Abnormal reinnervation of skeletal muscle in a tenascin-C-deficient mouse.

The possible involvement of tenascin-C in the reinnervation of a skeletal muscle was investigated in the tenascin-C-deficient mouse (T-/-) produced by Saga et al. (1992; Genes Dev 6:1821-1831). The pattern of reinnervation, observed after denervation of the triangularis sterni muscle, differs in T-/- and wild-type muscles in several traits. Axonal growth and stability of terminal arbors are impaired in the T-/- muscle: Some axons in mutant muscles grow beyond their original targets and reinnervate other synaptic sites, which may become dually innervated. In contrast to wild type, polyinnervation increases with time after denervation in T-/- muscles and is still present 7 months after nerve crush. The expression of a tenascin-C mRNA product disappears between 1 and 2 months after nerve crush. Of interest is that this transcriptional regulation in T-/- muscles occurs when major alterations in the morphology of regenerating endings become obvious. These observations strongly implicate tenascin-C in the formation, maturation, and stabilization of the neuromuscular junction.

Regulated expression of the proteoglycan SPOCK in the neuromuscular system.

SPOCK is prevalent in developing synaptic fields of the central nervous system (Charbonnier et al., 2000. Mech. Dev. 90, 317-321). The expression of SPOCK during neuromuscular junction (NMJ) formation was compared to agrin and acetylcholine receptor (AChR) distribution. SPOCK is detected within the myogenic masses during the early steps of embryonic development, and distributed in the cytoplasm of myotubes before coclustering with AChRs. In the adult, SPOCK is present in axons and is highly expressed by Schwann cells. SPOCK altered expression pattern after nerve lesioning, or cholinergic transmission blockade, strongly indicate that its cellular distribution at the NMJ depends on innervation.

Myoneurin, a novel member of the BTB/POZ-zinc finger family highly expressed in human muscle.

Initially characterized as Drosophila developmental regulators, the BTB/POZ and zinc finger proteins (BTB/POZ-ZF) constitute a growing family of proteins with gene expression regulatory functions since they have been shown to be involved in both transcriptional activation and repression of various genes in a broad range of species, including mammals. Here we report the cloning of a novel human transcript, coding for a 68-kDa deduced BTB/POZ-ZF protein. This molecule, called myoneurin on the basis of its prevalent expression in the neuromuscular system, contains an amino-terminal BTB/POZ domain and eight tandemly repeated zinc-finger motifs of the C(2)H(2) type. The murine myoneurin, identified in the mouse embryo, is highly homologous to the human protein.

Genomic organization and expression of the ubiquitin-proteasome complex-associated protein Rbx1/ROC1/Hrt1.

Rbx1/ROC1/Hrt1 (Rbx1) has been recently shown to be involved in the regulation of protein turn-over. Here, we report the organization of the human Rbx1 gene, established by both a cloning and a functional genomics approach. The human gene, composed of five exons, encompasses 22.3 kb on chromosome 22q 13. The expression of Rbx1 transcripts (0.5 kb) appears developmentally regulated during mouse embryonic development and is prevalent in the adult mouse genital tract. A Genbank database search for Rbx1 related sequences in various species, from plants to mammals, is indicative of a high degree of evolutionary conservation in mouse rat and zebra fish and also, for the main functional part of the molecule, in other living species, although their gene structures can be significantly altered.

Distinct location and prevalence of alpha-, beta-catenins and gamma-catenin/plakoglobin in developing and denervated skeletal muscle.

We studied the distribution of alpha-catenin, beta-catenin and gamma-catenin/plakoglobin in developing, adult and denervated mouse skeletal muscle. During primary myogenesis, all three catenins present a subsarcolemmal distribution within primary myotubes. During secondary myogenesis they accumulate at myotube-myotube contacts. In contrast to the other catenins, gamma-catenin is strongly expressed in the sarcoplasm. In adult muscle, all three catenins are localized on the presynaptic elements of the neuromuscular junction. In denervated muscles, alpha- and beta-catenins are upregulated like N- and M-cadherin, while the levels of gamma-catenin/plakoglobin remain unchanged. The developmental changes in localization and regulation of alpha- and beta-catenins in muscle compared to gamma-catenin/plakoglobin are suggestive of a privileged association of alpha- and beta-catenins with N- and M-cadherins, while gamma-catenin/plakoglobin appears to be expressed quite independently and must assume a different role during myogenesis.

The peripheral nerve and the neuromuscular junction are affected in the tenascin-C-deficient mouse.

A thorough examination of the structure and plasticity of the neuromuscular system was performed in tenascin-C mutant mice deficient in tenascin-C. The study of the peripheral nerve revealed a number of abnormal features. In the motor nerve, numerous unmyelinated and myelinated fibers with degraded myelin were present. Schwann cell processes often enclosed degenerative terminals. Transgene (beta-galactosidase) expression analyzed at the ultrastructural level was found to be unequally distributed in the mutant's neuromuscular tissues. At the NMJ, preterminal disorganization was prevalent. Some axon terminals exhibited abnormal overgrowth. A surprising lack of beta-galactosidase expression at some cellular sites known to possess tenascin-C in wild type mice correlated best with marked changes in the cytoarchitecture of the peripheral nerve and NMJ. In some other -but not all- cellular sites which normally express the molecule, immunofluorescence analysis suggested the presence of significant but low levels of tenascin-C-like immunoreactivity together with beta-galactosidase expression. Messenger RNA detection by RT-PCR confirmed the presence of low amounts of tenascin-C mRNA in skeletal muscle suggesting that the mice deficient in tenascin-C are not complete knock-outs of this gene, but low-expression mutants. Following in vivo injections of botulinum type-A toxin, we observed a greatly reduced sprouting response of the motor nerves in tenascin-C mutant mice. We also observed that N-CAM and beta-catenin were overexpressed in the mutant. Our results suggest that tenascin-C is involved both in stabilization and in plasticity of the NMJ.

M-cadherin distribution in the mouse adult neuromuscular system suggests a role in muscle innervation.

M-cadherin belongs to the Ca(2+)-dependent cadherin family of cell adhesion molecules and was first isolated from a mouse muscle cell line cDNA library. It is specifically expressed in muscle tissue during development and is supposed to play an important role in secondary myogenesis. In the present study the expression of M-cadherin mRNA and protein and its localization were investigated in adult mouse skeletal muscle and peripheral nerve. The mRNA was abundant in embryonic legs from embryonic day (E)14 to E18. It remained expressed in new-born and adult muscles. In the adult muscle M-cadherin immunoreactivity was only detected at the neuromuscular junction, associated with perijunctional mononucleated cells and on intramuscular nerves. Peripheral nerves were also M-cadherin-positive. The molecule was found at the surface of myelinated nerve fibres where it was concentrated at the node of Ranvier. When a nerve was crushed and allowed to regenerate, M-cadherin was over-expressed at the site of nerve injury and in the distal stump. M-cadherin was also upregulated on the sarcolemma of denervated muscle fibres. Taken together, these observations point toward a much wider tissue distribution of M-cadherin than previously thought. M-cadherin might be involved not only in specific steps of myogenesis but also in some aspects of synaptogenesis, axon/Schwann cell interactions and node of Ranvier structural maintenance.

Mouse G2-GPI AChE is processed as a membrane-bound ectoenzyme in transfected mouse sarcoma cells but is not a homophilic adhesion molecule.

Acetylcholinesterase (AChE) is mainly involved in synaptic transmission by hydrolyzing acetylcholine in the synaptic cleft. It has been suggested that it could also be involved in other functions such as cell-cell adhesion. In this study, we have expressed mouse G2-GPI AChE at the membrane surface of S180 cells. We obtained a transfected cell line which permanently expresses high levels of AChE at the cell surface. However, transfected cells behave as single cells in culture. We performed cell aggregation and adhesion tests and found no significant aggregation or adhesion, which suggests that AChE is not a homophilic adhesion molecule.

M-cadherin localization in developing adult and regenerating mouse skeletal muscle: possible involvement in secondary myogenesis.

In this work, we investigated the distribution of the Ca(2+)-dependent cell adhesion molecule, M-cadherin, in mouse limb muscle during normal development and regeneration. Using two unrelated anti-M-cadherin peptide antibodies, we found scarce M-cadherin immunostaining during primary myogenesis (E12-E14) with no accumulation at areas of cell-cell contact. In contrast, the staining sharply increased in intensity at E16, remained high during secondary myogenesis (E16-P0) but disappeared soon after birth. During secondary myogenesis, M-cadherin was specifically accumulated at the characteristic sites of insertion of secondary myotubes in neighbouring primary myotubes. M-cadherin was also accumulated at the areas of contact between fusing secondary myoblasts and myotubes in vitro. In the adult normal and regenerating muscle, we did not detect M-cadherin accumulations at the surface of myofibres. All together, these observations suggest that M-cadherin is specifically involved in secondary myogenesis.

Is intercellular communication via gap junctions required for myoblast fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-beta-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.

N-cadherin expression in developing, adult and denervated chicken neuromuscular system: accumulations at both the neuromuscular junction and the node of Ranvier.

N-cadherin, a member of the Ca(2+)-dependent cell adhesion molecule family plays essential roles in morphogenesis and histogenesis. N-cadherin has been shown in vitro to promote myoblast fusion and neurite outgrowth. We report here the cellular localization of N-cadherin during development and regeneration of the chick neuromuscular system. N-cadherin was uniformly expressed along the surface of myoblasts and myotubes of E6 limb muscles. Later, as synaptogenesis and secondary myogenesis proceeded, N-cadherin expression was down-regulated and restricted to some large-diameter fibres, then to the areas of contact between few myofibres and subsequently disappeared by embryonic day 17, suggesting that this cadherin may be implicated predominantly in fusion of primary myoblasts and, at lower degree, of secondary myoblasts. The presence of N-cadherin in muscle during the period of nerve trunk ingrowth and its down-regulation after synaptogenesis suggests that this molecule might be implicated in both processes. N-cadherin became accumulated at the neuromuscular junction only a few days after the first synaptic contacts were established and remained at the adult neuromuscular junction, suggesting a role of this molecule in the stabilization of the mature neuromuscular junction. In sciatic nerve, the level of N-cadherin expression remained unchanged from hatching to adult life. N-cadherin was widely distributed on the surface of myelinated fibres and on myelinating Schwann cells: in addition, it was concentrated at the node of Ranvier. At the ultrastructural level, the molecule was detected inside, at the surface and in the basal lamina of Schwann cells and also associated with endoneurial collagen. These observations suggest a role of N-cadherin in the structuring and stabilization of the myelin sheaths. After nerve injury, N-cadherin continued to be expressed by proliferating Schwann cells in the distal stump providing a substratum for regenerating axons. N-cadherin reappeared at the surface of denervated muscle fibres without disappearing from the former synaptic sites. It was detected not only in the sarcoplasm and on sarcolemma of denervated muscle fibres, but also in the basal lamina and in the extracellular matrix. The reexpression of N-cadherin at the surface of denervated muscle fibres suggests a role for this molecule in muscle reinnervation. The presence of N-cadherin in basal lamina and its association with collagen fibres raise questions about the release of N-cadherin in the extracellular space and the existence of a putative heterophilic ligand for N-cadherin.

N-cadherin and N-CAM-mediated adhesion in development and regeneration of skeletal muscle.

In this review, the experimental evidence supporting the fact that the cell adhesion molecules N-CAM and N-cadherin are involved in myogenesis has been surveyed. In order to give access to the function of these molecules, a strategy of in vivo localization and in vitro perturbation of their adhesive function by interfering antibodies and peptides was applied. Both molecules are expressed at the surface of myogenic cells during myogenesis in vivo and in vitro. The blockade of the N-CAM adhesion function leads to a mild reduction of the rate of myoblast fusion, while the inhibition of the N-cadherin function induces a drastic inhibition of fusion suggesting that N-cadherin-mediated adhesion is a critical step in the process of myoblast fusion. Both molecules are re-expressed during muscle regeneration suggesting that adult myogenesis is under the control of the same adhesive systems as embryonic and foetal myogenesis.

N-cadherin and N-CAM in myoblast fusion: compared localisation and effect of blockade by peptides and antibodies.

The expression and distribution of two cell adhesion molecules, N-cadherin and N-CAM, at the surface of cultured leg muscle cells from 11-day-old chicken embryos were studied and compared. N-cadherin, which was expressed by fusing myoblasts, was down-regulated on old myotubes while N-CAM was still present. Both molecules, as viewed by confocal microscopy, appeared to have coaccumulated at the areas of contact between fusing myoblasts. However, immunogold electron microscopy did not reveal significant colocalization of N-cadherin and N-CAM, and their segregation after antibody-induced patching suggested the absence of direct interactions between N-cadherin and N-CAM. The role of the Ca2+ dependent cell adhesion molecule N-cadherin in myogenesis was investigated. Myoblast fusion was inhibited (1) with a synthetic peptide containing the H-A-V sequence and (2) with a monoclonal anti-N-cadherin antibody, demonstrating that N-cadherin-mediated cell adhesion is required for myoblast fusion. Under the same conditions no effect of anti-N-CAM antibodies was observed. Taken together these observations suggest that N-cadherin, acting independently from N-CAM, is a major cell adhesion molecule involved in embryonic myoblast fusion in vitro.

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes.

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.

Modulation of expression and cell surface distribution of N-CAM during myogenesis in vitro.

We have studied the modulation of expression and surface distribution of the molecular forms of the neural cell adhesion molecule (N-CAM) during myogenesis in vitro. We found one minor and two major N-CAM forms-180, 145 and 125 kDa respectively in primary cultures of mouse muscle cells. The 180 and 145 kDa forms were present in myoblasts before fusion. At fusion, total N-CAM increased with no 180 kDa polypeptide, but with a new 125 kDa form, together with the 145 kDa form. We determined the localization of N-CAM on the myotube surface and compared it to that of the acetylcholine receptor. N-CAM but not the receptor was found on the myoblast surface before fusion. Both proteins were uniformly distributed on the cell surface of early myotubes. Then, bright spots appeared, rapidly followed by the formation of clusters of both the acetylcholine receptor and N-CAM, at the time contractile activity was established. However these clusters were never colocalized, except after synapse formation. N-CAM clusters, but not acetylcholine receptor clusters, were dispersed following Nocodazole-induced microtubule depolymerization. We further observed that patching of N-CAM by divalent anti-N-CAM antibodies had no effect on acetylcholine receptor clusters. These results suggest that there is no mechanochemical link between the receptor and N-CAM. In myotubes, part of the 125 kDa form was released from the cell surface by the phosphatidylinositol phospholipase C. This phosphatidylinositol anchored form was mostly present outside the clusters where the 145 kDa form seems to be concentrated. Another pool of 125 kDa was insoluble in non-ionic detergent and was extracted by 0.1% SDS only. We suggest that the SDS extracted 125 kDa N-CAM is present in basal lamina. Thus, specific N-CAM forms with different interactions with basal lamina or cytoskeleton and cell surface distribution are induced during myogenesis and may be responsible for decisive modifications of cell-cell interactions involved in myoblast fusion and synaptogenesis.

Cell modulation of hydrophobic tailed 16S acetylcholinesterase by intracellular calcium in rat superior cervical ganglion neurons.

In primary cell cultures of rat superior cervical ganglia (SCG) the tailed asymmetric 16S molecular form of acetylcholinesterase (AChE) possesses hydrophilic (high-salt soluble, HSS) and hydrophobic (detergent extracted, DE) variants. Hydrophobic tailed acetylcholinesterase is associated with membranes through a glycolipid anchor. In the presence of tunicamycin, an antibiotic which inhibits protein glycosylation, the cellular amount of the hydrophobic DE 16S AChE is increased. Exposure of the cells to the calcium ionophore A 23187 leads to a decrease in DE 16S AChE and a correlated increase in hydrophilic HSS 16S AChE. These results suggest the existence of an endogenous processing of tailed AChE, transforming the hydrophobic variant into an hydrophilic one controlled through glycosylation and intracellular calcium.

Interaction of an organophosphate with a peripheral site on acetylcholinesterase.

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.

Interaction of a bisquaternary ammonium compound with the peripheral organophosphorus (POP) site on acetylcholinesterase.

O-ethyl-S (2 diisopropylaminoethyl) methyl phosphorothiolate (MPT) is an active site-directed inhibitor of acetylcholinesterase (AChE). The inhibition of mouse muscle AChE by MPT as well as the inhibition of its individual molecular forms do not proceed as simple irreversible bimolecular reactions. The insolubilization of AChE into a semisolid matrix allows to characterize, after dialysis of all unbound ligand, a partially reversible phase of the inhibition by MPT. These results can be explained in terms of two different modes of inhibition by MPT: the classical irreversible phosphorylation of the active site and an inhibition phase involving the reversible binding of MPT at a site peripheral to the active site, the peripheral organophosphorus site (POP-site). We now find that BW 284 C 51, a reversible specific inhibitor of AChE which protects the active site against irreversible inhibition by low MPT concentrations, can prevent the occurrence of the partially reversible inhibition phase. Hence, BW may bind to a peripheral site that either overlaps or is linked to the POP-site.