Dissertations / Theses on the topic 'Shadow of prion protein gene'

Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes.¶The normal function of PRNP is elusive. The Prnp knock-out mice with disrupted ORF show only very subtle phenotype. A number of hypotheses were proposed on the function of mammalian PRNP. The extracellular, GPI-anchored, glycosylated mammalian PrP^C expressed in a heterogenous set of cells could: transport copper from extracellular to intracellular milieu, buffer copper from synapse, contribute to redox signalling, act neuroprotectively, mediate cell-cell contacts, affect lymphocyte activation, participate in nucleic acid metabolism, be a memory molecule, and be a signal-transduction protein.¶ Experimental evidence demonstrated a redundancy between the PRNP and another, unknown gene. The critical issue therefore is to discover new genes homologous with PRNP, candidates for this redundancy. Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof. Tatjana Simonic’s group (University of Milan, Italy), I discovered a new paralogue of PRNP. By searching manually, and in a targeted fashion, data deposited in public biological databases, I compiled support for the new human gene Shadow of prion protein (SPRN) including the direct evidence, homology-based evidence and ab initio gene prediction. The protein product called Shadoo (shadow in Japanese) is an extracellular, potentially glycosylated and GPI-anchored protein of a mature size of 100-odd amino acids. It is conserved from fish (zebrafish, Fugu, Tetraodon) to mammals (human, mouse, rat), and exhibits similarity of overall protein features with PrP. Most remarkably, the Sho is the first human/mammalian protein apart from PrP that contains the middle hydrophobic region that is essential for both normal and pathogenic properties of PrP. As this region is critical for heterodimerization of PrP, Sho may have potential to interact with PrP and is a likely candidate for the Protein X. Mammalian SPRN could be predominantly expressed in brain (Tatjana Simonic Lab, University of Milan, Italy).¶ Using the same approach to search public databases, I found, in addition, a fish duplicate of SPRN called SPRNB, and defined a new vertebrate SPRN gene family. Further, I also expanded a number of known fish genes from the PRNP gene family. The total number of the new genes that I discovered is 11. With the representatives of two vertebrate gene family datasets in hand, I conducted comparative genomic analysis in order to determine evolutionary trajectories of the SPRN and PRNP genes. This analysis, complemented with phylogenetic studies (Dr. Lars Jermiin, University of Sydney, Australia), demonstrated conservative evolution of the mammalian SPRN gene, and more relaxed evolutionary constraints acting on the mammalian PRNP gene. This evolutionary dialectic challenges widely adopted view on the “highly conserved vertebrate” PRNP and indicates that the SPRN gene may have more prominent function. More conserved Sprn could therefore substitute for the loss of less conserved, dispensable Prnp in the Prnp knock-out mice. Furthermore, the pathogenic potential of PRNP may be a consequence of relaxed evolutionary constraints.¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells.

The prion protein (PrP^c) is a normal host-encoded glycoprotein which accumulates as a disease specific protease-resistant isoform (PrP^sc) in the brains of infected hosts. In a number of species PrP polymorphisms and germline mutations are associated with the modulation of disease phenotype and the occurrence of familial prion disease. To investigate the biological consequences of manipulation of the prion protein in mice a flexible two step double replacement gene targeting strategy was developed. This method can be used to generate a series of mouse lines with alterations to the mouse prion protein gene (Prn-p). To facilitate gene targeting studies a restriction map of the 129/Ola Prn-p locus was constructed and a series of overlapping genomic clones were retrieved from a λ DASH II bacteriophage 129/Ola library. The double replacement strategy was used to generate PrP deficient mice and mice with subtle alterations to PrP codons 108 and 189. Murine PrP 108F/V_189L/T dimorphisms give rise to 2 distinct PrP allotypes, PrP-A and PrP-B and these are postulated to be responsible for the control of incubation time following challenge with a wide range of prion inocula. To test this proposal the endogenous 129/Ola PrP-A allotype [108L_189T] was converted by gene targeting to encode the PrP-B allotype [108F_189V]. Mice bearing codon 108 and 189 alterations were challenged with mouse adapted BSE isolate 301V. Gene targeting in 129/Ola derived HM-1 ES cells and breeding with 129/Ola mice enabled the investigation of the effect of PrP alterations in the absence of PrP overexpression artefacts or the influence of non-Prn-p genes. The dramatic acceleration of incubation time in mice homozygous for the Prn-p^a[108F_189V] gene targeted allele confirmed the major role of codons 108 and 189 in the control of BSE isolate 301V incubation time - and probably other prion isolates. This data provides the strongest evidence yet that the incubation time control, long attributed to the action of different alleles of Sinc (Prn-i), is determined by PrP codon 108L/F and 189T/V dimorphisms.

En el gen de la proteïna priònica, o PRNP, hem observat que el particular patró de variació que hem trobat basant-nos en dades de seqüenciació en humans es deu a selecció positiva, i que el mètode utilitzat per detectar selecció és crític. Utilitzant dades basades en SNPs es pot introduir un biaix al aplicar tests de neutralitat basats en diversitat de seqüències, i això pot portar a conclusions errònies. A més, hem vist que els polimorfismes en els codons 129 i 219 presenten gran diferències de freqüència en diferents poblacions humanes i també hem vist que aquestes posicions estan fixades en ximpanzés. La variació trobada en controls ha estat comparada amb el patró de variació existent en pacients per la mateixa regió. La reseqüenciació del gen PRNP en un gran nombre de mostres humanes i de ximpanzés ens ha permès obtenir un gran nombre d´informació d´aquest gen.
In the prion gene or PRNP, we have observed that the particular pattern of variation that we have found in this gene based on sequencing data in humans is due to positive selection, and that the method and the approach used to detect this selection critical. Ascertainment bias can be introduced by using SNP data and applying neutrality tests based on sequence diversity, therefore leading to anomalous conclusions being drawn. Moreover, we have seen that polymorphisms in codon 129 and 219 have big differences in frequency in different human populations and we have also seen that these positions are fixed in chimpanzees. The normal variation that we found in controls have been then compared with patients for the same region. The resequencing of PRNP in a very large sample of humans and chimpanzees has provided a great deal of information on this gene.

v It was found that most of the classical scrapie genotypes belong to R3 risk group, whereas atypical scrapie genotypes belonging to zero (0) and one (1) risk groups were frequently seen in sheep analyzed. In other words, Turkish sheep is found to have intermediate risk of classical scrapie and low atypical scrapie risk, in general. The data from the current study may help to establish a breeding program for classical scrapie control in Turkey and will be beneficial for both the animal and public health in the country. In addition, the outcomes of the study will fill the gap which is present in the geographic distribution data of PrP gene polymorphisms in Eurasia.Scrapie is an infectious fatal disease of sheep and goats which affects the central nervous system. In the present study, samples of 14 native Turkish sheep breeds (n=655) were analyzed with respect to their polymorphisms of PrP gene (at codons: 136, 141, 154 and 171) and their classical and atypical scrapie risk levels were identified. Turkish sheep are found to have the highest PrP genetic variability with 13 classical scrapie alleles and 14 atypical scrapie alleles compared to all previous studies. Classical scrapie-susceptible and wild-type ARQ allele was found as the most frequent allele in Turkish sheep examined. The most classical scrapie-susceptible allele, VRQ was detected at low frequencies in 5 of the breeds (Ç
ine Ç
apari, Dagliç
, Kivircik, Karayaka and Gö
kç
eada). One novel allele (TL141HQ) was observed in Sakiz breed for the first time in this study.

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.

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

INTRODUÇÃO: Os polimorfismos do gene da proteína priônica (PRNP) podem estar associados a doenças neurológicas não priônicas. Estudos em pacientes com doença de Alzheimer (DA) apontam para possível associação entre os polimorfismos do códon 129 do PRNP e DA. Essa associação não foi estudada na população brasileira. Neste estudo, descrevemos a associação entre os diferentes polimorfismos do PRNP e DA. MÉTODOS: Foi estudada amostra composta por 100 pacientes com DA, acompanhados no Ambulatório de Neurologia Cognitiva e do Comportamento e no Centro de Referência em Distúrbios Cognitivos do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, pareados para grupo controle com 111 indivíduos, em relação à frequência dos diferentes polimorfismos do PRNP e o desempenho cognitivo. Os polimorfismos do PRNP foram estudados pelo método de cromotografia líquida de fase reversa desnaturante (DHPLC). Foi realizada extratificação da amostra pelo genótipo da apolipoproteína E (apoE). RESULTADOS: A frequência dos polimorfismos do códon 129 foi: 45,5% M/M, 42,4% M/V e 12,2% V/V nos pacientes com DA; e 39,6% M/M, 50,5% M/V e 9,9% V/V nos indivíduos controles (p=0,503). O códon 117 apresentou variante alélica silenciosa em 5% dos pacientes com DA e 3% dos controles (p=0,780). A deleção de um ocatapeptídeo repetido ocorreu em 5% dos pacientes com DA e 4% dos controles (p=0,738). Todos os pacientes com DA e os controles eram N171N. Uma paciente do grupo com DA apresentou a mutação V180I. A análise bivariada e regressão logística não mostraram associação entre os diferentes polimorfismos do códon 129 e o desempenho cognitivo nos pacientes com DA, assim como nos indivíduos cognitivamente normais. A extratificação segundo genótipo da apoE não revelou diferença em relação aos polimorfismos do códon 129 do PRNP entre os grupos DA e controles. CONCLUSÕES: Não houve diferença de frequência dos diferentes polimorfismos do códon 129 do PRNP entre os pacientes com DA e idosos cognitivamente normais, bem como em relação aos demais códons polimórficos do gene. Não houve diferença em relação ao desempenho cognitivo nos pacientes com DA e nos controles segundo o polimorfismo do códon 129 do PRNP. Um paciente apresentou mutação do códon 180 (V180I), e recebeu o diagnóstico de doença de Creutzfeldt-Jakob genética
INTRODUCTION: The polymorphism in the prion protein gene (PRNP) may influence non prion neurological diseases. Some reports associate Alzheimers disease (AD) and the polymorphic codon 129 of the PRNP. This association has not been studied in Brazilian population. In this study we aimed to describe the association between the polymorphisms of codon 129 of the PRNP and AD. METHODS: One hundred AD patients were evaluated in the Cognitive and Behavioral Neurology Unit and Cognitive Disorders Reference Center of the Hospital das Clínicas of the University of São Paulo School of Medicine, matched for 111 controls, regarding to the PRNP polymorphism and cognitive measures. The PRNP polymorphisms were analyzed using denaturing high-performance liquid chromatography (DHPLC). Analyzes stratifying by apoE genotype was performed. RESULTS: The distribution of the codon 129 polymorphisms were: 45.5% M/M, 42.4% M/V and 12.2% V/V in AD patients; 39.6% M/M, 50.5% M/V and 9.9% V/V in the control group (p=0.503). The 117 codon analysis revealed silent allelic variant in 5% of AD patients and 3% of controls (p=0.780). The octarepeat deletion occurred in 5% of AD and 4% of controls (p=0.738). All AD patients and controls were N171N. One AD patient had a point mutation at codon 180 (V180I). Logistic regression failed to confirm any association between AD cognitive performance and the codon 129 of PRNP, as well as in the control group. There was no association between the codon 129 genotypes and genotypes and AD according to the apoE stratification. CONCLUSIONS: There were no differences in the frequency of the codon 129 polymorphism between AD. control group, according to the codon 129 polymorphisms. A point mutation at the codon 180 (V180I) was diagnosed in one patient

De nombreux travaux récents ont démontré l'importance de la stochasticité dans l'expression des gènes à différentes échelles. On passera tout d'abord en revue les principaux résultats expérimentaux pour motiver l'étude de modèles mathématiques prenant en compte des effets aléatoires. On étudiera ensuite deux modèles particuliers où les effets aléatoires induisent des comportements intéressants, en lien avec des résultats expérimentaux: une dynamique intermittente dans un modèle d'auto-régulation de l'expression d'un gène; et l'émergence d'hétérogénéité à partir d'une population homogène de protéines par modification post-traductionnelle.\\ Dans le Chapitre I, nous avons étudié le modèle standard d'expression des gènes à trois variables: ADN, ARN messager et protéine. L'ADN peut être dans deux états, respectivement ''ON'' et ''OFF''. La transcription (production d'ARN messagers) peut avoir lieu uniquement dans l'état ''ON''. La traduction (production de protéines) est proportionnelle à la quantité d'ARN messager. Enfin la quantité de protéines peut réguler de manière non-linéaire les taux de production précédent. Nous avons utilisé des théorèmes de convergence de processus stochastique pour mettre en évidence différents régimes de ce modèle. Nous avons ainsi prouvé rigoureusement le phénomène de production intermittente d'ARN messagers et/ou de protéines. Les modèles limites obtenues sont alors des modèles hybrides, déterministes par morceaux avec sauts Markoviens. Nous avons étudié le comportement en temps long de ces modèles et prouvé la convergence vers des solutions stationnaires. Enfin, nous avons étudié en détail un modèle réduit, calculé explicitement la solution stationnaire, et étudié le diagramme de bifurcation des densités stationnaires. Ceci a permis 1) de mettre en évidence l'influence de la stochasticité en comparant aux modèles déterministes; 2) de donner en retour un moyen théorique d'estimer la fonction de régulation par un problème inverse. \\ Dans le Chapitre II, nous avons étudié une version probabiliste du modèle d'agrégation-fragmentation. Cette version permet une définition de la nucléation en accord avec les modèles biologistes pour les maladies à Prion. Pour étudier la nucléation, nous avons utilisé une version stochastique du modèle de Becker-Döring. Dans ce modèle, l'agrégation est réversible et se fait uniquement par attachement/détachement d'un monomère. Le temps de nucléation est définit comme le premier temps où un noyau (c'est-à-dire un agrégat de taille fixé, cette taille est un paramètre du modèle) est formé. Nous avons alors caractérisé la loi du temps de nucléation dans ce modèle. La distribution de probabilité du temps de nucléation peut prendre différente forme selon les valeurs de paramètres: exponentielle, bimodale, ou de type Weibull. Concernant le temps moyen de nucléation, nous avons mis en évidence deux phénomènes importants. D'une part, le temps moyen de nucléation est une fonction non-monotone du paramètre cinétique d'agrégation. D'autre part, selon la valeur des autres paramètres, le temps moyen de nucléation peut dépendre fortement ou très faiblement de la quantité initiale de monomère . Ces caractérisations sont importantes pour 1) expliquer des dépendances très faible en les conditions initiales, observées expérimentalement; 2) déduire la valeur de certains paramètres d'observations expérimentales. Cette étude peut donc être appliqué à des données biologiques. Enfin, concernant un modèle de polymérisation-fragmentation, nous avons montré un théorème limite d'un modèle purement discret vers un modèle hybride, qui peut-être plus utile pour des simulations numériques, ainsi que pour une étude théorique.

Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes. ¶ ... ¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells