The cleavage of HuR interferes with its transportin-2-mediated nuclear import and promotes muscle fiber formation.

Although the function of posttranscriptional processes in regulating the expression of genes involved in muscle fiber formation (myogenesis) is well accepted, the mechanisms by which these effects are mediated remain elusive. Here, we uncover such a mechanism and show that during myogenesis, a fraction of the posttranscriptional regulator human antigen R (HuR) is cleaved in a caspase-dependent manner in both cell culture and animal models. Disruption of caspase activity in cultured myoblasts or knocking out the caspase-3 gene in mice significantly reduced HuR cleavage and the cytoplasmic accumulation of HuR in muscle fibers. The non-cleavable isoform of HuR, HuRD226A, failed to reestablish the myogenic potential of HuR-depleted myoblasts. HuR cleavage generates two fragments: HuR-cleavage product 1 (HuR-CP1) (24 kDa) and HuR-CP2 (8 kDa). Here, we show that one of these fragments (HuR-CP1) binds to the HuR import factor transportin-2 (TRN2) allowing HuR to accumulate in the cytoplasm. As this cytoplasmic accumulation is required for the promyogenic function of HuR, our data support a model, whereby during the transition phase from myoblasts to myotubes, a proportion of HuR is cleaved to generate HuR-CP1. By interfering with the TRN2-mediated import of HuR, this CP helps non-cleaved HuR accumulate in the cytoplasm thus promoting myogenesis.

The utrophin A 5'-UTR drives cap-independent translation exclusively in skeletal muscles of transgenic mice and interacts with eEF1A2.

The molecular mechanisms regulating expression of utrophin A are of therapeutic interest since upregulating its expression at the sarcolemma can compensate for the lack of dystrophin in animal models of Duchenne Muscular Dystrophy (DMD). The 5'-UTR of utrophin A has been previously shown to drive cap-independent internal ribosome entry site (IRES)-mediated translation in response to muscle regeneration and glucocorticoid treatment. To determine whether the utrophin A IRES displays tissue specific activity, we generated transgenic mice harboring control (CMV/betaGAL/CAT) or utrophin A 5'-UTR (CMV/betaGAL/UtrA/CAT) bicistronic reporter transgenes. Examination of multiple tissues from two CMV/betaGAL/UtrA/CAT lines revealed that the utrophin A 5'-UTR drives cap-independent translation of the reporter gene exclusively in skeletal muscles and no other examined tissues. This expression pattern suggested that skeletal muscle-specific factors are involved in IRES-mediated translation of utrophin A. We performed RNA-affinity chromatography experiments combined with mass spectrometry to identify trans-factors that bind the utrophin A 5'-UTR and identified eukaryotic elongation factor 1A2 (eEF1A2). UV-crosslinking experiments confirmed the specificity of this interaction. Regions of the utrophin A 5'-UTR that bound eEF1A2 also mediated cap-independent translation in C2C12 muscle cells. Cultured cells lacking eEF1A2 had reduced IRES activity compared with cells overexpressing eEF1A2. Together, these results suggest an important role for eEF1A2 in driving cap-independent translation of utrophin A in skeletal muscle. The trans-factors and signaling pathways driving skeletal-muscle specific IRES-mediated translation of utrophin A could provide unique targets for developing pharmacological-based DMD therapies.

Large-scale mRNA analysis of female skeletal muscles during 60 days of bed rest with and without exercise or dietary protein supplementation as countermeasures.

Microgravity has a dramatic impact on human physiology, illustrated in particular, with skeletal muscle impairment. A thorough understanding of the mechanisms leading to loss of muscle mass and structural disorders is necessary for defining efficient clinical and spaceflight countermeasures. We investigated the effects of long-term bed rest on the transcriptome of soleus (SOL) and vastus lateralis (VL) muscles in healthy women (BRC group, n = 8), and the potential beneficial impact of protein supplementation (BRN group, n = 8) and of a combined resistance and aerobic training (BRE group, n = 8). Gene expression profiles were obtained using a customized microarray containing 6,681 muscles-relevant genes. A two-class statistical analysis was applied on 2,103 genes with consolidated expression in BRC, BRN, and BRE groups. We identified 472 and 207 mRNAs whose expression was modified in SOL and VL from BRC group, respectively. Further clustering analysis, identifying relevant biological mechanisms and pathways, reported five main subclusters. Three are composed of upregulated mRNAs involved mainly in nucleic acid and protein metabolism, and two made up of downregulated transcripts encoding components involved in energy metabolism. Exercise countermeasure demonstrated drastic compensatory effects, decreasing the number of differentially expressed mRNAs by 89 and 96% in SOL and VL, respectively. In contrast, nutrition countermeasure had moderate effects and decreased the number of differentially-expressed transcripts by 40 and 25% in SOL and VL. Together, these data present a systematic, global and comprehensive view of the adaptive response of female muscle to long-term atrophy.

Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events.

In addition to showing differences in the levels of contractile proteins and metabolic enzymes, fast and slow muscles also differ in their expression profile of structural and synaptic proteins. Because utrophin is a structural protein expressed at the neuromuscular junction, we hypothesize that its expression may be different between fast and slow muscles. Western blots showed that, compared with fast extensor digitorum longus (EDL) muscles, slow soleus muscles contain significantly more utrophin. Quantitative RT-PCR revealed that this difference is accompanied by a parallel increase in the expression of utrophin transcripts. Interestingly, the higher levels of utrophin and its mRNA appear to occur in extrasynaptic regions of muscle fibers as shown by immunofluorescence and in situ hybridization experiments. Furthermore, nuclear run-on assays showed that the rate of transcription of the utrophin gene was nearly identical between EDL and soleus muscles, indicating that increased mRNA stability accounts for the higher levels of utrophin in slow muscles. Direct plasmid injections of reporter gene constructs showed that cis-acting elements contained within the utrophin 3'-untranslated region (3'-UTR) confer greater stability to chimeric LacZ transcripts in soleus muscles. Finally, we observed a clear difference between EDL and soleus muscles in the abundance of RNA-binding proteins interacting with the utrophin 3'-UTR. Together, these findings highlight the contribution of posttranscriptional events in regulating the expression of utrophin in muscle.

Distinct regions in the 3' untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells.

In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3' untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3'UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3'UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients.

Denervation enhances the physiological effects of the K(ATP) channel during fatigue in EDL and soleus muscle.

The objective was to determine whether denervation reduces or enhances the physiological effects of the K(ATP) channel during fatigue in mouse extensor digitorum longus (EDL) and soleus muscle. For this, we measured the effects of 100 microM of pinacidil, a channel opener, and of 10 microM of glibenclamide, a channel blocker, in denervated muscles and compared the data to those observed in innervated muscles from the study of Matar et al. (Matar W, Nosek TM, Wong D, and Renaud JM. Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during fatigue in skeletal muscle. Am J Physiol Cell Physiol 278: C404-C416, 2000). Pinacidil increased the (86)Rb(+) fractional loss during fatigue, and this effect was 2.6- to 3.4-fold greater in denervated than innervated muscle. Pinacidil also increased the rate of fatigue; for EDL the effect was 2.5-fold greater in denervated than innervated muscle, whereas for soleus the difference was 8.6-fold. A major effect of glibenclamide was an increase in resting tension during fatigue, which was for the EDL and soleus muscle 2.7- and 1.9-fold greater, respectively, in denervated than innervated muscle. A second major effect of glibenclamide was a reduced capacity to recover force after fatigue, an effect observed only in denervated muscle. We therefore suggest that the physiological effects of the K(ATP) channel are enhanced after denervation.

Role of intronic E- and N-box motifs in the transcriptional induction of the acetylcholinesterase gene during myogenic differentiation.

In this study, we examined whether an intronic N-box motif is involved in the expression of acetylcholinesterase (AChE) during myogenesis. We determined that AChE transcripts are barely detectable in cultured myoblasts and that their levels increase dramatically in myotubes. Nuclear run-on assays revealed that this increase was accompanied by a parallel induction in the transcriptional activity of the AChE gene. These changes in transcription were also observed in transfection experiments using AChE promoter-reporter gene constructs. Mutation of the intronic N-box at position +755 base pairs (bp) reduced by more than 70% expression of the reporter gene in myotubes. Disruption of an adjacent E-box, at position +767 bp, also reduced expression of the reporter gene following myogenic differentiation. Co-transfection experiments using AChE promoter-reporter gene constructs and a myogenin expression vector showed that expression of this regulatory factor increased expression of the reporter gene in myotubes. Although the AChE promoter contains multiple E-boxes, mutation of this intronic one was sufficient to prevent the myogenin-induced increase in reporter gene expression. Together, these results indicate that changes in AChE gene transcription occur during myogenesis and highlight the contribution of the intronic N- and E-box motifs in the developmental regulation of the AChE gene in skeletal muscle.

A novel mechanism for modulating synaptic gene expression: differential localization of alpha-dystrobrevin transcripts in skeletal muscle.

Alpha-dystrobrevin is a dystrophin-related and -associated protein that is involved in synapse maturation and is required for normal muscle function. There are three protein isoforms in skeletal muscle, alpha-dystrobrevin-1, -2, and -3 that are encoded by the single alpha-dystrobrevin gene. To understand the role of these proteins in muscle we have investigated the localisation and transcript distribution of the different alpha-dystrobrevin isoforms. Alpha-dystrobrevin-1 and -2 are concentrated at the neuromuscular junction and are both recruited into agrin-induced acetylcholine receptor clusters in cultured myotubes. We also demonstrate that all alpha-dystrobrevin mRNAs are transcribed from a single promoter in skeletal muscle. However, only transcripts encoding alpha-dystrobrevin-1 are preferentially accumulated at postsynaptic sites. These data suggest that the synaptic accumulation of alpha-dystrobrevin-1 mRNA occurs posttranscriptionally, identifying a novel mechanism for synaptic gene expression. Taken together, these results indicate that different isoforms possess distinct roles in synapse formation and possibly in the pathogenesis of muscular dystrophy.

Molecular mechanisms underlying the activity-linked alterations in acetylcholinesterase mRNAs in developing versus adult rat skeletal muscles.

The molecular mechanisms underlying the activity-linked plasticity of acetylcholinesterase (AChE) mRNA levels in mammalian skeletal muscle have yet to be established. Here, we demonstrate that denervation of adult muscle induces a dramatic (up to 90%) and rapid (within 24 h) decrease in the abundance of AChE mRNAs. By contrast, denervation of 14-day-old rats leads to a significantly less pronounced reduction (50% of control) in the expression of AChE mRNAs. Assessment of the transcriptional activity of the AChE gene reveals that it remains essentially unchanged in adult denervated muscles, whereas it displays an approximately two- to three-fold increase (p < 0.05) in denervated muscles from 2- to 14-day-old rats. In addition, we observed a higher rate of degradation of in vitro transcribed AChE mRNAs upon incubation with protein extracts from denervated muscles. Finally, UV-crosslinking experiments reveal that denervation increases the abundance of RNA-protein interactions in the 3' untranslated region of AChE transcripts. Taken together, these data suggest that the abundance of AChE transcripts in mature muscles is controlled primarily via posttranscriptional regulatory mechanisms, whereas in neo- and postnatal muscles, both transcriptional and posttranscriptional regulation appears critical in dictating AChE mRNA levels. Accordingly, the activity-linked transcriptional regulation of the AChE gene appears to demonstrate a high level of plasticity during muscle development when maturation of the neuromuscular junctions is still occurring.

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse.

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.

Myotubes originating from single fast and slow satellite cells display similar patterns of AChE expression.

Slow- and fast-contracting skeletal muscles of both rats and mice display significant differences in their patterns of acetylcholinesterase (AChE) expression. Although neural influences are known to account for a large proportion of these differences, intrinsic variations between fast and slow myogenic precursor cells have been implicated. In the present study, we have capitalized on the use of Immorto transgenic mice to obtain single myogenic precursor cells isolated from either slow or fast muscle fibers and determined whether these cells generated myotubes that produced distinct patterns of AChE expression as observed in vivo between slow and fast muscles. These two myotube populations displayed similar cell-associated and secreted AChE enzyme activity as well as comparable levels of AChE transcripts. Both myotube populations also expressed nearly identical molecular form profiles. By contrast, AChE activity and transcript levels were approximately two- and fivefold greater in fast skeletal muscles compared with slow ones. Together, these findings indicate that differences in AChE expression between fast and slow muscles are not due to inherent differences in myogenic precursor cells, thereby suggesting that other factors, such as innervation, play a predominant role in establishing the distinct patterns of AChE expression in these muscle types.

Stability and secretion of acetylcholinesterase forms in skeletal muscle cells.

Muscle cells express a distinct splice variant of acetylcholinesterase (AChE(T)), but the specific mechanisms governing this restricted expression remain unclear. In these cells, a fraction of AChE subunits is associated with a triple helical collagen, ColQ, each strand of which can recruit a tetramer of AChE(T). In the present study, we examined the expression of the various splice variants of AChE by transfection in the mouse C2C12 myogenic cells in vitro, as well as in vivo by injecting plasmid DNA directly into tibialis anterior muscles of mice and rats. Surprisingly, we found that transfection with an ACHE(H) cDNA, generating a glycophosphatidylinositol-anchored enzyme species, produced much more activity than transfection with AChE(T) cDNA in both C2C12 cells and in vivo. This indicates that the exclusive expression of AChE(T) in mature muscle is governed by specific splicing. Interaction of AChE(T) subunits with the complete collagen tail ColQ increased enzyme activity in cultured cells, as well as in muscle fibers in vivo. Truncated ColQ subunits, presenting more or less extensive C-terminal deletions, also increased AChE activity and secretion in C2C12 cells, although the triple helix could not form in the case of the larger deletion. This suggests that heteromeric associations are stabilized compared with isolated AChE(T) subunits. Coinjections of AChE(T) and ColQ resulted in the production and secretion of asymmetric forms, indicating that assembly, processing, and externalization of these molecules can occur outside the junctional region of muscle fibers and hence does not require the specialized junctional Golgi apparatus.

Expression of the utrophin gene during myogenic differentiation.

The process of myogenic differentiation is known to be accompanied by large increases ( approximately 10-fold) in the expression of genes encoding cytoskeletal and membrane proteins including dystrophin and the acetylcholine receptor (AChR) subunits, via the effects of transcription factors belonging to the MyoD family. Since in skeletal muscle (i) utrophin is a synaptic homolog to dystrophin, and (ii) the utrophin promoter contains an E-box, we examined, in the present study, expression of the utrophin gene during myogenic differentiation using the mouse C2 muscle cell line. We observed that in comparison to myoblasts, the levels of utrophin and its transcript were approximately 2-fold higher in differentiated myotubes. In order to address whether a greater rate of transcription contributed to the elevated levels of utrophin transcripts, we performed nuclear run-on assays. In these studies we determined that the rate of transcription of the utrophin gene was approximately 2-fold greater in myotubes as compared to myoblasts. Finally, we examined the stability of utrophin mRNAs in muscle cultures by two separate methods: following transcription blockade with actinomycin D and by pulse-chase experiments. Under these conditions, we determined that the half-life of utrophin mRNAs in myoblasts was approximately 20 h and that it remained largely unaffected during myogenic differentiation. Altogether, these results show that in comparison to other synaptic proteins and to dystrophin, expression of the utrophin gene is only moderately increased during myogenic differentiation.

An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers.

mRNAs encoding acetylcholinesterase (AChE; EC 3.1.1.7) are highly concentrated within the postsynaptic sarcoplasm of adult skeletal muscle fibers, where their expression is markedly influenced by nerve-evoked electrical activity and trophic factors. To determine whether transcriptional regulatory mechanisms account for the synaptic accumulation of AChE transcripts at the mammalian neuromuscular synapse, we cloned a 5.3-kb DNA fragment that contained the 5' regulatory region of the rat AChE gene and generated several constructs in which AChE promoter fragments were placed upstream of the reporter gene lacZ and a nuclear localization signal (nls). Using a recently described transient expression assay system in intact skeletal muscle, we show that this AChE promoter fragment directs the synapse-specific expression of the reporter gene. Deletion analysis revealed that a 499-bp fragment located in the first intron of the AChE gene is essential for expression in muscle fibers. Further analysis showed that sequences contained within this intronic fragment were (i) functionally independent of position and orientation and (ii) inactive in hematopoietic cells. Disruption of an N-box motif located within this DNA fragment reduced by more than 80% the expression of the reporter gene in muscle fibers. In contrast, mutation of an adjacent CArG element had no effect on nlsLacZ expression. Taken together, these results indicate that a muscle-specific enhancer is present within the first intron of the AChE gene and that an intronic N-box is essential for the regulation of AChE along skeletal muscle fibers.

Discordant expression of utrophin and its transcript in human and mouse skeletal muscles.

In order to determine the mechanisms regulating utrophin expression in human skeletal muscle, we examined the expression and distribution of utrophin and its transcript in biopsies from normal subjects as well as from Duchenne muscular dystrophy (DMD) and polymyositis (PM) patients. We first determined by immunoblotting that in comparison to biopsies from normal subjects, utrophin levels were indeed higher in muscle samples from both DMD and PM patients as previously shown. By contrast, levels of utrophin mRNAs as determined by both RT-PCR assays and in situ hybridization, were identical in muscle samples obtained from normal subjects versus DMD and PM patients. In these experiments, we also noted that while utrophin transcripts had a clear tendency to accumulate within the postsynaptic sarcoplasm of normal human muscle fibers, the extent of synaptic accumulation was considerably less than that which we recently observed in mouse muscle fibers. The distribution of utrophin transcripts in synaptic and extrasynaptic compartments of muscle fibers obtained from DMD and PM patients was similar to that seen along muscle fibers from normal subjects. Finally, we also monitored expression of utrophin and its transcripts during regeneration of mouse muscle induced to degenerate by cardiotoxin injections. In these regenerating muscles, we observed by both immunoblotting and immunofluorescence, a large increase (4- to 7-fold) in the levels of utrophin. In agreement with our results obtained with human muscle, the increase in utrophin levels in regenerating mouse muscle was not accompanied by parallel changes in the abundance of utrophin transcripts. Taken together, these results indicate that the levels of utrophin and its transcript in muscle are discordantly regulated under certain conditions thereby highlighting the important contribution of post-transcriptional regulatory mechanisms in the control of utrophin levels in skeletal muscle fibers.

Induction of utrophin gene expression by heregulin in skeletal muscle cells: role of the N-box motif and GA binding protein.

The modulation of utrophin gene expression in muscle by the nerve-derived factor agrin plausibly involves the trophic factor ARIA/heregulin. Here we show that heregulin treatment of mouse and human cultured myotubes caused a approximately 2.5-fold increase in utrophin mRNA levels. Transient transfection experiments with utrophin promoter-reporter gene constructs showed that this increase resulted from an enhanced transcription of the utrophin gene. In the case of the nicotinic acetylcholine receptor delta and epsilon subunit genes, heregulin was previously reported to stimulate transcription via a conserved promoter element, the N-box, which binds the multimeric Ets-related transcription factor GA binding protein (GABP). Accordingly, site-directed mutagenesis of a single N-box motif in the utrophin gene promoter abolished the transcriptional response to heregulin. In addition, overexpression of heregulin, or of the two GABP subunits in cultured myotubes, caused an N-box-dependent increase of the utrophin promoter activity. In vivo, direct gene transfer into muscle confirmed that heregulin regulates utrophin gene expression. Finally, electrophoretic mobility shift assays and supershift experiments performed with muscle extracts revealed that the N-box of the utrophin promoter binds GABP. These findings suggest that the subsynaptic activation of transcription by heregulin via the N-box motif and GABP are conserved among genes expressed at the neuromuscular junction. Because utrophin can functionally compensate for the lack of dystrophin, the elucidation of the molecular mechanisms regulating utrophin gene transcription may ultimately lead to therapies based on utrophin expression throughout the muscle fibers of Duchenne muscular dystrophy patients.

Calcitonin gene-related peptide decreases expression of acetylcholinesterase in mammalian myotubes.

Nerve-derived trophic factors are known to modulate expression of acetylcholinesterase (AChE) in skeletal muscle fibers, yet the precise identity of these factors remains elusive. In the present study, we treated mouse C2 myotubes with calcitonin gene-related peptide (CGRP). Compared to non-treated myotubes, cell-associated AChE activity levels were decreased by approximately 60% after 48 h of treatment. A parallel reduction in AChE total protein levels was also observed as determined by Western blot analysis. The reduction in AChE activity was due to a decrease in the levels of the G1 molecular form and to an elimination of G1. By contrast, levels of secreted AChE remained unchanged following CGRP treatment. Finally, the overall decrease in AChE activity was accompanied by a reduction in AChE transcripts which could not be attributed to changes in the transcriptional rate of the ACHE gene.

Acetylcholinesterase gene expression in axotomized rat facial motoneurons is differentially regulated by neurotrophins: correlation with trkB and trkC mRNA levels and isoforms.

We examined the potential influences of muscle-derived neurotrophins on the acetylcholinesterase (AChE) gene expression of adult rat motoneurons. Seven days after facial nerve transection, both AChE mRNA and enzyme activity levels were markedly reduced in untreated and vehicle-treated facial motoneurons, suggesting positive regulation of motoneuron AChE expression by muscle-derived factors. Because skeletal muscle is a source of neurotrophin-3 (NT-3), NT-4/5, and BDNF, these neurotrophins were individually infused onto the proximal nerve stump for 7 d, beginning at the time of axotomy. The trkB ligands NT-4/5 and BDNF prevented the downregulation of AChE mRNA and enzymatic activity, as determined by in situ hybridization, biochemical assay, and histochemical visualization of enzyme activity. In contrast, NT-3 had limited effects, and NGF was without effect. Because motoneurons normally express both trkB and trkC receptors and the trkC ligand NT-3 is the most abundant muscle-derived neurotrophin, we investigated possible reasons for the limited effects of NT-3. In situ hybridization and reverse transcription-PCR both revealed a downregulation of trkC mRNA in axotomized motoneurons, which contrasted the upregulation of trkB expression. Furthermore, isoforms of trkC were detected carrying insertions within their kinase domains, known to limit certain trkC-mediated signal transduction pathways. Because the changes in trkB and trkC mRNA levels were not significantly altered by neurotrophin infusions, it is unlikely they were induced by loss of muscle-derived neurotrophins. These results demonstrate that NT-4/5 and BDNF stimulate AChE gene expression in motoneurons and support the concept that muscle-derived trkB ligands modulate the cholinergic phenotype of their innervating motoneurons.

Polarized sorting of nicotinic acetylcholine receptors to the postsynaptic membrane in Torpedo electrocyte.

Several regulatory mechanisms contribute to the accumulation and maintenance of high concentrations of acetylcholine receptors (AChR) at the postsynaptic membrane of the neuromuscular junction, including compartmentalized gene transcription, targeting, clustering and anchoring to the cytoskeleton. The targeting of the AChR to the postsynaptic membrane is likely to involve a polarized sorting in the exocytic pathway. In this work, we used the electrocyte of Torpedo marmorata electric organ to study the intracellular trafficking of neosynthesized AChR and its delivery to the postsynaptic membrane. Gradient centrifugation and immunoisolation techniques have led to the isolation of two populations of post-Golgi transport vesicles (PGVs) enriched in proteins of either the innervated (AChR) or non-innervated (Na,K-ATPase) membrane domains of the cell. Immunolabelling of these vesicles at the EM level disclosed that very few PGVs contained both proteins. In AChR-enriched vesicles, high sialylation of AchR molecules, an expected post-translational modification of proteins exiting the trans-Golgi network, and the presence of a marker of the exocytic pathway (Rab6p), indicate that these vesicles are carriers engaged in the Golgi-to-plasma membrane transport. These data suggest that AChR and Na,K-ATPase are sorted intracellularly most likely within the trans-Golgi network. Furthermore, EM analysis and immunogold-labelling experiments provided in situ evidence that the AChR-containing PGVs are conveyed to the postsynaptic membrane, possibly by a microtubule-dependent transport mechanism. Our data therefore provide the first evidence that the targeting of receptors for neurotransmitters to synaptic sites could be contributed by intracellular sorting and polarized delivery in the exocytic pathway.

Molecular mechanisms and putative signalling events controlling utrophin expression in mammalian skeletal muscle fibres.

The absence of full-length dystrophin molecules in skeletal muscle fibres results in the most severe form of muscular dystrophy, the Duchenne form (DMD). Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified. Although utrophin is expressed along the sarcolemma in developing, regenerating and DMD muscles, it nonetheless accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibres. Due to the high degree of sequence identity between dystrophin and utrophin, it has been previously suggested that utrophin could in fact functionally compensate for the lack of dystrophin. Recent studies using transgenic mouse model systems have directly tested this hypothesis and revealed that upregulation of utrophin throughout dystrophic muscle fibres represents indeed, a viable approach for the treatment of DMD. Current studies are therefore focusing on the elucidation of the various regulatory mechanisms presiding over expression of utrophin in muscle fibres in attempts to ultimately identify small molecules which could systematically increase utrophin levels in extrasynaptic compartments of dystrophic muscle fibres. This review presents some of the recent data relevant for our understanding of the transcriptional regulatory mechanisms involved in maintaining expression of utrophin at the neuromuscular junction. In addition, the contribution of specific cues originating from motoneurons and the putative involvement of signalling events are also discussed.