Prions
Mad Cow Home or Best Links

Sheep-to-human and cow-to-human transmission demonstrated in vitro
New study: Isle of the Damned?
Swiss NMR group on mammalian prion structure
Prusiner: prion protein interactions with a synthetic fragment
Surprising prion binding to APP 1 and Nrf 2
Manuelidis: flogging a dead virino?

News Flash

Request a reprint of the Nature article from Byron Caughey or James Hope 
Sheep-to-human and cow-to-human transmission of disease has now been demonstrated directly in vitro. While this doesn't establish by itself that people get scrapie or mad cow disease, it does establish that there is no absolute species barrier to these particular heterologous protein-protein conversions.

1. The rates being reported (1:10) are rather hefty, ie, think about eating a scrapie sheep brain with 10x the inoculum or thinking about eating tainted beef burgers for ten years. A big (cumulative) inoculum can make up for a lot of initial inefficiency. It would be interesting to see the spread of efficiencies in 500 isolates of sporadic CJD, whether there was overlap.

2. Even if the initial stage measured here is slow and inefficient, subsequent stages use human-to-human recruitment and so could be just as rapid (or even more rapid, depending on induced strain) as something that started as human-to-human. That is, while it is somewhat difficult for a rogue bovine prion to recruit a human prion, the early human rogue prions might then go on to recruit normal human prions quite efficiently. The animal prion is soon irrelevent, except for the human template strain that it has created. Very small initial inocula may grow exponentially, because the initial catalyst of the conversion process makes more catalyst , not just inert reaction product.

3. Scrapie and BSE would not necessarily induce create the same strain in humans (and in fact the evidence from Collinge's study et seq. suggests that they don't, taking scrapie in with the 'sporadic'). Thus the observation that the conversion efficiencies were initially similar carries no information about whether subsequent human-to-human conversion efficiencies are similar or markedly different.

4. There are many dozens of scrapie strains as well of CJD and by no means were all of these was tested here. These scrapie strains could differ markedly in both types of efficiencies. If a relatively high efficiency scrapie strain was used that was not especially high titre in the diet, then there could be low CJD from scrapie. However, the evidence concerning scrapie-induced CJD is extremely meagre one way or the other.

5. It is not at all clear to what extent and for what processes the conversion test is optimized, ie, there is no demonstration of robustness. A pinch of salt, a different buffer, or a change in pH could all dramatically shift the relative efficiency measurements. The argument that the methodology of Bossers et al did give reasonable correlations between efficiencies and incubation times when restricted solely to sheep, has not been demonstrated to be applicable to the situation here.

6. Humans live a long time. It seems from iatrogenic CJD that miniscule inocula give rise to the disease in a couple of decades. It does not take long with exponential doubling for a initially low exposure to make up for a mere one-thirtieth the initial efficiency, especially if that much of that doubling is normal sequence rogue human converting normal human prion.

This experiment isn't the last word. It does not address many steps and aspects of the infectious process. I see no basis for making firm estimates of the extent of the epidemic from it.

The vast majority of UK residents are quite likely at some early to middling stage (secondarily affected with induced rogue human seed fibers) of the disease. How many will develop symptoms during their lifetimes is hard to say, I would guess in the millions. However therapy prospects look a whole lot better lately and the new in vitro conversion will speed testing, so perhaps it will be moot in the end (for most people).-- webmaster

Potential transmissibilities of BSE and scrapie to humans

Letter to Nature vol 388 page 285 July 17, 1997
Reprints:  Dr. Byron Caughey or Dr. James Hope
Abstract and summary quotes
More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt--Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis.

Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here the authors show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, they used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. They found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrPSc) and BSE (PrPBSE). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions.

Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.

Some summary quotes from the paper:

"We must emphasise that the cell-free assay is a test only of molecular compatibility between PrP-res and PrP-sen of different sequences. Other factors such as the dose, strain, and route of infection, the stability of the infectivity in the host and the efficiency of its delivery to the central nervous system are all likely to be important in vivo."

"Nonetheless some degree molecular compatibility is likely to be essential to the transmission of TSE diseases, especially given the remarkable correlation between PrP conversion efficiencies and transmissibilities.

The relatively low efficiency of conversion by PrPsc and PrPbse may explain at a molecular level the observed failure to transmit BSE to transgenic mice expressing human PrP and the lack of an epidemiological link of CJD to sheep scrapie. It is premature to draw firm conclusions from our results about the likelihood of BSE passing to humans, although the results suggest that BSE would be no more inherently transmissible to humans than is sheep scrapie.

[Nick Nuttall's story in the Times says
The scientists, whose work has been approved by the Ministry of Agriculture and Fisheries [MAFF], ...
This apparently means that in order to get authenic samples of BSE material, the authors may have had to submit the article to MAFF for pre-approval. In other cases, MAFF has withheld material, apparently fearing bad publicity might result from scientific studies. Statistical data was withheld from researchers for years. Data on BSE-to-dog still has not been released. -- webmaster]

New study: Isle of the Damned?

21 July 1997
When the Titanic went down, the band played on. They knew it was going down and so did the passengers. Today, as the whole island perhaps sinks into dementia, the captain issues yet another bogus press release and the band still plays on, but this time the idea is to keep the passengers distracted and clueless for as long as possible.

Commentary on the Raymond paper in the July 17, 1997 Nature, in the form of an mock interview that ridicules science reporting in the mass media from the Web newspaper that gives you all the news that's fit to print plus prints all the news that gives you fits, an exclusive interview with the webmaster:

[Reporter]: Tom, what was your reaction to the recent paper in Nature concerning in vitro transmission of scrapie and BSE to humans? Let's start with the editorial, 'Tracking turncoat prion proteins' -- was it even-handed?

[Tom]: The accompanying Nature News and Views was quite good, Nature vol 388, pp.228-229. Colin Masters and Konrad Beyreuther are veteran presenilin and APP researchers. They note that the template-directed (prion heterodimer) and nucleated-crystallization (seed fiber) models may both come into play, the former having the evolved docking interface causing hetero- to be generally weaker than homo-conversions and the latter containing the strain information. (Roland is quite correct to note that this cannot be so rigid so as not to be modulated by host.) These models don't address equillibria or kinetics, which vastly complicate them.

They make two interesting points about prion chaperoning (in yeast, hsp104 genetics can be played off against sup35): the elusive first nucleation site might be mass-action driven by high chaperone concentration brought about by local stress, and that 'the basis of strain variation might be found in the structural organization of these nucleating molecules themselves' (multi-chaperone theory). There are quite a few chaperones in mammals, with absolutely fascinating 3D structures, and these can be induced under different circumstances.

Masters and Beyreuther, from their own recent work on caspase in Alzheimer, would be well aware of the role of proteases in providing denatured grist for the chaperones and future amyloid, but had no space to discuss them here. I think this will be a very hot research area because this is where things first start to go wrong, though there aren't good therapeutic opportunities with protease inhibitors -- better to cap off existing nucleation sites once and for all.

They end up, reasonably enough, balanced on the fence as far as implications go for the Raymond et al study (seemingly much more ominous and better designed than the Collinge mouse experiment): 'If we're lucky, the species barrier ... will prove to be self-limiting. But if we're unlucky..."

[Reporter]: Who did what, in the main article? There are 15 authors from 8 labs.

[Tom]: Good question. The lead author GJ Raymond has quite a few previous related papers out with NIAID colleagues Caughey, Cheesbro, Lansbury, Kosisko, Priola, Bessen, and Bossers. They may just use the names in rotation. Other authors may just have provided material. Makes you wonder what's in those UPS trucks.

[Reporter]: Did you notice anything odd about the research in the main article?

[Tom]: Yes, the first oddity of the paper is that nvCJD and met/met-sporadic CJD data, in some unstated ratio and unstated number of sources, seem to be pooled when used as driver (Fig 4b group 4; Methods, end of Paragraph 1), even though we know these so far have been different strain types.

Val/val-sporadic and met/val-sporadic were not tested at all, nor were human-to-cow/sheep/mouse/hamster. I am not thinking here of the Swiss feeding human placentas to their cows, but rather of assessing asymmetry of the species barrier (VQ-to-cow was twice BSE-to-VQ). The heterozygous state also has some interesting unpredictability associated to it: would it be midway in conversion efficiency, ie at 69% of met/met? And how much trouble is it to run another gel?

This is one of many places in the article where the in vitro data has encouraging correlations with in vivo observations: the nvCJD have been met/met so far and the in vitro data favors this 3:1. The authors note that all codon-129 genotypes may eventually be affected.

[Reporter]: Was there anything else odd about the article?

[Tom]: Yes, the other oddity is the scatter in the BSE-to-bovine data. The legend to Figure 4a says, without elaboration, 'independent experiments.' This might mean BSE purified in different labs from different cows, or is it the level of reproducibilty of the conversion reaction on identical aliquots on different gels? I need to see the latter data before reading about normalizations and cross-comparisons.

The 20 data points are spread out fairly evenly from 2.5% to 24.5%, with some scrunching at the low end. I found a mean of 10.8 and a standard deviation of 6.1. The BSE-to-met and BSE-to-val data are confined to the 0-2% range with the former visually a more efficient conversion, say 3:1.

The authors now normalize by dividing by the mean of BSE-to-cow. Fine, this puts cow at 100% and has no effect on the met to val comparison which stays the same no matter what they are divided by. And it is true that BSE-to-cow and BSE-to-human is 10:1 using the means. However, the big spread in the BSE-to-cow data gives rise to a huge (never stated) uncertainty is this estimate. For example, using the lowest cow to the highest human, we see that the ratio is 1:1.

Now let's make similar calculations with the sheep data (also quite scattered), take a deep breath, and divide the cow data by the sheep data, and conclude that VQ sheep-to-human and BSE-to-met human are 1:1. However the formal statistical uncertainty alone in this estimate is huge: somewhere between 20:1 and 1:20 perhaps, with 95% confidence. The authors are never required to state the uncertainty -- this is the complaint I hear over and over about Nature, that sufficiently topical papers don't go out for serious peer review.

3c. That's just the formal uncertainty. What I really wonder about is the normalization of the number of nucleation sites in the donor across species. It's fine to have the same number of milligrams/ml of protein used, but suppose those milligrams are in long fibrils with few end points for scrapie and half of it is inert, while the BSE milligrams are in fibrils a quarter as long with four times the end points and all of it is active. This affects quantitation across the board.

Also problematic for cross-species comparisons is that the procedure was originally optimized for homologous sheep-sheep conversion and does not attempt to emulate in vivo solvation conditions, instead using the potent denaturant guanidinium HCl at multi- molar concentrations, a cation at the very top end of the Hofmeister series.

Clearly an alien process. Who can say what the effect would be of an extra pinch of salt, a change in pH, or a different buffer?

The authors do provide numbers supporting statistical signficance of conclusions that homo-conversions are higher than hetero-conversions. It is important to keep in mind that very few of the possible pairs of mammalian prions have been compared at this point, and that the result here refers to the particular unnatural reaction conditions used. At face value, like prefers like; the only explanation would seem to be an evolved docking interface, ie a native homo-oligomer. (Note that asymmetric conversion is expected and that anchored GPI anchor excludes many structures. By non-evolved docking surface i mean generic beta sheet extension )

It has become the fashion to blow off glycosylation, and only minor effects are seen under the conditions here. Still, I wonder if long-term we won't see some subtle effects on protein conformation, strain types and barriers from it.

Uncritically extending the methods of the authors, one might conclude that human-to-human and VQ-to-VQ were intrinsically slower than cow-to-cow, 1:2 for met-met, 1:6 for val/val, and 1:2 on scrapie, while mouse-mouse and hamster were about the same as cow-cow. Be this as it may, this is an interesting issue because after the first go-rounds, all TSE would be homo-conversion. Is there a rate (progression of pathology) intrinsic to (each strain) in each species? Humans might fortuitously be one of the slower species (but don't forget that long lifespan).

[Reporter]: So you think some caution is needed in making inferences from their data?

[Tom]: Yes. On balance, however, I was quite convinced by the many positive correlations of conversion efficiencies to in vivo experience that the method is on-track. Although it looks like they just ran a few gels, the authors have actually done a lot of hard work here to develop the conditions and purify experimental materials. They have commendably moved the subject forward in a significant manner: the in vitro method seems to correctly order, and even semi-quantitate, relative conversion efficencies.

It is a big jump from measuring a small part of the overall whole animal transmission process, the prion-to-prion part, to predicting incubation times as a function of dosage. The experiment here is roughly intra-cerebral injection, as opposed to dietary. Yet somehow, in scrapie polymorphisms, even this fragment of the process worked quite well in predicting disease onset. So perhaps we have gotten lucky and matters are not as difficult experimentally as they might have been. There can be optimism as well that therapy testing in vitro will be applicable as well.

[Reporter]: Is there bad news here as well?

[Tom]: Yes. On the down side, the results suggest at face value dramatically less of a species barrier than val/val mice, that quite a bit of sporadic CJD could be due to scrapie, that met-val and val-val will show up in nvCJD with sufficient dosages and incubation times, and that large scale met-met is on its way in rough proportion to consumed titre. The 10:1 ratio in BSE-to-human is cold comfort because of the doses and because after a biphasic time course, it's human-to-human.

[Reporter]: Thank you very much. Great story here. I've got you down as saying that nvCJD is 'clearly an alien process.'

[Tom]: But .... but...

------------------> Forwarded Story <--------------

MAD SCIENTIST SAYS ALIENS BEHIND ODD CJD !
....

Supplementary information

Nature did not post the ;letter itslef online but mysteriously included: a gif image (below) captioned " Critical amino acid residues in the PrP protein" which may be figure 5 of the paper and the "Legend to Figure 5", in the funky form of an offline Word 6.0 document. These are "not published in the printed journal" and so are supplemental:

Molecular assessment of the transmissibilities of BSE and scrapie to humans Structural barriers

The region of the prion protein involved in the transmission of disease between closely related species has been mapped to the central third of the molecule and the amino-acid sequence within this region has been shown to critically affect the conversion of PrP-sen to PrP-res9. Comparisions of the PrP sequences from those species examined in this conversion study again implicate the mid-third of protein (Fig. 5).

In particular, it is clear that a charged residue in ovine PrP-sen at position 171 (marked by „+¾ in Figure 5) can drastically reduce conversion efficiency using ov-PrPSc(VQ) or PrPBSE. This effect can be modified by differences at residue 136 in ovine PrP-sen (Fig. 4). It is more difficult to precisely define the residues which may account for the low efficiency conversion of human PrP-sen by ov-PrPSc(VQ) or PrPBSE. Since ov-PrPSc(VQ) and PrPBSE appear to convert human PrP-sen similarily, it is likely that the common differences in ovine and bovine PrP compared to human PrP will be most influential (Fig. 5, boxed residues) although, of course, the additive effects of other small differences or similarities within the sequence cannot be fully discounted.

The location of almost all of these residues strongly implicates the loop structures of PrP19 as critical regions for the conversion of PrP-sen to PrP-res and for the maintenance of at least part of the barrier to transmission of TSE's between species.

Critical amino-acid residues in the PrP protein

Legend to Figure 5
Amino-acid residues which significantly alter the conversion of PrP-sen to PrP-res are located primarily within the loop regions of PrP. The NMR-derived secondary structure of PrP-sen from residues 121 to 231 is shown19. Dark boxes represent regions of beta-sheet (S) while lightly shaded boxes represent regions of alpha helix (H). Except for a portion of the region between S2 and H2 which is not well-defined, the line represents loop structures. The central sequence of PrP seen within the NMR structure which is critical for conversion and cross-species transmission is shown below this secondary structure.

The sequence of PrP (residues 121-190, numbered with reference to the human PrP gene19,27 ) from species used in our study are shown while residues 113-120 are invariant in these proteins and are omitted. Dashes indicate amino-acids common to human PrP and their residue numbers in the species in question are shown above the sequence. The PrP molecules used in these studies are listed on the left.

The residues which appear to most dramatically affect the conversion of PrP-sen by PrPBSE and ov-PrPSc(VQ) are indicated. Boxed residues = residues which may account for the low efficiency conversion of human PrP-sen; * = residue which affects conversion of hamster PrP-sen; + = residue which most significantly affects conversion of different sheep PrP genotypes.

Critical Regions of Prion Protein for Species Barrier

References (numbering according to the main paper)

9. Kocisko, D. A., Priola, S. A., Raymond, G. J., Chesebro, B., Lansbury, P. T., and Caughey, B. (1995). Species-specificity in the cell-free conversion of prion protein to protease-resistant forms - a model for the scrapie species barrier. Proc. Natl. Acad. Sci., USA 92, 3923-3927.

19. Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wurthrich, K. (1996). NMR structure of the mouse prion protein domain PrP (121-231). Nature 382, 180-182.

27. Kretzschmar, H. A., Stowring, L. E., Westaway, D., Stubblebine, W. H., Prusiner, S. B., and Dearmond, S. J. (1986). Molecular cloning of a human prion protein cDNA. DNA 5, 315-324.

More media coverage on the new Nature paper. The Times writer does not mince words. Amazing factoid: the scientists had to get the article pre-approved for publication by the UK beef promotion agency MAFF.

Deja vu all over again: they are falling back on 'not to worry, people don't get scrapie, it's just scrapie in cows.' Except about all we know about all we know about people getting scrapie is that 85% of all CJD cannot be attributed to genetics or medical transmission.

This is really really bad news if it is only a 1:30 efficiency ratio for cow-to-human compared to human-to-human. Think for a moment about how low the doses must have been with intra-muscular growth hormone CJD and some of the surgical transfers.

While the initial stage may be slow and inefficient, subsequent stages can use human-to-human recruitment. That is, while it is difficult for a rogue bovine prion to recruit a human prion, the early human prions can then go on to recruit normal human prions quite efficiently. Very small initial inocula may grow exponentially, because the initial catalyst of the conversion process makes more catalyst, not just inert reaction.

The Norwegians are going to be totally freaked out over this. Scrapie is growing in Norway, with over 100 000 sheep slaughtered, and the National Institute of Public Health giving a green light to eating the Norwegain delicacy smalahove, sheep brain. (The Norwegian Board of Health is sceptical.) Are The Norwegians going to come clean about scrapie-to-reindeer or is that too sensitive vis-a-vis 'ethnic cleansing' of Lapps? -- webmaster

The BBC on BSE-to-humans

London correspondent 17 July 1997
According to the BBC, the Nature article found that it is thirty times more difficult to infect human proteins than cow proteins. The head of SEAC himself (Southwood ?) was interviewed.

However, the interviewer was unable to pin Southwood down to ANY definitive statement at all about the likelihood or number of future cases. All he would commit to is that it is less likely that there will be an epidemic.

MAFF has taken the attitude that this just proves how unlikely it is that there will be a nvCJD epidemic.

Lab tests prove BSE can pass to human tissue

July 17 1997  London Times    BY NICK NUTTALL, TECHNOLOGY CORRESPONDENT
BSE can be transmitted to humans beings, British scientists have found. The research shows that infectious proteins, or prions, from contaminated beef can, in laboratory tests, transmit to human tissue to trigger brain infections. The findings may also have implications for lamb infected with scrapie.

The researchers have found that the proteins involved with scrapie can also infect human proteins. James Hope, who led the research at the Institute for Animal Health in Newbury, Berkshire, said yesterday: "We have shown that change in a particular human protein can be induced by the bovine infectious agent."

However, he said the findings should be treated cautiously. "To extrapolate this to say that bovine spongiform encephalopathy has been, or is being, transmitted to humans discounts a lot of other factors that are involved in cross-species transmission," he said.

The scientists, whose work has been approved by the Ministry of Agriculture and is published in Nature, also found that scrapie can be transmitted to humans to trigger a change in proteins. However, scrapie can be traced back 200 years, yet there is no evidence that people eating lamb have been affected by scrapie. The scientists found that scrapie and BSE were equally good at infecting human proteins. "So you might actually infer that since the sheep and the bovine material convert the human ones at similar efficiency, BSE is not a risk factor for the disease. That would be the positive message," said Dr Hope, who is funded by the government's Biological and Biotechnological Sciences Research Council.

A more pessimistic conclusion is that scrapie-infected lamb is a threat to humans and should be treated in a similar way to beef. "It really depends on whether you wear rose-tinted or doom-laden glasses," Dr Hope said. He pointed out that outside the laboratory a multitude of additional factors ‚ such as the dose, strain, and route of infection ‚ would affect transmission.

Beef infected with BSE is thought to have triggered a new strain of Creutzfeldt-Jakob disease, which strikes people at a much younger age than the normal version, causing symptoms of dementia leading to death. The Department of Health has recorded 19 confirmed and probable cases of "new variant" CJD. The patients are thought to have become infected in the late 1980s, before strict controls came in to stop BSE-infected beef entering the human food chain.

Because of the disease's long incubation period, experts do not know if this is just the start of an epidemic that may kill thousands of people. Whether there is a serious epidemic will depend largely on how easily BSE can jump the species barrier and infect humans.

Brain diseases such as BSE, scrapie and CJD are thought to be caused by alterations to the molecular structure of proteins called prions. Normally harmless prions become defective, and cause other prions with which they come into contact to alter in the same way, thus setting up a chain reaction of infection.

Dr Hope and his team showed that prion proteins from both BSE and scrapie-infected animal brains were able to convert human prion proteins into the dangerous form. But the efficiency of this reaction was much lower than the conversion of human prion protein by the defective prion associated with "normal" CJD.

Dr Hope said: "We have shown that there is a molecular barrier between cows and humans, but it's not an absolute barrier. It's just a question of efficiency."

Study proves mad cow disease is contagious

July 16, 1997
By MAGGIE FOX, Reuter
LONDON - Mad cow disease can be transmitted to humans, but just barely, an international team of researchers said on Wednesday. Tests done with the prion proteins linked with mad cow disease showed the proteins could transmit the disease, James Hope of the Institute for Animal Health and colleagues in Britain, the Netherlands and the United States said.

Hope said they purified proteins from the brains of cows infected with bovine spongiform encephalopathy (BSE or mad cow disease), sheep infected with its cousin scrapie and humans who died of a new variant of Creutzfeldt-Jakob disease (CJD) that has been linked with BSE. They mixed them with a purified version of normal prion proteins from human brains.

"What we found is you can get conversion...but it was of a low efficiency," Hope said in a telephone interview. The normal proteins, when exposed to the abnormal ones, became abnormal themselves. They resisted protease, an enzyme that usually breaks down protein.

The findings shed a little more light on how people and animals catch such diseases, which remain mysterious. Victims have large numbers of the abnormal prion proteins in their brains, which take on a spongelike appearance and deteriorate until death. Most scientists think the proteins themselves affect other proteins, although some maintain a "slow virus" or other agent must be to blame.

Hope said he believed the proteins were infectious, although his group's findings could support either hypothesis.

"Our material is biochemically something like 95 percent pure," he said. "It is infectious either because you have a lot of the infective form of prion protein there or it is sequestering an as-yet unidentified molecular agent." Because the proteins were so pure, they would probably be more infectious than a real-life exposure, Hope added. "The material we were using in this conversion was highly infectious," he said. "If you were to inject it into a human or animal, they would get the disease."
But with humans, they would be exposed through eating meat or other animal parts. The infectious agent would have to pass through the body, and the dose would vary.
"One would not say that a small test tube is of the same complexity as a human being," Hope said. "So the likelihood is that it is even less infectious than these tests show."
Hope noted that scrapie had been reported in sheep for more than 100 years, but there was no link with CJD.
"We conclude that BSE is no more transmissible to humans than is sheep scrapie," he said. But, he added: "We can't absolutely say that because there is no risk of people getting sheep scrapie there will be no risk of people getting BSE. All we can say is that there is no absolute molecular barrier."
Hope said his group's techniques offered a useful way to test any possible drugs for treating CJD or BSE without using animals.
"You can look for drugs which prevent this conversion reaction," he said. "It's an ideal system to prove or disprove the prion hypothesis."
Nineteen people in Britain and one in France have succumbed to the new version of CJD, identified last year and linked with BSE. Doctors say it is far too early to know if a human CJD epidemic will mirror the BSE epidemic that decimated British herds in the 1980s.

Prion protein NMR structure and species barrier for prion diseases

Proc Natl Acad Sci U S A 94(14), 7281-7285 (1997)  [free fulltext]
Martin Billeter, Roland Riek, Gerhard Wider, Simone Hornemann, Rudi Glockshuber, and Kurt W¸thrich 
The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or "mad cow disease" and Creutzfeldt-Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121-231. A database search for mammalian prion proteins yielded 23İdifferent sequences for the fragment 124-226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103İpositions.

Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein-protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases for which there is evidence that they are related to a novel, so far unique, infectious agent, the prion (1, 2). TSEs have been reported to occur as infectious, inherited, and spontaneous diseases. Following the "protein-only hypothesis" (2, 3) the causative agent is a pathogenic conformation of the prion protein (PrP). PrP is ubiquitous in mammalian cells in a benign, cellular conformation (PrPC). In rare cases it may be transformed into the infectious scrapie conformation (PrPSc), which forms insoluble, protease-resistant aggregates in the brain of affected individuals (4, 5). The most widely discussed TSEs are Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy. Other human prion diseases include kuru, the Gerstmann-Str”ussler-Scheinker syndrome, and fatal familial insomnia.

With the background of the "mad cow crisis" in Europe, questions relating to the relative ease of infection between different individuals of the same species or between different species have attracted intensive interest, in particular regarding possible transmission of bovine spongiform encephalopathy from cows to humans through the food chain (6, 7).

A species barrier for prion infection has indeed been convincingly documented (4, 5) and found to vary widely depending on the pair of species involved and the direction of transmission. Typical laboratory tests involve inoculation of mice or hamsters by injection of infectious material directly into the brain, and work with transgenic animals has led to the identification of polypeptide segments in PrP that appear to dominate the species barrier (surveyed in refs. 4 and 8).

There is a general consensus that interspecies transfer, when compared with infection within one species, is inefficient and occurs, if at all, only after prolonged incubation times. However, uncertainty remains with regard to the stringency of the transmission barrier for certain pairs of species. In vitro studies of the cell-free conversion of PrPC to protease-resistant forms are overall in support of the aforementioned in vivo data (8).

The present paper correlates biological and biochemical data on the species barrier with the refined NMR structure of the recombinant C-terminal domain of the mouse PrP with residues 121-231 [PrP(121-231)] (9). The pairwise sequence identity for this domain of about 90% among mammalian species (10) implies that all these species should have identical three-dimensional PrP(121-231) folds (11); this is supported by the modeling of the three-dimensional structures of PrP(121-231) from different species in our laboratory (unpublished data). Within the framework of the protein-only hypothesis the contributions to the species barrier from the polypeptide segment 121-231 then must be related to part or all of the amino acid replacements between the PrP of a given pair of species.

The aforementioned in vivo inoculation studies and in vitro conversion experiments support the hypothesis that a critical step of infectious transmission of transmissible spongiform encephalopathies involves a specific intermolecular contact between PrPC and PrPSc (4, 5, 8). From additional work with mouse/human chimeras, interactions of PrPC with a so far unknown additional protein X also have been postulated to contribute to efficient conversion (12). Here, we map the polypeptide segment 124-226 of all known mammalian PrP sequences (10) onto the three-dimensional structure of mouse PrP(121-231) and evaluate potential effects on protein-protein interactions with PrPC.

Inoculation experiments with chimeric PrP suggested two protein binding sites in the PrP amino acid sequence. These experiments so far have been the only information available for mapping of species-related differences in PrPC. One of these binding sites would be located in the segment with residues 96-167, which would bind an infecting PrPSc particle. The second site would be composed of residues outside of this segment, which could possibly interact with the postulated protein X (4, 12).

The variable C-type residues in helix 1İhence would contribute to a protein surface area that might be involved in interactions with PrPSc, and the cluster of type A residues would be a potential binding site for protein X.İThe in vitro PrPC conversion experiments of Kocisko et al. (8) suggested that the hamster/mouse species barrier is dependent on the three amino acid exchanges M139I, N155Y, and N170S (Fig. 1). It is striking that the segment 96-167 as well as the group of residues 139/155/170 includes at least one residue from each of the classes A, B, and C (Fig. 1).

Other experimental data attribute an exclusive role for the species barrier between mouse and hamster to position 139İ(16): The substitution of Met in hamster PrP by Ile in mouse PrP appears to be sufficient to protect hamster from infection by mouse PrPSc. This would place the critical structural feature for the species barrier in the interface between helix 1İand the protein core (Figs. 2 and 4a). The residue 139İpresumably would become operational only after initial conformational transitions of PrPC induced by complexation with PrPSc and possibly protein X (9). The present structural observations can be expected to support future refinement of in vivo and in vitro experiments with chimeric PrP.

The class D exchange Q186 E in bovine PrP introduces a negative electrostatic charge in a surface location that is clearly separated in space from the two presumed binding sites characterized by A- and C-type residues, respectively (Fig. 2). Interestingly, recent results indicate the presence of Glu-186 also in the related species Watussi, Banteng, and Wisent (H.İSch”tzl, personal communication). The newly determined PrP sequences of the house dog, the Canadian wolf, and the dingo (H.İSch”tzl, personal communication) further extend the available information on D-type mutation sites, because the otherwise strictly conserved Asn-159 (Fig. 1) is replaced by Asp, and this negative charge is located in close spatial proximity to position 186İ(Fig. 2). This leaves the intriguing option that a third variable surface area in PrPC might emerge by further extension of the sequence database, and that this site might be functional in the species barrier with cattle, including possibly also transmission of disease from bovine spongiform encephalopathy-infected cows to other species.

Finally, although the NMR structure of mouse PrP(121-231) represents only part of PrPC and the ensemble of all accumulated data indicates that N-terminal parts of the sequence also may influence the species barrier (for example, see refs. 4 and 5), there is evidence that the data presented in this paper are highly relevant with regard to the intact system, and that PrP(121-231) has a key part in PrPC physiology (8, 12, 16). Structure predictions and experimental observations (9, 17) indicate that PrP(121-231) is probably the only polypeptide segment with a global fold in intact PrPC, and initial NMR studies with intact PrPC performed in our laboratory indicate that the segment 121-231 in the intact protein has the same fold as in PrP(121-231) (unpublished data).

Evolution of a Strain of CJD That Induces BSE-Like Plaques

Science  Volume 277, Number 5322,  4 July 1997, pp. 94-98
Laura Manuelidis,  William Fritch, You-Gen Xi 
Bovine spongiform encephalopathy (BSE) has become a public health issue because a recently evolved BSE agent has infected people, yielding an unusual form of Creutzfeld-Jakob disease (CJD). A new CJD agent that provokes similar amyloid plaques and cerebellar pathology was serially propagated. First-passage rats showed obvious clinical signs and activated microglia but had negligible PrP-res (the more protease-resistant form of host PrP) or cerebellar lesions. Microglia and astrocytes may participate in strain selection because the agent evolved, stabilized, and reproducibly provoked BSE-like disease in subsequent passages. Early vacuolar change involving activated microglia and astrocytes preceded significant PrP-res accumulation by more than 50İdays. These studies reveal several inflammatory host reactions to an exogenous agent.

The recent epidemic of bovine spongiform encephalopathy (BSE) has brought increased attention to its human counterpart, Creutzfeldt-Jakob disease (CJD), as well as to scrapie, an endemic infection of sheep. Since the first recognized case of BSE in 1985,İit is likely that more than one million cows have become infected by dietary exposure to scrapie-contaminated food (1). Domestic cats and various zoo animals have been similarly infected. Additional experimental propagation of the cow-derived agent in many species, including pigs, rodents, and primates (2, 3), suggests that the new BSE agent has acquired an enhanced ability to evade host defenses. In 1996,İthe BSE agent was linked to a variant disease in younger people (4) that was reproduced in primates by BSE inoculation (3). Because human CJD infections can be undetectable for more than 20İyears, it is likely that we will see more BSE-linked cases (5, 6). Thus, there is an emerging public health issue that requires a greater understanding of agent strains and their specific interactions with the host.

Many different infectious strains of the scrapie and CJD agents have been propagated in inbred mice encoding the same PrP amino acid sequence (7, 8). The main reasons why human "BSE" is considered to be caused by a new agent strain are based on (i) the youth of most victims (less than 30İyears old), (ii) the prolonged clinical course, (iii) severe involvement of the cerebellum, and (iv) the presence of many large plaques. Sporadic CJD, which accounts for ~90% of CJD worldwide and is infectious in a variety of species (9, 10), rarely shows cerebellar pathology or widespread plaques. Only two other human transmissible encephalopathies with a long clinical phase display cerebellar lesions and a plaque-rich phenotype. These are kuru, an infection transmitted by ritual cannibalism (9), and Gerstmann-Straussler-Sheinker disease (GSS), a group of rare "familial" forms of CJD (11).

One explanation for these different phenotypes focuses on mutations in host PrP. It has been suggested that a combination of mutations causes PrP to fold into an infectious conformation that encrypts and propagates strain-specific information (12). However, people infected with "BSE" do not have any of the plaque-associated PrP mutations such as Phe102 to Leu102 (13). Alternatively, a newly mutated exogenous virus may provoke unusual phenotypic responses in the host, including different forms of PrP-res (8). Additional host responses could signify recognition of the foreign agent hidden within cells. Because strong lymphocyte responses are evaded, it is often stated that these infections lack any inflammatory component (9, 14). We found that inflammatory cells can participate in the evolution of a new infectious strain. This strain causes a disease with notable similarity to both "familial" GSS and human "BSE" and evokes inflammatory reactions at early stages of infection.

To change an established CJD strain into one that could produce plaques and cerebellar lesions, we inoculated alternate species without manipulating normal host PrP sequences. Unusual inflammatory responses in microglia or astrocytes (or both) were used to evaluate host recognition. Such responses could subject the agent to more rigorous selective pressures favoring a mutated plaque-evoking strain. We therefore challenged several types of inbred mice, Chinese hamsters, and rats with a strain of CJD (designated SY) that was highly selected by serial passage 5İtimes in guinea pigs and then 24İtimes in Syrian hamsters (15). SY was successfully transmitted to all three species and had a relatively prolonged incubation period even after serial passage (>350 days). Only the rats showed remarkable pathology.

Plaques in P3 and P4 showed strong "Maltese cross" [Congo Red] birefringence comparable to that seen in "familial" GSS birefringence.

Commentary by Roland Heynkes

5 July 1997 
Existence or lack of inflammation are very week arguments for or against he virus/virino-theory. Furthermoore the observation of initial local inflammation reactions is not new and not from Manuelidis.
Williams ,A.E.; Lawson,L.J.; Perry,V.H.; Fraser,H. - Characterization of the
Microglial Response in Murine Scrapie - Neuropathology and Applied
Neurobiology 1994 Feb; 20(1): 47-55

Campbell,I.L.; Eddleston,M.; Kemper,P.; Oldstone,M.B.; Hobbs,M.V. -
Activation of cerebral cytokine gene expression and its correlation with
onset of reactive astrocyte and acute-phase response gene expression in
scrapie - Journal of Virology 1994 Apr; 68(4): 2383-7

Betmouni,S.; Perry,V.H.; Gordon,J.L. - Evidence for an early inflammatory
response in the central- nervous-system of mice with scrapie - Neuroscience
1996; 74(N1): 1-5
A CJD virus with only short persistence could not explain the infectivity of biopsy material. Furthermoore the virus theory can not explain the resitance of codon-129 heterozygotes, the radiation resistance of the infectious agent and the de novo introduction of a Gerstmann-Straeussler- Scheinker-syndrome in laboratory mice by site directed mutagenesis.

There is no proof for any increase of interspecies infectivity. Only the circumstances for the spread of the infectivity changed obviously so that lethal doses could be reached in several species.

There is no doubt about this and Bossers et al. recently demonstrated with the really important Kocisko in vitro technique, that observed levels of resistance against scrapie infections perfectly correlate with the degree of homology between the prion proteins of the host and within the infectious agent.

They had similar studies already in 1975 (Manuelidis,E.E. - Transmission of Creutzfeldt- Jakob disease from man to the guinea pig - Science 1975 Nov 7; 190(4214): 571-2) and they established a CJD model in guinea pig two decades ago (Manuelidis,E.E.; Kim,J.; Angelo,J.N.; Manuelidis,L. - Serial propagation of Creutzfeldt-Jakob disease in guinea pigs - Proceedings of the National Academy of Sciences of the United States of America 1976 Jan; 73(1): 223-7).

They did similar work with hamster (Manuelidis,E.E.; Angelo,J.N.; Gorgacz,E.J.; Manuelidis,L. - Transmission of Creutzfeldt-Jakob disease to Syrian hamster - Lancet 1977 Feb 26; 1(8009): 479) and mouse too (Manuelidis,E.E.; Gorgacz,E.J.; Manuelidis,L. - Transmission of Creutzfeldt-Jakob disease with scrapie-like syndromes to mice - Nature 1978 Feb 23; 271(5647): 778-9).

Finally they infected hamster with an infectious agent, that has been passaged through guinea pig (Manuelidis,E.E.; Gorgacz,E.J.; Manuelidis,L. - Interspecies transmission of Creutzfeldt-Jakob disease to Syrian hamsters with reference to clinical syndromes and strains of agent - Proceedings of the National Academy of Sciences of the United States of America 1978 Jul; 75(7): 3432-6).

And they where not the only team (Brownell,B.; Campbell,M.J.; Greenham,L.W.; Peacock,D.B. - Experimental transmission of Creutzfeldt-Jakob disease - Lancet 1975 Jul 26; 2(7926): 186-7).

Much earlier than Manuelidis the group of Gajdusek and Gibbs transmitted Kuru to chimpanzees, which are, as we know from the Weismann-report, also aboratory animals and CJD-models (Beck,E.; Daniel,P.M.; Alpers,M.P.; Gajdusek,D.C.; Gibbs,C.J.Jr. - Experimental "kuru" in chimpanzees. A pathological report. - Lancet 1966 Nov 12; 2(472): 1056-9)

So what is really new in this wondeful article?

Molecular Properties of Complexes Formed Between the Prion Protein and Synthetic Peptides

JMB Vol 270 Number 4, July 25 1997 pdf fulltext
Kiyotoshi Kaneko, Holger Wille, Ingrid Mehlhorn, Hong Zhang, Haydn Ball, Fred E. Cohen, Michael A. Baldwin, Stanley B. Prusiner
Complexes of the Syrian hamster cellular prion protein (PrPC) and synthetic Syrian hamster PrP peptides were found to mimic many of the characteristics of the scrapie PrP isoform (PrPSc). Either PrPC expressed in chinese hamster ovary (CHO) cells or a C-terminal fragment of 142 residues of recombinant PrP protein (rPrP) produced in Escherichia coli was mixed with an excess of a synthetic 56 amino acid peptide, denoted PrP(90-145). Complex formation required PrPC or rPrP to be destabilized by guanidine hydrochloride (GdnHCl) or urea and PrP(90-145) to be in a coil conformation; it was enhanced by an acidic environment, salt and detergent. If PrP(90-145) was in a [Beta]-sheet conformation, then no complexes were formed.

While complex formation was rapid, acquisition of protease resistance was a slow process. Amorphous aggregates with a PrPC/PrP(90-145) ratio of 1:1 were formed in phosphate buffer, whereas fibrils with a diameter of [sim]10[emsp14]nm and a PrPC/PrP(90-145) ratio of 1:5 were formed in Tris buffer. The complexes were stable only in the presence of excess peptide in either the coil or [Beta]-sheet conformation; they dissociated rapidly after centrifugation and resuspension in buffer without peptide. Neither a peptide having a similar hydrophobicity profile/charge distribution to PrP(90-145) nor a scrambled version, denoted hPrP(90-145) and sPrP(90-145), respectively, were able to induce complex formation. Although hPrP(90-145) could stabilize the PrPC/PrP(90-145) complexes, sPrP(90-145) could not. Studies of PrPC/peptide complexes may provide insights into how PrPC interacts with PrPSc during the formation of a nascent PrPSc molecule and into the process by which PrPC is

Identification of candidate proteins binding to prion protein.

Neurobiol Dis 1997;3(4):339-355 Andrews NC, ... Prusiner SB, Weissmann C, ... Taraboulos A, Stahl N

Prion diseases are disorders of protein conformation that produce neurodegeneration in humans and animals. Studies of transgenic (Tg) mice indicate that a factor designated protein X is involved in the conversion of the normal cellular prion protein (PrPC) into the scrapie isoform (PrPSc); protein X appears to interact with PrPC but not with PrPSc. To search for PrPC binding proteins, we fused PrP with alkaline phosphatase (AP) to produce a soluble, secreted probe. PrP-AP was used to screen a lambdagt11 mouse brain cDNA library, and six clones were isolated. Four cDNAs are novel while two clones are fragments of Nrf2 (NF-E2 related factor 2) transcription factor and Aplp1 (amyloid precursor-like protein 1). The observation that PrP binds to a member of the APP (amyloid precursor protein) gene family is intriguing, in light of possible relevance to Alzheimer's disease. Four of the isolated clones are expressed preferentially in the mouse brain and encode a similar motif.

Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein.

Cell 1995 Oct 6;83(1):79-90
Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M, Cohen FE, DeArmond SJ, Prusiner SB
Transgenic (Tg) mice expressing human (Hu) and chimeric prion protein (PrP) genes were inoculated with brain extracts from humans with inherited or sporadic prion disease to investigate the mechanism by which PrPC is transformed into PrPSc. Although Tg(HuPrP) mice expressed high levels of HuPrPC, they were resistant to human prions. They became susceptible to human prions upon ablation of the mouse (Mo) PrP gene. In contrast, mice expressing low levels of the chimeric transgene were susceptible to human prions and registered only a modest decrease in incubation times upon MoPrP gene disruption. These and other findings argue that a species-specific macromolecule, provisionally designated protein X, participates in prion formation. While the results demonstrate that PrPSc binds to PrPC in a region delimited by codons 96 to 167, they also suggest that PrPC binds protein X through residues near the C-terminus. Protein X might function as a molecular chaperone in the formation of PrPSc.

Mad Cow Home or Best Links