Prion Disease: Species Barrier Evaporates. 15 Sept 00
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Species-barrier prion replication in apparently resistant species.
Proof of risk of CWD to humans, cattle and sheep
Preclinical BSE: sheep blood shown infectious
Human blood Prpc: reality check on rodents
Preclinical diagnosis of scrapie by IHC: moving towards validation
Linear and conformational prion epitopes
Monomer-dimer equilibrium of native bovine Prpc not seen in recombinant protein
Chaperone BiP binds to mutant prion and mediates proteosome degradation
Ragged N terminus in types of CJD
Signal transduction normal role proposed

Species-barrier-independent prion replication in apparently resistant species.

Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10248-10253.
Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J.
Comment (webmaster): This is very interesting research with novel, if unwelcome, findings that contradict some conventional wisdom and conclusions from previous studies, and important implications for current public policy. Many of the authorities and agencies commenting on it have demonstrably and disturbingly failed to even read the abstract.

Subclinical disease is hardly a new concept -- it goes back to the time of Louis Pasteur. Everyone has heard of Typhoid Mary Mallon, born in 1869 in Ireland and died of a stroke in 1938 in New York, who infected numerous people with Salmonella typhi. Mallon, not believing in carriers and still working as cook under a pseudonym, infected 25 staff members at a maternity hospital (two fatally).

The lesson here for TSEs is that an aggressive typhoid surveillance program identified 237 other typhoid carriers by 1938, whereas Britain (unlike France) still refuses a testing program to identify non-clinical BSE. Like Typhoid Mary, policy makers in Britain cannot face up to implications of non-clinical disease and so keep spreading nvCJD across the world.

Typhoid is not necessarily progressive, that is, the infectious agent apparently reproduced in steady state without pathology during a 69 year life span. Few think that Mallon would have succumbed to this disease had she lived to 79 or even 99: Typhoid Mary had a subclinical infection, not a preclinical one.

Collinge's group reports a similar situation in prion disease, with the difference that progressive accumulation of infectious prion does occur, again in the absence of clinical sign. In mice, it appears that prion production can become decoupled from clinical sign, that is, infectious titres can be quite higher than in situations that have gone clinical, for example, Tg20 mice with high levels of normal prion can have early disease onset yet produce low levels of PrPSc. One sees from the data here the fallacy of relying on clinical sign and failure to conduct second passage (eg, Y145x) in many earlier experiments -- clinical sign is a completely unsuitable monitoring proxy for infectivity risk.

Some of the commentary has been ludicrous: there are 'no policy implications because the experiment was not done in cows'. If it had been done in cows, the complaint would be that only one breed was tested. If many breeds were tested, then it would not apply because not enough individuals of each were tested. If many individuals of many breeds were tested, then it could perhaps be considered by an unqualified committee at a future meeting, but there could be no implications for sheep unless many individuals representing thousands of genotypes were tested, and still there would be no implications for pigs, farmed fish, or chickens. All of this would take decades and cost a fortune -- rodents are used as model systems throughout medicine for exactly this reason.

Equally silly have been pronouncements that no changes are needed because current policy already assumes non-clinical BSE. The massive cull of over-30 month cows regardless of BSE status mainly has the effect of lowering overt clinical cases but what affect has it had on subclinical TSE? Thirty months was chosen to minimize effects on industry, not minimize BSE titre. Clinical cows are incinerated but not their at-risk herds, which still number some 1200 a year. No one knows how many are subclincial because testing of herdmates is forbidden. Why should this be forbiden, does MAFF fear subclinical cases would emerge?

Without non-clinical monitoring, policy is not based on the best available science. To the contrary, current policy is mainly directed at reassuring importers and nominally complying with EU rules. Reduction of infectious titre in the human food supply is a welcome byproduct to be sure, but how much is still getting through? Is BSE being eradicated or becoming endemic?

Wouldn't it be easier just to run the Prionics test on 47,000 random UK cows (like France is doing)?

Experimental details are best left to the paper, which is very clearly written. Worth noting are the extraordinary observation periods, up to 849 days, in which clinical scrapie was not observed despite a massive inoculum of nearly 10 million LD50's. Mice that never developed PrpSc had on average to be euthanized earlier for intercurrent disease, at 528 days; some of these might have gone on to become positive. There is a rare discussion of persistence of inoculum and why it is not applicable here. Several, but not all, of the mice showed spongiform encephalopathy with PrP amyloid plaques even though they lacked clinical sign; arguably these mice were on their way to full-blown clinical disease (but when? did Typhoid Mary have GI tract abnormalities?). In all 25 amyloidoses, researchers argue over whether amyloid per se is toxic or not; the authors here think not but offer no decisive data.

Second-passage experiments showed replication of prion took place: these went from one PrpSc positive and one PrpSc negative (spongiform status not stated) to CD-1 mice, Tg20 mice (overexpress mouse PrP, shortened incubation periods for mouse-adapted prions), and Syrian hamsters. The PrpSc donor infected all 3 groups of recipients with fairly short incubation times; the negative donor only affected hamster (mice were followed for 400+ days). End-point titration of PrPSc-negative donor brain gave a titer of a million LD50/g in hamsters despite the lack of transmission to Tg20 mice.

Strain-typing of PK-treated brain homogenate by western blot(see figure 3) dispells the myth that strain-type is always preserved: hamster and mouse differed sharply both with respect to fragment sizes and glycoform ratios (though hamster to mouse to hamster still is like original hamster).

Several commentators have said 'we have known all along about preclinical animals, there is nothing new here.' If this were true, it then makes no sense to have monitored only clinical animals. Subclinical carriers go undetected (even though many are readily and practicably detectable), even as they host prion replication and pose a very real threat on second passage to other species. Subclinical monitoring gives a much better picture of the epidemic status (France is finding about half of its cases in non-clinical cows) and whether risk reduction measures are actually working to keep BSE out of the food chain (and nvCJD out of iatrogenic).

Highlights of article (adapted):

Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. A strain of hamster prions often said to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease in the affeced mice. Prions pathogenic in both mice and hamsters are produced.

The existence of subclinical forms of prion infection have important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, based on clinical end-points, need to be fundamentally reassessed.

Prion diseases are both naturally and experimentally transmissible between different mammalian species but such transmission, as judged by appearance of clinical signs, is limited by a so-called "species barrier" (IH Pattison, 1965). This barrier may be of sufficient magnitude that transmissions, even when attempted by the most efficient, intracerebral, route of inoculation with high titer tissues, are extremely infrequent or absent. In contrast, same-species transmission of prions is typically highly efficient. Transmission is dose-dependent, with increasing mean incubation periods and a decreasing fraction of animals succumbing to the disease as increasing dilutions of inoculum are used. However, at higher titers, 100% of inoculated animals succumb to disease with a constant and remarkably consistent incubation period, which is not reduced by further increase of inoculum titer.

The biological effect of a species barrier is to increase mean incubation periods, increase the range of incubation periods, and reduce the fraction of animals succumbing to disease.... Second and subsequent passages of infectivity to the same species are associated with transmission parameters more closely resembling same-species transmissions [the primary amino acid sequence is now the same of course -- webmaster].. Species barriers have been quantified by the fall in mean incubation period on primary and second passage in the same species or by comparative end-point titration in the two species concerned.

The most intensively studied species barrier is the substantial barrier limiting transmission of prion diseases between hamsters and mice. In particular, the hamster scrapie strain Sc237 , which is similar to the strain classified as 263K [both originated in sheep but have had complex transmission histories; mice strains can differ at two amino acids. -- webmaster], regarded as nonpathogenic for mice (with no clinical disease in mice observed for up to 735 days postinoculation; Kimberlin 1978) and used in studies of species barriers in transgenic mice.

Transgenic mice expressing hamster PrP (in addition to endogenous mouse PrP [a situation sometimes interfering -- webmaster]), in sharp contrast to conventional mice, were highly susceptible to Sc237 hamster prions with consistent short incubation periods that were inversely correlated to hamster PrP expression levels. The prions propagated in the transgenic mice were only pathogenic for hamsters and not for conventional mice. Importantly, however, these studies [from the Prusiner lab] defined transmission by using clinical criteria and did not report PrPSc levels and types or prion titers in the brains of clinically unaffected animals. Such studies argued that species barriers resided in differences in the primary structure of the PrP in the inoculum and host, prion propagation proceeding most efficiently when these sequences were identical.

However, it has been recognized for many years that prion strain type has an important influence on ease of transmission of prion disease between species. Prion strains are associated with distinct PrPSc types that can be distinguished by Western blot analysis with distinct cleavage sites to proteinase K, implying distinct PrPSc conformations, and by differences in glycoform ratios of protease-digested PrPSc The importance of PrP primary structure homology to species barriers would be expected therefore to be only one factor in determining the efficiency of the interactions between PrP isoforms that determine prion propagation.

A striking example of the strain effect to species barriers has been provided by analysis of BSE prions. Classical CJD prions, propagated in humans expressing wild-type human PrP, transmit highly efficiently to mice expressing only human PrP with transmission characteristics consistent with complete absence of a species barrier.

Although in agreement with earlier studies no clinical signs of scrapie developed in such mice, our neuropathological, molecular, and passage studies reveal the presence of subclinical prion infection in such animals with high prion titers in brain. These results necessitate a re-evaluation and definition of prion transmission barriers.

In prion diseases, infectious titers in the brain rise progressively throughout prolonged, clinically silent periods that precede the onset of disease. Thus asymptomatic animals may harbor significant infectious titers in brain and other tissues. However, there may be subclinical, as distinct from such preclinical, forms of prion infection, where animals become asymptomatic carriers of infectivity and do not develop clinical disease in their lifetimes. Such carrier states are well recognized in other infectious diseases.

However, in prion diseases, where incubation periods are extremely prolonged, distinction between subclinical and preclinical states is more difficult. It certainly can be argued that animals dying after a typical lifespan without clinical signs of prion disease but harboring high levels of infectivity represent the late preclinical stage of "transmissions" where the "incubation period" exceeds the normal lifespan (Dickinson, 1975) [this definition makes subclinical redundant, but where does that leave Typhoid Mary? -- webmaster]... Here we use the term subclinical infection operationally to refer to animals in which prion replication is occurring but which have not developed clinical signs of prion disease during a normal lifespan.

However, whether or not this infectivity is classified as preclinical or subclinical, it has important public health implications. Iatrogenic transmission could occur from apparently healthy humans who may harbor high prion titers and many animal species (including sheep, pigs, and poultry) were exposed to BSE prions via contaminated feed and could have developed subclinical prion infection.

It is known that BSE prions retain their distinctive strain characteristics after passage in a number of other species including humans, arguing that such BSE passaged in species other than cattle also may be pathogenic to humans. The possibility that subclinical BSE might be present in other species and thereby present a threat to human health has been raised (Colline, 1996) but not yet rigorously investigated [because authorities refuse] screening apparently healthy cattle after slaughter to investigate whether significant levels of subclinical or preclinical BSE are present.

Secondly, because animals can harbor high levels of infectivity without developing clinical signs of prion disease, these results argue that PrPSc and indeed prions (whether or not they are identical) may not themselves be highly neurotoxic. Such results are in accordance with earlier findings of a lack of correlation between clinical disease and neuropathological features of prion disease, prion diseases in which PrPSc is barely or not detectable, and studies in mice with reduced levels of PrPC expression that have extremely high levels of PrPSc and prions in the brain and yet remain well for several months after their wild-type counterparts succumb (Weissmann, 1994).

Conversely, Tg20 mice, with high levels of PrPC, have short incubation periods and yet produce low levels of PrPSc after inoculation with mouse prions (Weissmann, 1996). In addition, brain grafts producing high levels of PrPSc do not damage adjacent tissue in PrP knockout (Prnpo/o) mice. The cause of neurodegeneration in prion diseases remains unclear. It remains possible that prion neurodegeneration is related, at least in part, to loss of function of PrPC. An alternative hypothesis is that a toxic, possibly infectious, intermediate is produced in the process of conversion of PrPC to PrPSc, with PrPSc, present as highly aggregated material, being a relatively inert end-product. The steady-state level of such a toxic monomeric or oligomeric PrP intermediate then could determine rate of neurodegeneration.

One possibility is that Sc237-inoculated mice propagate prions very slowly and that such a toxic intermediate is generated at extremely low levels that are tolerated by the mouse. The fact that the PrPSc-negative Sc237-inoculated mice were the ones culled earlier than those that were PrPSc positive, allows the assumption that they may have become PrPSc positive had they lived longer. A more detailed study of the time course of accumulation of infectivity will be necessary to investigate this further.

The transmission properties of prions from the subclinical Sc237-inoculated mice were remarkable. With respect to transmissions to additional CD-1 or Tg20 mice, the 100% attack rate and highly consistent incubation periods suggest transmission in the absence of a barrier. However, the incubation periods, notably in the Tg20 mice, which succumb to RML mouse prions in around 60 days, are very prolonged. The 100% attack rate argues against this being a consequence of low prion titer in the inoculum.

Incubation period at end point dilution in Tg20 mice of RML mouse prions is around 109 days. Remarkably, passage in hamsters of this isolate also showed a 100% attack rate and consistent incubation periods suggestive of transmission in the absence of a barrier. Again, incubation periods were extremely prolonged and differed markedly from the transmission properties of Sc237/263K prions in hamsters. Indeed, the incubation period seen would correspond to an Sc237 titer in Syrian hamsters of less than103 LD50/g brain, which is completely inconsistent with the titers measured; Sc237 incubation periods at end point dilution in Syrian hamsters are around 130 days. That a 100% attack rate was seen at a 127-day incubation period argues against persistent Sc237 inoculum, rather than newly formed prions, being responsible for the pathogenicity to hamsters. Together, these data suggested production of novel infectivity, pathogenic for both mice and hamsters on passage of Sc237 to CD-1 mice.

A recent report has suggested that hamster scrapie 263K may persist in the brains of inoculated C57BL/10 mice for prolonged periods without replication (Race and Cheesbro, 1998). It is possible that the prions detected in the brains of the C57BL/10 mice in the earlier study were not caused by persistence of inoculated 263K, but by propagation of prions with the properties we describe.

Our data are not consistent with infectivity in the PrPSc-positive Sc237-inoculated CD-1 mice being the result of persistence of residual Sc237 hamster scrapie inoculum. High levels of mouse PrPSc (and no hamster PrPSc) are detectable on Western blot, and prions pathogenic for mice are generated. Intracerebral inoculation is known to result in wide distribution of the inoculum outside the brain via the circulation and, presumably as a result of other clearance mechanisms, brain titers fall to undetectable levels within a few days. Prion titers present in the brains of these mice considerably exceed those inoculated. Together, these data argue strongly for prion replication in these mice.

The assessment of species barriers has relied on the development of a clinical disease in inoculated animals. On this basis there is a highly efficient barrier limiting transmission of Sc237 prions to mice. However, although not developing a clinical disease, and indeed living as long as mock-inoculated mice, Sc237-inoculated mice may accumulate high levels of prions in their brains. Previous studies on the species barrier between hamsters and mice (using the Sc237 or 263K strain) did not report whether PrPSc and/or infectivity were present in clinically unaffected animals or have attempted passage from mice only up to 280 days postinoculation. The barrier to primary passage appears in this case to be to the development of rapid neurodegeneration and the resulting clinical syndrome rather than a barrier to prion propagation itself.

The transmission characteristics of prions generated in the brains of Sc237-inoculated CD-1 mice argue that one or more distinct prion strains have been generated. The finding that Sc237-inoculated CD-1 mice in which PrPSc could not be detected on Western blot were the ones that had been culled after shorter periods than mice with detectable PrPSc argues that prion propagation is occurring in all of these mice, but is detectable only after prolonged incubation periods. More than one strain may be propagating in these mice, with preferential replication of a strain with higher pathogenicity for hamsters early in the incubation period. More extensive passage studies, including cloning of strains at end-point dilution in both mice and hamsters, will be required to investigate this further.

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease

EMBO J. 2000 Sep 1;19(17):4425-4430
Raymond GJ, Bossers A, Raymond LD, O'Rourke KI, McHolland LE, Bryant III PK, Miller MW, Williams ES, Smits M, Caughey B. 
Comment (webmaster): This is another excellent no-species-barrier paper with major public policy implications, though it had by design no press release or commenatary pieces in major weekly journals, which has resulted in lower impact than warranted. The wimpy title and abstract turn the data on its head, belittling the risk of CWD transmission to human established in the paper -- "these results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD" -- whereas the data says who would eat venison knowing the risk of conversion was 20% as efficient as CJD itself.

By way of explanation, one of the authors draws 100% of his salary from Colorado game tag sales, six are vulnerably located in small western towns, two at ag schools, three are USDA, one facility has an isolation moat to molligy townspeople, and eight have multiple risk factors.

Back at the ranch, the data shows that the risk of CWD transmission to humans is just as bad as BSE to humans, based on their previously reliable in vitro proxy test. The ethical problem here is that BSE transmission to human has accelerated to a horrific clip even as Colorado goes forward to sell more game tags this fall in an area with 15% confirmed CWD in deer. The real conclusion of the paper emerges 7 pages later, but how many hunters will read this far and put 2+2 together?

"Clearly, it is premature to draw firm conclusions about CWD passing naturally into humans, cattle and sheep, but the present results suggest that CWD transmissions to humans would be as limited by PrP incompatibility as transmissions of BSE or sheep scrapie to humans."

This paper is the best available science on the risk of transmission of CWD to humans, which is now no longer theoretical or questionaire-based but experimental. If we regulate BSE, why not regulate CWD -- and scrapie for that matter -- the same way? If a visitor to England for 6 months cannot donate blood in the US and Canada, a hunter in NE Colorado or SE Wyoming should not either. Do we encourage peope to hunt for meat in a herd of cows with 15% BSE? If scrapie is just as bad as BSE, why is the meat still being sold into the human food chain?

US TSE policy is paralyzed by economic conflicts, fears that past coverups of public health risks taken will unravel, and worries over accountability and liability. After all, the scrapie-to-human risk was established two years ago but no policy changes were made -- only because it would wreak havoc in the sheep industry (there is no way to certify a flock as scrapie-free ). People who bought hunting tags last year based on widely publicized reassurances that now have to be retracted (two of the authors) are going to raise questions. Then there is the vexatious issue of transmission of CWD to co-pastured cattle and problems this poses to the gigantic US beef industry. In short, nothing will be done -- the US, like England, only digs itself in deeper to put off the day of reckoning.

"Oh, what tangled webs we weave, when first we practise to deceive." -- Sir Walter Scott. 1808, Marmion, canto vi

Highlights of the paper (adapted):

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk (cervids), and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes.

Furthermore, the efficiency with which the protease-resistant PrP of one species induces the in vitro conversion of the normal PrP of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents.

Here we show that the CWD-associated PrP-res of cervids readily induces the conversion of cervid PrP sensitive molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrPCWD-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate [ie, higher, providing mild support for a sheep origin of CWD, probably at the Foothills Research Station in Ft. Collins, Colorado. -- webmaster].

A high percentage (up to 15%) of free-ranging deer and elk in parts of north-eastern Colorado and south-eastern Wyoming are infected with chronic wasting disease [The source for this statement is not properly cited: Miller,M.W., Williams,E.S., McCarty,C.W., Spraker,T.R., Kreeger,T.J., Larsen,C.T. and Thorne,E.T. (2000) Epidemiology of chronic wasting disease in free-ranging cervids. J. Wildl. Dis., 36, 676­690" is actually not due out until the October issue, ie J. Wildl. Dis., 36(4), 676­690 in press. -- webmaster]

Although it appears that natural transmission of CWD between cervids is important in the maintenance of the CWD epidemic, the origin of CWD and the mode of transmission between wild animals are not understood. Furthermore, it is not clear whether the disease can be transmitted to humans who hunt and eat these animals or to domestic livestock [see update] whose range may overlap with infected cervids. The transmissibility of CWD to animals should be tested experimentally in vivo, but such tests will take years to complete because of the long incubation periods commonly encountered in interspecies TSE transmissions. Because of these problems, and the fact that CWD transmissibility cannot be tested directly in humans, we have sought alternative clues to the potential interspecies transmissibility of CWD.

In TSE diseases, the host¹s protease-sensitive prion protein is converted to a protease-resistant isoform. Transgenic studies have demonstrated the importance of PrP sequence homology between the sources and recipients of TSE infections. Such PrP homology is also important in PrP-res formation, as demonstrated in scrapie-infected neuroblastoma cells) and cell-free reactions (Kocisko et al., 1995; Bossers et al., 1997, 2000; Raymond et al., 1997; Horiuchi et al., 2000).

In cell-free reactions, PrP-res directly induces the conversion of PrP-sen to a protease-resistant state that is biochemically indistinguishable from PrP-res (Kocisko et al., 1994). Although the products of this reaction have not yet been shown to be infectious, this apparent self-propagating activity of PrP-res correlates with infectivity in denaturation studies and shows striking strain and species specificities that reflect important biological parameters of these diseases.The efficiency of conversion reactions between PrP-res and PrP-sen of different species correlates with the relative TSE transmissibility between those species in vivo.

An important control point in interspecies TSE infections is the molecular compatibility between incoming PrP-res and the endogenous PrP-sen. In the present study, we have compared the efficiency with which PrP-res from CWD-infected cervids (PrPCWD) induced conversion of PrP-sen molecules from other species. Although multiple factors control whether CWD actually transmits to other species, our analysis provides an initial assessment of the relative likelihoods that incoming PrPCWD, if delivered to an appropriate anatomical site, can initiate new, potentially pathogenic PrP-res formation.

To test the ability of PrPCWD to induce the cell-free conversion of cervid PrP-sen molecules as a benchmark for other cross-species comparisons, we first needed to obtain suitable cervid PrP-sen and PrPCWD molecules. Cervid PrP has five known allelic variants.

For conversion reactions the [35S]PrP-sen molecules were labeled in the presence of a glycosylation inhibitor, tunicamycin, to simplify the banding pattern on SDS­polyacrylamide gels ; in previous studies, a lack of N-linked glycans had little or no influence on the species specificity of conversion reactions

Unlabeled PrPCWD was purified from brain tissue pools of either CWD-affected mule deer, white-tailed deer or elk, and immunoblotting analysis showed proteinase K-resistant bands of the expected size (19­30 kDa). Conversion reactions between the various cervid [35S]PrP-sen and normal cervid prion molecules generated 17­19 kDa, PK-resistant [35S]PrP products similar to those seen in conversion reactions with other PrP species , i.e. 6­8 kDa lower in apparent molecular weight than the full-length [35S]PrP-sen substrate. These products were also the size expected for unglycosylated, PK-treated PrP-res derived from brain.

The conversion efficiency (i.e. percentage of the input [35S]PrP-sen converted to the 17­19 kDa PK-resistant [35S]PrP bands) is shown in Figure 4. PrPCWD from elk induced the most efficient conversions of each of the cervid [35S]PrP-sen variants but deer PrPCWD preparations also gave strong conversions of these molecules. The conversions of the mule deer/white-tailed deer -GMNQ variant PrP-sen were least efficient amongst the inter-cervid conversion reactions even in the reactions induced by mule deer -PrPCWD predominantly from md/wd-GMNQ/GMSQ heterozygotes. Otherwise, there was no apparent statistically significant advantage in having exact sequence homology between the PrPCWD and the cervid [35S]PrP-sen molecules [ie, little by way of species barrier exists within these cervids -- webmaster]

CWD-induced conversions of human PrP-sen variants:

Considering that hunters and other humans are potentially exposed to CWD infectivity, we tested the efficiency of conversion reactions between PrPCWD and human PrP-sen molecules. Wild-type human PrP has two common allelic forms that encode either methionine or valine at codon 129. Although discrete 18 kDa, PK-resistant [35S]PrP products were sometimes visible in reactions of [35S]hu-M PrP-sen or [35S]hu-V PrP-sen with all of the three cervid PrPCWD preparations, the efficiencies of these conversions were 14-fold lower than the inter-cervid conversion reactions and 5-fold weaker than the homologous human-to-human CJD conversions [ie, deer to human is7% as efficient as deer to deer and 20% as efficient as human to human, so more than adequate to transmit disease. -- webmaster]. Methionine at codon 129 was slightly more efficient as a target than valine.

CWD-induced conversions of bovine PrP-sen: Since cattle can share range land with deer and elk in CWD endemic areas, we tested the efficiency of PrPCWD-induced conversions of bovine (bo) [35S]PrP-sen. A 20 kDa band was observed occasionally as a PK-resistant conversion product, but the efficiency of these conversions was an average of at least 5- to 12-fold weaker than the corresponding inter-cervid conversion reactions and the homologous conversion of bovine [35S]PrP-sen induced by BSE [ie, deer to cow could be 20% as efficient as cow to cow BSE, so more than adequate to transmit disease. -- webmaster].

CWD-induced conversions of ovine PrP-sen: Both domestic and bighorn sheep (Ovis canadensis) can also share habitats with CWD-infected cervids, so we tested PrPCWD-induced conversions of ovine PrP-sen. Ovine PrP exists in at least 11 allelic forms, several of which have been tested in previous cell-free conversion studies with scrapie and BSE probes. We used ovine-AQ, an allele associated with scrapie susceptibility in domestic sheep wildtype for bighorn sheep. The PrPCWD-induced conversions of sheep PrP-AQ were less than half as efficient as the corresponding homologous cervid and ovine PrP reactions but several fold more efficient than the corresponding PrPCWD-induced conversions of bovine and human [ie, deer to sheep has little by way of species barrier: 50% as efficient as within-species, and 3-7 times more efficient than deer to cow or deer to human. -- webmaster]

CJD, BSE, and scrapie conversion of deer and elk:

Conversions induced by ovine, human and bovine PrP-res measure the assymmetry of the species barrier . Of the ovine and bovine PrP-res types, ov-PrPSc(AQ) induced the strongest conversion of cervid PrP-sen molecules, but even these conversions were at least 5-fold less efficient than the homologous conversion of ov-AQ. Conversions of human [35S]PrP-sen molecules (hu-M and hu-V) by PrPBSE and ov-PrPSc(VQ) were reported in a previous study (Raymond et al., 1997). More replicates of the previous conversions as well as conversions of hu-M and hu-V PrP-sen induced by ov-PrPSc(AQ) are shown in Figure 4. These conversions of human PrP-sen by bovine and ovine PrP-res are all 10-fold less efficient compared with the corresponding homologous conversions and are close to the limit of detection for conversion product [these replicates are important: they are above the detection limit and definitely occurring. -- webmaster].

The cell-free PrP conversion reaction is a test of the molecular compatibility between PrP-res and PrP-sen of different sequences. While this compatibility seems necessary for TSE disease transmission in vivo, it is clearly not sufficient. Other factors such as dose, strain and route of infection, the stability of the infectivity inside and outside the host and the efficiency of its delivery to the nervous system are also important in determining actual transmission rates between species....The biggest challenge is the quantitative comparison of transmissibility. We anticipate that the cell-free conversion efficiency would correlate best quantitatively with relative intracerebral transmission titers between the relevant species. [so true; nevertheless the conversion test has been a reliable proxy so far -- webmaster]

Nonetheless, in side by side tests performed with multiple types of PrP-res, many fold (5 to >50) stronger conversion efficiencies have been observed with PrP-sen molecules from highly susceptible animals than with those from clearly resistant species/genotypes (Kocisko et al., 1995; Bossers et al., 1997, 2000; Raymond et al., 1997; Chabry et al., 1999). Intermediate efficiencies (2- to 4-fold weaker than homologous) have been observed with PrP-sen from animals that are susceptible but apparently less so than the original host species.

For instance, quantitative comparisons are available for relative transmissibilities of BSE to cattle and mice. In this case, BSE infectivity titers per gram are 103 higher when inoculated into cattle compared with mice (Bradley, 1999), while in cell-free reactions PrPBSE induces the conversion of bovine PrP-sen 3- to 4-fold more efficiently than murine PrP-sen (Raymond et al., 1997). The ratio of intracerebral infectivity titers of hamster 263K scrapie in Syrian hamsters versus RML mice is at least 107, while the 263K PrPSc-induced conversion of hamster PrP-sen is at least 7-fold more efficient than mouse PrP-sen under conditions similar to those used in the present study (Kocisko et al., 1995; Chabry et al., 1999). The ratio of intracerebral infectivity titers of Chandler mouse scrapie in RML mice versus Syrian hamsters is 105 while the mouse PrP-res-induced conversion of hamster PrP-sen is 5-fold less efficient than mouse PrP-sen (Kocisko et al., 1995; Chabry et al., 1999).

Based on the available information, it seems that the log of the relative intracerebral transmission titer per gram might be roughly proportional to the relative cell-free conversion efficiency on a linear scale. However, far more quantitative transmission data between various species will be required to establish the fit between these parameters. [This doesn't address transmission risks in the field; it is important to recall frequent horizontal transmission both at Foothills and Sybille research stations, even after rests and facility decontamination . -- webmaster]

The rank order efficiency of all the CWD-induced conversions of various aglycosyl PrP-sen molecules is e-GLSE, e-GMSE, md/wd-GMSQ, wd-SMSQ > md/wd-GMNQ, ov-AQ > bo > hu-M, hu-V ; this rank order would also apply to the relative susceptibilities of species to equivalent exposures to CWD infectivity.

CWD transmission to domestic or bighorn sheep is the next highest risk. However, bighorn sheep held with CWD-infected cervids have not developed TSE disease (M.W.Miller, unpublished data). For cattle and humans, it is likely that susceptibility to CWD would be severely, but perhaps not completely, limited by the lack of conversion compatibility of the respective PrP isoforms [conclusion not consistent with the data: deer to human is 20% as efficient as human CJD to human; miniscule iatrogenic exposures are proven to suffice over long time frames-- webmaster].

Discrete conversion products were sometimes observed, albeit very weakly, in human PrP-sen reactions with ovine PrPSc, PrPBSE or PrPCWD. This suggests that after exposure of humans to scrapie, BSE or CWD, potentially pathogenic conversion to human PrP-res might follow, if only rarely or inefficiently. [Doesn't the nvCJD epidemic provide a more realistic guide? -- webmaster].

Clearly, it is premature to draw firm conclusions about CWD passing naturally into humans, cattle and sheep, but the present results suggest that CWD transmissions to humans would be as limited by PrP incompatibility as transmissions of BSE or sheep scrapie to humans [which are efficient enough to give rise to a horrific unfolding epidemic -- webmaster].

Although there is no evidence that sheep scrapie has affected humans [absense of evidence is not evidence of absence -- webmaster], it is likely that BSE has caused new variant CJD in 74 people [make that 88 -- note the change just during the time the paper was in press -- webmaster]. Given the presumably large number of people exposed to BSE infectivity, the susceptibility of humans may still be very low compared with cattle, which would be consistent with the relatively inefficient conversion of human PrP-sen by PrPBSE. Nonetheless, since humans have apparently been infected by BSE, it would seem prudent to take reasonable measures to limit exposure of humans (as well as sheep and cattle) to CWD infectivity as has been recommended for other animal TSEs. [So will hunting tags be sold or not? -webmaster]

Preclinical BSE: sheep blood shown infectious

Thu, 14 Sep 2000  Lancet
Comment (webmaster): This short but very impressive Lancet article has been released over objections to imagined public reaction. The timing of it all is incredible: the CWD to humans 20% as bad as CJD to humans on Sept 1, no species barrier after all in subclinical mice on Sept 7 , and now infectious BSE blood transfusion from preclinical sheep on Sept 16.

There are 3 parts: the news & views, the article itself, and a Paul "Further studies of blood infectivity in an experimental model of TSE , with an explanation of why blood components do not transmit CJD in humans" Brown commentary saying the article should not have been published, that authorities have already taken whatever steps were practicable. MAFF and Food Safety gave a near-identical quote in response to the Hill-Collinge preclinical BSE study.

Actually, people facing a decision on elective surgery might want to know. Now they can make an informed choice based on their reading of the data (or that of a trusted consultant). The tradeoff is it could impact medical incomes and sale of blood products.

The "panic" rationale for non-disclosure has never made clear exactly who is panicking -- it doesn't seem to be the public or patients, perhaps it is the doctors and industry. The "father knows best" style of patronizing, authoritarian medicine has largely disappeared in the US -- people just don't want it.

The article (highlights, adapted):

Lancet 2000; 356: 999 - 1000
F Houston, J D Foster, Angela Chong, N Hunter, C J Bostock
We have shown that it is possible to transmit BSE to a sheep by transfusion with whole blood taken from another sheep during the symptom-free phase of an experimental BSE infection. BSE and nvCJD in human beings are caused by the same infectious agent, and the sheep-BSE experimental model has a similar pathogenesis to that of human nvCJD. Although UK blood transfusions are leucodepleted--a possible protective measure against any risk from blood transmission--this report suggests that blood donated by symptom-free nvCJD-infected human beings may represent a risk of spread of nvCJD infection among the human population of the UK.

The demonstration that nvCJD is caused by the same agent that causes bovine spongiform encephalopathy (BSE) in cattle has raised concerns that blood from human beings in the symptom-free stages of nvCJD could transmit infection to recipients of blood transfusions. There is no evidence that iatrogenic CJD has ever occurred [absence of evidence is not evidence of absence -- webmaster] as a result of the use of blood or blood products, but nvCJD has a different pathogenesis and could present different risks.

Available evidence, based on detection of infectivity in blood in rodent models, and absence of infectivity in naturally occurring TSEs, adds to the uncertainty in risk assessments of the safety of human blood. PrPSc has been reported in blood taken from preclinical TSE-infected sheep, but it does not follow that blood is infectious....BSE-infected sheep harbour infection in peripheral tissues and are thus similar to humans infected with nvCJD.

Blood was taken from UK Cheviot sheep challenged orally with 5 g BSE-affected cattle brain and transfused into Cheviot sheep from a scrapie-free flock of New Zealand-derived animals (MAFF/SF flock). MAFF/SF sheep do not develop spontaneous TSE [how long have they been under observation, any subclinical animals? -- webmaster] and the transfused animals are housed separately from other sheep [have other sheep _ever_ been housed in this facility? -- webmaster]. All sheep in the study have the PrP genotype AA136QQ171 which has the shortest incubation period of experimental BSE in sheep.

Nineteen transfusions from BSE-challenged sheep have been done, mostly with whole blood. Sheep have complex blood groups and only simple cross-matching can be done by mixing recipient serum and donor erythrocytes and vice versa. Therefore single transfusions only were made between sedated cross-matched animals to minimise the risk of severe reactions.

Negative controls were MAFF/SF sheep transfused with blood from uninfected UK Cheviot sheep [where would these come from? -- webmaster]. As a positive control, MAFF/SF sheep were intravenously injected with homogenised BSE-affected cattle brain.

We have seen BSE clinical signs and pathological changes in one recipient of blood from a BSE-infected animal, and we regard this finding as sufficiently important to report now rather than after the study is completed, several years hence. The blood donation resulting in transmission of BSE to the recipient was 400 mL of whole blood taken from a healthy sheep 318 days after oral challenge with BSE.

BSE subsequently developed in this donor animal 629 days after challenge, indicating that blood was taken roughly half way through the incubation period. 610 days after transfusion, the transfused sheep (D505) itself developed typical TSE signs: weight loss, moderate pruritus, trembling and licking of the lips, hind-limb ataxia, and proprioceptive abnormalities.

This is the first experimental transmission of BSE from sheep to sheep and so we have nothing with which to compare this incubation period directly. In cross-species transmissions, bovine BSE injected intracerebrally gives incubation periods of about 450 days in these sheep,5 and the donor animal had an oral BSE incubation period of 629 days (see above). There are no similar data available on other infection routes.

Immunocytochemistry with the antibody BG4 on tissues taken from sheep D505 showed widespread PrPSc deposition throughout the brain and periphery. Western blot analysis of brain tissue with the antibody 6H4 showed that the PrPSc protein had a glycoform pattern similar to that of experimental BSE in sheep and unlike that of UK natural scrapie, indicating that the TSE signs resulted from transmission of the BSE agent.

All other recipients of transfusions and positive and negative controls are alive and healthy. The positive controls, which involve a species barrier, are expected to have lengthy incubation periods. With one exception, all transfused animals are at earlier stages post-transfusion than was D505. The exception is a sheep which is healthy 635 days after transfusion with BSE-blood donated at less than 30% of the BSE incubation period of the donor sheep.

Although this result was in only one animal, it indicates that BSE can be transmitted between individuals of the same species by whole-blood transfusion. We have no data on blood fractions or on levels of infectivity in blood of preclinical nvCJD cases, but whole blood is not now used in UK transfusions. The presence of BSE infectivity in sheep blood at an early stage in the incubation period suggests that it should be possible to identify which cells are infected, to test the effectiveness of leucodepletion, and to develop a diagnostic test based on a blood sample.

Comment (Paul Brown, Lancet 2000; 356: 955 - 956): If large numbers of apparently healthy people are now silently incubating infections with bovine spongiform encephalopathy (BSE), the implications for public health include the possiblity that blood from such individuals may be infectious. Established facts about infectivity in the blood of human beings and animals with transmissible spongiform encephalopathies (TSEs) are as follows:

Blood, especially the buffy-coat component, from animals experimentally infected with scrapie or CJD and from either a clinical or preclinical incubation phase, is consistently infectious when bioassayed by intracerebral or intraperitoneal inoculation into the same species;

In naturally infected animals (sheep and goats with scrapie, mink with transmissible mink encephalopathy, and cows with BSE), all attempts to transmit disease through the inoculation of blood have failed;

Blood from four of 37 human beings with clinically evident sporadic CJD has been reported to transmit the disease after intracerebral inoculation into guinea pigs, mice, or hamsters. But each success has been questioned on technical grounds and has not been reproducible;

Epidemiological data have not revealed a single case of CJD that could be attributed to the administration of blood or blood products among patients with CJD, or among patients with haemophilia and other congenital clotting or immune deficiencies who receive repeated doses of plasma concentrates.

No comparable information about nvCJD is available. However, since lymphoreticular organs, such as tonsils have been shown to contain the prion protein (which is an excellent index of infectivity), whereas it is not detectable in patients with sporadic CJD, there is some reason to worry that blood from individuals incubating nvCJD might be infectious. Data from studies into the ability of blood from experimentally infected rodents and primates with nvCJD to transmit the disease will not be available for months or years.

In this issue of The Lancet, F Houston and co-workers report convincing evidence that blood >from a seemingly healthy sheep incubating BSE (infected by the oral route with brain from a diseased cow) was able to cause the disease when transfused into another sheep. This observation is entirely consistent with past experience in experimentally infected rodents. It extends current knowledge about blood infectivity in experimental models to a host/TSE strain pair that is closer to the human nvCJD situation than the earlier rodent studies. It is also the first successful transfusion of BSE from blood taken during the all-important incubation period of infection. This result is part of a larger study (n=19) that includes both positive and negative control animals, all still healthy and in various early stages of the incubation period.

Is it appropriate to publish an experimental result from a single animal in a study that is not far enough along even to have validated its positive controls? Especially a result that does not in any fundamental way change our current thinking about BSE and vCJD and which would not seem to have any practical consequences for public health? The UK National Blood Transfusion Service has already implemented leucodepletion of donated blood, and imports all plasma and plasma derivatives from BSE-free countries. No further measures would seem possible--short of a draconian decision to shut down the whole UK blood-donor system. What, therefore, is the rationale for this publishing urgency? The answer, evidently, is a perceived need to "defuse", by an immediate and accurate scientific report, public reaction to possibly inaccurate media accounts. The full study, when it appears, will be an important addition to our knowledge of TSEs, but science should not be driven to what in certain medical quarters might be termed a premature emission through fear of media misrepresentation.

Detection of BSE infectivity in brain and spleen of experimentally infected sheep.

Vet Rec 1996; 138: 546-48
Foster JD, Bruce M, McConnell I, Chree A, Fraser H.

Different levels of prion protein (PrPc) expression on hamster, mouse and human blood cells.

Br J Haematol 2000 Aug;110(2):472-80 [access blocked]
Holada K, Vostal JG
Laboratory of Cellular Hematology, CBER, FDA, Bethesda, MD, USA.
Comment (webmaster): This is good work but bad news. If human blood cells express far more copies of normal prion on their surface than rodents, are rodent studies then adequate or even suitable for studying risks of TSE transmission by blood products? While neither conversion nor infectivity was studied here, where there is smoke there is fire: greater availability of normal prion means greater opportunities for recruitment by an exongenous template seed. Thus rodents may well understate risks, as their cells don't express nearly as many cell surface prions on various blood components as humans do.

Sheep and cattle were not studied but would be an important extension of this work. One wonders why 18 years into the BSE epidemic this has not yet been done -- flow cytometry was widely available by the 1960's. Of what relevence to human infection are complicated rodent studies of the role of the immune system? Public policy obviously cannot be based on studies of mouse platelets or hamster peripheral blood cells after this. And yet that is what may be done.

"The host prion protein, PrPc, and its conformationally changed isoform, PrPsc, play an essential role in the transmissible spongiform encephalopathy (TSE) infections. The prion hypothesis postulates that PrPsc is the TSE infectious agent and that it serves as a template to convert host PrPc to additional PrPsc. Blood of experimentally TSE-infected rodents has been shown to contain TSE infectivity. If blood-borne TSE infectivity requires association with PrPc, differences in the distribution of PrPc in blood could affect the amount and distribution of blood-borne infectivity in different hosts.

We have compared the distribution of PrPc on the peripheral blood cells of humans, hamsters and mice using quantitative flow cytometry. Human lymphocytes, monocytes and platelets displayed much greater quantities of PrPc than corresponding mouse cells.

Mouse platelets did not express any detectable PrPc. A similar low level of PrPc was found on both human and mouse red blood cells. None of the hamster peripheral blood cells displayed detectable amounts of PrPc.

If PrPc contributes to the propagation or transport of TSE infectivity in blood, the species differences in PrPc distribution reported here need to be considered when extrapolating the results of rodent TSE transmission studies with blood and blood components to humans."

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

J Clin Microbiol 2000 Sep;38(9):3254-3259 [access blocked]
O'Rourke KI, Baszler TV, Besser TE, Miller JM, Cutlip RC, Wells GA, Ryder SJ,
Parish SM, Hamir AN, Cockett NE, Jenny A, Knowles DP
...PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing.

In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue.

The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie.

Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy.

The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97.6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported.

Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.

Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders.

Ann Neurol 1999 Nov;46(5):774-7
Saiz A, Graus F, Dalmau J, Pifarre A, Marin C, Tolosa E
The detection of 14-3-3 protein in cerebrospinal fluid by immunoblotting is useful for the diagnosis of Creutzfeldt-Jakob disease (CJD). We found 14-3-3 protein in 10 of 80 (12.5%) patients with paraneoplastic neurological disorders (PNDs), whose presenting symptoms may mimic those of CJD.

In 47 of 48 CJD patients, the 14-3-3 protein was detected as a single band, and it was detected as a double band in 1 patient. The double-band pattern was observed in 9 of 10 14-3-3 protein-positive patients with PNDs. The 14-3-3 protein assay may be positive in PND patients, but the immunoblotting pattern distinguishes most PND samples from those of CJD.

ELISkA quantifies 14-3-3 proteins in the cerebrospinal fluid of suspected Creutzfeldt-Jakob disease patients.

Ann Neurol 2000 Sep;48(3):395-8
Kenney K, Brechtel C, Takahashi H, Kurohara K, Anderson P, Gibbs CJ Jr 
The detection of 14-3-3 protein by Western immunoblot is a sensitive and specific cerebrospinal fluid marker of Creutzfeldt-Jakob disease (CJD). We developed a quantitative enzyme-linked immunosorbent assay (ELISA) that reliably detects 14-3-3 in cerebrospinal fluid. In a prospective study of 147 cerebrospinal fluid samples, the mean 14-3-3 concentration among pathologically confirmed CJD patients (28.0+/-20.6 ng/ml, n = 41) is significantly higher than the mean in the cerebrospinal fluid of those with other neurological disorders (3.1+/-2.9 ng/ ml, n = 84). At a cutoff value of 8.3 ng/ml, the ELISA has a sensitivity of 92.7% and a specificity of 97.6%. The 14-3-3 ELISA supports a diagnosis of CJD in patients who fulfill clinical criteria for possible CJD.

Current clinical diagnosis in Creutzfeldt-Jakob disease: identification of uncommon variants.

Ann Neurol 2000 Sep;48(3):323-9 Zerr I, Schulz-Schaeffer WJ, Giese A, Bodemer M,... Gefeller O, Kretzschmar HA, Poser S
According to the recently established molecular basis for phenotypic heterogeneity of sporadic Creutzfeldt-Jakob disease (CJD), six different phenotypes are characterized by the size of the protease-resistant fragment of the pathological prion protein (types 1 and 2) and homozygosity or heterozygosity for methionine or valine at codon 129 of the prion protein gene (designated by MM1, MM2, MV1, MV2, W1, and W2).

In the present investigation, we analyzed the value of commonly used clinical tests (electroencephalogram [EEG], detection of 14-3-3 protein in cerebrospinal fluid [CSF], and hyperintensity of the basal ganglia in magnetic resonance imaging) for the clinical diagnosis in each CJD phenotype. The detection of periodic sharp and slow wave complexes in the EEG is reliable in the clinical diagnosis of MM1 and MV1 patients only.

The CSF analysis for 14-3-3 protein showed high sensitivity in all analyzed subgroups with the exception of MV2 patients. Valine-homozygous patients had a negative EEG, but most had detectable levels of neuronal proteins in the CSF. The sensitivity of the magnetic resonance imaging was 70%, irrespective of the subgroup, but was particularly reliable in the clinical diagnosis of MV2 patients. The widening spectrum of diagnostic techniques in CJD is not only useful in the increased accuracy of the clinical diagnosis but should also lead to the identification of more atypical cases of sporadic CJD.

Follicular dendritic cells and dissemination of CJD

J Virol 2000 Sep;74(18):8614-22
Manuelidis L, Zaitsev I, Koni P, Yun Lu Z, Flavell RA, Fritch W
The contribution of immune system cells to the propagation of transmissible encephalopathies is not well understood. To determine how follicular dendritic cells (FDC) may act, we challenged lymphotoxin beta null and wild-type (wt) controls with a Creutzfeldt-Jakob disease (CJD) agent.

There was only a small difference in incubation time to clinical disease even after peripheral challenge with low infectious doses (31 in a total of 410 days). Brain pathology with extensive microglial infiltration, identified by keratan sulfate, as well as astrocytic hypertrophy, was also equivalent in all groups despite the fact that null mice had neither FDC nor splenic metallophilic macrophages that filter particulate antigen.

Because FDC accumulate pathologic prion protein (PrP) in infected but not wt mice, we studied the cellular distribution of PrP by confocal microscopy. The majority of pathologic PrP collected on the plasma membrane of FDC, as identified by the Ca(+2)-binding protein S100A.

This surface distribution suggested that agent aggregated with pathologic PrP might spread by cell-to-cell contacts. While several types of leukocytes may be involved in agent dissemination, cells of myeloid lineage were found to be infectious. Moreover, perivascular tracks of microglia and abnormal PrP after intraperitoneal inoculation were consistent with hematogenous spread.

In summary, FDC are not required for CJD agent spread from the periphery, although FDC may enhance spread through surface accumulation of pathologic PrP. While it is still not clear where the infectious agent replicates, macrophages can sequester appreciable levels of infectivity and hence act as reservoirs for prolonged latent infection.

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus

J Mol Biol 2000 Aug 18;301(3):567-73 [access blocked]
Li R, Liu T, Wong BS, Pan T, Morillas M, Swietnicki W, O'Rourke K, Gambetti P, Surewicz WK, Sy MS
We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP.

The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP.

The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231).

Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145.

By Western blotting with PepSpot, only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes.

Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.

Comment (webmaster): The 3D nmr structure of the human prion (1QM0)has been available for many months and is in any event nearly identical to rodent. However, both this structure and the protein targets here represent non-native prion protein lacking important covalent modifications such as the early arginines, glycosylation, and GPI sites, as well as signficant structural modifications induced by copper binding by the repeat. We are talking here of a minimum of 7 significant changes between recombinant and real protein.

It is thus physically impossible for the backbone and side chains of recombinant and native protein to have the same conformation. The abstract misleadingly implies that normal human prion was used as antigen and target; in fact recombinant protein from E. coli was used for both. No mention is made of how the antibody panel reacts with purified native Prpc or human PrpSc; conditions that generated the Prionics monoclonal to PrpSc remain a mystery.

It is naive to assume a simplistic Anfinsen principle, that a fully denatured recombinant protein will refold to native conformation or even to a lowest free energy state, especially in the absence of chaperone (see review of 17 amyloids by JW Kelly). Since the normal function of the prion protein remains largely unknown, no independent assay of regain of native function (usually the best measure of correct folding) can be undertaken.

Nevertheless, the finding that the linear Mabs 5B2 and 8B4 but not the more distal 2GB block access of C terminus-specific Mab 6H3 is quite interesting since in nmr, no physical proximity could be observed and the domains are at opposite ends of the monomer. It is difficult to distinguish simple physical blockage with retention of target conformation from indirect blockage by conformational change at a distance induced by antibody binding.

If a prion dimer were present, the usual Jacob-Monod half-rotation of dimers could place the N and C termini of different monomers in proximity even though they were at opposite ends in the monomer. Though "not invented here," it would have been useful for 5C3, 2C9 and 7A12 to have included competition from 3F4 because it is well-characterized and the subject of a recent xray crystalographic study of 104-113.

Monomer-dimer equilibrium of native Prpc not seen in recombinant protein

JBC Papers in Press. Published on August 30, 2000 [free pdf]
Rudolf K. Meyer, Ariel Lustig, Bruno Oesch, Rosmarie Fatzer; Andreas Zurbriggen and Marc
Highlights of article (adapted):

By three lines of evidence (ELISA, crosslinking experiments and size exclusion chromatography) under native conditions at least part of the native bovine PrPc exists as a monomer-dimer equilibrium. Such protein-protein interactions were absent in recombinant protein showing for the first time a biochemical difference in respect to the native, glycosylated form.A bovine PrPc specific immuno sandwich ELISA was developed, calibrated with recombinant PrP and used to partially purfiy a distinct PrPc fraction. When serial dilutions of brain homogenate or partially purified PrPc were measured, using the peptide antibody C15S, a nonlinear dose response curve was obtained. This nonlinearity was shown not to be due to an artifact of the procedure but to a monomer ­ dimer equilibrium of PrPc with preferential binding of the antibody to the dimer. The disassociation constant was measured as 3.9 x 10 8 M ­1 at 37 °C, free energy ­ 48.6 kJ M ­1, enthalpy 9.5 kJ M ­1, and entropy (0.2 kJ K ­1 M ­1 ).

Additional evidence of dimer formation was shown by Western blotting of partially purified PrPc crosslinked by the homobifunctional crosslinker BS 3 [the lysine-specific cross-linking agent, bis(sulfosuccinimidyl)-suberate]. Size exclusion chromatography showed an additional shoulder not observed with recombinant PrP at a position between ovalbumin (44k) and gamma globuline (158k). The difference in respect of dimer formation between native PrPc and recombinant PrP might be explained by the lack of glycosylation of the latter.

Most of the biochemistry of PrPc is known from recombinant PrP, as PrPc is comparatively rare even in the brain and only few micrograms have yet been purified. Recombinant PrP is a monomer. Membrane interaction, copper binding and superoxide dismutase activity have been described, all from bacterial expression systems.

PrP Sc is part of, or even identical to the prion, the infective agent of transmissible spongiform encephalopathies. It is not very soluble and mostly aggregated in prion rods or amyloid deposits. A seeding model was proposed, in which a spontaneous reversible thermodynamically controlled conformational change of PrPc to PrP Sc was postulated. PrP Sc is stabilized only when bound to a crystal-like seed or aggregate of PrP Sc . Seed formation is extremely slow, but once a seed is present monomers can be added rapidly. However, increasing experimental evidence argues for a more specific interaction of PrPc with PrP Sc: the conversion of PrPc to PrP Sc was inhibited by antibody binding to PrPc in vitro and was interpreted as steric blocking of a binding site to PrP Sc; the side of this interaction was localized to 91-146. The hypothetical protein X, a PrP-binding protein present in brain homogenates, would enable dimerization and could be a requirement for PrPc - PrP Sc interaction. The PrPc - protein X complex would then bind PrP Sc , creating a replication-competent assembly.

Brain material (thalamus) was derived from normal Swiss cattle. Brain tissue from the fish Salmo truta [apparently Salmo trutta, brown trout -- webmaster] and from PrP null mice was used as negative controls and for preparing dilutions. Fragments of brain tissue (0.5 g) were homogenized in 10 ml/g (wet weight) of a 320 mM sucrose solution with an Ultra-Turrax T25 (Janke and Kungel, Staufen, Germany). In a first step, heat labile proteins were precipitated by heat treatment. Most proteins, but little of the PrP under consideration, were precipitated by this procedure. The homogenate was cleared by a short (5 minutes) centrifugation at 7000 x g.

In brain homogenate, about ten times more PrPc was detected with the dot blot assay than with the ELISA. But the amount of partially purified PrPc detected both by the dot plot assay and by the ELISA corresponded well with each other. The ELISA detected only about 10% of the PrPc present in untreated thalamus homogenate and only this fraction was actually partially purified. The nature of the remaining PrPc , not detected by the ELISA, was not analyzed further. The use of bovine brain, instead of e.g. mouse brain, had the advantage that large amounts of precisely specified tissue (thalamus) was available. Most likely the purified PrPc was soluble, either secreted or being released during homogenization. A soluble form of PrPc was described for human cerebrospinal fluid

The affinity of the antibody to PrP monomers was only about 4% as compared to PrP dimers. C15S may preferentially bind to dimers if both antibody-binding sites are used because of close proximity of two epitopes. Bovine PrP has 10 lysines excluding the signal sequence, i.e. there are ample possibilities for BS 3 crosslinking with a spacer length of 11.5 Å. Even in the partially purified fractions, PrPc is outnumbered by more than 8000 (by weight) by unrelated proteins which argues against unspecific crosslinking of PrP molecules simply by chance. Because of the monomer-dimer equilibrium, faster- 17 - moving dimers will dissociate when they separate from the monomer pool, resulting in a deformation of the monomer peak toward higher molecular weights and not necessarily in the formation of an additional peak.

A PrPc dimerization has been described for mouse neuroblastoma cells transfected by hamster, but the PrP dimer observed [1995 J. Biol. Chem. 7, 3299] was covalently crosslinked. We speculate that the crosslinking was enzymatically induced by the tissue culture cells used.

Our results showing a spontaneous PrPc monomer ­ dimer equilibrium support the concept of PrP dimer and heterodimer formation in prion propagation.

Comment (webmaster): This is a thorough, persuasive result and a long-overdue experimental approach. Far too much time has been spent chasing artefacts of recombinant protein. Bovine thymus is readily availble on a scale of tens of kilograms.

The prion protein does not present remarkable solubility problems or new issues in protein purification; it is only that few neurologists are trained in protein chemistry. Many simple procedures have not even been tried, for example, exogenous administration of gangliosides displaces GPI-anchored proteins. GPI-anchored proteins are clustered in rafts so heterologous cross-linking here might be informative as well.

We could grumble that the authors did not proceed with mass spectometry to characterize the trypic fragments carrying the cross-links -- this would have shown the geometry of the dimer association (conceivably that proposed earlier by Warwick). Intra-molecular cross-linking with BS3 is also commonly done and would be useful for determine distance relations of copper-loaded prion repeat region to the globular domain.

It is only the soluble prion protein that can form dimers (so the finding here comes as no surprise). The underlying symmetry explanation for this was given by Jacob and Monod 38 years ago and has been validated by now in thousands of proteins. In brief, two GPI-anchored monomers cannot make the 180 degree turn of standard homodimers; spoon-packed dimers are not self-terminating, etc. However, a GPI-anchored monomer might be able to dock with a free monomer. If the repeat region is not important to the dimer interface, a proteolytic fragment, eg 90-230, could participate as well.

Mixed-species dimers are quite possible (recall hemoglobin); these are quasi-homodimers since amino acid sequences and 3D structures differ only slightly. Note that the well-studied amyloid-forming protein, transthyretin, is a soluble tetramer. Oligomeric interfaces do con comprise the underlying cross-beta fibril structure in any amyloid and should not be confused with this sequence-independent interface (which, unlike a dimer, is not self-terminating -- precisely the problem in amyloid disease).

True dimer interfaces are highly evolved complementary surfaces implying physiological significance (the authors note above persistence in an 8000-fold excess of other proteins). GPI proteins in general are poised for release; indeed, if meant to stay on the cell surface, why cleave a perfectly good intrinisic membrane signal peptide. As noted, such products have been reported previously; this would apply in spades to doppel which is armored for the outside world with a second disulfide pair.

The dimer-specific monoclonal is also a welcome development. This could be quite useful in looking at the role of the dimer in disease processes. Indeed, the authors intimate that the fabled protein X might simply be another prion monomer. It might well be best to start with a dimer for in vitro conversion processes.

The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome

JBC Papers in press. published on September 1, 2000 as Manuscript M005543200 [free pdf]
Taocong Jin, Yaping Gu, Gianluigi Zanusso, ManSun Sy, Anil Kumar, Mark Cohen, Pierluigi Gambetti, and Neena Singh
Comment (webmaster): Point mutations lead to prion disease by mechanisms that remain murky. It is generally not known whether normal function is affected (perhaps inducing compensatory over-production) nor which sites exhibit cross-recruitment (in Q217R, only the abnormal allele product accumulates in amyloid.). It has not been feasible to systematically classify mutations, say by parallel multi-well testing of all known sites.

Here, Q217R, a mutation in helix C some 3 residues distal to the second cysteine and 14 residues proximal to GPI signal peptide attachment point, gives rise to molecular sub-populations (with and without anchor-attachment) degraded by different routes in different cellular compartments. The ER chaperone BiP unsurprisingly is involved, though not Grp94 or Hsp47. The neuroblastoma transfection system does not quite lead to PrpSc (time frames of days vs decades) but may otherwise be a satisfactory proxy system.

Despite the progress reported here, it has not been established which sub-population gives rise to PrpSc in vivo, though the data does not favor the non-GPI portion directed by BiP a 654 aa protein, AAF42836] to the proteasome. In a protein with so many potentially incomplete post- or co-translational modifications (eg, N-terminal arginines) in the ER, important roles for quantitatively minor protein species are hard to rule out.

Highlights of article (adapted):

We examine the role of molecular chaperones in the folding of normal and mutant PrP Q217R in transfected neuroblastoma cells. Although most of the PrP 217R escapes the endoplasmic reticulum (ER) quality control system and aggregate in post-Golgi compartments, a significant proportion of PrP 217 retains the C-terminal glycosylphosphatidyl inositol signal peptide (PrP232), and does not exit the ER. [(1997) J.Biol.Chem. 272, 28461-28470]

We have now studied the folding and turnover of PrP232 to understand the mechanism by which abnormal PrP forms cause cellular toxicity in our cell culture model, and in the human brain carrying the GSS Q217R mutation. In this report, we show that PrP232 remains associated with the chaperone BiP for an abnormally prolonged period of time, and is degraded by the proteasomal pathway. This study is the first demonstration that BiP is chaperoning the folding of PrP, and plays a role in maintaining the quality control in the PrP maturation pathway.

Several lines of evidence indicate that the PrPc to PrP Sc conversion is mediated by chaperones [(1998) Nature 392, 23-24]. Molecular chaperones Hsp104 and GroEL have been shown to promote the conversion reaction of mammalian PrPc in a cell-free system and the conversion of prion-like proteins in intact yeast cells. Several chemical chaperones have been shown to act as conversion inhibitors. Data from transgenic animal models argue for the requirement of an accessory protein for the refolding of PrPc to PrP Sc . This protein, temporarily named protein X, remains to be identified, and may very well be a cellular chaperone.

Mutations in the prion protein gene (PRNP) are believed to destabilize the mutant PrP, which then easily converts to PrP Sc). Our series of studies using human neuroblastoma cell models of inherited prion diseases are indeed consistent with this hypothesis. Although these models do not lead to the spontaneous formation of PrP Sc , they show that PrP variants carrying a human pathogenic mutation either become insoluble and are trapped in intracellular compartments , or are rapidly degraded through the proteasomal system. If proteasomal function is impaired, the mutant PrP accumulates in an insoluble and weakly PK-resistant form in the endoplasmic reticulum (ER) and post-ER compartments [J. Biol. Chem. 274, 23396-23404].

These findings argue that chaperones play a double role in inherited prion diseases. They are likely to be involved not only in the conversion of PrP to PrP Sc as in other forms of prion diseases, but may also play a role in the destabilizing effect that the PRNP mutations have on the mutant PrP. Interaction of mutant PrP with chaperones might promote its degradation and delay the conversion to PrP Sc, helping to explain the conundrum of why inherited prion diseases often manifest clinically only late in life although the mutation is present from conception.

PRNP-transfected neuroblastoma cells were used to examine the role of ER-localized molecular chaperones in the processing of normal and mutant PrP. Q217R presents in the seventh decade and is characterized by a slowly progressive dementia associated with cerebellar and extrapyramidal signs. The histopathological hallmark of the Q217R GSS variant is the presence of PrP 217 containing amyloid deposits and neurofibrillary degeneration in the brain tissue

In this study, we provide the first evidence that the ER chaperone BiP binds to PrP. It binds transiently to PrPc and to some forms of PrP 217, but it remains associated for an extended period of time to PrP232, which is subsequently degraded by the proteasomal pathway. At variance with the anchored forms of PrP 217 that are aggregated, insoluble in non-ionic detergents, and partially resistant to proteinase-K (PK) digestion, a relatively large portion of PrP232 remains soluble and sensitive to PK digestion. We postulate that the association of PrP232 with BiP sequesters PrP232 and prevents the formation of homoaggregates, maintaining PrP232 relatively soluble and PK-sensitive.

. Plasmids containing normal and mutant (T34G) hamster BiP cDNA were used in this study along with anti-KDEL (a monoclonal antibody to the peptide KDEL that recognizes Grp94, BiP, and two other proteins of 64 and 47kDa), anti-hamster BiP, anti-calreticulin, and anti-Grp94, anti-PrP (3F4), anti-calnexin, and anti-GPI-SP mouse serum raised in our facility against a 22 residue synthetic peptide representing the GPI domain.

Highly abnormal forms of PrP 217 are retained in the endoplasmic reticulum and are associated with the chaperone BiP. Mature PrPc migrates as three distinct bands, which correspond to the PrPc with two, one, and no glycans respectively. In contrast, PrP 217 shows an additional band of 32kDa (PrP232), which migrates at 30k (rather than at 27k as PrPc ) following deglycosylation. We have previously shown that PrP232 lacks the GPI anchor and retains the 22 amino acid GP.

PrP232, as well as two other forms of PrP 217 migrating at 29-30kDa, remain Endo-H sensitive, indicating that they are not transported out of the ER. To determine if the PrP232 that is retained in the ER is bound to a molecular chaperone, Trans 35 S-methionine labeled PrPc and PrP 217 cells were lysed under non-denaturing conditions and immunoprecipitated with anti-KDEL, or anti-PrP antibody 3F4 prior to analysis by SDS-PAGE.

Anti-KDEL is a monoclonal antibody raised to the carboxy-terminal ER-retention signal KDEL of proteins, and reacts with BiP (Grp78), Grp94, and Hsp47. After immunoprecipitation with anti-KDEL, four major bands are detected in both PrPc and PrP 217 lysates, which include a band of 94kDa corresponding to Grp94 (not shown), a 78kDa band (BiP), and two additional bands of 64kDa and 47kDa . However, in the PrP 217 lysates, an additional band that migrates at 32kDa is detected.

Immunoprecipitation of lysates with the anti-PrP antibody 3F4 shows that only the 32kDa PrP isoform of PrP 217 lysates co-migrates with a similar form detected in anti-KDEL immunoprecipitates....These experiments clearly show that the 32kDa band recovered following immunoprecipitation with antibodies to KDEL contains PrP232, and that PrP232 is associated with BiP in the Q217R transfected neuroblastoma cells. PrP232 is associated with BiP, but not with any of the other ER chaperones. Pulse chase experiments show PrP232 remains associated with BiP for an abnormally prolonged period of time, and is degraded by the proteasomal pathway. We did not detect ubiquitination of PrP232 or other PrP232-related forms. Following inhibition of proteasomal degradation, PrP232 does not form an aggresome-like structure in the cytosol.

BiP promotes proper folding of newly synthesized polypeptides by keeping the precursors in a soluble state. It interacts transiently with proteins which are properly folded. In contrast, it binds misfolded or unassembled proteins in relatively stable complexes and may mediate their retrograde translocation for proteasomal degradation, as it does with PrPc.

BiP interaction with 217R is more complex. In our neuroblastoma cell model, this mutant PrP is expressed in two major variants. The first variant, here referred to as PrP 217 , includes three glycoforms and carries a GPI anchor as PrPc . The second variant, PrP232, which accounts for approximately 11% of the total Q217R mutant PrP, is glycosylated but lacks the GPI anchor and carries the uncleaved GPI anchor signal peptide. Unlike PrPc , PrP 217 is unstable and approximately 50% of it becomes insoluble and aggregates in a compartment distal to the cis-Golgi. Furthermore, the instability leading to aggregation is very likely due to misfolding since it is largely corrected by exposure of the nascent PrP 217 to low temperatures known to favor correct folding.

However, despite misfolding, PrP 217 interacts with BiP only briefly in a way that is indistinguishable from that of PrPc, and, like PrPc , exits the ER. In contrast, BiP interaction with PrP232 is sustained and results in the retention of this form in the ER, but eventually PrP232 is translocated into the cytosol and degraded in the proteasomal system. Only BiP, among a variety of protein folding chaperones present in the ER, shows this association with PrP232.

The mechanism leading to the different BiP interaction and cellular routing between PrP 217 and PrP232 is unclear. It might be due to the lack of the GPI anchor in conjunction with the presence of the GPI signal peptide that makes it impossible for PrP232 to enter the secretory pathway, or related to intrinsic conformational differences. The conformation of some of the mutant PrP might be altered to such an extent that the anchor signal peptide cannot be cleaved, blocking the anchor addition and resulting in the formation of the PrP232 variant. Alternatively, the sustained association of PrP232 with BiP by itself might interfere with the anchor addition process. Y145stop is also degraded by the proteasomal pathway; however it is not bound to BiP

In addition to PrP, several other secretory and membrane proteins implicated in causing disease are degraded by the proteasomal pathway when they are mutated, and aggregate in intracellular compartments following the inhibition of proteasomal function, such as huntingtin, ataxins 1 and ataxin 3, and alpha -synuclein.The central theme in these disorders, as in prion diseases, is the conversion of a normally beta-sheet rich structure that is insoluble, aggregates, and forms intracellular deposits.

Although BiP prevents the aggregation and facilitates degradation of PrP232, it may in fact render PrP232 more pathogenic by maintaining it in a partially unfolded state in the ER, especially if BiP promotes the re-folding of PrP232 into an alternative, more pathogenic form, instead of its aggregation when proteasomal function is compromised.

Ragged N terminus types of CJD

PNAS Vol. 97, Issue 18, 10168-10172, August 29, 2000
Parchi P, Zou W, Wang W,... Ironside JW, Gambetti P, ... James W. Ironside, P Gambetti, Shu G. Chen
Comment (webmaster): Proteolytic clipping plays a central role in many amyloid disorders, most notably Alzheimer. In CJD, N-terminal sequencing is a much needed improvement over gel classification. Gels lack sufficient resolving power to characterize molecular species involved found in PrpSc, which is quite heterogeneous at the peptide level -- after PK treatment -- depending both on the disease and codon 129. Fortunately, only the N terminus seems to have ragged ends. Table 1 of the paper is very instructive and strongly supports the type 1,2 classification based on glycine 82 or serine 97 as the principle N terminus, resp.

The first site is odd, coming near the end of the penultimate repeat (residue 74 is comparable): PQGGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ GGGTHSQWN KPSKPKTNMKHM AGAAAAGAVVGGLGG YMLG; the latter is in a rather variable short domain, probably a hinge region connecting adjacent structured domains. Position 97 has the fewest evolutionary constraints of any residue in mature prion protein: ser, gly, asn are interchangable in all clades examined; S and N alleles occur in squirrel monkey; S and G in bovine (D10614 vs S55629).

An allele at serine 97 in humans would be most interesting -- many sequencing studies to date have not properly considered this part of the gene. Since cow can be either serine or glycine at codon 97 (as well as 5 or 6 repeats), species barrier issues also arise in the region of ragged post-PK N termini. Ominously, the S97 cow is also matches human in octarepeat number (though the terminal repeat still has 9 residues and gly 94 is missing in ruminants). Sheep (all alleles), goats, deer, and elk are serine at codon 97.

One important point made in the paper is that sporadic CJD is clearly not a single entity -- it is split into two very distinct classes which in turn are split by the 3 alternatives at codon 129. We have no idea what underlies the two classes (scrapie sheep consumption vs spontaneous?) nor whether their proportions have changed in recent times. While 36 cases is already a lot of work, the authors correctly note that an even larger series is needed given so many classes present.

Note that little is learned about native precursor to the protease-resistant core, that is, were differing ragged N-termini already present? It is most unlikely that humans encode a protease with the same specificity as PK in an appropriate and relevent cellular compartment -- protease K has such a long history in TSE research that we forget it has no physiological relevence to vertebrates. However, PK can only make peptides shorter, so in vivo bounds are set; note all amino acids distal to the nick are small (serine and glycine) with the exception of a minor occurence of tryptophan that does not fit the profile of protease K specificity. This trp is thus likely to have been the in vivo N terminus, with no PK effect on it.

Different PK end products cannot really be attributed to different conformations, much less cross-beta, in a crude brain extract with an unknown starting compound and many possible additional ligands present. The conflict over copper's role in strain types was not resolved; lipid binding could also be a factor. Activity of proteases is determined more typically by bond specificity and accessibility (sometimes just a breathing mode, not a two-state conformation).

Highlights of article (adapted):

14 groups of human prion diseases in the forms of sporadic, familial, and iatrogenic cases, as well as kuru and the new variant CJD. In human prion diseases, two major types of PrP(Sc), type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrP(Sc) in 36 cases of prion diseases representing 14 groups of human prion diseases (sporadic, familial, iatrogenic cases, kuru and nvCJD).

We demonstrated using N-terminal sequencing and mass spectrometry at the ProSeq Microsequencing Facility (Boxford, MA) two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrP(Sc), respectively, and numerous secondary cleavages distributed along the region spanning residues 74-102.

Accordingly, we identify three regions in PrP(Sc): one N-terminal (residues 23-73) that is invariably PK-sensitive, one C-terminal (residues 103-231) that is invariably PK-resistant, and a third variable region (residues 74-102) where the site of the PK cleavage, likely reflecting the extent of the beta-sheet structure, varies mostly as a function of the PrP genotype at codon 129.

Two major types of PrPSc are distinguished by a difference in size of the proteinase K (PK)-resistant core fragment. After gel electrophoresis, type 1 migrates at 21 kDa, and type 2 migrates at 19 kDa. The difference in size results from the cleavage of PrPSc at different sites, which, in turn, is likely to reflect the distinct conformation or a different ligand interaction of the two PrPSc types. (Both the type 1 and 2 fragments have an intact C terminal GPI.) he N-terminal species starting at the Gly-82 residue (G82) was found in all cases associated with type 1 PrPSc whereas the species beginning at Ser-97 (S97) was present in all cases linked to type 2 PrPSc. Overall, the N terminus was more ragged in PrPSc type 2 than in type 1. Furthermore, in type 2, the N termini of the variant forms were farther away from and more often located at both sides of the common S97 N terminus. G74, G78, G82, G86, G90, G92, S97, W99, S103 were the termini seen; the mutations studied were E200K, D178N (both form mutant-only PrpSc), 4- and 5-extra repeats.

The type 1 and type 2 PrPSc core fragments generated by PK have been shown recently to be reliable markers of disease phenotype and to be in large part functions of the genotype at codon 129 of the PrP gene, the site of a common methionine/valine (M/V) polymorphism. Although the PrPSc typing based on electrophoretic mobility is simple and effective in the routine diagnosis of prion diseases, it has a low resolution and provides limited information on the precise size and possible variety of the PrPSc fragments resistant to proteases.

In sporadic type 1, methionine homozygosity favors a PK secondary cleavage that is more N-terminal than the principal cleavage at G82, whereas the presence of the valine codon shifts the secondary cleavages toward the C-terminal side of G82. However M/V is not a simple superposition but introduces a new site at S97, though the presence of one or two 129 V alleles results in a more ragged N terminus of PrPSc in both type groups.

nvCJD, type 2 129 M/M, was indistinguishable from sporadic CJD with corresponding PrPSc type and 129 genotype.

Collinge's four types of PrPSc has the type 3 variant associated with iatrogenic CJD with at least one 129 V allele and type 4 is with new variant CJD. Type 4 and type 3 differ only in the glycosylation pattern but have the same gel mobility that is lower than that of the PrPSc type 1 and type 2 associated with sporadic CJD. We found that in both nvCJD and iatrogenic CJD with 129 V/V, the gel mobility of PrPSc was indistinguishable from that of the type 2 associated with sporadic CJD. Based on these data, we suggested that Collinge et al. had incorrectly matched our type 1 and type 2 with theirs and that our type 2 actually corresponds to their type 3, whereas their type 1 and 2 likely represent an artifactual subdivision of our type 1. More recently, Collinge and coworkers have reported that the PrPSc identified by them as type 1 may result from a conformation acquired by PrPSc in the presence of copper. [(1999) Nat. Cell Biol. 1, 55-59]

Signal Transduction Through Prion Protein

Science 289, Number 5486, Issue of 15 Sep 2000, pp. 1925-1928
S. Mouillet-Richard,M. Ermonval, C. Chebassier, J. L. Laplanche, S. Lehmann, J. M. Launay,  O. Kellermann 
Comment (webmaster): Despite 6,821 prion papers at medline, normal function is not satisfyingly pinned down, but this is not at all unusual genome wide or even among the 25-odd amyloid disorders. This is a sophisticated cell biology paper with solid antecedents describing an interesting scenario for why normal prion protein is located on the cell surface. The data support a role in a specific signalling cascade, though what the extracellular primary signal and regulatory target of tyrosine kinase Fyn might be are left as directions for future research, as are possible effects of depletion from sequestration in PrpSC. One possibility [below] is a role in calcium homeostatis; another is Cbl is the substrate for Src-like kinases like Fyn and activated in response to the engagement of cell surface receptors, with Fyn phosphorylating tyrosine 731 in Cbl that may regulate activation of phosphatidylinositol 3-kinase. [JBC 274 2097-2106, January 22, 1999]

We are left wondering whether signaling could really be the principal normal function, or only in a neural subset of all the cell types expressing normal prion (which includes anucleate red blood cells), which protein domains participate in signals and which have unrelated functions, what the inter-relationship with the putative copper binding might be, and whether the ancient tandem duplicate doppel (not included in controls) retains the proposed signaling role.

Many signal transduction proteins are part of large protein superfamilies, but the human genome project is sufficiently advanced to say that this is not the case at all for the prion protein. These raises question of how it is possible for the prion protein to interact with standardized components of the signalling cascade lacking a common domain. Note Science maintains a large complex site on signalling; medline has 957 matches to Fyn.

The authors are quoted in the San Francisco Chronicle as saying, ``The location of this protein at the cell surface was indeed evocative of a signaling molecule,'' said lead researcher Odile Kellerman of the Pasteur Institute in Paris. ``However, there was no clear-cut evidence until now that (a prion) could be activated and transduce a signal within the cell.''

One of the most intriguing puzzles remaining is to identify message-bearers outside the nerve cells that can activate the prions. The trigger ``may be some molecule that is secreted by neurons in their environment, a protein present at the surface of neighboring cells or even some components of the extracellular matrix,'' Kellerman said, referring to the region outside the nerve cell.

It is churlish to expect too much from one paper -- good progress was made here in the cell system, the cascade, and specific identification of caveolin-1 isoforms and tyrosine kinase Fyn.

Highlights of article (abridged):

The cellular prion protein PrPc is a GPI-anchored cell-surface protein whose biological function remains enigmatic. We used the murine 1C11 neuronal differentiation model to search for PrPc-dependent signal transduction through antibody-mediated cross-linking. A caveolin-1-dependent coupling of PrPc to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger PrPc-dependent Fyn activation was restricted to its fully differentiated serotonergic or noradrenergic progenies. Moreover, the signaling activity of PrPc occurred mainly at neurites. Thus, PrPc may be a signal transduction protein.

PrP-deficient mice are viable and develop normally, but they display minor defects that differ according to the null strain. In contrast, mice expressing an NH2-terminally truncated version of PrPc in a null background show neuronal degeneration soon after birth, suggesting that PrPc may play an important role in the maintenance and/or regulation of neuronal functions . Recent data have focused on the copper-binding ability of PrPc, and an involvement of PrPc in the regulation of the presynaptic copper concentration and of synaptic transmission has been proposed. The attachment of PrPc to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor may also be consistent with a role in cell-surface signaling or cell adhesion. Indeed, the 37-kD laminin receptor binds PrPc.

Because PrPc may act as a cell-surface receptor, we investigated whether signal transduction pathways coupled to PrPc after antibody-mediated cross-linking. Our experimental strategy relies on the neuronal differentiation model 1C11 (9). The 1C11 clone is a committed neuroectodermal progenitor with an epithelial morphology that lacks neuron-associated functions. Upon induction, 1C11 cells develop a neural-like morphology, acquire neuronal markers, and convert into either serotonergic cells or noradrenergic cells. The choice between the two differentiation pathways depends on the set of inducers used.

Within 4 days, serotonergic cells implement a complete serotonergic phenotype, including 5-HT synthesis, storage, transport, catabolism, as well as three functional serotonergic receptors of the 5-HT1B/D, 5-HT2B, and 5-HT2A subtypes. The noradrenergic phenotype of noradrenergic cells is complete within 12 days, with a progressive onset of catecholamine synthesis, storage, transport, catabolism, and an 1D-adrenoceptor. The differentiation events involve almost 100% of the cells and follow a well-characterized time course.

PrPc is constitutively expressed in the 1C11 progenitor and throughout differentiation (10). Thus, the 1C11 cell line offers the opportunity to study PrPc in relation to the onset of neuronal functions and within an integrated neuronal context. The effects of antibody-mediated ligation of PrPc at the cell surface of the 1C11 progenitor or its fully differentiated serotonergic and noradrenergic progenies were followed. PrPc cross-linking did not induce any specific phosphoinositide (PI) hydrolysis or nitric oxide production, nor did it promote the activation of p21ras or phospholipase A2 in the 1C11 cell line within 30 min of cross-linking (11).

In contrast, ligation of PrPc with specific antibodies induced a marked decrease in the phosphorylation level of the tyrosine kinase Fyn in both serotonergic and noradrenergic cells. The effect became measurable 10 min after antibody-mediated ligation. Similar decreases were obtained with two distinct antibodies to PrP (1A8 and SAF 61), but not with irrelevant antibodies directed against the membranous serotonin transporter. Fyn immunoprecipitates were subjected to immune complex kinase assay. As expected (12), the dephosphorylation of Fyn monitored in serotonergic and noradrenergic cells after PrPc cross-linking increased kinase activity. The activation of the Fyn kinase was abolished in the presence of competing Fab fragments of SAF61 antibodies.

In 1C11 precursor cells, the phosphorylation level of the tyrosine kinase Fyn was not sensitive to PrPc cross-linking. However, 1C11 cells contain similar amounts of PrPc as found in noradrenergic cells. Also, PrPc is equally well released by PI- specific phospholipase C (PI-PLC) from undifferentiated and differentiated cells. Moreover, the levels of Fyn expression, as assessed through immunoprecipitation, were roughly the same in the 1C11 clone and its differentiated progenies. Therefore, the signaling competence of the 1C11 cell line toward PrPc activation appeared to depend on the conversion of 1C11 cells into either serotonergic or noradrenergic cells.

Because Fyn is an intracellular protein but PrPc is anchored to the outer membrane, the PrPc-dependent signaling mechanism causing Fyn activation is likely to involve intermediate factor(s). To identify candidate proteins, we performed coimmunoprecipitation experiments with antibodies to PrP using metabolically labeled lysates of serotonergic or noradrenergic cells. In addition to heterogeneously glycosylated PrPc molecules, two bands of Mw 21 and 22 kD, respectively, were revealed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. When the samples receive a pre-treatment of enzymatic deglycosylation with N-glycosydase F (PNGase) , the monoglycosylated PrPc molecules (migrating to 28 to 30 kD) and the highly glycosylated isoforms (migrating as a band spanning 33 to 40 kD) all resolve into a single species of 25 kD. In contrast, p21 and p22 do not shift in molecular weight after PNGase treatment. Microsequence analysis allowed us to unambiguously identify p21 and p22 as caveolin-1 (cav-1) and caveolin-1 (cav-1), respectively. These two isoforms of caveolin-1 differ in their respective initiation sites only (13).

In the gel analysis of an immunoprecipitate from 1C11 undifferentiated cells, the bands corresponding to p21 and p22 were barely detectable. This observation could not be explained by a defect in caveolin-1 expression in 1C11 precursor cells because all cell types studied here express caveolin. The level of caveolin expression, as measured by Western blot, increased only faintly in differentiated cells compared with 1C11 cells. The capacity of PrPc to interact with caveolin-1 therefore appeared to depend on the differentiation of 1C11 cells toward either a serotonergic or a noradrenergic program.

To assess whether caveolin-1 takes part in the PrPc-mediated Fyn activation, we introduced antibodies to caveolin-1 to serotonergic or noradrenergic cells before PrPc cross-linking with the use of the cell bombardment technique. Immunosequestration of caveolin-1 in live cells blocked the PrPc-dependent activation of Fyn. Thus, caveolin-1 appears to be one of the protagonists involved in PrPc coupling to the tyrosine kinase Fyn.

Antibodies to clathrin, which were used as a control, were unable to cancel the Fyn response, although the amplitude of Fyn activation was somewhat reduced under these conditions. Such a partial reduction upon clathrin immunosequestration may reflect indirect interference with the caveolin-dependent signaling pathway through changes in membrane properties. However, it may also indicate a direct involvement of clathrin in the PrPc-mediated signal.

The difference in signaling competence of 1C11 versus serotonergic and noradrenergic cells prompted us to examine whether the response to PrPc cross-linking was related to the sequential acquisition of neurotransmitter-associated functions. PrPc cross-linking was applied at specific time points during each differentiation program. Antibody-mediated ligation of PrPc failed to induce any activation of the tyrosine kinase Fyn by day 2 of either program. Because neurite outgrowth and the onset of neuronal markers are seen as early as 1 day after either induction, simple engagement of 1C11 cells in a neural-like program was not sufficient to confer responsiveness to PrPc stimulation. Instead, not until day 4 of the serotonergic and day 12 of the noradrenergic differentiation programs were the signaling cascades triggered by PrPc cross-linking. Thus, full differentiation of 1C11 cells and the induction of a functional bioaminergic uptake are a prerequisite for responsiveness to PrPc activation.

Because the two identified partners of the cascade (Fyn and caveolin) are available in the cells from the beginning of differentiation, several hypotheses can be made. First, PrPc signaling may involve other yet-to-be-identified cellular partner(s) whose expression is strictly related to the differentiation stage of the cells. Second, the interaction of all cellular partners within a signaling complex may depend on the overall acquisition of neuronal and neurotransmitter-associated functions, specific to the ultimate stage of differentiation. Because caveolin-1 appears to interact with PrPc from day 2 of either the serotonergic or the noradrenergic program, it may be that the recruitment of Fyn to the PrPc-caveolin-1 complex occurs only at terminal stages of differentiation. Third, the onset of the overall functions of bioaminergic neurons may be a prerequisite to the proper structural organization of the signaling partners within subcellular compartments or microdomains.

Because of the neuronal polarity of serotonergic and noradrenergic cells, we performed cell fractionation to separate neurite extensions from cell bodies and we evaluated the relative contributions of either compartment to PrPc signaling. In serotonergic and noradrenergic cells, PrPc was abundant at the surface of cell bodies. Prion proteins were also clustered along the neurites. The fraction of PrPc located at the cell body only weakly contributed to the activation of Fyn upon antibody-mediated ligation. In contrast, cross-linking of neuritic PrPc induced a marked increase in Fyn kinase activity. The amplitude of the response was similar to that observed with total cell lysates (Fig. 3D). In serotonergic serotonergic and noradrenergic noradrenergic cells, the signaling activity of PrPc may essentially be attributable to those PrPc molecules located on neurites.

Thus, in the differentiating 1C11 cell system, coupling of PrPc to the tyrosine kinase Fyn is closely related to the maturation of the cells. Neurotransmitter-associated functions, as well as the structural morphology of the cells, appear to be involved. It is likely that the sequential onset of functional bioaminergic receptors and transporters is accompanied by a spatial organization of membrane components within specialized domains, possibly including cell-surface receptors, membrane adaptators, and signaling molecules. How could this PrPc-dependent signal functionally interact with other transduction pathways or contribute to cell homeostasis? Brief exposure of fully differentiated serotonergic or noradrenergic cells to antibodies to PrP does not induce any noticeable morphological change. PrPc is not required for the expression of a critical cell function. Instead, PrPc may be involved in the modulation of neuronal functions at the cellular level.

The identification of PrPc as a signaling molecule opens new directions for unraveling PrPc function. It also implies the existence of extracellular signals capable of triggering the activation of this protein and provides a foundation for uncovering such signals. In the context of prion infection, an important question to resolve is how PrPSc accumulation may interfere with the signaling activity of PrPc. The 1C11 cell system, which supports prion replication in vitro, may help to illuminate this issue.

Altered Intracellular Calcium Homeostasis in Cerebellar Granule Cells of Prion Protein-Deficient Mice.

J Neurochem 2000 Oct;75(4):1487-1492 
Herms JW, Korte S, Gall S, Schneider I, Dunker S, Kretzschmar HA  
Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium ([Ca(2+)](i)) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization.

Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.

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