Perhaps the best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid.
These lectures have been excerpted from: S. B. Prusiner (1998) Prions. Proc. Natl. Acad. Sci. USA 95: 13363-13383 (html)(pdf) and C. Weissmann (1999) Molecular genetics of transmissible spongiform encephalopathies. J. Biol. Chem. 274: 3-6 (html)(pdf) with modifications.
ABBREVIATIONS USED
BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; sCJD, sporadic CJD; fCJD, familial CJD; iCJD, iatrogenic CJD; vCJD, (new) variant CJD; CNS, central nervous system; FFI, fatal familial insomnia; FSE, feline spongiform encephalopathy; FSI, fatal sporadic insomnia; GSS, Gerstmann-Sträussler-Scheinker disease; HGH, human growth hormone; Hu, human; MBM, meat and bone meal; Mo, mouse; r, recombinant; SHa, Syrian hamster; Tg, transgenic; TME, transmissible mink encephalopathy; wt, wild-type.
Prions (proteinaceous and infectious agents) cause invariably fatal neurodegenerative diseases (spongiform encephalopathies) in man and other animals (Table S1). In man the disease is usually a rapid, fatal, dementia (that is, loss of thinking ability), whereas in animals it is usually manifest as a fatal ataxia (that is, loss of motor function). The "protein-only" hypothesis (Table S2) proposes that the disease is caused by an altered conformation of a "normal" protein; that is, both the normal protein and the infectious protein have identical amino acid sequences, but the three-dimensional shapes of the two proteins are different. The normal protein, found predominantly on the outer surface of neurons, is called PrPC and the altered, infectious protein is called PrPSc. PrPSc can be identified experimentally as a proteinase-resistant, aggregated form of PrPC that accumulates mainly in the brain of affected organisms (Figure R1).
Familial Spongiform Encephalopathies Are Associated with Mutations in the PrP Gene
The structure of the Prnp gene and its mRNA are shown in Figure T2. Although most cases of human prion disease are sporadic, about 10% are familial (genetically inherited) and linked to one of a number of mutations in the Prnp gene (Figure R2) It is believed that these mutations allow spontaneous conversion of PrPC into PrPSc with a frequency sufficient to cause disease within the lifetime of the individual. Sporadic CJD could be attributed to rare instances of spontaneous conversion of PrPC into PrPSc or rare somatic mutations in the Prnp gene. In both cases the initial conversion would be followed by autocatalytic propagation. Mice overexpressing a murine PrP transgene with a mutation corresponding to the human GSS mutation Pro-102 to Leu spontaneously contract a lethal scrapie-like disease, and it has been reported that this disease can be transmitted to mice expressing the same mutant transgene at lower levels, which do not lead to spontaneous disease.
The Conversion Reaction
Conversion of PrPC to PrPSc in scrapie-infected cells is a late post-translational process, occurring after PrPC has reached its normal extracellular location or thereafter. Why is spontaneous formation of PrPSc an extremely rare event and how does scrapie infection promote conversion? The "refolding model" (Figure R3) proposes that conversion requires that PrPC be unfolded to some extent and refolded under the influence of a PrPSc molecule, a process that would have to overcome a high activation energy barrier and might require a chaperone and an energy source. The "nucleation model" proposes that PrPC is in equilibrium with PrPSc (or a precursor thereof) and that PrPSc is only stabilized when it adds onto a crystal-like seed or aggregate of PrPSc. If a stable aggregate needs to consist minimally of a substantial number of PrPSc molecules, then its spontaneous formation would be a very rare event. However, once a seed is present, monomer addition could ensue at a rapid rate. Trapping of PrP by essentially irreversible aggregation would drive the bulk conversion process. The proposed process is akin to the assembly of (protease-sensitive) flagellin to (protease-resistant) flagellar filaments. Interestingly, the same flagellin molecule can assemble into two types of flagella, depending on the provenance of the seed, thereby providing an analogy for conformationally determined prion strain specificity.
Discovery of the Prion Protein
The discovery of the prion protein transformed research on scrapie and related diseases. It provided a molecular marker that was subsequently shown to be specific for these illnesses as well as the major, and very likely the only, constituent of the infectious prion.
PrP 27-30 was discovered by enriching fractions from SHa brain for scrapie infectivity. This protein is the protease-resistant core of PrPSc and has an apparent molecular mass of 27-30 kDa. Although resistance to limited proteolysis proved to be a convenient tool for many but not all studies, use of proteases to enrich fractions for scrapie infectivity created a problem when the NH2-terminal sequence of PrP 27-30 was determined. The ragged NH2 terminus of PrP 27-30 yielded three sets of signals in almost every cycle of the Edman degradation. Only after these signals were properly interpreted and placed in correct register could a unique sequence be assigned for the NH2 terminus of PrP 27-30. The determination of the amino acid sequence of the NH2 terminus of PrP 27-30 made subsequent molecular cloning studies of the PrP gene possible.
The finding that PrP mRNA levels were similar in normal uninfected and scrapie-infected tissues caused some investigators to argue that PrP 27-30 was not related to the infectious prion particle. An alternate interpretation prompted a search for a prion protein in uninfected animals that was found to be protease sensitive and soluble in nondenaturing detergents, unlike PrP 27-30. This isoform was designated PrPC (Figure S2). Deduced amino acid sequences from PrP cDNA as well as immunoblotting studies revealed that PrP 27-30 was NH2-terminally truncated and was derived from a larger molecule, designated PrPSc, that was unique to infected animals.
Mice Lacking PrPC Are Resistant to Scrapie
The "protein only" hypothesis predicts that in the absence of PrPC mice should be resistant to scrapie and fail to multiply the infectious agent. Mice devoid of PrP (Prnpo/o) were generated by homologous recombination (so-called knockout mice: Figure T3) and found to be essentially normal (Figure T4). When challenged with mouse prions, mice devoid of PrP were completely protected against scrapie disease, and prions failed to accumulate in spleen and brain, in contrast to wild-type mice. Re-introduction of mouse Prnp transgenes into Prnpo/o mice resulted in several lines with varying expression levels of PrPC, which were susceptible to mouse prions; the higher the PrPC content of the brain, the shorter the incubation times.
The Species Barrier Is Abolished by Introducing the PrP Transgene of the Prion Donor into the Recipient
Prions are transmitted from one species to another much less efficiently, if at all, than within the same species and only after prolonged incubation times. This prolongation is often referred to as the "species barrier". Prions synthesized de novo reflect the sequence of the host PrP gene and not that of the PrPSc molecules in the inoculum derived from the donor. On subsequent passage in a homologous host, the incubation time shortens to that recorded for all subsequent passages. In the case of prion transmission from hamsters to mice, this so-called species barrier was overcome by introducing hamster Prnp transgenes into recipient wild-type mice (Table S3). Importantly, the properties of the prions produced in these transgenic mice corresponded to the prion species used for inoculation, that is infection with hamster prions led to production of hamster prions, but infection with mouse prions gave rise to mouse prions. Within the framework of the "protein only" hypothesis this means that hamster PrPC but not mouse PrPC (note: the mouse and hamster Prnp genes encode PrPC's that differ by 10 amino acids) is a suitable substrate for conversion to hamster PrPSc by hamster prions and vice versa. Interestingly, susceptibility of the mouse to prions from other species, such as hamster, mouse, or man, is increased when the "foreign" PrP transgenes are introduced into a PrP knockout mouse (Prnpo/o mouse), suggesting that the resident mouse gene inhibits the propagation of the "foreign" prions.
The demonstration that disruption of the PrP gene confers resistance to scrapie and that reintroduction of a PrP-encoding transgene restores susceptibility paved the way to reverse genetics of PrP, that is the introduction of deletions or mutations into the Prnp gene and determination of the capacity of the modified gene to confer susceptibility to scrapie to a PrP knockout mouse. Transgenes encoding PrP with deletions extending to codon 93, but not to 106, restored susceptibility to scrapie.
Miniprions
Transgenic mice containing mutated or truncated mouse Prnp genes have been constructed. Deletions removing nearly one half of the PrPC protein (a miniprion called PrP106), removing amino acids 23 to 89 together with amino acids 141 to 176, did not prevent PrPSc formation. In fact, this severely deleted Prnp gene created a new "species" of prion--one that would infect transgenic mice carrying the mutated transgene, but not mice overexpressing a normal Prnp transgene. Tg(PrP106)Prnp0/0 mice that expressed PrP106 developed neurological dysfunction about 300 days after inoculation with RML prions previously passaged in CD-1 Swiss mice. The resulting prions containing PrPSc106 produced CNS disease in about 66 days upon subsequent passage in Tg(PrP106)Prnp0/0 mice (Table S5). The Tg(MoPrP-A) mice overexpressing MoPrP are resistant to RML106 miniprions but are highly susceptible to RML prions. These mice require more than 250 days to produce illness after inoculation with miniprions but develop disease in about 50 days when inoculated with RML prions containing full-length MoPrPSc.
The unique incubation times and neuropathology in Tg mice caused by miniprions are difficult to reconcile with the notion that scrapie is caused by an as-yet-unidentified virus. When the mutant or wt PrPC of the host matched PrPSc in the inoculum, the mice were highly susceptible (Table S5). However, when there was a mismatch between PrPC and PrPSc, the mice were resistant to the prions. This principle of homologous PrP interactions, which underlies the species barrier (Table S3), is recapitulated in studies of PrP106 where the amino acid sequence has been drastically changed by deleting nearly 50% of the residues. Indeed, the unique properties of the miniprions provide another persuasive argument supporting the contention that prions are infectious proteins.
PrPSc conformation enciphers diversity
Persuasive evidence that strain-specific information is enciphered in the tertiary structure of PrPSc comes from transmission of two different inherited human prion diseases to mice expressing a chimeric MHu2M PrP transgene. In FFI, the protease-resistant fragment of PrPSc after deglycosylation has a mass of 19 kDa, whereas in fCJD(E200K) and most sporadic prion diseases it is 21 kDa (Table S6). This difference in molecular size was shown to be due to different sites of proteolytic cleavage at the NH2 termini of the two human PrPSc molecules reflecting different tertiary structures. These distinct conformations were understandable because the amino acid sequences of the PrPs differ.
Extracts from the brains of FFI patients transmitted disease to mice expressing a chimeric MHu2M PrP gene about 200 days after inoculation and induced formation of the 19-kDa PrPSc, whereas fCJD(E200K) and sCJD produced the 21-kDa PrPSc in mice expressing the same transgene. On second passage, Tg(MHu2M) mice inoculated with FFI prions showed an incubation time of about 130 days and a 19-kDa PrPSc, whereas those inoculated with fCJD(E200K) prions exhibited an incubation time of about 170 days and a 21-kDa PrPSc. The experimental data demonstrate that MHu2MPrPSc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet the amino acid sequence of MHu2MPrPSc is invariant.
The results of our studies argue that PrPSc acts as a template for the conversion of PrPC into nascent PrPSc. Imparting the size of the protease-resistant fragment of PrPSc through conformational templating provides a mechanism for both the generation and propagation of prion strains.
Hallmark of prion diseases
The hallmark of all prion diseases--whether sporadic, dominantly inherited, or acquired by infection--is that they involve the aberrant metabolism and resulting accumulation of the prion protein. The conversion of PrPC into PrPSc involves a conformation change whereby the alpha-helical content diminishes and the amount of beta-sheet increases. These findings provide a reasonable mechanism to explain the conundrum presented by the three different manifestations of prion disease.
Understanding how PrPC unfolds and refolds into PrPSc will be of paramount importance in transferring advances in the prion diseases to studies of other degenerative illnesses. The mechanism by which PrPSc is formed must involve a templating process whereby existing PrPSc directs the refolding of PrPC into a nascent PrPSc with the same conformation. A knowledge of PrPSc formation not only will help in the rational design of drugs that interrupt the pathogenesis of prion diseases but it may also open new approaches to deciphering the causes of and to developing effective therapies for the more common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Indeed, the expanding list of prion diseases and their novel modes of transmission and pathogenesis, as well as the unprecedented mechanisms of prion propagation and information transfer, indicate that much more attention to these fatal disorders of protein conformation is urgently needed.