Prion Proteins and Acquired Dementia

Authors: Kirk M. Maxey, M.D., Jacques Grassi, Ph.D., and Jeff Johnson, Ph.D.

Transmissible spongiform encephalopathy (TSE) is a rare form of neurodegenerative dementia. TSE, or prion disease, has risen from obscurity in the early part of the 20^th century to a position of sensational prominence today.¹ The small handful of known TSEs (Figure 1) are atypical in that they can arise spontaneously, be inherited via somatic mutation, or be acquired by exposure to an infectious protein. The latter mode of transmission is unprecedented and still poorly understood. The infectious agent (prion; PrP^Sc or PrP^res) is a misfolded variant of the normal cell surface glycoprotein PrP^c.^2,3 The majority of human TSE is attributable to spontaneous and genetic causes (Figure 1). The total infectious cases of TSE worldwide since 1950 is perhaps 3,000, about the same as the automobile accident death toll in the US during the first 30 days of 2004. Yet the first week of the New Year saw federal and international regulatory agencies and commodity markets convulsed by a single case of a TSE detected in a farm animal; a dairy cow tested positive for bovine spongiform encephalopathy (BSE) in the northwestern US. There was no comparable attention focused on the accidental deaths, or to a host of other health concerns (Table 1). The unusual history of TSE and the unique associated molecular pathogenesis provides some perspective (Side Bar 1).

The human prion protein (PrP) is initially expressed as polypeptide of 253 amino acid residues from a single gene on human chromosome 20 containing the entire open reading frame on a single exon (Figure 2).⁴ As it translocates through the ER and Golgi, it acquires several post-translational modifications, including removal of the N-terminal 23 residue signal peptide, glycosylation at asparagines 181 and 197 in the C-terminal third of the protein, and formation of a single disulfide bond between cysteine residues 179 and 214.^5,6 The final modification is the proteolytic cleavage of the C-terminal 24 amino acids followed by addition of a glycosyl phosphatidylinositol (GPI) anchor.^7,8 Mature cellular PrP is a 209 amino acid glycoprotein tethered to the cell surface where it forms detergent-insoluble lipid raft complexes that are enriched in cholesterol and sphingosine, as depicted in Figure 3.⁹ PrP that fails to properly traverse this post translational processing pathway can enter the cytosol, where it is highly neurotoxic.¹⁰ Once in place facing the extracellular space, PrP turns over relatively rapidly by two separate routes. One is endocytosis, through the invagination of the lipid raft complex, and the other is via shedding by proteolytic cleavage just above the carboxy-terminal attachment site of the GPI anchor.^11,12 The fate and function of shed, soluble, or secreted PrP is just as mysterious as that of the membrane anchored form. However, PrP^c shedding has been documented in human platelets, and PrP^c is readily detected in secretions such as breast milk. No functional role has yet been determined for the PrP^c protein. PrP^c-/-null mutant mice are viable and healthy, with only subtle behavioral abnormalities.

Despite being highly conserved, with greater than 90% homology among most mammalian species, the human prion gene has a number of normal polymorphic variants and several disease-inducing mutations (Figure 2). In the more common dementias such as Alzheimers, familial disease is associated with several different proteins, each with a variety of functional mutations. This has aided the development of both disease process and normal function models. In familial TSE, the mutation has uniformly been found only in the prion protein itself. The high frequency of human prion polymorphism seems to have been driven, at least in part, by cannibalism and its associated transmission of prion disease.¹³ Homozygotes, having two identical copies of the prion gene, show increased susceptibility and shorter incubation times when infected with prions. Of the 143 recorded victims of vCJD in the UK, all were homozygous for 129-methionine, which is the ancestral amino acid at that position. Yet nearly 65% of the British population are either 129-valine homozygotes or 129-M/V heterozygotes. Among the survivors of the kuru epidemic, the 129-valine allele has been so strongly selected that it is actually more frequent (55%) than 129-methionine, and virtually all of the older kuru surviors are 129-M/V heterozygotes.¹³ At least four other common human polymorphisms (E210K, N171S, G142S, and the 84-91 PHGGSWGQ octarepeat deletion) may also have been selected for their protective value in TSE resistance.

Recent efforts to understand the pathogenesis of TSE have led to investigations into the processing and elimination of misfolded proteins within the cell, as well as the potential involvement of RNA in PrP^Sc propagation. Aggregated intracytoplasmic PrP^Sc is a hallmark of TSE and is known to induce neuronal cell death. In normal cells, misfolded PrP in the ER is retrogradely transported to the cytosol where it is rapidly degraded by the proteosome.²¹ Proteosomal inhibition by pharmacological intervention results in the appearance of both detergent-soluble PrP^c and PrP^Sc-like proteins, characterized by detergent insolubility and resistance to proteinase K digestion. Transient inhibition of the proteasome results in continued accumulation of PrP^Sc-like molecules, indicating that once the process of misfolding has been initiated, PrP^Sc-like proteins continue to propagate, even after removal of the inhibitor. Unfortunately, PrP^Sc is not the only form of PrP to necessitate concern, as accumulation of even small amounts of PrP^c in the cytosol is strongly neurotoxic. Might RNA also be involved in the conversion of PrP^c to PrP^Sc? A recent report indicates that relatively large pieces of single-stranded RNA increase PrP^Sc amplification several fold over control reactions performed in the absence of RNA.²² The most active of the RNA species (>300 bp) co-purified with ribosomal RNA, which is found at the site of protein synthesis in cells. Perhaps the “protein only” understanding of TSE will need to be revised, but it is far too early to draw concrete conclusions.

References

1. Prusiner, S.B. Science 278, 245-251 (1997).
2. Prusiner, S.B. N. Engl. J. Med. 344(20), 1516-1526 (2001).
3. Stahl, N. et al. FASEB J. 5, 2799-2807 (1991).
4. Puckett, C., et al. Am. J. Hum. Genet. 49, 320-329 (2004).
5. Lehmann, S., et al. Biomed. Pharmacother. 53, 39-46 (1999).
6. Rudd, P.M., et al. Biochemistry 40(13), 3759-3766 (2001).
7. Stahl, N., et al. Cell 51, 229-240 (1987).
8. Stahl, N., et al. Biochemistry 31, 5043-5053 (1992).
9. Critchley, P., et al. Biochem. Biophys. Res. Commun. 313, 559-567 (2004).
10. Ma, J., et al. Science 298, 1781-1785 (2002).
11. Parkin, E.T., et al. J. Biol. Chem. 279(2), (2004).
12. Stahl, N., et al. Biochemistry 29(38), 8879-8884 (1990).
13. Mead, S., et al. Science 300, 640-643 (2003).
14. Gordon, W.S. Vet. Res. 58, 516-520 (1946).
15. Alper, T., et al. Biochem. Biophys. Res. Commun. 22, 278-284 (2004).
16. Prusiner, S.B. Proc. Natl. Acad. Sci. USA 95, 13363-13383 (1998).
17. Brown, P., et al. Lancet 340, 24-27 (1992).
18. de Villemeur, T.B., et al. Neurology 47, 690-695 (1996).
19. Miller, M.W., et al. Nature 425, 35-36 (2003).
20. Salman, M.D. J. Vet. Med. Sci. 65(7), 761-768 (2003).
21. Dimcheff, D.E., et al. Trends Cell Biol. 13(7), 337-340 (2003).
22. Deleault, N.R., et al. Nature 425, 717-720 (2003).

Side Bar 1: A Historic Perspective on Prion Diseases

A chronic affliction called scrapie has been endemic in the sheep populations of northern Europe for centuries. Curiously, although the sheep of New Zealand are descended from these European sheep, they are free of the disease. Sheep with scrapie show abnormal behavior, including repetitive ‘scraping’ of body parts against firm objects. They also show the typical spongiform neuropathology. Scrapie is the oldest known TSE, and much of what is known about the disease process has been learned by transmitting the scrapie infectious agent to mice, hamsters, and other experimental animals. Microscopic examination of the brains of scrapie-affected sheep reveals massive neuronal loss, gliosis, and sponge-like vacuolization in the tissue sections. Also present are abnormal amyloid protein clumps and fibrils. These scrapie-associated fibrils (SAF) are the necessary and sufficient agent for transmission of the disease. Gordon,¹⁴ Alper,¹⁵ and others showed that SAF seemed largely devoid of nucleic acids, and remained infectious after radiation and heat treatments known to inactivate DNA and RNA viruses. Stanley Pruisiner, recognized with a Nobel Prize in medicine in 1998, built on this work and in 1982 cloned a single protein that comprised the SAF fibrils.¹⁶ The new protein was widely expressed in neurons, where its function remains unknown. Remarkably, exposure of the normal cellular protein to a misfolded, aggregated, and partially digested template of the same peptide from SAF had the capacity to foster spongiform encephalopathy. Pruisiner coined the name “prion” for this protein, from the words “proteinaceous and infectious”. (While a direct coupling of the word fragments would yield ‘proin’, the letters have been conformationally rearranged to ‘prion’, mirroring the key transformation of this disease process.) The normal cellular protein is designated PrP^c (prion protein-cellular) and the oligomeric, truncated, and conformationally rearranged infectious form is designated PrP^Sc (prion protein-scrapie).

The “protein only” hypothesis of TSE states that oral, systemic, or intracerebral exposure of susceptible animals to PrP^Sc alone is the cause of the disease. Although it is widely accepted today, this idea created considerable controversy when initially proposed. Infectious proteins were a novel concept and ran counter to prevailing history and dogma in infectious disease. Yet an unusual mechanism of transmission alone would never have attracted the current level of media attention.

Human Transmission of Prion Disease

There are a number of highly publicized epidemics of human transmission of prion disease. Ritual cannibalism among the Fore tribe in Papua, New Guinea, prior to Western contact was the initiating event in an epidemic of the prion disease kuru. The source of the original infective prions is unknown but it is reasonable to surmise that a sporadic case of Creutzfeld-Jacob Disease (CJD) was passed to several close relatives who consumed infected brain tissue. Kuru was killing around 1% of the Fore population each year at the height of the epidemic. The initiating event is thought to have occurred in the early 20^th century, as the local oral historians place the first kuru-like death around 1920. The epidemic was terminated by Western missionaries who discouraged ritual cannibalism. Because they were only partially successful, cannibalism and the attendant prion disease persisted into the 1950’s, when the nature of transmission and the spongiform pathology was documented. Cannibalism was then outlawed as a public health measure, and kuru declined rapidly thereafter.

Iatrogenic transmission of prion disease by medical professionals has been possible since the introduction of neurosurgical procedures and has been documented since the 1960s. Hospital acquired infections consist mainly of the transmission of CJD-infective prions (iCJD), from contaminated surgical tools and equipment and from human-derived tissues and extracts. About ninety individuals who received human growth hormone (HGH) extracted from human cadaver pituitaries were infected with CJD between 1963, when the first HGH extracts were produced, and 1977, when the purification procedure in the US was modified in a way that (serendipitously) reduced prion contamination.^17,18 The majority of HGH-derived cases occurred in France, where the newer procedure was not adopted, while 22 of the deaths were recorded in the US. The epidemic was terminated by the introduction of recombinant HGH preparations and the abandonment of human-derived brain extracts as awareness of prions dawned in the 1980s.

By far the largest epidemic of human transmission of prion disease has its roots in the industrialization of livestock production. High protein feed supplements produced from the bone and offal of sheep, pigs, chickens, and cattle have been in widespread use since the 1950s. Collectively called meat and bone meal (MBM), the supplements were first rendered (heated) and extracted with hydrocarbon solvents to recover the valuable animal fats. Although not understood at the time, these steps probably helped to denature any prions in the rendered MBM and eliminated its ability to transmit disease. The initiation of this epidemic, widely known as the bovine spongiform encephalopathy (BSE) crisis or mad cow disease, came when environmental groups in Britain successfully pressured the feed industry to abandon hydrocarbon solvent processing of MBM in the early 1980s.¹ It may also have been aided by the contributions of small-scale producers of MBM lacking the capacity to perform solvent extraction. The resulting high-fat MBM supplements contained infectious prions, probably scrapie prions from sheep, although this cannot be determined precisely. In Great Britain, 40,000 cattle per year were clinically diagnosed as having BSE at the height of the bovine epidemic in 1992-93. Based on a 5-year incubation time and the known low precision of the diagnostic methods, perhaps 300,000 British cattle were being infected each year in 1987-88. The human cases of prion disease attributable to this epidemic peaked in 1996, and have been referred to as new variant CJD (vCJD). The disease has afflicted 143 individuals, mostly teenagers and young adults living in Britian, who presumably consumed beef products from the BSE-infected cattle. The terminating event of the epidemic was a governmental ban on feeding any MBM to cattle that was put into effect in the UK in 1988. This ban was reinforced in 1996 with additional precautions against entry of bovine nervous system tissue into human food, and banning animals over 30 months old from human consumption. A more limited ban only on ungulate-derived MBM recently instituted by the US would seem insufficient to explain the virtual absence of the epidemic in America. As noted earlier, North America has been largely free of BSE, with only two cases reported in 2003, both in cattle born in Canada. The fact that hydrocarbon solvent processing of MBM in the US continues unabated is circumstantial evidence that this may be protective.

North America does, however, have its own epidemic of prion disease, known as chronic wasting disease (CWD) in deer and elk. CWD was documented in both captive and wild herds of cervids well before the BSE crisis, and there is no question that it is horizontally transmitted from animal to animal as an infectious disease. Contaminated MBM is unlikely to have played any role in CWD. The physical contact and high population density around artificial feeders is an important factor in CWD transmission.¹⁹ The originating event is unknown, but the disease does have a suspiscious epidemiologic center around the veterinary facilities of Colorado State University in northern Colorado, where the disease was first detected in captive mule deer in the 1960s.²⁰ CWD has spread as far as Montana, New Mexico, and Wisconsin over a period of 30 or 40 years, and now appears endemic in wild deer and elk populations. Its risk to public health seems to be low, with no known cases of transmission to humans. Deer infected with CWD have been experimentally co-mingled with domestic cattle for up to 5 years without transmission of the disease across this species barrier.

Assay Methodology for Prion Measurement

Diagnostic techniques used to characterise the presence of TSEs must reveal in suitable samples the presence either of lesions characteristic of TSEs or of PrP^Sc, or of the transmissible agent itself. For the diagnosis of TSEs, conventional methods require a brain tissue sample, histopathology, immunohistochemistry, and experimental inoculation in rodent models. Histopathology consists of histological detection (essentially in CNS tissues) of the lesions characteristic of TSEs (spongiosis, astrogliosis, PrP amyloid plates) and remains a reference method for confirming a clinical diagnosis. It is highly specific since it allows direct observation of the signs of the disease. However, it is now known to be less sensitive than other techniques. The sensitivity of observations under the microscope can be increased by means of immunohistochemical techniques that use antibodies specific for PrP to detect the accumulation of PrP^res in amyloid plaques. The most sensitive method for the diagnosis of TSEs is unquestionably the experimental infection of laboratory animals: following an injection of a homogenate prepared from the tissue in question, the animal is monitored for the emergence of clinical signs. The development of the experimental TSE is then confirmed by means of classical techniques (histology, immunohistology, western blot). For obvious practical reasons, these experiments are generally performed on rodents (mice, hamsters), but in certain extreme cases experimental infections have been performed with sheep or cattle. The principal drawbacks of these methods is that they are labour-intensive and time-consuming. Between 300 and 700 days are needed to perform an experimental infection test in mice, and between two and 10 years in cattle. The availability of transgenic mice expressing the same PrP as that of the donor species will shorten the time required, but in all cases these tests will last at least three to six months.

None of the methods referred to above is really suitable for high-throughput screening or lends itself to automation. After the first “mad cow crisis” in Europe in 1990s and the realization that BSE could be transmitted to humans, there was a pressing need to develop new, simpler, and faster diagnostic methods. These methods should enable either large-scale epidemiological studies for more accurate assessment of the characteristics of the outbreak, or systematic testing, at the slaughterhouse for example, of all animals before their entry into the human food chain. A new generation of so-called “rapid” diagnostic tests was therefore developed, based on the immunological detection of PrP^Sc, the sole direct marker identified for TSEs which accumulates in peripheral lymphoid tissues during the incubation period and afterwards in the brain of affected animals at the clinical stage of the disease. To date, there are no clearly identified antibodies that specifically recognize PrP^Sc in its native form. Since PrP^c is always present in tissues expressing PrP^Sc, all these tests are based on the differences in the biochemical properties between PrP^c and PrP^Sc and of the availability of antibodies recognizing the denatured form of PrP^Sc. Most tests take advantage of the relative resistance of PrP^Sc to degradation by proteolytic enzymes, particularly proteinase K. Other tests employ the aggregation properties of PrP^res when it is extracted in the presence of detergents. All tests include a PrP^Sc denaturation step to allow its detection by antibodies that often recognize PrP^c or denatured PrP (whether derived from PrP^c or PrP^Sc). Certain tests detect PrP^Sc directly by means of the increase that occurs in its immunoreactivity following denaturation. To date, three tests are sharing the market of routine testing for BSE in cattle (about 10 million tests per year). These are: i) the Prionics Check test which is based on a western blot determination of PrP^Sc, ii) the Enfer test which is an ELISA technique involving a direct coating of PrP^Sc onto polystyrene (in presence of PK) and subsequent detection of immobilized PrP^Sc, after denaturation, with an enzyme labelled antibody, and iii) the Bio-Rad test (formally developed at CEA) which combines a rapid purification and concentration of PrP^Sc and detection of denatured PrP^Sc by a sandwich immunoassay. These three tests were officially validated in 1999 by the European commission and proved to allow a very satisfying post-mortem diagnosis of BSE in cattle. They are now systematically used for monitoring risk populations in Europe (fallen stocks, emergency slaughtered animals) and for routine testing animal over thirty months entering into the human food chain (Europe and Japan). Since 2001, rapid tests have identified more than 3,000 BSE-positive cattle and removed more than 600 BSE-infected animals from human consumption.