J. Neurochem. (2011) 119, 251–261.
Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrPSc), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrPC). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrPSc. In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.
bovine spongiform encephalopathy
protein-misfolding cyclic amplification
Human prion diseases are a complex group of fatal subacute neurodegenerative diseases. They are clinically characterized by progressive dementia associated with various neurological symptoms and, neuropathologically, by spongiform change, neuronal loss, reactive astrocytosis and microglial proliferation in the CNS. Unlike other brain proteinopathies, human prion diseases such as sporadic, infectious, and genetic forms can be transmitted experimentally to non-human primates and rodents. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form and accounts for more than 80% of all cases, with a yearly incidence of 1–1.5 cases per million. Genetic forms (also called inherited or familial prion diseases) are of autosomal dominant inheritance and genetically linked to mutations in the prion protein (PrP) coding sequence such as the E200K mutation in genetic CJD, the P102L mutation in Gerstmann–Sträussler–Scheinker syndrome, the D178N/M-129 haplotype in familial fatal insomnia (Kovacs et al. 2005). Accidental transmissions to humans are responsible for infectious forms (also termed acquired forms) such as iatrogenic and variant Creutzfeldt-Jakob diseases (iCJD and vCJD respectively) (Will et al. 1996; Brown et al. 2006). An almost constant biochemical hallmark is the accumulation, mostly in the extra-cellular space, of scrapie prion protein (PrPSc), an abnormal, partially protease-resistant isoform of the host-encoded cellular prion protein (PrPC). PrPC is a membrane glycophosphatidylinositol (GPI)-anchored glycoprotein of 253 amino acids in humans that is highly preserved among species (Weissmann 2004). Post-translational modifications of the protein, such as its N-terminal truncation, may vary between mammalian species (Laffont-Proust et al. 2005, 2006). PrPC is particularly expressed by neurons and glial cells and has two N-glycosylation sites at positions 181 and 197, which are variably occupied and produce di-, mono- and unglycosylated forms (Kretzschmar et al. 1986; Moser et al. 1995). The N-terminal moiety of PrP contains an octapeptide repeat and is not structured in aqueous solution on nuclear magnetic resonance studies (Zahn et al. 2000). The C-terminal half of the protein includes two anti-parallel β-sheet and three α helices. According to the protein only hypothesis, the mechanism for prion propagation involves the conversion of PrPC into PrPSc in a post-translational process (Prusiner 1998). Compared with PrPC, PrPSc is enriched in β-sheet content (43% vs. 3% for hamster PrPSc), shows insolubility in non-denaturing detergents and partial resistance to proteolysis. Aggregated PrPSc accumulates in the brain of affected individuals and forms the infectious prion particles.
A striking phenomenon of human prion diseases is extreme variability in their clinical presentation, neuropathological pattern, and molecular subtype. Factors that encipher such diversity are not completely understood, and include host genetics and the properties of prion strains. Indeed, despite the absence of a nucleic acid genome, various prion strains can be propagated in experimental models (Prusiner 1998). Strains isolated from animal prion disorders were originally defined by the pattern of disease they produce upon transmission to syngenic animals (Bruce et al. 1976; Morales et al. 2007). In a given host, prion strains differ primarily with regard to incubation time and the location of abnormal PrP, as well as the distribution of brain lesions. Although uncertainties remain regarding the molecular basis of strain diversity, increasing evidence indicates that strain-specific properties are encoded within structural differences in the conformation/aggregation state of the PrPSc molecules (Bessen et al. 1995; Collinge et al. 1996; Telling et al. 1996; Caughey et al. 1998; Thomzig et al. 2004; Castilla et al. 2008b; Sim and Caughey 2009; Tixador et al. 2010). In human prion disorders, distinct PrPSc types have been identified on the basis of their biochemical properties and many efforts have been made to correlate them to clinico-pathogical phenotypes. The occurrence of vCJD epidemics and the emergence of novel prion disorders in animals with zoonotic potential have prompted the prion community not only to provide a comprehensive classification of the wide phenotypic and molecular spectrum of human disorders, but also to isolate the prion strains responsible for the different subtypes. The aim of this minireview was to provide a status report on vCJD, one of the best-characterized human prion disorders, and to discuss recent results that shed light on the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.
Open questions concerning the variant Creutzfeldt-Jakob epidemics
Variant CJD was first described in 1996 in the UK (Will et al. 1996). The disease occurs in younger patients than sCJD and has demonstrated clinical and neuropathological specificities. Most of all, the presence of florid amyloid plaques that are consistently observed throughout the brain with a greater number in cerebral and cerebellar cortices, contributes to the final diagnosis. There is a body of evidence to support a causal link between vCJD and the bovine spongiform encephalopathy (BSE) agent (Collinge et al. 1996; Lasmézas et al. 1996; Bruce et al. 1997; Scott et al. 1999) with human infection probably caused by dietary exposure (Ward et al. 2006). Among non-UK countries with vCJD cases, France was the first affected and has identified the largest number of cases (25 as of 31 December 2010). According to year of onset, the number of cases reached peak incidence in 1999 in UK and 2004 in France. These data are in good agreement with the estimated dietary exposure to BSE linked to bovine exports from the UK to France. Since 1999 in the UK and 2004, in France the number of cases has regularly decreased (Brandel et al. 2009). No case has been detected since the end of 2008 in France, and only five cases of probable vCJD has been diagnosed since 2009 in the UK where the number of deaths from probable and definite vCJD has markely decreased (three in 2009 and 2010, one in 2011).
Except for one debated case reported very recently (Kaski et al. 2009; Brandel et al. 2010), the same genetic pre-disposition [methionine homozygosity (MM) at codon 129 of PRNP] has been observed in patients with probable or definite vCJD from the UK, France and all other involved countries so far. The larger part of the general population, 10% of which is valine-homozygous (VV) and 50% of which is heterozygous (MV), may be protected from the epidemics. However, concerns have been raised about the occurrence of vCJD in these genetic backgrounds. Variant CJD in MV and VV patients might have a longer incubation period that may favor secondary transmission. Indeed, vCJD MM patients show widespread involvement of the lymphoreticular system (tonsils, appendix, spleen and lymph nodes) (Hill et al. 1997b; Bruce et al. 2001) even during the pre-clinical disease stage, as the agent reaches the CNS through the abundant sympathetic innervation of gut-associated lymphoid structures (Haik et al. 2003, 2004b). The presence of circulating infectivity was confirmed by the report of three cases of vCJD linked to transmission via transfusion of non-leukocyte-reduced red cells from donors incubating the disease (Llewelyn et al. 2004).
Accidental blood contamination provided the first evidence to support the view that the genotypic barrier of transmission toward vCJD infection may not be complete. Indeed, a case of pre-clinical infection was reported in a MV patient who died of a non-neurological disease five years after receiving a blood transfusion from a vCJD donor. PrPSc with a profile on Western blot typical of vCJD was detected in the patient’s lymphoid tissues, but not in the central nervous system (Peden et al. 2004). Additional evidence was raised by a retrospective appendix and tonsil survey that aimed to estimate the prevalence of asymptomatic carriers that will eventually develop the disease in the general population; this survey identified two cases exhibiting PrP accumulation in the appendix who were identified as valine homozygous individuals (Ironside et al. 2006). This observation indicates that the vCJD agent is able to replicate in a PrP non-methionine homozygous context at least at the periphery.
Transgenic mice expressing human PrP have been considered to model the primary passage of BSE to humans. BSE did not transmit to gene targeted transgenic mice expressing a physiologic level of human PrP regardless of the genotype at codon 129 suggesting a significant species barrier that may account for the low number of human vCJD cases (Bishop et al. 2006). Transgenic mice over-expressing human PrP with a valine at codon 129 are sensitive to BSE transmission (Hill et al. 1997a), but the resulting BSE prion fails to adapt in this genetic background on second passage and does not produce PrPSc.
These results are in contrast with those obtained when BSE is transmitted to mice over-expressing PrP M129 suggesting that the presence of a valine at codon 129 strictly restricts the propagation of the BSE agent (Wadsworth et al. 2004). This hypothesis has been recently supported by results obtained using a non-cellular model of prion conversion known as protein misfolding cyclic amplification (PMCA). Using PrPSc from a brain isolate as seed, and excess PrPC molecules contained in normal tissue homogenate as substrates, PMCA is able to amplify very low quantities of PrPSc by repeating cycles of alternating steps of incubation and sonication. Together with PrPSc amplification, this method generates new infectious particles and can reproduce some prion strain behaviors such as genotypic barriers of transmission and strain mutation/adaptation (Saborio et al. 2001; Castilla et al. 2005; Saa et al. 2006; Castilla et al. 2008a; b). Using substrates prepared from normal human brain, human platelets and the transgenic mice expressing human PrP produced by Bishop et al. (2006), Jones et al. (2009) showed that PrPSc from vCJD patients was amplified efficiently in 129 MM substrate, to a lesser degree in 129 heterozygous substrate and weakly (if at all) in 129 VV substrate. Similar results were obtained when BSE PrPSc and substrates from transgenic mice were used (Jones et al. 2007, 2008, 2009). Extrapolating from these results, one would suggest that the replication of the bovine agent is variously slowed down when MV PrP is expressed, and more dramatically slowed when VV PrP is expressed.
Together, these studies are consistent with a possible replication of the bovine agent in, at least, codon 129 heterozygous individuals. In an attempt to anticipate a second wave of vCJD cases in exposed populations, two important points should be considered: the duration of the incubation period, and whether the disease will remain recognizable at the clinical and pathological levels. The Kuru epidemics taught us that the incubation period after an oral exposure in humans can exceed 50 years, and that codon 129 heterozygosity is associated with extended incubation periods (Collinge et al. 2006). In iatrogenic CJD secondary to extractive growth hormone treatment, we demonstrated that the occurence of heterozygous French cases was delayed in exposed patients. It is worth noting that in both diseases, the clinico-pathological phenotype is mostly retained in MV 129 patients (Brandel et al. 2003). From these infectious forms, one would conclude that vCJD in MV 129 patients will fully reproduce the classical vCJD phenotype. However, recent transmission data are not fully consistent with this hypothesis (see below).
Variant Creutzfeldt-Jakob disease: from biochemical simplicity to biological complexity
Variant CJD has been now observed in 12 countries with the same clinico-pathological presentation. In a recent study, we demonstrated that vCJD from France and the UK are indistinguishable with similar brain lesion profiles and a consistent pattern of PrPSc deposition (Brandel et al. 2009). Most of all, PrPSc in vCJD exhibits a peculiar profile on Western blot, with an unglycosylated band migrated at 19 kDa and a higher representation of diglycosylated PrP molecules (PrPSc 2B or 4) (Collinge et al. 1996; Parchi et al. 1997). Using antibodies specific for the PrP90–94 sequence, which is spared in type 1 but not type 2 PrPSc, a minority of type 1 with a high proportion of diglycosylated forms has been found in association with type 2B PrPSc (Yull et al. 2006). The type 2B biochemical signature is a useful hallmark of BSE agent replication in other species including non-human primates (Hill et al. 1997a; Lasmezas et al. 2005). It has been observed in all French and British patients so far (Brandel et al. 2009). In addition, in contrast with sporadic and genetic CJD (Puoti et al. 1999; Haik et al. 2004c; Head et al. 2004), the same biochemical signature has been detected in all brain areas studied (Brandel et al. 2009). This highly uniform biochemical and pathological signature of vCJD is fully consistent with the presence of a unique infectious strain.
Unexpectedly, strain-typing experiments in various transgenic models have provided divergent results. Within the context of homotypic transmission, Beringue et al. inoculated transgenic mice over-expressing human M129 PrP with vCJD isolates from France and one from the UK [World Health Organization (WHO) reference brain sample, code NHBY0/0003] (Minor et al. 2004). All French vCJD isolates propagated as variant-like CJD strains with type 2B PrPSc and abundant florid amyloid plaques (Beringue et al. 2008). However, transmission of the WHO reference sample led to the propagation of either vCJD or an sCJD-like prion. The sCJD-like strain replicated faster and produced distinct brain PrPSc and lesion patterns compared with the vCJD strain. In addition, these mice also showed early replication of the vCJD agent in their lymphoid tissue indicating that both strains were initially present in the inoculum (Beringue et al. 2008). This result was in contrast with those obtained in a transmission study of the same WHO reference vCJD isolate on transgenic mice produced by gene-targeting (Bishop et al. 2006). Therefore, PrP over-expression probably favored either the emergence of a minor component of a strain/PrP conformation mixture or the occurrence of strain mutation by interconversion into an alternative conformation during the replication process. These findings are reminiscent of those obtained in the transmission of BSE to V129 PrP over-expressing mice and in successive transmissions of vCJD in V129 and M129 transgenic hosts (Asante et al. 2002; Wadsworth et al. 2004). They have been very recently confirmed in mice over-expressing mouse/human protein chimeras (Giles et al. 2010). Transmission of five vCJD isolates from the UK National CJD Research and Surveillance Unit of Edinburgh to these highly sensitive mice resulted in two different strains, one sCJD-like and one vCJD-like, which could be variously selected by serial passages as a function of the amino-acid present at position 111. The sCJD-like strain was of the type 1 and did not transmit to mice expressing bovine PrP, in contrast with the vCJD strain (Giles et al. 2010). The occurrence at first and serial passages of mice with both PrPSc types and the observation that some mice with sCJD-like prion occasionally showed some lesions indicative of vCJD (amyloid plaques) support the idea that vCJD infectivity contains a minor component with sCJD-like behavior. It can be speculated that this minor infectious component may correspond to the type 1B PrPSc detected in all brain areas of vCJD and in BSE in cattle (Yull et al. 2006).
Finally, despite one of the most standardized clinico-pathological phenotypes among human prion diseases, which occur in a unique genetic PRNP background and consistently produce a typical PrPSc signature, vCJD transmission studies reveal the divergent potential of prions. Such divergence also raises concerns about our ability to recognize, at the pathological and biochemical levels, the diseases that may result from strain divergence of the bovine agent in infected humans. It should be emphasized that the expression of one prion strain rather than another may vary with serial passages in the same species. However, the recipients of blood transfusion from donors incubating vCJD who developed a prion disease demonstrated typical features of vCJD (Wroe et al. 2006; Head et al. 2009b).
Sporadic Creutzfeldt-Jakob disease: from biochemical complexity to biological simplicity
In contrast with vCJD, the clinico-pathological presentations of sCJD vary considerably. Primirarly based on presenting and the most prominent symptoms during diseases duration, various distinct forms were identified a long time ago, including the Heidenhain variant (the typical dementing CJD form with visual symptoms, myoclonia, and frequent epilepsia), the Brownell-Oppenheimer ataxic variant (revealed by cerebellar ataxia with late dementia), the thalamic variant, and the panencephalopathic variant. In an attempt to determine the molecular basis of such a phenotypic variability, many studies in the past 10 years have explored the correlation of sCJD phenotypes with PRNP polymorphisms and PrPSc biochemical properties. Two molecular factors have been found useful to establish an initial classification of sporadic subtypes: the methionine-valine polymorphism at codon 129 of PRNP and the migration pattern of PrPSc on one-dimensional electrophoresis (Parchi et al. 1996; Hauw et al. 2000). Following limited proteinase K (PK) digestion, PrPSc appears as three separated bands on a western blot (36, 30, and 19–21 kD which correspond respectively to the di-, mono-, and non-glycosylated PrP forms). In sCJD, two classifications have been documented. As a function of the size of the non-glycosylated band of the PK resistant C-terminal core of the protein, the classification from London identified three subtypes (1, 2, and 3) (Collinge et al. 1996; Hill et al. 2003) and the other from Cleveland identified two subtypes (1 and 2) (Parchi et al. 1996). Type 1 of the classification by Parchi et al. includes types 1 and 2 from the classification by Collinge et al. (Brandner 2011; Parchi et al. 2011). For clarity and to allow comparison between transmission studies, we will employ the classification from Cleveland, which remains the most widely used and proven robust and reliable in a recent inter-laboratory assessment study within the NeuroPrion consortium (Parchi et al. 2009a).
PrPSc type 1 has a relative molecular mass of 21 kDa with a primary cleavage by PK at residue 82, and PrPSc type 2 has a relative molecular mass of 19 kDa and a primary cleavage by PK at residue 97 (Parchi et al. 2000). PrPSc type 2A is observed in sCJD and differs from vCJD type 2B by a lower proportion of di-glycosylated forms (Head et al. 2004). By combining codon 129 genotype and PrPSc typing, Parchi et al. (1999) provided the first molecular basis for a classification of sCJD subtypes. Three major (MM1 + MV1, MV2 with amyloid plaques, VV2) and three minor molecular subtypes (MM2 cortical, MM2 thalamic, VV1) matching well with the most commonly encountered clinico-pathological phenotypes of sCJD cases were described. However, increasing evidence has now indicated that the nosology of sCJD is probably more complex. Within some molecular subtypes, additional pathological variants have been reported such as MM, VV and MV1 patients with amyloid plaques (Hauw et al. 2000; Kawauchi et al. 2006; Lo et al. 2006; Kobayashi et al. 2008) and there are important phenotypic variations among patients of the same molecular subtype at the clinical and pathological levels, including the level of neuronal loss and the aspect of PrP deposits (Faucheux et al. 2009, 2011). At the biochemical level, several reports have documented a higher level of complexity for molecular typing results in sCJD. Indeed, Notari et al. (2004) showed that migration properties of PrPSc 1 or 2 differ slightly as a function of codon 129 genotype and experimental conditions for PK digestion. Beyond the type 1/type 2 dichotomy, additional biochemical properties of PrPSc have been recently reported, including: occurrence of N- and C-terminally cleaved PrPSc fragments, possibly reflecting the presence of multiple PrPSc conformers with distinct PK resistance properties (Zanusso et al. 2004; Notari et al. 2008); and variation in the PK resistance of PrPSc as a function of genotypic subgroups, which correlated with some clinico-pathological phenotypes (such MM1, which was found to differ from VV1) (Uro-Coste et al. 2008). Moreover, a novel glycotype of PrPSc has been identified that lacks diglycosylated PrP and is associated with an original clinico-pathological presentation (Zanusso et al. 2007). The Cleveland group reported a novel form of sporadic prion diseases, with normal PrP coding sequence, initially named protease-sensitive prionopathy and now known as variably protease-sensitive prionopathy, which affects all three genotypes at codon 129 and has also been identified in the UK, the Netherlands and Italy (Head et al. 2009a ; Jansen et al. 2010; Zou et al. 2010). This form is characterized, compared with classical sCJD, by a higher sensitivity of PrPSc to protease, the presence of additional cleaved PrPSc fragments (18, 12–13, and 8 kDa) resembling those observed in GSS patients with the A117V mutation, and a particular immunostaining pattern with granular PrP-positive deposits such as dot-like or plaque-like cerebellar formations (Gambetti et al. 2008; Zou et al. 2010).
Molecular classifications of human prion diseases that extensively used PrPSc typing were based on the postulate that the same abnormal PrP is present throughout the CNS; however, increasing evidence indicates that this is not the case. First, Puoti et al. (1999) documented that the co-occurrence of various PrPSc types can be observed in the brain of one individual with sCJD, together in the same brain region and/or separately in different areas and that this phenomenon significantly affects PrP tissue deposits and vacuolization. We obtained similar findings in a larger series of French sCJD and observed that, in these sCJD patients with both PrPSc types, the codon 129 genotype was a prominent factor that significantly affected the PrPSc type distribution in the CNS. Following series has further documented this phenomenon that shows a frequency higher than 40% in MM patients (Head et al. 2004; Schoch et al. 2006; Uro-Coste et al. 2008; Cali et al. 2009; Parchi et al. 2009b). The presence of both PrPSc types in one individual seems to influence the clinico-pathological phenotype with intermediate features between CJD type 1 and CJD type 2 of the same genotype (Cali et al. 2009). It should be emphazised that such a co-occurrence of PrPSc types has also been documented in genetic and iatrogenic prion diseases (Haik et al. 2004c). The significance of coexisting PrPSc types is still unclear, and it may correspond to co-occuring prion strains in an individual. In addition to this heterogeneity of PrPSc N-terminal cleavage, we recently demonstrated that PrPSc glycosylation varies between brain structures in a highly significant manner (Levavasseur et al. 2008). The diversity of PrPSc glycoform ratio is controlled by codon 129 genotype and PrPSc type in a brain region-specific manner. For instance, in the thalamus of VV2 patients, we observed a glycoprofile indistinguishable from PrPSc 2B, which characterized vCJD patients. This result led us to perform a retrospective analysis of sCJD cases that occurred before significant BSE exposure to eliminate the hypothesis of vCJD occurrence in a VV genetic background with atypical presentation. We finally concluded that these VV2 patients were genuine sCJD cases (Levavasseur et al. 2008).
Given both the striking phenotypical heterogeneity of sCJD and the increasing molecular diversity between and within molecular subtypes with strong variations among brain regions in the same individual, transmission studies could be expected to lead to the identification of a large panel of prions and frequent strain divergence, as repeatedly observed in transmission studies of the monomorphous vCJD. On the contrary, recent investigations of sCJD strain properties in various species have provided convergent results supporting a very limited number of sCJD strains (Fig. 1).
PrP expression is required for prion propagation and neurotoxicity, and the cross-species barrier of transmission is determined (at least in part) by the PrP sequence homology between the infectious prion and the host (Büeler et al. 1993; Brandner et al. 1996; Prusiner 1998; Mallucci et al. 2003). Transmission of sCJD to wild-type mice has proven to be poorly efficient. The use of various lines of transgenic mice expressing full-length or chimeric human and mouse PrP have provided evidence that the identity of codon 129 genotype between host and agent does not systematically control the efficiency of transmission and that some sCJD isolates behave differently from each other (Scott et al. 2000; Asante et al. 2002; Korth et al. 2003; Taguchi et al. 2003; Kobayashi et al. 2007, 2010). A significant advance has been made recently by researchers from Edinburgh, who systematically studies the strain properties of the six molecular sCJD subtypes by inoculating a panel of mice produced by gene targeting and expressing different forms of PRNP (MM, MV, VV) (Bishop et al. 2010). In these mice, only four distinct strains could be identified after two passages. MM1 and MV1 isolates demonstrated similar biological properties after transmission in the three genetic backgrounds (strain M1). The strain associated with an MV2 patient could not be distinguished from the VV2 strain (strain V2). VV1 behaved as a distinct agent (strain V1). The MM2 isolate transmitted poorly to the three mouse lines, including MM mice (strain M2) (Bishop et al. 2010). These results confirm and extend previous results obtained in two distinct species. First, despite a low PrP sequence homology between man and vole, the bank vole (Clethrionomys glareolus) has been found to be susceptible to primary transmission of prions from CJD patients with incubation periods comparable with those of transgenic mice expressing human PrP (Nonno et al. 2006). In this model, MM1 and MV1 CJD also behaved as similar agents, and differed from MM2 CJD. MV2 and VV2 did not transmit. Second, a recent transmission study in primates provides some evidence, notably from squirrel monkeys, supporting the view that MM1 and MV1 isolates on the one hand, and VV 2 and MV2 isolates on the other hand, have similar transmission properties (Parchi et al. 2010). In addition, in squirrel, MM1+2 and MM sCJD cases with PrPSc type 1 induced the same lesion patterns. Altogether, these transmission studies provide convergent results supporting the occurrence of sCJD in two strains (M1 and V2), as well as two additional potential strains (M2 and V1) that remain to be confirmed (Fig. 1). It is worth noting that these results, obtained in vivo, are consistent with sCJD PMCA results (Jones et al. 2008) and partly match those of a study that focused on the PK resistance profiles of PrPSc and identified two main different biochemical signatures in sCJD patients (PrPSc MM1/MV1, PrPSc MV2/VV2), whereas VV1 and MM2 PrPSc showed distinct biochemical properties (Uro-Coste et al. 2008). In contrast with in vivo results, PrPSc from MV1+2 and VV1+2 demonstrated similar biochemical properties resembling those of MV2 and VV2, respectively. In addition, employing a conformational stability immunoassay that used chaotropic salts to probe the PrPSc conformation by determining its resistance to denaturation, Cali et al. also observed in MM patients that when PrPSc type 1 and type 2 are present together in the same anatomical region or in an in vitro mixture, PrPSc type 1 behaves as PrPSc type 2 (Cali et al. 2009; Gambetti et al. 2011).
Theorical considerations and concluding remarks
One model of prion strains known as conformational selection proposes that strains are rarely clonal and are composed of a PrPSc mixture (Collinge 1999, 2010; Collinge and Clarke 2007). Among a relatively high number of possible different PrPSc types/conformations in mammals, only a subset can replicate in a given species as a function of PrP sequence, natural clearance capacity, and the action of unknown PrP conversion co-factors. In this model, a prion strain may operate as a quasi-species and represent an ensemble of molecular species maintained under host selection. A strain in a given host would have a major molecular component identified by its biochemical properties (and recognized using western blot typing after protease digestion) but would also include minor molecular sub-species. Transmission from one species to another would relate to substantial overlap of permissible PrPSc conformations between the PrP structures from the infectious source to the new host. Interspecies transmission may favor the replication of a minor component selected by the new host (apparent mutation) or the occurrence of a distinct PrPSc (direct mutation) notably if the host PrPC is not compatible with all PrPSc species from the source (Collinge and Clarke 2007; Collinge 2010). Such mechanisms might have occurred during transmission of the bovine agent to humans (see Fig. 2), resulting in the occurrence of additional minor PrPSc sub-species. Because at this time this transmission has only occurred in methionine homozygous genotype, which is known to favor the formation of type 1 PrPSc in sporadic disease, a type 1 PrPSc sub-species would have been generated in vCJD brains. In experimental transmission, the use of transgenic mice over-expressing M129 PrP with a reduced incubation period when challenged with sCJD may have contributed to the revelation of this sub-strain during serial passages of vCJD isolates. That such a CJD-like sub-strain emergence has only been observed in some vCJD isolates illustrates that strain mutation is an occasional phenomenon that can be strikingly influenced by as small a difference as a single conservative substitution of host PrP, as demonstrated by studies using Tg1014 chimeric mice (Giles et al. 2010).
Recent compelling data from PrPSc studies in sCJD indicate not only that human strains may be regarded as a mixture of PrP species, but also that this mixture may vary among brain regions as a function of PrP genotype. It remains uncertain why CJD subtypes converge into a limited number of strains despite the increasing diversity of sCJD subtypes and evidence of PrPSc heterogeneity within individual patients. Methodological limits certainly play a role. Indeed, one of the two studies that systematically address the relationship between molecular subtypes and strain is based on the inoculation of engineered mice with only one isolate (presumably typical of each subtype), and cannot account for the diversity within each subtype. Another limit of the study is provided by the fact that these mice appear to be insensitive to some sCJD strains, regardless to the genotype at codon 129. This is also the case in the bank vole model, in which transmission of MV2 and VV2 is inefficient. Concerning studies in non-human primates, high numbers of each isolate were used but in a very limited number of monkeys (1 or, rarely, 2 per isolate) making it difficult to observe inter-individual variations or strain mutation/divergence, which have been proven to occur randomly. It should be emphasized that, in all experimental models including transgenic mice expressing human or chimeric PrP, homospecific cofactors that may contribute to the emergence/propagation of sCJD in humans are potentially missing (Telling et al. 1995; Kaneko et al. 1997; Fasano et al. 2006).
Unlike vCJD, which is clearly caused by exogenous prion infection with a cross-species transmission that may have promoted strain mutation, the origin of sCJD is still being discussed. Various hypotheses have been debated, including the spontaneous conversion of PrP as a rare stochastic event, somatic mutation and (for some rare molecular subtypes) an infectious origin through, for instance, infection by agents responsible for atypical BSE (Prusiner 1998; Casalone et al. 2004; Comoy et al. 2008; Kong et al. 2008). According to the quasi-species model, the limited number of sCJD strains that has already been identified may suggest that among the range of possible PrPSc conformations in the human brain, only a small subset are able to replicate. Limits may rely on genetic factors such as the codon 129 genotype, which has no measurable effect on the folding or dynamics of wild type PrPC and probably impacts PrPSc formation (Hosszu et al. 2004; Lee et al. 2010); other potential candidates; and cell- or region-specific cofactors, such as those that regulate PrP glycosylation and the expression of cellular chaperones. Identifying these factors may help us to better understand how sporadic strains are generated and provide promising tools for researchers to limit prion replication when sCJD PrPSc formation has already occurred. However, in addition to the technical limitations mentioned above, one fundamental issue is whether we can reliably study a sporadic disease using infectious models of transmission. In other words, it cannot be excluded that the entire transmission process (including inoculum preparation, the use of high doses and of non-natural routes, and host response) may contribute to the selection of very few conformational sub-species among sCJD PrP conformers. Such consideration reinforces the need of additional experimental tools for human strain typing based on molecular or cellular models such as refined PMCA systems or cell cultures.
The increasing molecular diversity of PrPSc in the brain of sCJD patients suggests that conformational selection applies differently among brain regions and that the formation/degradation equilibrium of PrPSc species has not reached a steady state in all cases. Therefore, it would come as no surprise that inoculating distinct brain regions of one individual with sCJD/sCJD1+2 in transgenic mice over-expressing PrP will reveal the presence of different prion strains (see Fig. 2). In addition, the transmission studies of additional isolates from each classical PrPSc types and of atypical PrPSc species will also probably increase the complexity of the spectrum of sCJD strains.
Such comprehensive recognition of sCJD strains is important for therapeutic research and public health because the efficacy of anti-prion molecules and inactivation procedure may vary among prion strains (Haik et al. 2004a; Cronier et al. 2007; Giles et al. 2008). It will pave the route to a better understanding of how protein misfolding occurs as a sporadic event and propagates through the CNS as different strains, a matter of interest to those beyond the prion field who study other brain proteinopathies such as Alzheimer’s disease, other tauopathies and Parkinson disease, which also occur primarily as sporadic diseases and in which prion- or prion strain-like phenomena have been very recently reported (Meyer-Luehmann et al. 2006; Li et al. 2008; Aguzzi 2009; Clavaguera et al. 2009; Frost et al. 2009; Olanow and Prusiner 2009).
This research was supported by INSERM, CNRS, the European Commission FP6 programme, the Institut National de Veille Sanitaire (InVS), the Alliance-Biosecure Foundation, and ANR (ANR Blanc).