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 Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof.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.

Several lines of evidence suggest that the expression of the cellular prion protein (PrPC) is altered with age and in sporadic Alzheimer’s disease, however, published results have been contradictory. Furthermore, a relationship between the expression of PrPC and Tau has started to emerge. We have revealed a specific relationship between the expression of PrPC and Tau in neuroblastoma cell lines and transgenic mouse models. In addition, we identified that the expression levels of PrPC are reduced in multiple brain regions following the progression of sporadic Alzheimer’s disease. Furthermore, the reduction in PrPC expression significantly correlated with the reduction in Tau expression and coincided with an increase in Tau pathology. In addition, data from neuroblastoma cell lines implicated the glycosylphosphatidylinositol (GPI)-anchor and in part the localisation of PrPC to lipid rafts in mediating these alterations to Tau. We hypothesise that the reduction in PrPC expression reflects a primary mechanism in Alzheimer’s disease pathogenesis and indirectly triggers the reduction in Tau expression which subsequently contributes to neuronal destabilisation and disruption to neuronal function. Soluble oligomeric forms of amyloid-beta are the primary pathogenic species in Alzheimer’s disease and strongly correlate with the presence and severity of cognitive decline. PrPC acts as a high affinity neuronal receptor for amyloid-beta oligomers and triggers pathogenic signaling cascades which induce synaptic impairment and further exacerbate neuronal destabilisation. We demonstrated that Flotillin-1 and the lipid raft localisation of PrPC are essential for the binding of amyloid-beta oligomers to PrPC. Furthermore, the metabotropic glutamate receptor, mGluR5 plays a pivotal role in the aberrant signaling of PrPC, and this PrPC/mGluR5 complex provides a mechanistic link between extracellular amyloid-beta oligomers and intracellular Tau phosphorylation, by Fyn kinase, Pyk2 and possibly by inactivation of the protein phosphatase, PP2A. Considering there is now strong evidence that Tau is the mediator of amyloid-beta induced toxicity, the reduction in Tau levels mediated by PrPC may be a protective mechanism. amyloid-beta oligomers interact with a multitude of neuronal receptors in addition to PrPC. It is likely that activation of multiple receptor complexes and signalling cascades are responsible for synaptic impairment and Tau phosphorylation induced by amyloid-beta, however, these complexes remain to be fully determined. Investigating amyloid-beta oligomer induced Tau phosphorylation in vitro has proven challenging, however, we suggest that a functional, mature, neuronal model is necessary to induce the complex mechanisms linking extracellular amyloid-beta oligomers and the phosphorylation of intracellular Tau. A greater understanding of the complex relationship between amyloid-beta, PrPC and Tau will aid in our understanding of the molecular mechanisms underlying Alzheimer’s disease and in the discovery of novel therapeutic targets for this progressive neurodegenerative disease.

No description available.

Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.

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.

No description available.

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Scrapie und die Bovine Spongiforme Enzephalopathie (BSE) sind stets tödlich verlaufende transmissible spongiforme Enzephalopathien (TSE) bei kleinen Wiederkäuern und Rindern. Nach der „Prion-Theorie“ ist die pathologische Isoform eines zellulären Proteins, des Prion-Proteins, der Hauptbestandteil, wenn nicht sogar die einzige Komponente der TSE-Erreger. Gemäß dieser Theorie lagert sich die pathologische Isoform, PrPSc, an die zelluläre Form, PrPC, an und führt so zu einer Konformationsänderung von PrPC. Bisher lassen sich TSE-Erreger nur im Tierversuch sowie in wenigen überwiegend von Nagetieren stammenden Zelllinien vermehren. Das Ziel der vorliegenden Arbeit war deshalb die Identifikation und Charakterisierung neuer TSE-empfänglicher Zelllinien, wobei vor allem die Infektion oviner und boviner Zelllinien mit Scrapie- bzw. BSE-Feldisolaten im Vordergrund stand. Hierzu wurde zunächst der Einfluss unterschiedlicher Kultur- und Infektionsbedingungen (Nährmedien, Umsetzraten, Temperatur, Herstellung der Inokulate, Inokulationsprotokoll) auf den Infektionserfolg studiert. Basierend auf den dabei gewonnenen Daten wurde ein Infektionsprotokoll erstellt, das im weiteren Verlauf für alle in dieser Studie untersuchten Zelllinien verwendet wurde. Durch die Zellbank des Friedrich-Loeffler-Instituts stand eine Vielzahl unterschiedlicher eukaryotischer Zelllinien zur Verfügung, von denen 53 aus geeigneten Organen und Geweben und größtenteils vom Wiederkäuer stammende Zelllinien ausgewählt wurden. Anschließend wurden diese Zelllinien kultiviert und wiederholt hinsichtlich ihrer PrPC-Expression analysiert. Dabei zeigte sich, dass das PrPC-Expressionniveau für jede Zelllinie sehr individuell und weder spezies- noch gewebespezifisch ist. 34 der 53 Zelllinien exprimierten PrPC in detektierbarer Menge und wurden in den weiteren Infektionsstudien verwendet. Dabei wurden sie mit verschiedenen TSE-Stämmen bzw. -Isolaten (bovines BSE-Material, ovines und caprines Scrapie-Material, mausadaptierte BSE- und Scrapie-Stämme) inokuliert. Der Großteil dieser Zelllinien (30 von 34) zeigte sich gegen alle eingesetzten Prion-Erreger resistent. Zwei Zelllinien konnten transient für 10 (MGbov900) bzw. 32 (Bov11) Passagen mit bovinem BSE-Material infiziert werden. Damit war die prinzipielle Möglichkeit einer BSE-Infektion dieser Zellen gezeigt. Aus bisher ungeklärten Gründen wurde die Prion-Infektion jedoch von beiden Zelllinien wieder verloren und erneute Infektionsversuche blieben erfolglos. Zwei weitere Zelllinien konnten persistent infiziert werden. Die Zelllinie N2a229, eine Sublinie der in der Prion-Forschung weit verbreiteten Neuroblastomzelllinie N2a, war für den mauspassagierten Scrapie-Stamm RML empfänglich. Des Weiteren wurde eine bovine Zelllinie (Bov5; 154PES) identifiziert, die für zwei ovine Scrapie-Feldisolate empfänglich war. Es handelt sich dabei um die erste nicht transgene Zelllinie, die mit einem Scrapie-Feldisolat infiziert werden konnte. Die beiden verwendeten Isolate (S71/04 und S95/04) stammten von Schafen aus einem klassischen Scrapie-Ausbruch aus dem Jahr 2004. In den infizierten Bov5Sc-Zellen steigerte sich die initial schwache PrPSc-Akkumulation über mehrere Passagen zu einem starken Signal, das durch verschiedene Prion-spezifische Antikörper im Dot-Blot, im Western-Blot und mit dem „Zell-ELISA“ nachgewiesen werden konnte. Die Infektion ist bereits seit über 200 Passagen stabil. Sie war mit den besagten Scrapie-Isolaten mehrfach wiederholbar und durch die Selektion von infizierten Einzelzellen konnten hochpositive Sublinien erhalten werden. Infizierte Bov5Sc-Zellen führten nach Inokulation in transgene Rinder- oder Schaf-PrPC überexprimierende Mäuse zu Scrapie-Erkrankungen und der Bildung von PrPSc im Gehirn der Mäuse. Die Infektion von Rinderzellen mit einem ovinen TSE-Isolat bedeutete die Überwindung einer Speziesbarriere. In weiteren Untersuchungen konnte gezeigt werden, dass eine Adaptation des Erregers an die Zellen stattgefunden hatte, welche sich z. B. in einem veränderten Glykosylierungs-profil darstellt. Verglichen mit zwei murinen TSE-infizierten Zelllinien zeigten die Bov5Sc-Zellen ähnliche Eigenschaften hinsichtlich ihrer Proteinase K-Resistenz, aber eine deutlich verlängerte Reaktionszeit gegenüber PrPSc inhibierenden Substanzen. Neben den Infektionsstudien an den „Zellbank-Zelllinien“ wurden transgene Zelllinien hergestellt, die auf der Basis von RK13-Zellen (Nierenzellen aus dem Kaninchen) und Hpl3-4-Zellen (neuronale Zelle aus PrP-defizienten-Mäusen) das PrPC von Maus, Schaf, Nerz, Hund sowie zwei chimären Konstrukten aus Maus/Rind/Maus bzw. Maus/Schaf/Maus exprimierten. Ein Großteil dieser transgenen Zelllinien war gegenüber einer Infektion und insbesondere einer Infektion mit TSE-Feldisolaten resistent. Durch Infektionsstudien mit dem mausadaptierten murinen Scrapie-Stamm RML konnten jedoch interessante Einblicke in die Hintergründe der zellulären TSE-Empfänglichkeit gewonnen werden. So zeigte sich, dass die Expression eines chimären PrP-Konstruktes aus Maus und Schaf (Mushp) nur in RK13-Zellen, nicht aber in Hpl3-4-Zellen zu einer für den Scrapie-Stamm RML empfänglichen Zelllinie führte. Dagegen konnten murines PrPC exprimierende Hpl3-4-Zellen erfolgreich mit RML, nicht aber mit dem Stamm Me7 infiziert werden. Diese Versuche unterstützen die Annahme, dass für eine Zellinfektion weitere zelluläre Komponenten eine Rolle spielen und zeigen, dass nur die richtige Kombination aus exprimiertem PrPC und zellulärem Hintergrund die Empfänglichkeit einer Zelllinie für einen speziellen TSE-Erreger bestimmen kann. Weitere Studien mit empfänglichen bzw. infizierten bovinen Zelllinien werden zu einem besseren Verständnis der zellulären Pathogenese bei BSE und Scrapie führen, woraus sich möglicherweise auch ein therapeutischer und diagnostischer Nutzen ziehen lässt.

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.

No description available.