Researchers Homing In On Mechanisms Of Encephalopathic Diseases

Encephalopathic Diseases One of the great biomedical quests of the last 20 years surrounds a handful of rare and mysterious brain diseases that affect humans and a small group of other mammals. An ongoing international research effort is aimed at understanding the mechanism of these neurodegenerative diseases. Recently, this area has been of particular relevance because one of the disorders-mad cow disease-has become a major, well-publicized health problem in Europe. Four encephalopathic diseases occur in people: Creutzfeldt-Jakob disease (CJD), Gerstmann- Straussler- Scheinker disease (GSS), fatal familial insomnia, and kuru, an ailment once associated with ritualistic cannibalism among Fore highlanders of Papua New Guinea. Related animal diseases include sheep scrapie and bovine spongiform encephalopathy (BSE, or mad cow disease). Other encephalopathic disorders are also found in domestic cats, mink, antelope, and mule deer. The fatal diseases cause the brain to become pitted, taking on a distinctive spongiform, or Swiss-cheese-like, appearance. Over a long period in humans and a somewhat shorter period in animals, those afflicted eventually lose motor coordination and develop dementia, among other symptoms. Researchers hypothesize that these diseases can be contracted in one of three ways: infection (through brain tissue transplantation in humans or sheep-scrapie-contaminated feed in cows, for example); inheritance of mutated genes that encode a misshapen protein; or a random genetic mutation. "From the pathogenesis point of view, everyone agrees, the accumulation of the abnormal protein in the brain is the cause of the disease," remarks Maurizio Pocchiari, a neurologist at the Instituto Superiore Di Sanit! in Rome who studies CJD. Most researchers investigating these spongiform diseases believe that a mutated protein called a prion is the root of the pathology, while a small minority say an exogenous agent like a virus is ultimately responsible. The crux of the debate lies with the role of the protein-as the transmissible agent-or as a player in a viral-mediated disease. Although the first case of CJD was described more than 60 years ago, interest in the field really took off in the mid-1960s, when kuru and CJD were proved to be transmissible to primates (D.C. Gajdusek et al., Nature, 209:794-6, 1966; C.J. Gibbs et al., Science, 161:388-9, 1968). In the following decade, researchers looked for a slow-acting virus or virus-like agent responsible for the diseases. The indestructibility of the sheep-scrapie agent to ultraviolet or ionizing radiation prompted researchers to postulate that the agent might lack nucleic acids, the building blocks of DNA and RNA. DEBUNKING DOGMA: San Francisco's Stanley Prusiner first decribed prions. In 1982, Stanley B. Prusiner, a professor of neurology and biochemistry at the University of California, San Francisco, published a landmark paper (S. Prusiner, Science, 216:136-44) that described a new type of pathogenic agent-consisting only of protein. He dubbed this agent a prion, suggesting that these proteins caused sheep scrapie. Fifteen years ago, Prusiner's hypothesis was difficult for the rest of the scientific community to swallow. "At the time, the notion was heretical," wrote Prusiner in a 1995 review in Scientific American (S.B. Prusiner, "The Prion Diseases," January 1995, pages 48-57). "Dogma held that the conveyers of transmissible diseases required genetic material composed of nucleic acid in order to establish an infection in the host." In 1985, teams led by Prusiner, molecular biologist Charles Weissmann from the University of Zurich, and geneticist Leroy Hood of the California Institute of Technology (now at the University of Washington, Seattle) cloned a prion gene that encodes a protein called PrP. This protein is normally found on the surface of most mammalian cells, especially brain neurons. Its function still is poorly understood. According to the prion explanation for the spongiform diseases, people and animals develop the disorders because abnormally shaped PrP proteins convert normal PrP proteins into the pathological form. Skeptics of the protein-only explanation, a much smaller group, contend that PrP is actually a viral receptor that sits on the cell surface. If the receptor is mutated, that cell is more susceptible to a viral infection, say the prion skeptics. The virino hypothesis, first proposed in the late 1970s by researchers at the Institute for Animal Health in Edinburgh, blends the virus and prion ideas. It says that a virino is simply a viral nucleic acid encased in prion protein. 'NOT NAILED': Infectivity experiments are needed, notes Minnesota's Ashley Haase. "I think the evidence is far and away supportive of prions and the protein-only theory of infectivity, but I don't think it's been nailed," comments Ashley Haase, head of the microbiology department at the University of Minnesota. "What we know is that an abnormal form of a protein found in normal cells forms amyloid plaques and is associated with spongiform changes in the brain." Carleton Gajdusek, a neuroscientist at the National Institute of Neurological Disorders and Stroke, maintains that how researchers on both sides of the prion debate explain the diseases' mechanisms is a matter of semantics. Gajdusek, widely considered the founder of this field of neurodegenerative research, won the 1976 Nobel Prize in physiology and medicine for his work on kuru transmission. "There are no competing theories about these diseases," he says. "There is, however, a huge controversy about the language of the field." If the causative agent is described using the classic definition of a virus-any obligate internal intracellular parasite requiring the informational and energy system of the host-all groups actually are referring to the same entity, Gajdusek asserts. In a 1994 paper (F. Cohen et al., Science, 264:530-1, 1994), structural biologist Fred Cohen from UC-San Francisco, in collaboration with the Prusiner group, suggested a mechanism for the neurodegenerative pathology of prion-related diseases. In a sort of slowed-down molecular chain reaction, the conversion of normal PrP to the misshapen prion configuration leads to the formation of new prions, nerve cell dysfunction, and death, and in some cases the accumulation of sheets of amyloid protein called plaques. These are not dissimilar to those seen in Alzheimer's disease (AD) patients, but AD plaques are composed of a different type of protein. The acceptance by the mainstream scientific community of Prusiner's once-controversial ideas is evidenced by the many important prizes he has won in the last several years, including a Gairdner Foundation International Award (R. Kaufman, The Scientist, June 14, 1993, page 3), the Albert Lasker Basic Medical Research Award (B. Spector, The Scientist, Oct. 17, 1994, page 1), and, most recently, the Wolf Prize for medicine (Notebook, The Scientist, Jan. 22, 1996, page 30). PRION PROPONENT: To not accept the prion theory is to "stick one's head in the sand." remarks MITs Phillip Sharp. "To not accept the fact that a protein is the major nucleator and affecter of the disease is to stick one's head in the sand," remarks Phillip Sharp, professor and chairman of the biology department at the Massachusetts Institute of Technology, who won the 1993 Nobel Prize for physiology and medicine. On the other hand, biophysicist Robert Rohwer, who once worked with Gajdusek, is convinced that there is a virus. "You can explain the behavior of these agents as a classical virus interacting with a PrP prion receptor that determines susceptibility. This is exactly analogous to the polio virus story," states Rohwer, director of the molecular neurovirology unit at the Veterans Affairs Medical Center in Baltimore. In the late 1980s, studies conducted in Prusiner's lab by Karen Hsiao, now a professor of neurology at the University of Minnesota, showed that a mutation in the PrP gene is genetically linked to GSS (K.K. Hsiao et al., Nature, 338:342-5, 1989; K.K. Hsiao et al., Science, 250:1587-91, 1990). Subsequently, transgenic mice expressing the mutated PrP gene developed a neurodegenerative disease that was pathologically similar to human GSS. Next, the researchers injected experimental animals with extracts from the brains of the sick mice. After eight months, some of the inoculated animals developed clinical symptoms similar to scrapie (K.K. Hsiao et al., Proceedings of the National Academy of Sciences, 91:9126-30, 1994). "These results support the view that mutated PrP causes neurodegeneration and forms into prions," says Stephen DeArmond, a professor of neuropathology at UC-San Francisco and a close collaborator of Prusiner's. But this did not yet prove that prions were infectious or could pass disease to other animals. More than 10 mutations of the human PrP gene, however, have so far been linked to other inherited prion diseases. Rohwer, a self-described "consistent critic of the prion hypothesis," concedes that the strongest evidence for a prion is Hsiao's work, although he has some reservations about the results. For instance, he says, GSS mice "do not elaborate the amyloid that is proposed to be the source of infectivity." KEY RESEARCH: NIH's Byron Caughey has led pivotal prion experiments. "Nobody's doubting that by having the abnormal PrP gene in transgenic animals that they're getting a neurodegenerative disease," notes biochemist Byron Caughey of the National Institutes of Health's Rocky Mountain Laboratories in Hamilton, Mont. "The more difficult question is: 'Is infectivity being generated in their brains, and if so, is it due solely to the presence of mutant PrP?' That remains under debate." The pivotal experiment needed to answer the prion infectivity question is to induce a conversion of the normal PrP to the abnormal form in a test tube, with no possibility of "contamination" by a separate, non-PrP agent. The next critical step would be to inject the pure, abnormal PrP preparation into an animal to see if it develops the signs of a spongiform disorder. Observers agree that some of the most significant recent experiments in the field are those by Caughey, biochemist Peter Lansbury from MIT, and their colleagues (D.A. Kocisko et al., Nature, 370:471-4, 1994). "We have shown for the first time that you can make the abnormal PrP in vitro," says Caughey. "But we haven't yet been able to assay whether we're making a new infectious agent in that process. To believe in the 'protein-only' theory, one needs to show that by converting the normal PrP to abnormal PrP, you can create a new infectious agent." Caughey's lab and others are ironing out kinks in their experimental protocol. His technique, in its current form, needs at least as many abnormal proteins as normal ones to jump-start the conversion reaction. "Right now it's impossible to separate the newly converted abnormal proteins from the existing ones," explains Caughey. "I think our assay indicates that the abnormal PrP can induce its own formation, which is a central, underlying requirement for it being an infectious protein, but direct proof of its infectious nature is lacking," he adds. "There seems to be no doubt that aberrant protein molecules are associated with infectivity," comments James Darnell, head of the laboratory of molecular cell biology at Rockefeller University. "The bottom line is that infectivity with any kind of pure prion protein aggregate has yet to be demonstrated." Rohwer declares that if Caughey "can increase the efficiency of his assay, he can answer the infectivity question one way or another. If he can demonstrate net production of infectivity, then I'll be happy to join the prion forces. I'll be the first one to send a congratulatory letter to Dr. Prusiner. However, that is not my expectation." DeArmond says that everyone is well aware of what definitive experiments need to be done. But, he adds: "It may not be as simple as that. Some of our more recent data has shown that another molecule or mechanism might also be involved in the conversion [G.C. Telling et al., PNAS, 91:9936-40, 1994; G.C. Telling et al., Cell, 83:79-90, 1995]." According to DeArmond, two hypotheses could explain this complexity. One, expounded by the skeptics, is that the other factor is an agent independent of the host, like a virus. The other hypothesis is that there are host factors. "My own lab thinks that there is a nucleic acid involved, but it's a normal host gene-not an exogenous agent-that controls the mechanism which actually converts the normal protein to the abnormal type," he says. For now, the researchers have dubbed this suspect host cofactor "protein X." A few aspects of the diseases' mode of action still perplex all spongiform disorder researchers. "One of the most contentious questions-and one of the most difficult to explain using a protein-only type of mechanism for the infectious agent-is the origin of multiple strains," notes Caughey. This idea came about because spongiform disorders cause slightly different symptoms and pathology, such as different distribution patterns of plaque accumulation in the brain. "Most infectious agents have multiple strains, because strains are encoded by mutable nucleic acids that are specific to the infectious agent. But if you have no nucleic acid genome to mutate, how can you explain the basis of propagation of distinct strains when the agent is only a protein?" asks Caughey. "What you have to postulate to explain this is that this protein can exist in multiple forms, which can in turn induce their own strain-specific propagation in the host." This could arise from multiple conformations of the protein or packing arrangements of PrP in the abnormal protein aggregate, he suggests. "A recent paper from our lab [R.A. Bessen et al., Nature, 375:698-700, 1995] showed that abnormal PrPs derived from two different agent strains in hamsters converted the same normal hamster PrP to different strain-specific abnormal PrPs in vitro." In some cases, it is difficult for abnormal prion proteins made by one animal species to cause disease in another. Prion researchers call this the host species barrier. What determines cross-species transmissibility is of prime concern, especially now because of BSE's possible link to the recent cluster of CJD cases in the United Kingdom. Prion skeptics argue for the presence of some viral nucleic acid that codes for the control of this rigid transmission. But DeArmond points out that studies in transgenic mice over the last five years show that the phenomena of multiple strains and species barriers can be explained by the structure of the abnormal prion protein and its interaction with the normal prion protein and other host factors (S.B. Prusiner et al., Cell, 63:673-86, 1990). In an attempt to improve the rate of cross-species transmission of CJD prions, "Glenn Telling, a fellow in Stanley Prusiner's lab, made a series of transgenic mouse lines: one which only expresses human normal PrP proteins and one that expressed a chimeric mouse-human PrP," notes DeArmond. "One hundred percent of the chimeric mice came down with human prion disease. What this suggests is that there is some other factor, a single protein or a series of proteins necessary for transmission." Prusiner and DeArmond's labs postulate that these data lend credence to the idea that a helper protein influences the formation of the abnormal protein. The UC-San Francisco researchers think that this protein recognizes the mouse portion of the human-mouse protein and binds to that domain, thus facilitating the folding of the protein into the abnormal, pathological shape. "I believe this is the first time transgenic mice technology has been used to dissect functional domains of a protein," notes DeArmond. Amid all of the prion work, a few other groups have not stopped looking for viral particles and nucleic acids in the human and animal encephalopathies. "There's still a segment in the scrapie [research] community that thinks there's a small genome that hasn't been found yet and is somehow necessary to effect all of these changes," says Haase. "What speaks against it is mainly the absence of evidence for it. The infectivity experiment is the last dragon for Stanley [Prusiner] or someone to slay. "But to be fair, we say the same thing to Stanley: 'Show me this protein in a test tube and show that it causes infectivity.'" One of these viral-seeking groups is headed by Laura Manuelidis, head of the neuropathology section at the Yale University School of Medicine. In May 1995, Manuelidis's group published evidence implicating a virus in CJD (L. Manuelidis et al., PNAS, 92:5124-8, 1995). In their experiments, CJD brain fractions that were rich in prion proteins but low in nucleic acids were barely infectious. In contrast, samples with proteins attached to nucleic acids were very infectious, but lost their infectivity when these structures were disrupted. Pocchiari and virologist Heino Diringer from the Robert Koch Institute in Berlin claim to have found a virus-like particle in humans with CJD. "But that does not mean what we found is the infectious agent," says Pocchiari. "Structurally, it looks like a virus." "Neither theory is proven, no matter what anyone tells you," Manuelidis told The Scientist earlier this year (K.Y. Kreeger, Feb. 5, 1996, page 14). "The protein doesn't transmit the disease, and nobody's identified a specific viral nucleic acid." EVIDENCE: Prio data abound, says Case Western's Pieluigi Gambetti. "I agree she did find some nucleic acid," remarks Pierluigi Gambetti, a professor of pathology at Case Western Reserve University School of Medicine who studies families with fatal insomnia. "There will always be an element that could be used to insert some doubt to make a claim for a virus, but in my opinion there is so much evidence to support the protein-only hypothesis." Manuelidis's group is about to publish another study (M. Dron, L. Manuelidis, Journal of Neurovirology, in press) that takes the 1995 work one step further. "We've isolated several sequences from infectious CJD brain fractions that are not in the normal parallel brain fractions or in the nucleic acid or protein databases. The data are encouraging, but the real significance of each of these sequences needs to be determined," says Manuelidis. "Viruses are far more than the sum of their parts," observes Rohwer. "Whole viruses can exhibit properties that are very different from native proteins and nucleic acids. They've shown us that time and time again." Whatever the ultimate conveyor of transmissibility might be, Caughey notes, scientists' ideas converge on explanations for pathogenesis: "PrP is critical in the whole pathogenic process and can modulate whether or not disease occurs."

Researchers Homing In On Mechanisms Of Encephalopathic Diseases

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