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Title: Propagation of RML Prions in Mice Expressing PrP Devoid of GPI Anchor Leads to Formation of a Novel, Stable Prion Strain}
Article:
Abstract:
PrPC, a host protein which in prion-infected animals is converted to PrPSc, is linked to the cell membrane by a GPI anchor. Mice expressing PrPC without GPI anchor( tgGPI- mice), are susceptible to prion infection but accumulate anchorless PrPSc extra-, rather than intracellularly. We investigated whether tgGPI− mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrPSc. We found that RML and ME7, but not 22L prions propagated in tgGPI− brain developed novel cell tropisms, as determined by the Cell Panel Assay( CPA). Surprisingly, the levels of proteinase K-resistant PrPSc( PrPres) in RML- or ME7-infected tgGPI− brain were 25–50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrPSc and SFL PrPSc are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice.
Introduction:
Prions, the causative agents of transmissible spongiform encephalopathies, contain as their main component PrPSc, a multimeric conformer of the ubiquitous host protein PrPC. PrPC can carry one or two N-linked glycans, whose structure is highly variable[1]–[3], or remain unglycosylated; moreover the glycosylation state varies in different tissues and cell lines, and even in different brain regions[4]–[6]. Most PrPC is attached to the outer surface of the plasma membrane by a glycosylphosphatidylinositol( GPI) anchor[7], but transmembrane forms have been identified[8],[9]. In prion-infected cells PrPC is converted to PrPSc at the membrane[10] and within the endosomal-lysosomal compartment[11], and accumulates mainly in intracellular compartments[12],[13]. While PrPC is readily cleaved off the cell surface by PIPLC[7], PrPres is not, even though it retains its GPI anchor[14]. PrPSc presents as a partially proteinase K( PK)-resistant form, designated rPrPSc or PrPres, or as a PK-sensitive entity, sPrPSc or PrPsen[15]–[17]. Mouse prions occur in the form of a wide variety of strains, which have the same PrP sequence but differ in their incubation time in various mouse lines and in the pattern of lesions they cause in brain[18]. It is believed that strain-ness is encoded by the conformation of PrPSc and in many instances there are differences in the physicochemical properties of the cognate PrPres[19]–[24]. Interestingly, different prion strains populate distinct regions of the brain[25]–[27] and are selective in regard to the cultured cell lines they can chronically infect[28]–[33]. This raises the unanswered question as to which cellular property underlies prion strain tropism. The seeding or nucleation model for prion propagation[34]–[36] predicates that PrPC monomers add to PrPSc and in doing so assume the conformation of its subunits, thus allowing faithful propagation of strains. A prerequisite for chronic infection is that PrPC monomers with a conformation allowing efficient accretion be present at a concentration sufficient to allow synthesis of PrPSc at a rate higher than that of its depletion[37]–[39]. PrPC is conformationally very flexible[40]–[42] but certain conformations may be more frequent in some cell types but less so or absent in others, which could account for the tropism of prion strains. This proposal begs the question as to why certain conformations would be favored in some cells but not in others. One possibility is that PrPC conformation is modulated by the association of PrPC with a cell-specific component, RNA and phospholipids being two possibilities among several[43],[44]. PrP has a strong binding affinity for polyanions, in particular RNA and homoribopolynucleotides[45],[46]. Binding of RNA causes conformational changes of PrP[47],[48] and in vitro conversion of purified PrPC or recombinant PrP to infectious PrPSc by Protein Misfolding Cyclic Amplification( PMCA) requires the presence of a polyanion and/or phospholipid[44],[49]–[51]. Another possibility is that the highly variable glycosylation of PrPC observed in different cells and tissues[52] might affect its susceptibility to conversion to PrPSc, because of an effect on its conformation[53] or its interactions with putative auxiliary host proteins such as the conjectured protein X[54]. Distinct PrPC glycoforms promote PMCA-mediated prion formation in a species-specific manner[55]. There have been many attempts to explore the role of glycans in the fidelity of strain propagation, in particular by generating amino acid replacements at the glycosylation sites[53],[56]–[58]. However, in these experiments failure to propagate prions or to propagate them faithfully could be due to sequence changes, which are known to affect efficiency of propagation and cause strain shifts[59], rather than the lack of glycans. We were therefore interested in investigating the effect of post-translational modifications of PrPC on the fidelity of strain propagation in the absence of an amino acid sequence change. Some years ago Chesebro et al. generated transgenic mice lacking the PrP GPI signal sequence on a PrP null background( tgGPI− mice)[60]. In mice hemizygous for the transgenes, PrP devoid of the GPI anchor( GPI−-PrP) was expressed at about one quarter the level of PrP in wild-type controls, was largely unglycosylated and absent from the plasma membrane. Nonetheless, these mice, inoculated with RML or 22L prions accumulated high levels of infectivity and PK-resistant PrP in the form of extracellular amyloid plaques in their brains, but exhibited no striking clinical signs up to 600 and 400 days post infection( dpi), respectively. GPI−-PrPC to GPI−-PrPSc conversion is believed to occur extracellularly[11],[60]–[62]. Mice homozygous for the transgenes expressing GPI−-PrP at a twofold higher level than their hemizygous counterparts developed neurological signs from 300 to 480 dpi[63]. Cultured cells expressing GPI−-PrP secreted the bulk of the anchorless PrP into the medium, none was found at the cell surface but some was associated with membrane fractions[64]. Cells expressing epitope-tagged GPI−-PrP exposed to 22L prions did not become chronically infected, however during the acute infection phase, tagged PrPres was transiently formed[61],[65]. To determine whether prions formed from PrPC that had the same sequence but different post-translational modifications would retain their strain characteristics, we inoculated tgGPI− mice with various prion strains or isolates and determined the cell tropism of the resulting isolates by the Cell Panel Assay( CPA)[33]. We found that the CPA properties of 22L prions remained unchanged, whereas those of RML, 139A, 79A and ME7 prions became distinctly different when propagated in tgGPI− mice. RML, 139A and 79A prions are derived from drowsy goat scrapie and are related[66],[67] while ME7 and 22L are distinct and unrelated strains derived from sheep scrapie[68]. Because the changes in properties could have been due to the absence of the GPI anchor and/or to diminished glycosylation, RML and ME7 prions propagated in GPI− mice were transferred to wild-type mice and their properties were again assessed. As judged by the CPA, ME7 prions recovered their original properties after one passage, showing that their adaptation to the GPI− environment was readily reversible. RML-derived prions, however, did not recover their original cell tropism, even after three cycles of propagation in wild-type mice, and therefore constituted a novel, stable strain, which we designated “SFL”( “Scripps Florida Laboratory”). Because both RML and SFL prions were stably propagated in wild-type mice, we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPI− mice, either because of the different structure or reduced expression level of the hypoglycosylated GPI-less PrPC, or because of a different replication mode in the extracellular space.
Discussion:
Prion populations are considered to be “quasi-species”, i. e. to consist of a major component and a multiplicity of variants present at low levels, of which one may be selected as the major component if the population is exposed to a different environment[39],[74],[75]. We have reported previously that when 22L prions were transferred from brain to PK1 or R33 cells, their properties, as measured by the CPA, changed gradually in the course of many doublings, suggesting that a 22L variant present at low levels in the brain-derived population was being selected in the cellular environment. Conversely, when the cell-adapted 22L variants were again propagated in brain, the resulting population gradually re-acquired the properties of the original brain-derived 22L. When 22L prions were cloned by endpoint dilution into PK1 cells they were initially swa sensitive and incapable of developing swa resistance( “swa incompetent”), but as the populations were further propagated they became swa competent while remaining swa sensitive, suggesting that during propagation swa-resistant prions arose at a low level by “mutation” and could be selected when the population was challenged with swa[74]–[76]. In all these cases the changes were reversed when the prions were propagated in the original environment; assuming that properties of prions are encoded by the precise conformation of the cognate PrPSc, we concluded that the conformational states underlying adaptation to the cellular environment are separated by low activation energy barriers which allowed reversible conformational switches leading to “strain variants” or “sub-strains”[75]. In contrast to these earlier results with 22L prions, we now report that when RML prions from wild-type brain were propagated in brain producing anchorless PrP, a GPI−-PrPSc conformer adapted to that environment evolved, and when this was transferred to wild-type mice, a novel conformation of wild-type PrPSc, which we designated SFL, distinct from that of the original RML, was stably maintained. Because SFL does not revert to RML, it must be either better adapted to propagation in wild-type brain, prevented from reverting by a high activation energy barrier or both( Figure 5). Of note, methods commonly used to distinguish strains, such as incubation period, western blotting or conformational stability assays could not differentiate between RML and SFL, whereas this was readily achieved with the Extended Cell Panel Assay( ECPA)[77]. ME7 also acquired distinct properties when propagated in tgGPI− brain, however it reverted to what appears to be its original form when passaged through wild-type brain. Strain switching has been observed previously in transfer between animal species, when 139A prions were transferred from mouse to hamster and back to mouse[78]; in that case, a different amino acid sequence in the intermediate host may have led to the adoption of a more favorable conformation, which was preserved when the prions were returned to the original host. Mutated PrPSc could be formed if accretion of GPI−-PrPC to wild-type seed entailed adoption of a conformation slightly different from that of the seed, or if wild-type PrPSc contained a variety of mutant seeds –possibly at a low level– to one or some of which the resident GPI−PrPC preferentially accreted, adopting its conformation[76]. Considering the large variety of “classical” murine prion strains[79], the plethora of strains generated by Prusiner and his colleagues[80],[81] and the many variants we have observed, all encoded by a single murine PrP sequence, the number of stable conformational states must be vast. The structural elucidation of PrPSc and its variants continues to be the Holy Grail of the prion field.
The agent causing transmissible spongiform encephalopathies , the prion , consists mainly if not entirely of PrPSc , an aggregated conformational variant of PrPC . PrPC is a host-specified glycoprotein which is attached to the outer surface of the plasma membrane by a glycosylphosphatidylinositol ( GPI ) anchor . PrPSc is thought to propagate by seeded conversion , in which PrPC accretes to an aggregate of PrPSc and thereby assumes its conformation . Prions occur as distinct strains , which have the same PrP sequence but different PrPSc conformations . Many strains are very stable but some can “mutate” , especially after transient propagation in another animal species . Here “mutation” is attributed to a conformational change of the underlying PrPSc protein rather than a change in the nucleotide sequence of a gene . In transgenic mice that express anchorless PrP , conversion to PrPSc occurs extracellularly rather than in a cell-associated compartment . Surprisingly , RML , a very stable prion strain , when propagated in tgGPI− mice and then returned to wild-type mice , was recovered as a novel strain , designated SFL , which was stable over many transmissions . Thus , faithful strain propagation was abrogated either as a consequence of the modified PrP or because of a different replication mode in the extracellular compartment of tgGPI− brain .