Etude du rôle biologique de la protéine prion et de sa région polybasique par étude transcriptomique et par développement de souris transgéniques

Les Encéphalopathies Spongiformes Transmissibles (EST), ou maladies à prions, constituent un ensemble d atteintes neurodégénératives fatales qui touchent assez bien l homme que l animal. L implication de la PrPC dans les maladies prions et sa conversion en forme pathogène PrPSc ont fait l objet d étude pour plusieurs décennies et sont maintenant bien documentées. Cependant la fonction physiologique de cette protéine n'est pas encore claire. Nous avons cherché à contribuer à mieux comprendre la fonction biologique de la protéine prion cellulaire PrPC. Cette thèse est composée de deux projets . Le premier était l étude de la région polybasique de la protéine prion par développement de souris transgéniques exprimant des protéines prion mutées pour cette région. Le second consiste en une étude du rôle de la protéine prion PrPC au cours de l'embryogenèse, notamment par analyse transcriptomique. Nos travaux sur les souris transgéniques exprimant une PrP modifiée au niveau de la région polybasique montrent que les modifications apportées à cette région ne sont pas toxiques et que la délétion 23-27 ne semble pas affecter la sensibilité à l infection aux prions. Nos analyses montrent par ailleurs un rôle crucial et complémentaire au cours de l embryogénèse de la famille des protéines prions et plus précisément dans le contrôle de la maintenance et de la différentiation des cellules trophoblastiques, suggérant une implication de ces protéines dans la biologie des cellules souches.Transmissible Spongiform Encephalopathies, or Prion diseases, are a group of fatal neurodegenerative diseases that affect Humans and animals. The role of PrPC in prion diseases and its conversion into its pathological isoform PrPSc have been studied for many years and are well described. However, the biological function of this protein remains unclear. Our aim was to contribute to a better understanding of this physiological role. This thesis was subdivided in two projects. The first one aimed at analyzing the polybasic region of the protein by the creation of transgenic mice expressing mutated alleles and the second one aimed at studying the role of the prion proteins during early mouse embryogenesis, notably using transcriptomic analyses. Analysis of our transgenic mice expressing PrP alleles mutated in the polybasic region reveals the absence of in vivo toxicity of such alleles and that the deletion of aa23-27 does not affect the mouse susceptibility towards prions. Our transcriptomic studies revealed a crucial and complementary role of the prion proteins during embryogenesis and more precisely in the self-renewal and differentiation of the trophoblastic cells. It suggests an implication of these proteins in the biology of stem cells.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Molecular networks implicated in speech-related disorders:FOXP2 regulates the SRPX2/uPAR complex

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.</p

Transcriptomic analysis brings new insight into the biological role of the prion protein during mouse embryogenesis

Chantier qualité GAThe biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes

PrP and Shadoo are required for trophoblastic development

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Mutated but not deleted ovine PrPCN-terminal polybasic region strongly interferes with prion propagation in transgenic mice

Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the 23-31 N-terminal polybasic region of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitution and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP deleted for residues 23-26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine BSE prions, whereas mutation caused apparent resistance to disease.IMPORTANCE Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein, by identifying the key amino acid residues involved

Molecular networks implicated in speech-related disorders: FOXP2 regulates the SRPX2/uPAR complex

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders