Neural cell adhesion molecule (NCAM) and myoblast fusion

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.     

Expression of the neural cell adhesion molecule NCAM in endocrine cells

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.     

A role for the neural cell adhesion molecule, NCAM, in myoblast interaction during myogenesis

The Ca2(+)-independent neural cell adhesion molecule, NCAM, is expressed by both nerve and muscle cells and has been shown to mediate both nerve-nerve and nerve-muscle cell interaction. A role for NCAM in muscle-muscle cell interaction has been proposed but not demonstrated. Here we report evidence that NCAM is expressed by embryonic chick muscle cells during in vitro development and functions together with Ca2(+)-dependent adhesion molecules in mediating myoblast interaction during the formation of multinucleate cells.     

Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling

The neural cell adhesion molecule (NCAM) participates in adhesion and neuritic outgrowth during nervous system development. In the adult brain, NCAM is considered to be involved in neuronal sprouting and synaptic remodeling. the NCAM concentration of brain tissue has proved to be a useful marker of these processes, especially when viewed in comparison with the concentration of a marker of mature synapses, e.g. D3-protein (SNAP-25) or synaptophysin. The present review focusses on studies of adult brain in which NCAM concentration estimates and NCAM/D3 ratios have been used to evaluate the rate of synaptic remodeling in brain damage and degenerative diseases.Special issue dedicated to Dr. Robert Balázs.     

Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 m thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make theee-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.     

Metalloprotease-induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180(1)) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM-transfected L-fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate-induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM-dependent neurite branching and outgrowth. Moreover, NCAM-dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease-induced ectodomain shedding of NCAM down-regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity.     

Induction of neural cell adhesion molecule (NCAM) in Xenopus embryos

Using a classical neural induction protocol (H. Spemann and H. Mangold (1924). Roux' Arch. Entwicklungsmech. Org. 123, 389-517), it has been demonstrated that the sustained presence of NCAM in Xenopus embryos, as detected by immunohistochemistry, was confined to the experimentally induced nervous system and the primary host nervous system. Furthermore, in vitro NCAM expression by dorsal blastopore lip and animal pole tissue was detected only when the two tissues were cultured in contact. These and other results show that readily detected and sustained levels of NCAM expression in Xenopus can be used as a marker for neural tissue and an early positive indicator that neural induction has occurred. They suggest that the observed levels of NCAM are a consequence of and not a prerequisite for induction. Using NCAM expression in vitro to determine the minimum time necessary for this induction to occur in vivo, it was found that NCAM was first detected in cultured animal pole that had been removed at stage 10.75 or later. Thus, an inductive step necessary and sufficient for stimulation of NCAM expression in animal pole tissues had not occurred or was reversible prior to the first 2 to 2.5 hr of gastrulation.     

Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.     

F3/contactin,a neuronal cell adhesion molecule implicated in axogenesis and myelination

A general feature of the cell adhesion molecules belonging to the immunoglobulin family (Ig-CAMs) is to display a modular structure that provides a framework for multiple binding sites for other recognition molecules. Among this family, F3/contactin is a glycan phosphatidyl-inositol (GPI)-anchored molecule expressed by neurons that displays the distinctiveness to exert heterophilic but no homophilic binding activities. The Ig domains of F3/contactin were shown to interact with the L1 family of Ig-CAMs, including L1, NrCAM, and neurofascin. Binding between F3/contactin and NrCAM is known to modulate axonal elongation of the cerebellar granule cells and to control sensory axon guidance. F3/contactin mediates neuron-glial contacts through its association with extracellular matrix components (tenascin-R, tenascin-C) and RPTPbeta/phosphacan, influencing axonal growth and fasciculation. Another major role of F3/contactin is to organize axonal subdomains at the node of Ranvier of myelinated fibers in interplay with other Ig-CAMs, through its binding with caspr/paranodin at paranodes and the voltage-gated sodium channels in the nodal region. The F3/contactin deficient mice display a severe ataxia correlated with defects in axonal and dendritic projections in the cerebellum. These mice also display defects in nerve influx conduction due to the disruption of the axo-glial contacts at paranodes. Finally, the recent identification of a Drosophila homologue of F3/contactin indicated that this family of GPI-anchored CAMs plays a conserved function in axonal insulation.     

The neural cell adhesion molecule NCAM regulates neuritogenesis by multiple mechanisms of interaction

The neural cell adhesion molecule NCAM and its glycosylation with polysialic acid (polySia) are crucially involved in proliferation, migration and differentiation of neural progenitors. Modification with polySia, homophilic and heterophilic interactions set the function of NCAM, but little is known on their interplay. We have shown recently that removal of polySia induces neuronal differentiation via heterophilic NCAM interactions at cell contacts between SH-SY5Y neuroblastoma cells. Here we analyze the additional impact of NCAM-positive fibroblasts as a ligand-presenting cellular environment, a model often used to demonstrate the neuritogenic effect of homophilic NCAM interactions. Native SH-SY5Y cells did not respond to interactions with fibroblast NCAM. However, after induction of neuronal differentiation by retinoic acid the previously ineffective NCAM signals activated extracellular signal-regulated kinase (ERK) and promoted neuritogenesis. Removal of polySia increased neuritogenesis in retinoic acid-treated cells additive to the NCAM substrate effect. The change in responsiveness to substrate NCAM was associated with a rearrangement of polysialylated NCAM away from its enrichment at homotypic cell-cell contacts and with the appearance of non-polysialylated NCAM, i.e. changes facilitating NCAM interactions with the substrate. Thus, heterophilic and homophilic NCAM interactions are integrated into the cell's response yet they have the capacity to independently trigger neuritogenesis. The actual occurrence of each of these interactions, however, depends on the cellular context, targeted cell surface presentation of NCAM and the dynamic regulation of its modification by polysialic acid. In summary, this study reveals how the complex interplay of NCAM interactions and polysialylation provides an elaborate system to regulate neuritogenesis.     

Differential expression of the neural cell adhesion molecule NCAM 140 in human pituitary tumors

We have analyzed the expression of the intracellular marker protein neuron specific enolase (NSE), synaptophysin (SYN) and of the cell surface marker NCAM (neural cell adhesion molecule) in both normal hypophysis and in pituitary adenomas in order to explore their potential use as diagnostic tools. All adenomas (4 prolactinomas, 3 growth hormone (GH) producing adenomas and 4 inactive adenomas) showed SYN and NSE immunoreactivity on tissue sections and this was confirmed by immunoblots. NCAM 140 (an isoform of NCAM with molecular mass 140 kDa) was detected by immunoblotting in normal human adenohypophysis, in all GH adenomas, and in three out of four inactive adenomas, but not in prolactinomas. Using highly sensitive techniques, NCAM immunoreactivity was observed by electron microscopy in all adenomas. These data indicate that NCAM 140 is a constituent of the cell surface of endocrine cells in both normal human adenohypophysis and its tumors. Since prolactinomas express very low levels of NCAM 140 compared to other hypophyseal tumors its virtual absence could be used for differential diagnosis. A combined analysis of NCAM, SYN and NSE could be useful to characterize inactive adenomas which are not immunoreactive for pituitary hormones and which may contain no or only low levels of the alpha chain of the glycoprotein hormones.     

Neural cell adhesion molecule (NCAM) association with PKCbeta2 via betaI spectrin is implicated in NCAM-mediated neurite outgrowth

In hippocampal neurons and transfected CHO cells, neural cell adhesion molecule (NCAM) 120, NCAM140, and NCAM180 form Triton X-100-insoluble complexes with betaI spectrin. Heteromeric spectrin (alphaIbetaI) binds to the intracellular domain of NCAM180, and isolated spectrin subunits bind to both NCAM180 and NCAM140, as does the betaI spectrin fragment encompassing second and third spectrin repeats (betaI2-3). In NCAM120-transfected cells, betaI spectrin is detectable predominantly in lipid rafts. Treatment of cells with methyl-beta-cyclodextrin disrupts the NCAM120-spectrin complex, implicating lipid rafts as a platform linking NCAM120 and spectrin. NCAM140/NCAM180-betaI spectrin complexes do not depend on raft integrity and are located both in rafts and raft-free membrane domains. PKCbeta2 forms detergent-insoluble complexes with NCAM140/NCAM180 and spectrin. Activation of NCAM enhances the formation of NCAM140/NCAM180-spectrin-PKCbeta2 complexes and results in their redistribution to lipid rafts. The complex is disrupted by the expression of dominant-negative betaI2-3, which impairs binding of spectrin to NCAM, implicating spectrin as the bridge between PKCbeta2 and NCAM140 or NCAM180. Redistribution of PKCbeta2 to NCAM-spectrin complexes is also blocked by a specific fibroblast growth factor receptor inhibitor. Furthermore, transfection with betaI2-3 inhibits NCAM-induced neurite outgrowth, showing that formation of the NCAM-spectrin-PKCbeta2 complex is necessary for NCAM-mediated neurite outgrowth.     

Metalloprotease‐induced ectodomain shedding of neural cell adhesion molecule (NCAM)

Transmembrane forms of neural cell adhesion molecule (NCAM140, NCAM180¹) are key regulators of neuronal development. The extracellular domain of NCAM can occur as a soluble protein in normal brain, and its levels are elevated in neuropsychiatric disorders, such as schizophrenia; however the mechanism of ectodomain release is obscure. Ectodomain shedding of NCAM140, releasing a fragment of 115 kD, was found to be induced in NCAM‐transfected L‐fibroblasts by the tyrosine phosphatase inhibitor pervanadate, but not phorbol esters. Pervanadate‐induced shedding was mediated by a disintegrin metalloprotease (ADAM), regulated by ERK1/2 MAP kinase. In primary cortical neurons, NCAM was shed at high levels, and the metalloprotease inhibitor GM6001 significantly increased NCAM‐dependent neurite branching and outgrowth. Moreover, NCAM‐dependent neurite outgrowth and branching were inhibited in neurons isolated from a transgenic mouse model of NCAM shedding. These results suggest that regulated metalloprotease‐induced ectodomain shedding of NCAM down‐regulates neurite branching and neurite outgrowth. Thus, increased levels of soluble NCAM in schizophrenic brain have the potential to impair neuronal connectivity. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

Although recent advances in broad-scale gene expression analysis have dramatically increased our knowledge of the repertoire of mRNAs present in multiple cell types, it has become increasingly clear that examination of the expression, localization, and associations of the encoded proteins will be critical for determining their functional significance. In particular, many signaling receptors, transducers, and effectors have been proposed to act in higher-order complexes associated with physically distinct areas of the plasma membrane. Adult muscle stem cells (satellite cells) must, upon injury, respond appropriately to a wide range of extracellular stimuli: the role of such signaling scaffolds is therefore a potentially important area of inquiry. To address this question, we first isolated detergent-resistant membrane fractions from primary satellite cells, then analyzed their component proteins using liquid chromatography-tandem mass spectrometry. Transmembrane and juxtamembrane components of adhesion-mediated signaling pathways made up the largest group of identified proteins; in particular, neural cell adhesion molecule (NCAM), a multifunctional cell-surface protein that has previously been associated with muscle regeneration, was significant. Immunohistochemical analysis revealed that not only is NCAM localized to discrete areas of the plasma membrane, it is also a very early marker of commitment to terminal differentiation. Using flow cytometry, we have sorted physically homogeneous myogenic cultures into proliferating and differentiating fractions based solely upon NCAM expression.     

Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion

Molecular force measurements quantified the impact of polysialylation on the adhesive properties both of membrane-bound neural cell adhesion molecule (NCAM) and of other proteins on the same membrane. These results show quantitatively that NCAM polysialylation increases the range and magnitude of intermembrane repulsion. The repulsion is sufficient to overwhelm both homophilic NCAM and cadherin attraction at physiological ionic strength, and it abrogates the protein-mediated intermembrane adhesion. The steric repulsion is ionic strength dependent and decreases substantially at high monovalent salt concentrations with a concomitant increase in the intermembrane attraction. The magnitude of the repulsion also depends on the amount of polysialic acid (PSA) on the membranes, and the PSA-dependent attenuation of cadherin adhesion increases with increasing PSA-NCAM:cadherin ratios. These findings agree qualitatively with independent reports based on cell adhesion studies and reveal the likely molecular mechanism by which NCAM polysialylation regulates cell adhesion and intermembrane space.     

Cell adhesion molecule (NCAM): Its role in the development and functioning of cultured hippocampal neurons

The dynamics of NCAM expression on the neuronal membranes in dissociated rat hippocampal cell culture during 1–12 days ofin vitro development were studied. Using immunocytochemistry and electron microscopy, quantitative estimation of NCAM re-distribution on the plasma membrane of the neurons in the course of their development and maturation was carried out. By means of computer simulation, localization of NCAM molecules on the membrane of cultured neurons was modeled. It was shown that changes in the level and pattern of NCAM expression are one of the possible mechanisms providing synaptic plasticity and learning and memory processes.     

Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.     

Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo

Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.     

Synaptic strength as a function of post- versus presynaptic expression of the neural cell adhesion molecule NCAM

To evaluate the contributions of the pre- versus postsynaptic expression of NCAM in regulation of synaptic efficacy, we cultured dissociated hippocampal cells from NCAM-deficient and wild-type mice in homo- and heterogenotypic combinations. Double recordings from synaptically coupled neurons maintained in heterogenotypic cocultures showed that synaptic strength of excitatory but not inhibitory synapses depended on expression of NCAM post- but not presynaptically. This correlated with higher levels of potentiation and synaptic coverage of NCAM-expressing neurons compared to NCAM-deficient neurons in heterogenotypic cocultures. Synaptic density was the same in homogenotypic cultures of NCAM-deficient and wild-type neurons as well as in heterogenotypic cocultures in which glutamate receptors were blocked. These observations indicate that the relative levels of postsynaptic NCAM expression control synaptic strength in an activity-dependent manner by regulating the number of synapses.