|
||||||||||||||||||
 |
From: TSS ()
A field on fire The biochemistry of mad cows Prion diseases are neurodegenerative diseases1 that have been linked together because they may potentially have the same cause.These include the diseases scrapie of sheep and BSE (bovine spongiform encephalopathy) of cattle, and also several human diseases that include sporadic CJD (Creutzfeldt-Jakob) disease and a variety of inherited forms.The inherited forms of prion diseases are linked to mutations within the gene for the prion protein.Around 85% of all human cases of prion disease are sporadic CJD, which is a disease affecting people of around 60 years of age. The cause of this disease remains unknown. Unfortunately, the name of this disease causes some confusion, as it is similar to vCJD (variant CJD), a related disease of much younger people. David R. Brown (Bath, UK) The Biochemist — August 2005. © 2005 Biochemical Society 6 vCJD first emerged in the mid-1990s and has been linked to the BSE epidemic that reached its peak a few years earlier. It is now largely accepted that BSE is the cause of vCJD. The number of deaths from vCJD stands at 150 and the number of deaths in 2004 was the lowest since the disease was first diagnosed 10 years ago. The BSE epidemic arose as a result of the feeding of rendered animal remains to dairy cattle. As cattle were consuming BSEcontaminated meat, the number of cases escalated rapidly to reach a maximum of around 32 000 in one year. Despite the bans that came into force to prevent the feeding of animal remains to cattle, BSE persists, although the numbers are much lower. BSE occurs in many countries outside of the UK, ranging across Europe and including Japan and North America. Currently there is no clear explanation for the persistence of BSE. Given the reduction of vCJD cases, many researchers feel that this disease is disappearing along with BSE. Others researchers do not necessarily agree. The link between BSE and vCJD is not fully proven and there is a view held that vCJD and BSE are related diseases but not causally linked. The most widely accepted alternative hypothesis for the cause of vCJD (and the low levels of BSE seen in many countries) is the effect of an environmental factor, possibly altered trace-element absorption. However, at present this remains only a hypothesis. Research into prion diseases has always been controversial. Stanley Prusiner did much to advance the protein-only hypothesis, i.e that the abnormal isoform of the PrP (prion protein) can be transferred between individuals and initiate prion disease. Despite its initially contentious nature, this theory has largely been accepted, and Stanley Prusiner won the Nobel Prize for his work in 1997. One of the strongest pieces of evidence was the finding from Charles Weissmann’s group that PrP-knockout mice are resistant to mouse-passaged scrapie2. Again, many researchers still don’t accept that only abnormal PrP is required for the prion disease. Recently, Prusiner’s group published a paper in Science in which recombinant PrP in a fibrillar form was introduced into transgenic mice and caused a prion-like disease3. This should have been the final proof, but the argument rages on because the mice were ‘transgenic’. The most widely confirmed finding regarding the biochemistry of prion proteins in recent years is the discovery that the PrP binds metals. More than 50 research groups have now confirmed that PrP is a copperbinding protein4. Links between TSEs (transmissible spongiform encephalopathies) and copper date back to the 1960s, but the first biochemical evidence came from two papers looking at copper binding to 1997 in Nature, initiated the upsurge in interest in this idea, as it showed that Cu binding to PrP could have effects in vivo5. Recent work on this aspect is reviewed in the accompanying article by John Viles. The metalbinding capacity of PrP has recently been used to extract large amounts of PrP from the brains of cows. This has allowed the first NMR structural analysis of native PrP. In contrast to the expanding role of metals in prion research, other directions in the field have often been faddish, with new ideas being taken up and hotly explored for a number of years and then largely abandoned. In 1999, a protein homologue of PrP was discovered and termed doppel. It was thought that this protein might play a key role in disease progress, but now this has largely been dismissed. Similarly, transmembrane forms of PrP were identified in in vitro translation systems and it was suggested these might occur in vivo. Again, this lead has failed to provide any real insight and the finding has largely been dismissed. Central to understanding how the disease process works is determining the mechanism of protein conversion from the cellular form to the diseasespecific form. It has been known for some time that the disease-specific form is highly resistant to proteases, can polymerize to form either fibrils or complex aggregates and is high in B-sheet content. Much work has focused on creating in vitro methods for generating the abnormal form of PrP, from either recombinant or native sources6. Many assays that involve the use of recombinant protein also use high concentrations of denaturants, such as 2 M guanidine, and although producing elegant models of fibril formation, they are highly artificial and are unlikely to reflect the normal mechanism of PrP polymerization. Thus new methods using native conditions have been generated. In particular, refolding PrP with Mn can generate PrP aggregates able to form the nucleus of PrP polymerization. Using native PrP, Claudio Soto’s group have generated a polymerization technique that involves the addition of PrPSc extracted from brain and sonication7. This technique vastly amplifies the amount of protease-resistant PrP present in the samples. Promising studies have suggested that this technique could also amplify trace amounts of PrPSc in blood. Transgenic mice have been used as a powerful tool in prion disease research. In particular, mice expressing either bovine or human PrP genes without background mouse PrP were used to verify the link between BSE and vCJD. In addition, PrP knockout mice have been used to investigate the cellular relevance of PrP expression. In more recent years, transgenic approaches have been used in an attempt to understand the pathway of peripheral infection in prion disease and, in particular, the role of the immune system8. Lymphoid tissues can also accumulate infectious prions and, in particular, follicular dendritic cells of the spleen are crucial to the initial amplification of prions when they first enter the body. Following this initial step, prions progress into the central nervous system in mice via the sympathetic innervation. Other studies have suggested that complement components in mice are involved in the progression of prions towards the brain through the immune system. It has also recently been shown that, in mice, chronic inflammatory conditions in the periphery alter the distribution of prions in peripheral tissues, and this is dependent on expression of lymphotoxin-. There is, however, a slight problem with such elaborate schemes. Mice do not normally contract a prion disease and the immune systems of humans and mice may have subtle differences that would make the findings in mice irrelevant to disease progression in humans. In such a short article it is not possible to summarize fully the extraordinary extent to which PrP and prion diseases are being studied in terms of basic biochemistry and cell biology. Important advances have been made into the mechanisms of cellular internalization of the protein, factors that regulate its expression, the structural content (and how the structure changes with metal binding), potential binding partners for the protein, and the protein’s function. The evidence that the prion protein normally acts as an antioxidant continues to mount, but there are other theories that are also important. In particular, PrP expression modifies intracellular signaltransduction pathways and cellular adhesion. In the study of prion diseases, a major concern for biochemists is how the conversion to an abnormal isoform, rich in B-sheets, makes the protein more resistant to processes that should degrade it. The resistance of abnormal PrP to degradation by heat or other harsh conditions is controversial (biochemically) because the data suggest that the bonds in the protein cannot be hydrolysed in the same ways as the bonds in other proteins. As some of the findings proposed are chemically impossible, then the supposed super-resistance of this protein to extreme conditions needs to be reinvestigated. However, interactions with metal surfaces have been found to increase the potential infectivity of the protein, making ways to detect the protein on metal surfaces and to clean surgical instruments a high priority. A developing field of interest is the possible link between the cause of sporadic TSEs and potential risk factors in the environment. This has led to a major programme of research, funded by the European Commission under the title ‘FatePride’. The link between trace elements in the environment and scrapie is a central interest of this project. This was initiated by a finding that manganese substitution in the prion protein metal-binding domain can cause protein conversion to an abnormal isoform. This project requires the comparison of highly accurate geochemical maps that indicate the level of bioavailable metals with regional levels of TSEs, such as scrapie. Unfortunately, this project has been greatly inhibited by officials of DEFRA selectively withholding data on the location of scrapie-infected farms from the geochemists involved in the project. Investigation of the cell-death mechanism of prion disease follows a circular path. In 1994, it was first shown with cell culture experiments that expression of native PrP by cells was necessary for the neurotoxicity of the toxic form of PrP. In 1996, studies with transplantation of brain tissue into transgenic mice showed that PrPSc generated in the brain could not kill neurons lacking cellular PrP. Recently, conditional PrP-knockout mice were developed. When expression of PrP was switched off during the time-course of the disease, production of abnormal PrP halted, and the mice recovered, showing no further signs of neuronal cell death9. It is now 10 years since the panic created by the first cases of vCJD were identified. Has work in this field really advanced? The major advances have been in terms of cell biology and biochemistry, particularly related to the normal activity of the PrP. There has also been a great improvement in the diagnostic tools used to verify that a demented person has vCJD and not some other neurodegenerative disorder, but this diagnosis still only occurs after the patient is beyond help. It is perhaps fortunate that there has been no massive epidemic of vCJD, as the field has not advanced quickly enough to deal with such an epidemic if it emerged. The research in this field is plagued, more than in many other fields, by obsessive rivalry, dirty politics, miscommunication, the withholding of data, poor collaboration and bullying of young researchers. Perhaps much of this is driven by the fear of research funding disappearing faster than new cases of vCJD appear, but it is not helped by the current trend among the science funders to divert funding away from basic research and into diagnosis and therapy. Although these tools will ultimately be needed10, breaking the back of successful basic research on the prion protein will not assist anyone. As far as we know, the true vCJD epidemic could be seen in people of the same age as most CJD patients, placing the true epidemic 20 years in the future. Figure 3. PrP is expressed on the surface of cells as a glycosylphosphatidylinositol-anchored protein. Confocal images of cells expressing green-fluorescentprotein- labelled PrP are shown. References 1. Brown, D.R. (2005) Neurodegeneration and Prion Disease, Springer, New York 2. Weissmann, C. (2004) Nat. Rev. Microbiol. 2, 861–871 3. Legname, G., Baskakov, I.V., Nguyen et al. (2004) Science 305, 673–676 4. Brown, D.R. (2002) Prion Disease and Copper Metabolism, Horwood Publishing, Chichester 5. Brown, D.R., Kefeng, Q., Herms, J.W. et al. Nature (London) 390, 684–687 6. May, B.C., Govaerts, C., Prusiner, S.B. and Cohen, F.E. (2004) Trends Biochem. Sci. 29, 162–165 7. Soto, C., Saborio, G. P. and Anderes, L. (2002) Trends Neurosci. 25, 390–394 8. Aguzzi, A. and Sigurdson, C.J. (2004) Nat. Rev., 4, 725–735 9. Mallucci, G., Dickinson, A., Lineham, J. et al. (2003) Science 302, 871–4 10. Mallucci, G. and Collinge J. (2005) Nat. Rev. 6, 23–34 The Biochemist — August 2005. © 2005 Biochemical Society 8 David Brown was born in Australia, where he completed his university training at Sydney University, graduating in 1990 with a Ph.D. in neuroscience. In 1993 he left Australia and subsequently worked in the US, Germany and the UK. He has worked on prion diseases for the last 12 years. In 2001 he established a laboratory at the University of Bath, where he is currently Reader in Biochemistry. David Brown is a member of the Spongiform Encephalopathy Advisory Committee. bssdrb@bath.ac.uk FULL TEXT WITH GRAPHICAL CHARTS; http://www.biochemist.org/bio/02704/0006/027040006.pdf TSS
|
