PRIONS are creepy. Although not alive by any accepted definition, these unusual particles ––believed to cause diseases such as mad cow and chronic wasting disease––still manage to propagate by deforming normal proteins, triggering a chain reaction with typically fatal results. They are lethal in vanishingly small doses, and as opposed to viruses or bacteria, which succumb to environmental conditions or antibiotics, prions are resistant to extreme temperatures, radiation exposures and many chemical treatments that kill any life. For all of these reasons, the fight against prion-related diseases largely relies on understanding and stopping the movement of the pathogens. It‘s a battle of containment.

Since the first cases of CWD surfaced among deer in southwestern Wisconsin in 2002, the state Department of Natural Resources has waged that war using hunters and sharpshooters to kill deer in a targeted exclusion zone. At the same time, UW-Madison launched a significant research effort to study and explain the strange protein that seems to be at the root of the outbreak. The good news is that recent research has filled in some blanks about the movement and behavior of prions. The bad news is that the results suggest that prion control will be extremely difficult.

Just this summer, in one of the most notable studies to date, Joel Pedersen, a CALS professor of soil science, and Judd Aiken, a virologist in the UW School of Veterinary Medicine who studies several types of prions, reported a drastic increase in prion transmission when prions attach to clay particles in soil. Pedersen says the finding could explain how the disease is transmitted among deer in southwest Wisconsin: Saliva, urine or feces dropped by deer can make soil infectious.

“Our studies on prion disease transmission underscore the need for wildlife managers to consider soil as a potentially important environmental reservoir of infectivity,” Pedersen says.

A Warning––and a Disaster

More than 200 years ago, Scottish shepherds found that some of their animals seemed to itch uncontrollably, and noting that the affected sheep compulsively scraped up against rocks and trees, they nicknamed the condition “scrapie.” It would be the first of a host of related degenerative diseases observed in mink, deer, elk, cats, bovines and humans that followed a similar destructive pattern. In each case, some infectious agent got into an animal’s nervous system and set to work chewing up its brain, causing an invariably fatal disease.

Not until the 1960s did scientists theorize that the infectious agent might be a prion, an otherwise normal protein that somehow folds itself into the wrong shape and somehow––there’s that word again––distorts other prion proteins in a cell. Able to propagate despite having no DNA or RNA, prions seemed to defy the laws of biology, and some considered them too good––or too bad‚––to be true until California biologist Stanley Prusiner isolated them in the lab in 1982. That finding was “intellectually shaking,” recalls Elizabeth Craig, a CALS professor of biochemistry who studies proteins. “Self-propagation was associated with DNA and RNA. It was shocking to find it without either one.”

Prions grew considerably more mainstream in the 1990s, when an outbreak of mad cow disease in the United Kingdom forced that nation to cull millions of cattle to control the infection. Since the start of the epidemic, about 150 Britons have died of a human prion disease called variant Creutzfeld-Jakob disease, apparently as a result of eating infected beef.

UW-Madison scientists have played a critical role in the exploration of the prion diseases, and indeed, had the warnings of a Wisconsin virologist been heeded, the United Kingdom might have been spared the mad cow disaster, or at least controlled it much sooner. Beginning in 1990, Richard Marsh MS’66 PhD’68 cautioned that dairy cows might get infected from feed that contained byproducts from prion-infected cows. (In many countries, inedible byproducts and the carcasses of diseased cattle are treated with heat or chemicals before being blended into animal feed.) Marsh, who had grown up on a mink farm, reached this conclusion after studying a 1985 outbreak of prion disease at a mink farm in Stetsonville, Wis., which killed 60 percent of the animals.

But the notion that prions could be transmitted in feed was treated as heresy until cases of mad cow multiplied in the United Kingdom. The government’s slow response allowed the disease to spread among cattle and humans. Only when the United Kingdom banned cattle byproducts from cattle feed––as Marsh had advocated––was mad cow brought under control. In 1997, the same ban was enacted by the Food and Drug Administration in this country.

The United States never endured the human and economic costs of mad cow disease due to a combination of tighter regulation on cattle feed and a bit of luck. But several states from Wyoming to New York have seen spot outbreaks among wild deer and elk of CWD, which causes a range of neurological symptoms, including staggering, slobbering and eventually death.

Although no Wisconsinite is known to have been infected by eating venison, the DNR estimates that 5 percent of does and 10 percent of bucks in the deer exclusion zone in southwestern Wisconsin are infected with the disease. Wildlife managers have tried to contain CWD by increased hunting pressure, hoping that reducing the deer population in the infected zone will slow or halt transmission and at least prevent the disease from spreading further. Hunters must have kills from the exclusion zone tested for prion infections, and because the tests are not perfect, an animal that passes must be carefully butchered to avoid any residual threat of infection. Yet despite the DNR’s efforts, the deer population in the exclusion zone has grown since CWD was first identified there in 2002.

Follow the Soil

A more complete scientific understanding of how the disease is transmitted could suggest better methods for containment. While feeding practices were the root of the mad cow problem, CWD in deer and elk “is transmitted horizontally, from animal to animal, apparently through an environmental reservoir,” says Pedersen. The most likely source happens to be Pedersen’s area of expertise: soil.

An environmental chemist, Pedersen joined a team of UW researchers who in 2003 landed more than $5 million in grants from the U.S. Department of Defense to probe the disease. His role was to come up with a system to test how prions move around in various types of soils, which might help scientists pinpoint how deer were getting and passing on the disease. In 2006, Pedersen, Aiken and Christopher Johnson, a graduate student in the UW’s cellular and molecular biology program, reported that prions could transmit the disease in the laboratory even when bonded to clay particles. In follow-up experiments, Pedersen mixed sand and prions in a test tube to see how well prions are absorbed at different pH values.

Although he notes that his test-tube soils are an “idealized system,” Pedersen says the data suggest that prion absorption depends on soil pH, with alkaline soils promoting the particles’ movement. The results could have implications for the disposal of infected deer meat. Currently, the carcasses are decomposed in chemical digesters, but authorities are considering dumping them in landfills to reduce costs. The study results will help outline what landfill conditions would promote prion movement, says Pedersen, especially if lime is used to hasten decomposition. “If you are concerned about prion migration through a landfill, you don’t want them moving, so more absorption is good,” and alkaline conditions should be avoided.

However, some movement could be beneficial in a natural environment, Pedersen adds. “Where prions enter the soil through excretion or the decomposition of a carcass, prions that migrate to deeper soil would be less accessible to organisms like deer, so it might be a good thing if they are mobile.”

Soil may be doing more than just transporting prions, however. Pedersen and Aiken have recently reported that prion infectivity increased by a factor of 680 when the aberrant proteins were bound to a common type of clay, meaning a prion dose of just 0.2 micrograms (less than one one-hundred millionth of an ounce) could infect a significant number of lab animals.

“I think we have made an important step toward establishing that soil is a plausible reservoir for infection, and that the dose does need not to be high,” Pedersen says

Pedersen cautions that the infectivity study was done with hamsters and must be repeated with ruminants, which have a different type of digestive system. “If the results hold,” he says, “we may want to minimize situations where transmission is likely to occur via soil, such as where deer tend to congregate and exchange fluids with soil.” To avoid such situations, the Department of Natural Resources bans baiting and feeding deer in the exclusion zone.

But how could tiny clay particles make prions more dangerous? Perhaps by protecting them from destruction in the digestive system, Pedersen says. If that hypothesis proves correct, it could have human-health implications. Various clays, such as bentonite or kaolin, are added to some foods during processing. If clay does boost the infectivity of any prions we happen to eat, it could also help prions that infect deer, sheep or cattle jump to another species, such as us.

“This is speculation,” says Pedersen, “but it’s not outside the realm of the imagination that this enhancement of infectivity could perhaps enhance interspecies transmission.”

Knowing When to Fold

Beyond urgent efforts to control prions and the diseases they trigger, CALS researchers are also engaged in fundamental research aimed at understanding the nature of the particles themselves. Proteins capable of forming prions are in many ways like the thousands of other proteins found in every cell, where they play many structural and chemical roles. Defined by a particular combination of amino acids, which link like beads on a chain to form a long molecule, each protein folds itself into a unique shape that allows it to do its job inside a cell. Prions form when the folding goes wrong.

Protein folding resembles a microscopic version of origami on steroids, and so errors can arise. Normally such mistakes cause no problems; a lone protein that takes on the wrong shape can’t do any harm by itself. But some proteins that get misfolded become capable of triggering a chain reaction, in which they recruit other healthy proteins with the same amino acid sequence and convert them into prion form. As their numbers get larger, these malformed proteins band together in long strings that can destroy a cell’s functions.

Elizabeth Craig has found that both the formation and propagation of prions may be abetted by other proteins called chaperones. Ironically, scientists began studying these molecules for their ability to repair damaged proteins, a function that helps some microbes survive in extremely hot temperatures. Doubling the irony, one job of chaperones is to protect proteins from misfolding. Nonetheless Craig is finding that with chaperones, more of a good thing is not always a good thing.

Her experiments have shown that chaperones can help a prion convert a normal protein into a new prion. Furthermore, they have also shown that chaperones can accelerate the chaining activity that links prion proteins into long strings. “When a prion is growing into a long fibril,” she says, “new subunits can only be added at the ends. If you cut the fibril in half, now you have four ends.” This is exactly what she has seen in yeast experiments.

“They are doing what the chaperones are supposed to do, altering protein conformation, but this cutting turns out to be essential for allowing the prion to spread by making more ends, where additional prion protein molecules can attach,” she says.

As research into these bizarre proteins proceeds, scientists see hints that protein malformations play a role in other conditions. In the brain-destroying disease Alzheimer’s, for example, misfolded beta amyloid proteins form filaments and big, snarly tangles that seem to eat the brain.

Even the very existence of prions has changed the way biologists frame their experiments. The mere reality of a protein that can exist in two forms––one able to infect cells and convert its healthy proteins––introduces a range of complexities into experimental biology, Craig says.

“When we thought about disease transmission or inheritance of traits, we always used to think only about (DNA or RNA),” she says. “Now we have to think about protein as well.” And if prions break the laws of replication, who is to say that some other bizarre pathogen isn’t out there, waiting to overturn biology again? As Craig says, “It makes you a little humble.”

SIDEBAR — Protein Origami

Like other proteins in the body, prions are essentially long chains of amino acids, which fold up into particular shapes that allow them to function in cells. But with prions, the folding process goes berserk. Rogue proteins somehow become folded into the wrong shape and begin to recruit other healthy proteins around them to adopt the same shape. Often, a deadly chain reaction ensues, in which misfolded prions link together to form long fibers, creating plaques that destroy cells.

This article was posted in Agriculture, Environment, Fall 2007, Features and tagged Biochemistry, Genetics, Human-wildlife interactions, protein origami, sidebar, Soil science, Wildlife biology.