Molecular Aspects of Disease Pathogenesis in the Transmissible Spongiform Encephalopathies

Suzette A. Priola and Ina Vorberg

1. Introduction

The transmissible spongiform encephalopathy (TSE) diseases are a group of rare, fatal, and transmissible neurodegenerative diseases that include kuru and CreutzfeldtJakob disease (CJD) in humans, scrapie in sheep, transmissible mink encephalopathy (TME; 82), and chronic wasting disease (CWD) in mule deer (141) and elk (142). Over the last 20 yr, they have gone from a fascinating but relatively obscure group of diseases to one that is a major agricultural and economic problem as well as a threat to human health. The shift in the relative impact of the TSE diseases began in the late 1970s when the United Kingdom altered the process by which animal carcasses were rendered to provide a protein supplement (i.e., meat and bone meal) to sheep, cattle, and other livestock. Several years later a new disease was recognized in the British cattle population. The pathological and immunohistochemical characteristics of the disease clearly placed it among the TSEs. The new disease was named bovine spongiform encephalopathy (BSE) by the scientific community (2,16,138) and "mad cow disease" by the less-than-scientific press. At its peak in the UK, several thousand cattle a year were diagnosed with BSE, and millions of cattle were slaughtered. Introduction of the specified offals ban as well as banning the practice of feeding ruminants to other ruminants has led to a drastic decrease in the number of yearly BSE cases in the UK (less than 500 in 2003), and the epidemic is clearly on the wane. However, BSE has now spread throughout the rest of Europe, as well as to Japan, Russia, Canada, and Israel and thus remains a worldwide problem.

A primary concern following the identification of BSE in 1985 was that it might cross species barriers to infect humans. Initially, it was thought that transmission of BSE to humans was unlikely, given that humans appeared to be resistant to scrapie, an

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

animal TSE that had been endemic in British sheep for centuries. However, a few years after BSE was first recognized, a previously unknown form of CJD (variant CJD or vCJD) was identified in young people in Great Britain (140). The hypothesis that vCJD was the consequence of exposure of humans to BSE (140) has now been supported by several different studies (20,35,36,111), and over 140 cases of vCJD have been confirmed.

Variant CJD has increased public awareness in low-risk BSE countries of the dangers of exposure of humans to animal TSE diseases. For example, in the United States the presence of CWD in wild populations of deer and elk has raised concerns about the possible transmission of CWD to humans through consumption of venison. Indeed, recent in vitro studies have suggested that humans might be susceptible to CWD (110). There is also concern that CWD might cross species barriers to infect livestock that share range land with infected deer and elk and thus be transmitted to humans via infected cattle or sheep. In either case the end result could be reminiscent of what has happened in the UK: the occurrence of a new form of TSE disease in humans.

The impact of BSE has thus spread far beyond the agricultural arena and is now considered a significant threat to human health. Variant CJD has made it clear that exposure of humans to TSE-contaminated products can lead to disease. This in turn has fueled concerns about the possible spread of vCJD via contaminated blood products that has led to stringent new blood donor criteria in the United States and abroad. The only way to eliminate these concerns is to develop sensitive and specific diagnostic tests for TSE infection as well as effective postexposure therapeutics. Currently these are two very active areas of TSE research, although the unique nature and long-term pathogenesis of these diseases has made development of TSE diagnostics and therapeutics especially difficult. However, new methodologies have increased our understanding of the biochemistry of these diseases. This new understanding may in turn eventually lead to novel approaches in TSE diagnostics and therapeutics.

2. Prion Protein

One of the most intriguing aspects of the TSEs is that no virus or bacteria has ever been associated with infectivity. This, coupled with the extreme resistance of TSE infectivity to inactivation by a variety of harsh treatments, led to the hypothesis that the infectious agent in these diseases was an infectious, self-replicating protein (1,51). In the early 1980s a protein was found that was closely associated with TSE infectiv-ity, and this protein was termed prion protein or PrP (11). Further analysis revealed that PrP was in fact a normal host cellular protein (11,41,78) that accumulates during TSE infection as an abnormal isoform. This accumulation occurs primarily in nervous system tissues but often also in tissues of the lymphoreticular system. It was this abnormal form of PrP that was first identified as scrapie-associated fibrils from scrapie-infected brain tissue (84,85). The fact that abnormal PrP is only found in TSE-infected tissues and is very closely associated with tissue preparations enriched for infectivity has led to the proposal that abnormal PrP itself is the infectious protein agent (100). Although this hypothesis remains a point of contention in TSE research, the key role of PrP in disease pathogenesis is undeniable (17,21).

Normal PrP, an approx 253-amino acid glycoprotein that is expressed on the cell surface in a wide variety of tissues, is both detergent soluble and sensitive to digestion with proteinase K (PrP-sen)*. PrP-sen is a cell surface glycoprotein containing two N-linked glycosylation sites (26,52). Variable glycosylation of these two sites results in both PrP-sen and PrP-res migrating as three discrete bands consisting of unglycosylated, partially glycosylated, and fully glycosylated PrP (Fig. 1). A glycophosphotidyl-inositol (GPI) membrane anchor is also added post-translationally to PrP-sen, allowing it to be inserted into the plasma membrane (10,57,58). By contrast, the abnormal form of PrP associated with disease (PrP-res) forms insoluble aggregates and is partially resistant to digestion with proteinase K such that approx 60-70 N-termi-nal amino acids are cleaved. This leads to a size shift characteristic for the PrP-res molecule (Fig. 1). PrP-res is post-translationally derived from mature PrP-sen (12,27). Since both PrP-sen and PrP-res are central to the pathogenesis of the TSE diseases, an understanding of their basic biosynthesis is critical.

The normal function of PrP is unclear. Two different strains of mice in which PrP has been ablated develop normally but demonstrate subtle nervous system abnormalities (37,133), whereas a third strain of PrP knockout mice showed a loss of Purkinje cells in the brain and developed a cerebellar dysfunction (119). PrP-sen has been reported to be involved in mitogen-induced activation in lymphocytes (23). Recent studies have provided compelling evidence that normal PrP may be involved in fyn kinase signaling (86) as well as pro- and antiapoptotic mechanisms (15,87). These latter observations are particularly intriguing since conversion of PrP to its abnormal form could conceivably lead to a loss of function that may help to explain the mechanism of neurodegeneration during TSE infection. At present it is difficult to determine which, if any, of these observations reflect the true function of PrP.

2.2. Cell Biology of PrP-res Formation

Historically, it has been quite difficult to infect cells in vitro with a TSE agent, hindering attempts to study the cellular biology of PrP-res formation. Despite the almost ubiquitous expression of PrP-sen in many different cell types throughout the body, PrP-res accumulation in vivo appears to be limited to cells of the nervous and lymphoreticular systems. Similarly, even though most cell lines express PrP-sen, the susceptibility of tissue culture cells to TSE infection is also restricted. Susceptibility of cells to infection is probably dependent not only on the cell type and sequence of the cellular PrP-sen protein (Fig. 2) but also on the strain and infectious titer of the TSE agent. Occasionally, even when cells had been successfully infected with a TSE agent, the low percentage of infected cells meant that PrP-res was not even detectable

*The normal form of PrP has been referred to as either PrPC for PrP-cellular or PrP-sen, which designates its sensitivity to proteinase K. The abnormal form of PrP has been referred to as either PrPSc for PrP-scrapie or PrP-res, which designates its partial resistance to proteinase K. Since the terms PrP-sen and PrP-res reflect a general operational definition rather than a disease-specific phenotype, they will be used throughout this discussion.

PrP and TSE Disease •Normal PrP

•Proteinase K sensitive (PrP-sen) •Detergent soluble •Expressed in many different tissues •Primarily alpha helix/loop structure

•Abnormal PrP

•Partially Proteinase K resistant (PrP-res) •Detergent insoluble, aggregated •TSE specific •Found in CNS.LRS •Mostly beta sheet structure

Fig. 1. Properties of normal and disease-associated prion protein (PrP). The panel on the left is a Western blot of normal PrP (PrP-sen) and abnormal PrP (PrP-res) isolated from mouse scrapie-infected neuroblastoma cells (107). PrP is a cell surface glycoprotein that migrates as three distinct glycosylated bands: a fully glycosylated band (F) representing complex glycans at both N-linked glycosylation sites, a partially glycosylated band (P) representing partially processed glycans at one or both N-linked glycosylation sites, and an unglycosylated band (U). Following digestion with proteinase K (PK), PrP-res shifts 6-7 kDa in size compared with PrP-sen. This shift is diagnostic for PrP-res and TSE diseases in general. Molecular mass markers in kilodaltons are indicated on the right. CNS, central nervous system; LRS, lymphoreticular system; TSE, transmissible spongiform encephalopathy.

(Fig. 3) until after the cells were cloned (106). Despite these technical difficulties, a limited number of mouse scrapie strains have been successfully used to infect mouse and rat cells (22,107,114,120). Additionally, although persistent attempts to infect cells with BSE and CJD have not been successful, rabbit epithelial cells overexpressing the sheep PrP protein have recently been infected with sheep scrapie (134). All these infected cell lines make PrP-sen, accumulate PrP-res, and replicate scrapie infectivity. As a result, the cell biology of PrP-res formation has now been studied in some detail.

In mouse scrapie-infected mouse neuroblastoma (Sc+-MNB) cells, in which PrP-sen is the precursor to PrP-res, the biosynthesis of PrP-res differs dramatically from that of PrP-sen (12,26,27,30,128), and only a small proportion of the available PrP-sen is converted to PrP-res (27,30). The conversion of PrP from the protease-sensitive to the protease-resistant form probably occurs either on the plasma membrane or along an endocytic pathway (4,13,27,30,83), possibly in a cellular compartment containing

Single Cell

Fig. 2. Scrapie infection of tissue culture cells. A single cell (large square) and its DNA genome (light gray rectangle) is pictured. The major obstacles to successfully infecting cells in vitro are represented by the dark gray rectangles. A prion protein (PrP)-res (black triangles) positive, high-titer brain homogenate from a scrapie-infected animal is layered onto cells that encode PrP-sen (black circles). Following an incubation period of several hours, medium is added, and the cells are cultured until confluent and then passaged many times to remove any remaining brain homogenate. At different passages post infection, the cells are then assayed for scrapie infectivity in vivo and/or PrP-res formation in vitro (107). Several factors appear to influence the likelihood of obtaining persistently infected cells including the scrapie titer of the brain homogenate and multiplicity of infection, the PrP sequence, the expression level of PrP-sen, the scrapie strain, and the cell type. Persistently infected cells may account for less than 1% (106) or up to 10% (134) of the total cell population. When low numbers of cells are persistently infected, PrP-res can only be detected after the cells have been cloned (see Fig. 3).

Single Cell

Fig. 2. Scrapie infection of tissue culture cells. A single cell (large square) and its DNA genome (light gray rectangle) is pictured. The major obstacles to successfully infecting cells in vitro are represented by the dark gray rectangles. A prion protein (PrP)-res (black triangles) positive, high-titer brain homogenate from a scrapie-infected animal is layered onto cells that encode PrP-sen (black circles). Following an incubation period of several hours, medium is added, and the cells are cultured until confluent and then passaged many times to remove any remaining brain homogenate. At different passages post infection, the cells are then assayed for scrapie infectivity in vivo and/or PrP-res formation in vitro (107). Several factors appear to influence the likelihood of obtaining persistently infected cells including the scrapie titer of the brain homogenate and multiplicity of infection, the PrP sequence, the expression level of PrP-sen, the scrapie strain, and the cell type. Persistently infected cells may account for less than 1% (106) or up to 10% (134) of the total cell population. When low numbers of cells are persistently infected, PrP-res can only be detected after the cells have been cloned (see Fig. 3).

cholesterol-rich membranes (129). In Sc+-MNB cells, PrP-res eventually accumulates in the secondary lysosomes (27,30) with little, if any, present on the cell surface (12,25,83). In vivo, PrP-res can also accumulate in the extracellular spaces as amorphous deposits or more organized fibrils or amyloid plaques (64-67). Despite its exposure to endolysosomal hydrolases, PrP-res has a half-life of greater than 48 h (12,27). This metabolic stability of PrP-res could explain its accumulation in vivo, especially in the nondividing cells of the central nervous system.

Both the normal and abnormal forms of PrP are derived from the same gene and have the same amino acid sequence. No post-translational modifications have been identified that can account for the different biochemical properties of PrP-sen and PrP-res. Thus, it is likely that the difference between the two molecules is a conformational one. Analysis of PrP-res by infrared spectroscopy (29,32,89) and the recent

Sen Res

Cell Passage (PI)

Incubation time

Sen Res

Passage 12

Passage 8

Passage 3

Passage a=scrapie positive/total animals, (days to death + standard deviation)

Fig. 3. Uncloned scrapie-infected tissue culture cells do not necessarily have detectable levels of PrP-res. The left side of the figure shows a Western blot of PrP-sen expressed in mouse neuroblastoma cells infected with scrapie compared with PrP-res expression in these cells at passages 3, 8, and 12 post infection. For the bioassay summarized in the table on the right, cell lysates were injected intracranially into mice susceptible to scrapie, and the animals were monitored for disease (107). Note that although no PrP-res was detectable by Western blot, the fact that all mice at each passage tested succumbed to scrapie clearly demonstrates that the cells are persistently infected with mouse scrapie. These data illustrate the higher sensitivity of the in vivo bioassay versus Western blotting when monitoring scrapie infection. (+), PrP-res from scrapie-infected brain.

determination of the structure of PrP-sen by nuclear magnetic resonance (NMR) (42,112,113,143) and X-ray crystallography (71) support this hypothesis and demonstrate that PrP-res has a much higher p-sheet content than PrP-sen (Fig. 4). A critical question within the TSE diseases that remains unresolved is how the conformational conversion of PrP-sen to PrP-res occurs.

2.3. Conversion of PrP-sen to PrP-res

The conversion of PrP-sen to PrP-res is a process that is critical to disease patho-genesis, especially if PrP-res is by itself the infectious agent. Two primary models have been used to describe the self-propagation of PrP-res. The heterodimer model (9,51,101) proposes that a monomer of PrP-res interacts with a monomer of PrP-sen and converts it to more PrP-res by inducing a conformational change. The two molecules, now both PrP-res, separate, and the process continues, leading to accumulation of PrP-res and disease. In the seeded polymerization model (45,63), a "seed" of PrP-res composed of an organized array of multiple PrP-res molecules interacts with PrP-sen and converts it to PrP-res by inducing a conformational change. The newly formed PrP-res molecule remains a part of the polymer, and the process repeats, eventually leading to accumulation of PrP-res and disease. Recent data suggest that only aggregates of PrP-res can induce the conversion of radiolabeled PrP-sen to PrP-res (31). In

Fig. 4. PrP-res formation involves a conformational change in PrP-sen. During disease pathogenesis, PrP-sen (upper panel) undergoes a conformational change from a primarily a-helical structure (gray boxes) to a primarily p-sheet structure (black boxes) following conversion to PrP-res (lower panel). A linear representation of the NMR structure of processed PrP-sen from amino acid residues 23-230 is shown (113). Solid lines indicate loop structures; a dashed line represents disordered structure. Following digestion with proteinase K, which removes the N-terminal 60-70 amino acid resides, protease-resistant PrP-res lacks most of the disordered N-terminal region. The structure of PrP-res is theoretical and is based on predictions from infrared spectroscopy (32). Note that significant regions of p-sheet are predicted in PrP-res downstream of the small regions of p-strand present in PrP-sen.

Fig. 4. PrP-res formation involves a conformational change in PrP-sen. During disease pathogenesis, PrP-sen (upper panel) undergoes a conformational change from a primarily a-helical structure (gray boxes) to a primarily p-sheet structure (black boxes) following conversion to PrP-res (lower panel). A linear representation of the NMR structure of processed PrP-sen from amino acid residues 23-230 is shown (113). Solid lines indicate loop structures; a dashed line represents disordered structure. Following digestion with proteinase K, which removes the N-terminal 60-70 amino acid resides, protease-resistant PrP-res lacks most of the disordered N-terminal region. The structure of PrP-res is theoretical and is based on predictions from infrared spectroscopy (32). Note that significant regions of p-sheet are predicted in PrP-res downstream of the small regions of p-strand present in PrP-sen.

yeast, another organism in which prion-like activity has been found (139), only aggregated forms of yeast prions can induce the formation of new yeast prions (47,91,124,130). Dissociation of PrP-res to a soluble form leads not only to a loss of its "converting activity" but also to a loss of infectivity (31,46,117). Finally, monomeric PrP-res as resistant to proteinase K as aggregated PrP-res has never been detected, although this does not rule out the possibility that less proteinase K-resistant monomeric PrP-res is present and active at low levels. Therefore, most of the currently available evidence supports the seeded polymerization model.

3. The Role of PrP in TSE Diseases

Any model of PrP-res formation must be able to explain at a molecular level (1) how PrP-res could induce its own formation (i.e., replicate); (2) how PrP can control not only disease incubation time but also species barriers to infection; (3) the occurrence of different scrapie strains in animals with one PrP genotype; and finally (4) human familial TSE diseases that are both infectious and heritable. Fortunately, several recently developed assays including a polymerase chain reaction (PCR)-like assay of PrP-res formation (116), two different cell-free conversion systems (72,137), persis tently infected and acutely exposed Sc+-MNB cells (107,137), as well as transgenic mice (21,108,121), have allowed researchers to study PrP-res formation in greater detail than ever before and have led to valuable new insights into the conversion process.

3.1. "Replication" of PrP-res

The most direct evidence that PrP-res can induce its own formation comes from an experimental system in which radiolabeled tissue culture-derived PrP-sen can be converted into a proteinase K-resistant form using PrP-res isolated from TSE-infected brains (72-74). In this cell-free conversion system (Fig. 5A), the primary components are immunoprecipitated PrP-sen and enriched preparations of PrP-res. A similar system using less purified components and PrP-sen tagged with a unique antibody epitope instead of radioactively labeled PrP-sen, has also recently been developed (137) (Fig. 5B). Via an interaction that most likely involves the C-terminal portion of PrP (59,73), PrP-res binds selectively to PrP-sen, and conversion of PrP-sen into PrP-res occurs. Some evidence suggests that binding and conversion are two distinct events (40,61,99), a hypothesis that is supported by data suggesting that initial PrP-PrP interactions may map to a different region of PrP than the region that appears to influence conversion (60,74,97,99,123). The fact that PrP-res can induce PrP-sen to convert to the abnormal form in the absence of a living cell provides compelling evidence that PrP-res can "replicate" itself in the absence of any metabolic activity and demonstrates that a viable virus or bacteria is not needed for this process to occur. However, it is still unclear whether or not this newly formed PrP-res is infectious.

Fig. 5. (opposite page) Formation of PrP-res in vitro. (A) The cell-free assay for PrP-res formation in vitro (72). Radiolabeled PrP-sen (35SPrP-sen) is isolated by immunoprecipitation from eukaryotic tissue culture cells and mixed with PrP-res isolated from an animal infected with a TSE disease. Following a 2-d incubation at 37°C, the mixture is treated with proteinase K (PK) and assayed by autoradiography for PK-resistant, radiolabeled PrP-res that has shifted in molecular weight the expected 6-7 kDa (right panel). This assay can be performed using any species of PrP-sen or PrP-res as long as the necessary tissue culture cells and infected brain homogenates are available. (B) A cell lysate-based assay for PrP-res formation (137). The principle is similar to that of the cell-free conversion assay, except that radioactivity is not used and purified PrP-sen and PrP-res are not needed. A TSE-infected brain homogenate containing both PrP-sen and PrP-res (gray circles and squares, respectively) is mixed with a cell lysate containing an epitope-tagged PrP-sen molecule (flagged black circle) at a ratio of 1:8. The epitope in PrP-sen must be recognized by an antibody that does not recognize the PrP in the infected brain homogenate. Following a 4-d incubation at 37°C and treatment with PK, formation of new PrP-res (flagged black square) is assayed using an antibody that will only recognize epitope-tagged PrP

Fig. 5. (continued) molecules. A Western blot of the input PrP-res from an uninfected (mock) brain homogenate or from brain homogenates from mice infected with four different scrapie strains (87V, 22L, ME7, and RML) is shown on the left. Note that each strain has a distinctive pattern of three glycosylated PrP-res bands. A Western blot of newly formed epitope-tagged PrP-res for each of the four strains is shown on the right. It was developed using an antibody that does not recognize the PrP-res molecules shown in the left panel.

Fig. 5. (continued) molecules. A Western blot of the input PrP-res from an uninfected (mock) brain homogenate or from brain homogenates from mice infected with four different scrapie strains (87V, 22L, ME7, and RML) is shown on the left. Note that each strain has a distinctive pattern of three glycosylated PrP-res bands. A Western blot of newly formed epitope-tagged PrP-res for each of the four strains is shown on the right. It was developed using an antibody that does not recognize the PrP-res molecules shown in the left panel.

3.1.1. Cofactors in PrP-res Formation

Although these cell-free conversion systems could be seen as demonstrating that only PrP-sen and PrP-res are needed for conversion to occur, the fact that the preparations used are not absolutely pure precludes this interpretation. If other molecules are involved they might only need to be present in very low levels to have an effect. The role of other molecules in the conversion process is unclear. Glycosaminoglycans (GAGs) have been proposed as one possible cofactor (see ref. 94 for review). GAGs bind PrP (24,44), inhibit formation of PrP-res (28), are often associated with PrP plaques in the brain of infected individuals (126), and can inhibit TSE disease in vivo (70). Chaperones have been shown to enhance the formation of PrP-res in the cell-free conversion system, although none of the chaperones tested are found in cellular compartments where PrP-res formation most likely occurs (40). Other as yet unidentified cell factors have also been proposed as potential cofactors in the conversion process (132). It is certainly possible that any or all of the proposed cofactors could play a role in formation of PrP-res, but further studies are necessary before any definite conclusions can be drawn.

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