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From: TSS ()
Detection of Type 1 Prion Protein in Variant Creutzfeldt-Jakob Disease Helen M. Yull,* Diane L. Ritchie,* Jan P.M. Langeveld,† Fred G. van Zijderveld,† Moira E. Bruce,‡ James W. Ironside,* and Mark W. Head* From the National CJD Surveillance Unit,* School of Molecular and Clinical Medicine, University of Edinburgh, Edinburgh, United Kingdom; Central Institute for Animal Disease Control (CIDC)-Lelystad, † Lelystad, The Netherlands; Institute for Animal Health, Neuropathogenesis Unit, ‡ Edinburgh, United Kingdom Molecular typing of the abnormal form of the prion protein (PrPSc) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrPSc molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrPSc that accumulates in the brain in variant Creutzfeldt- Jakob disease also contains a minority type 1 component. This minority type 1 PrPSc was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrPSc types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrPSc molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain. (Am J Pathol 2006, 168:151–157; DOI: 10.2353/ajpath.2006.050766) snip... Discussion In the apparent absence of a foreign nucleic acid genome associated with the agents responsible for transmissible spongiform encephalopathies or prion diseases, efforts to provide a molecular definition of agent strain have focused on biochemical differences in the abnormal, disease-associated form of the prion protein, termed PrPSc. Differences in PrPSc conformation and glycosylation have been proposed to underlie disease phenotype and form the biochemical basis of agent strain. This proposal has found support in the observation that the major phenotypic subtypes of sCJD appear to correlate with the presence of either type 1 or type 2 PrPSc in combination with the presence of either methionine or valine at codon 129 of the prion protein gene.2 Similarly, the PrPSc type associated with vCJD correlates with the presence of type 2 PrPSc and is distinct from that found in sCJD because of a characteristically high occupancy of both N-linked glycosylation sites (type 2B).6,11 The means by which such conformational difference is detected is somewhat indirect; relying on the action of proteases, primarily proteinase K, to degrade the normal Figure 6. Type 1 PrPSc is a stable minority component of PrPSc from the vCJD brain. Western blot analysis of PrP in a sample of cerebral cortex from a case of vCJD during digestion with proteinase K is shown. Time points assayed are indicated in minutes (T0, 5, 10, 30, 60, 120, 180). Duplicate blots were probed with 3F4, which detects both type 1 and type 2 PrPSc, and with 12B2, which detects type 1. The insert shows a shorter exposure of the same time course study from a separate experiment also probed with 3F4. Both blots included samples of cerebral cortex from a case of sporadic CJD MM1 (Type 1) and molecular weight markers (Markers) indicate weights in kd. Figure 7. A minority type 1-like PrPSc is found in vCJD tonsil, vCJD transmitted to mice and in BSE. Western blot analysis of PrPSc in a concentrated sample of tonsil from a case of vCJD (Tonsil), in a concentrated brain sample of a wild-type mouse (C57BL) infected with vCJD and in a sample of cattle BSE brain (BSE) is shown. Tissue extracts were digested with proteinase K. Duplicate blots were probed with either 3F4 or 6H4, both of which detect type 1 and type 2 PrPSc, and with 12B2, which detects type 1. The blots included samples of cerebral cortex from a case of sporadic CJD MM1 (Type 1) and molecular weight markers (Markers) indicate weights in kd. Type 1 PrPSc in Variant Creutzfeldt-Jakob Disease 155 AJP January 2006, Vol. 168, No. 1 cellular form of PrP and produce a protease-resistant core fragment of PrPSc that differs in the extent of its N-terminal truncation according to the original conformation. A complication has recently arisen with the finding that both type 1 and type 2 can co-exist in the brains of patients with sCJD.2,5-8 More recently this same phenomenon has been demonstrated in patients with iatrogenically acquired and familial forms of human prion disease. 9,10 The existence of this phenomenon is now beyond doubt but its prevalence and its biological significance remain a matter of debate. Conventional Western blot analysis using antibodies that detect type 1 and type 2 PrPSc has severe quantitative limitations for the co-detection of type 1 and type 2 PrPSc in individual samples, suggesting that the prevalence of co-occurrence of the two types might be underestimated. We have sought to circumvent this problem by using an antibody that is type 1-specific and applied this to the sole remaining human prion disease where the phenomenon of mixed PrPSc types has not yet been shown, namely vCJD. These results show that even in vCJD where susceptible individuals have been infected supposedly by a single strain of agent, both PrPSc types co-exist: a situation reminiscent of that seen when similarly discriminant antibodies were used to analyze experimental BSE in sheep.14,17 In sporadic and familial CJD, individual brains can show a wide range of relative amounts of the two types in samples from different regions, but where brains have been thoroughly investigated a predominant type is usually evident.2,6,10 This differs from this report on vCJD, where type 1 is present in all samples investigated but always as a minor component that never reaches a level at which it is detectable without a type 1-specific antibody. It would appear that the relative balance between type 1 and type 2 is controlled within certain limits in the vCJD brain. A minority type-1-like band is also detected by 12B2 in vCJD tonsil, in BSE brain and in the brains of mice experimentally infected with vCJD, suggesting that this balance of types is agent, rather than host or tissue, specific. Interestingly the “glycoform signature” of the type 2 PrPSc found in vCJD (type 2B) is also seen in the type 1 PrPSc components, suggesting that it could legitimately be termed type 1B. PrPSc isotype analysis has proven to be extremely useful in the differential diagnosis of CJD and is likely to continue to have a major role in the investigation of human prion diseases. However, it is clear, on the basis of these findings, that molecular typing has quantitative limitations and that any mechanistic explanation of prion replication and the molecular basis of agent strain variation must accommodate the co-existence of multiple prion protein conformers. Whether or not the different conformers we describe here correlate in a simple and direct way with agent strain remains to be determined. In principle two interpretations present themselves: either the two conformers can be produced by a single strain of agent or vCJD (and, therefore, presumably BSE) results from a mixture of strains, one of which generally predominates. Evidence for the isolation in mice of more than one strain from individual isolates of BSE has been presented previously.18,19 One practical consequence of our findings is that the correct interpretation of transmission studies will depend on a full examination of the balance of molecular types present in the inoculum used to transmit disease, in addition to a thorough analysis of the molecular types that arise in the recipients. Another consequence relates to the diagnostic certainty of relying on PrPSc molecular type alone when considering the possibility of BSE infection or secondary transmission in humans who have a genotype other than methionine at codon 129 of the PRNP gene. In this context it is interesting to note that this minority type 1B component resembles the type 5 PrPSc described previously to characterize vCJD transmission into certain humanized PRNP129VV transgenic mouse models.12,20 This apparently abrupt change in molecular phenotype might represent a selection process imposed by this particular transgenic mouse model. Irrespective of whether this proves to be the case, the results shown here point to further complexities in the relationship between the physico-chemical properties of the prion protein, human disease phenotype, and prion agent strain. Acknowledgments snip... Type 1 PrPSc in Variant Creutzfeldt-Jakob Disease 157 AJP January 2006, Vol. 168, No. 1 ...TSS
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