultures of PrPC deficient (Prnp-/-) and wild-type (Prnp+/+) mice were performed, and the culture was properly standardized and characterized. Through self-renewal, proliferation and cell migration assays, it was suggested that PrPC promotes these cellular events regardless of STIP1, and possibly the laminin protein is a critical target for migration via PrPC.

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.

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.

Transmissible spongiform encephalopathies (TSEs) are rare, uniformly fatal neurodegenerative disorders that can affect many mammalian species, including humans. A hallmark of these diseases is the conversion of cellular prion protein (PrPC) into an abnormally folded form. This misfolded PrPC is infectious, since it can provide a template for pathogenic conversion of PrPC in a new host. In addition to any toxicity of the misfolded protein, loss of normal PrPC function could be involved in the neurodegenerative processes. However, the physiological role of PrPC is still poorly understood and this project has aimed to address that lack of knowledge. Out of the many putative functions ascribed to PrPC, the most commonly proposed is that it protects cells from stress. In contrast, I have found that stable transfection of the prion protein gene into SH-SY5Y neuroblastoma cells increases cell death in response to serum removal from the culture medium. Following treatment with several chemical toxins, two out of four stably transfected clones did, generally, display greater viability than untransfected cells that do not express detectable levels of PrPC. However, knockdown of PrPC expression by RNA interference had no effect on this stress resistance, indicating that it may not have been mediated directly by PrPC. Given the lack of robust stress protection afforded by PrPC transfection, proteomic analyses of the cells were carried out to identify alternative processes that were perturbed as a result of PrPC expression. The results obtained suggested roles for PrPC in cytoskeletal organisation and cell cycle regulation. Various proteins involved in cytoskeletal organisation were confirmed by western blotting to be differentially expressed in some or all of the stably transfected clones. Additionally, the expression changes to proteins involved in cell cycle regulation resulted in slower proliferation of the clones compared with untransfected cells, a difference that was reduced following RNA interference-mediated knockdown of PrPC. Taken together, these data suggested that specific growth factor-activated pathways were differentially regulated in the stably transfected clones. One candidate pathway was nerve growth factor (NGF) signalling, which promotes neuronal survival and differentiation as well as regulating various processes outside of the nervous system. PrPC-transfection resulted in altered expression of receptors for NGF, suggesting that the stably transfected clones were, indeed, responding differently to NGF stimulation. However, the molecular mechanism responsible for these expression changes remains to be determined, since co-immunoprecipitation experiments did not identify any physical interactions between PrPC and the NGF receptors. Nonetheless, a role for PrPC in modulating NGF signalling has the potential to explain many of the diverse phenotypic observations in PrPC-null mice and might indicate that loss of PrPC function is an important part of TSE pathogenesis.

Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, São José dos Pinhais, 2011 / Inclui bibliografias / Scrapie é uma doença neurodegenerativa, progressiva e fatal de ovinos e caprinos, pertencente ao grupo das Encefalopatias Espongiformes Transmissíveis (EETs), ou doenças priônicas. O acúmulo de uma isoforma normal (PrPSc) da proteína prion celular (PrPC) / Scrapie is a fatal, neurodegenerative disease that affects sheep and goats and belongs to the Transmissible Spongiform Encephalopathies (TSEs) or prion diseases. It is caused by the deposition of an abnormal isoform (PrPSc) of the host-encoded cellular pr

Made available in DSpace on 2014-12-05T18:40:14Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) luiz_berbert_ioc_mest_2010.pdf: 5652664 bytes, checksum: 7e9002e7c7fc1786ff895512aa437d50 (MD5) Previous issue date: 2014-10-07 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro,RJ, Brasil / A proteína Príon celular (PrPc) é uma glicoproteína constitutivamente expressa no sistema nervoso, bem como no sistema imune, porém, seu papel fisiológico e em processos infecciosos é pouco compreendido. Dados prévios da literatura sugerem disfunções fisiológicas no sistema imune em modelos murinos transgênicos que superexpressam essa proteína podendo levar os mesmos a um processo de imunodeficiência mediante infecçãos. Para testar essa hipótese, este projeto tem o objetivo de avaliar o impacto da infecção experimental pelo T. cruzi em camundongos que superexpressam (TG20) ou não expressam PrPc (KO), analisando parâmetros de infecção correlacionados com a infiltração linfocitária no tecido cardíaco. Nossos resultados demonstram que a superexpressão de PrPc em camundongos gera inicialmente susceptibilidade do modelo à infecção se comparado ao grupo que não expressa a proteína, conforme observado nas taxas de sobrevida e parasitemia A presença de células T, B, bem como células CD11b+ de infiltrado inflamatório é numericamente maior no grupo TG20, porém, esse evento não parece estar relacionado diretamente com alterações observadas no baço e linfonodos desses animais durane cinética de infecção; em relação à expressão de moléculas de ativação linfócitos nestes modelos, observamos aumento das mesmas nas subpopulações linfocitárias do grupo TG20. Nos timo dos modelos TG20 foram observadas alterações fenotípicas, bem como aumento de espressão de laminina e fibronectina e seus receptores nos timócitos. Os dados deste trabalho demonstram que Prpc tem um grande impacto na fisiologiada infecção pelo T. cruzi no modelo murino que superexpressa essa proteína, correlacionando esse evento a uma imunodeficiência previamente observada no mesmo modelo não infectado / Cellular Príon Protein (PrP c ) is a glycoprotein constitutively expressed in nervous an d immune systems, but its physiological role is not well und erstood. Previous data suggest that murine models which overexpress PrP c show physiological disfunctions that could be related with a possible immunodeficiency, which might lead to poor immune response against infections. To support this hypothesis, this project has the aim to evaluate the impact of experimental T. cruzi infection in mice that overexpress or do not express PrP c , correlating infection parameters to lymphocyte infilt ration on heart tissue. Our results demonstrated that PrP c overexpression in mice leads to animal susceptibility during acute infection when compared to KO group, comp aring survival and parasitemia rates on these groups. Inflammatory infiltrate cells such as T and B lymphocytes, as well as CD11b + cells are present in higher numbers in TG20 model, but it seems to be not correlated with spleen and lymph nodes alteractions duri ng infection kinectics; activation molecules are highly express in TG20 periferical lympho cytes. TG20 mouse thymus showed phenotypic alteractions, as well as higher expressio n of laminin and fibronectin and its receptors in thymocytes, which could be possibily re lated to immature thymocyte exportation and their presence in heart tissue. Those data suggest thar PrP c has an important impact during T. cruzi infection in murine model that overexpress PrP c , correlating these alteractions to previous immunodeficien cy in non infected TG20 murine models

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...