VETERINARY TIMES Volume 33, number 2, 27th January 2003
TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Susan Haywood BVSc, PhD, MRCVS and David R. Brown M.Sc, Ph.D.
Discuss a re-evaluation of the TSE enigma and explore the role of environmental factors in prion diseases
Just over two decades ago the spongiform encephalopathies, as they were then known, were confined to a disparate group that included scrapie in sheep, the rare Creutzfeld-Jakob disease in man (CJD) and the even more exotic Kuru in a supposedly cannibalistic tribe in Papua, New Guinea.
All that changed in 1986 when Bovine Spongiform Encephalopathy (BSE) was identified in UK cattle.
Very soon after, the transmissible spongiform encephalopathies (TSE’s) or prion diseases came to include transmissible mink encephalopathy (TME), chronic wasting disease in mule deer (CWD), reports of TSE’s in zoo animals and felines and latterly variant vCJD in young people.
All these are progressively degenerative diseases of the central nervous system that prove ultimately fatal.
They are characterised by a long incubation period, failure to elicit an immune response and an aetiology which involves a hitherto unknown class of infectious agents of remarkable stability and persistence.
They have in common a pathology that, in addition to neuronal death and spongiform changes, includes the presence of amyloid plaques in the CNS.
The TSE’s have raised considerable public concern with respect to the unknown extent of the infection in the food chain, the possible transmissibility to humans and most particularly the relationship of BSE to vCJD.
Despite extensive research and an equally wide-ranging BSE Public Inquiry chaired by Lord Phillips (1), there is much that is unanswered or mainly speculative and it is time for a re-evaluation of the collated information, together with more recent investigations which have an important bearing on the pathogenesis on this unique class of diseases.
Scrapie, prion hypothesis and interraction with the host genome
Prior to 1986 most research centred on Scrapie. This disease has been endemic in UK for over 250 years and is found in most countries except Australia and New Zealand.
As all Vet students know scrapie is characterised by neuronal vacuolation, reactive astrogliosis and occasional plaque formation. The scrapie agent was deemed infectious in that it could be passaged to other hosts, but was biologically unique in its heat resistant properties, small size and apparent lack of nucleic acid.
As many as 22 strains have been isolated characterised by differing incubation periods in the natural host and lesion profiles produced in inoculated experimental mice.
Natural infection is by lateral spread and possibly maternal transmission, otherwise it can be transmitted by intracerebral inoculation to goats, mice and hamsters but not cats or mink.
The route of natural infection has been established as via the gastrointestinal tract to the lymphatic tissues (Peyers patches) then to spleen and thymus during which passage the scrapie agent replicates principally within the follicular dendritic cells of the lymphoid tissues.
The subsequent passage to the brain was thought to be via the ganglia of the autonomic system - a process taking many months.
The cause has been much disputed, but in 1982 Prusiner (2) showed that the scrapie agent was a small proteinaceous infectious particle, lacking nucleic acid, which he named a prion. He later demonstrated that prion protein PrPC is a component of the normal cell and encoded by the host genome.
The scrapie-associated prion protein PrPSc has a similar amino acid structure but an altered 3D configuration. This proposed conformational change conferred on it a resistance to protease digestion and the ability to convert normal PrPC to PrPSc, by a form of chain reaction.
The abnormal isoform accumulates in the brain in insoluble aggregates: a characteristic of prion diseases in general. Prusiners hypothesis has become known as the ‘protein only’ or prion hypothesis, of TSE disease causation.
Genetic factors, however, can affect susceptibility or otherwise to disease, and by 1986 it was understood that susceptibility to scrapie was controlled by a complex interaction between host genes and the particular strain of PrPSc .
The PrP (sinc) gene has been identified in sheep and found to have polymorphisms at codons 136,154 and 171 which are ‘disease linked’. In Suffolk sheep it has been found that valine (V), arginine (R) and glutamine (Q) or (VRQ) encoded at these sites are most susceptible to Scrapie, whilst alanine (A), arginine (R), arginine (R) or (ARR) are most resistant.
An ARQ associated genotype is also linked with susceptibility in the Suffolk, but not in the Cheviot, breed in which the same pattern confers resistance (3). The underlying rationale of these sometimes conflicting observations is unclear and indeed Phillips warns that “understanding of these polymorphisms is fundamental to efforts to breed scrapie resistant flocks”.
BSE not a form of scrapie
Identified in 1986, BSE rapidly spread to affect UK herds although the incidence was very limited within individual herds.
The source was assumed, on epidemiological grounds, to be from commercial feed which contained rendered animal protein. Certainly, cases dramatically declined subsequent to the time when the ‘ruminant-derived protein ban’ came into force in 1988, establishing this theory beyond reasonable doubt.
Initially, the infectious prion was thought to be a modified scrapie prion which had crossed species, but this was later dismissed by Phillips on the ground that BSE differed essentially from scrapie in disease-profile, incubation and transmissibility.
The report states with confidence that “the BSE agent is not an unmodified form of scrapie. Rather, it seems to be a novel TSE agent that arose from a prion mutation in cattle, sheep or another species in the 1970’s or earlier” (since zoo animals had contracted the disease ).
The report goes on to say: “The infectious agent is a post-translationally modified prion protein, a self replicating protein. Other as yet unknown factors may contribute to the development of BSE in infected animals”.
The source of the supposedly infected feed, however, was never identified and the disease never reproduced experimentally in this way. Also the limited nature of the infection, localised often to just one animal in a herd, was puzzling, the more especially since this could not be explained by host predisposition, as with sheep to scrapie, since no variant polymorphisms have been identified in cattle.
The explanation rests on the unsatisfactory ‘packet theory’ of infection whereby single high titre doses were unevenly dispersed in the feed; but this has never been confirmed and is at odds with the known high infectivity of PrPSc affected tissues.
Once again, and referred to by Phillips, other environmental factors may play an aetiological role.
Link between BSE, and vCJD remains to be confirmed
Human prion disease, including CJD, had been recognised by this time as falling into 3 categories: sporadic (85%), familial (<15%) as a result of a point mutation in PrP gene and iatrogenic (<1% due to medical introduction as a result of vaccines and so on).
Epidemiological studies from around the world had moreover failed to identify a causal link between scrapie and CJD. With the emergence of BSE a CJD Surveillance Unit was established with the remit of studying cases of CJD that could have been linked to BSE.
In 1995, two cases of CJD were reported in young people. By March 1996 that had risen to 10 cases. Neuropathological findings revealed the presence of large amyloid plaques in the brains of these unfortunate people, more akin to Kuru than sporadic CJD, and which was now renamed variant CJD .
Several studies showed similarities between BSE, vCJD and TSE’s in zoo animals and felines. Another study showed that in transgenic mice in which the host PrP gene was replaced by that of human PrP gene (effectively bypassing the species barrier), when challenged with either BSE or vCJD it showed similar patterns of disease distinct from sporadic or iatrogenic CJD (4) .
This is supportive evidence to the effect that BSE and vCJD are caused by a similar strain of prion but does not conclude that vCJD is caused by BSE, as Phillips implies.
Circumstantial evidence linking the consumption of beefburgers by young people in support of the transinfection theory, whilst persuasive, has never been proven, in that the putative ‘infectious’ burgers have never been identified, nor indeed fed, to experimental animals.
Groups supposedly more at risk such as farmers, vets, abattoir workers and butchers have not shown an increased risk of vCJD.
It is quite surprising that the one experiment that would confirm a link between BSE and vCJD has not been carried out. If BSE and vCJD are the same strain of disease and take on different characteristics dependent on the host organism, then infecting cows with vCJD should lead to the cows developing BSE.
This would prove BSE and vCJD to be the same disease. However, those who could have carried out the experiments have classed them as “unethical” because of the need to inject human brain into an animal.
It is incontrovertible that, the experiments that are the main support for the hypothesis that vCJD and BSE are the same disease also require that vCJD be injected into the brain of the putative source ‘host’ animal.
Until this is performed, hypotheses of the causal relationship of BSE and other TSE’s, including vCJD, remain conjectural and the role of other, possibly environmental, factors must be reconsidered.
The prion ‘protein only’ hypothesis may require modification
The prion hypothesis suggests that an abnormal isoform of the prion protein PrPSc alone causes the TSEs. This hypothesis still remains controversial because of the lack of formal proof. In the UK in particular, many scientists still don’t accept that this protein is the sole cause of the disease.
Indeed ‘manufactured’ PrPSc failed to induce prion disease in mice (5). It is clear, however, that conversion of the normal brain PrPC to the disease specific PrPSc is necessary for the establishment of the characteristic neuropathology of the TSEs.
PrP is a copper-binding protein with a role in copper metabolism
On the contrary it is almost universally accepted that the normal protein, PrPc is a metal binding protein.
PrPc was first suggested to be a copper binding protein in the early 1990’s and confirmed by David Brown and colleagues in 1997(6) This has been followed by close to a 100 publications that have reafffirmed that PrPc is a copper binding protein.
Further work by Brown and others showed that PrPc displays SOD-like activity and indeed, it has been proposed that neurodegeneration in prion disease is a direct consequence of a failure of neuronal antioxidant activity. PrPc has a role in cellular copper transport and the sequestration of copper (6).
Early work proposed a link between copper metabolism and prion disease in that the copper chelator cuprizone causes neuropathological changes in rodents similar to that seen in experimental scrapie. In this context it is interesting that the abnormal isoform, PrPSc, is almost devoid of copper and SOD-like activity is severely reduced.
PrPSc binds manganese in place of copper
A number of researchers have shown that the isoform of the protein associates with other metals. Brown and co-workers have shown that manganese can replace copper in native PrPC to create the isoform PrPSc,. which is protease resistant and lacks antioxidant function, (although there is no evidence that it is infectious).
Most particularly, manganese has been shown to be associated with PrPSc in the brains of both human CJD patients (7) and mice inoculated with scrapie (8).
There is additional evidence that there are changes in metal metabolism in the brain with a loss of brain copper (but an increase in the liver) coincident with the increase of brain manganese in the CJD patients and the scrapie infected mice.
Additionally, manganese elevations in rodent scrapie are both presymptomatic and systemic. The cause of this remains unclear, but it is currently being investigated as to whether elevated blood manganese can be used as a diagnostic test for TSEs.
All in all, there is a solid and expanding amount of literature showing that metal imbalance and TSEs are linked.
TSE’s may be an environmental/industrial disease
Coming at these diseases from another direction, Mark Purdey, a farmer from Somerset, has published evidence that hotspots of TSEs exist in regions of the world where there is environmental imbalance between copper and manganese.
Farms in Iceland prone to scrapie have soils with dramatically increased manganese levels. A similar situation exists in Colorado where deer develop chronic wasting disease (9).
These findings led both Mark Purdey and David Brown to hypothesise that sporadic TSEs might be a result of animals becoming exposed to conditions where manganese in their diets is elevated and copper is deficient.
Manganese and copper are ubiquitous metals and it is hard to imagine how the changes in these metals might initiate such diseases. Since the mid 20th Century, however, industrialisation has been fairly intensive, especially in the ferromanganese industry and considerable amounts of manganese are present in pollution.
Indeed lead fuel replacements also use manganese. In Slovakia, there are high levels of both inherited and sporadic forms of CJD around areas of intense ferromanganese industry. Therefore there is the potential for new disease to arise as a result of intense industrialisation.
One issue with BSE and vCJD remains unanswered: why such a high incidence in the UK? One possibility is that the UK is just the first, and others will follow. BSE that was once thought to be specific to UK is now Europe wide.
In the UK, the majority of BSE developed as a result of the feeding of offal from BSE-infected animals back to other animals. This unfortunately has masked the possibility of tracing the origins of BSE back to any source.
Similarly, the mobility of humans around the UK also makes it difficult to trace vCJD back to any factor, especially not one that might be ‘environmental’.
Although the BSE Inquiry clearly stated that alternative possibilities for the origin of BSE should be investigated and given support, there has been little support forthcoming, despite the fact that most of the evidence linking TSEs and manganese has appeared since that time.
This is in part as a result of the subsequent Horn report “Review of the origin of BSE” (10). This report failed to note that comparing maps of BSE incidence to a map of manganese hotspots across the UK when the epidemic was well established was inappropriate, since BSE was clearly spread at this stage by recycling infected offal.
A more detailed analysis, looking at the location of the very first cases of where BSE were reported (as viewed on the DEFRA web page,) and the map provided in the Horn report, indicates that the original BSE farms lie directly in a manganese hot spot!
Others, however, have not allowed themselves to be side tracked in this way but concentrated on the scientific evidence. In particular, authorities in the Environmental Protection Agency in Colorado have begun investigating the link between manganese, copper and chronic wasting disease incidence in deer.
This disease was originally thought to be a copper deficiency disease before the prion hypothesis came to be recognised and CWD was recognised as a TSE.
Although manganese might not be ‘the cause’ it is clear from the biochemical studies that have been carried out that metals do play a role in the pathogenesis of TSEs. Therefore, even if manganese is just a risk factor, it is important that this factor be kept in the equation, because it might just be the key that unlocks the truth about these diseases.
Finally, the most tantalising question remains: how does the PrPSc isoform propagate and, even more so, become infectious? It has been shown that the manganese-bound isoform PrPSc has a configuration that makes it protease resistant and that this particular conformation tends to form spontaneous amyloid aggregates.
It is suggested that when this self-propagation reaches an optimum size, it recruits normal cellular PrPC expression to convert to PrPSc which then replicates uncontrollably: a sort of hijacking of the synthetic machinery which then operates in a runaway fashion, free of normal checks and balances.
It is interesting that metalloprotein chemistry of this nature (but involving metals other than manganese) is now being implicated for the analogous plaque formation in Alzheimer’s and certain other neurodegenerative conditions. If so, then the prion diseases are not a biological freak of nature but a subclass of a category of diseases relating to dysregulation of metalloprotein chemistry associated with metal imbalance.
(1) The BSE Inquiry Report: (2000).
(2) Prusiner S.(1982) Novel Infectious agents Cause Scrapie. Science 216,136-144
(3) Hunter N, Foster J, Goldman W, Stern M, Hope J & Bostock C.(1996) Natural Scrapie in a Closed flock of Cheviot Sheep occurs only in specific PrP genotypes. Archives of Virology 141:809-824
(4) Hill AF, Debruslais M, Joiner S, Sidle KC, Gowand I, et al (1997) The same prion strain causes vCJD and BSE. Nature, 389:448-50
(5) Hill AF, Antoniou M, Collinge(1999) J.Protease resistant prion protein produced in vitro lacks detectable infectivity. J Gen Virol. 80:11-14.
(6) Brown D R (2001) Copper and prion disease. Brain Research Bulletin. 55 (2): 165-173.
(7) Boon-Seng Wong, Shu G Chen, Colucci M, Xie Z, Pan T, Liu T Li R Gambetti P, Sy M-S & Brown D R. (2001) Aberrant metal binding by prion protein in human prion disease. Journal of Neurochemistry, 7:1,400-1,408.
(8) Thackrey A M, Knight R, Haswell S J, Bujdoso R & Brown D R (2002) Metal imbalance and compromised antioxidant function are early changes in prion disease. Biochem J. 362: 253-258.
(9) Purdey M. (2000) Ecosystems supporting clusters of sporadic TSE’s demonstrate excesses of the radical generating divalent cation manganese and deficiencies of antioxidant co-factors; Cu, Se, Fe, Zn. Does a foreign cation substitution at ar PrP’s Cu domain initiate TSE? Med. Hypoth. 54:278-306.
(10) Horn, G. (2001) Review of the Origin of BSE.
Dr Susan Haywood is a Senior Fellow in the Department of Veterinary Pathology, University of Liverpool with a Wellcome Trust-funded research project into copper pathobiology and proteomics in sheep.
Dr David Brown is a Lecturer in biochemistry at the Department of Biology and Biochemistry, Bath University and BBSRC Senior Fellow who has been researching prion diseases for the last ten years. He has a distinguished international reputation in the prion disease field and is invited to lecture on the subject around the world.
He is also the editor of a forthcoming book on the subject “Prion Diseases and Copper Metabolism” to be published soon by Horwood Press.