Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. Polymorphisms that alter the prion protein of sheep or humans have been associated with transmissible spongiform encephalopathy susceptibility or resistance. In contrast, there is no strong evidence that non-synonymous mutations in the bovine prion gene (PRNP) are associated with classical BSE disease susceptibility. However, two bovine PRNP insertion/deletion polymorphisms, one within the promoter region and the other in intron 1, have been associated with susceptibility to classical BSE. These associations do not explain the full extent of BSE susceptibility, and loci outside of PRNP appear to be associated with disease incidence in some cattle populations. To test for associations with BSE susceptibility, we conducted a genome wide scan using a panel of 3,072 single nucleotide polymorphism markers on 814 animals representing cases and control Holstein cattle from the United Kingdom BSE epidemic. A higher resolution, 50K SNP, genome-wide scan was used with a sub-set (N=330) of animals to test for association with BSE incidence. Further, the bovine prion gene was tested, 19 haplotype tagging SNPs and 2 insertion/deletion alleles for association with BSE disease incidence. The Holstein case and control cattle analyzed here consisted of two sets, one set with known family relationships and the second set with BSE cases with paired controls. The family set comprised of half-sibling progeny from six sires, revealed 27 SNPs representing 18 chromosomes associated with incidence of BSE disease, confirming previously reported chromosomal regions. Further analysis of the BSE case and paired control samples using the 50K SNP chip a polymorphic locus was identified on chromosome 1 at 29,147,078 bp with a moderate significant association (p=3.09E-5) and over-represented in BSE affected animals. Further, analysis of the prion gene and the paired BSE case and control animals revealed a haplotype within the region of high LD to associate with BSE un-affected animals (p-value =0.000114). It is clear from our analyses that several regions of the genome are statistically associated with the incidence of classical BSE in European Holstein cattle. / Animal Science

No description available.

Stuttgart, Univ., Diss., 2002.

No description available.

Le transfert de prions des aliments dans la muqueuse et leur passage de la muqueuse vers le système nerveux constituent des chaînons manquants dans la compréhension de la pathogenèse des ESST. Ces événements cruciaux se déroulent à des stades très précoces de linfection et restent sans signes cliniques. Lexplication de ces phénomènes permettrait de mieux appréhender les mécanismes infectieux et de mettre en place des traitements. élucidation Ce travail repose sur des observations largement étayées : - lors dune infection orale par des prions, les plaques de Peyer, spécialisées dans léchantillonnage et le traitement déléments transitant dans liléon, sont des zones privilégiées de passage dagents infectieux de la lumière vers la muqueuse intestinale. - lagent responsable des maladies à prions est retenu et répliqué par des cellules du système immunitaire comme les cellules folliculaires dendritiques. Laccumulation de prions est en effet mise en évidence dans les organes lymphoïdes secondaires dorganismes infectés. - en cas dinfection par voie orale par des prions, le processus de neuroinvasion débute dans le système nerveux périphérique et se propage vers le système nerveux central par les fibres sympathiques et parasympathiques. Ces différentes observations sont à la base de notre hypothèse de travail : les cellules dendritiques capteraient les prions au niveau de la lumière des plaques de Peyer, migreraient dans les tissus lymphoïdes drainants et les transmettraient aux cellules folliculaires dendritiques et/ou aux fibres nerveuses périphériques. En véhiculant les prions, elles seraient à la base à la fois de la lymphoinvasion et de la neuroinvasion. Pour vérifier notre hypothèse nous avons investigué plusieurs pistes : létude comparative du phénotype et la localisation des cellules dendritiques des plaques de Peyer chez les souris saines et les souris infectées par les prions. la localisation des zones de contact entre cellules dendritiques et des fibres nerveuses au sein des organes lymphoïdes secondaires de souris infectées versus saines. lélaboration et létude dun modèle in vitro murin de transmission de prions des cellules dendritiques aux cellules nerveuses périphériques. Le phénotype et la localisation des cellules dendritiques des plaques de Peyer durant la phase préclinique dinduction orale dune ESST ont été analysés selon plusieurs paramètres. Les résultats seront décrits et discutés dans le chapitre 1. Lépithélium associé aux follicules lymphoïdes des plaques de Peyer est une zone de transcytose déléments qui transitent dans le tractus intestinal. On y distingue des entérocytes, des cellules M et des cellules dendritiques. Une attention particulière a été portée aux cellules dendritiques localisées dans lépithélium à différents temps durant une infection par des prions. Une analyse quantitative et phénotypique de cette population particulière de cellules a été réalisée dans la perspective de classer cette population parmi les sous-populations de cellules dendritiques connues. Lexpression membranaire de la protéine prion cellulaire et la détection de la forme totale (PrPc + PrPsc) à la surface de ces cellules dendritiques des plaques de Peyer a été examinée. Les zones de contacts entre DC et FDC, lieux possibles de dissémination des prions au sein des organes lymphoïdes, ont été étudiées de façon quantitative. En vue de connaître les sites potentiels de neuroinvasion, nous avons établi dans le chapitre 2 une topographie des fibres nerveuses au sein des plaques de Peyer, des ganglions mésentériques et de la rate. Nous avons comparé les observations faites sur des souris saines, des souris infectées avec prions et des souris transgéniques. Ensuite, nous avons tenté de mettre en évidence des contacts entre les cellules dendritiques et les fibres nerveuses. La localisation des connexions neuro-immunes dans les organes lymphoïdes a particulièrement retenu notre attention. Les connexions entre cellules dendritiques et les fibres nerveuses décrites dans le chapitre 2 fournissent des informations de localisation. Elles permettent donc denvisager des sites potentiels de neuroinvasion mais elles ne renseignent pas sur la transmission des prions dun type cellulaire à lautre. En particulier la dissémination de lagent pathologique des DC aux neurones périphériques. Le chapitre 3 détaille nos travaux consacrés à lélaboration dun modèle in vitro reproduisant des interfaces entre des DC et des cellules nerveuses. Dans un premier temps, nous avons évalué la validité du modèle par rapport à la situation in vivo. Dans un second temps, les cellules dendritiques infectées par les prions ont été cultivées au contact de neurones issus des ganglions de la racine dorsale, ceux-là mêmes qui sont infectés in vivo, pour étudier la transmission des prions des DC aux neurones du système nerveux périphérique.

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.

Prion is an infectious isoform of a normal cellular protein which is capable of converting the non-prion form of the same protein into the alternative prion form. Mammalian prion protein PrP is responsible for prion formation in mammals, causing a series of fatal and incurable prion diseases. (1) We constructed, for the first time, a two-component system to phenotypically monitor the conformational status of PrP in the yeast cells. In this system, the prion domain of Sup35 (Sup35N) was fused to PrP90-230, and the initial formation of the PrPSc-like conformation stimulated prion formation of Sup35N, which in turn converted soluble Sup35 into the prion isoform, leading to a detectable phenotype. Prion-like properties of PrP were studied in this novel yeast model system. Additionally, we employed this system to study amyloidogenic protein Aβ42 aggregation in the yeast model. It has been suggested that the ability to form transmissible amyloids (prions) is widespread among yeast proteins and is likely intrinsic to proteins from other organisms. However, the distribution of yeast prions in natural conditions is not yet clear, which prevents us from understanding the relationship between prions and their adaptive roles in various environmental conditions. (2) We modified and developed sequence and phenotype-independent approaches for prion detection and monitoring. We employed these approaches for prion-profiling among yeast strains of various origins. (3) Lastly, we found a prion-like state [MCS+] causing nonsense suppression in the absence of the Sup35 prion domain. Our results suggested that [MCS+] is determined by both a prion factor and a nuclear factor. The prion-related properties of [MCS+] were studied by genetic and biochemical approaches.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of invariably fatal neurodegenerative diseases of both humans and animals, thought to be caused by the abnormally folded prion protein PrPSc. Prion disease research continues to be faced by a number of difficult challenges. First, the unequivocal diagnosis of most prion diseases currently requires the post-mortem collection of central nervous system tissue, either for histological examination or Western blot analysis; second, a viable treatment for clinical stage disease has not yet been identified; third, the exact details of disease pathogenesis have not been elucidated; and fourth, the normal function of PrPC is not definitively known. The primary objective of the studies presented here was to diagnose prion disease in live animals, using Magnetic Resonance Imaging (MRI). Increases in T2 relaxation time and apparent diffusion coefficient (ADC) were observed very early following the infection of Syrian golden hamsters with the 263K strain of scrapie. These changes were evident well before the appearance of either clinical symptoms or the typical histological changes characteristic of prion disease, suggesting that they are the result of the progressive accumulation of fluid, and that this may constitute a novel early marker of prion disease pathogenesis. Following the establishment of this model system, a secondary objective was composed: to test the viability of a potential treatment (pentosan polysulphate) using a number of different treatment regimens. It was determined that pentosan polysulphate (PPS) was ineffective as a treatment unless it was administered intra-cerebrally very early in infection, although it was shown to slow the appearance of the histological hallmarks of prion disease. In response to the results of these studies, a potential model was proposed, relating PrP, aquaporin-4 (AQP4) regulation, and oedema. Although speculative, this model may have implications for both normal PrPC function and disease pathogenesis. / February 2009

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of inevitably fatal neurodegenerative diseases that occur in mammalian species. Ovine susceptibility to scrapie, the prototypical TSE, is predominantly modulated by nonsynonymous polymorphisms within exon 3 of the ovine prion protein gene (PRNP). Investigation of PRNP exon 3 for two hair-sheep breeds revealed a novel predicted amino acid substitution (P116) associated with the ovine ARQ allele (P116A136R154Q171). Additionally, two novel ovine PRNP genotypes (PARQ/ARR; PARQ/ARQ) also were detected, and most of the hair sheep sampled possessed PRNP exon 3 genotypes associated with some degree of resistance to scrapie and/or experimental BSE (bovine spongiform encephalopathy). Unlike sheep, expression of bovine spongiform encephalopathy (BSE) in cattle and other bovids has not been associated with nucleotide variation within bovine PRNP exon 3. However, BSE susceptibility has been tentatively associated with specific insertion-deletion (indel) polymorphisms within the putative bovine PRNP promoter, and to a lesser extent intron 1, for a few German cattle breeds. Evaluation of the patterns of nucleotide variation associated with bovine PRNP exon 3 provided evidence that strong purifying selection has intensely constrained bovine exon 3 over the long-term evolutionary history of the subfamily Bovinae, as well as evidence for significant purifying selection in regions of bovine PRNP exon 3 that are considered to be of functional, structural, and pathogenic importance in other mammalian species. Evaluation of the frequencies of known indel polymorphisms within the putative bovine PRNP promoter for a panel of U. S. cattle sires revealed no significant differences in the distribution of promoter alleles and/or genotypes between U. S. cattle sires and BSEaffected German cattle. Notably, a nonsynonymous PRNP exon 3 polymorphism (T50C) identified in American bison (Bison bison) was tentatively associated with Brucella spp. seropositivity. Specifically, a significant overabundance (P = 0.021) of Yellowstone National Park bison possessing the CC genotype were Brucella spp. seropositive. Furthermore, the T-allele and TT genotype were observed at significantly higher frequencies in three bison populations that were either founded from Brucella spp. seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis.

No description available.