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Laurent Schaeffer

Publications and source records attributed to Laurent Schaeffer.

14 recordsLinked to original sources
PubMed2006

NOV/CCN3 impairs muscle cell commitment and differentiation.

NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

Animals↗
PubMed2005

Calcineurin-NFAT signaling, together with GABP and peroxisome PGC-1{alpha}, drives utrophin gene expression at the neuromuscular junction.

We examined whether calcineurin-NFAT (nuclear factors of activated T cells) signaling plays a role in specifically directing the expression of utrophin in the synaptic compartment of muscle fibers. Immunofluorescence experiments revealed the accumulation of components of the calcineurin-NFAT signaling cascade within the postsynaptic membrane domain of the neuromuscular junction. RT-PCR analysis using synaptic vs. extrasynaptic regions of muscle fibers confirmed these findings by showing an accumulation of calcineurin transcripts within the synaptic compartment. We also examined the effect of calcineurin on utrophin gene expression. Pharmacological inhibition of calcineurin in mice with either cyclosporin A or FK506 resulted in a marked decrease in utrophin A expression at synaptic sites, whereas constitutive activation of calcineurin had the opposite effect. Mutation of the previously identified NFAT binding site in the utrophin A promoter region, followed by direct gene transfer studies in mouse muscle, led to an inhibition in the synaptic expression of a lacZ reporter gene construct. Transfection assays performed with cultured myogenic cells indicated that calcineurin acted additively with GA binding protein (GABP) to transactivate utrophin A gene expression. Because both GABP- and calcineurin-mediated pathways are targeted by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), we examined whether this coactivator contributes to utrophin gene expression. In vitro and in vivo transfection experiments showed that PGC-1alpha alone induces transcription from the utrophin A promoter. Interestingly, this induction is largely potentiated by coexpression of PGC-1alpha with GABP. Together, these studies indicate that the synaptic expression of utrophin is also driven by calcineurin-NFAT signaling and occurs in conjunction with signaling events that involve GABP and PGC-1alpha.

Animals↗
PubMed2005

Histone deacetylase 9 couples neuronal activity to muscle chromatin acetylation and gene expression.

Electrical activity arising from motor innervation influences skeletal muscle physiology by controlling the expression of many muscle genes, including those encoding acetylcholine receptor (AChR) subunits. How electrical activity is converted into a transcriptional response remains largely unknown. We show that motor innervation controls chromatin acetylation in skeletal muscle and that histone deacetylase 9 (HDAC9) is a signal-responsive transcriptional repressor which is downregulated upon denervation, with consequent upregulation of chromatin acetylation and AChR expression. Forced expression of Hdac9 in denervated muscle prevents upregulation of activity-dependent genes and chromatin acetylation by linking myocyte enhancer factor 2 (MEF2) and class I HDACs. By contrast, Hdac9-null mice are supersensitive to denervation-induced changes in gene expression and show chromatin hyperacetylation and delayed perinatal downregulation of myogenin, an activator of AChR genes. These findings show a molecular mechanism to account for the control of chromatin acetylation by presynaptic neurons and the activity-dependent regulation of skeletal muscle genes by motor innervation.

Acetylation↗
PubMed2005

[Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene].

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.

Animals↗
PubMed2004

14-3-3 gamma associates with muscle specific kinase and regulates synaptic gene transcription at vertebrate neuromuscular synapse.

The muscle-specific receptor tyrosine kinase (MuSK) is part of a receptor complex, activated by neural agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). To gain insight into the function of the MuSK complex, we have developed a proteomic approach to identify new MuSK partners. MS analysis of MuSK crosslink products from postsynaptic membranes of the Torpedo electrocytes identified the adaptor protein 14-3-3 gamma. The 14-3-3 gamma protein was found localized at the adult rat NMJ. Cotransfection experiments in COS-7 cells showed that MuSK codistributed with the 14-3-3 gamma protein at the plasma membrane. Furthermore, 14-3-3 gamma was copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. The 14-3-3 gamma protein did not colocalize with agrin-elicited acetylcholine receptor (AChR) aggregates in cultured myotubes, suggesting that it is not involved in AChR clustering. Expression of 14-3-3 gamma specifically repressed the transcription of several synaptic reporter genes in cultured myotubes. This repression was potentiated by MuSK expression. Moreover, the expression of 14-3-3 gamma in muscle fibers in vivo caused both the repression of synaptic genes transcription and morphological perturbations of the NMJ. Our data extend the notion that, apart from its well documented role in AChR clustering, the MuSK complex might also be involved in the regulation of synaptic gene expression at the NMJ.

14-3-3 Proteins↗
PubMed2004

MUSK, a new target for mutations causing congenital myasthenic syndrome.

We report the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.

Adult↗
PubMed2004

Conditional gene knock-down by CRE-dependent short interfering RNAs.

Short interfering RNAs (siRNAs) are short (21-23 nt) double-stranded RNAs that direct the sequence-specific degradation of corresponding mRNAs, resulting in suppression of gene activity. siRNAs are powerful tools for gene functional analysis in mammals. Chemically synthesized siRNAs permit transient gene repression but preclude inhibition of stable gene products as well as long-term phenotypic analyses. Permanent gene suppression can be achieved by transcribing siRNAs as stem-loop precursors from Pol III promoters. This approach, however, has a major limitation: inhibition cannot be controlled in a time- or tissue-specific manner. Thus, the approach cannot be applied to genes essential for cell survival or cell proliferation. To overcome these limitations, we have designed a CRE-lox-based strategy that allows one to repress gene activity in a time-dependent manner in cells, and in a time- or tissue-dependent manner in animals. Our approach promises to improve dramatically the procedures for functional genetics in mammals.

Animals↗
PubMed2004

Thrombin reduces MuSK and acetylcholine receptor expression along with neuromuscular contact size in vitro.

In the course of studies on thrombin and its inhibitor(s) in synaptic plasticity, we addressed the question of their roles in the formation of neuromuscular junctions (NMJ) and used a model of rat neuron-myotube cocultures. We report that the size of acetylcholinesterase (AChE) patches used as a marker of neuromuscular contacts was decreased in the presence of either thrombin or SFLLRN, the agonist peptide of the thrombin receptor PAR-1, whereas it was increased with hirudin, a specific thrombin inhibitor. In an attempt to relate these neuromuscular contact size variations to molecular changes, we studied muscle-specific tyrosine kinase receptor (MuSK), acetylcholine receptor (AChR) and rapsyn expression in the presence of thrombin. We showed that thrombin did not change rapsyn gene and protein expression. However, the expression of MuSK and surface AChR proteins was diminished in both myotube cultures and neuron-myotube cocultures. These reductions in protein expression were associated with a decrease in MuSK and AChR alpha-subunit gene expression in myotube cultures but not in neuron-myotube cocultures. Moreover, the expression of the AChR epsilon-subunit gene, specifically enhanced by neuron-released factors, was not modified by thrombin in neuron-myotube cocultures. This suggests that thrombin did not affect the expression of synaptic AChRs enhanced by neuron-released factors but rather reduced the level of extrasynaptic AChRs. Taken together, these results indicate that thrombin in balance with its inhibitor(s) could modulate the formation of neuromuscular contacts in vitro by affecting the expression of two essential molecules in NMJ postsynaptic differentiation, MuSK and AChR.

Animals↗
PubMed2004

Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation.

In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.

Animals↗
PubMed2003

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G-protein-coupled protease-activated receptor-1.

Regulation of thrombin activity may be required during skeletal muscle differentiation since the thrombin tissue inhibitor protease nexin-1 appears at the myotube stage before being localized at the neuromuscular synapse. Here, we have used a model of rat fetal myotube primary cultures to study the effect of thrombin on acetylcholine receptor (AChR) expression, which is enhanced at the myotube stage. Our results show that thrombin decreases both the number of surface AChRs (AChRn) and AChR alpha-subunit gene expression. Using the agonist peptide SFLLRN, we establish that the AChRn decrease is mediated by the G protein-coupled thrombin receptor "protease-activated receptor-1" (PAR-1). Moreover, the specific thrombin inhibitor hirudin increases AChRn by inhibiting the thrombin intrinsically present in the cultures. We further demonstrate that the activation of PAR-1 by thrombin induces intracellular calcium movements that are blocked by 2-APB, an inhibitor of inositol 1,4,5-triphosphate (IP3)-induced calcium release. These calcium signals are more intense in nuclei than in the cytoplasm and are consistent with the intracellular distribution of IP3 receptor that we find in the cytoplasm in a cross-striated pattern and at a high level in the nuclear envelope zone. Finally, we show that the blockade of these IP3-induced calcium signals by 2-APB prevents the AChRn decrease induced by thrombin. Our results thus demonstrate that thrombin downregulates AChR expression by activating PAR-1 and that this effect is mediated via an IP3 signaling pathway.

Acetylcholine↗
PubMed2003

Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction.

Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.

Agrin↗
PubMed2003

Synapse-specific gene expression at the neuromuscular junction.

Agrin is the key neural factor that controls muscle postsynaptic differentiation, including the induction of synapse-specific transcription via neuregulins. In 1995, the promoter element responsible for the targeting of AChR delta and epsilon gene transcription to the skeletal muscle subsynaptic area was identified. This element, named N-box, recruits the Ets-related transcription factor GABP to AChR delta and epsilon promoters, and both the N-box and GABP are required to obtain transcriptional stimulation by neuregulins. The physiological importance of the N-box has been definitively established with the discovery of myasthenic families carrying single-point mutations in the N-box of the AChR epsilon gene promoter and showing reduced levels of AChR epsilon subunit expression. The control of synapse-specific transcription by agrin and neuregulins through the N-box and GABP is not restricted to the case of AChR genes. The same regulation holds true for the ACh esterase and utrophin genes, thus showing that nerve-induced transcriptional activation of several synapse-specific genes is triggered by a common mechanism involving agrin, neuregulins, and ultimately the N-box and Ets-related transcription factors.

Agrin↗
PubMed2002

Expression of mutant Ets protein at the neuromuscular synapse causes alterations in morphology and gene expression.

The localized transcription of several muscle genes at the motor endplate is controlled by the Ets transcription factor GABP. To evaluate directly its contribution to the formation of the neuromuscular junction, we generated transgenic mice expressing a general Ets dominant-negative mutant specifically in skeletal muscle. Quantitative RT-PCR analysis demonstrated that the expression of genes containing an Ets-binding site was severely affected in the mutant mice. Conversely, the expression of other synaptic genes, including MuSK and Rapsyn, was unchanged. In these animals, muscles expressing the mutant transcription factor developed normally, but examination of the post-synaptic morphology revealed marked alterations of both the primary gutters and secondary folds of the neuromuscular junction. Our results demonstrate that Ets transcription factors are crucial for the normal formation of the neuromuscular junction. They further show that Ets-independent mechanisms control the synaptic expression of a distinct set of synaptic genes.

Acetylcholinesterase↗