Transcriptome analysis of prion disease animal models

Prion diseases are incurable and fatal neurodegenerative disorders that affect both humans and animals. The causative agent is an infectious protein called prion (PrPSc), which is the pathological form of a normal protein (PrPC) present on the cell membrane. The molecular mechanisms underlying prion replication and subsequent degeneration of the Central Nervous System (CNS) are still poorly understood and therefore innovative approaches are needed to build diagnostic, therapeutic, taxonomic, and disease surveillance tools. We are going to adopt an unbiased genomic approach and conduct whole transcriptome analyses using microarray gene expression methods in brain and/or blood of infected animals versus healthy controls. We hope to identify a set of genes that can be used for early diagnosis and/or as targets for therapeutic strategies. Within the Trans2Care project we intend to promote collaboration and exchange of knowledge to facilitate all partners’ research objectives, and possibly find a common way to accelerate the process aimed at improving our healthcare system

Modification of a commercial dna extraction kit to simultaneously recover rna, safely and rapidly, and to assess molecular biomass of the total and the active part of microbial communities, from soils with diverse mineralogy and carbon content : S11.04-P -15

We have modified a commercial DNA extraction kit for soil to simultaneously co-extract RNA. In this new procedure RNA and DNA are separated by two selective purifications in cascade without the need of DNAase or RNAse digestion. Consequently DNA and RNA are respectively purified from the whole co-extraction solution. Nucleic acids extraction is based on the action of SDS coupled with an efficient bead-beating step, but it does not require any solvent. Avoiding the use of solvents, which are damaging for human health and environmental quality, was one of our most important motivations to develop this protocol. In a second time, we have optimized this protocol to improve the DNA and RNA yield, but kipping those yields below the saturation limit of the kit to assess and quantify the variations of molecular biomass of the total (DNA) and the active (RNA) part of microbial communities in natural samples. We have also introduced a first step of homogenization of soil sample in liquid nitrogen to improve the reliability of the fungal 18S gene sequence quantification. Finally, we have shown that this protocol can be applied to a wide diversity of soils whatever their mineralogy and metal content (2 Ferralsols, 1 Vertisol, 2 Andosols from Madagascar), texture or biomass content (1 poor sandy soil from Congo and one carbon rich temperate soil sample submitted or not to a 1 month cold stress). * E Tournier, L. Amenc and AL. Pablo contributed equally to this study. (Texte intégral

A system-level approach for deciphering the transcriptional response to prion infection

Motivation: Deciphering the response of a complex biological system to an insulting event, at the gene expression level, requires adopting theoretical models that are more sophisticated than a one-to-one comparison (i.e. t-test). Here, we investigate the ability of a novel reverse engineering approach (System Response Inference) to unveil non-obvious transcriptional signatures of the system response induced by prion infection. Results: To this end, we analyze previously published gene expression data, from which we extrapolate a putative full-scale model of transcriptional gene-gene dependencies in the mouse central nervous system. Then, we use this nominal model to interpret the gene expression changes caused by prion replication, aiming at selecting the genes primarily influenced by this perturbation. Our method sheds light on the mode of action of prions by identifying key transcripts that are the most likely to be responsible for the overall transcriptional rearrangement from a nominal regulatory network. As a first result of our inference, we have been able to predict known targets of prions (i.e. PrPC) and to unveil the potential role of previously unsuspected genes. Contact: [email protected] Supplementary Information: Supplementary data are available at Bioinformatics onlin

Novel markers for neurodegeneration

Prion diseases are incurable and fatal neurodegenerative disorders that affect both humans and animals. The causative agent is an infectious protein called prion (PrPSc), which is the pathological form of a normal protein (PrPC) present on the cell membrane. The molecular mechanisms underlying prion replication and subsequent degeneration of the Central Nervous System (CNS) are still poorly understood and therefore innovative approaches are needed to build diagnostic, therapeutic, taxonomic, and disease surveillance tools. We adopted an unbiased genomic approach and conducted whole transcriptome analyses using microarray and RT-qPCR gene expression methods in brain of infected macaques versus healthy controls. We identified a set of genes that could become novel biomarkers for early diagnosis and/or therapeutic strategies for prion diseases and other neurodegenerative disorders

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” PrPSc 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 PrPSc recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a β-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 PrPSc, and the other to classical PrPSc. Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrPSc 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

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

The intrinsically disordered and amyloidogenic protein α-synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nanoelectrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as oxidation

Early structural features in mammalian prion conformation conversion

The conversion to a disease-associated conformer (PrP (Sc) ) of the cellular prion protein (PrP (C) ) is the central event in prion diseases. Wild-type PrPC converts to PrP (Sc) in the sporadic forms of the disorders through an unknown mechanism. These forms account for up to 85% of all human (Hu) occurrences; the infectious types contribute for less than 1%, while genetic incidence of the disease is about 15%. Familial Hu prion diseases are associated with about forty point mutations of the gene coding for the PrP denominated PRNP. Most of the variants associated with these mutations are located in the globular domain of the protein. In a recent work in collaboration with the German Research School for Simulation Science, in Jülich, Germany, we performed molecular dynamics simulations for each of these mutants to investigate their structure in aqueous solution. Structural analysis of the various point mutations present in the globular domain unveiled common folding traits that may allow to a better understanding of the early conformational changes leading to the formation of monomeric PrP (Sc) . Recent experimental data support these findings, thus opening novel approaches to determine initial structural determinants of prion formation. © 2012 Landes Bioscience

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Roupala cataractarum Sleumer

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