University of California, San Francisco researchers have made a finding regarding prions in mice that they say warrants similar study in cattle.
In their investigation, published in the March 19 issue of Proceedings of the National Academy of Sciences, the researchers report that mice exposed to the lethal prion (PREE-on) pathogen develop high levels of the infectious agent in some skeletal muscle. Until now, scientists have thought skeletal muscle was neither susceptible to high levels of infection, nor infectious. The researchers have not investigated whether high levels of prions amass in the skeletal muscle of cattle, sheep, deer or elk infected with prions.
Substantial evidence has suggested that prions only accumulate at high levels in brain, spinal cord and lymphatic tissue. As a result, Great Britain – where the epidemic of the prion disease known as bovine spongiform encephalopathy (BSE)1. developed in cattle in the mid 1980s – and countries of the European Union have banned brain, spinal cord and lymphatic tissues from the human food supply.2. This measure is the most extreme step these countries have taken in light of the considerable evidence that consumption of BSE-contaminated beef products causes a human form of the neurodegenerative disease, known as new variant Creutzfeldt-Jakob disease (nvCJD).
However, researchers have not reported whether the skeletal muscle of BSE-infected cattle transmits to mammals. And the UCSF researchers say that in light of their finding in mice this investigation should be carried out in both livestock and game.
“Whether prions accumulate in the skeletal muscle of other animals remains to be established. But our findings indicate the need to carry out a comprehensive and systematic investigation of the distribution of prions in the skeletal muscle of animals who develop prion diseases,” says the senior author of the study, Stanley B. Prusiner,3. MD, UCSF professor of neurology and biochemistry and director of the UCSF Institute for Neurodegenerative Diseases.
Notably, no cases of BSE have been reported in the United States,4. which, since 1989, has banned the importation of cattle, cattle products and sheep from Great Britain and all other European countries with cases of BSE, and which maintains strict surveillance of cattle within its borders. And in Great Britain strong measures to curtail the incidence of BSE have significantly decreased the incidence of BSE. (In Great Britain, cases of BSE have dropped from 36,680 in 1992 to fewer than 1,500 cattle in 2000 and are much lower in all European countries except Portugal and Ireland.)
Moreover, the rate of transmission of BSE to humans appears to be low, presumably due to a species barrier. (As of Feb. 4, 2002, 106 had died of nvCJD and eight were still living with the disease in Great Britain,5. and three people had died in France. One person died of CJD in Ireland but as he lived in England for a time, the source of his disease is unclear.)
In the UCSF study, the level of prions varied significantly among the different skeletal muscles of the mice, reaching high levels only in the hind limb. The researchers do not know what factors cause the variation. Nor do they know if the distribution would be expected to be the same within the skeletal muscles of other species or among different strains of prions.
“It’s possible that if prions do develop at high levels in the skeletal muscle of other animals that the distribution within the muscles varies even within breeds, varieties and lines of a species, as well as with the strain of prions,” says the lead author of the study, Patrick J. Bosque , MD, assistant professor of neurology at the University of Colorado Health Sciences Center. Bosque conducted the work while he was a postdoctoral fellow in the Prusiner laboratory.
While there is no clear evidence that deer and elk with chronic wasting disease or sheep with different strains of scrapie have ever transmitted these prion diseases to humans, the researchers say the investigation should extend to these animals, as well.
In their study, the researchers note that several variables related to the nature of prions could affect whether the findings in mice would be seen in livestock or humans. In the mouse study, particular strains of mouse prions were studied, and the animals were infected through inoculation. However, the efficiency with which various prion strains accumulate in muscle may vary. Moreover, the efficiency with which various strains cross the “species barrier,” a barrier that precludes the transmission of most diseases between species, also varies. Finally, oral transmission of prions – the mode by which transmission would naturally occur – is inefficient compared to transmission by inoculation.
To determine whether cattle develop prions in their skeletal muscle, says Bosque, scientists need to investigate the various skeletal muscle groups of slaughtered animals suspected of having BSE, using highly sensitive and quantitative techniques. Obtaining consistent results from some 50 BSE-infected cattle representing the various breeds would probably be sufficient to draw a conclusion, he says.
Possible new diagnostic tool
Bosque says that if high levels of prions propagate and accumulate early in muscle, conducting muscle biopsies on live animals could prove an effective diagnostic tool in asymptomatic animals. Muscle biopsy might even be used to diagnose humans suspected of having any of the various forms of prion diseases, he says. As brain biopsies are a high-risk procedure and are not 100 percent accurate, most patients are diagnosed definitively only on autopsy.
The discovery that prions propagate in the skeletal muscle of mice could put researchers a step closer to understanding how the inscrutable prion carries out its scourge. The only known component of the prion (PrPSc)6. is an aberrant form of a normal protein, known as the cellular prion protein (PrPC), which exists in a healthy state in humans and many animals. The protein becomes lethal when some of the spirals that make up a portion of the protein molecule lose their normal conformation and flatten into so-called beta sheets. (Prions can arise spontaneously, be inherited through a genetic mutation or develop through infectious transmission.) Once the conversion occurs, the prion moves on to normal prion proteins, pinning and flattening their spirals, as well. The accumulation and aggregation of the flattened beta sheets within a cell leads to structural damage that causes cell degeneration. The destruction spreads quickly through the brain, generally causing death in less than a year.
One explanation for prions propagating at high levels only in the brain and lymphatic tissue has been that there simply might be more normal prion protein in these regions, and therefore more protein that would be susceptible to conversion. Another possibility has been that some essential, as-yet-unidentified, molecules other than the prion might be involved in the process of converting normal prion proteins, and that these other factors are found only in sufficient quantity in brain, spinal cord and lymphatic tissues.
The current study sheds light on these possibilities. In their study, the researchers inoculated the brains of normal mice with either of two strains of mouse prions, known as the Me7 and the Rocky Mountain Laboratory strains. They discovered that the animals did, in fact, develop high levels of prions in some skeletal muscle, specifically in muscles from the hind limb.
Then, to determine whether prions were actually produced in the muscle or merely migrated and accumulated there, the team developed mice genetically engineered to express either mouse or Syrian hamster PrPC (normal cellular prion protein) exclusively in hind limb muscle. When they inoculated the muscles with prions, the mice developed high levels of new prions in that muscle. In contrast, when prions were injected into mice genetically engineered to express PrPC exclusively in liver cells, the liver cells did not develop high levels of new prions.
This finding demonstrates, the researchers say, that factors other than the amount of normal prion protein expressed in a given tissue contribute to prion propagation. And this conclusion supports the possibility that only certain tissues contain these elusive factors. Prusiner has long proposed that the prion requires the help of an as-yet-unidentified “protein X” to latch on to the normal, corkscrew-shaped tendrils of the prion protein and pin them flat.
“One possibility,” says Prusiner, “is that protein X is not expressed, or is only expressed at very low levels, in certain muscle tissues. And that could be why we are seeing this wide disparity from brain to muscle to liver, as well as between one muscle group and another.”
Moreover, the finding could explain why previous studies have not detected high levels of prions in skeletal muscle, the researchers say. “Previous studies might not have been done using sensitive enough assays. But in other studies scientists perhaps looked at the wrong skeletal muscle,” says Bosque.
Other co-authors of the UCSF study were Chongusuk Ryou, PhD, postdoctoral scholar in the Prusiner lab; Glenn Telling, PhD, formerly a postdoctoral scholar in the Prusiner lab and currently associate professor of microbiology, immunology and molecular genetics, and neurology, and a member of the Sanders Brown Center on Aging, University of Kentucky; David Peretz, PhD, a postdoctoral fellow in the Prusiner lab and a member of the UCSF Institute of Neurodegenerative Diseases, and Giuseppe Legname, PhD, UCSF adjunct assistant professor of neurology and a member of the UCSF Institute of Neurodegenerative Diseases.
The study was funded by grants from the National Institutes of Health and by a gift from the G. Harold and Leila Y. Mathers Foundation.
Link to normal prion protein image and photograph of Stanley B. Prusiner, MD: http://pub.ucsf.edu/imagedb/imsearch.php?keywords=prusiner
1. The outbreak of BSE in Great Britain is believed to have occurred as a result of cattle being fed ground sheep offal that was infected with scrapie, a prion disease.
2.http://www.fda.gov/oc/opacom/hottopics/bse.html
3. Prusiner won the Nobel Prize in Physiology or Medicine in 1997 for discovering that a class of neurodegenerative diseases known as spongiform encephalopathies was caused by prions.
4. http://www.cdc.gov/travel/madcow.htm
5. http://www.cjd.ed.ac.uk/index.htm
6. The prion, for proteinacious infectious particle, is unlike any other disease –causing agent. All other pathogens – bacteria, viruses, protozoans or fungi – contain nucleic acid that allows them to transmit their pathogenic code. The prion, in contrast, is devoid of nucleic acid, and instead in composed of amino acids. Prion diseases are rare in humans. One in a million people each year develop a sporadic form of the condition, known as Creutzfeldt-Jakob disease (CJD), for which there is no known cause. Approximately 5 to 15 percent of all cases are inherited. The disease can also occur in humans through infectious transmission, from ingestion of prion-contaminated meats and contamination through biological and pharmaceutical products, as seen with the development of new variant CJD in 1990. Other infectious prion diseases include kuru, which arose among New Guinea natives engaged in ritualistic cannibalism, and iatrogenic CJD, caused by prion-contaminated cadaveric growth hormone and dura mater grafts. Prion diseases also develop in sheep, deer, elk and mink.
Journal
Proceedings of the National Academy of Sciences