Identification of genes involved in ceramide-dependent neuronal apoptosis using cDNA arrays

BACKGROUND: Ceramide is important in many cell responses, such as proliferation, differentiation, growth arrest and apoptosis. Elevated ceramide levels have been shown to induce apoptosis in primary neuronal cultures and neuronally differentiated PC 12 cells. RESULTS: To investigate gene expression during ceramide-dependent apoptosis, we carried out a global study of gene expression in neuronally differentiated PC 12 cells treated with C(2)-ceramide using an array of 9,120 cDNA clones. Although the criteria adopted for differential hybridization were stringent, modulation of expression of 239 genes was identified during the effector phase of C(2)-ceramide-induced cell death. We have made an attempt at classifying these genes on the basis of their putative functions, first with respect to known effects of ceramide or ceramide-mediated transduction systems, and then with respect to regulation of cell growth and apoptosis. CONCLUSIONS: Our cell-culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. Of the 239 genes that met the criteria for differential hybridization, 10 correspond to genes previously involved in C(2)-ceramide or TNF-α signaling pathways and 20 in neuronal disorders, oncogenesis or more broadly in the regulation of proliferation. The remaining 209 genes, with or without known functions, constitute a pool of genes potentially implicated in the regulation of neuronal cell death

A defective Krab-domain zinc-finger transcription factor contributes to altered myogenesis in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis—a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneratio

Combined mRNA and microRNA profiling reveals that miR-148a and miR-20b control human mesenchymal stem cell phenotype via EPAS1

International audienceMesenchymal stem cells (MSC) are present in a wide variety of tissues during development of the human embryo starting as early as the first trimester. Gene expression profiling of those cells has focused primarily on the molecular signs characterizing their potential heterogeneity and their differentiation potential. In contrast, molecular mechanisms participating to the emergence of MSC identity in embryo are still poorly understood. In this study, human embryonic stem cells (hES) were differentiated toward MSCs (ES-MSC) to compare the genetic patterns between pluripotent hES and multipotent MSC cells by performing a large genome-wide expression profiling of mRNAs and microRNAs (miRNAs). After whole-genome differential transcriptomic analysis, a stringent protocol was used to search for genes differentially expressed between hES and ES-MSC, followed by several validation steps to identify the genes most specifically linked to the MSC phenotype. A network was obtained that encompassed 74 genes in 13 interconnected transcriptional systems which are likely to contribute to MSC identity. Pairs of negatively correlated miRNAs and mRNAs, which suggest miRNA-target relationships, were then extracted and validation sought using PremiRs. We report here that under-expression of miR-148a and miR-20b in ES-MSCs, as compared to ES, allows an increase in expression of the EPAS1 transcription factor that results in the expression of markers of the MSC phenotype specification

Modélisation pathologique des maladies monogéniques par l'utilisation des cellules souches embryonnaires humaines (preuve de concept appliquée à la dystrophie myotonique de type 1)

Parmi leurs applications prometteuses, les lignées de cellules souches embryonnaires humaines (hES) présentent un potentiel inestimable pour améliorer la compréhension des mécanismes moléculaires et cellulaires impliqués dans le développement de maladies monogéniques. Cette application de modélisation pathologique est devenue possible grâce à l utilisation de lignées hES porteuses de la mutation causale d une maladie monogénique, obtenues au cours d un diagnostique pré-implantatoire. L équipe dans laquelle j ai effectué mes travaux de thèse a démontré que des lignées hES et leurs progénies, porteuses de la mutation causale de la dystrophie myotonique de type 1 (DM1), exprimaient des défauts moléculaires caractéristiques de la pathologie, permettant ainsi leur analyse de façon plus pertinente par rapport à des cultures primaires dérivées de biopsies de patients et validant l utilisation de ce modèle cellulaire. Dans ce contexte, dans la première partie de mon travail de thèse, mon objectif a été de mettre au point des conditions de culture permettant la différenciation des cellules hES normales et mutantes vers le lignage neural afin d obtenir des populations homogènes de progéniteurs neuraux et de cellules souches neurales, puis de les caractériser sur le plan phénotypique et fonctionnel. Par une étude transcriptomique, j ai ensuite comparé le profil d expression de ces progéniteurs neuraux à une autre population homogène de précurseurs mésenchymateux. J ai ainsi identifié des gènes et des voies de signalisation spécifiques à chacune de ces populations. (Article 1). Dans la seconde partie de mes travaux, ma contribution au projet de modélisation pathologique de DM1 a été d utiliser ces progéniteurs neuraux et les cellules souches neurales mutés pour explorer les mécanismes physiopathologiques responsables des symptômes neurologiques observés dans cette pathologie. J ai ainsi identifié une anomalie dans une voie de signalisation cellulaire perturbée, la voie la voie mTORC1, basée sur l observation selon laquelle les cellules NSC porteuses de la mutation DM1 proliféraient plus lentement que les cellules contrôles (Article II). J ai également étudié l expression la protéine Tau,connue pour son implication dans la maladie d Alzheimer, et mis en évidence des modifications suggérant une altération du transport axonal dans les neurones issus des lignées hES mutantes. Ces résultats, associés à ceux réalisés dans l équipe, permettent d apporter la preuve de concept de l intérêt d un tel modèle cellulaire pour la modélisation pathologique des maladies monogéniques.Among their promising applications, human embryonic stem cells lines (hES) have huge potential to improve the understanding of molecular and cellular mechanisms involved in the development of monogenic diseases. This application of modeling pathologic became possible using hES cell lines carrying the causal mutation of a monogenic disease, obtained during pre-implantation diagnosis. The team where I did my thesis work demonstrated that hES cell lines and their progeny, carrying the causal mutation in myotonic dystrophy type 1 (DM1), expressing the molecular defects characteristic of the pathology, allowing more relevant analysis than primary cultures derived from biopsies of patients and validates the use of this cell model. In this context, in the first part of my thesis, my goal was to develop culture conditions for hES cell differentiation into normal and mutant neural lineage in order to obtain homogeneous populations of neural progenitors and neural stem cells and to characterize their phenotypic and fonctional preperties. Next, using a transcriptomic method, I compared the expression profile of neural progenitors to another homogeneous population of mesenchymal precursors. Thus, I identified genes and signaling pathways specific to each of these populations. (Article 1). In the second part of my work, my contribution to the pathological modeling of DM1 was to use these mutant neural progenitor cells and neural stem cells to explore the pathophysiological mechanisms involved in neurological symptoms observed in this pathology. Thus, I have identified a cell signaling pathway defects in mTORC1 pathway based on the observation that NSC cells carrying DM1 mutation proliferated more slowly than control cells (Article II).At last, I also studied the expression of Tau protein, a protein involved in Alzheimer s disease and I have highlighted changes suggesting impairement of axonal transport in neurons derived from hES cell lines mutant. These results, together with those performed in the team, can provide proof of concept for the benefit of such a cell model for modeling disease monogenic diseases.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Gene expression profiling of human satellite cells during muscular aging using cDNA arrays

International audienceIt is well established that biological aging is associated with functional deficits at the cellular, tissue, organ and system levels, but the molecular mechanisms that control lifespan and age-related phenotypes are still not well understood. In order to investigate the molecular mechanisms underlying myoblast aging, we have used quantitative hybridization of a cDNA array of 2016 clones from a human skeletal muscle 3'-end cDNA library to monitor gene expression patterns of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and at different stages of proliferation (early, presenescent and senescent). We have shown that expression profiles in satellite cells vary with donor age, with an up-regulation of genes involved in muscle structure, muscle differentiation and in metabolism in the newborn, and a down-regulation of genes involved in protein renewal in adults. We have also observed that myoblasts isolated from subjects of different ages have typical expression profiles at the beginning of their proliferative lifespan. However, this phenomenon progressively disappears as the cells approach senescence. In addition, even though some of the modifications are similar to those observed in other cell types, we have observed that many changes in gene expression are characteristic of the myoblasts, confirming the hypothesis that the program of replicative senescence is specific for each cell type. Finally, we have identified four potential new markers of presenescence for human myoblasts, which could be useful in developing therapeutic strategies

Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

International audienceHuman embryonic stem cells can be differentiated along different lineages, providing the possibility of a precise analysis of genes profiles associated with specific commitments. Subtractive gene expression profiling between differentiated and undifferentiated cells provides lists of potential actors in this commitment. This combines, however, genes that are specifically associated with development and others that are over expressed because of nonlineage-specific differentiation systems. As a way to establish gene profiles associated with the neural and/or to the mesodermal commitments of human embryonic stem cells more precisely, we have carried out a 2-step analysis. We first performed a subtractive analysis of gene profiles of each of these lineages as compared to the undifferentiated stage. Then, we extended the analysis by comparing the 2 sets of results with each other. This strategy has allowed us to eliminate large numbers of genes that were over expressed in both sets of results and to uniquely associate different gene networks with either the neural or the mesodermal commitments

Modifications in the myogenic program induced by in vivo and in vitro aging

International audienceIn this study, we have used high density cDNA arrays to assess age-related changes in gene expression in the myogenic program of human satellite cells and to elucidate modifications in differentiation capacity that could occur throughout in vitro cellular aging. We have screened a collection of 2016 clones from a human skeletal muscle 3'-end cDNA library in order to investigate variations in the myogenic program of myotubes formed by the differentiation of myoblasts of individuals with different ages (5 days old, 52 years old and 79 years old) and induced to differentiate at different stages of their lifespan (early proliferation, presenescence and senescence). Although our analysis has not been able to underline specific changes in the expression of genes encoding proteins involved in muscle structure and/or function, we have demonstrated an age-related induction of genes involved in stress response and a down-regulation of genes involved both in mitochondrial electron transport/ATP synthase and in glycolysis/TCA cycle. From this global approach of post-mitotic cell aging, we have identified 2 potential new markers of presenescence for human myotubes, both strongly linked to carbohydrate metabolism, which could be useful in developing therapeutic strategies

Changes in transcriptome after in vivo exposure to ionising radiation reveal a highly specialised liver response

International audiencePurpose: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity.Materials and methods: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR).Results: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes.Conclusions: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death

International audienceTo define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immuno-staining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adeno-viruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation