© Images.com/Corbis
Thursday, March 15, 2007

Prying Into Prions

Challenges, Gloom and Hope for Treating Deadly Diseases

By: Scott P. Edwards

Prion diseases such as "mad cow" turn the brains of otherwise healthy people into a spongy mush, inevitably killing them. Now researchers are beginning to understand how mutant prion proteins cause this destruction.

Among the most mysterious and virulent diseases known, prion diseases turn the brains of otherwise normal, healthy people into a spongy mush—and, without fail, kill them. While mad cow disease, Creutzfeldt-Jakob disease, fatal familial insomnia, and other prion diseases are exceedingly rare, scientists have spent the past 25 years searching for answers to how an enigmatic protein could cause such destruction. Researchers are now beginning to understand the role that mutant prion proteins play in these brain-wasting diseases, and though the road to a cure is full of obstacles, a bit of the gloom is starting to lift.   

Chain Reaction of Misfolded Proteins

The idea that a protein could be the cause of an infectious disease was revolutionary until the early 1980s, when Stanley Prusiner, M.D., of the University of California–San Francisco School of Medicine introduced the concept of prions, for which he won the Nobel Prize in 1997. Prions, says Prusiner, are “tiny protein molecules that seem to cause a variety of slow-acting and inevitably fatal diseases in animals and humans.” Prusiner named these proteins “prions,” short for “proteinaceous infectious particles.”

Found in the brains of all living animals, prions contain no DNA or RNA, the building blocks of the genetic code. In their normal form, prions are natural components of the body, although their function is still largely unknown. But when they become twisted or folded in large numbers, over time, they lead to diseases that are characterized by holes in the brain where neurons have died and by rapid loss of mental abilities.

Every protein in the body has a specific conformation, or shape, and many of them contain amino acids that allow them to change the shape of other proteins. Prions exist in two distinct conformations in the brain: a “wild type” that everyone possesses and a densely folded, infectious type. The infectious form of the protein recruits wild-type proteins to misfold, causing them to be infectious as well. Once this occurs, a massive chain reaction takes place in which increasing numbers of prions in the brain misfold and become infectious, triggering the cascade of deadly neurodegenerative effects.

prion molecule 
 These molecular models show how a normal prion protein (top) is folded into a helix, while the infectious disease form (bottom) is misfolded.

Credit: © Eurelios/Phototake 

In about 85 percent of human prion disease, the misfolding of proteins, called cellular prion proteins (PrPc), into a disease form, or isoform (PrPSc), occurs sporadically.  Another 14 percent of human disease occurs from familial forms, associated with mutations of the gene that encodes prion protein. The remaining one percent of human prion disease is transmitted by eating food made from an infected animal, such as a cow.  Whether sporadic, inherited, or transmitted, prion diseases all induce the buildup in the brain of toxic plaques that cause the widespread death of neurons.

In 2003, Stanford University geneticist Gregory S. Barsh, M.D., discovered that abnormal prion proteins, unlike their normal wild-type counterparts, are folded in such a way that enzyme target sites are hidden. In wild-type prions, enzymes degrade the proteins at the end of their lives. The folds in the abnormal prions, however, make them resistant to enzyme destruction.

Certain other neurodegenerative diseases feature a toxic buildup of other types of protein—notably Alzheimer’s, in which the protein beta-amyloid forms plaques. Some researchers are investigating the idea that beta-amyloid might have a folding procedure similar to that of the abnormal prion.

The best-known prion disease is mad cow disease, formally known as bovine spongiform encephalopathy (BSE). Other animal prion diseases include scrapie, which affects sheep and goats, and chronic wasting disease, which afflicts deer and Rocky Mountain elks. Even rarer forms of the disease affect minks, monkeys, lemurs, household cats, and feline species in zoos. Among human prion diseases the most common are Creutzfeldt-Jakob disease, new variant Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. (To learn more about prion diseases, read “Prion Diseases: Rare but Deadly.” )

New Developments Show Promise

While not one effective treatment for prion diseases exists (the only available treatments aim to ameliorate symptoms), several recent research studies show promise. Among them are prion depletion, therapeutic vaccines, and inhibition of the protein fragmentation process.

In February 2007, researchers led by Giovanna Mallucci, Ph.D., of London’s Institute of Neurology, reported a study showing that depleting prions in laboratory animals early in the disease process, before neuronal loss is widespread, may Her study ... showed that cognitive and behavioral impairments, as well as neurological pathology, could be reversed in mice if prion depletion is done early enough in the disease process. be an effective therapy. In mouse models, Mallucci and her colleagues found that an enzyme called Cre recombinase, which is used to modify genes and chromosomes, selectively inhibited the gene for PrPc so that no more of the protein is produced. Research efforts are under way to develop a drug that can safely induce this process. 

“By depleting prions in the brain,” says Mallucci, “the process of conversion [from normal to abnormal prion protein] is blocked. Depleting PrPc removes the substrate for ongoing replication and prevents continued production of the toxicity.”

Mallucci says that for this approach to have any positive effect, prions would need to be depleted in mice up until about 60 percent of the way through the incubation period—before neuronal loss is established and when pre-degenerative changes can be reversed. Her study, published in the February issue of Neuron, showed that cognitive and behavioral impairments, as well as neurological pathology, could be reversed in mice if prion depletion is done early enough in the disease process. While scientists say the physiological role of the normal prion protein is unclear, Mallucci’s team found that PrPc depletion had no effect on behavior or cognition in uninfected mice.

“Our findings of early reversible neurophysiological and cognitive deficits occurring prior to neuronal loss open new avenues in the prion field. To date, prion infection in mice has conventionally been diagnosed when motor deficits reflect advanced neurodegeneration. Now, the identification of early dysfunction helps direct the study of mechanisms of neurotoxicity and therapies to early stages of disease, when rescue is still possible,” Mallucci and her colleagues wrote in their paper in Neuron.

In addition, she says that her research may also lead to preclinical testing of therapeutic strategies that could be helpful for early intervention in humans with prion diseases, particularly variant Creutzfeldt-Jakob disease. This could not only halt clinical progression of the disease but also reverse cognitive abnormalities, she adds.

Howard Federoff, M.D., Ph.D., a prion researcher at the University of Rochester Medical Center, recently reported that a therapeutic vaccine can slow the progression of prion diseases in mice. Federoff and his colleagues engineered a virus Serio writes that a single protein plays a major role in deadly prion diseases by smashing up clusters of infectious proteins, creating the “seeds” of destruction. that carries the genetic code for producing antibodies that bind to and attack prion proteins. They injected this virus into the brains of mice, which allowed immune cells in their brains to produce antibodies based on the code. They then injected infectious mouse prion proteins into the bellies of these mice, which traveled to their brains and caused other normal proteins to misfold and form toxic plaques.

Both the test mice and control mice that did not receive the therapeutic vaccine died. The vaccinated mice, however, survived about 30 percent longer (260 days compared to 200 days for the control mice). Says Federoff, “We delayed the emergence of disabling clinical signs and substantially delayed death.” The antibodies produced by the engineered virus likely work by binding to infectious prions and preventing the formation of the toxic plaques. According to Federoff, this approach might be effective for people who have variant Creutzfeldt-Jakob disease.

While conventional thinking holds that prion diseases worsen by converting ever more good proteins to misfolded bad forms, Tricia Serio, Ph.D., and her colleagues at Brown University say that fragmentation of prion complexes is another crucial step in the disease process. In the February 2007 issue of PLoS Biology, the peer reviewed, public access journal of the Public Library of Science, Serio writes that a single protein plays a major role in deadly prion diseases by smashing up clusters of infectious proteins, creating the “seeds” of destruction.

According to Serio, Hsp104, a molecule required for prion replication, may play the role of “protein crusher.” “To understand how fragmentation speeds the spread of prions, think of a dandelion,” Serio said. “A dandelion head is a cluster of flowers that each carries a seed. When the flower dries up and the wind blows, the seeds disperse. Prion protein works the same way. Hsp104 acts like the wind, blowing apart the flower and spreading the seeds.” Her team found in laboratory studies that Hsp104 crushes complexes of a yeast protein called Sup35 that is similar to human prion protein. Prions still multiply without this crush-induced fragmentation, she cautions, but they do so at a much slower rate. If a drug could safely inhibit this fragmentation process, it could substantially slow the spread of the infectious prions that cause Creutzfeldt-Jakob disease and other human prion diseases.

A paper in the February 2007 online edition of the Proceedings of the National Academy of Sciences offers further insight into how normal prions are converted and produce amyloid plaques, possibly leading to means to intervene in this process. Studying this same Supp35 protein that Serio’s team used, researchers at the Scripps Research Institute, led by Ashok Deniz, Ph.D., found that the protein in its native state lacks a specific structure and forms intermediate shapes during the conversion of normal proteins to amyloid plaques found in Creutzfeldt-Jakob disease and variant Creutzfeldt-Jakob disease, as well as Alzheimer’s, which is not a prion disease.  

Deniz says this intermediate stage of the process is critically important, since “no single native unfolded protein is capable of initiating the amyloid cascade [by itself] because of this constant shape-shifting.” This knowledge, Deniz says, may help scientists in the search for potential new therapeutic agents that intervene during this shape-shifting phase.           

Challenges, Gloom, and “Reasonable Expectations”

Despite such imaginative and innovative efforts, human therapies are still a distant possibility, and gloom persists. In the years since Prusiner coined the term “prion” in the early 1980s, scientists have indeed learned a great deal about prion diseases. We now know that prions contain no nucleic acid (in contrast with most infectious diseases, which are spread through either a virus or bacteria, which contain DNA and RNA information) and that infectivity is associated with PrPSc. That said, however, numerous challenges face the scientific community in developing effective, appropriate treatments for prion diseases. Chief among them:

  • Prion structure. Scientists know the 3-D structure of the normal prion protein, but not the structure of its disease form (PrPSc). If they can find out more about the PrPSc structure, they may be able to design effective drugs to counteract it, says Richard Johnson, M.D., of the Johns Hopkins University School of Medicine, who served as a consulting neurologist on a Public Health Service epidemiological study of human prion diseases and has testified before Congress about them.
  • Drug delivery. Because prion diseases manifest themselves in the brain, drugs to treat them must be able to cross the blood-brain barrier. As scientists develop potential new drugs, they must find ways to deliver them to specific brain areas, and at high enough levels to prevent disease or to clear diseased proteins.
  • Misfolding. Scientists know that the abnormal prion proteins form plaques in living animals and that they behave differently than normal proteins do, but they do not yet understand why the prion protein misfolds in the first place.
  • Diagnosis. Most cases of prion diseases are not diagnosed in their early stages. Because they have long incubation periods—some up to 50 years—the buildup of abnormal prions in the brain has a cumulative effect. Thus, by the time most patients are diagnosed, dementia and other irreversible damage have set in.
  • Rapid progression. Once symptoms appear, the disease progresses rapidly. From week to week, says Johnson, patients lose their ability to work and remember names, among other symptoms. At this point it is too late for effective treatment. “Drugs at this stage,” he adds, “would only preserve dementia, which is not what we’re looking for.”

Federoff says that perhaps the most difficult challenge facing scientists is “developing reasonable expectations to be able to alter these diseases.” Because of these challenges, a sense of pessimism pervades the prion research community. “Think about a misfolded protein,” says Johnson. “What kind of drug could unfold it or make it not fold in the first place? It’s hard to picture a drug that could do this.”

Despite the sense of gloom among researchers, Federoff, for one, remains optimistic. Success, he says, depends on scientists’ ability to more regularly diagnose prion diseases at their early stages, when there is greater potential for restoring or abating underlying problems in the brain. These therapeutic strategies, he adds, may be partially or fully effective.

“Take, for example, cancer therapies that extend life an additional three, six, 12 months,” he says. “Any significant extension of functionality is an advance. We’re not talking about cures, but extending life and functionality that will resonate with patients.”

That is a goal worth working toward.



About Cerebrum

 
Bill Glovin, editor
Carolyn Asbury, Ph.D., consultant

Scientific Advisory Board
Joseph T. Coyle, M.D., Harvard Medical School
Kay Redfield Jamison, Ph.D., The Johns Hopkins University School of Medicine
Pierre J. Magistretti, M.D., Ph.D., University of Lausanne Medical School and Hospital
Robert Malenka, M.D., Ph.D., Stanford University School of Medicine
Bruce S. McEwen, Ph.D., The Rockefeller University
Donald Price, M.D., The Johns Hopkins University School of Medicine

Do you have a comment or question about something you've read in CerebrumContact Cerebrum Now.