Rona Barron - ORCID: 0000-0003-4512-9177 https://orcid.org/0000-0003-4512-9177Item is not available in this repository.12

Barron, Rona

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Queen Margaret University eResearch

The nature of the prion

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     Edinburgh Research Explorer                                      The nature of the prionCitation for published version:Barron, R 2009, 'The nature of the prion', pp. 22-23, Bioforum Europe, 12.Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Preprint (usually an early version)Published In:Bioforum EuropeGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015TSE InfectivityThe Nature of the Prion The TSE Infectious AgentTSE diseases (also known as Prion dis-eases) are fatal infectious neurodegener-ative diseases of animals which include Creutzfeldt-Jakob Disease (CJD) in hu-mans, scrapie in sheep and goats, BSE in cattle, and Chronic Wasting disease (CWD) in deer. The true nature of the TSE infectious agent is unknown but it has been proposed that the agent is com-posed solely of a misfolded form of the host prion protein, PrP. This abnormal form (PrPSc) is resistant to digestion with proteinase K (PK), is deposited in infected tissue, and co-purifies with infectivity. For these reasons, all current Rapid TSE Diagnostic assays are based on the de-tection of PrPSc either by its PK resis-tance, its altered conformation, or its af-finity for specific ligands. Current tests can only be performed on post-mortem tissue, but new pre-mortem tests in de-velopment are also based on the detec-tion of PrPSc in accessible tissues and body fluids (such as blood) by amplifying or concentrating the low levels of PrPSc which may be present. All of these assays rely on the assump-tion that the correlation between TSE in-fectivity and PrPSc is always 100 %. How-ever if disease can exist in the absence of large amounts of PrPSc, reliance on PK-resistant PrP as a sole measure of infec-tivity may in some instances underesti-mate the biological properties of diagnostic samples, and undermine cur-rent efforts to contain or eradicate TSEs.Does PK-resistant PrP Correlate  with Infectivity?In our laboratory, transgenic mice which have a single amino acid mutation in the endogenous PrP gene (P101L) were inocu-lated intracerebrally with TSE infectivity from a case of human familial prion dis-ease (Gerstmann-Straussler-Scheinker disease; GSS) or a strain of scrapie propa-gated in hamsters (263K). These mice (Tg 101LL) developed clinical signs of TSE dis-ease and also showed classical TSE asso-ciated spongiform degeneration of the brain on post-mortem examination. How-ever they contained extremely low or un-detectable levels of PK-resistant PrP in the brain [1]. The lack of PrPSc would suggest that these tissues were not infectious, however disease could be transmitted very efficiently from these tissues to Tg 101LL and wild type mice. We performed further analyses of these tissues to determine the exact levels of the infectious agent and PrPSc that were present, and compared these with a well characterised laboratory strain of TSE agent, ME7. In 4 of 5 tissues examined the titres of infectivity were equivalent to or higher than ME7, but the levels of PK-resistant PrP were undetect-The abnormal prion protein (PrPSc) is currently the only biomarker used for the rapid diagnosis of Transmissible Spongiform Encephalopathy (TSE) disease (or Prion disease). PrPSc is deposited in infected tissue and co-purifies with infectivity, and is thought to be the sole component of the infectious agent. However, evidence now exists of TSE disease in the absence of this marker protein, raising questions about the nature of the infectious agent and the future of TSE diagnosis. Dr. Rona Barron,  Roslin Institutei m m u n o l o g y • BIoforum Europe 3/2008able by standard assay, and therefore esti-mated to be less than 1 % of that seen in ME7. These data implied that either the number of PrPSc molecules per unit of in-fectivity varies by over 1000-fold between strains, or that most PrPSc seen in end stage disease is not infectious.Is PK-sensitive PrPSc  the Infectious Agent?Analyses of other PrP transgenic models with the 101L mutation have revealed the presence of an abnormal, disease as-sociated conformer of PrP which appears to be sensitive to PK [2, 3]. It has been suggested that such PK-sensitive PrPSc (sPrPSc) conformers may be responsible for the observed high levels of TSE infec-tivity in tissues containing no PK-resis-tant PrP. We analysed tissues from our mice showing high titres of infectivity and low levels of PrPSc for the presence of sPrPSc by limited PK digestion studies, and immunoprecipitation studies using monoclonal antibodies specific for ab-normal conformers of PrP. We found no evidence of sPrPSc in any of the tissues [1]. We also analysed these tissues using a Conformation Dependant Immunoas-say (CDI) [4] which does not require PK digestion of samples to identify PrPSc. This assay measures the change in anti-body binding following denaturation in guanidine. An increase in the denatured/native ratio due to increased availability of an antibody epitope indicates the pres-ence of abnormal PrP conformers. Again, this assay revealed no evidence of any abnormal PrP conformers in the high in-fectivity/low PrPSc samples.The 101L-PrP transgenic models in which sPrPSc is observed were created by a method which results in overexpression of the PrP transgene, and it is possible that the abnormal sPrPSc observed in these mice is produced in response to the 8–16 fold overexpression of PrP in the mice. Our Tg 101LL line was produced by gene targeting, which introduces the mu-tation into the endogenous murine PrP gene resulting in wild type expression of the mutated gene. The disease in Tg 101LL mice is therefore due to infection of the mice with an exogenous TSE agent, implying that the conformers of abnormal PrP produced due to overexpression of PrP and TSE infection must be different.Are All Abnormal Forms  of PrP Infectious?Our data indicate that the PrPSc observed in terminal disease tissues may be a pathological by-product of the infection rather than the infectious agent. Other studies have also reported transmission of disease from tissues containing no PK-resistant PrP [5], indicating that this ob-servation is not unique to the Tg 101LL line. In addition, following inoculation with an isolate of atypical human GSS we have shown the presence of PrP amyloid plaques in the brains of Tg 101LL mice which show no clinical signs of disease or spongiform degeneration in the brain [6]. The presence of PrP amyloid plaques in these apparently healthy mice implies that these amyloid deposits also do not contain the TSE infectious agent. The true nature of the TSE infectious agent is still the subject of much debate. Our data suggest that PK-resistant PrP, sPrPSc and PrP amyloid are not the in-fectious agent, but it is possible that an as yet unidentified specific conformer of abnormal PrP holds the key to TSE in-fectivity. However we cannot yet rule out the possible involvement of other molecules. Although our data suggest that PrPSc is not a marker of the TSE infectious agent, it is currently the best marker for diagnosis of TSE disease, and current testing is identifying and removing in-fected animals from the food chain. How-ever, it is vital that markers of TSE infec-tivity other than PrPSc are identified and validated in models such as those we have described and characterised here. We anticipate that such research will lead to the development of more robust diagnostic assays for TSE disease which will have important implications for both animal and human health.This work has been sponsored by BBSRC and DEFRA, and was performed by D. King, S. Campbell, J. Manson, K. Chapman (University of Edinburgh), A. Bellon (Scripps), and Anthony William-son (Scripps)References[1] Barron et al.: J Biol Chem 282, 35878–35886 (2007)[2] Nazor et al.: EMBO J 24, 2472–2480 (2005)[3] Tremblay et al.: J Virol 78, 2088–2099 (2004)[4] Safar et al.: Nature Med 4, 1157–1165 (1998)[5] Lasmezas et al.: Science 275, 402–405 (1997)[6] Piccardo et al.: PNAS, USA 104, 4712–4717 (2007) www.eMagazineBIOforum.comCONTACTDr. Rona Barron PhDneuropathogenesis unitRoslin InstituteTel. +44 131 667 5204rona.barron@roslin.ed.ac.ukFig.: 1 Immunohistochemistry of sections from 101LL/263K infected brain, and control ME7 infected brain. Both tissues contain similar titres of the infectious agent (108.7 IU/g tissue), but 101LL/263K infected tissue contains < 1 % of the amount of PrPSc found in ME7 tissue.i m m u n o l o g yBIoforum Europe 3/2008 • 

The nature of the prion

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