Vero Prion Review

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ART I C L E Prions: Introducing a Complex Scientific

Controversy to a Biology Classroom

IGOR V . ZAIT S E V

It has been almost 50 years since Thomas Kuhn, in The Structure ofScientific Revolutions, posited that science does not progress by thesteady accumulation of knowledge, but rather by a system of com-petition among paradigms. They vie for supremacy through greaterparsimony, explanatory power, and popularity among the commu-nity of scientists (Kuhn, 1962). The current controversy concerningthe identity of prions (PrPs) (proteins devoid of the nucleic acid) asthe infectious agents of transmissible spongiform encephalopathies(TSE) elucidates all the issues involved in just such a debate.

While modern biology high school and university textbookscover many scientific controversies that have been resolved decadesand even centuries ago, they fail to cover current scientific disputes.This article is intended to address such an omission by introduc-ing the prion controversy in biology classes in high schools andcolleges.

In 1982, biochemist and neurologist Stanley Prusiner proposeda hypothesis concerning infectious proteins. He identified them asabnormal prions, proteinaceous infectious particles capable ofconverting normal prions (naturally present proteins in mammals)into an abnormal form causing a fatal disease of the central nervoussystem (CNS) in both animals and humans. Heretofore, it had been

accepted that infections could be caused only by protozoans, fungi,bacteria, rickettsia, viruses, or viroids. Only nucleic acids, informa-tional polymers, were known to be able to duplicate themselves,not proteins. For the discovery of prions which Prusiner positedcan cause TSE, he received a Nobel Prize in 1997.

Were Prusiners hypothesis correct, our understanding of theorganic world would be changed forever. However, Laura Manuelidis(2007), one of most dedicated scientists in this field and the headof neuropathology at the Yale School of Medicine, contends thatprions thereby became canonized, although careful review of datarevealed many discrepancies. Indeed, even NobelPrize winners can err (Allchin, 2008), includ-ing Prusiner, and prions thus remain in the

realm of a hypothesis (Manuelidis, 2007).Despite overwhelming opposing

data published in The Lancet, Science,Virology, The Journal of Virology, Journal of Cellular Biochemistry, ViralImmunology, Journal of NeuroVirology,Proceedings of the National Academyof Sciences,and many other scientificpublications, most, if not all, biologytextbooks in the U.S. present prions asthe primary cause of TSE. While thereare scientists convinced of the ability ofabnormal prions to cause infections, thereare other scientists who, based on their obser-

vations and experimental data, do not think that prions couldbecome infectious. Manuelidis suggests that prions may simply bepart of the late stage of a disease, not part of the cause (Mihailova,2007), and PrP infectivity is questionable, and perhaps non-exis-tent (Manuelidis, 2007).

Cannibalism & the Rise of TSE

Since students seem more engaged when instructors incorporateexamples from popular culture into classroom discussions (Pryor2008), one might start a consideration of prions with mention oKurt Vonneguts science fiction novel, Cats Cradle (1963), beforeintroducing Deadly Feasts (1997), a shocking nonfiction case his-tory of the discovery and epidemiology of the fatal disease TSECertainly, truth is stranger than fiction if one were to contrasRichard Rhodes documented study and any of Vonneguts sciencefiction novels. Cats Cradle, concluding in an apocalyptic climaxconcerns the ability of a nucleant that can turn water into ice, justas an abnormal prion can allegedly turn its host prions into abnormal forms, resulting in this fatal brain pathology. Deadly Feastsbegins with a description of a burial ceremony that the womenof the Fore tribe used to practice in New Guinea, the last wild

place on earth. Sixty or more native women with their babies andsmall children, the family of a deceased woman, would gather tobury her in their stomachs rather than abandon her to rot in theground. Why should we throw away good meat? It is not right,one woman told an anthropologist (Rhodes, 1997). The mournerswould eat body parts, including the bones, which they charred inthe open fire to soften them, even the feces would be eaten, mixedwith edible ferns and cooked in banana leaves (Rhode, 1997). Forewoman recalled that cannibalizing the corpses of their kindredstarted within the lifetime of the oldest grandmother. I eat you,was a Fore greeting (Rhode, 1997). The deceased who died of

leprosy or diarrhea were not consumed.

By 1950, a disease called kuru (KOO-roo), whichmeans shivering with cold or fear, had killed women

in every Fore village. One of the most pronouncedsymptoms would be unprovoked laughterBecause of this, the disease became known aslaughing death. The victims would lose theirability to walk, shiver uncontrollably but notfrom cold or fear; their speech would becomeblurred. Finally, their ability to swallow wouldbe so impaired that their relatives would have

to chew food for the dying victims and forceit down their throats. Such symptoms were

considered to have been caused by bewitchmentNevertheless, the flesh of women killed by sorcery

was considered clean enough to be eaten by other

TSE continues to

spread throughout the

world, killing people who eat

the tissue of cattle infected

with BSE, children treated

with human growth hormone,

patients in surgery ... herds of

sheep, cattle, deer, elk,

and mink.

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women. Fore men were blamed for practicing sorcery on theirwomen and children, and were hated and feared by natives of NewGuinea. Medical researchers who encountered Fore womens symp-toms at first thought they were dealing with a new form of encepha-litis. The part of the brain of these Fore patients primarily damagedwas the cerebellum. Postmortem examinations revealed multipleholes in this part of the brain. Surprisingly, there was no inflamma-tion as in encephalitis. Interestingly enough, after the cessation ofcannibalism, kuru gradually disappeared (Gajdusek, 1977).

Another mysterious disease involving brain damage withoutapparent inflammation was discovered in Germany in 1913 by a young physician, Gerhard Creutzfeldt. He had a patient whosecheerful personality had abruptly changed. No longer wantingto eat or bathe, she began screaming that she was possessed of adevil and that she was dead and wanted to sacrifice herself. At thesame time, the woman had sudden outbursts of laughter, distractedspeech, tic-like jerks in her arm, fluttering facial muscles, alteredreflexes, and tremors that started up whenever shemade a voluntary movement. In her last hours,the womans swallowing was impaired and shewent into a deep stupor. Creutzfeldt, recognizingthat this was a new disease, reported his findingsin a German medical journal. Alfons Jakob read

Creutzfeldts paper and contacted him becausehe too had lost patients with similar symptoms.Thus, this disease was named Creutzfeldt-Jakobdisease (CJD). One of the most surprising charac-teristics of the kuru and CJD was that there was noapparent inflammation to the damaged brain. In1957, a neuropathologist at the National Instituteof Health, Igor Klatzo, was the first to associatekuru with CJD, in correspondence with virologistDaniel Carleton Gajdusek. Thereafter, research vet-erinarian William Hadlow realized that scrapie, thedegenerative brain disease he had studied in sheep,was also very similar to kuru and CJD in humans. Studying sheepbrain sections under the microscope, Hadlow identified cerebellar

holes and sponginess as also occur in the brains of kuru and CJDvictims, while it also affected the cerebral cortex in CJD, but not inkuru victims.

Scrapie was first documented about 1750. Scrapie-infectedsheep symptoms involve behavioral changes such as biting at theirlegs, itching, pulling wool from their sides, and abnormally react-ing to noise. Infected animals develop tremors and incoordinationthat progress to decumbency and death. It had been known since1930 that scrapie was infectious. The most common method oftransmission is from dams to their own and other offspring. Thereis no treatment for scrapie and animals die from one to six monthsafter the onset of symptoms.

If kuru and CJD have the same nature as scrapie, those fatal

human neurodegenerative diseases might also be infectious, and,of course, would raise public health concerns. In Gajduseks lab, inthe 1960s, brain tissue from kuru victims had been homogenizedand inoculated into those of chimpanzees. Within a couple of years,the animals showed the first signs of inactivity, shaking and trem-bling, and uncoordinated movements, quickly followed by furtherphysical deterioration. The brains of the sacrificed laboratory ani-mals were sectioned, and with further histological analysis, it wasevident that their brain pathology was indistinguishable from thatof the kuru victims. Indisputably, the disease had been shown tobe transmissible. If it could be passed on to chimpanzees, it couldbe passed on to humans. The connection between kuru and can-nibalism was no longer in question. CJD was not confined to New

Guinea, but was occurring throughout the world. British cows hada long history of having been fed protein supplements made fromscrapie-infected sheep offal and even infected dead cows. RichardRhodes called it an industrial cannibalism (Rhodes, 1997). Itshould not have been surprising that those cows developed symp-toms somewhat similar to those of infected sheep, which today isknown as mad cow disease or bovine spongiform encephalopathy(BSE). Consumption of contaminated beef led to spreading deadlyinfection to humans. Neither cooking, nor chemical disinfectantsnor irradiation deactivate the infectious agent and, at the presentime, there is no treatment for TSE. Scientists are racing to identifythe precise agent of the fatal disease, as the controversy of possiblesources is still unresolved. TSE continues to spread throughout theworld, killing people who eat the tissue of cattle infected with BSEchildren treated with human growth hormones, patients in surgery(transplants, transfusion, use of contaminated surgical instruments), herds of sheep, cattle, deer, elk, and mink. (See Table 1.)

Prions or Not Prions That Is theQuestion

Primary experiments have shown that the causal agent of TSEis capable of passing through bacterial filter. It causes no appar-ent inflammation, raises no fever, nor indicates any other signs orsymptoms of an immune response. Moreover, this agent was highlyresistant to such insults as boiling and even autoclaving. It wasalso resistant to disinfection with chloroform, carbonic acid, strongformaldehyde, and UV light. No bacteria grew in scrapie-infectedtissue, and none was visible under the light microscope.

Could the causal agent of TSE be a virus, a piece of bad newswrapped in protein, as Peter Medawar once quipped (Rhodes1997)? Many scientists reject this, citing its resistance to UV light

which is known to kill microorganisms by damaging their nucleicacids. An experiment done in the 1960s on the homogenate ofa scrapie brain with enzymes, known to damage nucleic acidsdemonstrated no reduction in infectivity while homogenates withenzymes, known to damage proteins, reduced infectivity by morethan ninety percent. So is the infectious agent a protein? At first, apositive answer to this question sounds like science fiction, sinceas far as we know, infections are not caused by proteins, but bymicroorganisms. We know that in order for them to multiply, nucle-ic acid is required. According to the current fundamental principleof biology, proteins cannot replicate themselves, causing infectionsas nucleic acid can. Prions lack any nucleic acid and, thereforeviolate the universal rule of protein synthesis. Francis Crick did

Table 1. Some forms of transmissible spongiform

encephalopathies in mammals.

SPECIES DISEASE ABBREVIATIONS

mankuru

Creutzfeldt-Jakob Disease

CJD

sheep scrapie

mink transmissible mink encephalopathy TME

cattle bovine spongiform encephalopathy BSE

deer

elkchronic wasting disease CWD


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not want to overlook the possibility of an exception, noting thatdiscovery of an exception will shake the whole intellectual basisof molecular biology (Rhodes, 1997). Even so, one must not jumpas yet to a quick conclusion. The infectious agent could be either avirus whose genome is protected from UV light inside a sturdy coatof protein, or a virus that is super efficient at repairing radiationdamage to its genome.

At this time, let us recall what Kurt Vonneguts Cats Cradle isall about. In this science fiction tale, a scientist creates a nucleant

capable of turning all the water on the planet, including the waterin human cells and blood, into ice. In 1995, Byron Caughey andPeter Lansbury borrowed Vonneguts scientific fantasy for the titleof a paper, The Chemistry of Scrapie Infection: Implications of theIce 9 Metaphor (Lansbury & Caughey, 1995). Vonneguts ficti-tious nucleant is a seed capable of converting nearby fluid waterto crystalline form. Gajdusek visualized a similar infective processat work in the TSE. He proposed that a nucleant crystal of abnor-mal PrP was the TSE infectious agent. According to his hypothesis,the abnormal prion is capable of converting a normal prion intothe abnormal conformation. Prusiner coined the name prion andenthusiastically pursued the proof of this hypothesis and, thus, wasrewarded a Nobel Prize.

The functions of prions in a healthy individual are still unclear.Some researchers suggest that they might play a role in the celldeath and neural excitability. All mammals produce host prionsin both diseased and healthy individuals. Prions are expressedacross the entire CNS, especially in the hippocampus, striatum,and frontal lobe. The unique sequences of amino acids in a prionmake it possible for these molecules to have two different, stabletertiary structures. One type, called a cellular (healthy) prionprotein (PrPC), has functional structure folds with many -helices.The abnormal prion protein (PrPSc) has many -plated sheets. Theyare the same protein but just folded differently. PrPSc is amyloidfibrils assembled in large structures. Prusiners team of researchscientists suggest that PrPScconverts -helices into -plated sheets(Pan et al., 1993) and thus transmits TSE (Prusiner, 1998). Let us

consider some scientific data that contradicts rather than supportsPrusiners prion hypothesis:

1. It has been established that one of the major routes of trans-mission of TSE is along the gastrointestinal tract in whichan infectious agent invades the mucosa and, thereby, infectsthe Peyers patches. However, recently it has been shownthat the PrP is rapidly destroyed by alimentary track fluids(Jeffrey et al., 2006). If so, it makes the ability of infectiousPrP to implant further, crossing the blood-brain barrier,almost impossible. The viral hypothesis does not contra-dict this new finding since we know that enteroviruses arecapable of withstanding acid and proteolytic secretions.

2. The presence of hundreds of different strains of TSE indifferent species also challenges the prion hypothesis. Thepresence of strains is one of the characteristic features ofan infectious agent with a nucleic acid. These strains havebeen successfully passed from one species of animals toanother, and even back to the original species (e.g., sheep tomice, then mice to sheep), preserving their singular strainidentity, despite PrP differences between sheep and mice(reviewed in Manuelidis, 2007). The presence of differentforms of PrP in those species during infection does notsatisfy the first of Kochs postulates that states that a patho-gen must be invariably present, in a constant form, in everycase of the disease. Consequently, how can protein particlesbehave as various strains while they display different identi-ties in a single strain of the disease?

3. Infectivity and PrP titer are not proportional across strainsMany different animal models in different laboratoriesshow that PrP presence is a poor marker for the level oinfectivity, and the lack of PrP does not preclude infection(reviewed in Manuelidis, 2007). Some slow CJD strainsshow no detectable PrP. Baker et al. (2002) showed thatliving microglia from a CJD-infected brain had no detect-able prions, yet contained maximal levels of infectivityAnother study shows that PrP neither encodes nor altersagent-specific characteristics (Arjona et al., 2004). Blockingthe formation of prions by an antimalaria drug does notlengthen CJD victims lives (Collinge, 2009). These are afew examples from many studies that do not confirm prioninfectivity. Initial misleading data, suggesting that PrP is theinfectious agent, had been the result of technical difficultiesto purify PrP from nucleic acid present in infected animaltissue; thus, infectious preparations often contain a largeamount of nucleic acids.

4. The host can recognize a TSE agent and recruit its innateimmune system to respond as early as 20-30 days afterinoculation while PrP begins to accumulate only at 90days post-inoculation, and is incapable of activating thesame immune pathways (Manuelidis, 2007). Lu et al

(2004) detected innate immune host responses before theoccurrence of PrP changes. These host responses are con-sistent with a foreign pathogen, but not host encoded PrPThe epidemic outbreak of BSE also strongly implicates anexogenous infectious agent (Liu et al., 2008).

5. Virus-like particles in TSEs had been detected in manyexperimental animal tissue samples by many different laboratories (David-Ferreira et al., 1968; Bignami & Parry, 1971Lampert et al., 1971; Baringer & Prusiner, 1978; Sklaviadiset al., 1992; Liberski & Brown, 2007; Manuelidis et al.,2007). Treatment with low concentrations of SDS removedresidual PrP from these particles, but did not reducetheir infectivity. On the other hand, disruption of nucleic

acid-protein complexes destroyed 99.5% of their infectiv-ity (Manuelidis et al., 1995). It has been shown that cellsinfected with scrapie and CJD agents produce intracellular25-nm virus-like particles (Manuelidis et al., 2007). Theirsize is similar to the size of small RNA viruses. Disruptionof these viral particles destroys infectivity.

The abundance of scientific data and arguments among animpressive segment of scientists, contradicting and challengingPrusiners hypothesis, warrants continued reconsideration. Byincluding current unresolved scientific controversies, such as thehypothetical nature of prions, for the first time in biology coursesstudents could be introduced to one of the most contentious unresolved disputes in the culture of a discipline so scrupulous thatfinding the true answer can be as hard as nibbling on granite,

as they say in Russia. The theoretical importance of the topic ofinfectious proteins might be compared to the eighteenth-centurydebates on spontaneous generation, although the task of identify-ing the nature of the infectious agent of fatal TSE has proven farmore complicated than what had been resolved by Francesco Redand Louis Pasteur. This mysterious agent, like a molecular ghost,is still invisible to us even at the most sophisticated levels oftechnology and molecular biology. The answers may be found asresearch scientists devise new ways to evaluate the TSE infectionGiving students the opportunity to think about and discuss thistopic will greatly expand their background and skills, as the scien-tific community still searches for answers.

THE AMERICAN BI OLOGY TEACHER PRIONS 52


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I have observed a high level of student interest during suchinterruptive classroom discussions. The class becomes particularlyfascinated with the unusual transmission and nature of this deadlydisease. Students who were shy begin to ask questions. The classas a whole begins to question the fact that its textbooks have com-pletely ignored the theoretical importance of the complex scientificcontroversy.

Many recent studies show that case studies increase studentparticipation and improve student understanding of subject mat-

ter. However, one of the negative aspects is that it could becometime-consuming. Introducing the current controversy on TSE, ateacher could kill two birds with one stone by covering the topicof prions from the curriculum while reviewing material for the finalexamination.

References

Alexeeva, I., Elliott, E.J., Rollins, S., Gasparich, G. E., Lazar, J. & Rohwer, R. G. (2006).

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hamsters with scrapie.Journal of Clinical Microbiology , 44(1), 91-97.

Allchin, D. (2008). Nobel ideals and Nobel errors. The American Biology Teacher,

70(8), 502-505.

Arjona, A., Simarro, L., Islinger, F., Nishida, N. & Manuelidis, L. (2004). Two Creutzfeldt-

Jakob disease agents reproduce prion protein-independent identities in cellcultures. Proceedings of the National Academy of Sciences , 101(23), 8768-8773.

Baker, C.A., Martin, D. & Manuelidis L. (2002). Microglia from CJD brain are infectious and

show specific mRNA activation profiles. Journal of Virology, 76, 10905-10913.

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Bignami, A. & Parry, H. (1971). Aggregations of 35-nanometer particles associated with

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David-Ferreira, J., David-Ferreira, K., Gibbs, C. & Morris, J. (1968). Scrapie in mice:

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Jeffrey, M. et al. (2006). Transportation of prion protein across the intestinal mucosa of

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Neurobiology, 33(4), 395-397.

Liu, Y., Sun, R., Chakrabarty, T. & Ma nuelidis, L. (2008). A rapid accurate culture assayfor infectivity in Transmissible Encephalopathies. Journal of Neurology, 14(5),

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Appendix

Below are low-power light microscope images of sections of

the laboratory mouses brain that has been affected by one

of the strains of transmissible spongiform encephalopathy(TSE), a deadly disease in animals and humans. This strain

had been passed from an infected cow to a human and then

on to the mouse, preserving its original identity throughout

transmission. The sections of the brain were taken from the

same TSE-infected mouse. They have

been stained using immunohisto-

logical techniques to reveal changes

occurring due to the infection.

The top section is a part of the

mouses cerebrum. There are numer-

ous activated microglial cells (red)

that indicate an ongoing processknown as microgliosis. On the sec-

tion below, another staining of

the cerebrum reveals numerous

accumulations of abnormal prion

proteins (also in red). This indicates a

TSE infection.

In the third section of the brain, there

is a brain stem and a cerebellum.

While the brain stem is filled with

abnormal prion proteins, there is no

indication of their presence in the

cerebellum. The same pattern is alsoevident in affected cows and humans

with this TSE strain. The fact that the

pattern remains the same in such

transmission is characteristic of an

infectious agent with nucleic acid.

Images courtesy of Laura

Manuelidis.

BIO IGOR V. ZAITSEV, Ph.D., is Assistant Professor in the Science Department, City

University of New York, Borough of Manhattan Community College, New York

NY 10007; e-m ail: [email protected].

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