Size distribution dependence of prion aggregates infectivity

We consider a model for the polymerization (fragmentation) process involved in infectious prion self-replication and study both its dynamics and non-zero steady state. We address several issues. Firstly, we extend a previous study of the nucleated polymerization model [M.L. Greer, L. Pujo-Menjouet, G.F. Webb, A mathematical analysis of the dynamics of prion proliferation, J. Theoret. Biol. 242 (2006) 598; H. Engler, J. Pruss, G.F. Webb, Analysis of a model for the dynamics of prions II, J. Math. Anal. Appl. 324 (2006) 98] to take into account size dependent replicative properties of prion aggregates. This is achieved by a choice of coefficients in the model that are not constant. Secondly, we show stability results for this steady state for general coefficients where reduction to a system of differential equations is not possible. We use a duality method based on recent ideas developed for population models. These results confirm the potential influence of the amyloid precursor production rate in promoting amyloidogenic diseases. Finally, we investigate how the converting factor may depend upon the aggregate size. Besides the confirmation that size-independent parameters are unlikely to occur, the present study suggests that the PrPsc aggregate size repartition is amongst the most relevant experimental data in order to investigate this dependence. In terms of prion strain, our results indicate that the PrPsc aggregate repartition could be a constraint during the adaptation mechanism of the species barrier overcoming, that opens experimental perspectives for prion amyloid polymerization and prion strain investigation.     

Inactivation of prions by acidic sodium dodecyl sulfate

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.     

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.     

Modulation of prion-dependent polyglutamine aggregation and toxicity by chaperone proteins in the yeast model

In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.     

Molecular dynamics simulations of early steps in RNA‐mediated conversion of prions

The rate‐limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis‐folded state in which the protein not only forms cell‐toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA‐mediated mis‐folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.     

The aminoglycoside G418 hinders de novo prion infection in cultured cells

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.     

Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI ⁺] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI ⁺] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ ⁺] prion propagation. In contrast, weak [PSI ⁺] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ ⁺] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI ⁺]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI ⁺]/[RNQ ⁺] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI ⁺] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.     

Influence of water, fat, and glycerol on the mechanism of thermal prion inactivation

Extending the recent analysis of the safety of industrial bovine fat-derived products for human consumption (Müller, H., Stitz, L., and Riesner, D. (2006) Eur. J. Lip. Sci. Technol. 108, 812-826), we investigated systematically the effects of fat, fatty acids, and glycerol on the heat destruction of prions. Prion destruction was qualitatively and quantitatively evaluated in PrP 27-30, or prion rods, by the inactivation of infectivity as well as by the degradation of the polypeptide backbone. Under all conditions analyzed, inactivation of prion infectivity was achieved more efficiently than backbone degradation by several orders of magnitude. The presence of fat enhanced prion inactivation and offers a mild treatment for prion decontamination. In contrast, the presence of fat, fatty acids, and especially glycerol protected the PrP 27-30 backbone against heat-induced degradation. Glycerol also protected against heat-induced inactivation of prion infectivity. A phase distribution analysis demonstrated that prions migrated to the interphase of a fat/water mixture at room temperature and accumulated in the water phase at higher temperatures. In a systematic study of the mechanism of prion destruction, we found an intermediate structure of PrP that has fewer fibrils in beta-sheet formation, lower resistance to protease digestion, greater aggregation, and reduced solubility compared with PrP 27-30 but retains residual infectivity. These findings suggest that prion infectivity depends on beta-sheet-rich fibrillar structure and that inactivation proceeds in a stepwise manner, which explains the tailing effect frequently observed during inactivation.     

Biological Roles of Prion Domains

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.     

Striatal pathology underlies prion infection-mediated hyperactivity in mice

Although prion diseases are most commonly modeled using the laboratory mouse, the diversity of prion strains, behavioral testing and neuropathological assessments hamper our collective understanding of mouse models of prion disease. Here we compared several commonly used murine strains of prions in C57BL/6J female mice in a detailed home cage behavior detection system and a systematic study of pathological markers and neurotransmitter systems. We observed that mice inoculated with RML or 139A prions develop a severe hyperactivity phenotype in the home cage. A detailed assessment of pathology markers, such as microglial marker IBA1, astroglial marker GFAP and degeneration staining indicate early striatal pathology in mice inoculated with RML or 139A but not in those inoculated with 22L prions. An assessment of neuromodulatory systems including serotonin, dopamine, noradrenalin and acetylcholine showed surprisingly little decline in neuronal cell bodies or their innervations of regions controlling locomotor behavior, except for a small decrease in dopaminergic innervations of the dorsal striatum. These results implicate the dorsal striatum in mediating the major behavioral phenotype of 139A and RML prions. Further, they suggest that measurements of activity may be a sensitive manner in which to diagnose murine prion disease. With respect to neuropathology, our results indicate that pathological stains as opposed to neurotransmitter markers are much more informative and sensitive as markers of prion disease in mouse models.Key words: PrP, neurodegeneration, protein misfolding, home-cage, transmissible spongiform encephalopathy     

Prions affect the appearance of other prions: the story of [PIN(+)

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.     

Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion "strains," [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.     

Synthesis of High Titer Infectious Prions with Cofactor Molecules

Recently, synthetic prions with a high level of specific infectivity have been produced from chemically defined components in vitro. A major insight arising from these studies is that various classes of host-encoded cofactor molecules such as phosphatidylethanolamine and RNA molecules are required to form and maintain the specific conformation of infectious prions. Synthetic mouse prions formed with phosphatidylethanolamine exhibit levels of specific infectivity ∼1 million-fold greater than “protein-only” prions (Deleault, N. R., Walsh, D. J., Piro, J. R., Wang, F., Wang, X., Ma, J., Rees, J. R., and Supattapone, S. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E1938–E1946). Moreover, cofactor molecules also appear to regulate prion strain properties by limiting the potential conformations of the prion protein (see Deleault et al. above). The production of fully infectious synthetic prions provides new opportunities to study the mechanism of prion infectivity directly by structural and biochemical methods.     

Prions in yeast

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the "protein only" model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions.     

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.     

Insoluble aggregates and protease-resistant conformers of prion protein in uninfected human brains

Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.     

Biological Roles of Prion Domains

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.Key Words: amyloid, amyloidosis, epigenetic, evolution, inheritance, mammals, misfolding, protein, stress, yeast     

Defining the Conformational Features of Anchorless,Poorly Neuroinvasive Prions

Infectious prions cause diverse clinical signs and form an extraordinary range of structures, from amorphous aggregates to fibrils. How the conformation of a prion dictates the disease phenotype remains unclear. Mice expressing GPI-anchorless or GPI-anchored prion protein exposed to the same infectious prion develop fibrillar or nonfibrillar aggregates, respectively, and show a striking divergence in the disease pathogenesis. To better understand how a prion''s physical properties govern the pathogenesis, infectious anchorless prions were passaged in mice expressing anchorless prion protein and the resulting prions were biochemically characterized. Serial passage of anchorless prions led to a significant decrease in the incubation period to terminal disease and altered the biochemical properties, consistent with a transmission barrier effect. After an intraperitoneal exposure, anchorless prions were only weakly neuroinvasive, as prion plaques rarely occurred in the brain yet were abundant in extracerebral sites such as heart and adipose tissue. Anchorless prions consistently showed very high stability in chaotropes or when heated in SDS, and were highly resistant to enzyme digestion. Consistent with the results in mice, anchorless prions from a human patient were also highly stable in chaotropes. These findings reveal that anchorless prions consist of fibrillar and highly stable conformers. The additional finding from our group and others that both anchorless and anchored prion fibrils are poorly neuroinvasive strengthens the hypothesis that a fibrillar prion structure impedes efficient CNS invasion.