Iceland meeting of 22-27 August, 1998
Erice workshop book finally appears
Prion immunoreactivity in appendix
Prion protein expression, superoxide dismutase activity, and iNOS.
High risk sheep alleles found in New Zeeland/Australia
An unusual case of CJD: "parkinson plus"
APO E not correlated with CJD
Is beef ban followed: sensitive test for bovine mDNA
New sheep alleles
Mule Deer sequence update
N. Hunter review of scrapie
Biochem J 1998 Sep 1;334( Pt 2):423-429 Brown DR, Besinger AThe function of the prion protein (PrPc) remains uncertain. It has been suggested that prion protein expression may aid cellular resistance to oxidative stress by influencing the activity of Cu/Zn superoxide dismutase (Cu,Zn SOD). The activity of Cu,Zn SOD was investigated in mice with different levels of PrPc expression. Increasing levels of PrPc expression were linked to increased levels of Cu,Zn SOD activity. Western-blot and Northern-blot analysis indicated that mice either lacking or overexpressing PrPc had levels of Cu,Zn SOD mRNA equivalent to those expressed in wild-type mice. Mice overexpressing the prion protein had lower levels of resistance to oxidative stress but higher expression levels of glutathione peroxidase, probably due to increased levels of hydrogen peroxide produced by increased Cu,Zn SOD activity. When cells were metabolically labelled with radioactive copper, increased radioactivity was immunoprecipitated with Cu,Zn SOD from mice with higher levels of PrPc. In addition, diethyldithiocarbamate, a copper chelator that inactivates Cu,Zn SOD by capturing copper from the molecule, is more able to inactivate Cu,Zn SOD expressed in animals with higher levels of PrPc. As recent studies have suggested that PrPc may regulate some aspect of copper metabolism, it is suggested that PrPc expression may regulate Cu,Zn SOD activity by influencing copper incorporation into the molecule.
J Neurovirol 1998 Aug;4(4):445-450 Ju WK, Park KJ, Choi EK, Kim J, Carp RI, Wisniewski HM, Kim YSThe neuronal cell damage caused by inducible nitric oxide synthase (iNOS) in brain has been reported to be associated, at least in part, with many neurodegenerative diseases including Alzheimer's disease. We recently observed vacuolation and astrocytosis in the brains of ME7 scrapie strain-infected C57BL mice. To investigate if these phenomena might have a relationship to iNOS, the level of iNOS expression was measured immunohistochemically and molecular biologically in the brains of scrapie-infected C57BL mice. The number and size of astrocytes were increased and immunoreactivity of glial fibrillary acidic protein (GFAP) was significantly enhanced. iNOS immunoreactivity was observed in the astrocytes of the scrapie-infected group, but not in the control group. iNOS mRNA levels were increased in scrapie-infected mice compared to the levels in non-infected mice of the same age. Our results suggest that iNOS induction in reactive astrocytes is a part of the neurodegenerative mechanisms in scrapie infection.
J Gen Virol 1998 Aug;79( Pt 8):2079-2082 Hunter N, Cairns DAs natural scrapie occurs only in sheep of specific PrP genotypes, one proposed aetiology was that scrapie is simply a genetic disease. However, Cheviot and Suffolk sheep of scrapie-susceptible genotypes are found in Australia and New Zealand, both generally accepted to be scrapie-free countries. A study of more common Australia and New Zealand sheep breeds (Merinos and Poll Dorsets) was carried out in order to obtain more generally applicable estimates of Australia and New Zealand sheep PrP genotype frequencies. We have confirmed that animals of highly susceptible PrP genotypes are found in Australia and New Zealand. Interestingly, the Poll Dorset sheep, although born in New Zealand, were brought to the UK as young adult animals and subsequently remained free of clinical scrapie despite 21% of the sheep having scrapie-susceptible genotypes. These results have implications for the genetic control of occurrence of the equivalent human diseases.
Neurology 1998 Aug;51(2):617-619 Vingerhoets FJ, Hegyi I, Aguzzi A, Myers P, Pizzolato G, Landis TA patient with histopathologically verified sporadic Creutzfeldt-Jakob disease (CJD) presented initially with diplopia, sleep disturbances, and L-dopa-responsive parkinsonism. After more than a year of slow progression, he did not become demented, and failed to fulfill the clinical criteria for possible CJD. No clinical examinations currently proposed to detect CJD showed the disease. CJD should be in the differential diagnosis of "parkinson plus" syndromes until a different etiology has been found or a histopathologic examination performed.
Neurology 1998 Aug;51(2):548-553 Chapman J, Cervenakova L, Petersen RB, Lee HS, Estupinan J, Richardson S, Vnencak-Jones CL, Gajdusek DC, Korczyn AD, Brown P, Goldfarb LGThe APOE genotype has been shown to influence the risk of developing sporadic and familial AD. This effect is isoform-dependent, the APOE epsilon4 allele increasing susceptibility and the APOE epsilon2 allele providing protection. Amyloid formation is an important part of the pathogenesis in AD as well as in spongiform encephalopathies; apoE deposition in amyloid plaques has been documented in both conditions. We examined the frequency of the APOE alleles in patients with various forms of transmissible spongiform encephalopathies, or prion diseases, including sporadic and iatrogenic Creutzfeldt-Jakob disease; familial Creutzfeldt-Jakob disease associated with PRNP 178N/129V and 200K/129M point mutations and a 24-nucleotide repeat expansion; fatal familial insomnia caused by the 178N/129M mutation; Gerstmann-Straussler-Scheinker disease associated with 102L/129M mutation; and kuru.
None of the groups we studied had a significant excess of APOE epsilon4 allele when compared with appropriate controls. CONCLUSION: Our results do not support the contention that the APOE epsilon4 allele is a risk factor for developing Creutzfeldt-Jakob disease or related disorders.
Gene 1998 Feb 27;208(2):131-138 Komatsu Y, Horiuchi M, Ishiguro N, Matsui T, Shinagawa MApolipoprotein E (ApoE) plays a central role in lipid transport and is suggested to be involved in neuronal repair. Human ApoE epsilon 4 allele is known as a risk factor for Alzheimer's disease, and an association of the human ApoE genotype with the human prion disease, Creutzfeldt-Jakob disease, is suggested, albeit controversial. We analyzed the sheep ApoE gene to determine whether any association between the sheep ApoE genotype and the sheep prion disease, scrapie, existed. The sheep ApoE cDNA contained an open reading frame (ORF) consisting of 948 base pairs (bp) that encoded 316 amino acids (aa). The sheep ApoE gene was composed of four exons separated by three introns, and the ORF was encoded by three exons, designated exons 2, 3, and 4. Nucleotide sequence analysis also showed the presence of one G/T nucleotide polymorphism in the ORF that resulted in an Ala/Ser amino-acid substitution at codon 258. PCR-restriction fragment length polymorphism analysis of genomic DNA showed the presence of three sheep ApoE genotypes that were the result of the homologous and heterologous combinations of the two alleles. We analyzed the sheep ApoE genotypic and the allelic frequencies in scrapie and control Suffolk sheep, but they did not significantly differ from those in the control sheep, even though PrP genotype-matched populations were compared. The ApoE genotype appeared not to be associated with the progression of the disease when looking at the age at death. These results indicated that in Suffolk sheep, none of the ApoE genotypes was associated with scrapie.
Clin Neuropathol 1998 Jul;17(4):229-234 Heldt N, Boellaard JW, Brown P, Cervenakova L, Doerr-Schott J, Thomas C, Scherer C, Rohmer FWe report a kindred of French/Alsatian origin with symptoms of Gerstmann-Straussler-Scheinker disease over 3 generations. In the propositus, cerebellar signs and memory disturbance were the presenting features, followed by other neurological manifestations. Biopsy of the cerebral cortex showed numerous multicentric and "kuru"-type amyloid plaques that on immuno-light and electron microscopy stained with antibody to prion protein. Molecular genetic analysis revealed an A117V mutation in the open reading frame of the prion protein gene. Questions as to pathology and spread of this mutation are discussed.
J Food Prot 1998 May;61(5):513-518 Tartaglia M, Saulle E, Pestalozza S, Morelli L, Antonucci G, Battaglia PAA ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and the amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds.
Some highlights from the 'TSE' meeting in Iceland last week [Alex Bossers reporting]:
- B-cells, T-cells?? NO, FDCs (follicle dendritic cells) seem to be the key cells in TSEs. Charles Weismann presented some nice experiments that the FDCs play the major role in replicating TSE, while B-cells in the neighbourhood of FDCs were able to take up but not to replicate the agent. Experiments in the UK also point in the direction of the FDCs. Finally different research groups seem to agree with each other. So far this work was not presented as in press yet.
- John Collinge has plans to screen for nvCJD by tonsillar biopsies. All nvCJD cases analyzed thus far are positive by IHC in the tonsils while the other types of CJD are 'negative' in tonsils. His plans include screening all routinely taken tonsils for nvCJD to get an indication of the nvCJD incedence and the amount of cases still to come.
- Prusiners group also has bovine-PrP expressing mice, however these mice had incubation times after BSE inoculation even longer than the most to BSE susceptible normal mice (R3).
- Prusiners group again: Defining structural differences between TSE strains (same PrP template as basis) using phage-displayed MCA's. MCA's to different regions of PrP-Sc were labelled and incubated with the different strain isolates. Different binding patterns were found for the different strains! ==> more conformations in one (molecule) seed.
Prusiner made an interesting comment on unusual aspects of M Bruce's R3 mice strain at the Inquiry hearing, beginning at Day 27 page 22. Basically he doesn't believe the graph on page 500 of Bruce's Nature paper in the fall of 1997: "These R3 mice... behave toward BSE and variant CJD in a way that is very much different than anything that has ever been seen except with transgenic mice.... human, mouse, bovine, feline prion.. quite remarkable, that these curves are superimposable..we are talking about four different amino acid sequences of PrP. ..."
The Iceland result is not surprising per se in that like-like primary sequences in one strain of mouse could have longer incubation times than like-unlike in another strain: there are many possible explanations. However it does point out the impossibility of quantitatively measuring in vivo species-barrier efficiencies -- there will always be some mouse strain out there where it will be still lower.
The monoclonal antibody phage display is a welcome addition. However, different strains do not necessarily have the same underlying covalent structure (or even a unique covalent structure), even though they may have started out with identical covalent primary structure for full-length prion apo-protein (same PrP template as basis?). This is due to different endopeptidase boundaries, ragged N and C exopeptidase termini, differing arginine modification, and the reported 400 different glycosylations.
The prion strain literature repeats this mistake so many times that, just like the giant alligators in the New York City sewer system, it has become an established fact. (More oft-repeated rubbish: the statement that only 1 in 100,000 monomers is infectious when really the '1' means one infectious unit in a particular inefficient 1991 indicator strain bioassay, not that only 1 of the monomers was in an infectious configuration. And how many times have you seen the falsehood that 'the prion protein is the only documented case of an infectious protein.')
If the two strains don't have the same covalent structure, their 3D conformations cannot possibly be the same because (1) they aren't even comparable and (2) the Schroedinger equation forbids it even in stretches where they are identical through boundary conditions. Unless they can show that the final covalent structures are the same globally, their outcome follows from fall-term Chemistry 101.
What they showed instead was that the mAb phage display had the ability to detect differences in the strains. And hopefully they learned some specifics about what stretches were exposed or not exposed and so something about the overall structural pictures. Strain differences are just window-dressing upon a standard underlying architecture.
If there is such a thing as immuno-gold mAb phage display, they might be able to sort out another exceeding vexatious issue in trans-species and heterozygous TSE, namely whether individual congophilic fibrils have identical strain units. Recall that in P102L, A117V, D1787N, F198S, and N217R have amyloid consisting solely of mutant allele, whereas Y145 stop, repeat insertions, E200K, V210I, and sporadic M129V are also able to recruit the 'other' allele [compilation of Cardone and Pocchiari, pg 247, Erice workshop]. I expect in some cases that the individual fibers have sorted themselves out over time through reversible equilibria and are pure monomer of one strain type conformer or the other. So several strains can co-exist with different efficiencies that change over passage, hyper and drowsy in mink etc.
Congophilic fibrils must play an essential role in defining the rogue conformer; GG Glenner had it right 20 years ago. This is implied by very recent results with Sup and Ure in yeast. What are the odds that the only 2 proteins found to have prion-like properties are the only 2 proteins in the genome to have the Asn/Gln-rich domains with prionlike fibril potential? Zero when the organism has 7000 genes.
At the Erice workshop [pg 220] Caughey and Raymond write, "... only ordered aggregates of PrP-res, albeit widely variable in size, can induce the conversion to the protease-resistant form in the cell-free system." They neatly summarize the counter-arguments to worries that not all deposits of Prp_res in vivo are birefringent or have EM-visible amyloid fibrils by (1) short oligomers are not detectable [up to 60], (2) masking by other fibrils or a plethora of other amyloid proteins, and (3) lack of fibril alignment needed for coherent optical rotation. To which I would add, (4) differential fragility to preparative procedures.
Sup was shown to be an infectious protein , with hindsight in 1965. Yeast people view protein infectivity merely as cytoplasmic inheritance associated with cell fusion, which is a very good way of looking at prion infectivity as well. Protein-only is a no-brainer in yeast because researchers have complete control over viruses, transposons, and the like.
Perhaps further taxonomy is needed in the congophilic conformational diseases. The subclasses would consist of the poly-glutamine ladder type (spinocerebral ataxia, sup, ure, ...) and the hairpin formers (Alzheimer, insulin, prion?, ...). Both classes end up as cross-beta fibrils according to fiber diffraction but there may be two structural subclasses that we are not yet distinguishing.
SCIENTIFIC PROGRAMME Thursday, 20-21 August 1998 [No abstracts are available at this time from conference web site. However, participants evidently received a set of abstracts. The webmaster reviews a few of these that were received by fax below:] 14. The uptake of recombinant prion proteins by human and murine cells is mediated by the 37 kDa laminin receptor precursor. C.I. Lasm»zas, J.-M. Peyrin, S. Ha‘k*, R. Rieger, J-P Deslys, S. Weiss D. Dormont. This would be important given the previously reported yeast two-hybrid binding provided 'mediated' means something direct. As I noted just recently, the 37k form is not the usual mature form of this protein. However, the abstract indicates vague associations with no real protein chemistry done (eg characterizing cross-linking) and no case is made that this is significant in vivo. 15.RNA aptamers directed against scrapie associated fibrils. C.I. Lasm»zas, S. Weiss The aptamer selection process is brought to bear against scrapie-associated fibril, which they note was found in the French nvCJD case, the idea being that some G-quartet among the billions and billions created could provide the necessary specificity. This would be like the Prionics monoclonal only with RNA. 18. Trans-dominant inhibition of PrPSc accumulation in scrapie-infected mouse neuroblastoma cells by the deletion mutant PrPcD 114-121. C. HĖlscher, H. Delius, A. Błrkle. FS This goes Shmerling et al one better with a much shorter and more focused deletion than 32-121 or 32-134 . 119MA-GAAAAGAV-VGGLGGYM129 shows the deletion right in the heart of the core region. And in culture too! Trans-dominant means that the normal allele cannot be recruited by exogenous rogue conformer, possibly because the deletion is binding something in the process, perhaps unable to join the growing fiber or allow its extension. Follow-up to J Virol. 72 1153-1159 1998. 23. Suramin reversibly uncouples PrPc from PrPSc biogenesis and generates a prion protein with properties of both PrPc and PrPSc. C.Spielhaupter, S. Gilch, I. Westner, M. Groschup, H.M. SchĒtzl. Suramin is a non-selective P2 -purinoceptor antagonist, a polysulfonated binaphthyl urea that competes with heparin and a general growth factor receptor and G protein antagonist. By 'properties of both' they mean 'insolubility, PK sensitivity, and N-terminus retention.' 56. PrP messenger RNA: Regulation of PrP expression through differential RNA processing in sheep tissues and implications for scrapie susceptibility. W. Goldmann, G. O•Neill, F. Cheung, F. Charlson, K. Donnelly, D. Cairns, N. Hunter. This does not relate to whether exon 2 is spliced out or not but rather tissue specific polyadenylation (ruminants only), with two speices of mRNA found differing by 2.3kb in length and in translatability. "Additional RNA processing in other positions of the untranslated regions has been found resulting in a oomplex system of post-transcriptional modulation of PrP expression." They report high variability (3' UTR polymorphisms) within and between breeds possibly associated with scrapie susceptibility. These authors have not yet corrected a major sequence screw-up at GenBank concerning the 3' UTR in sheep. There are apparently 82 errors in 3246 bp or 2.5% error rate, in M31313 or AJ223072. D38179 and U67922 agreed completely as independent determinations. 28. Gene expression in scrapie: cloning of a new scrapie responsive gene and the identification of increased levels of seven other mRNA transcripts. F. Dandoy-Dron1, F. Guillo1, L. Benboudjema, J.-P. Delys, C. Lasm»zas, D. Dormont, M.G. Tovey1, M. Dron Presumably adds to J Biol Chem. 1998 Jul 17; 273(29): 18015-18018. and J Biol Chem. 1998 Mar 27; 273(13): 7691-7697. These are after-the-fact proteins probably not relevent to the core issues but possibly important therapeutic targets.The new ones ar ScRG-1, preferentially expressed in glial cells, of 98 amino acids, a signal peptide and a known human homologue. 11. The molecular layer of the human cerebellum in Creutzfeldt-Jakob disease: A Golgi and electron microscope study. S. Baloyannis, V. Costa. 13. New insights into the life cycle of prions. S. Weiss. 14. The uptake of recombinant prion proteins by human and murine cells is mediated by the 37 kDa laminin receptor precursor. C.I. Lasm»zas, J.-M. Peyrin, S. Ha‘k*, R. Rieger, J-P Deslys, S. Weiss D. Dormont. 16. Nucleic acid induced aggregation of mouse recombinant prion protein to amyloids and other polymeric structures. P.K. Nandi,E. Leclerc 17. Expression of full length chicken prion protein and binding with filamentous phage M13 particles in vitro. H. Wang, B.S. Wong, E.A. Gould, I.M. Jones. 19. Evidence of a homologous ssDNA in nemavirus is the genome of the scrapie agent. H. Narang, Ken Bell International, Newcastle-upon-Tyne, UK. 20. Unusual characteristics of several scrapie isolates derived from hay mites. R.I.Carp1, S. Sigurarson2, H.C. Meeker1, R. Rubinstein1, R.J. Kascsak1, M. Papini1, P. Kozlowski , H.M. Wisniewski 21. Studies on the proteinase K sensitivity of cellular prion protein (PrPc) of various species. A. Buschmann, M.H. Groschup. 22. Role of immunogen and immunized species in the linear and comformational epitope response to prion protein PrP. R.J. Kascsak1, R. Rubenstein1, R. Kascsak1, R.I. Carp1, R. Meloen, J. Langeveld . 24. Suppression of PrPSc and HIV-1 gp120 induced neuronal cell death by sulfated colominic acids. H.C. SchrĖder1, H. Ushijima2, S. Perovic1, J. Leuck, P.G. Rytik, W.E.G. Młller 25. MS-8209, an amphotericin B derivative, affects both scrapie agent replication and PrPres accumulation in Syrian hamster scrapie. K.T. Adjou1, R. Demaimay1, J.-P.Deslys1, C.I. Lasm»zas1, V. Beringue1, S. Demart1, F. Lamoury1, M. Seman2, D. Dormont 26. Follicular dendritic cells in the spleen support replication in a mouse scrapie model. K.L. Brown, K. Stewart, I. McConnell, H. Fraser, M.E. Bruce. 27. TSE agents propagation after oral contamination. T.Maignien, C. Lasm»zas, V. Beringue, J.-P. Deslys, D.Dormont. 29. Electrophysiological and morphological studies of single subicular neurons in brain slices from mice infected with scrapie. J.R.T. Greene, V.H. Perry, S. Betmouni. 45. Severe, early and selective neuronal vulnerability in experimental transmissible spongiform encephalopathies. M. Guentchev1, M. Groschup2, R. Kordek3, P.P. Liberski3, H. Budka 46. The Echigo-1 panencephalopathic type of Creutzfeldt-Jakob disease passage in hamsters and neuropathological characterisation. P.P. Liberski1, J.A. Hainfellner2, A. Wali1, R. Kordek1, H. Budka 47. Atypical case of sporadic Creutzfeldt-Jakob disease in young adult patient. W. Kozubski, M. Wender, P.P. Liberski, J. Szczech, D. Lenart-Jankowska. 48. Epidemiological determinants of the pattern & magnitude of the vCJD epidemic in Great Britain. A. C. Ghani, N.M. Ferguson, C.A. Donelly, T.J. Hagenaars, R.M. Anderson. 49. Transmissions of "new variant" and sporadic CJD to mice. M.E. Bruce, R.G. Will, J. Ironside, I. McConnell, D. Drummond, A. Suttie, A. Chree. 50. Human susceptibility to Creutzfeldt-Jakob disease agents. J.-P. Deslys1, A. Jaegly1, F. Mouthon1, C. Lasm»zas1, T.B.de Villemeur3,4, A. Alp»rovitch2, D. Dormont 51. PrP gene dosage and allelic specificity in the transmissible spongiform encephalopathies. J.Manson, R. Moore*, P. McBride, J. Hope, J. Selfridge*, D. Melton. 52. The afferent projections to the mediodorsal and anterior thalamic nuclei: possible prion transmission in FFI. J.L. Velayos, M. Oliva, F. Alfageme. 53. A quantitative study of synapse loss and neuronal degeneration in the scrapie-infected murine hippocampus. M. Jeffrey1, D.A. Brown2, W.G. Halliday1, J.R. Fraser 54. Immunohistochemical characteristics of an antiserum to an N-terminal prion protein peptide. E. Monks1, E.D. Weavers1, A. Fullam-Church1, M. Rogers 57. The influence of the AHQ PrP allele on susceptibility to clinical scrapie in British flocks of Swaledale, Welsh Mountain and Finn Dorset sheep. M. Dawson, L.J. Hoinville, T.C. Martin, C.A. Hoek, A.H. Murray. 58. Tonsillar biopsy as a tool for a pre-clinical diagnosis of scrapie. B.E.C. Schreuder, L.J.M. van Keulen, M.E.W. Vromans, J.P.M. Langeveld, M. Smits. 59. Cerebrospinal fluid profile in sheep with scrapie by high resolution proton magnetic resonance spectroscopy: Possible use as a diagnostic tool ? M. P»pin1, S. Maillet2, J. Vion-Dury2, S. Confort-Gouny2, P. Russo1, R. Sanchis1, F. Schelcher3, P. Cozzone 60. Mathematical models of the epidemiology and control of scrapie within a sheep flock. M.E.J. Woolhouse, S.M. Stringer, L. Matthews, N. Hunter, R.M. Anderson. 61. Transgenic mice expressing the bovine prion protein - A useful tool for detection of BSE infectivity. A. Buschmann, E. Pfaff, H. Mueller, K. Reifenberg, M.H. Groschup. 62. Anti-oxidant status in cattle naturally affected with bovine spongiform encephalopathy. T. Martin, S. Cawthraw, M. Dawson. 63. Studies on BSE and scrapie agents: Paradoxical inactivation data. D.M. Taylor, K. Fernie, P.J. Steele. 64. Transmission dynamics and the clustering of BSE cases. C.A. Donnelly, N.M. Ferguson, A.C. Ghani, T.J. Hagenaars, R.M. Anderson.
Mol Biotechnol 1998 Jun;9(3):225-234 Hunter N[Note there is also a good review of BSE in sheep at the Maff Web site from a recent SEAC meeting. -- webmaster]
Scrapie and other transmissible spongiform encephalopathies (TSEs) are characterized by similar pathology, biochemistry and genetics. The PrP protein and its conversion to the disease-related isoform, PrPSC, are crucial for the development of all TSEs. Although scrapie is more often studied in laboratory rodents, it is not a natural disease of these animals, and much can be learned from the normal hosts, sheep. Disease incidence is linked to polymorphisms and mutations of the PrP gene. The complex relationships between PrP genotype and the survival of sheep subjected to scrapie infection are now being investigated in terms of the different structure of the PrP protein molecules produced by each allele. It is these structures and their differing abilities to convert to PrPSC that hold the key to understanding why TSEs occur.
John Collinge's group had addressed the subject of the risk from nvCJD appendectomy cases before the case of Tony Barrett surfaced in : Hill et al (1997) "Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy" The Lancet, 349, 9045, 11January: The following extract from their paper is relevant:
"Recently, we reported that nvCJD is associated with a specific pattern of protease-resistant prion protein (PrP) on Western blot analysis. This marker can already be used to aid differential diagnosis on brain biopsy samples. However, since PrP is widely expressed outside the central nervous system, we investigated whether an alternative and more accessible tissue might be biopsied to allow a diagnosis of nvCJD before death and to avoid brain biopsy. PrP is expressed in the lymphoreticular system and prion replication is known to occur in the spleen and other lymphoreticular tissues in experimental rodent scrapie models; prion infectivity has also been reported in human lymphoreticular tissues.
"We have studied PrP in tonsillar tissue obtained at necropsy using both immunohistochemistry on periodate-lysine-paraformaldehyde and formalin-fixed tissue, and Western blot analysis of frozen tissue. The patient was a 35-year-old woman who died after a 14-month illness with depression at onset followed by ataxia, hyper-reflexia, memory loss and dementia; a diagnosis of nvCJD was made by neuropathology. Abnormal PrP staining was present within tonsillar germinal centres.
Western blot analysis revealed the presence of protease resistant PrP; confirming the diagnosis of prion disease. Furthermore, the sizes and intensity ratios of the three PrP bands (representing diglycosylated, monoglycosylated, and unglycosylated PrP) were similar to those seen in brain from the same patient (designated a type 4 pattern) suggesting that it may be possible to make the specific diagnosis of nvCJD by this technique. However, tonsillar tissues from types 1-3 CJD were not available for comparison. Such study of protease-resistant PrP in lymphoreticular tissue may be particularly relevant in iatrogenic CJD after peripheral prion inoculation (for instance after treatment with human cadaveric pituitary-derived growth hormone where lymphoreticular involvement would be expected.
"In experimental murine and sheep scrapie, prion replication occurs initially in spleen and is only detectable in the central nervous system considerably later in the incubation period. Abnormal PrP immunostaining has been reported in tonsils of experimental scrapie-infected sheep long before the occurrence of clinical signs. It is possible therefore that Western blot analysis of human tonsil material may allow early clinical, or possibly pre-clinical, diagnosis of CJD and nvCJD although extensive prospective studies of suspected cases will be necessary to assess the clinical usefulness of this investigation.
Tonsil tissue can be easily obtained by biopsy under local anaesthetic in most patients and complications are most unusual. Although many adults will have had childhood tonsillectomy, lymphoreticular tissue may still be obtained from the lingual tonsillar remnants. As a result of the extreme resistance of prions to normal sterilisation procedures, current UK recommendations are that neurosurgical instruments from patients with CJD are destroyed. While infective titres of prions in tonsil may be much lower than in brain, similar precautions to avoid cross contamination and iatrogenic transmission of CJD would seem prudent at present. The development of a disposable tonsil-biopsy kit should be feasible. "
'New' natural occuring sheep PrP-ORF polymorphisms. Additionally to the 8 described (at M112T, A136V, M137T, L141F, R154H, Q171R, Q171H, R211Q) reported are: in Cyprus Q171K low incedence, no scrapie A136T low incedence, no scrapie in Iceland (and probably also in Norway!?) M137T (finally it has been found outside the Netherlands!) S138N 5-9% of Icelandic sheep, no scrapie association yet !!! R151C ! very rare, except for one flock, protective to scrapie? What will happen to the disulfide bridges?????Comment (webmaster):
For sheep to have 13 amino acid substitutions at 9 positions is quite provocative, given that humans have 17 known point substitutions at 17 positions with *15* clearly associated with CJD and 2 modulating progression (one beneficially); that is, hardly any of the human mutations have been innocuous. Using these odds at face value, which I don't recommend, the sheep alleles mostly cause scrapie. Now humans live a lot longer so small effects show up whereas sheep may get a horizontal jump-start. Given these striking data, the burden of explanation must be on sheep-ologists to tell us why the situation is so turned around in their species.
The curious point is that many thousands of control individuals have been sequenced, not just people from neurological 'flocks' and yet basically no neutral polymorphisms have shown up (other than at 3rd codon position, repeat deletions, and short insertions). Of course, sheep breeding practises make interpretation of gene frequencies impossible, that is, do rare mutations become founder-effect polymorphism? Note sheep have two double and one triple-valued allele -- this is unlike humans where all are single-valued.
Good point on R151C. Disulphides have not yet been specifically disabled in transgenic mice as far as I can recall (but see ref below) Prion protein disulphides are strictly conserved in all species back to at least 310 million years; further, this is the first change to cysteine ever observed in any species in any part of the sequence.
R151C might not be causative if the standard problem is an ER anomaly which these might cross correctly under Glockshuber's views that S-S are always formed outside the cell. [However disulphides can form internally despite the reduced glutathione if the folding protein's 3D environment drives it strongly enough.] Note that there is a disulphide isomerase in the ER; this plus steric considerations and overall energetics could prevent mixed-up disulphides. Which is not to say that R151C is necessarily neutral as it is a highly non-conservative change.
M112T A136V A136T M137T M137T S138N L141F R151C R154H Q171R Q171H Q171K R211Q (All are single-step genetic code changes)
Herrmann LM, Caughey B Neuroreport 1998 Aug 3;9(11):2457-2461The conversion of normal, protease sensitive prion protein (PrP-sen) to the abnormal protease-resistant form (PrP-res) is of central importance in the pathogenesis of scrapie and other transmissible spongiform encephalopathies. In the present study, the effects of reduction of the disulfide bond on the PrP-sen to PrP-res conversion in a cell-free system were examined. The addition of the disulfide reducing agent dithiothreitol inhibited the cell-free conversion reaction with an IC50 of 2-2.5 mM. Separate pretreatment of either PrP-sen or PrP-res with dithiothreitol and an alkylating agent also inhibited the conversion reaction. Results of this study show that preservation of the disulfide bond is important in the conversion of PrP-sen to PrP-res.
28 Aug 98 Correspondent: K. O'RourkeAF009180, Odocoileus hemionus hemionus prion gene, allele S2, and AF009181, allele S1: These entries have been revalidated. One error was found at base 348 in this truncated sequence. In addition to the coding changes, there were 2 silent changes between N1 and S2, 5 silent changes between N1 and S1, and 5 (not 1) silent changes between S1 and S2. Mule deerwere seen with the N1S2, S2S2 or S1S1 genotypes in a limited sample , no N1N1, no N1S1. A large sample will be studied in the fall.
Alleles 138S2 and 138N1 encode Ser and Asn at codon 138, respectively. Allele 138S1 differs from allele 138S2 by a silent mutation.
Comment [webmaster]: Published and unpublished sequences around the epitope IHFG show that the ser-asn allele occurs separately in many lineages at codon '138' making it doubtful for familial CWD (though allele state could modulate CWD along the lines of codon 129. Curious that mule deer have so many more silent changes than what is seen in humans.
samsrpl ihfg n dyedryyrenm canis lupus familiarishallstromii samsrpl ihfg n dyedryyrenm canis lupus familiaris samsrpl ihfg n dyedryyrenm canis lupus canadensis samsrpl ihfg n dyedryyrenm mustela vison samsrpl ihfg n dyedryyrenm mustela putorius samsrpl ihfg n dyedryyrenm equus familiaris caballus samsrpl ihfg n dyedryyrenm equus przewalskii caballus samsrpl ihfg n dyedryyrenm equus quagga boehmi samsrpl ihfg s dyedryyrenm sus scrofa samsrpl ihfg n dyedryyrenm camelus dromedarius samsrpl ihfg n dyedryyrenm lama glama samsrpl ihfg s dyedryyrenm tursiops truncatus samsrpl ihfg n dyedryyrenm gazella subgutturosa samnrpl ihfg n dyedryyrenm bos taurus samsrpl ihfg n dyedryyrenm bos primigenius taurus samsrpl ihfg s dyedryyrenm bos javanicus samsrpl ihfg s dyedryyrenm bos taurus samsrpl ihfg s dyedryyrenm bison bonasus samsrpl ihfg s dyedryyrenm tragelaphus strepsiceros samsrpl thfg n dyedhyyrenm tragelaphus angasi samsrpl ihfg s dyedryyrenm budorcas taxicolor samsrpl ihfg n dyedryyrenm capra hirtus samsrpl ihfg n dyedryyrenm capra ibex nubiana samsrpl ihfg n dyedryyrenm ovibos moschatusmoschatus samsrpl ihfg n dyedryyrenm ovis aries samsrpl ihfg n dyedryyrenm addax nasomaculatus samsrpm ihfg n dwedryyrenm hippotragus niger samsspl ihfg n dyedryyrenm cervus elaphuscanadensis samsrpl ihfg n dyedryyrenm cervus elaphus samsrpl ihfg s dyedryyrenm cervus nippon dybowskii samsrpl ihfg n dyedryyrenm odocoileus hemionus samsrpl ihfg n dyedryyrenm giraffa camelopardalis samsrpl ihfg n dyedryyrenm ateles geoffroyi samsrpl ihfg n dyedryyrenm ateles paniscus samsrpl ihfg n dyedryyrenm cebus apella samsrpl ihfg n dyedryyrenm callithrix jacchus samsrpl ihfg n dyedryyrenm aotus trivirgatus samsrpl ihfg n dyedryyrenm saimiri sciureus samsrpl ihfg n dyedryyrenm cercopithecus mona samsrpl ihfg n dyedryyrenm cercopithecus neglectus samsrpl ihfg n dyedryyrenm cercopithecus patas samsrpl ihfg n dyedryyrenm macaca sylvanus samsrpl ihfg n dyedryyrenm colobus guereza samsrpl ihfg n dyedryyrenm macaca fascicularis samsrpl ihfg n dyedryyrenm cercopithecus dianae samsrpl ihfg n dyedryyrenm callicebus moloch samsrpl ihfg n dyedryyrenm theropithecus gelada samsrpl ihfg n dyedryyrenm cercopithecus aethiops samsrpl ihfg n dyedryyrenm papio hamadryas samsrpl ihfg n dyedryyrenm macaca fuscata samsrpl ihfg n dyedryyrenm presbytis francoisi samsrpl ihfg n dyedryyrenm mandrillus sphinx samsrpl ihfg n eyedryyrenm cercocebus aterrimus samsrpl ihfg n dyedryyrenm macaca nemestrina samsrpl ihfg n dyedryyrenm macaca mulatta samsrpl ihfg n dyedryyrenm cercocebus torquatus atys samsrpl ihfg n dyedryyrenm macaca arctoides samsrpi ihfg n dyedryyrenm pongo pygmaeus samsrpi ihfg s dyedryyrenm pan troglodytes samsrpi ihfg s dyedryyrenm symphalangus syndactylus samsrpi ihfg s dyedryyrenm homo sapiens samsrpi ihfg s dyedryyrenm gorilla gorilla samsrpi ihfg s dyedryyrenm hyalobates lar samsrpi ihfg s dyedryyrenm hylobates syndactylus samsrpl ihfg n dyedryyrenm oryctolagus cuniculus samsrpm ihfg n dwedryyrenm cricetulus migratorius samsrpm ihfg n dwedryyrenm sigmodon fulviventer samsrpm ihfg n dwedryyrenm sigmodon hispiedis samsrpm ihfg n dwedryyrenm meriones unguiculatus samsrpm...ihfg n dwedryyrenm rattus rattus samsrpm...lhfg n dwedryyrenm cricetulus griseus samsrpm...lhfg n dwedryyrenm mus musculus samsrpm...lhfg n dwedryyrenm mesocricetus auratus samsrpm...mhfg n dwedryyrenm mus musculus samsrpv ihfg n eyedryyrenq trichosurus vulpecula
Prions and Brain Diseases in Animals and Humans, ed. D. Morrison NATO ISI Series Plenum Press ISBN 0-306-45825-X ... August 19-23, 1996 Erice workshop
Lee,I.Y., Westaway,D., Smit,A.F., Cooper,C., Yao,H., Prusiner,S.B. and Hood,L.GenBank entries U29185, U67922, U29186 corresponding to this article refer to an unpublished item called: Structure and Organization of Chromosomal Regions Carrying the Mammalian Prion Gene from Three Species