Biochemical Properties of Highly Neuroinvasive Prion Strains

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrP^Sc. Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrP^Sc over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrP^Sc over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS.     

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89–143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89–143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102–109 and 117–136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.     

Infectious prions and proteinopathies

Transmissible spongiform encephalopathies (TSEs) are caused by an infectious agent that is thought to consist of only misfolded and aggregated prion protein (PrP). Unlike conventional micro-organisms, the agent spreads and propagates by binding to and converting normal host PrP into the abnormal conformer, increasing the infectious titre. Synthetic prions, composed of refolded fibrillar forms of recombinant PrP (rec-PrP) have been generated to address whether PrP aggregates alone are indeed infectious prions. In several reports, the development of TSE disease has been described following inoculation and passage of rec-PrP fibrils in transgenic mice and hamsters. However in studies described here we show that inoculation of rec-PrP fibrils does not always cause clinical TSE disease or increased infectious titre, but can seed the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). These data are reminiscent of the “prion-like” spread of misfolded protein in other models of neurodegenerative disease following inoculation of transgenic mice with pre-formed amyloid seeds. Protein misfolding, even when the protein is PrP, does not inevitably lead to the development of an infectious TSE disease. It is possible that most in vivo and in vitro produced misfolded PrP is not infectious and that only a specific subpopulation is associated with infectivity and neurotoxicity.     

Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Prion-like propagation of cytosolic protein aggregates

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.     

Life cycle of cytosolic prions

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.     

Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.     

Prion-like propagation of cytosolic protein aggregates: Insights from cell culture models

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.Key words: prion, Sup35, huntingtin, polyglutamine, Tau, co-aggregation, amyloid, α-synuclein     

Epigenetic Dominance of Prion Conformers

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer''s and Parkinson''s diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.     

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.     

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP^C) into an infectious conformation (PrP^Sc). PrP^C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP^Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.     

In vitro analysis of SpUre2p, a prion-related protein, exemplifies the relationship between amyloid and prion

The yeast Saccharomyces cerevisiae contains in its proteome at least three prion proteins. These proteins (Ure2p, Sup35p, and Rnq1p) share a set of remarkable properties. In vivo, they form aggregates that self-perpetuate their aggregation. This aggregation is controlled by Hsp104, which plays a major role in the growth and severing of these prions. In vitro, these prion proteins form amyloid fibrils spontaneously. The introduction of such fibrils made from Ure2p or Sup35p into yeast cells leads to the prion phenotypes [URE3] and [PSI], respectively. Previous studies on evolutionary biology of yeast prions have clearly established that [URE3] is not well conserved in the hemiascomycetous yeasts and particularly in S. paradoxus. Here we demonstrated that the S. paradoxus Ure2p is able to form infectious amyloid. These fibrils are more resistant than S. cerevisiae Ure2p fibrils to shear force. The observation, in vivo, of a distinct aggregation pattern for GFP fusions confirms the higher propensity of SpUre2p to form fibrillar structures. Our in vitro and in vivo analysis of aggregation propensity of the S. paradoxus Ure2p provides an explanation for its loss of infective properties and suggests that this protein belongs to the non-prion amyloid world.     

Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer''s disease.     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP(Sc) produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.Key words: prion, Sup35p, Ure2p, Rnq1p, [PSI⁺], [URE3], [PIN⁺], amyloid fibrils     

Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS

Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Sträussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease.     

Prion disease: exponential growth requires membrane binding

A hallmark feature of prions, whether in mammals or yeast and fungi, is exponential growth associated with fission or autocatalysis of protein aggregates. We have employed a rigorous kinetic analysis to recent data from transgenic mice lacking a glycosylphosphatidylinositol membrane anchor to the normal cellular PrP(C) protein, which show that toxicity requires the membrane binding. We find as well that the membrane is necessary for exponential growth of prion aggregates; without it, the kinetics is simply the quadratic-in-time growth characteristic of linear elongation as observed frequently in in vitro amyloid growth experiments with other proteins. This requires both: i), a substantial intercellular concentration of anchorless PrP(C), and ii), a concentration of small scrapies seeding aggregates from the inoculum, which remains relatively constant with time and exceeds the concentration of large polymeric aggregates. We also can explain via this analysis why mice heterozygous for the anchor-full/anchor-free PrP(C) proteins have more rapid incubation than mice heterozygous for anchor-full/null PrP(C), and contrast the mammalian membrane associated fission or autocatalysis with the membrane free fission of yeast and fungal prions.