Ln Vited Revie W Molecular Clues to Pathogenesis in Prion Diseases

Histol Histopathol (1997) 12: 583-594 Histology and Histopathology

ln vited Revie w

Molecular clues to pathogenesis in prion diseases

M. Laurent and G. Johannin Service d' lmagerie Cellulaire, Université Paris-Sud, Orsay Cedex, France

Summary. The infectious agent of the transmissible Introduction spongiform encephalopathies (TSE)resembles a virus in that it propagates in vivo and has distinct strains. Although recent and public developments about However, compelling evidence strongly suggests that a 'mad cow disease' and Creutzfeldt-Jakob disease go far posttranslational structural alteration in a glycoprotein beyond purely scientific concern, it would be wrong to P~PC(the normal, cellular isoform of the so-called prion believe that prion diseases - which is the generic name protein) is responsible for pathogenesis of these diseases. for al1 these transmissible neurodegenerative diseases - According to this hypothesis - now close to being have been only recently discovered. The first case of a generally accepted -, iatrogen, sporadic and familia1 sheep which presented al1 the symptoms of scrapie forms of TSE would have the same molecular (excitability, itching, ataxia and finally paralysis and mechanism: the conversion of PrPC into a protease- death) was recognized in the middle of the 18th century resistant isoform prPSc kinetically behaves as an (Reibel, 1994; Weissmann, 1994a). Of course, other autocatalytic process which, combined with the high unidentified cases might have occurred long before. turnover rate of the normal isoform, may endow the Scrapie is the prototype of what has proved to be a group system with bistability properties and subsequent of lethal diseases affecting animals such as sheep, cows, threshold behavior between normal and pathogenic minks, moufflons, ostriches, cheetahs, cats and also steady-states. Normal prion protein seems to be humans (Reibel, 1994). In 1936, Cuillé and Chelle necessary for long-term survival of Purkinje showed that scrapie was due to an agent found in the neurons, regulation of circadian rhythms and, more brain and in the spinal chord of diseased animals and controversially, for normal synaptic function. At least that the disease could be transmitted to sheep and goats part of the pathology might be due to the unavailability by inoculating them with the lombard cord of affected of normal isoform rather than to the accumulation of animals (Weissmann, 1994a). This agent was shown PrPSc. NMR structure of the normal mouse prion protein early on to be non-conventional because it has particular reveals a short, unexpected B-sheet which might be a properties such as uncommon resistance to treatments nucleation site for the conformational transition between that usually destroy nucleic acids found in viruses and P~PCand P~PSC.Prion diseases may challenge the edged bacteria. Moreover, incubation periods for the disease distinction that we use to make between informational are extraordinarily long (measured in months in animals (DNA) and functional (proteins) macromolecules. and years in man). In 1982, Prusiner proposed naming Pathogenic mechanism of prions might also be involved this agent as 'prion' to distinguish it from conventional in other proteins to achieve and pass on their pathogenic agents such as bacteria and viruses (Prusiner, conformation. Hence, structural inheritance at the 1982). molecular leve1 might be the missing link for In humans, four slow degenerative diseases of the the understanding of the structural inheritance central nervous system have been described: kuru; processes featured at the cellular level. Moreover, Creutzfeldt-Jakob disease (CJD); Gerstmann-Straussler- evolutionary paradigm postulating a primitive RNA Scheinker disease (GSS); and fatal familia1 insomnia world is weakened by the mechanism of prion (FFI). Kuru, historically contracted at the beginning of diseases. this century in Papua New Guinea's Fore highlanders, may have originated from the consumption of the Key words: Prion diseases, Prion protein, Spongiform remains of a CJD sufferer (Dormont and Bursaw, 1996) encephalopathies, Bistability, Structural inheritance and was propagated through ritual cannibalism. Inoculation studies by Gajdusek's group (Gajdusek et 0Mnt reguests to: Dr. Michel Laurent, SeMce d'lrnagerie Cellulaire, al., 1966) had demonstrated the infectious nature of kuru URA 116 CNRS, BU 440, Universit4 Paris-Sud, 91405 Orsay Cedex, and then of CJD and GSS by transmitting the disease to France chimpanzees through the injection of diseased brain Molecular clues to prion diseases

tissue. The infectious nature of FFI was shown much biology concerning the edged distinction that we make more recently (Tateishi et al., 1995). Although between informational and functional macromolecules, incubation periods are particularly long, once the first namely DNA and proteins respectively. clinical symptoms such as loss of memory or motor disturbances appear, progression to death may take only Nature of the transmissible agent a few months. The pathological changes vary in location and intensity but the brain tissue typically develops The unsual properties of prion diseases gave rise spongelike holes as neurons die while glia proliferate. early to speculations about the nature of the trans- No inflammatory nor immunological counterpart is missible agent responsible for neurodegenerative observed. diseases. In the 60s, the leading theory supposed that an The current epizooty of bovine spongiform unidentified, slow-acting virus was the real culprit. encephalopathy (BSE or 'mad-cow disease') which Although viruses can be very hard to find, no-scrapie- principally occurs in Great Britain, is believed to result associated virus or nucleic acid has ever turned up, in from a change in the process of making feed spite of many attempts to find traces of it, during the last supplements given to cattle. These supplements coming 30 years (Kellings et al., 1992). However, Alper7s from cattle (50%) and sheep (15%) offal were probably experiments (Alper et al., 1966, 1967) weakened this contaminated by a scrapie-like agent. In 1980, the theory by showing that scrapie infectivity resists stopping of the fat-extracting process by organic solvents inactivation by both ultraviolet and ionizing radiations, probably increased contamination of the feed which usually destroy nucleic acids. The possibility that supplements. Hence, at least in the corresponding the scrapie agent might be devoid of nucleic acid was species, the agent responsible for ESB and kuru can reinforced by Prusiner's group who later showed that diffuse through the digestive tract. Kuru has almost reagents - such as nucleases, psoralens, hydroxylamine disappeared with the cessation of ritual cannibalism, and ~n2+ions - that specifically modify or damage strongly suggesting that this disease was transmitted nucleic acids, do not alter scrapie infectivity in orally, as proposed for BSE. Iatrogen forrns of CJD have homogenates or partially purified preparations (Diener et been observed as a result of the use of contaminated al., 1982; McKinley et al., 1983; Gabizon et al., 1987). surgical material, cornea or eardrum transplants or Although it is known that some viruses can resist therapeutic use of hypophyse-derivated products from treatments that normally destroy nucleic acids, two human origin. However, prion diseases may also arise additional observations go against the vira1 hypothesis. spontaneously without any apparent cause (so-called Modern subtraction hybridization experiments were sporadic) or be genetic (5 to 15% of CJD are thought to unable to reveal the presence of DNA or RNA sequences be familial). Whatever its iatrogen, or sporadic or in infected brain tissues which would be absent in familia1 origin, the disease can usually be transmitted to normal ones (Mestel, 1996). Moreover, the occurrence animals by intracerebral inoculation. The possibility of a of some familial prion diseases makes it much more vertical transmission (from the cow to the calf for unlikely to be a virus, without definitively excluding instance) is as yet hypothetical, although such an such a possibility. One would have to imagine that occurrence has been reported in the case of captive mule although the virus would infect al1 the members of the deer and elk in zoological gardens (Reibel, 1994). family, only those who have a putative mutation would However, much of the current upheavals come from a develop the disease. recent report (Will et al., 1996) indicating that ten cases On the other hand, there is now a wealth of of CJD were detected in Great Britain whose clinical and biochemical and genetic evidence supporting the pathological features seem significantly distinct from contention that a protein (so-called prion protein or PrP) those of the common (although rare) disease, especially is the key for infectivity. Prusiner first reported the concerning the fact that the patients were unusually existence in scrapie brains of a protein that was required young. Moreover, clinical symptoms (with more ataxia for infectivity and that did not seem to be present in than dementia) and histo-immunochemical features seem healthy brains (Prusiner et al., 1981; Prusiner, 1982). to place these new cases closer to kuru or iatrogen CJD According to a scenario originally outlined by Griffith rather than sporadic CJD. Correlatively to the current (1967), this protein (so-called prpSc, Sc for scrapie) epizooty of BSE and the long incubation periods of might direct its own replication. The fact that the prion prion diseases, these data make us wonder if these protein was encoded by a chromosome gene (and not by human cases might be linked to eating BSE-tainted beef. a virus) was suggested by the observation that the leve1 In any case, many intriguing questions about the of PrP messenger RNA remained unchanged throughout molecular mechanism of prion diseases remain to be the course of scrapie infection (Oesch et al., 1985; solved before an answer can be unambiguously given Basler et al., 1986). This result led to the identification concerning the problem of the species barrier betweeen of the normal gene product, a protein designated prpC, animals and humans. Beyond the irnportant problems of and also to the determination of its primary structure, in public health, spongiform encephalopathies are of hamsters and mice (and later in other mammal species) fundamental interest for biologists because they might by sequencing of molecular clones recovered from come to challenge the current dogma of molecular cDNA libraries (Chesebro et al., 1985; Oesch et al., Molecular clues to prion diseases

1985; Basler et al., 1986; Locht et aL, 1986). The amino substitutions (Tyr to His at site 155 and Asn to Ser at site acid sequences of both the normal P~PCand pathological 143). The fact that this event is the only one of its kind prpSc proteins were found to be identical. The direct in the gene tree might suggest that these changa could implication of the PrP protein in infectivity was also have predisposed humans towards a prion strain demonstrated by severa1 groups, using distinct types of present in cattle. However, since correlation is not evidence: Weissman showed that "knockout" mice causation, this observation does not demonstrate that lacking the PrP gene are resistant to scrapie and fail to BSE can be transmitted to humans. propagate the infectious agent when they are inoculated The PrP protein is synthetized as a precursor of 254 with diseased brain tissue from the same species (Büeler amino acids in Syrian hamsters and mice or 253 amino et al., 1992). Moreover, they showed that susceptibility acids in humans. An N-terminal signal sequence of 22 to scrapie is a function of P~PClevels in the host (Büeler amino acids is cleaved in the endoplasmic reticulum et al., 1993). Hsiao et al. (1990) showed that transgene (Hope et al., 1986; Turk et al., 1988; Safar et al., 1990) mice overexpressing the PrP gene with a mutation and 23 amino acids are removed from the COOH corresponding to that found in the familia1 form of terminus on addition of a glycosyl-phosphatidylinositol human GSS, spontaneously contract a lethal scrapie-like (GPI) anchor at Ser 231 (Stahl et al., 1987, 1990a). Two disease. Finally, Bessen et al. (1995) took advantage of additional glycosylations occur at Asn 181 and Asn 197 the fact that two hamster-ada ted mink strains give rise respectively and a disulphide bond is formed between to two distinguishable prPS: molecular species (so- Cys 179 and Cys 197 (Fig. 1). NMR three-dimensional called strain-specific species). They found that in a cell- structure of the mouse PrP domain 121-131 (Riek et al., free system, prpSc from the two strains could convert the 1996) reveals that the protein contains three a-helices same prpC protein into two distinct sets of products that and a two-stranded antiparallel p-sheet. These results exhibit the same physico-chemical properties as those of disagree with previous model calculations (Huang et aL, natural prpSc molecules associated with the two strains. 1994) that predicted a fow helix bundle model without In the same vein, it has been shown that synthetic any P-structured element in the corresponding domain of peptides which mimic some of the conformational the normal prion protein. The second and third a-helix features of prpSc, are able to induce cellular PrP to are linked by the single disulphide bond in the protein acquire properties of the scrapie isofosm (Kaneko et al., and their twisted V-shape forms the scaffold onto which 1995). Even if it has not yet been established that the in the first helix and the short p-sheet are anchored (Fig. 2). vitro converted form of the protein can also generate Since it has been proposed (Pan et al., 1993; Huan et infectivity, these results support the idea that the al., 1996) that the transition from prpC to PrPSC is pathogenic isoform prpSc of the prion protein ma impose its conformation upon the native protein PrPd: Today, although some authors still believe that the infectious agent is a specific nucleic acid associated with or packaged in some host-derived protein (Manuelidis et al., 1987; Xi et al., 1992; Mestel, 1996), the most accepted theory in the field - known as 'the protein-only' hypothesis - postulates that the conversion of the constitutive host protein P~PCto a pathogenic, isomenc form P~PS~constitutes the molecular basis of prion diseases.

PrP gene and protein The entire open reading frame of PrP gene is contained within one exon (Basler et al., 1986), localized in the short arm of human chromosome 20 and the homologous region of mouse chromosome 2. This suggests that the PrP gene probably exists in the common ancestor of mamrnals and its appearance thus Fig. 1. Biosynthesis and maturation of the normal pnon protein. precedes the speciation of mammals. Accordingly, DNA Maturation of the precursor of the prion protein involves three distinct sequences related to a PrP cDNA clone have been events: cleavage of the sequence signal at the N-terrninus, glycosylation (CHO) at Asn 181 and Asn 197 and removal of 23-C-terminal detected in invertebrates including nematode, drosophila aminoacids on addition of a gmyl-phosphati~inositol(GPI) andior and possibly yeast (Westaway and Prusiner, 1986). at Ser 231. A disulphide bond is formed between Cys 179 and Cys 214. Phylogenetic relationships among the PrP gene of Positions of the regular secondary structures (H, a-helice and S, b- vertebrates (Krakauer et al., 1996) show that the ancestor strand), are shown for the mature mouse prion profein: S1 (12&131), of the hominoids (excluding the orangutan) and of cattle S2 (161-164), H1 (144-154), H2 (179, 193) and H3 (200-217). (Bos taurus) uniquely shared (among 33 species of Calculations have not yielded conclusive resulta (Huang et d., 1994) for vertebrates so far analyzed) two pairs of derived the segment 23-108, which contains the prion-characteristic octapeptlde Molecular clues to prion diseases

accompanied by an increase in the f3-sheet content of the making undetectable a putative difference between prpC protein, it seems tempting to speculate that the short f3- and any infectious subspecies of prpSc. sheet found in the normal protein might be a nucleation site for a conformational transition that could include the Is it the depletion of P~PCor the accumulation of lo~pconnecting the p-sheet to the first helix which is P~P~Cthat is responsible for neurotoxicity in prion essentially hydrophobic. diseases? Primary sequences of normal prpC and infectious P~P&isoforms of the prion protein are indistinguishable Normal prion protein is constitutively expressed at but the two forms differ in their physicochemical the outer surface of the plasma membrane, where it is properties and in their secondary structure (Pan et al., anchored by its GPI moiety (Stahl et al., 1987, 1990a,b). 1993; Stahl et ai., 1993; Cohen et al., 1994), although The hysiological function of the normal host protein the NMR or cristallographic threedimensional structure PrP 6' has long been obscure and still remains of the prpSc isoform has not yet been observed. In controversial. Although substantial amounts of normal particular, prpSc is partially resistant to proteinase K PrP protein are found not only in brain but also in heart under conditions where prpC is readily cleaved and and skeletal muscle (Büeler et aL, 1992) and, to a lesser solubilized (Meyer et al., 1986). More precisely, extent, in most other organs except liver and pancreas proteinase K only removes 23 amino acids from the NH2 (Weissmann, 1994a), it is quite justified to search for the terminus of the P~PS~isoform, leading to a 27-30 kD putative function of the normal prion protein in neuron fragment which posseses al1 the characteristics of cells since prion diseases are characterized principally infectivity. Moreover, prpSc, unlike prpC, gives rise to by neurodegenerative disorders, although pathological cerebral amyloid formation, a highly ordered protease- changes in peripheral organs have also been reported in resistant aggregate characterized by its insolubility and scrapie-infected animals (Ye and Carp, 1995). fibrillar structure (Alper et al., 1967; Jarrett and Uninoculated old mice harboring high copy numbers Lansbury, 1993). The resistance of prpSc to proteinase K of wild-type PrP transgenes were found to develop treatment was found for al1 the forms of transmissible trunca1 ataxia, hindlimb paralysis, and tremors spongiform encephalopathies. Although no chemical (Westaway et al., 1994). The normal PrP protein was dserence has been detected so far between prpC and claimed to be necessary for normal synaptic function prpSc, its possible occurrence cannot be complete1 (Collinge et al., 1994), although other findings report eñcluded because the ratio of infectious units to PrP& that the loss of the normal form of prion protein does not molecules is about 1:100000 (Bolton et al., 1991), alter neurona1 excitability and synaptic transmission in the hippocampus in mice (Lledo et al., 1996). Tobler et al. (1996) showed that mice devoid of normal prion protein have altered circadian activity rythms and sleep. Hence, one can wonder if FFI, one of the inherited prion diseases in human for which similar alterations in sleep and circadian rythms of many hormones are observed, might be related to the normal function of the prion protein rather than to a dysfunction resulting from the accumulation of the prpSc isoform. Furthermore, mice homozygous for a disrupted PrP gene grew normally after birth (histology of the cerebellum is still normal at 18 weeks and may be so later) but they began to show, at about 70 weeks of age, progressive symptoms of ataxia and motor disturbances correlated with an extensive loss of Purkinje neurons (Sakaguchi et al., 1996). These results disagree with previous studies performed with other PrP null-mice (Büeler et al., 1992, 1993) which exhibit no ataxic phenotype at up to 93 weeks. However, differences related to breeding conditions or to the embryonic stem cell lines used or to the transfection Flg. 2. Three-dimensional structure of the mouse prion domain process may explain that in some transgenic mice, PrP(l21-231) as determined by NMR. Ribon diagram indicating the susceptibility to prion is abrogated, but that some positions of the three a-helices (cyiinders) and the antiparallel two- physiological functions of normal PrP protein are stranded a-sheet (arrows). The second and third helices are linked by a retained. Hence, if the results of Sakaguchi et al. (1996) disulphide bond (ctteckered pattern). Dashed lines indicate structures are confirmed, they would indicate that the loss of PrP that are not well defined (adapted from Riek et al., 1996). The first a- helice migM be part of a single binding site for the pathogenic isofom and not the accumulation of the prpSc isoform should be P~P%,and sequence dirences between species in this part of the the primary cause of the death of Purkinje cells, at least stnicture might be involved in the barrier of prion disease transmission in PrP null-mice. In any case, pathogenic effects between species. resulting from the absence of the normal isoform of the Molecular clues to prion diseases

protein should appear only after a very prolonged time of the crucial experiments which make the 'protein only' delay. However, one fundamental question would remain hypothesis, originally outlined by Griffith (1967), a unexplained in such a case: why incubation time is much more and more solid hypothesis for prion diseases. The shorter in scrapie-infected non-transgenic mice than in agent res onsible for the disease would be the modified PrP null-mice. If prpSc is athogenic only because it form P~P%cof the normal host prion protein prpC, the promotes conversion of PrP 8 to prpSc and subsequently pathogenic form prpSc multiplying by converting the diminishes the leve1 of available normal prpC isoform, normal form into a co y of itself (Griffith, 1967). delayed onset of disease would have to be observed in Normal host protein PrP8 and modified fom prpSc are scrapie-infected mice compared to PrP null-mice. Hence, believed to be conformational isomers. According to the even if the loss of prpC may contribute to the scrapie above discussion, we do not know yet if prpSc is histopathology and disease, it is probable that prpSc also intrinsically pathogenic or whether its pathogenicity has, directly or by acting synergically in a complex with results from the fact that the conversion mechanism prpC, a route of pathogenicity that is distinct from that renders the normal host isoform prpC unavailable, or resulting from the loss of the normal function of the PrP whether accumulation of prpSc and loss of prpC both protein. According to this possibility, it was shown contribute, in kinetically distinct ways, to pathogeneeity. (Westaway et al., 1994) that overexpression of wild-type lbo alternative mechanisms have been proposed for prpC in uninoculated transgenic mice is pathogenic, but the conversion of prpC to prpSc in prion strains. The that the novel neurological syndrome observed in these 'nucleation model' (Gajdusek, 1988; Jarret and cases (necrotizing myopathy involving skeletal muscle, Lansbury, 1993; Kocisko et al., 1994, 1995) supposes demyelinating polyneuropathy and foca1 vacuolation of that severa1 prpSc monomers polymerize to form a the central nervous system) widens the spectmm of pnon nucleus. Once the nucleus has been formed, further disease pathogenesis. accretions occur through the binding of prpC to the The role, in scrapie pathology, of the loss of the polymer, a conformational rearrangement (PrP C-~rPc) normal function of the prpC protein was also studied by taking place to adjust the incoming molecule prpC to the grafting experiments recently performed by Weissma~ template. The trapping of prpC would be an essentially and Aguzzi's groups (Brandner et al., 1996; Isenrna~et irreversible aggregation process which would drive the al., 1996). They grafted neural tissue overexpressing bulk conversion reaction. The alternative model known P~PCinto brains of knockout mice for the PrP gene, i.e. as the 'refoldin model' (Prusiner, 1991) proposes that in mice which are resistant to scrapie and do not constitutive PrPE monomers would be unfolded to some propagate infectivity. The mice were then infected with extent and subsequently refolded under the influence of scrapie prions by intracerebral inoculation. The grafted prpSc molecules (Fig. 3). As an example, Liautard tissue accumulated high levels of prpSc associated with suggested that prions could be misfolded molecular typical histopathological changes of scrapie and it chaperones (Liautard, 1991, 1992). In both models, the became infectious. However, although important pathogenic, conformational isomer prPc would thereby amounts of prpSc migrated from the grafted tissue to the impose its conformation upon the native protein prpC in host cerebral tissue, no pathological change was a globally autocatalytic process requiring the presence of observed in this host tissue 16 months after inoculation. preexisting prpSc (Weissmann, 1995). According to Hence, brain tissue devoid of normal prpC protein, in Come et al. (1993), the heterodimer prpC-prpSc would addition to being resistant to scrapie infection, is not act as a catalyst for the conversion. darnaged by exogenous prPc. Once again, these results Most, if not all, inherited prion diseases in humans disagree with those of Sakaguchi et al. (1996) on the (which are autosomal dominant disorders) are linked to particular point concerning the long-term survival of one of a number of mutations in the PrP gene. For Purkinje neurons in PrP-null mice. Most importantly, Prusiner (1991), the mutations increase the frequency of they seem to indicate that it is not the accumulation of the spontaneous conversion of prpC into PrpSc, allowing P~PS~but rather the unavailability of normal prpC the expression of the disease to occur within the lifetime isoform, for some unknown intracellular process that is of the individual. NMR structure of the prion protein directly linked to spongiosis and neural death. However, (Riek et al., 1996) reveals that al1 six residues of the one cannot exclude that graft-derivated prpSc which domain PrP(121-231) for which mutation is believed to have migrated in the host brain cannot be internalized in be associated with familia1 prion diseases (or PrP null-cells, especially if P~PSCcan be endocytosed predisposition to prion diseases for those who think that only by way of association with F'rpC. Therefore, it is the PrP protein is not the true infectious agent) are not possible to conclude at this time that prpSc is located in (or close to) regular secondary structure inherently non-toxic, particularly in mice that are not elements (except in the first helix). Mutation of one of devoid of the PrP gene. these residues might therefore destabilize the three- dimensional structure of the protein or modify its ligand- The 'protein only' hypothesls and Its dynamlc binding properties. Sporadic CJD would also arise from consequences the spontaneous conversion of prion protein from the normal to the pathogenic isoform, due either to a PrP Prusiner, Weissmann and Caughey conducted many gene somatic mutation or to rare instances involving Molecular clues to prion diseases

modification of wild-type prpC protein. In both cases the of the protein. Normal prpC undergoes endocytosis initial conversion is thought to be followed by auto- (Caughey et al., 1989; Borchelt et al., 1990) and possibly catalytic propagation (Weissmann, 1994a). recycling (Harris et al., 1993). The synthesis and To understand how the prion invasion accords with degradation of prpC are rapid with half-life estimated to the 'protein only' hypothesis, two linked phenomena 0.1 and 5 hours, respectively (Caughey et al., 1989; have to be considered: 1) The prpC + prpSc conversion Borchelt et al., 1990). In contrast, no degradation is an autocatalytic process. 2) In a c 1, the normal prion pathway is known for the prpSc isoform and that is protein prpC tums over whereas Pr & isoform does not probably why this molecular species accumulates in (Fig. 4). This is precisely one of the important cells when it is formed. differences that was evidenced between the two isoforms Theoretical kinetic analysis of the dynamic process including the turnover of the normal prion protein (Laurent, 1996a,b) shows that the system exhibits bistability properties, indicating that the very slow accumulation of abnormal form of the protein in the brain would in fact be the consequence and not the cause of the disorders. The cause would be a transition between two alternate, stable steady states of the system. The two alternate steady states are associated, respectively, with low (normal steady state) and high (pathogenic steady state stationary concentrations of the pathogenic isoform PrPLc of the prion protein (Fig. 5). Dynamics of prion infection differs from those of virus replication, healthy organisms being able to eliminate spontaneously infrathreshold amounts of foreign prpSc protein. Under controlled conditions, addition of suprathreshold quantities of prpSc or also, paradoxically, of the normal prpC protein, provokes a transition towards the pathogenic steady-state. Hence the possible presence of a small amount of the P~PSCprotein in lymphocytes (which was reported as prevalent in the human population (Manuelidis and Manuelidis, 1993) does not necessarily constitute an indication of a non- symptomatic but infectious pathogenic state. Moreover,

Fig. 3. Models for the catalysed conformational conversion of PrPc to PrPSc. a. The simplest model of Prusiner (1991) in which the native protein PrPc binds to endogenous or exogenous PrPsc which acts as a catalyst in the PrPc-PrPsc conversion. b. In this slightly dierent model, Flg. 4. Normal and pathogenic pathways for the prion protein. PrP PrPsc isoform forms a complex with normal PrPc protein and the mRNA is constitutively expressed in the brains of adult animals and its heterodimer PrF-PrP* is the catalyst for the PrF-PrPsc conversion. concentration remains unchanged in the brain of scrapie-infected In both cases, the initial conversion is followed by autocatalytic animals (compared to normal tissue). The synthesis and degradation of propagation. Sporadic form of prion diseases are thought to result from PrPc are rapid although the accumulation of PrPsc is slow. PrPSc the occurrence of the spontaneous conversion of PrPc to PrPsc which formation is a posttranslational, autocatalytic process (see Fig. 3) and would be a very rare event (but see Laurent, 1996a,b). In the case of no degradation pathway is known for this protease-resistant isoform. certain mutations in the PrP gene, spontaneous conversion may occur Accumulation of PrPSc is the major pathognomonic feature of prion about a million times more frequently than in the case of the wild-type diseases, with frequent formation of amyloid plaques consisting mainly protein (Weissmann, 1994). of aggregates of PrP*. Molecular clues to prion diseases

infectious prion particles should not be seen as Prion stralns and species barrier necessarily composed of the abnormal isoform of the protein, as usually stated. Theoretical analysis shows The weak point of the 'protein only' hypothesis has that particles containing overproduced normal PrP long been the phenomenon of scrapie strains. About 20 protein might also be pathogen, as was experimentally different prion strains have been isolated so far in mice, reported (Westaway et al., 1994; Mestel, 1996). depending on how rapidly infection takes place in However, mice expressing high levels of PrP transgenes different mouse strains and also how the patterns of do not seem to accumulate high levels of protease symptoms and brain lesions are observed (DeArmond et resistant PrP protein (Westaway et al., 1994). al., 1994). Obviously, the problem of prion strains is Convergent data (Büeler et al., 1992, 1993; Sakaguchi et closely related to the so-called species barrier, which al., 1995) also demonstrate that the disease pro resses hinders (at least under some circumstances) prion much more slowly in heterozygous mice (Prn-pOfi mice transmission from one host species to another. having one normal allele for the PrP gene and one allele According to the 'protein only' hypothesis which in which the PrP ene is disrupted) than in wild-type assumes that the difference between prpC and P~PS~is controls (Prn-pt F+). The difference in incubation solely conformational, strain differences would times between transgenic and wild-type mice is about correspond to different conformational states of the a factor of 2 in the case of the mouse-adapted pathogenic prpSc isoform. Hence, each stable prpSc CJD agent (Sakaguchi et al., 1995) and exceeds a strain would convert host prpC into a copy of itself. factor of 10 in the case of the scrapie agent (Büeler Although recent experiments of Bessen et al. (1995) and et al., 1992, 1993). If we compare incubation times Collinge et al. (1996) agree with such a possibility, the for P~~-~OIO(homozy ous for the disrupted PrP gene) authors assumed (but do not demonstrate) that the ~rn-~Ol+and Prn-pt 9' + mice, one can conclude that relative electrophoretic mobility on western blots or normal prpC afford some kind of partial protection strain-specific susceptibilty to hydrolysis of prpC by against scrapie or CJD disease. However, in the proteinase K that they observed for different prion framework of the 'protein only' hypothesis, these strains, was due to differences in conformation. data indicate that the susceptibility to scrapie or CJD Usually, transmission of prions from one species to is a function of the leve1 of normal prpC in the host. another is very inefficient (if at all) and this phenomenon This observation agrees fairly well with the dynamic occurs only after prolonged incubation times. For analysis. instance, Prusiner (1987) and Telling et al. (1994) Within the limits of the accuracy of these reported that only 10% of mice intracerebrally conclusions which are those of the 'protein only' inoculated with human prions developed some hypothesis, compounds that would act on the turnover dysfunction of the central nervous system after rate of the normal P~PCprotein appear as a possible incubation times exceeding 500 days. The species barrier therapeutic strategy against prion diseases. between mice and Syrian hamsters for prion

Flg. 5. Dynamic process of Rate of prpC synthesis prion propagation induding the .------turnover of the normal prion low medium high protein. Size of arrows and circles respectively reflect the flux intensities and lhe relative stationary concentration of the PrPc and PrPsc species. For low rates of PrPc biosynthesis, ppc -!S?- normal form PrPc is dominant and the flux rnerely represents the flux of degradation of that ;@ species. On the other hand, for a high rate of PrPc bio- PPC 0- M* synthesis, the metabolic flux is preponderanly oriented toward the forrnation of the isoform PrPsc, the stationary rate of which is high. For intermediate rates, two distinct stationary state may exist, one being normal, the other being pathogenic and the transitions between these states exhibit threshold features (Laurent, 1996a,b). The same results are obtained when the rate of degradation of the normal form of prion protein is allowed to vary.

Molecular clues to prion diseases

a mechanism which might also be involved in other Structural inheritance and the primltive world normal proteins to achieve and pass on their functional conformation. As Weissmann (1994b) noted: From a molecular point of view, the examples "Phenomena discovered in higher eucaryotes acquire evoked above rely on a mechanism in which a protein additional respectability if they are also found in yeast'. passes its structural information to another protein This criterium seems to be satisfied for prions insofar as molecule without any direct involvement of the genomic other examples pertaining to yeast seem to indicate that material. In other words, it means that a protein could such a process might be less exceptional than it appeared have the property of bearing both functional and at first glance. Yeast has a variety of examples of informational roles. Such a process might be connected extrachromosomic inheritance controlling phenotypic to the phenomena of structural inheritance observed at characters via a non-mendelian way. %O determinants, the cellular level, especially in ciliates (Beisson and namely URE3 and psi, seem relevant to a process similar Sonnebom, 1965; Fleury and Laurent, 1994). to prions. URE3 corresponds to the spontaneous A set of experiments, first carried out on development of an unusual nitrogen metabolism Paramecium (Beisson and Sonneborn, 1965), and properties in yeast cells. The URE3 determinant is extended to miscellaneous ciliate species, has shown that associated to a non-mitochondrial cytoplasmic element, the polarity inversion of a row of cilia was a phenotypic and the URE3 phenotype would be best explained as if character transmitted from one generation to the next the protein called Ure3 had flipped into an abnormal without any modification of the genoma. Although the configuration (Wickner, 1994). The strains possessing cellular material which is present at the periphery of the the URE3 determinant show exactly the same cell (the cortex) is not the support of any genetic deregulation features as the nuclear mutants ure2. The material, it seems to contain the necessary information strains ure2 are unable to propagate the URE3 for the clonal transmission of this inversion. The origin phenotype, as transgenic mice deleted of the gene PrP of this inversion, -thereafter named 'structural cannot be infected by the prion protein. Conversely, the inheritance' in opposition to genomic inheritance- must surexpression of the Ure2 protein leads to a large be searched for at the level of some organization coupled increase of the intracellular rate of the URE3 to structural determinants rather than at the level of a determinant and the Ure3 protein from URE3 cells is molecular code, as genetics used to teach it. At the much more resistant to a protease than is the normal present time, our knowledge about the molecular basis protein (Masison and Wickner, 1994). The protein of this mechanism is rather scarce. However, while the determinant of the psi phenotype has been recently concept of structural inheritance is one of the major characterized as the product of the SUP35 gene, which contributions to contemporary biology made by the has structural analogies with the PrP protein (Cox, study of ciliates, it does not necessarily constitute a 1994). As a working hypothesis, these results are in simple atypical curiosity about protozoa. In effect, such favour of considering URE3 or psi as submitted to the mechanisms have been invoked at the intracellular level, mechanism involved in prion diseases, although it has for bacteria (Hedges, 1985; Shapiro, 1993) as well as not yet been shown that the abnormal proteins can mammals (form of the cytoskeleton (Solomon, 1981; actually infect normal yeast cells. Theurkauf, 1994), form of the cells (Albrecht-Buehler, A very puzzling obsewation has been reported about 1977), number of microtubule organizing centres (Shay the p53 protein. This protein is a multifunctional protein et al., 1978)). Moreover, with regard to some which plays a central role in modulating gene pluricellular species which, like Stenostomwn, duplicate transcription, policing cell cycle check-points, activating by elongation followed by division, some processes of apoptosis, controlling DNA replication and repair and structural inheritance have been clearly established maintaining genomic stability (Elledge and Lee, 1995). (Frankel, 1989). Thus, a process of non-nucleic Under certain circumstances, the mutant protein can inheritance can concem not only a specific protein, but drive wild type into a mutant conformation (Milner and also the organization of complex macromolecular Medcalf, 1991). This effect is observed when p53 structures such as cilia, centrioles etc. Hence, structural mutant and wild-type are cotranslated. The inheritance at the molecular level might be the missing cotranslational effect of mutant p53 upon wild-type link for the understanding of the structural inheritance conformation was attributed to some interaction between processes featured at the ceilular level. nascent polypeptides and oligomerization of the full- Even though the hypothesis about the ability of a length proteins. Oligomers of p53 proteins can be protein to impose its own conformation to a distinct one induced to conformational change in a cooperative is not definitively demonstrated, strong arguments favor manner. The mechanism herein evoked strongly such a possibility for prion protein and other proteins as reminds one of the 'chaperone' hypothesis, previously well. Therefore, what seems possible nowadays could quoted about the prion protein (Liautard, 1991, have been realized in the past, and it seems quite 1992). By the way, we should remark that it has also conceivable that a protein could have been able to been shown that molecular cha~eronescan control mediate a process of structural inheritance at the very their own assembly (Cheng et al.', 1990; Lissin et al., beginning of the evolution of living organisms. By 1990). showing that a protein can support a process of structural Molecular clues to prion diseases

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