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 However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished.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.

Transmissible spongiform encephalopathy (TSE) strains are defined by their biological properties on transmission to wild-type mice, specifically by their characteristic incubation periods and patterns of vacuolar pathology (‘lesion profiles’) in the brain. Whilst a single TSE strain has been identified in variant Creutzfeldt-Jakob disease (vCJD), the phenotypic heterogeneity observed in sporadic CJD (sCJD) implies the existence of multiple strains of agent. These distinct strains are proposed to be enciphered by the different conformers of abnormal prion protein (PrP), recognised as different protease resistant PrP (PrPres) types by Western blotting (type 1 or type 2) and are thought to be substantially influenced by the different prion protein gene (PRNP) codon 129 polymorphism (MM, MV and VV). To test the relationship between disease phenotype and agent strain, this study carried out a full characterisation of the sCJD agent by primary transmission of brain tissue from 27 sCJD cases (comprising all six possible combinations of PRNP codon 129 genotype and PrPres type) in panels of wild-type mice using the standard strain typing properties of incubation period and lesion profiles, plus a full analysis of PrP in the mouse brain and the PrPres molecular subtypes present. Results were directly compared with the transmission characteristics of brain tissue from 10 vCJD cases. The characterisation of the agent strain in sCJD and vCJD was extended to include analysis of subsequent mouse-to-mouse passages. In an additional investigation, wild-type mice were experimentally challenged with a wide-range of lymphoid tissues, neural tissues and biological fluids from vCJD and sCJD patients in order to investigate the extent of peripheral involvement in CJD and to determine whether the agent is subject to any tissue-specific modifications. Analysis of all 27 sCJD sources demonstrated the existence of two strains of agent, one associated with the MM1/MV1 subgroups and the other associated with the MM2 subgroup, which could be distinguished by their transmission properties in the mice. The lack of transmission in mice challenged with VV1, MV2 and VV2 tissues provided evidence of at least one further sCJD strain. In contrast, all 10 vCJD sources resulted in consistent incubation periods and lesion profiles, suggesting that all 10 patients investigated were infected with the same strain of agent. Overall, the observation that PrPres type in sCJD and vCJD was maintained on transmission is consistent with the proposition that PrPres type plays a role in enciphering strain-specific information. Experimental transmissions from peripheral tissues extended the evidence for a peripheral infection in vCJD. However, comparison of incubation periods and lesion profiles from transmission of brain and peripheral tissues showed no evidence of tissue-specific modification in the biological properties of the agent. Furthermore, the detection of low levels of infectivity in a sCJD buffy coat sample provides supporting evidence for a peripheral involvement in sCJD. This study highlights the complex relationship between disease phenotype, PRNP codon 129 genotype, PrPres type and agent strain in sCJD and vCJD. Overall, this study confirms that multiple strains of agent are associated with sCJD, some of which successfully propagate in wild-type mice but none of which are identical to the agent responsible for vCJD. Importantly, the sCJD strains identified here by their biological properties partially correlated with the current sub-classification system for sCJD which is based on the clinical and pathological phenotype of the disease.

<p>Patients suffering from different forms of neurodegenerative diseases, such as: Creutzfeldt Jacob Disease (CJD), Alzheimer disease (AD), mild cognitive impairment (MCI), frontotemporal dementia and Parkinson’s disease (PD) were examined with Positron Emission Tomography (PET) and the combination of different radiotracers: ¹⁵O-water, N-[¹¹C-methyl]-L-deuterodeprenyl (DED), [¹⁸F] 2-fluorodeoxyglucose: (FDG), N-methyl-[¹¹C]2-(4-methylaminophenyl)-6-hydroxybenzothiazole (PIB) and L-[¹¹C]-3,4-dihydroxiphenyl-alanine (DOPA). The radiotracers and the combinations of different radiotracers were selected with the intention to detect, in the brain, patterns of neuronal dysfunction, astrocytosis, axon degeneration or protein aggregation (amyloid), in the brain which are pathognomonic for specific diseases and may contribute to improve clinical differential diagnoses. Examinations in healthy volunteers were performed to allow comparisons with patients. In addition, animal studies were conducted to complement the information. In some cases, the PET findings could be compared with the results of autopsies.</p><p>In contrast to the micropathology, in which only a limited part of a tissue (obtained post-mortem or by biopsy) is inspected, one PET acquisition provides an image of the whole system (e.g.: the brain and the cerebellum). This form of imaging pathology is “<i>in vivo</i>”, where the examination is innocuous for the patient. </p><p>This thesis is an attempt to stimulate the development of new tracers, new tracer combinations and methods that directly or indirectly describe the anatomo-physiopathological changes produced in the brain in neurodegenerative diseases. A better description of different diseases can be obtained, confirming or questioning the clinical diagnoses and widening our understanding of the mechanisms underlying neurodegeneration. Different pathologies can produce similar symptoms and thus causing confusion regarding clinical diagnosis. The used PET combinations improved the accuracy of the diagnoses. The incipient knowledge emerging from a new neuroimaging pathology in combination with other disciplines may open the way to new classifications of dementias and neurodegenerative diseases based on an “<i>in vivo</i>” pathology. </p>

The Transmissible Spongiform Encephalopathies (TSEs) or “prion” diseases are a group of fatal neurodegenerative diseases the aetiology of which is not fully understood. These diseases are characterised by a number of pathological changes in the central nervous system (CNS) including; vacuolation of the neuropil, gliosis and deposition of PrPSc; the abnormal form of the host glycoprotein PrP. Although the major pathology in these diseases is associated with the CNS the immune system is central to the pathogenesis of many natural and experimental TSEs including natural scrapie in sheep, chronic wasting disease in free ranging and captive deer and variant CJD (vCJD) in humans. Unlike many infectious diseases where deficiencies in immune function are opportunistic for the invading pathogen a competent immune system is required for efficient TSE infection via peripheral routes. As infection of the lymphoid tissues in many TSEs can occur many months before the detection of infectivity in the CNS, the determination of those cells in the lymphoid system has been the focus of much research and a number of studies now point towards the importance of the follicular dendritic cell (FDC), a long-lived radio resistant cell, in TSE pathogenesis. The involvement of FDCs in peripheral TSE pathogenesis relates to the inability of ionising radiation to influence pathogenesis, the association of PrP protein with FDCs in both uninfected and infected lymphoid tissues, and the demonstration that TSE pathogenesis is severely impaired in mice devoid of these cells. The aims of this thesis were to further understand the role of FDCs in the pathogenesis of a range of mouse-adapted experimental TSE strains and to determine if peripherally acquired TSE infections are influenced by host age or by stimulation of the immune system. Using chimaeric mouse models where a mismatch in the expression of PrP protein between FDCs and lymphoid/myeloid cells was produced, further evidence for a critical role for in the pathogenesis of the ME7 TSE strain was produced. Although these findings produced strong evidence that FDCs were important for the ME7 strain the possibility that different TSE strains may target different cell types in the peripheral lymphoid system was explored using a range of mice with specific immunological defects. Infection of these mice with several experimental TSE strains showed that the presence of mature FDCs was also important for the pathogenesis of the strains tested. Clinical cases of vCJD have been confined almost exclusively to young adults, although the reasons behind this apparent age-related susceptibility are not fully understood. The capacity of the immune system to mediate immune responses to pathogens declines with age as a result of impaired lymphocyte and FDC function. As FDCs are critically involved in the pathogenesis of many TSEs, including vCJD, it was hypothesised that an aging immune system may impair disease pathogenesis. Peripheral infection of senescent mice failed to produce clinical disease during lifespan, although evidence of disease transmission, was detected in a proportion of aged mice. These findings demonstrate that this inefficient disease transmission, as a consequence of age, may lead to considerable levels of sub-clinical disease within the population. Finally the influence of immune system stimulation, by the generation of a humoral immune response, on peripheral TSE pathogenesis was investigated. These findings demonstrated that immunisation can influence pathogenesis, but only during the early stages of infection prior to spread to the CNS. These data imply that modulation of the immune system does not alter TSE pathogenesis once disease has been initiated in the CNS. Finally, these studies have found some preliminary evidence that TSE infection may induce FDC activation suggesting that TSE infection may influence the immune response. Together, these data show that a functional immune system and specifically, the presence of mature FDCs, are central to the pathogenesis of peripherally acquired TSE infections.

Patients suffering from different forms of neurodegenerative diseases, such as: Creutzfeldt Jacob Disease (CJD), Alzheimer disease (AD), mild cognitive impairment (MCI), frontotemporal dementia and Parkinson’s disease (PD) were examined with Positron Emission Tomography (PET) and the combination of different radiotracers: 15O-water, N-[11C-methyl]-L-deuterodeprenyl (DED), [18F] 2-fluorodeoxyglucose: (FDG), N-methyl-[11C]2-(4-methylaminophenyl)-6-hydroxybenzothiazole (PIB) and L-[11C]-3,4-dihydroxiphenyl-alanine (DOPA). The radiotracers and the combinations of different radiotracers were selected with the intention to detect, in the brain, patterns of neuronal dysfunction, astrocytosis, axon degeneration or protein aggregation (amyloid), in the brain which are pathognomonic for specific diseases and may contribute to improve clinical differential diagnoses. Examinations in healthy volunteers were performed to allow comparisons with patients. In addition, animal studies were conducted to complement the information. In some cases, the PET findings could be compared with the results of autopsies. In contrast to the micropathology, in which only a limited part of a tissue (obtained post-mortem or by biopsy) is inspected, one PET acquisition provides an image of the whole system (e.g.: the brain and the cerebellum). This form of imaging pathology is “in vivo”, where the examination is innocuous for the patient. This thesis is an attempt to stimulate the development of new tracers, new tracer combinations and methods that directly or indirectly describe the anatomo-physiopathological changes produced in the brain in neurodegenerative diseases. A better description of different diseases can be obtained, confirming or questioning the clinical diagnoses and widening our understanding of the mechanisms underlying neurodegeneration. Different pathologies can produce similar symptoms and thus causing confusion regarding clinical diagnosis. The used PET combinations improved the accuracy of the diagnoses. The incipient knowledge emerging from a new neuroimaging pathology in combination with other disciplines may open the way to new classifications of dementias and neurodegenerative diseases based on an “in vivo” pathology.

No description available.

One of the key molecular events in the transmissible spongiform encephalopathies or prion diseases is the conformational conversion of the cellular prion protein PrPC into the misfolded and pathogenic isoform, PrPSc. Prion diseases are fatal neurodegenerative conditions affecting humans and other animal species, which present with diverse clinical and neuropathological phenotypes. In humans, prion diseases can occur as sporadic, familial or acquired forms. Sporadic Creutzfeldt–Jakob disease (sCJD) accounts for the majority of cases. The current classification system of human prion diseases recognizes several distinct clinico-pathological entities including sCJD, variant Creutzfeldt-Jakob disease (vCJD), Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia and variably protease-sensitive proteinopathy. Prion protein gene (PRNP) mutations and polymorphisms, and PrPSc types have a profound effect on these clinico-pathological phenotypes. Prion diseases of sheep and goats, cattle, and cervids are all actual animal health problems and present potential risks to human health. Thus far the only known zoonotic prion disease is bovine spongiform encephalopathy, which has resulted in vCJD in humans. The recognition of new forms of prion diseases in animal and humans has generated increased awareness of the animal and public health risks associated with prion disease. However the mechanisms involved in prion replication, transmission, and neurodegeneration remain poorly understood. This thesis uses in vitro PrP conversion assays (protein misfolding cyclic amplification and real time quaking-induced conversion) to model different aspects of human prion replication: Molecular susceptibility, genetic compatibility, spontaneous formation and the effect of molecules that might enhance or prevent conversion were each investigated in order to obtain a better understanding of the molecular mechanism of the prion replication. I have addressed the hypothesis that the major determinant factors in prion disease pathogenesis (PRNP genetics, PrPSc types and species barriers) are intrinsic to the prion protein conversion process and their effects can be faithfully recapitulated by in vitro conversion assays. The results shows that in vitro conversion assays used in this thesis can model the combined effects of different PrP type and genotypes, can replicate aspects of cross-species transmission potential and provide information about molecular barrier to zoonotic transmission, can model de novo PrPSc formation, and can assess the potential impact of chaperones on conversion of the human prion protein. In summary, this work provides evidence that the origin, propagation and transmission of prions can be meaningfully investigated in cell-free systems.

No description available.

The human prion protein (PrP) gene (PRNP) codon 129 (M/V) polymorphism is a susceptibility factor for variant Creutzfeldt-Jakob Disease (vCJD) and a major determinant of clinico-pathological phenotype in sporadic CJD. The role of codon 129 in defining susceptibility and strain transmission properties has been investigated in three lines of transgenic mice that express human PrP. The human PRNP gene has directly replaced the murine version, by gene targeting, and variation at codon 129 has given the three genotype lines (HuMM, HuMV, and HuVV). The genetics of these three mouse lines are otherwise identical, and therefore differences in transmission properties can be directly attributable to the codon 129 genotype. vCJD inoculation has shown that all three codon 129 genotype mice are susceptible with a ranking of transmission efficiency of HuMM>HuMV>HuVV. HuMM mice develop the most widespread neuropathology with features similar to human vCJD. Subclinical infection was noted in each mouse line. These data suggest that the vCJD strain is transmissible to humans of each of the three codon 129 genotypes, implying that non-MM cases of human infection with bovine spongiform encephalopathy (BSE) may exist but with long subclinical incubation periods. Inoculation of material from blood transfusion associated vCJD showed no change in transmission properties suggesting that the threat of a future epidemic of human-to-human vCJD infection has not been increased by adaptation of the vCJD strain. However the route of infection, for example via blood transfusion or surgery, may be more efficient that the original oral route of BSE infection. sCJD is classified into six subgroups according to clinico-pathological features, and defined by codon 129 genotype and electrophoretic mobility type (1 or 2) of disease associated PrPSc (MM1, MM2, MV1, MV2, VV1, VV2). Typical cases from each subgroup have shown specific transmission properties suggesting that the subgrouping is defining separate disease strains. The commonest subgroup (MM1) was the most transmissible and the HuVV mouse line the most susceptible host. These data outline the transmission risk from all sCJD types to recipients of each codon 129 genotype should an infection event occur, and show the significant role of recipient codon 129 genotype in defining the clinical or subclinical state and the success or failure of transmission. This is important for determining individual risk following known exposure, and for modelling the potential of iatrogenic infection from sCJD patients.

No description available.