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I would like to take an opportunity to review some of the prion-related disorders now popularized in the lay press, which include the so-called mad cow disease. I have several reasons for studying these disorders. First, the prion disorders were initially thought to be exclusively veterinary, i.e. scrapie in sheep. Subsequently the disorders have been described with some fanfare in cannibals from New Guinea in the form of Kuru and in the sporadic form of Creutzfeld Jacob Disease (CJD). The world will soon see many cases of the latter disorder appearing in people. Second, I believe that studying Mad Cow disease will offer an opportunity to study basic biochemical mechanisms that have far reaching implications. This disorder is really one of protein conformation and an unrelated, but somewhat similar, disorder in Alzheimer’s disease. Information on the protein chemistry and control and mechanisms involved with this family of diseases will offer a likelihood for therapeutic intervention. Third, the protein catabolism associated with these diseases involves the newly described Ubiquitin system, knowledge of which will have tremendous impact on such broad-ranging topics as apoptosis (cell death), as well as future modalities for cancer therapy.
The history of prion disorders must start with scrapie, described over 200 years ago. Sheep affected with this disorder tend to scrape themselves repeatedly on poles or logs, thus rubbing the fur down to bare bone. This interesting disorder had no treatment and involved animals were often simply destroyed. In the 1930’s and 1940’s, an interesting subpopulation of Libyan Jews were studied and found to have a 30-fold increased risk for Creutzfeld Jacob Disease (CJD). Subsequent studies in this population show that they have an amino acid variation in the 200th amino acid codon involving the described prion. This group of people had an unusual dietary delicacy of sheep brains and sheep eyes. Review of this information may eventually show that this 30-fold increase in CJD was environmental as well as genetic. The Fore Tribe of New Guinea represents a unique population of people who have added much to the story. This group of people have, as a tribe, lost over 2,500 individuals to the disorder Kuru, mostly women and children. To gather information on the unusual characteristics of this disorder, Gadjusek traveled to New Guinea in the 1950’s and 1960’s to obtain specimens and do environmental evaluation. Subsequent research done at the NIH resulted in brain tissue being injected in chimpanzees, and transmission of Kuru-like disease to the chimpanzee. Review of the environmental risk revealed that women and children would honor their dead by eating brain tissue at the burial ceremony. In addition, these fiercely warrior-like people would treat the vanquished tribes in a similar fashion. The male warriors almost exclusively imbibed the skeletal muscle. It is felt this would account for the unique distribution of the disorder in younger people and women. This information evolved in the setting of the observation that scrapie was remarkably similar to Kuru in biologic activity and disease manifestation. The term “slow virus” was actually coined regarding scrapie in 1954, when researchers tried to explain the slow nature of presentation of the disorder. Certainly sporadic cases of Creutzfeld Jacob Disease (CJD) were also associated with observation of a similar disorder, Gerstmann-Straussler-Scheinker Disease. An additional similar disorder turned out to be fatal familial insomnia (FFI). The latter disease is particularly interesting, since it involves deposition of prions in a specific region of the thalamus, leading to inability to sleep. Not surprisingly, those affected wear out and die. There was also a sudden interest in CJD, when transmissibility of the disorder was identified in the mid 1980’s. The best-known example is that involving human growth hormone, HGH. In 1985, the first reports of CJD and HGH recipients were made. These recipients dated from 1963-1980 and had been exposed to batch lots of human pituitary extracts. Up to 20,000 pituitaries were batch-lotted to purify HGH during that earlier time frame. As a latent finding, these patients had a relatively high frequency of CJD development, and in fact at least 20 recipients have died in the United States and 84 worldwide by the most recent numbers of 1999. A larger crisis probably was averted by the introduction of recombinant DNA human growth hormone in 1986 – thus eliminating the need for human source in production. Other examples of CJD development have occurred, including the use of corneal transplants, neurosurgical tools, electrodes, and dura patches. Much of this remained as a medical anomaly until the mechanism was elucidated.
“There is one particular thing that is nothing whatsoever in any way, shape, form like any other…”
– Nothing Like a Dame, from South Pacific
by Oscar Hammerstein.
Many of the answers to these prion disorders turned out to come from the pioneering work of Stanley Prusiner, a research neuroscientist now with UCSF. His work in defining the prion hypothesis – he even was able to name the problem – is a remarkable example of convergent medical technology. He became interested in mechanisms of neurologic disease when he cared for a patient dying with Creutzfeld Jacob Disease. In 1972, beginning a residency in Neurology, he was quite impressed at the rapid clinical neurologic deterioration of a patient while the body itself remained unaffected. The lack of a febrile response, white blood cell or pleocytosis or immune response, seemed quite peculiar since he had been told the patient was dying of a “slow virus”. The interest in CJD quickly shifted to scrapie, when the unusual radiobiological data of Alper became known on the scrapie agent.
Prusiner set off on the task of bioassays and preparations, trying to purify the infectious agent of scrapie. He faced tremendous logistic difficulties. After several years, he determined that the scrapie agent was not a virus at all. As the infectious material was known to be sensitive to proteinases, Dr. Prusiner coined the term “prion” as a contraction of “proteinaceous” and “infectious”. Once the material was purified, the terminus of the amino acid sequence was determined. This allowed for molecular cloning. DNA probes determined the amino acid sequence of the protein. Results showed that a single protein of 27,000 molecular weight is responsible for the transmission of scrapie. Furthermore, a post-translational change is responsible for the pathologic nature of the protein. That is to say, the amino acid sequence does not change in disease; rather, it is the protein conformation that is altered. This was therefore a unique finding in that an infectious agent was a protein, and that the mechanism of disease was a conformational change. This represented a paradigm change in approach to this combined genetic and infectious disease. Subsequent studies using highly sophisticated techniques have shown that the prion protein in normal format has a dominant alpha-helix structure. The prion protein, when it becomes abnormal, becomes a more dominant beta-pleated structure. The latter beta-pleated structure would also explain the unusual dichroism that is seen in the brain deposited material in animals affected with scrapie. (This is the same kind of beta-pleated sheet, by the way, that is involved in other disorders of protein conformation, which include Alzheimer’s and amyloidosis.) The standard tools of molecular biology were utilized to make so-called “knock-out” mice. These mice are not able to make prion protein, but can make all other components of the genome. A true knock-out mouse cannot be given scrapie. This seems to show that the infectious isoform is necessary, as is the endogenous normal protein, for the change of a prion disorder to evolve. It subsequently turns out that the basic amino acid sequence of the proposed host will play a definite role in susceptibility to prion disorders. This has been shown for the Libyan Jews, Kuru tribe people, and even in mad cow disease victims. In fact, in the variant CJD patients, it has been found that they all have an amino acid substitution that involves a methionine in the prion, and the pairing of a methionine containing prion in a specific location with the mother and father (homozygous) is required for the disease to take hold. Those who has a methionine mixed with the valine or just valine in both prions are seemingly resistant to mad cow. Subsequent work, however, has not completely excluded that there may be just an increased time lag in getting the variant CJD. Amino acid sequencing of the prion, as well as other proteins (such as apolipoprotein E), may play a role in susceptibility as well as the time for incubation before the prion protein can subsequently manifest. We are too new into the problems of variant CJD to assume that those who do not have the methionine/methionine prion are the only folks who will be susceptible to mad cow disease. Additionally, the species barrier – initially felt to protect the human population from the veterinary disease – probably is related to concurrent amino acid sequencing in the prion as well as the glycolization of the prion protein and/or other structures. In the first pass of a species jump, one can historically note a prolonged time lag before the disease will actually show up in the new species.
Having discovered that the prion problem represents a conformational issue, then Prusiner and his workers have begun the challenging task of identifying at what level prevention and therapeutics may play a role. Certainly the reaction of a normal prion to an abnormal prion involving just conformational change may involve opportunity to stabilize the prion protein or destabilize the variant prion protein.
This entire process is said to require various chaperone proteins, and modulation of these agents has not been well discussed in my literature review to date. I think it is particularly of note that the variant prion protein associated with these disorders is quite resistant to protease activity. Again, the concept arises that the ubiquitin system, which is critically involved with protease activation and basic housecleaning in the cell proteins, may be at some risk in the prion related diseases. The technical aspects of ubiquitin metabolism does require several unique steps, highlighting which proteins are to be submitted to the standard proteases in the cell and thus purged from the cellular environment. Ubiquitin, as it attaches to the protein, requires a specific Ring (“Really Interesting New Gene”) lock-and-key region. One would wonder if conformational changes in the alpha-helix to beta-pleated sheep would act as a mechanism, thus covering up the ubiquitinization site. Ubiquitin interaction with other proteins, i.e. Alzheimer’s disease and amyloidosis, probably play a similar role in allowing these proteins to accumulate and have remarkably long biological half-lives. My own opinion is that these disorders of ubiquitin function will turn out to be remarkably important in explaining the resistance of variant prion proteins to proteases.
I would now like to turn our attention to the problem of mad cow disease. This is in the family of transmissible spongiform encephalopathies (TSE). These disorders have involved multiple animals and include sheep, goats, mule, deer, elk, mink, domestic cats, zoo species, cattle, and man. There are multiple levels in which mad cow disease – bovine spongiform encephalopathy (BSE) – can be addressed. This seems more than just bad luck. In 1979 Margaret Thatcher, a conservative, was elected and soon thereafter decided that the meat industry in England should be responsible for its own standards in the rendering of carcasses from bovine source. To improve fat as well as improve reclaimed protein, standards were changed. Lower heat standards as well as less utilization of solvents (hexane) resulted in increased delivery of reclaimed protein. Around this same time frame, I believe that bone meal mixtures with this material were found to be a relatively inexpensive and easily produced protein source in feeds for various animals. In fact, the scrapie infected sheep were probably involved in this rendering process change and may have been introduced into the food chain at that point. Other potentially important steps include the increased mechanization of the killing line associated with the intensive harvest of bovine material. The so-called captive bolt gun was utilized in the same time frame to instill an air embolism into the brain of the cow in question, leading up to its subsequent harvesting for slaughter. This air embolism, however, shattered brain tissue and spread neurologic tissue throughout the vasculature, since the heart would keep pumping for a number of minutes and potentially spread the neurologic tissue into the skeletal muscles. Additionally, the increased mechanization also included a technique involving splitting the skull and specialized claw devices for harvesting eye and brain, as well as mechanized vertebral removal. The latter materials were added to the offal, which can be used as foodstuffs and still carry the “all beef” label.
These changes in the handling of intensively harvested meat products are temporally related to the first reported cases of “Mad Cow Disease” in the UK in 1986. Mad Cow, of course, is a metaphor for bovine spongiform encephalopathy (BSE). This early description in 1986 caused some consternation primarily in the veterinary field, but soon thereafter in 1987 it was postulated that this problem may be related to the use of bone meal as a feed source for ruminants. In 1988, the UK required that animals showing evidence of BSE be treated as a notifiable disease. Bone meal products were banned from feed to other ruminants in England. Additionally, a slaughter and compensation program was instituted. This program, however, only gave 50% of the value for animals found with BSE and 100% if an animal was slaughtered and found to be negative for BSE. This latter differential may have resulted in farmers having motivation to avoid turning in BSE contaminated stock. Also in 1988, BSE was found to be transmissible to mice in experimental models. Interestingly, in 1989, a group of scientists were gathered in the UK to act as BSE advisory scientists. At this meeting, recollections from Dr. Laura Manuelidis of the Yale School of Medicine are interesting. At that time, she states that the ruling British bureaucrats who managed the meeting of Ministry of Agriculture, Food and Fish (MAFF) were resistant to “unwelcome scientific advice about an epidemic spread of BSE worldwide, and especially about undeniable possibility of transmission of BSE agent to humans”. Human involvement, in fact, was “dismissed”. She states that the process of not initiating a kill of “healthy” cows, as well as a ban of use of offal in foods, represented halfhearted efforts based on economic rather than scientific data. She speculates that the payment to farmers for sick or almost sick cows at half the market price would encourage farmers to rapidly push suspect cows to market for full price. These commercial interests dominated the meetings and the feeling that a “species barrier” would protect the human population apparently dominated the final conclusions from the meeting. Additionally, the entire meeting was dominated by restrictions that findings and results from the meeting were to be kept in secrecy. After that meeting, in which more than 1,000 farms had been revealed to have infection with BSE in the UK, Dr. Manuelidis remained particularly concerned to prevent further problems that might spread to the USA. Though she was sworn to secrecy, she did feel “obligated as a physician to inform the US Department of Agriculture (USDA) upon my return to the United States, and strongly urged my USDA contact to ban further imports of feed”. The USDA acted promptly and instituted a ban of UK cattle to the USA, as well as UK feed products including bone meal. Until 1989, quite a bit of bone meal animal feed had been imported into the United States. The economics of this export process had been quite dominant and the cattle feed business, with exportation of bone meal product, had accounted for many thousands of tons of material sent all over the world – including to the United States. Given the latency of the disorder, this information might be quite revealing, especially if the areas of importation were reviewed and true prion screening programs were instituted in potentially involved cattle. Certainly in 1989, the MAFF were deeply involved with controlling all studies as well as specimens.
Scientists wishing to study the problem were restricted in access to contaminated brain tissue, and it was often stated that this is “just a cattle scrapie” as well as a veterinarian problem, not a medical problem. In that same time frame of 1989, Sir Richard Southwood in the Department of Health for the UK stated, “this is not a human problem”. The remarkable episode in 1989 of the Agriculture Minister and his daughter eating a burger on television to reassure the populace has been replayed in the minds of the British public. By 1990, it was also found that other animals were becoming involved with apparent BSE. Five antelopes were found in that year to have a problem, and “Max” the cat developed BSE to the great consternation of the pet-loving public of Britain. Soon thereafter, slaughtering compensation programs switched to 100% compensation for any BSE involved animal. This was also the first year for documentation of laboratory controlled BSE transfer to pigs. The presence of BSE in a pet in 1990 rapidly resulted in a ban of bovine offal from pet food. The economics of the problem were hitting the British farm industry, with a 40% drop in beef product purchases. By 1992, 1,000 cows per week were developing BSE on a clinical basis and needing to be destroyed. Interestingly, the European Union has consistently had a time lag in its handling of the BSE problem. In 1994, the EU prohibited mammalian protein feed to ruminants. Increased concern that the species barrier could be broken and involving humans led to published concerns in 1994 and 1995 by Dr. Manuelidis, that this problem could be transmitted to people. Additional concern that BSE contamination in milk, gelatin, amino acid preparations, and plasma as well as blood products were initially being raised in 1995. The agriculture and veterinary reassurances were still forthcoming when in December of 1995, the British Agriculture Minister stated, “I am absolutely certain that British beef is wholly safe”. However, the beef hit the fan – in March of 1996 – when the first reported cases of variant CJD occurred and were reported to a stunned British public. When the announcement March 21, 1996, occurred that a remarkable low-age population of British citizens had come down with what appeared to be BSE in humans, the government soon banned the use of bovine offal including brain, spinal cord and eyes as binders in hamburgers. McDonald’s was quick to respond and ban any British beef and burgers. Within a month, in April of 1996, the CDC in the USA initiated a tracking for all cases of CJD in the USA. However, in the same time frame, the USDA issued a report saying that transmission of BSE to humans was “virtually impossible”. Increasing concern about the entire issue led to the ban of bone meal and meat products to be used as a fertilizer. Since 1996, 4.6 million cattle have been destroyed in Britain. This has left the remarkable problem of 663,000 tons of material left to be burnt. The storage of this material alone has led to a crisis in space in the 15 designated centers in England as incineration plans have evolved. Initial incinerators in England did not have the capacity to handle this waste disposal. Application to landfills is felt to be potentially risky, since the prion protein itself is found to be remarkably stable biologically. Interesting solutions to disposal of this volume of material has resulted in plans to incinerate the waste products and cement plants, and even coal-burning electrical energy plants. As the destruction of potentially contaminated cattle grows in the European, the problem is likely to expand at an exponential rate. Human cases are estimated to range from 30,000 to 500,000 in the United Kingdom alone.
Exports in the beef industry have suffered and as the problem of BSE being present in various countries in the EU, we have seen the development of remarkable increase in media coverage. In reviewing the media coverage issues, I found it interesting that early reference suggested that the fear of the Mad Cow Disease was somehow a plot from either the chicken or poultry council or some vegetarian syndicate. The so-called Vegeteens have used this as part of their own agenda to re-emphasize “eating cows is the real madness”. I have found some interesting headlines, which have included reference to “farmageddon”. Certainly the tabloids have been increasingly creative with “bad moos for Britain”; then there was the “steak in the heart of Britain”, and the reference to “making hash of British meat industry”. A recent article included reference to the “cow-cowering Europeans”, and I suspect the British populous had some relief in that the problem has spread to the continent and called attention to German sausage – “the wurst case dilemma”. Interestingly, the idea of genetically modified foods have had concurrent controversy in Europe. This has led to reference that “franken foods” may actually save the European beef industry. American soybeans have suddenly become heavily imported to Europe to substitute for the bone meal that had become commonplace as a ruminant feed.
My own perception is that this crisis will evolve into a remarkable case study in public health awareness of betrayal of trust issues. Additionally, however, the issues of protein conformational disorders will have a remarkable impact on basic science understanding in regard to protein metabolism. Of note, the ubiquitin system may be related to the remarkable resistance of the variant prion. I suspect that the conformational change associated with this deadly prion production results in difficulty with protease activation and may lead to resistance in the prion catabolism in a normal fashion. Inappropriate build-up in the material in the brain does have a remarkable three-dimensional biochemistry in the production of beta-pleated sheets of amyloid-like material. I am suspicious that this problem is related to the metabolism problems associated with other protein deposition diseases, especially those of Alzheimer’s disease and amyloidosis. Understanding the breakdown mechanisms requisite for this protein deposition will likely lead to more than recommended treatments for CJD victims. I have found reference to work being performed in Geneva, Switzerland, at Serona Pharmaceutical in developing a potential drug which will result in “beta-pleated sheet breaker peptides”. Efforts to return prions to normal shape, and thus allow catabolism of protein, will likely have impacts far beyond mad cow disease in humans. One can anticipate that beta sheet breaker peptides can be applied in Alzheimer’s disease. In fact, a new startup company, Axonyx, is a New York startup which will be applying this technique in Alzheimer’s disease. Future treatments, if they evolve from increased understanding of protein related disorders, could therefore have dramatic impact on far more common disorders such as Alzheimer’s.