Relationship between prion propensity and the rates of individual molecular steps of fibril assembly.

Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be experimentally highly tractable; but quantitative understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with solution and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally determined by the nature of the soluble precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quantitative understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biological pathways.

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

The indel polymorphisms of the prion protein gene (PRNP ) in Chinese yak

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Prion protein gene (PRNP) polymorphisms in native Chinese cattle

Le polymorphisme au sein de quatre regions du gene codant pour la proteine prion bovine (PRNP) confere la susceptibilite a l'encephalopathie bovine spongiforme (BSE). Ceux-ci comprennent un polymorphisme d'insertion/deletion (indel) de 23 pb dans le promoteur, un indel de 12 pb dans l'intron 1, un octapeptide repete ou un indel de 24 pb au sein du cadre de lecture, et un polymorphisme mononucleotidique (SNP) dans la region codante. Dans ce travail, les auteurs ont examine la frequence des genotypes, des alleles et des haplotypes pour ces indel au sein de 349 bovins d'origine chinoise, de meme que la sequence nucleotidique de ce gene chez 50 de ces animaux. Leurs resultats montrent que l'allele ayant la deletion de 12 pb et l'haplotype combinant la deletion de 23 pb et la deletion de 12 pb, lesquels ont ete suggeres comme etant importants pour la susceptibilite a la BSE, sont rares au sein des bovins du sud de la Chine. Une difference significative a ete observee entre les bovins affectes par la BSE et les bovins chinois sains pour ce qui est de l'indel de 12 pb. Au total, 14 SNP ont ete observes dans la region codante du gene PRNP chez les bovins chinois. Trois de ces SNP etaient associes a des changements d'acides amines (K3T, P54S et S154N). La substitution E211K qui a ete rapportee recemment chez un cas atypique de la BSE chez un bovin americain n'a pas ete detectee dans ce travail.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

Influence of specific HSP70 domains on fibril formation of the yeast prion protein Ure2.

Ure2p is the protein determinant of the Saccharomyces cerevisiae prion state [URE3]. Constitutive overexpression of the HSP70 family member SSA1 cures cells of [URE3]. Here, we show that Ssa1p increases the lag time of Ure2p fibril formation in vitro in the presence or absence of nucleotide. The presence of the HSP40 co-chaperone Ydj1p has an additive effect on the inhibition of Ure2p fibril formation, whereas the Ydj1p H34Q mutant shows reduced inhibition alone and in combination with Ssa1p. In order to investigate the structural basis of these effects, we constructed and tested an Ssa1p mutant lacking the ATPase domain, as well as a series of C-terminal truncation mutants. The results indicate that Ssa1p can bind to Ure2p and delay fibril formation even in the absence of the ATPase domain, but interaction of Ure2p with the substrate-binding domain is strongly influenced by the C-terminal lid region. Dynamic light scattering, quartz crystal microbalance assays, pull-down assays and kinetic analysis indicate that Ssa1p interacts with both native Ure2p and fibril seeds, and reduces the rate of Ure2p fibril elongation in a concentration-dependent manner. These results provide new insights into the structural and mechanistic basis for inhibition of Ure2p fibril formation by Ssa1p and Ydj1p.

A role of cellular prion protein in programming T-cell cytokine responses in disease.

The cellular prion protein (PrPC) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPC has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8–24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP0/0 T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF- and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T-cell activation and effector function.

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

Determining the role of mononuclear phagocytes in prion neuroinvasion from the skin

Many prion diseases are acquired by peripheral exposure, and skin lesions are an effective route of transmission. Following exposure, early prion replication, upon FDCs in the draining LN is obligatory for the spread of disease to the brain. However, the mechanism by which prions are conveyed to the draining LN is uncertain. Here, transgenic mice were used, in which langerin(+) cells, including epidermal LCs and langerin(+) classical DCs, were specifically depleted. These were used in parallel with transgenic mice, in which nonepidermal CD11c(+) cells were specifically depleted. Our data show that prion pathogenesis, following exposure via skin scarification, occurred independently of LC and other langerin(+) cells. However, the depletion of nonepidermal CD11c(+) cells impaired the early accumulation of prions in the draining LN, implying a role for these cells in the propagation of prions from the skin. Therefore, together, these data suggest that the propagation of prions from the skin to the draining LN occurs via dermal classical DCs, independently of langerin(+) cells.

The effect of sucrose on protein stability and fibrillation of prion and S6 protein

Aggregation and fibrillation of proteins have a great importance in medicine and industry. Misfolding and aggregation are the basis of many neurodegenerative diseases like Alzheimer and Parkinson. Osmolytes are molecules that can accumulate within cells and act as protective agents and they can inclusively act as protein stabilizers when cells are exposed to stress conditions. Osmolytes can also act as protein stabilizers in vitro. In this work, two different proteins were studied, the ribosomal protein from Thermus thermophilus and the mouse prion protein. The existence of an unstructured N-terminal on the prion protein does not affect its stability. The effect of the osmolyte sucrose on the fibrillation and stabilization of these two proteins was studied through kinectic and equilibrium measurements. It was shown that sucrose is able to compact the native structure of S6 protein in fibrillization conditions. Sucrose affects also folding and unfolding kinetic of S6 protein, delaying unfolding and increasing folding rate constants. The mechanism of stabilization by sucrose is non-specific because it is distributed for all protein structure, as it was demonstrated by a protein engineering approach. Sucrose delays the process of formation and elongation of S6 and prion protein from mouse. This delay is the result of the compaction of the native structure refered above. However, cellular toxicity studies have shown that fibrils formed in the presence of sucrose are more toxic to neuronal cells.

Étude numérique des premières étapes d'agrégation du peptide amyloïde GNNQQNY, impliqué dans une maladie à prion.

Les protéines amyloïdes sont impliquées dans les maladies neurodégénératives comme Alzheimer, Parkinson et les maladies à prions et forment des structures complexes, les fibres amyloïdes. Le mécanisme de formation de ces fibres est un processus complexe qui implique plusieurs espèces d’agrégats intermédiaires. Parmi ces espèces, des petits agrégats, les oligomères, sont reconnus comme étant l’espèce amyloïde toxique, mais leur mécanisme de toxicité et d’agrégation sont mal compris. Cette thèse présente les résultats d’une étude numérique des premières étapes d’oligomérisation d’un peptide modèle GNNQQNY, issu d’une protéine prion, pour des systèmes allant du trimère au 50-mère, par le biais de simulations de dynamique moléculaire couplée au potentiel gros-grain OPEP. Nous trouvons que le mécanisme d’agrégation du peptide GNNQQNY suit un processus complexe de nucléation, tel qu’observé expérimentalement pour plusieurs protéines amyloïdes. Nous observons aussi que plusieurs chemins de formation sont accessibles à l’échelle du 20-mère et du 50-mère, ce qui confère aux structures un certain degré de polymorphisme et nous sommes capable de reproduire, dans nos simulations, des oligomères protofibrillaires qui présentent des caractéristiques structurelles observées expérimentalement chez les fibres amyloïdes.

Regulation of GABA(A) and glutamate receptor expression, synaptic facilitation and long-term potentiation in the hippocampus of prion mutant mice

Background: Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP(sc)) of the natural cellular prion protein (PrP(c)) encoded by the Prnp gene. Although several roles have been attributed to PrP(c), its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP(c) studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation. Principal Findings: Here we explore the role of PrP(c) expression in neurotransmission and neural excitability using wild-type, Prnp -/- and PrP(c)-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp -/- mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp -/- and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA(A) and AMPA-kainate receptors are co-regulated in both Prnp -/- and Tg20 mice. Conclusions/Significance: Present results demonstrate that PrP(c) is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA(A) and AMPA-Kainate neurotransmission. New PrP(c) functions have recently been described, which point to PrP(c) as a target for putative therapies in Alzheimer's disease. However, our results indicate that a "gain of function" strategy in Alzheimer's disease, or a "loss of function" in prionopathies, may impair PrP(c) function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.

Prion Protein is Expressed on Long-term Repopulating Hematopoietic Stem Cells and is Necessary for their Self-renewal

We show that the prion protein (PrP) is expressed on the surface of bone marrow cell populations enriched in long-term repopulating hematopoietic stem cells. Affinity purification of the PrP-positive and PrP-negative fractions from these populations, followed by competitive reconstitution assays, show that all long-term repopulating hematopoietic stem cells express PrP. Hematopoietic stem cells from PrP null bone marrow exhibit impaired self-renewal in serial competitive transplantation experiments, and premature exhaustion when exposed to cell cycle-specific myelotoxic injury. Therefore, PrP is a novel marker for hematopoietic stem cells and regulates their self-renewal.