Sheep: more in vitro prion conversion
Conversion of normal prion via a localized domain
Protease-resistant, detergent-insoluble prion not necessarily infectious
Deliberate domino iatrogenic amyloidosis
AD amyloid peroxide causes Alzheimer's damage?
Clusterin prevents aggregation of neuropeptide 106-126 in vitro.
Population dynamics of scrapie in a sheep flock
Prion polymorphisms in sheep with natural scrapie in Norway
Monoclonal antibodies in human prion disease
E200K + valine at codon 129 = type 2 protease-resistant prion
Brain injury does not modify TSE
Alex Bossers thesis, Chap 6 1 June 1999Congratulations to Alex Bossers on completion of his thesis! This has appeared as a elegantly printed 145 page book (which is not on his web site at this moment). Most of the chapters have appeared as journal articles. Chapter 7 carries an account of the in vitro cross-species conversion studies published in Nature. 1997 July 17; 388(6639):285-8 with the Rocky Mtn lab. That experiment raised the question, when are the follow-up studies going to be published, especially the ones exploring the conversion of normal human prion to rogue, by CWD.
Chapter 6 partly addresses this mystery; it has been submitted to J. Virology but not yet appeared. This looks at the 9 sheep allele conversion efficiencies, as driven by 3 homozygous PrP genotypes (wt , AA136VV, QQ171RR). Availability of these homozygous scrapie sheep was crucial for the interpretability of results. Target normal prion protein of the right sequence was produced in cell lines. Naturally, conversion efficiencies varied enormously for different scrapie isolates even of the same genotype, yet relative efficiencies did not, allowing intra but not inter normalization by each homozygous case.
Wildtype, A136V, and L141F were most efficiently converted, which correlates with observed scrapie susceptibility. Q171R, R154H, and M112T were least efficiently converted, correlating with resistance to scrapie. (Note however that these _are_ still converted with some 15% efficiency; one 154HH and one 171RR scrapie sheep are known.) This extends the Nature paper: the in vitro efficiency of protease-resistant protein production works as a rough proxy for quantitating the species barrier.
Recall sheep and cow had the same efficiency in converting human, supporting transmissibility of scrapie to humans. While more work is needed, this is the best available science upon which rational policy must be based. (Molecular studies are much more powerful than questionaires.)
Q171R is the first experimental counter-example to like-like: it drives conversion more efficiently in the heterozygous situation (wt is 138%, 136V 125%, 137T 117%). Only one scrapie sheep isolate was available here. [This phenomenon had been anticipated some time back (May 1997) at http://www.mad-cow.org/~tom/species_barrier.html -- the STC matrix is not always symmetric nor diagonal dominant, though these remain the rule for geometrical reasons given in the 1960's by Jacob-Monod.]
Note that this amounts to a negative species barrier. This question has come up before in first passage of BSE to kudu. More generally, exposing many hundreds of zoo species to BSE could create highly undesirable infections agents in some of them.
Pairs like A136V and wildtype behaved more conventionally. Here the normalized homozygous:heterozygous ration switched from 60% (wt, 136VV driven) to 53% (136VV, wt driven). In addition, the convertibility order of each allele was generally independent of driving seed (table 2).
137T is like wildtype in its high conversion efficiency. No explanation is offered for why wildtype makes the most efficient target; this is not supportive of notions of other authors suggesting mutations would be more susceptible being less thermodynamically stable. However, note that wt is only 1 step removed at most from the driving rogue prion whereas the others are 2 steps removed, eg, 136V driven by 171R, so wt reactions have less of a sequence species barrier.
No spontaneous protease resistant protein was seen under the conditions used. This is of special interest in regards to R211Q which is somewhat comparable to a GSS site. This is apparently a common polymorphism in goats and suggested as the source of the rare allele in sheep.
Scrapie sheep homozygous at 154H would be of considerable interest: is 154H an intrinsically resistant target in vitro or would it be converted efficiently if a homozygous seed were present.
Variability in different isolates of the same prion genotype could have many explanations. Here, the source flock susceptibility allele is considered as a potentially important background complication.
An interesting point is made in regards to breeding sheep against scrapie, that it might be better to mix several resistant genotypes into the flock to benefit from heterozyogous inefficiencies. This would be have advantages in not requiring the sheep to be so inbred that other desirable traits are lost.
All in all, a lot of good information to ponder here.
Comment (Alex Bossers 3 Jun 1999 ): One addition : Chap 6 describes the conversions of three different types of PrP-Sc, namely homozygous wt (A136 R154 Q171), homozygous 136V (V136 R154 Q171), and homozygous 171H (A136 R154 H171). So we did not test 171R PrP-Sc since no biological sample was preserved from that single case. The PrP-Sc 154H sample has been requested by us months ago. This would finalize the scientific support for breeding policies.
The EMBO Journal Vol. 18,pp. 3193-3203, 1999 Motohiro Horiuchi and Byron CaugheyComment (webmaster): This is an important extension of earlier papers (Kocisko et al. 1994, Chabry et al 1998, DebBurman et al 1997, etc] on in vitro conversion to PK resistance. It is difficult to review briefly because of its length and the complexity of describing controls.
Basically, they got away from guanadine, using 200mM KCl and pH 6.0 instead. The advantage of more physiological conditions is that antibodies can be used to physically block certain epitopes, here carboxy-terminal, that in turn blocks conversion. However, antibody proteins are so large that blockage only at epitope 219-232 is hard to interpret. (Their panel was a little sparse in the main globular domain as well.) Figure 8 is a good place to start -- the authors are the first to throw in space-filling carbohydrates [unfortunately -N-acetylglucosamine-N-acetlyglucosamine-mannose-(mannose)2] in a model.
While KCl worked with 5x better efficiency than nothing, conversion efficiency was still half that of GuHCl. Hybrid conditions (say, lower GuHCl) was not reported. While it might seem churlish to complain about successful conversion conditions, there is still quite a bit of material not converting for which there is no explanation. There is also some PK res not centrifuging out and PK sens that does -- what are these?
They also get big benefits from using a mouse with a stop codon just before the GPI attachment site. This results in (apparently) near- native protein free in the extracellular medium. S-35 met labels all protein of course; supernatants and lysates thus have built-in specificiy controls.
They find a temporal separation of binding of PK sens to PK res and conversion of sens. In fact, KCl is not needed to have sens bind to res (however it is barely converted after 48 hrs). With KCl, binding is slower but conversion is better. There are many more experiments establishing the specificity of res as a seed for the conversion process.
It was not shown that bound sens was actually an intermediate in the conversion process sequence of events. The authors note an inconsistency with the lack of inhibition by 3F4 to the fibrilogenic epitope. What might be going on is that 106-126, a separate domain, is forming the cross-beta with the globular domain as mainly a decorative appendage. Recruitment of sens is initially done through a dimer oligomerization patch on the globular domain; later, the recruited monomer rearranges itself to join the growing fibril.
This approach is providing progress; while getting control of conditions is a grind, there are important applications to species barriers and therapy (as noted by the authors).
In the transmissible spongiform encephalopathies, normal prion protein (PrP-sen) is converted to a protease-resistant isoform, PrP-res, by an apparent self-propagating activity of the latter. Here we describe new, more physiological cell-free systems for analyzing the initial binding and subsequent conversion reactions between PrP-sen and PrP-res. These systems allowed the use of antibodies to map the sites of interaction between PrP-sen and PrP-res.
Binding of antibodies (219-232) to hamster PrP-sen residues 219-232 inhibited the binding of PrP-sen to PrP-res and the subsequent generation of PK-resistant PrP. However, antibodies to several other parts of PrP-sen did not inhibit. The 219-232 epitope itself was not required for PrP-res binding; thus, inhibition by 219-232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP-sen structure.
The selectivity of the binding reaction was tested by incubating PrP-res with cell lysates or culture supernatants. Only PrP-sen was observed to bind PrP-res. This highly selective binding to PrP-res and the localized nature of the binding site on PrP-sen support the idea that PrP-sen serves as a critical ligand and/or receptor for PrP-res in the course of PrP-res propagation and pathogenesis in vivo.
J Virol 1999 Aug;73(8):6245-6250 Chabry J, Priola SA, Wehrly K, Nishio J, Hope J, Chesebro BConversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.
Comment (webmaster): This would seem to be a case of capping the recruiting fibril with a monomer that does not serve to further recruit larger peptides. A graphic illustrating how this might work was posted here on 14 Aug 97 at l While a very interesting experimental result with many implications, and one with potential to arrest fibril extension in vivo, it might present therapeutic risks if 119-136 forms amyloid on its own. That is why covalently modified residues must cap the caps.
J Biol Chem, Vol. 274, Issue 25, 17981-17986, June 18, 1999 Gideon M. Shaked, Gilgi Fridlander, Zeev Meiner, Albert Taraboulos, and Ruth GabizonIt has been postulated that prion diseases propagate by the conversion of detergent-soluble and protease-sensitive PrPC molecules into protease-resistant and insoluble PrPSc molecules by a mechanism in which PrPSc serves as a template. We show here that the chemical chaperone dimethyl sulfoxide (Me2SO) can partially inhibit the aggregation of either PrPSc or that of its protease-resistant core PrP27-30.
Following Me2SO removal by methanol precipitation, solubilized PrP27-30 molecules aggregated into small and amorphous structures that did not resemble the rod configuration observed when scrapie brain membranes were extracted with Sarkosyl and digested with proteinase K.ŬInterestingly, aggregates derived from Me2SO-solubilized PrP27-30 presented less than 1% of the prion infectivity obtained when the same amount of PrP27-30 in rods was inoculated into hamsters.
These results suggest that the conversion of PrPC into protease-resistant and detergent-insoluble PrP molecules is not the only crucial step in prion replication. Whether an additional requirement is the aggregation of newly formed proteinase K-resistant PrP molecules into uniquely structured aggregates remains to be established.
Comment (webmaster): DMSO is a potent solvent with many effects. It should not be assumed that proteins can be reversibly denatured by adding and removing solvents under harsh conditions. The treatments here may simply have irreversibly denatured infectious fibrils; renaturing conditions for fibrils were not found and monomers came out of solution too rapidly, equilibrium state not being attained. It is no surprise then that the amorphous aggregates lost infectivity.
15 Jun 99 MedlineComment (webmaster): This cure isn't readily adapted to CJD -- it would require a brain transplant. The curious factoid here is that the amyloid-producing liver is given in turn to a patient with liver cancer. So the first recipient remains genetically abnormal but that gene is not expressed to any extent; the second patient is genetically normal except for the new liver but presumbably acquires deliberate iatrogenic amyloidosis.
Hepatogastroenterology 1998 Sep-Oct;45(23):1375-80 Monteiro E, Perdigoto R, Furtado ALFamiliar amyloid polyneuropathy (FAP), an autosomal dominant inherited multisystemic disorder was first observed by Corino de Andrade, a Portuguese neurologist, in 1939. This disease of Portuguese origin was probably spread by fishermen, mainly to Sweden and Japan. There are neural and systemic amiloid deposits. The amyloid protein is the variant transthyretin (TTR) in which methionine (MET) is a substitute for valine in position 30 (TTR MET 30). It is mainly produced by the liver (90%) and, in small amounts, by the choroidal plexus. Symptoms usually start in the 3rd and 4th decade of life and the patients usually die within 10-15 years.
From the therapeutic options--plasmapheresis, immunoadsorption and liver transplantation; the latter seems to be the only one, which stops the production of TTR MET 30 in a permanent way, by means of the liver. The lack of any other effective therapy and the success of the first liver transplantation performed in Sweden arouse great hope. So far, around 300 patients have been transplanted all over the world. A hundred and thirty of them were transplanted in Portugal. A Kaplan Meier survival curve of the Portuguese patients shows a survival rate of 78% at 5 years.
However, in spite of the progression of the disease being halted, the irreversibility of some neurological lesions seems to persist. This fact raises the problem of the timing of the transplantation. It seems that the patients should be transplanted as soon as the symptoms start, since mortality and severe morbidity seems to mainly involve those in whom symptomatic disease has lasted longer than six years. As the explanted liver is a morphologic normal liver, a sequential (domino) transplant has been carried out in 16 cases so far done on patients with either hepatocellular carcinoma or liver metastatic disease.
Transplantation 1998 Jun 15;65(11):1496-8 Stangou AJ, Heaton ND, Rela M, Pepys MB, Hawkins PN, Williams RIn transplantation, novel methods are required to augment the supply of donor organs. We report the first domino liver transplant in which a patient with familial amyloid polyneuropathy (FAP) received an orthotopic split liver graft, and her explanted liver was donated to another patient. Three successful liver transplants were thus achieved from the one cadaver liver.
A cadaveric donor liver was split and the left lobe was grafted into a child with biliary atresia. The right lobe was transplanted into a woman with FAP associated with the transthyretin Met30 variant. Her own otherwise healthy liver was donated to a patient with cirrhosis and hepatocellular carcinoma.
Fifteen months after transplantation, all three recipients are well with normal liver function. The domino recipient developed inferior vena cava stricturing at the level of anastomosis after surgery with resultant ascites, requiring dilatation and LeVeen shunt insertion. Serum amyloid P component scintigraphy showed amyloid regression in the domino donor and to date has not identified any amyloid deposits in the recipient, who also remains free of tumor recurrence.
Domino transplantation using the livers from patients with FAP may be justified for patients whose disease condition precludes a long spell on the waiting list, including those with hepatic malignancies and those for whom palliation rather than long-term cure is the aim.
Biochemistry, May 27, 1999 Xudong Huang, Craig S. Atwood, Mariana A. Hartshorn, ..., Ashley I. BushComment (webmaster): This paper could be a missing link that explains the central mystery in amyloid disorders: why cross-beta fibrils are toxic. The authors find that Alzheimer Abeta amyloid can reduce ferric iron and cupric copper. These then are re-oxidized in a process whereby molecular oxygen is reduced to hydrogen peroxide.(via a hydroxyl free radical intermediate that is very reactive and damaging). This would explain the benefit of vitamin E therapy.
No structural explanation is offered for how iron or copper bind amyloid or whether this is specific to the 1-42 sequence or related to cross-beta architecture. Rat homologue has 3 substitutions relative to human (R5G, Y10F, and H13R) which markedly lowers its pathogenicity. Note prion repeat region binds copper and zinc but is lost to proteolysis in prion amyloid. There cannot be different unrelated pathological mechanisms for each of 22 proteins forming fibrils with a common architecture -- cross-beta itself must be able to bind and activate these reactive metal ions.
Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid Abeta peptides has been linked to peroxide generation in cell cultures by an unknown mechanism. We now show that human Abeta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II) with the generation of the hydroxyl radical [TBARS assay], setting up conditions for Fenton-type chemistry. Molecular oxygen is then trapped by Abeta and reduced to H2O2 in a reaction that is driven by substoichiometric amounts (1:10) of Fe(II) or Cu(I). Efficiencies match in vivo pathology: A1-42 exceeds A1-40, which exceeds rat A1-40. These findings indicate that the accumulation of Abeta could be a direct source of oxidative stress in Alzheimer's disease.
Abeta is a metalloprotein that binds Zn(II), Cu(II), and Fe(III). A histidine-mediated zinc binding site was mapped to a contiguous sequence between positions 6 and 28 of the Abeta sequence.
Cu and Fe concentrated (0.4 mM and 1 mM, respectively) in amyloid deposits in AD may serve to both assemble A aggregates and engender reactive oxygen species generation. These reactions may contribute to the oxidation damage and A oligomerization that is in evidence in AD.
Transgenic mice that express amyloid neuropathology have been reported to concentrate redox-active Fe in histological amyloid deposits, suggesting that the recruitment of Fe is a general feature of beta-amyloid architecture. For metal ions to cycle between reduced and oxidized states requires an outside source of oxidizing agent because there is net reduction of molecular oxygen.
Biochem Biophys Res Commun 1999 Jun 7;259(2):336-340 McHattie S, Edington NThe prion/amyloid neuropeptide 106-126 spontaneously aggregates to form fibrillar structures in vitro. The aggregation in vitro could be prevented in a dose-related manner by clusterin, and the specificity of this action was confirmed by reversal with antibody to clusterin. The relevance of these observations is discussed in relation to previous observations that clusterin and PrP co-localise in naturally occurring cases of BSE.
Michel D, Chatelain G, North S, Brun G Biochem J 1997 Nov 15;328 ( Pt 1):45-50Clusterin/apoJ is an intriguing gene frequently isolated by differential screening in laboratories from different areas of molecular biology, since it is overexpressed in numerous cases of degenerative diseases such as Alzheimer's disease and scrapie. While the dramatic increase of clusterin expression in injured tissues is well established, the molecular basis of the gene induction remains unclear.
The only DNA region strictly conserved between clusterin gene proximal promoters from different vertebrate classes is a14-bp DNA element specifically recognized by the HSF1 transcription factor which can mediate heat-shock-induced transcription in transient expression assays. Conversely, the avian clusterin proximal promoter, point-mutated at the level of this element, no longer transmits heat-shock activation.
These findings provide a possible explanation for the high sensitivity of clusterin expression to environmental changes and allow the classification of clusterin as an extracellular version of heat-shock protein.
Eur J Neurosci 1996 Mar;8(3):589-97 Chiesa R, Angeretti N, Lucca E, Salmona M, Tagliavini F, Bugiani O, Forloni GThe synthetic peptide homologous to PrP fragment 106-126 (PrP 106-126) induced in vitro neuronal apoptosis and glial proliferation. We used Northern blot analysis and the RNA polymerase chain reaction to assess the expression of several genes associated with programmed cell death and proliferation. Blots of total RNA extracted from neuronal and astroglial cells exposed to PrP 106-126 for between 1 h and 7 days were hybridized with probes recognizing c-fos, c-jun, c-myc, p53, hsp-70 and bcl-2 mRNA.
Except for a slight decrease in bcl-2 mRNA in neuronal cells, no change in other transcripts was evident. Since clusterin (apolipoprotein J) mRNA levels are increased in prion-related encephalopathies and clusterin immunoreactivity has been located in association with PrPSc in Gerstmann-Straussler-Scheinker brain, the expression of clusterin was determined in neuronal and astroglial cells chronically exposed to PrP 106-126.
Although the induction of clusterin has been involved in the apoptotic mechanism in other experimental conditions, its expression was unchanged in PrP 106-126-treated neurons, while a three-fold induction of clusterin mRNA was observed in astrocytes exposed to PrP 106-126. To investigate whether the clusterin up-regulation was simply associated with the astroglial proliferative stimulus of PrP 106-126 or was specifically induced by the peptide, we measured clusterin expression in astrocytes finding PrPSc is directly involved in the clusterin up-regulation seen in prion-related encephalopathies and is associated with astroglial cells.
J Gen Virol 1999 Jun;80 (Pt 6):1551-6 Doh-ura K, Mohri S, Tashiro H, Kawashima T, Kikuchi H, Iwaki TThe pathogenesis of neuroinvasion in Creutzfeldt-Jakob disease and other transmissible spongiform encephalopathies following the peripheral uptake of a disease agent is still not fully understood. The possibility of neuroinvasion either being established or being accelerated by an insult to the brain has not previously been tested. The experiment described herein was designed to examine this possibility by wounding the brain following an intraperitoneal challenge with a mouse-adapted strain of human transmissible spongiform encephalopathy, Fukuoka-1 strain. The results showed that brain injury introduced in any period before the appearance of cerebral abnormal prion protein deposition modified neither the clinical features, including the incubation period, nor the histopathology of the mice. Our findings suggest that neurovirulence of the agent may not be sufficiently promoted by brain injury.
Philos Trans R Soc Lond B Biol Sci 1999 Apr 29;354(1384):751-6 Woolhouse ME, Matthews L, Coen P, Stringer SM, Foster JD, Hunter NA detailed analysis of an outbreak of natural scrapie in a flock of Cheviot sheep is described. A total of 137 cases was reported over 13 years among 1307 sheep born into the flock. The epidemiology of scrapie can only be understood with reference to sheep demography, the population genetics of susceptibility to scrapie, pathogenesis during a long incubation period, and the rate of transmission (by both vertical and horizontal routes), all of which interact in complex ways.
A mathematical model incorporating these features is described, parameter values and model inputs are derived from available information from the flock and from independent sources, and model outputs are compared with the field data. The model is able to reproduce key features of the outbreak, including its long duration and the ages of cases.
The analysis supports earlier work suggesting that many infected sheep do not survive to show clinical signs, that most cases arise through horizontal tranmission, and that there is strong selection against susceptible genotypes. However, important aspects of scrapie epidemiology remain poorly understood, including the possible role of carrier genotypes and of an environmental reservoir of infectivity, and the mechanisms maintaining alleles giving susceptibility to scrapie in the sheep population.
Journal of General Virology (1999), 80, 10731077 M. A. Tranulis, A. Osland, B. Bratberg, M. J. Ulvund Fax +47 51 62 72 90.Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.
Naturwissenschaften 1999 May;86(5):212-20 Bodemer WDetection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies.
Knowledge of the conformational transition rendering a non-pathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies.
Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis--a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.
Ann Neurol 1999 Jun;45(6):812-6 Hainfellner JA, Parchi P, Kitamoto T, Jarius C, Gambetti P, Budka HA novel phenotype of familial Creutzfeldt-Jakob disease (CJD) with mutated codon 200 of the prion protein gene (PRNP) coupled with the valine codon 129 (E200K-129V haplotype) has two features never observed in subjects carrying the pathogenic mutation coupled with the methionine codon 129 (E200K-129M haplotype):
(1) plaque-like prion protein (PrP) deposits in the cerebellum and
(2) type 2 protease-resistant prion protein (PrP(res)). This observation further underlines the role of codon 129 on the mutated PRNP allele in modulating the phenotype of familial prion diseases.