Browsing by Person "King, Declan"

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Accumulation of prion protein in the brain that is not associated with transmissible disease
(National Academy of Sciences, 2007-03-13) Piccardo, Pedro; King, Declan; Ghetti, Bernardino; Manson, Jean C.; Barron, Rona
Prion diseases or transmissible spongiform encephalopathies are characterized histopathologically by the accumulation of prion protein (PrP) ranging from diffuse deposits to amyloid plaques. Moreover, pathologic PrP isoforms (PrPSc) are detected by immunoblot analysis and used both as diagnostic markers of disease and as indicators of the presence of infectivity in tissues. It is not known which forms of PrP are associated with infectivity. To address this question, we performed bioassays using human brain extracts from two cases with phenotypically distinct forms of familial prion disease (Gerstmann-Sträussler-Scheinker P102L). Both cases had PrP accumulations in the brain, but each had different PrPSc isoforms. Only one of the brains had spongiform degeneration. Tissue from this case transmitted disease efficiently to transgenic mice (Tg PrP101LL), resulting in spongiform encephalopathy. In contrast, inoculation of tissue from the case with no spongiform degeneration resulted in almost complete absence of disease transmission but elicited striking PrP-amyloid deposition in several recipient mouse brains. Brains of these mice failed to transmit any neurological disease on passage, but PrP-amyloid deposition was again observed in the brains of recipient mice. These data suggest the possible isolation of an infectious agent that promotes PrP amyloidogenesis in the absence of a spongiform encephalopathy. Alternatively, the infectious agent may be rendered nonpathogenic by sequestration in amyloid plaques, or PrP amyloid can seed amyloid accumulation in the brain, causing a proteinopathy that is unrelated to prion disease. Formation of PrP amyloid may therefore not necessarily be a reliable marker of transmissible spongiform encephalopathy infectivity.
Characterization of an unusual transmissible spongiform encephalopathy in goat by transmission in knock-in transgenic mice
(Microbiology Society, 2013-08-01) Wilson, Rona; King, Declan; Hunter, Nora; Goldmann, Wilfred; Barron, Rona
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle, and its transmission to humans through contaminated food is thought to be the cause of the variant form of Creutzfeldt–Jakob disease. BSE is believed to have spread from the recycling in cattle of ruminant tissue in meat and bone meal (MBM). However, during this time, sheep and goats were also exposed to BSE-contaminated MBM. Both sheep and goats are experimentally susceptible to BSE, and while there have been no reported natural BSE cases in sheep, two goat BSE field cases have been documented. While cases of BSE are rare in small ruminants, the existence of scrapie in both sheep and goats is well established. In the UK, during 2006–2007, a serious outbreak of clinical scrapie was detected in a large dairy goat herd. Subsequently, 200 goats were selected for post-mortem examination, one of which showed biochemical and immunohistochemical features of the disease-associated prion protein (PrPTSE) which differed from all other infected goats. In the present study, we investigated this unusual case by performing transmission bioassays into a panel of mouse lines. Following characterization, we found that strain properties such as the ability to transmit to different mouse lines, lesion profile pattern, degree of PrP deposition in the brain and biochemical features of this unusual goat case were neither consistent with goat BSE nor with a goat scrapie herdmate control. However, our results suggest that this unusual case has BSE-like properties and highlights the need for continued surveillance.
Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy
(American Society for Microbiology, 2013-11-15) Piccardo, Pedro; King, Declan; Telling, Glenn; Manson, Jean C.; Barron, Rona
Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar “prion-like” mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrPTSE) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrPTSE from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrPTSE. In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrPTSE is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrPTSE in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.
High Titers of Transmissible Spongiform Encephalopathy Infectivity Associated with Extremely Low Levels of PrPSc in Vivo
(American Society for Biochemistry and Molecular Biology, 2007-10-08) Barron, Rona; Campbell, Susan L.; King, Declan; Bellon, Anne; Chapman, Karen E.; Williamson, R. Anthony; Manson, Jean C.
Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrPSc) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrPSc. This work questions the correlation between PrPSc level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrPSc as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.
How does host PrP control TSE disease?
(Springer, 2004-05) Manson, Jean; Barron, Rona; Tuzi, Nadia L.; Baybutt, Herbert; Bishop, Matthew; Cancellotti, Enrico; Hart, Patricia; Jamieson, L.; Aitchison, L.; Gall, E.; Bradford, Barry; King, Declan
PrP is central to the TSE disease process and has been hypothesised to be the infectious agent. Polymorphisms in the PrP gene of a number of species are associated with different incubation times of disease following exposure to an infectious agent and mutations in the human PrP gene can apparently lead to spontaneous genetic disease. Strains of TSE agent are proposed to be generated and maintained through differences in glycosylation or conformation of PrP and the barrier to infection between species is thought to be due to the differences in the sequence of PrP between different species. In order to test these hypotheses, we have introduced specific modifications into the endogenous mouse Prnp gene by gene targeting. The mutated PrP gene is in the correct location under the control of the endogenous Prnp regulatory sequences and thus expressed in the same tissues and amounts as the wild type Prnp gene. This strategy therefore allows the effect of specific mutations in the PrP gene to be assessed. We have introduced mutations into the Prnp gene which prevent glycosylation at each or both of the two N-linked glycosylation sites of PrP and are using TSE infection of these mice to investigate the role of PrP glycosylation in strain targeting and strain determination. We have investigated the role of the sequence of the host PrP gene in determining susceptibility by inserting point mutations or replacing the murine PrP gene with that of human or bovine PrP. This has produced a model of TSE disease which contains high levels of infectivity in the absence of PrPSc and we are using this model to determine the nature of the infectious agent. We have thus established that the gene targeting approach can produce models for TSE disease which address fundamental questions associated with these diseases. We aim to use these models to address central issues including the origin of strains, the species barrier and the nature of the infectious agent.
Mechanism of PrP-Amyloid Formation in Mice Without Transmissible Spongiform Encephalopathy
(Wiley, 2011-06-03) Jeffrey, Martin; McGovern, Gillian; Chambers, Emily V.; King, Declan; Gonzalez, Lorenzo; Manson, Jean C.; Ghetti, Bernardino; Piccardo, Pedro; Barron, Rona
Gerstmann–Sträussler–Scheinker (GSS) P102L disease is a familial form of a transmissible spongiform encephalopathy (TSE) that can present with or without vacuolation of neuropil. Inefficient disease transmission into 101LL transgenic mice was previously observed from GSS P102L without vacuolation. However, several aged, healthy mice had large plaques composed of abnormal prion protein (PrPd). Here we perform the ultrastructural characterization of such plaques and compare them with PrPd aggregates found in TSE caused by an infectious mechanism. PrPd plaques in 101LL mice varied in maturity, with some being composed of deposits without visible amyloid fibrils. PrPd was present on cell membranes in the vicinity of all types of plaques. In contrast to the unicentric plaques seen in infectious murine scrapie, the plaques seen in the current model were multicentric and were initiated by protofibrillar forms of PrPd situated on oligodendroglia, astrocytes and neuritic cell membranes. We speculate that the initial conversion process leading to plaque formation begins with membrane-bound PrPC but that subsequent fibrillization does not require membrane attachment. We also observed that the membrane alterations consistently seen in murine scrapie and other infectious TSEs were not present in 101LL mice with plaques, suggesting differences in the pathogenesis of these conditions.
Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures
(Oxford University Press, 2021-07-08) King, Declan; Skehel, Paul A.; Dando, Owen; Emelianova, Katie; Barron, Rona; Wishart, Thomas M.
Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures is an on-going question. Here, we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines.
Polymorphisms at codons 108 and 189 in murine PrP play distinct roles in the control of scrapie incubation time
(Microbiology Society, 2005-03-01) Barron, Rona; Baybutt, Herbert; Tuzi, Nadia L.; McCormack, James; King, Declan; Moore, Richard C.; Melton, David W.; Manson, Jean C.
Susceptibility to transmissible spongiform encephalopathies (TSEs) is associated strongly with PrP polymorphisms in humans, sheep and rodents. In mice, scrapie incubation time is controlled by polymorphisms at PrP codons 108 (leucine or phenylalanine) and 189 (threonine or valine), but the precise role of each polymorphism in the control of disease is unknown. The L108F and T189V polymorphisms are present in distinct structural regions of PrP and thus provide an excellent model with which to investigate the role of PrP structure and gene variation in TSEs. Two unique lines of transgenic mice, in which 108F and 189V have been targeted separately into the endogenous murine Prnp a gene, have been produced. TSE inoculation of inbred lines of mice expressing all allelic combinations at codons 108 and 189 has revealed a complex relationship between PrP allele and incubation time. It has been established that both codons 108 and 189 control TSE incubation time, and that each polymorphism plays a distinct role in the disease process. Comparison of ME7 incubation times in mouse lines that are heterozygous at both codons has also identified a previously unrecognized intramolecular interaction between PrP codons 108 and 189.
Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC
(Public Library of Science, 2012-11-05) Vascellari, Sarah; Orrù, Christina D.; Hughson, Andrew G.; King, Declan; Barron, Rona; Wilham, Jason M.; Baron, Gerald S.; Race, Brent; Pani, Alessandra
Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrPSc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrPSc propagation involves conversion from its normal isoform, PrPC, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrPSen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10−8 and 10−13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrPC. Although the brains of 263K-affected mice had little immunoblot-detectable PrPRes, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrPRes strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc.
PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions
(Springer Nature, 2016-06-26) Barron, Rona; King, Declan; Jeffrey, Martin; McGovern, Gillian; Agarwal, Sonya; Gill, Andrew C.; Piccardo, Pedro
Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the “prion-like” spread of aggregated forms of the beta-amyloid peptide (Aβ), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.
Quantitative imaging of tissue sections using infrared scanning technology
(Wiley, 2015-10-29) Eaton, Samantha L.; Cumyn, Elizabeth; King, Declan; Kline, Rachel A.; Carpanini, Sarah M.; Del-Pozo, Jorge; Barron, Rona; Wishart, Thomas M.
Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi-quantitative results. Visualising infrared (IR) ‘tags’, with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR-based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR-based scanning technology yields comparable area-based quantification to those obtained from a modern high-resolution digital slide scanner. Secondly, IR-based dual target visualisation and expression-based quantification is rapid and simple. Thirdly, IR-based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR-based QFIHC provides an alternative method of rapid whole-tissue section low-resolution imaging for the production of reliable and accurate quantitative data.
Transmission of murine scrapie to P101L transgenic mice
(Microbiology Society, 2003-11-01) Barron, Rona; Thomson, Val; King, Declan; Melton, David W.; Manson, Jean C.
The PrP protein is central to the transmissible spongiform encephalopathies (TSEs), and the amino acid sequence of this protein in the host can influence both incubation time of disease and targeting of disease pathology. The N terminus of murine PrP has been proposed to be important in the replication of TSE agents, as mutations or deletions in that region can alter the efficiency of agent replication. To address this hypothesis and to investigate the mechanisms by which host PrP sequence controls the outcome of disease, we have assessed the influence of a single amino acid alteration in the N-terminal region of murine PrP (P101L) on the transmission of TSE agents between mice. Mice homozygous for the mutation (101LL) were inoculated with TSE strains 139A and 79A derived from mice carrying a Prnpa allele, and 79V and 301V derived from mice carrying a Prnpb allele. Incubation times in 101LL mice were extended with all four strains of agent when compared with those in the corresponding mouse genotype from which the infectivity was derived. However, the degree to which the incubation period was increased showed considerable variation between each strain of agent. Moreover, the presence of this single amino acid alteration resulted in a 70 day reduction in incubation time of the 301V strain in Prnpa mice. The effect of the 101L mutation on murine scrapie incubation time appears therefore to be strain specific.
Variable tau accumulation in murine models with abnormal prion protein deposits
(Elsevier, 2017-11-07) Piccardo, Pedro; King, Declan; Brown, Deborah; Barron, Rona
The conversion of cellular prion protein (PrP) into a misfolded isoform is central to the development of prion diseases. However, the heterogeneous phenotypes observed in prion disease may be linked with the presence of other misfolded proteins in the brain. While hyperphosphorylated tau (p.tau) is characteristic of Alzheimer's disease (AD), p.tau is also observed in human prion diseases. To explore this association in the absence of potential effects due to aging, drug treatment, agonal stage and postmortem delay we analyzed p.tau and PrP immunopositivity in mouse models. Analyses were performed on mice inoculated with prion agents, and mice with PrP amyloid in the absence of prion disease. We observed that p.tau was consistently present in animals with prion infectivity (models that transmit disease upon serial passage). In contrast, p.tau was very rarely observed or absent in mice with PrP amyloid plaques in the absence of prion replication. These data indicate that the formation of p.tau is not linked to deposition of misfolded PrP, but suggest that the interaction between replication of infectivity and host factors regulate the formation of p.tau and may contribute to the heterogeneous phenotype of prion diseases.