P Chafey

Unité Inserm U1077, Caen, Lower Normandy, France

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Publications (28)265.75 Total impact

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    ABSTRACT: Doublecortin (DCX) is a microtubule-associated protein involved in neuronal migration, which causes X-linked lissencephaly and subcortical laminar heterotopia (SCLH) when mutated. Here we show that DCX interacts with the ubiquitin-specific protease Drosophila fat facets related on X chromosome (DFFRX). This interaction was confirmed by targeted mutagenesis, colocalization, and immunoprecipitation studies. DFFRX is thought to deubiquitinate specific substrates including beta-catenin, preventing their degradation by the proteasome. Interestingly, unlike beta-catenin, no ubiquitinated forms of DCX could be detected, and indeed we show that DCX interacts with a novel recognition domain in DFFRX, located outside of its catalytic site. We also show that DFFRX associates with microtubules at specific subcellular compartments, including those enriched in DCX. These results thus suggest that in addition to vesicular trafficking, DCX may play a role in the regulation of cell adhesion via its interaction with DFFRX in migrating and differentiating neurons.
    Molecular and Cellular Neuroscience 02/2005; 28(1):153-64. · 3.84 Impact Factor
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    ABSTRACT: Recent human genetic approaches showed that mutations in three genes encoding OPHN1, PAK3, and alphaPIX cause nonspecific X-linked mental retardation. These three proteins act to modulate Rho GTPase signaling pathways and may participate in neuronal morphogenesis by regulating the actin cytoskeleton. Here we showed that the Oligophrenin-1 gene is expressed in the developing spinal cord and later in brain areas that are characterized by high synaptic plasticity. At the cellular level OPHN1 is expressed in both glial and neuronal cells where it colocalizes with actin, notably at the tip of growing neurites. This interaction seems to be direct through a novel uncharacterized domain in the carboxyl-terminal end of OPHN1. Overexpression experiments in fibroblasts showed that the OPHN1 RhoGAP domain regulates in vivo the actin cytoskeleton by inhibition of Rho pathways. Interestingly the amino-terminal domain of OPHN1 inhibits the RhoGAP activity through an as yet unknown mechanism, suggesting that OPHN1 may be tightly regulated in vivo.
    Molecular and Cellular Neuroscience 09/2003; 23(4):574-86. · 3.84 Impact Factor
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    ABSTRACT: Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.
    Human Molecular Genetics 07/2003; 12(12):1415-25. · 7.69 Impact Factor
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    ABSTRACT: Type I lissencephaly is a cortical malformation disorder characterized by disorganized cortical layers and gyral abnormalities and associated with severe cognitive impairment and epilepsy. The exact pathophysiological mechanisms underlying the epilepsy and mental retardation in this and related disorders remain unknown. Two genes, LIS1 and doublecortin, have both been shown to be mutated in a large proportion of cases of type I lissencephaly and a milder allelic disorder, subcortical laminar heterotopia (SCLH). Studying the protein products of these genes and the biochemical pathways in which they belong is likely to yield important information concerning both normal and abnormal cortical development. The relationships between the LIS1 and Doublecortin proteins are not yet well defined, but both are believed to play a critical role in cortical neuronal migration. Lis1 is expressed from very early development in the mouse and in both proliferating cells and post-mitotic neurons of the cortex. This protein is likely to have multiple functions since it is a subunit of the enzyme platelet-activating factor acetylhydrolase, which degrades platelet activating factor, and has also been shown to be involved in microtubule dynamics, potentially influencing nuclear migration through its interaction with the dynein motor protein complex. Doublecortin on the other hand is exclusively expressed in post-mitotic neurons and is developmentally regulated. In young developing neurons Doublecortin has a specific subcellular localization at the ends of neuritic and leading processes. This localization, combined with our previous data showing that it is a microtubule-associated protein and that it interacts with adapter complexes involved in vesicle trafficking, suggests a role in the growth of neuronal processes, downstream of directional or guidance signals. The observations summarized here favor the suggestion that whereas LIS1 may play a role in nuclear migration, Doublecortin is instead restricted to functions at the leading edge of the cell.
    Cerebral Cortex 07/2003; 13(6):620-6. · 8.31 Impact Factor
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    ABSTRACT: Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.
    Molecular and Cellular Neuroscience 10/2001; 18(3):307-19. · 3.84 Impact Factor
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    ABSTRACT: X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.
    Nature Genetics 03/2000; 24(2):167-70. · 35.21 Impact Factor
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    ABSTRACT: Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.
    Neuron 07/1999; 23(2):247-56. · 15.77 Impact Factor
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    ABSTRACT: MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.
    Molecular Biology of the Cell 08/1998; 9(7):1891-902. · 4.60 Impact Factor
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    ABSTRACT: Antibodies to dystrophin have increased accuracy in the diagnosis of Duchenne/Becker muscular dystrophy (D/BMD). Both typical and 'atypical' presentations of this disease can be confirmed by demonstrating qualitative and quantitative defects in the expression of dystrophin protein. However, owing to the propensity for dystrophin degradation in vitro, caution needs to be applied while performing and interpreting antibody-based dystrophin analysis. Here we identify two cases where in vitro protein degradation caused diagnostic confusion. We demonstrate the use of utrophin/dystrophin related protein (DRP) as sensitive control for sample degradation, since it is more labile than dystrophin. We suggest that the concomitant or sequential usage of antibodies specific for dystrophin along with utrophin/DRP can help reduce the misdiagnosis of D/BMD.
    Biochemical and Biophysical Research Communications 01/1998; 241(2):232-5. · 2.41 Impact Factor
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    ABSTRACT: In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.
    Molecular and Cellular Biology 12/1996; 16(11):6065-74. · 5.37 Impact Factor
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    ABSTRACT: The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin, a cytoplasmic component of the dystrophin-glycoprotein complex, by in vitro biochemical studies such as overlay assay or immunoprecipitation. Using the two-hybrid system, we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin, therefore confirming for the first time this interaction via an in vivo approach. In addition, we have reduced the interaction domain to the distal half of alpha1 syntrophin.
    FEBS Letters 04/1996; 383(1-2):124-8. · 3.58 Impact Factor
  • Geochimica et Cosmochimica Acta 01/1996; 60(20). · 3.88 Impact Factor
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    ABSTRACT: Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.
    Molecular and Cellular Biology 11/1995; 15(10):5453-60. · 5.37 Impact Factor
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    ABSTRACT: Cells of the embryonic mesenchymal cell line C3H10T1/2 have revealed the potential that the four regulatory factors belonging to the MyoD family have to activate myogenesis. In the present study we have further investigated the myogenic phenotype of C3H10T1/2 cells stably transfected with either Myf5, MyoD, myogenin or MRF4 cDNAs. We have studied the influence of each transfected cDNA on expression of the four endogenous muscle regulatory genes and on the ability of these embryonic myogenic derivatives to express adult muscle genes. No trace of endogenous transcripts distinct from the exogenous one was found in any of the four converted populations at the myoblast stage. This indicates that cross-activation within the MyoD family does not occur at the myoblast stage in these cells. Similarly, evidence was obtained that auto- or cross-activation of the Myf5 gene occurs neither at the myoblast stage nor at the myotube stage and that no autoactivation of the MRF4 gene occurs. Our results together with previous observations indicate that in C3H10T1/2 myogenic derivatives: (1) Autoactivation at the myoblast stage is restricted to MyoD (2) Expression from each cDNA alone is sufficient to establish and maintain the myoblast phenotype (3) The endogenous Myf5 gene is not mobilized. We have also observed that endogenous transcripts for MyoD and myogenin begin to accumulate at the onset of differentiation in the four myogenic derivatives, whereas accumulation of endogenous MRF4 transcripts starts after myotubes have formed and occurs at a much lower level (100- to 500-fold lower) than in differentiated cultures of myosatellite cells.(ABSTRACT TRUNCATED AT 250 WORDS)
    Differentiation 03/1994; 55(3):185-92. · 2.86 Impact Factor
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    ABSTRACT: Due to their quiescent nature and spatial complexity, many target tissues for gene therapy will require novel strategies. An alternative to ex vivo gene transfer, providing many technical advantages and possibly allowing sufficient transfer of the therapeutic gene, is direct in vivo delivery of the vehicle. For a favorable outcome, this procedure is dependent on a high-titer vector, fully competent before post-mitotic cells. In view of the restrictions with the use of retroviruses, we investigated the potentials of adenovirus. Adenoviruses have as primary targets of infection the differentiated epithelial cell. The large DNA genome of the virus hints to a large cloning capacity. Furthermore, the wild type adenovirus has been largely used in man as a vaccine against adenovirus-induced respiratory disease. Taken together, the biological characteristics of adenovirus and the precedent of administration to humans are suggestive of adenovirus-based gene therapy for diseases involving a variety of quiescent tissues. The use of a replication-defective adenovirus carrying a gene encoding a nuclearly-targeted beta-galactosidase Ad.RSV beta gal demonstrated that replication-defective adenovirus offers an efficient means to transfer a gene for extended periods of time in the liver, muscle, lung and brain (1-6).
    Gene Therapy 02/1994; 1 Suppl 1:S53-4. · 4.32 Impact Factor
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    ABSTRACT: Duchene muscular dystrophy (DMD) is a fatal progressive X-linked muscle disorder, caused by mutations in the dystrophin gene. We have investigated adenovirus-mediated transfer of a dystrophin minigene in a mutant mouse lacking dystrophin, the mdx mouse. We report here that six months after a single intramuscular injection of a recombinant adenovirus containing a human dystrophin minigene, a large number of dystrophin-positive fibres are still detected in the injected muscles. Moreover, although the minigene encodes a truncated protein, its expression is able to protect the fibres efficiently against the degeneration process that affects the dystrophin-deficient mdx myofibres.
    Nature Genetics 11/1993; 5(2):130-4. · 35.21 Impact Factor
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    ABSTRACT: The dystrophin whose defect is responsible for Duchenne and Becker muscular dystrophies is present in muscle, brain and cerebellum. We describe here the detection of dystrophin in human cultured skin fibroblasts, L809 cells and murine 3T6 cell line. Dystrophin transcripts initiated at the muscle specific first exon can also be amplified by cDNA-PCR from various fibroblastic cells. The expression of the dystrophin gene in fibroblasts could account for some abnormalities observed in patient's fibroblast cultures.
    Biochemical and Biophysical Research Communications 05/1993; 192(1):69-74. · 2.41 Impact Factor
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    ABSTRACT: Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle.
    Nature 03/1993; 361(6413):647-50. · 38.60 Impact Factor
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    ABSTRACT: The pattern of expression of two distal transcripts initiated in the 62nd intron of the dystrophin gene was investigated under different circumstances; (i) during the development of different rat tissues these transcripts and Dp71, a protein encoded by one of them, increased with brain development and decreased with muscle development; (ii) in cultured glial and neuronal cells, the distal promoter was coactivated with tissue-specific upstream promoters, the muscle-type promoter in glial cells and the brain-type promoter in neuronal cells, which suggests that activity of the upstream promoter does not interfere with activity of the distal promoter; (iii) in lymphoblasts of DMD patients with various deletions of the dystrophin gene, the most distal of which included the 56th intron, the production of the distal transcript was not perturbed.
    Neuromuscular Disorders 01/1993; 3(5-6):519-24. · 3.46 Impact Factor
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    ABSTRACT: A transcript generated by the distal part of the Duchenne Muscular Dystrophy (DMD) gene was initially detected in cells where the full size 14-kilobase (kb) messenger RNA is not found at a significant level. This transcript, approximately 4.5 kb long, corresponds to the cysteine-rich and carboxyl-terminal domains of dystrophin. It begins with a novel 80- to 100-nucleotide exon containing an ATG start site for a new coding sequence of 17 nucleotides in-frame with the consecutive dystrophin cDNA sequence from exon 63. This result suggests the existence of a third promoter that would be localized about 8 kilobases upstream from exon 63 of the DMD gene. The distal transcript is widely distributed but is absent in adult skeletal and myometrial muscle. It is much more abundant in fetal tissues. With an antibody directed against the dystrophin carboxyl terminus, the protein corresponding to this transcript was detected as a 70- to 75-kDa entity on Western blots. It was found in all tissues analyzed except in skeletal muscle. It was not found in lymphoblastoid cells from a Duchenne patient with a complete deletion of the dystrophin gene. The role and subcellular localization of this protein is not known. It may explain extramuscular symptoms exhibited by some Duchenne patients.
    Proceedings of the National Academy of Sciences 09/1992; 89(16):7506-10. · 9.81 Impact Factor

Publication Stats

2k Citations
265.75 Total Impact Points

Institutions

  • 1990–2001
    • Unité Inserm U1077
      Caen, Lower Normandy, France
  • 1999
    • Institut de Génétique Moléculaire de Montpellier
      Montpelhièr, Languedoc-Roussillon, France
  • 1995
    • Université René Descartes - Paris 5
      Lutetia Parisorum, Île-de-France, France
  • 1994
    • Institut de Cancérologie Gustave Roussy
      Île-de-France, France