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Misfolded and Misunderstood


There is perhaps no deadlier, more diverse, and least understood disease-causing agent than the prion protein. Thought to be responsible for more than 15 fatal mammalian neurological diseases known as transmissible spongiform encephalopathies, prions spread throughout the host in a misfolded and aggregated state, have incubation periods of up to four years, and are resistant to chemical digestion. Prions are believed to play a role in Creutzfeldt-Jakob disease, a human neurodegenerative disease that has several subtypes, as well as Gerstmann-Sträussler-Scheinker syndrome and Kuru, also known as "laughing sickness" for the uncontrollable bouts of laughter sufferers sometimes display. Bovine spongiform encephalopathy, otherwise known as "mad cow disease," has posed a serious health threat and had a devastating effect on our food supply — several million cattle in the United Kingdom alone have been killed over the last 20 years due to infection. Other diseases include scarpie, which affects sheep and goats, and chronic wasting disease, which has been found in deer, elk, and moose. In addition to a diversity of disease forms, prion disorders can come about due to genetics, transmission, or can appear sporadically, or through a combination of the three.

Aside from the sheer complexity of prion diseases, there is also still some disagreement in the scientific community about the theory of prion protein disease. Skeptics say that it is just that — a theory. This debate can be traced all the way back to 1982 when Stanley Prusiner, now director of the Institute for Neurodegenerative Diseases at the University of California, San Francisco, first came out with the prion protein theory as an explanation for mad cow disease and CJD. Prusiner, who would go on to win the Nobel Prize in 1997 for his prion research, coined the term prion as a portmanteau of "proteinaceous" and "infectious." But some researchers took issue with the idea from the start — most famously Yale University's Laura Manuelidis, a professor of neuropathology who made the case that a virus causes CJD and that prions are merely a result of viral infection.

"Prion diseases have historically been very controversial, and still to date there are some scientists who are quite skeptical about the protein hypothesis, despite the enormous amount of evidence," says Ilia Baskakov, director of the Prion Institute at the University of Maryland's Biotechnological Institute. "It creates a lot of useless discussion in the field. … The theory about viral infection causing CJD is no more than an amusing chapter from the history of science."

Baskakov, together with prion-hypothesis skeptic Robert Rohwer, director of the Molecular Neurobiology Laboratory at the Veterans Affairs Maryland Health Care System, published an article in Acta Neuropathologica in January that showed how TSE could be induced without an outside catalyst like a virus. Using Golden Syrian hamsters as a model system along with synthetic E. coli recombinant prion proteins, the researchers were able to demonstrate that wild-type prion proteins can induce prion disease in normal, wild-type hosts.

In addition, researchers at East China Normal University and Ohio State University confirmed that altered forms of prion protein produce prion disease, as they reported in a paper published in Science in February. The research team introduced a form of recombinant prion into mice, with the same aggregated, protease-resistant, and self-perpetuating characteristics of pathogenic prion protein isoform. Characterization of the diseased mice revealed classic signs of prion disease and sufficient data that they had in fact succumbed to prion disease.

"The prion protein is an interesting protein. It can misfold and spontaneously go wrong, whereas most biological processes have an error rate and editing mechanism to fix them, so we don't quite know what the rules of engagement are for the prion protein — it's quite a mystery," says David Westaway, director of the Centre for Prions and Protein Folding Diseases at the University of Alberta. "The biology is subtle, so even though it is quite small, it's now becoming clear that the prion protein has different bioactivities — different fragments of the protein may be doing different things. It may start in one cell and move to another. We [have] to figure out a way to take apart that multi-component activity."

In the lab

Despite all of the available 'omic analysis tools, protein folding simulation and informatics software, uncovering the mechanisms of prion disease is still a battle that will primarily be fought at the bench. "Certainly biotechnology has played a role in this research, but at the same time there's still a lot of other findings and other studies that need to happen in the wet lab," says Ryan Maddox, an epidemiologist at the Center for Disease Control's National Center for Emerging and Zoonotic Infectious Diseases. "The thing about prion diseases, in general, is that it's a very unique infectious agent and there's a lot of different kinds of research going on to just learn more about it."

And because there is no shortage of proteins that interact with the prion protein — for example, there might be pages of candidates that some analysis tools will spit out for a particular prion protein — lots of work still needs to be done before researchers can really benefit from high-throughput methods to study all of the protein-protein -interactions that could play a role in a particular disease. "Noise in the data is perhaps the biggest challenge as there are a lot quiescent prion proteins hanging around that are probably going to require very special tools to dial into what proteins are relevant to how the disease process is perpetuated," Westaway says. "Something that's unique in biology is to have an infectious disease appear out of nowhere. That's because the infectious pathogen is encoded in the genome. ...Keep in mind that prion proteins go back hundreds of millions of years, so it's not like having a retrovirus in your genome that gets activated. It's something different and a very interesting challenge that I suspect will require more of the wet lab-type analysis than from any other type of approach."

Understanding the long history of prions in the genomes of various mammals, as well as gene paralogs, is also playing a role in elucidating this complex disease agent. Earlier this year, a team of researchers from McGill University used homology detection methods, including pGenThreader, Hidden Markov Models, and PSI-TBlastN, to obtain a complete annotation of the prion protein gene family in the genomes of 42 vertebrates. The group found that Doppel, one such prion protein paralog, was likely present in the last common ancestor of present-day tetrapods, and a transcribed pseudogene derived from Shadoo, a newly discovered glycoprotein that is conserved across primates and may play a role in regulating the meiosis gene SYCE1. They also showed that the gene which encodes for the prion protein at the human locus is conserved across at least 16 mammals and evolves like a long non-coding RNA, fashioned from fragments of ancient elements.

Pierluigi Gambetti, director of the National Prion Disease Pathology Surveillance Center at Case Western Reserve University, is focused on answering the big questions about prion disease. He is attempting to gain insight into the individual pathogenic steps that occur between the nascent formation of the normal prion protein and the development of its many disease forms. "A signature of prion diseases are the vacuolization degeneration and sponge formation, but researchers do not yet know why they form. We have no idea how to prevent it, how they form, and also where the disease starts — is it everywhere at the same time or specifically at one place, then spreading out to other regions of the brain?" Gambetti says. "Those are the areas where we need much more knowledge, biotechnology development, and detailed understanding of the confirmation of the normal prion protein, locate where it forms and how it forms, if we want to block this. … It's like trying to fix a car engine, but you actually don't know how the engine functions, so you are partially blind to what you're doing."

It was thought that a reliable principle to follow is that when the disease is different, the prion protein is different, which would then lead one to conclude that a patient has a different genotype. But Gambetti's research has helped make clear such convenient simplicity does not always work. He and his colleagues at the prion center recently identified the first new prion disease since the discovery of Creutzfeldt-Jakob disease in 1920. This new disease is called variably protease-sensitive prionopathy. This new sporadic form of prion disease differs from CJD in that its duration is longer and includes some signs of psychiatric pathology and Parkinson's disease. "The striking difference was not only clinical but pathological as well — we wondered whether we could theoretically come up with six different diseases, and we got very close to it; the mechanism was essentially proven, but had interesting exceptions," Gambetti says. "We felt that each of them had a different disease–it did not turn out to be exactly like that."

This newly discovered disease can affect individuals with any of the three types of the prion protein gene identified as 129VV (valine-valine), MV (methionine-valine), or MM (methionine-methionine) that result from the special feature of the prion protein gene encoding for either one of the two amino acids methionine or valine. "You have patients that are MV that are type 1 and patients that are MM but that are indistinguishable, so you can see that the prion protein gene is kind of silent, it doesn't contribute to a variation in the disease," Gambetti says. "You have a situation in which the gene, although different, does not modify the disease. … That is an additional complication, from the prion protein gene, when we move to the whole genome, we are playing a completely different and more complicated game."

Slow pace

The difficulty in studying prion diseases is the length of time it takes to complete some experiments and obtain definitive data due to an incubation period that moves at a snail's pace — which is probably not helping researchers come to a quick and sound resolution to end all the disagreement. While new assay technologies have definitely helped ramp up the pace of prion research, there are still many tests that researchers must conduct that take years to complete. "We have had a lot of development with new in vitro biochemical assays which have partially replaced animal bioassays, but on occasion, we do still have to go back to animals and it takes years, because with some experiments you need to show that you are actually working with transmissible forms of prions, especially if you need to do two or three serial passages," Maryland's Baskakov says.

The next big piece of data many prion disease researchers would like to have in hand is a full description of the atomic level structures of the various infectious forms. This is key in the development of not only therapeutics, but also better tests with increased sensitivity and accuracy. "This will require developing more robust protocols for generating the infectious form in vitro first, because right now the current protocol is difficult to work with and it doesn't generate sufficient amounts of infectious material for us to look at structure," he says. "Eventually some breakthroughs will have to generate a sufficient amount of material for structural studies, but so many steps have to happen, probably in five or 10 years, that will be the target."

As far as the CDC investigators like Maddox are concerned, the most pressing concern is keeping this area of research well-funded, even when there is not a serious threat to the public. "There has been a decrease, or the threat of a decrease, in research funding because, while we have good news in the variant CJD outbreak not resulting in the number of affected individuals that many initially predicted, the outcome of this is the perception among some funding agencies that money should be spent elsewhere," he says. "Funding is still important and is currently allowing collaborations between prion researchers and scientists working on other neurologic disorders, with the goal of a mutually beneficial exchange of knowledge and the identification of commonalities between different diseases that may lead to more effective screening and treatment."

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