Stanley Prusiner (1942) received the 1997 Nobel Prize in Physiology or Medicine for discovering the agents responsible for the transmissible spongiform encephalopathies—diseases so named because the brains of afflicted subjects contain numerous holes or vacuoles, which give them a spongy (“spongiform”) appearance under the microscope. These diseases include scrapie in sheep, bovine spongiform encephalopathy (“mad cow disease”) in cattle, and Creutzfeldt-Jacob disease (CJD) and kuru in humans. Each of these diseases is invariably fatal.
Pruisner’s discovery was iconoclastic in the extreme because the etiologic agents of these diseases, which can multiply and kill, are comprised entirely of protein! Since they can replicate, despite carrying no genetic information, they defy the very foundation of biology. Pruisner dubbed them “prions”—an acronym he derived from “proteinaceous infectious particle.” The spongiform encephalopathies are now more commonly called “prion diseases.”
How might an infectious agent, which contains no genetic information, replicate? Pruisner provided the answer. Prion proteins (PrPSc) (Sc for scrapie, the prototype prion disease) are misfolded forms of corresponding normal cellular proteins (PrPC), which are generally present in all vertebrates, and which are particularly plentiful in the brain. The PrPSc isoforms act as templates that cause the normally configured proteins to refold into the PrPSc configuration. Once underway, this conversion process might escalate exponentially. In that way PrPSc isoforms “replicate,” and their accumulation in the brain leads to the characteristic prion disease pathology. See Aside 1.
[Aside 1: Prion diseases were once referred to as “slow virus diseases,” where “slow” referred to the course of the disease, rather than the agent. All the prion diseases have a clinically unapparent incubation period that may last for as long as 50 years. But once symptoms emerge, the duration of the clinical stage is only a matter of months, and invariably ends in death. The length of the incubation period appears to be inversely correlated with the level of PrPC. The actual cause of cell death in prion diseases is not known.]
The medical relevance of Pruisner’s discovery of prions, and of their mode of replication, may be much more significant than merely their association with the relatively rare infectious prion diseases. That is so because similar aggregates of misfolded proteins have since been observed in the much more widespread Alzheimer’s and Parkinson’s diseases, as well as Lou Gehrig’s (ALS) and Huntington’s diseases. Misfolded PrP-like proteins associated with Alzheimer’s disease include amyloid-β and tau. In Parkinson’s disease, these aggregates are comprised of α-synuclein. [The entire family of the PrP-like misfolded proteins are referred to as amyloids.] Thus, Pruisner’s discovery may have significant implications for the diagnosis and treatment of much more prevalent neurodegenerative diseases and dementias. See Aside 2.
[Aside 2: Potential therapies include the targeting of toxic species of PrP with monoclonal antibodies or with other ligands that bind to amyloid aggregates. Apropos that, in November 2016, the drug solanezumab, which targets amyloid, failed in a clinical trial to determine whether it might help people with mild dementia. Critics of the “amyloid hypothesis” (who still remain; see below) cited the failed trial as evidence against the amyloid premise. For a review of evidence in support of the amyloid hypothesis, and for analyses of the meaning of the failed solanezumab trial, see references 1 and 2. For a current review of the field, see reference 3.]
If Alzheimer’s and Parkinson’s diseases, as well as other neurodegenerative disorders such as ALS, indeed resemble the transmissible prion diseases, as disorders of protein conformation, then they also suggest new disease paradigms. Some of these diseases are infectious, while others are sporadic, or genetic. However, and importantly, some of these diseases may be transmitted by several of these various ways that determine the frequency and distribution of a disease in a population. For instance, Pruisner’s group demonstrated that CJD can be an infectious as well as a familial disease (see below). In the latter instance, it results from a particular mutation in the cellular PrP gene. Indeed, more than 20 mutations in PrP are now known which underlie inherited prion diseases. See Asides 3 and 4.
[Aside 3: It is not surprising that Pruisner’s proposal of an entirely new type of infectious agent—one comprised entirely of protein—met with considerable skepticism. Reflecting on the rather vicious ridicule that some of the naysayers subjected him to, Pruisner wondered “how the course of scientific investigation might have proceeded had transmission studies not been performed until after the molecular genetic lesion had been identified (4).”]
[Aside 4: Might Alzheimer’s disease be transmissible? There is no epidemiological evidence to suggest that possibility. However, bearing in mind that infectious prion diseases, such as CJD, can be transmitted during medical or surgical procedures (e.g., corneal transplant), it is reasonable to suggest that Alzheimer’s too might be transmitted by a physician’s or surgeon’s treatment. Other known routes of iatrogenic CJD transmission include injection of pituitary hormones obtained from cadavers, and intracerebral exposure to contaminated neurosurgical instruments. Apropos the latter possibility, amyloid-β adheres stubbornly to metal surfaces, and prions are highly resistant to sterilizations that would inactivate a true virus. Yet, because of the already high prevalence of Alzheimer’s disease in the population, and a possible decade-long non-clinical incubation period, the risk of iatrogenic transmission in that instance is not yet known. New (and expensive) methods have been developed for removing amyloids from surgical instruments, but they are not widely used because of the uncertainty of the danger of iatrogenic transmission.]
Here now is the story of Pruisner’s discovery of prions, with a nod toward how he persevered in the face of the widespread disbelief and scorn that his discovery engendered.
Pruisner first became interested in neurological diseases as a third-year medical student at Penn, during his rotation on the neurology service (5). However, that exposure to neurological diseases did not immediately affect his career goals. Instead, in his fourth year, he satisfied his desire to do research by investigating oxidative metabolism of brown fat cells. Nonetheless, his research on fat cells excited him enough to envision a career as a physician-scientist. “I was astonished that people actually got paid to solve puzzles every day—what a fantastic way to make a living (5).”
Pruisner continued his medical training as an intern at the University of California, San Francisco (UCSF). Providentially perhaps, he found his internship to be demanding enough to dissuade him of any thoughts of a career practicing clinical medicine. So, Pruisner spent the next three years at the NIH researching enzyme regulation in bacteria; an experience that he found gratifying enough to decide that a career in medical research would be his goal.
Next, Pruisner had to choose a research area. He was still interested in neurological diseases. “With its billions of neurons, its ability to affect all aspects of human activity and its endless mysteries, the brain seemed a perfect subject for research… (5).” To gain the background he would need for his new calling, Pruisner decided to carry out an “abbreviated residency” in neurology at UCSF.
The next step in Pruisner’s path would be choosing a solvable research problem. Here now is an example of one of those fortuitous happenings that can make a scientific career and, with a bit of luck, lead to a singularly important scientific breakthrough. “It was during my residency at UCSF that I encountered a patient with a rare progressively debilitating illness called Creutzfeldt-Jacob disease (or CJD), and the mysteries surrounding this illness launched my scientific studies for the next four decades (5).”
Pruisner was intrigued as well as perplexed by his CJD patient. She had suddenly developed severe intellectual and memory deficits, and myoclonus (jerky movements in her muscles). But more puzzling: “She exhibited no signs of an infectious disease…she did not have a fever, and she had no increase in white blood cells in either her blood or cerebrospinal fluid (5).” Yet CJD is a transmissible disease, as shown earlier by Carleton Gajdusek’s finding that the illness could be passed to a chimpanzee by injecting it with a brain extract from a dead human CJD patient. See Aside 5.
[Aside 5: Gajdusek also experimentally transmitted kuru, via homogenates of human patient biopsies, to chimpanzees. Moreover, his epidemiological studies showed that kuru was transmitted among the Fore people of New Guinea via ritual cannibalism. For more on this, and for an account of how Bill Hadlow first suggested that scrapie and kuru might have a similar underlying basis, see reference 6.]
Gajdusek also found that CJD symptoms did not emerge in injected chimpanzees until months after the inoculation; a finding that agreed with the prevailing view in the scientific community that the scrapie-like diseases are caused by “slow viruses”—a term originally coined by Bjorn Sigurdsson in 1954 while he was working on scrapie in Icelandic sheep. Gajdusek referred to the scrapie agents as “unconventional viruses,” although he had no knowledge of how they might differ from “conventional viruses.”
Pruisner became fascinated by the prospect of once-and-for-all defining the nature of the agents responsible for the transmissible spongiform encephalopathies. However, more experienced, and cautious colleagues at UCSF saw the scrapie project as fraught with too many pitfalls, and tried to steer Pruisner away from it. But he would not be deterred.
One of Pruisner’s UCSF colleagues alerted him to papers by radiation biologist Tikvah Alper, who incidentally trained with Lisa Meitner (7). Alper noted other bizarre properties of the scrapie agents. In brief, she found them to be extremely resistant to UV light and X-rays, which should have inactivated any “conventional” virus by damaging its DNA or RNA genome. And: “Since a single X-ray photon should be sufficient to kill a single scrapie agent, Alper was able to calculate the minimal size of the agent. She estimated that it was less than one-hundredth the size of a typical virus (5).”
Alper’s findings clearly suggested the provocative idea that the scrapie agent does not contain a nucleic acid genome. Yet Pruisner, like everyone else, held to the belief that some sort of “novel” virus was responsible for her unusual results. “What else could the “scrapie agent” be? There was nothing else (5).”
Nonetheless, in the back of Pruisner’s mind, he did not completely dismiss “…the most startling interpretation: All the data might be pointing to an infectious particle devoid of nucleic acid and thus with no apparent way to replicate (5).” Pruisner indeed was intrigued by this radical possibility. And, notwithstanding the advice of more experienced colleagues, Pruisner, a former chemistry major, believed that the scrapie problem would be easy. “It’s just a problem in protein chemistry (5).” And, if it were true that the scrapie agents contain no genetic information, “then it would be worth an enormous effort to decipher the structure (5).”
Pruisner’s first step would be to isolate the scrapie agent from brain homogenates; a feat not accomplished by Alper, nor by anyone else. To monitor his progress towards purification, Pruisner planned to assay his fractions by means of a biological assay, making use of the 1961 finding by British scientist, Richard Chandler, that scrapie disease could be transmitted from one mouse to another via injection of brain homogenates. Pruisner would employ the so-called “endpoint dilution” procedure, in which the titer of a sample is the last dilution (e.g., 1/2, 1/4, 1/8, etc.) able to induce scrapie infection.
But, as predicted by others, complications soon materialized: “…so little was known about the physical nature of the mysterious scrapie agent that hundreds of fractions would have to undergo titration measurements (5).” Additionally, Pruisner’s assay would require 60 mice to measure the titer of each sample. What’s more, since the scrapie incubation period could be a year or longer, some titrations might very well require that long as well. Consequently, Pruisner might have to maintain thousands of mice during this entire time. And, in the end, the assay might not be sensitive enough to show small increases in purity.
The above issues alone may explain why no one before Pruisner tried to systematically investigate the scrapie agent’s molecular nature. But, there is more. Even if Pruisner’s assays were sufficiently sensitive, the critical experiments would need to be repeated before they might be published. And, his findings would still need to be confirmed by others before they might be accepted. Furthermore, before Pruisner could make headway on this hugely expensive project, he would need to acquire a grant to support it. The NIH—the usual source for large biomedical research grants—appeared unlikely to provide that support, since its Virology Study Section held the view that a slow virus causes scrapie and that the issue should be approached as a virological problem, rather than by Pruisner’s chemical approach. Nonetheless, Pruisner was undeterred, “The hubris of youth was all that propelled me forward (5).”
The NIH indeed rejected Pruisner’s application for support of his scrapie project. However, Pruisner did obtain modest funding at UCSF from the Howard Hughes Medical Institute, which enabled him to set out on his project. Meanwhile, William Hallow and Carl Eklund, at the Rocky Mountain Laboratory in Hamilton, Montana, had been studying scrapie pathogenesis in sheep and goats, and had made some failed “hit-or-miss” attempts to define the molecular nature of the agent. When they met Pruisner, his more systematic approach impressed them, and they then taught him “an immense amount” about scrapie. Importantly, they helped him to characterize the scrapie agent’s sedimentation behavior—a key step towards purifying it.
Pruisner then began to produce his first experimental results, which were curious in the least: “I had anticipated that the purified scrapie agent would turn out to be a small virus and was puzzled when the data kept telling me that our preparations contained protein but not nucleic acid (8).”
But while Pruisner’s findings raised the possibility that he might be on to something new and exciting, not all was going well for him. He lost his funding at UCSF from the Howard Hughes Medical Institute. Worse yet, UCSF told him that he would not be promoted to tenure. But, the tenure decision was reversed, and because he had by now developed a starting point for his studies, and because his early results suggested that the project might yield intriguing new findings, he was awarded modest support from the NIH, as well as more substantial funding from the R. J. Reynolds Company (really).
Pruisner’s rate of progress was significantly enhanced when he found that he could shorten the length of time needed for his assays by moving from mice to hamsters, in which 70 days were required, rather than the 360 days needed in mice. And, he also, redesigned his measurement method. By 1982, in addition to the results of his biochemical analysis (which implied that the scrapie agent was comprised entirely of protein), he also found that scrapie infectivity could be reduced by treatments that alter proteins, but not by treatments that alter nucleic acids. Believing that he now had sufficient data to support his premise that the scrapie agent is comprised only of protein, he published his findings in a paper in Science (9).
Pruisner introduced the term “prion” for the first time in the 1982 Science paper (9). But in doing so, he set off a “firestorm (8).” Most virologists were skeptical of his findings, and some competitors, who had been working on scrapie and CJD, were incensed by his claims. “At times the press became involved since the media provided the naysayers with a means to vent their frustration at not being able to find the cherished nucleic acid that they were so sure must exist. Since the press was usually unable to understand the scientific arguments and they are usually keen to write about any controversy, the personal attacks of the naysayers at times became very vicious (8).” Nonetheless, Pruisner was confident that he was right: “Despite the strong convictions of many, no nucleic acid was found; in fact, it is probably fair to state that Detlev Riesner (Aside 6) and I looked more vigorously for the nucleic acid than anyone else (8).”
[Aside 6: Detlev Riesner had been studying viroids when he met Pruisner. These agents, which mimic viruses, are small, naked, single-stranded, circular RNAs, that infect plants. They were discovered by Theodore O. Diener, a Swiss plant pathologist. Since the small size of viroids is consistent with Alper’s X-ray data, which showed that the scrapie agent too is extremely small, Pruisner sought out Riesner to inquire whether viroids might be the causative agents of CJD. The meeting between the two researchers led to a long-time collaboration, and both contributed to the discovery that the scrapie agents do not contain nucleic acids.]
By the next year Pruisner had isolated the scrapie prion protein, and the following year Leroy Hood (who helped found the human genome program) determined a portion of its amino acid sequence. Meanwhile, skeptics kept searching for a nucleic acid-containing scrapie agent. And while they never succeeded in their efforts to overturn the wealth of evidence Pruisner was accumulating in support of the prion hypothesis, the mystery of how prions might replicate still remained to be solved. Toward that end, Pruisner collaborated with Charles Weissmann to clone the cellular gene encoding the prion protein.
“Once cDNA probes for PrP became available, the PrP gene was found to be constitutively expressed in adult uninfected brain. This finding eliminated the possibility that PrPSc stimulated the production of more of itself by initiating transcription of the PrP gene… (8).” Moreover, with the isolated PrP proteins in hand, it was clear that PrPSc was not the translational product of an alternatively spliced mRNA. [“The entire open reading frame of all known mammalian and avian PrP genes resides within a single exon (8).”] Furthermore, PrPSc was not the result of a posttranslational modification of PrPC.
Pruisner and coworkers next considered the possibility that PrPC and PrPSc differed only in their conformations. However, bear in mind that the molecular biology dogma of the day held that the amino acid sequence of a protein specifies only one biologically active conformation of the protein. Consequently, the idea that PrPC and PrPSc differed only in their conformations “was no less heretical than that of an infectious protein (8).” Nonetheless, the results of structural studies indeed bore out the conformation premise. See Aside 7.
[Aside 7: For aficionados: “Fourier transform infrared (FTIR) and circular dichroism (CD) studies showed that PrPC contains about 40% α-helix and little β-sheet, while PrPSc is composed of about 30% α-helix and 45% β-sheet. Nevertheless, these two proteins have the same amino acid sequence!” The PrPSc structure enables amyloids to form their characteristic tightly interacting, many stranded and repetitive intermolecular β-sheets. Readers interested in these, and additional structural studies might begin with Pruisner’s Nobel lecture (4).]
Pruisner and coworkers then carried out a series of telling experiments that began to unlock the mystery of prion replication. The first step was to generate mice that lacked both copies of the mouse PrPC gene. Importantly, this treatment rendered these mice completely resistant to mouse PrPSc. However, when hamster PrPC genes were incorporated into the genomes of these mice, and were expressed in them, these transgenic mice then were susceptible to hamster PrPSc. However, the mice remained resistant to mouse PrPSc. Thus, in mice, the hamster PrPC transgene product was required to promote the replication of hamster scrapie prions, whereas the mouse PrPC protein was required to promote the replication of the mouse scrapie prions.
These results show that the scrapie PrP isoform and the normal cellular PrP protein each play crucial roles in the transmission and pathogenesis of prion disease. Importantly, these results are completely consistent with the “misfolding hypothesis,” in which the scrapie isoform catalyzes the conversion of the normal cellular PrP protein into the scrapie conformation.
The finding that expression of the hamster PrPC promotes (and indeed is required for) replication of the hamster PrPSc, but does not promote replication of the mouse PrPSc, is an example of the “species barrier” to prion infection—in which the passage of prions between species is generally restricted. Differences in the amino acid sequence homology between the PrPSc of one species and the PrPC of another species, which might impair their interaction, readily explain the species barrier. See Aside 8.
[Aside 8: Interestingly, despite prions not having genomes, prion “mutation” can occur, in the sense that prions encoded by the same PrP gene may assume different conformations, thereby giving rise to a kind of prion “polymorphism,” which may enable prions to cross species barriers by a process of conformational selection.]
In 1990, in another series of key experiments, Pruisner’s research group discovered a mutation of the human PrP gene (a leucine substitution at codon 102), which appeared to be linked to Gerstman-Straussler-Scheinker syndrome (a very rare, exclusively inheritable, progressive spongiform encephalopathy in humans). Next, they generated a recombinant mouse PrP gene that encoded the leucine substitution at codon 102. Importantly, transgenic mice, which expressed the recombinant PrP gene, developed a scrapie-like disease with many of the pathological features of Gerstman-Straussler-Scheinker syndrome. What’s more, inoculates, which contained brain extracts from those mice, transmitted the disease to inoculated mice. Thus, Pruisner’s group demonstrated that a prion protein, containing a single amino acid substitution, can be the cause of a human familial prion disease.
Only a portion of Pruisner’s contributions up until he received his 1997 Nobel award were noted in the above narrative. For a more complete review of his work until then, see his 1997 Nobel lecture (4). Pruisner is still investigating neurodegenerative and dementing diseases at the UCSF School of Medicine, where he also serves as the director of its Institute for Neurodegenerative Diseases.
As already noted, Pruisner’s career is also remarkable for his having persevered in authenticating his iconoclastic protein-only prion hypothesis, despite the continuing and widespread skepticism and ridicule from colleagues in the scientific community. In that regard, his story is reminiscent of Howard Temin’s after announcing his discovery of reverse transcription by the RNA tumor viruses; which eventually would be re-designated “retroviruses” (10).
Pruisner himself was unprepared for the level of resistance to his discovery of prions: “…this created a rather harrowing and arduous journey for more than a decade…Many argued that I was spewing heresy and I had to be wrong (5).” What’s more, the ferocity of some of the personal attacks against Pruisner, particularly those in the media, were affecting his family.
But what might have explained the extent of the enmity on the part of some naysayers? Could it have simply been that the prion hypothesis conflicted with molecular biology dogma of the day? Or could it have been, as Pruiner suggested, that some critics perhaps were reacting to their frustration at not being able to find the nucleic acid, which they were sure had to be there?
Resigned to the criticism, Pruisner stated: “When there is a really new idea in science, most of the time it’s wrong, so for scientists to be skeptical is perfectly reasonable.” And, stoically, Pruisner’s answer to his critics was to keep producing data. “The incredulity of my colleagues only strengthened my conviction that scientists have a responsibility to convince their skeptics of the validity and importance of discoveries that run counter to prevailing opinions, and they can do so only by performing experiments that challenge their own hypotheses. Sometimes the road of testing and retesting is long and arduous—such was the case for me (5).” Moreover, and to Pruisner’s credit as a scientist, he also attributed his tenacity to his fascination with prions.
By the late 1980s, enough scientific data (particularly the knockout mouse studies) had emerged to begin garnering a measure of acceptance for the protein-only prion hypothesis. Then, in 1996, with the appearance in Britain of the first human cases of mad cow disease (incidentally identified as such using techniques originally developed by Pruisner), prions were suddenly a hot topic in the media. And, the very next year Pruisner was awarded the Nobel Prize.
Did the spotlight on mad cow disease and prions influence the Nobel committee’s decision? Pruisner conceded that “It didn’t hurt.” And, he graciously admits to the part that luck may have played in his exceptional career. “Extremely intelligent men and women can toil for years in the vineyards of science and never be fortunate enough to make a great discovery. And then there are a few people who are recipients of mammoth doses of good luck. The infectious pathogen that we now call a prion might well have turned out to be an atypical virus—not nearly as interesting as an infectious protein…or another group instead of mine might have discovered prions; that sort of preemption happens all the time in science (5)”
Yet some of Pruisner’s critics remained skeptical of the prion hypothesis even after he was awarded the Nobel Prize. For example, consider the following excerpts from a 1998 Science paper by Bruce Chesebro (11): “Although the notion that “protein only” can account for the infectious agent has received considerable publicity as a result of the Nobel prize award to S. Prusiner for the discovery of prions, the fact remains that there are no definitive data on the nature of prions… There are arguments both for and against the hypothesis that abnormal PrP itself is the transmissible agent, but on either side of this controversy no argument is as yet completely convincing … Clearly, we are in the very early stages of exploration of this subject. It would be tragic if the recent Nobel Prize award were to lead to complacency regarding the obstacles still remaining. It is not mere detail, but rather the central core of the problem, that remains to be solved.”
References:
1. Abbott A. 2016. The red-hot debate about transmissible Alzheimer’s. Nature 531: 294–297 doi:10.1038/531294a
2. Abbott A and Dolgin E. 2016. Failed Alzheimer’s trial does not kill leading theory of disease. Nature doi:10.1038/nature.2016.21045
3. Collinge J. 2016. Mammalian prions and their wider relevance in neurodegenerative diseases. Nature 539:217–226.
4. Pruisner SB, Prions, Nobel Lecture, December 8, 1997.
5. Pruisner S. Madness and Memory: The Discovery of Prions-A New Biological Principle of Disease, Yale University Press, 2014.
6. Carlton Gajdusek, Kuru, and Cannibalism, Posted on the blog April 6, 2015.
7. Max Delbruck, Lisa Meitner, Niels Bohr, and the Nazis, Posted on the blog November 12, 2013.
8. Stanley B. Prusiner – Biographical”. Nobelprize.org. Nobel Media AB 2014. Web. 24 Nov 2016. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/1997/prusiner-bio.html>
9. Prusiner, SB. 1982. Novel proteinaceous infectious particles cause scrapie. Science 216:136–144.
10. Howard Temin: “In from the Cold,” Posted on the blog December 14, 2013.
11. Chesebro B. 1998. BSE and Prions: Uncertainties About the Agent. Science 279:42-43.
DOI: 10.1126/science.279.5347.42
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