Annie Cartaud

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)J. Biol. Chem. 272, 8117–8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24–48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of β-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5′ from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-β-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.

14-3-3 γ 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 γ. The 14-3-3 γ protein was found localized at the adult rat NMJ. Cotransfection experiments in COS-7 cells showed that MuSK codistributed with the 14-3-3 γ protein at the plasma membrane. Furthermore, 14-3-3 γ was copurified by affinity chromatography with MuSK from transfected COS-7 cells and myotubes. The 14-3-3 γ 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 γ 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 γ 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.

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (muscle‐specific kinase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear.

Identification of dystrophin isoforms in Torpedo postsynaptic membranes

IDENTIFICATION OF DYSTROPHIN ISOFORMS IN TORPEDO POSTSYNAPTIC MEMBRANES A. CARTAUD( ! ), M.A. LUDOSKY( 1 ), F.STETZKOWSKI-MARDEN( 1 ), F. TOME(2), T. KHURANA(3) and J. CARTAUD( I ). (1) Ins~i~u~ J. Monod. U n i v e r s i ~ Paris VII, Paris, France. (2) INSERM U1.,53, Paris. France and (3) Hov,,arci Hughes Medical Inst i tute, Boston, USA. Dystrophin is a cytoskeletal protein of the spectrin superfamily localized beneath the sarcolemma oF skeletal muscles. A homologous protein to dystrophin and sharing the same MW -the dystrophin related protein or DRP = h~o been recently identified and have a broader tissue distribution. Dystrophin or a closely related protein has also been identified at postsynaptic sites in synapses of the central and peripheric nervous system (I) as well as in Toroedo electric tissue (2.3). Despite the known amino acid sequence of these proteins, their function remains unclear, in particular at the synapse. The structural properties of the Torpedo electrocyte. in particular its dense cholinergic innervation allowed us to investigate the immunological properties of the synaptic dystrophin and the possible interactions of this protein with other components of the postsynaptic membrane. Immunofluorescence and immunoblotting experiments using various antibodies directed either against human dystrophin, DRP or Toroedo dystrophin suggest that authentic dystrophin is the major isoform at the electromotor synapse whereas DRP. is the major isoform present at the neuromuscular junction (in mammals and Toroedo). The presence of dystrophin at an early stage of development of TorDedo embryos suggests that it may be critically involved in interactions with other components of the membrane during synaptogenesis.