The Science and Science Fiction of Prions

The artistamp commemorates the scourge of Mad Cow disease when it was destroying the livestock in UK beginning in 1986 (Wikimedia, Michael Thompson)

 

A made-for-TV terror plot.

It is season 7 of 24, a bioweapon developed and tested in a brutalized African nation under the heel of a delusional despot, turns up in the US in the shipyards of Alexandria, Virginia on the outskirts of Washington, D.C.  Gallant Jack Bauer hijacks a flatbed truck hauling a shipping container with the bioweapon inside. The shipping container is damaged during the chaos of the hijacking and one of the canisters containing the bioweapon begins to leak.  Jack takes a deep breath and enters the shipping container to stop the leak. He is nonetheless exposed to the weapon. The time is 10:55 PM. (If you haven’t seen the show, a digital clock indicating the timeline appears before and after each commercial break).

 

Two episodes later, at precisely 12:25 AM, Jack has his first seizure. Someone injects Jack with what sounds like “Pendoprine.” The drug controls his shaking but Jack will need to inject himself every two hours.

 

Some hours later, with the mother lode of pathogen destroyed, the would-be terrorists capture Jack hoping to harvest his brain and spinal cord to begin to rebuild their weapon stocks. Wracked with painful seizures, Jack still manages to escape.

 

Knowing that his body remains the sole source of the deadly pathogen, Jack contemplates self-incineration. But luckily for fans of the show, the need to tie up a number of loose ends left in the many plots and subplots that characterize each season of 24 distracts Jack from this final act of self-sacrifice.

 

At the end of the last episode, just before dawn, and having accepted his fate, an ambulance whisks away our nearly comatose hero to serenely await his certain and imminent demise. At the eleventh hour (figuratively), Jack’s estranged daughter step’s forward to offer her stem cells for an experimental treatment that could cure her father, restoring hope for another season of 24. Halleluja!

 

Prion primer

 

So what is the bioweapon from which Jack Bauer was so determined to save us? It is a “weaponized prion.” Although most of us may have heard the word “prion” in connection with stories about mad cow disease, it is less likely that we appreciate the truly bizarre nature of this unique pathogen.

 

Mad cow disease certainly scares the bejeezus out of beef farmers. And it’s not just because cattle with the disease rapidly become “downers,” the designation ranchers give to cattle that can’t stand up.  The impact on the pocketbook is catastrophic.  In  2003 the discovery of a single mad cow in Washington State, one that apparently originated in Alberta, caused the hasty erection of trade barriers that ultimately cost the Canadian economy more than $5 billion!

 

Why does Jack have seizures? A thin slice of brain of a cow that has died of an infectious prion disease has one feature that jumps out, obvious to even an untrained observer using a low power microscope. Holes reminiscent of the holes one sees in a natural sponge riddle the brain. The technical term for this pathology is “spongiform encephalopathy.”

 

 

Spongiform change in the brain occurs as the result of the wholesale death of neurons, the cells that are the functional heart of our nervous system— the ones that store our memories, collate the inputs from our senses and control our movements. It is the same pathology seen in the brains of sheep with a bizarre disease called “scrapie”, so-called because scrapie-infected sheep are so inclined to rub against things like fences and trees that they scrape off most of their wool. It is the same pathology seen in experimentally infected apes, hamsters, and mice.  It is the same pathology seen in the brains of people who have died of an exceedingly rare and deadly disorder called Creutzfeldt-Jakob Disease or CJD.

 

 

The other feature of prion-infected brain tissue that requires more sophistication to detect are “plaques”, dense deposits of molecules (specifically, protein molecules) in and around the deteriorating tissue.

 

When scientists purified these deposits and examined them using a powerful electron microscope they found the plaques contained rod-shaped particles. The particles are made up entirely of protein. And most importantly, this purified material is potently infectious and can cause spongiform encephalopathy when injected into healthy animals!

 

 

“Prion” was the term coined by Nobel Laureate Stanley Prusiner as a mashup of the words “pro(teinaceous) in(fectious particle)” to distinguish this novel pathogen from all other known pathogens such as viruses, bacteria, fungi or parasites. It’s arguable whether viruses are actually “alive” since they do not have all the genes necessary to sustain life on their own. But even at this genetically stripped-down level, every one of these pathogens has a nucleic acid (DNA or RNA) genome. Prions have no DNA or RNA, only protein.

 

 

Long chains of amino acids link together to form proteins.  You can read and understand what I’ve written here because each word has a unique sequence of the 26 standard letters in our alphabet. Because there are 20 possible amino acids, so too does every protein have a unique sequence determined by the sequence of the gene that encodes the protein. Prusiner’s group deciphered short stretches of the amino acid sequence of the the major protein deposited in prion plaques. With this vital information in hand they were able to find the gene that directed cells to make the protein.  Surprisingly, a foreign organism did not produce the prion protein. The genes of the victim itself encoded the deadly protein!

 

We humans and all of our vertebrate cousins on the evolutionary tree make the “prion protein” called PrP.  As a scientist who has discovered a new protein, the first question one wants to answer is “What does this protein do?”  One fairly straightforward approach is to use genetic engineering to remove the gene encoding the protein in a mouse.  When so-called “knock-out” mice display defects in the development or function of organs, tissues or cells, then one has an idea of what the protein does in the body.  From this perspective, it was disappointing for the scientific community to discover that mice that do not have any PrP are healthy and happy.1  In spite of more than 20 years of intense investigation we still have no unequivocally accepted idea of what PrP is doing.

 

What we have learned is that PrP has a largely solitary existence tethered to the surface of most cells in our bodies. It is easily destroyed by certain chemicals or mild heat and readily digested by the kind of protein degrading enzymes you might find in stain-fighting laundry detergents or meat tenderizers. Infectious PrP is exactly the same protein— it has the same sequence of amino acids linked together—but it folds into a different shape and thereby acquires distinct properties. In contrast to normal PrP, PrP in the diseased brain is found in long crystalline aggregates of millions of PrP molecules. And it is highly resistant to destruction by heat or digestion by enzymes.

 

The “Prion Hypothesis” is the startling idea that explains how this alternative form of PrP is infectious.  It posits that when PrP in a prion comes in contact with a solitary PrP molecule minding its own business on the cell surface, it serves as template for the structural rearrangement of the normal PrP into the pathological form. Thus a single infectious particle grows by recruiting normal PrP one molecule at a time. The rods fragment and the infection spreads, slowly but surely through tissues and organs.

The animation shows how infectious PrP (the aggregate on the right) induces normal PrP (coming in from the left) to fold into the infectious form.

 

This method of propagation helps to explain puzzling features of the transmissible spongiform encephalopathies. First, these are slow diseases. Under ideal conditions, say in a laboratory mouse, it may take a couple of months following infection for an animal to show signs of disease. In humans it may take decades. This is counter to how we typically experience infectious disease on our lives. When exposed to someone with a cold, we can generally expect to feel the first symptoms with in a couple of days. And those symptoms can include fever, an auspicious sign that our immune system has detected a foreign invader and is mounting a vigorous defense.

 

And there lies the second feature that stumped scientists and physicians about this disease. Here you have an infectious disease, causing wholesale decay of parts of the brain and the immune system is completely oblivious! But there is no foreign invader to present to raise the alarm and activate the immune response. It is, after all, our own protein taking on a new structure and new properties that is wreaking havoc in the brain.

 

 

Plausibility Analysis

 

So how plausible are the events leading up to and following Jack Bauer’s  fictional encounter with “weaponized” prions?

 

Making prions into a bioweapon requires overcoming several substantial obstacles. Attempts to manufacture infectious prions from PrP made in genetically engineered bacteria have disappointed scientists (from a purely basic science perspective that is).  For now the central nervous system tissue of a diseased individual is the only source of highly infectious prions.

 

It is another curious feature of the transmissible spongiform encephalopathies that prions made in the brains of one species, hamster for example, are only weakly effective in causing disease in a different species, like mouse. We think this is because mouse PrP has a slighty different amino acid sequence than hamster PrP. But once hamster prions have taken hold in the mouse brain, the next generation of mouse-adapted prions are highly infectious in mice.

 

This so-called “species barrier” suggests that with current state-of-the art technology, any prion weapon would be adapted by passage through a human host and manufactured by extraction of the victims’ brains and spinal cords. Indeed, the R & D effort would need a steady stream of human (literally) guinea pigs as test subjects and as incubators for pathogen amplification.

 

Let’s imagine that the repressive regime that created the weaponized prion in the TV show were able to provide the right research environment to develop and manufacture a highly purified super prion. How would the pathogen be delivered to the target population?

 

In the episode, a stream of vapor escaping from a damaged pressurized cylinder exposes Jack Bauer to the bioweapon. I am guessing that the idea was to release the prions in some form of aerosol that would cover a broad area. Because prions are particulate, I have to assume that they have done something to reduce the size of the particles to a size that could be encapsulated by a tiny droplet and float indefinitely in the air like a miniature, deadly dandelion seed.

 

PMCA (prion misfolding cyclic amplification) is one of the most recent innovations in prion diagnosis.  Intense sound waves sheer pre-existing prions into many smaller particles that can convert normal PrP into infectious prions much more efficiently than just a few large particles. Perhaps a similar method could be used to create compact, highly infectious particles for the bioweapon.

 

The next question is, “How do prions get into the body?” Let’s say that, even though Jack held his breath when inside the shipping container with the leaky canister, some prion laced aerosol managed to come to rest on his olfactory epithelium, the tissue in our noses that holds the tips of olfactory neurons. These neurons contain the odorant receptors that detect volatile molecules in the air. Electrical signals travel along axons, long thin cell extensions that penetrate like wires through a thin section of the skull and connect the olfactory epithelium to the olfactory bulbs of the brain.

 

It is known from experiments using isolated cells growing in Petri dishes in the lab, that cells take up prion particles. Suppose some of these particles moved along the axon of an olfactory neuron toward the brain. The rate of such movement is on the scale of much less than one centimeter per day. So by this route, it might take a day or more for the first infectious particles to reach the brain.

 

The other way to become infected with prions is to ingest them. That’s how cattle get infected. One of the consequences of modern industrial cattle farming is the use of high-protein artificial feed that makes cattle grow faster and produce more milk. One of the components of this feed is “bone meal” which is basically the ground up leftovers of the slaughtering and rendering industries, including bones, brains and spinal cords.

 

Because sheep scrapie is fairly common it is likely that sheep prions were probably the first pathogens to contaminate high-tech cattle feed. Most likely, cattle ate infected material for some time before the sheep prion jumped the species barrier and became a cow prion. Once the bones, brains and spinal cords of infected cattle came back into the feedstocks, the die was cast.

 

The culture of modern industrial farming forced the practice of cannibalism upon livestock animals. Cannibalism among humans is also a cultural dictate. The Fore tribe in Papua New Guinea used to practice ritual cannibalism as a means of honouring their deceased ancestors. At some point in Fore history there was likely one of those one-in-a-million cases of CJD in an older person whose body was later consumed by his or her extended family members. The men of the tribe would eat muscle tissue and women and children would feast on the leftovers including the brain, by far the richest source of prions.

 

Thank you Randall Munroe http://xkcd.com

 

The Fore called this disease “kuru.” The disease was thoroughly studied by Carleton Gadjusek2 who found that the mean incubation time for the disease was 14 years. For health reasons, the Fore banned cannibalism, but cases of kuru continued to show up for decades. Gadjusek was a virologist and without any real evidence suggested that a “slow virus” caused kuru.  We now know that this very long incubation time likely represents the time it takes prions to propagate in peripheral organs and begin that slow crawl through the central nervous system to the brain where it causes damage.

 

Even if prions bypass the rigmarole of finding their way from the point of entry to the brain, there is still a considerable lag between infection and the onset of symptoms. Scientists inject infectious prions directly into the brains of experimental mice and yet it still takes two to four months for the mice to show disease symptoms.

 

In humans, CJD occurring without any known infection or genetic predisposition is extremely rare (1 case per million) and occurs in old age.  A few people have been unwittingly infected with CJD. Sometimes a surgical procedure or a penetrating brain injury tears or punctures the leathery covering of the brain called dura mater.  Surgeons can patch the hole by grafting dura mater harvested from cadavers.  If the donor cadaver happens to have belonged to someone who died with undiagnosed CJD… well you get the picture. Even after this direct exposure of the brain to prions, it takes about 18 months for the recipient to become symptomatic.

 

Prion disease is very slow to develop even if prions don’t have to take a roundabout route to the brain. Yet Jack Bauer is showing severe movement impairment only 90 minutes after exposure! We need to accept that somewhere in the back-story of the script, the terrorists have undertaken a sophisticated development program to overcome the inherent sluggishness of prion disease.

 

Then there’s the treatment Jack gets to reduce the spasms he experiences. In fact the only symptom of prion disease that is routinely treated are the involuntary spasms (myoclonus) that characteristically develop in CJD patients.  These are usually eased with antiepileptic drugs. So that’s a pretty medically sound tidbit from the show.

 

The scene where the conspirators, having had their weaponized prion stock destroyed, prepare to harvest Jack Bauer’s brain and spinal cord to restart their bioweapon program. This is an entirely plausible scenario. We can appreciate that all the new prion particles accumulating in Jack’s brain maintain the unique structural characteristics of the super prion.

 

In the last scenes of the season finale, Jack’s condition is worsening by the minute. Let’s accept (for the moment) that there is an “experimental stem cell treatment” that could save his life. Won’t Jack be dead before anyone can intervene?

Unlike the brisk pace of decline depicted in the TV show, the time from first symptoms to death for real patients with CJD is around a year. In the TV show it looks like Jack will be dead within hours. Standard medicine offers nothing to slow the progression of the disease. But there is at least one remarkable exception to this general rule.

 

In 2001, an athletic 17-year-old in Dublin named Jonathan Simms, began to show symptoms of a sinister new disease showing up in Europe called “variant” CJD3 or vCJD, after eating prion-contaminated beef during the devastating outbreak of mad cow disease that reached its peak in 1992.

 

Knowing his son had, at most, only a year to live, Jonathan’s father, Don, quit his job as an electrician and began devouring scientific and medical literature. He found a scientist who had discovered that a common drug called pentosan sulfate could prevent the conversion of normal PrP to the pathogenic form in the test tube. But when he heard that the molecules of the drug would not be able to pass from the blood stream into the brain, Don found an oncologist who had developed a pump designed to infuse anti-tumor drugs directly into the brain to treat cancers. He put these two together and then fought the medical bureaucracy for permission to have his son’s brain infused with pentosan sulfate.

 

Amazingly Jonathan Simms died only earlier this year (March, 2011) having survived 10 years with the disease. From this inspiring story of love and persistence on the part of a devoted father we have learned that extraordinary measures can extend life of CJD patients.4

 

So let’s accept that doctors could keep Jack alive long enough to execute their plan. Could the experimental stem cell treatment that apparently cured Jack Bauer’s disease between seasons 7 and 8 of 24 actually work?  On the positive side, scientists report that implantation of neural stem cells into prion-infected mice delays the onset of symptoms and extends the life span if the procedure takes place before the mice are sick. The effects are very subtle but significant. On the negative side, if the transplantation is undertaken after symptoms have already started, the benefits are nil. This is not good news for Jack Bauer.

 

Even if the stem cells implanted in Jack’s brain “took” and started to replace his lost neurons, the new neurons would have PrP expressed on their surfaces and would not be exempt, in the long-term, from the effects of the prions already in Jack’s brain. There is perhaps one ray of hope filtering through the gap between science and science fiction.

 

Remember those mice I mentioned that have no PrP and are happy and healthy but uninformative? One tremendously informative feature of these mice is that they stay healthy and happy even after having been injected with massive doses of infectious prions! The lesson this experiment teaches is that the injected prions are not themselves toxic. Instead, there is something that happens during the conversion of normal PrP into the prion form that kills neurons.

 

Given early diagnosis, the donor stems cells could be infected with a benign virus that would deliver something called “small interfering RNAs” that could specifically eliminate PrP expression.  Neurons developing from these stem cells would be immune to the effects of pre-existing prions. It is within the realm of existing science to propose that such viruses could also be used to directly infected into Jack Bauer’s beleaguered brain and rescue his remaining neurons.

 

Conclusion

 

24 is pure TV fantasy (obviously). In fact the whole premise, not just it parts, is improbable. A weaponized prion that has the properties described in the show might be produced with a huge investment in research infrastructure, recruitment of highly trained scientists with corruptible ethics, and a huge amount of “luck” in overcoming the obstacles I’ve laid out.5 Truly horrific acts of terrorism are perpetrated without the level of innovation required to pull off this particular scenario.

 

Prions remain intriguing entities. Besides being the only non-living pathogen we know of scientists continue to moil over many outstanding issues. What function does PrP perform in the healthy animal? What is the mechanism by which the prion particle serves as a template to recruit normal PrP? Why is the conversion reaction toxic and why does it only kill neurons in particular parts of the brain but do not cause problems for other tissues in the body like muscles or lymph nodes where conversion also takes place? How can the disease be definitively diagnosed in living patients and will we be able to offer any real treatment options down the road?

 

This is a very rare disease in humans. But unlike most similarly rare diseases, prion disease attracts a truly intense level of scientific scrutiny.  There is nothing mundane in the prion world. Doubtless the bizarre features of prion disease will continue to stir the imaginations of scientists (and script writers) for years to come.

— John Glover

 

UPDATE NOVEMBER 19, 2011:

A few years ago I did a sabbatical in the lab of Neil Cashman at the Brain Research Centre at the University of British Columbia. Part of the reason I was there was because I wanted to do some experiments with infectious prions and Neil has one of the few facilities with an appropriate biosafety level to do so. Working in that room involved entering an antechamber, shedding my wallet and phone, donning a disposable paper suit and booties and a surgical mask, and putting on two pairs of latex gloves, one over the other. One of the pieces of the equipment in the room was used for sonicating prion-containing brain homogenates. I had wondered what this thing would look like as I was aware that sonication is an excellent way to create aerosols, the same mode of delivery of the bioweapon I talk about in the article. I was glad to see that the apparatus was covered to isolate it from the rest of the lab, and that the tubes containing the brain homogenates would remain sealed during the procedure. My personal experience informed my analysis of the plausibility of aerosol-borne prions.
Maryam Rahnama, a grad student at SFU and a Crux reader, alerted me to an article that I had missed, showing that mice exposed to nebulized (turned into an aerosol) brain homogenates from other mice with infectious prions, caused disease in the exposed mice. In my article, I left out much of the detail about how scientists understand the role of certain types of immune cells in the amplification of prions outside the central nervous system. In the experiments described in this paper, mice that lack the cells required for amplification of prions in the peripheral organs get prion disease just as rapidly as the mice who are intact indicating that the route to the brain is direct. They conclude that the olfactory epithelium or the eye are the most likely routes of entry since these are spots where the central nervous system comes in closest contact with air.
I haven’t bothered my readers with endless references to primary scientific literature but this paper is published by the Public Library of Science (PLoS) and freely accessible without a subscription. The URL is: www.plospathogens.org/article/info:doi/10…/journal.ppat.1001257
Thanks Maryam!

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Footnotes:

 

1. A number of research groups simultaneously created PrP knockout mice. Some knockout mice were pretty much normal while others suffered neurodegeneration. We now know the engineering strategy used in the mice that got sick resulted in an unexpected rearrangement of genes. Tracking down this “error” and figuring out why the mice were sick took years. As often happens in science, the illness in the mice where the engineering had gone awry ended up being more informative than the wellness of the mice in which the engineering went as expected. It turns out that these mice have acquired the ability to express a distant cousin of PrP in the brain that is normally expressed only in testes.

 

2. Gadjusek, an American virologist, won the Nobel Prize in 1976 for figuring out that kuru was an infectious disease and arguably saved thousands of Fore from disease by providing a strong scientific argument for the banning of ritual cannibalism. However Gadjusek was a complex and singularly distasteful figure. The video “The Genius and the Boys” documents the inglorious aspects of his behaviour.

 

3. Each of us has two copies of every gene in our bodies as the result of having pairs of chromosomes.  Both PrP-encoding genes in all known cases of vCJD encode the amino acid methionine at position129 (the 129th amino acid in the PrP amino acid sequence).  The more common version of PrP has the amino acid valine at position 129. Scientists discovered that PrP with methionine at position 129 is more prone to being recruited by the prion particles present in meat from mad cow afflicted cattle. This doesn’t mean that people with the more common version will never get the disease. It is possible that the disease in these people will have a very long incubation time before the onset of symptoms.

 

Interestingly, scientists have recently gone back to sequence the prion gene from tissue samples of kuru victims and from other Fore tribe members who participated in ritual cannibalism but did not get kuru. Scientists discovered a new version of the PrP gene encoding a different amino acid at position 127 in more than half of the survivors. This version of the gene was never found among those who died. This novel form of PrP is resistant to being recruited by prion particles. The variation is found nowhere else in the world except in the Fore, and then only in the geographic region where they practiced ritual cannibalism most diligently. This may represent a chance mutation occurring in a single person within the last couple of centuries (very recently in evolutionary time scales) and then subjected to intense selection by the cultural practices of the people in a position to inherit the gene.

 

 

4. Including Jonathan Simm’s story in what seems like a trivial exercise (analyzing a TV show) is not meant to demean the tragedy of Jonathan Simms or his family. There have been no large-scale trials of the pentosan sulfate treatment that would prove its efficacy in treating CJD patients more generally. One big problem is that in its early stages, when treatment might actually have some benefit, CJD looks like any number of other neurodegenerative conditions.  A definitive diagnosis can only be made by post-mortem examination of the brain. The inability to make a diagnosis at an appropriate time makes it unethical to treat patients who may not benefit. The Jonathan Simms story is still moving for other reasons.  There are other extraordinary stories of people driven by parental love to penetrate the mysteries of science and actually create new ideas at the forefront of medicine. One of these is the story of Lorenzo Odone and his parents Augusto and Michaela, dramatized in the 1992 movie Lorenzo’s Oil. You can read a good article about Lorenzo’s Oil here: http://www.distrofiamuscular.net/lorenzo_oil_dmd.pdf.

 

 

5. In 2002 the US Department of Defense appropriated $42.5 million to support scientific research on prions. Did the DoD want scientists to work through the bioweapon scenario like the one played out in 24? Not really. At least not in the publicly available documents. It turns out that the DoD is more concerned about the risk that food and blood, two commodities the military needs in vast quantities, could be contaminated by prions. Given the outbreak of mad cow disease in Great Britain, the later appearance of variant CJD, and the banning of blood donations from people with even a whiff of risk of prion infection, this concern seems justified. They wanted to promote science that would produce sensitive product safety tests, early diagnosis procedures, and novel treatments for affected personnel. Find out more here: http://cdmrp.army.mil/pubs/press/2002/prionpreannc.shtml