ɑ-Synuclein strains and seeding in Parkinson's disease, incidental Lewy body disease, dementia with Lewy bodies and multiple system atrophy: similarities and differences.

Several age-related neurodegenerative disorders are characterized by the deposition of aberrantly folded endogenous proteins. These proteins have prion-like propagation and amplification properties but so far appear nontransmissible between individuals. Because of the features they share with the prion protein, PrP, the characteristics of pathogenic protein aggregates in several progressive brain disorders, including different types of Lewy body diseases (LBDs), such as Parkinson's disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB), have been actively investigated. Even though the pleomorphic nature of these syndromes might suggest different underlying causes, ɑ-synuclein (ɑSyn) appears to play an important role in this heterogeneous group of diseases (the synucleinopathies). An attractive hypothesis is that different types of ɑSyn protein assemblies have a unique and causative role in distinct synucleinopathies. We will discuss the recent research progress on ɑSyn assemblies involved in PD, MSA and DLB; their behavior as strains; current spreading hypotheses; their ability to seed centrally and peripherally; and their implication for disease pathogenesis.

Binding, internalization and fate of Huntingtin Exon1 fibrillar assemblies in mitotic and nonmitotic neuroblastoma cells.

AIMS: The aggregation of Huntingtin (HTT) protein and of its moiety encoded by its Exon1 (HTTExon1) into fibrillar structures inside neurons is the molecular hallmark of Huntington's disease. Prion-like transmission of these aggregates between cells has been demonstrated. The cell-to-cell transmission mechanisms of these protein aggregates and the susceptibility of different kinds of neuronal cells to these toxic assemblies still need assessment. METHODS: Here, we documented the binding to and internalization by differentiated and undifferentiated neuroblastoma cells of exogenous fibrillar HTTExon1 and polyglutamine (polyQ) polypeptides containing the same number of glutamines. We assessed the contribution of endocytosis to fibrillar HTTExon1 uptake, their intracellular localization and fate. RESULTS: We observed that undifferentiated neuroblastoma cells were more susceptible to fibrillar HTTExon1 and polyQ than their differentiated counterparts. Furthermore, we demonstrated that exogenous HTTExon1 aggregates are mainly taken up by endocytosis and directed to lysosomal compartments in both mitotic and quiescent cells. CONCLUSIONS: These data suggest that the rates of endocytic processes that differ in mitotic and quiescent cells strongly impact the uptake of exogenous HTTExon1 and polyQ fibrils. This may be either the consequence of distinct metabolisms or distributions of specific protein partners for amyloid-like assemblies at the surface of highly dividing versus quiescent cells. Our results highlight the importance of endocytic processes in the internalization of exogenous HTTExon1 fibrils and suggest that a proportion of those assemblies reach the cytosol where they can amplify by recruiting the endogenous protein after escaping, by yet an unknown process, from the endo-lysosomal compartments.

α-Synuclein strains cause distinct synucleinopathies after local and systemic administration.

Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions. The molecular hallmark of synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson's disease and dementia with Lewy bodies. The discovery of α-SYN assemblies with different structural characteristics or 'strains' has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson's disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood-brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.

Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape.

Quantitative resistance mediated by multiple genetic factors has been shown to increase the potential for durability of major resistance genes. This was demonstrated in the Leptosphaeria maculans/Brassica napus pathosystem in a 5year recurrent selection field experiment on lines harboring the qualitative resistance gene Rlm6 combined or not with quantitative resistance. The quantitative resistance limited the size of the virulent isolate population. In this study we continued this recurrent selection experiment in the same way to examine whether the pathogen population could adapt and render the major gene ineffective in the longer term. The cultivars Eurol, with a susceptible background, and Darmor, with quantitative resistance, were used. We confirmed that the combination of qualitative and quantitative resistance is an effective approach for controlling the pathogen epidemics over time. This combination did not prevent isolates virulent against the major gene from amplifying in the long term but the quantitative resistance significantly delayed for 5years the loss of effectiveness of the qualitative resistance and disease severity was maintained at a low level on the genotype with both types of resistance after the fungus population had adapted to the major gene. We also showed that diversity of AvrLm6 virulence alleles was comparable in isolates recovered after the recurrent selection on lines carrying either the major gene alone or in combination with quantitative resistance: a single repeat-induced point mutation and deletion events were observed in both situations. Breeding varieties which combine qualitative and quantitative resistance can effectively contribute to disease control by increasing the potential for durability of major resistance genes.

Partial selfing, ecological disturbance and reproductive assurance in an invasive freshwater snail.

Although reproductive assurance (RA) might play a central role in the evolution of the selfing rate, this hypothesis has never been seriously investigated in an hermaphroditic animal. We studied the mating system of the freshwater snail Physa acuta in which the availability of mating partners might be highly variable, because this species is an efficient colonizer occupying unstable habitats. A total of 11 populations differing in ecological disturbance regime (water level, openness) and snail densities were monitored over 2 years. The outcrossing rate was estimated in ca 10 families per population using microsatellite markers and the progeny-array approach. Components of fecundity and survival were recorded for each progeny. Predominant outcrossing (t(m)=0.94) was detected, with a few individuals (4%) purely selfing. The outcrossing rate did not explain among-family variation in fitness components. None of the predictions formulated under the RA hypothesis were verified: (i) selfing was related neither to disturbed habitats, nor to temporal density fluctuations, (ii) it was positively related to population density, (iii) it co-occurred with multiple paternity, and (iv) it did not induce delayed reproduction. Explanations for these negative results are discussed in light of other arguments supporting the RA hypothesis in P. acuta, as well as alternative theories explaining the occurrence of partial selfing, as either a genetically fixed or plastic trait.

Population biology of the invasive freshwater snail Physa acuta approached through genetic markers, ecological characterization and demography.

Abstract The respective role of factors acting on population functioning can be inferred from a variety of approaches, including population genetics and demography. We here investigated the role of four of these factors (mating systems, population size, bottlenecks and migration) in the hermaphroditic freshwater snail Physa acuta. Twenty-four populations were sampled either around Montpellier (local scale), or at the scale of France (global scale). At local scale, eight populations were sampled twice, before and after summer drying out. The genetic structure of these populations was studied using microsatellite loci. Populations were classified according to openness (ponds vs. rivers) and water regime (permanent vs. temporary) allowing predictions on genetic patterns (e.g. diversity within populations and differentiation). At local scale, progeny-arrays analysis of the selfing rate was conducted, and size distributions of individuals were followed over two years. Results with regard to the four factors mentioned above were: (i) Estimates of population selfing rates derived from inbreeding coefficients were only slightly higher than those from progeny-arrays. (ii) More variation was detected in rivers than in ponds, but no influence of water regime was detected. One reason might be that permanent populations are not going less often through low densities than those from temporary habitats at the time scale studied. (iii) There was limited evidence for genetic bottlenecks which is compatible with the fact that even marked reduction in water availability was not necessarily associated with demographic bottlenecks. More generally, bottlenecks reducing genetic variation probably occur at population foundation. (iv) Lower genetic differentiation was detected among rivers than among ponds which might be related to limitations on gene flow. Demographic and temporal genetic data further indicates that flooding in rivers is unlikely to induce marked gene flow explaining the strong genetic differentiation at short geographical scale in such habitats. Finally, the demographic data suggest that some populations are transitory and subject to recurrent recolonization, a pattern that was also detected through genetic data.

Effects of acibenzolar-s-methyl and ethirimol on the composition of a laboratory population of barley powdery mildew.

ABSTRACT To assemble a laboratory population of Blumeria graminis f. sp. hordei for a competition experiment, controlled crosses were performed among 30 parent isolates, which were characterized by their pathotype (i.e., the phenotype observed on a differential set containing lines including host resistance alleles) and their response to the fungicides ethirimol and triadimenol. Variability in the response to the chemical inducer of host-resistance acibenzolar-S-methyl (BTH: benzo[1,2,3]thiadiazole-7carbothioic acid-S-methyl ester) was introduced by using isolates collected in fields repeatedly treated with this chemical. Based on their pathotype and response to ethirimol, 137 isolates recovered from the crosses were chosen to assemble a laboratory population. This protocol produced variability in the laboratory population for traits chosen only in the parents (triadimenol) or both in the parents and the progenies (ethirimol). It was therefore postulated that the variability in the response to BTH, if present in the parents, was also present in the laboratory population. No association between these traits was observed. The effect of BTH on the evolution of the laboratory population was compared with the effect of the fungicide ethirimol. The laboratory population was exposed to selection pressures and its evolution was followed over 10 generations. Ethirimol treatments always induced a decrease in sensitivity, whereas no consistent trend was observed for sensitivity to triadimenol (not selected). This result indicates that the application of ethirimol induced a selection pressure. For BTH, (toxicological) sensitivity tests have not detected any consistent evolution, but pathotype diversity indicated that in cases when BTH was applied with ethirimol, BTH induced a further selection pressure in addition to that of ethirimol.

Crystal structures of the yeast prion Ure2p functional region in complex with glutathione and related compounds.

The [URE3] phenotype in yeast Saccharomyces cerevisiae is due to an altered prion form of Ure2p, a protein involved in nitrogen catabolism. To understand possible conformational changes at the origin of prion propagation, we previously solved the crystal structure of the Ure2p functional region [Bousset et al. (2001) Structure 9, 39-46]. We showed the protein to have a fold similar to that of the beta class of glutathione S-transferases (GSTs). Here we report crystal structures of the Ure2p functional region (extending from residues 95-354) in complex with glutathione (GSH), the substrate of all GSTs, and two widely used GST inhibitors, namely, S-hexylglutathione and S-p-nitrobenzylglutathione. In a manner similar to what is observed in many GSTs, ligand binding is not accompanied by a significant change in the conformation of the protein. We identify one GSH and one hydrophobic electrophile binding site per monomer as observed in all other GSTs. The sulfur group of GSH, that conjugates electrophiles, is located near the amide group of Asn124, allowing a hydrogen bond to be formed. Biochemical data indicate that GSH binds to Ure2p with high affinity. Its binding affects Ure2p oligomerization but has no effect on the assembly of the protein into amyloid fibrils. Despite results indicating that Ure2p lacks GST activity, we propose that Ure2p is a member of the GST superfamily that may describe a novel GST class. Our data bring new insights into the function of the Ure2p active region.

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p.

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.

Structure of the globular region of the prion protein Ure2 from the yeast Saccharomyces cerevisiae.

BACKGROUND: The [URE3] non-Mendelian element of the yeast S. cerevisiae is due to the propagation of a transmissible form of the protein Ure2. The infectivity of Ure2p is thought to originate from a conformational change of the normal form of the prion protein. This conformational change generates a form of Ure2p that assembles into amyloid fibrils. Hence, knowledge of the three-dimensional structure of prion proteins such as Ure2p should help in understanding the mechanism of amyloid formation associated with a number of neurodegenerative diseases. RESULTS: Here we report the three-dimensional crystal structure of the globular region of Ure2p (residues 95--354), also called the functional region, solved at 2.5 A resolution by the MAD method. The structure of Ure2p 95--354 shows a two-domain protein forming a globular dimer. The N-terminal domain is composed of a central 4 strand beta sheet flanked by four alpha helices, two on each side. In contrast, the C-terminal domain is entirely alpha-helical. The fold of Ure2p 95--354 resembles that of the beta class glutathione S-transferases (GST), in line with a weak similarity in the amino acid sequence that exists between these proteins. Ure2p dimerizes as GST does and possesses a potential ligand binding site, although it lacks GST activity. CONCLUSIONS: The structure of the functional region of Ure2p is the first crystal structure of a prion protein. Structure comparisons between Ure2p 95--354 and GST identified a 32 amino acid residues cap region in Ure2p exposed to the solvent. The cap region is highly flexible and may interact with the N-terminal region of the partner subunit in the dimer. The implication of this interaction in the assembly of Ure2p into amyloid fibrils is discussed.

Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2.

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

PCR cloning and detection of point mutations in the eburicol 14alpha-demethylase (CYP51) gene from Erysiphe graminis f. sp. hordei, a "recalcitrant" fungus.

Molecular studies of some micro-organisms are hampered by the difficulty of obtaining sufficient amounts of nucleic acids. A cloning strategy based on PCR has therefore been used to clone the eburicol 14alpha-demethylase (CYP51) gene of the obligate fungus Erysiphe graminis f. sp. hordei (Egh) using minute amounts of genomic DNA. The CYP51 gene encodes the enzymatic target of a major group of fungicides. Sequencing CYP51 from different Egh isolates revealed the occurrence of two alleles for this gene. An allele-specific PCR assay was developed to detect each CYP51 allele.