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Neuron-glia cell adhesion molecule (Ng-CAM) has previously been shown to be present exclusively on neurons and to mediate adhesion between neuronal membranes and glial cells. In the present study, its chain structure, binding functions, and relation to N-CAM (the other known CAM on neurons) were investigated further. Three polypeptide components of chicken Ng-CAM (Mr 200,000, 135,000, and 80,000) have been isolated. By using specific antisera against each component, the Mr 135,000 and Mr 80,000 components were found to cross-react antigenically with the Mr 200,000 component but not with each other. The conclusion that the Mr 135,000 and 80,000 components are structurally related to different regions of the Mr 200,000 component was further supported by the finding that 32P could be incorporated in vitro into the Mr 200,000 and 80,000 components but not into the Mr 135,000 component. Ng-CAM appears to be involved in both neuron-glia adhesion and neuron-neuron adhesion by distinguishable mechanisms that appear to involve different sites or conformations of the molecule. Polyclonal antibodies and a monoclonal antibody against Ng-CAM both inhibited adhesion between glia and neurons derived from brain, cerebellum, and retina. In contrast, antibodies against N-CAM (which inhibit neuron-neuron adhesion) did not inhibit neuron-glia adhesion. These findings confirm the proposed function of Ng-CAM in neuron-glia adhesion. In addition, however, Ng-CAM was found to be involved directly or indirectly in neuron-neuron adhesion. Non-cross-reactive polyclonal anti-Ng-CAM and anti-N-CAM antibodies each inhibited the aggregation of neurons from whole brain and cerebellum and the inhibition was greater when both antibodies were present together. In contrast, monoclonal anti-Ng-CAM antibodies were found that inhibited neuron-glia adhesion but did not inhibit neuronal cell aggregation. The amount of Ng-CAM expressed on neurons was not directly predictive of the effect of anti-Ng-CAM antibodies on their homotypic aggregation. Although Ng-CAM and N-CAM can be expressed simultaneously on individual neurons, the ratio of N-CAM to Ng-CAM ranged from 1.5 for cerebellar cells to 10.0 for retinal cells. While, as expected, retinal cell aggregation was inhibitable only by anti-N-CAM, cerebellar cells, which expressed at least as much Ng-CAM as brain cells, showed significantly less inhibition by anti-Ng-CAM antibodies. These findings raise the possibility that Ng-CAM may actually interact with N-CAM to yield non-linear effects. That Ng-CAM and N-CAM may function differently in vivo was suggested by their distribution in sections of brain regions. Within the cerebellum, for example, immunofluorescent anti-N-CAM staining was relatively uniform in all layers; in contrast anti-Ng-CAM staining was absent on dividing external granule cells and was present in greatest abundance on processes of post-mitotic migratory cells in the molecular layer. These observations are consistent with the hypothesis that Ng-CAM mediates neuron-glia adhesion and is thereby also involved in neuronal migration along radial glial cells.

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N-CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti-Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'-phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.

Previous studies in this laboratory have led to the identification of the neural cell adhesion molecule, N-CAM, a homophilic ligand that mediates adhesion between neurons as well as between neurons and striated muscle precursors. By means of a similar immunological approach but with different assays, we have now identified a cell adhesion molecule on neurons (Ng-CAM) that mediates the heterotypic adhesion between neuronal membranes and glial cells. In this paper, we compare certain aspects of the structure and function of Ng-CAM and embryonic N-CAM from the chicken. Ng-CAM was localized by specific antibodies on neurons but not on glia, and double-staining methods showed that individual neurons contained both Ng-CAM and N-CAM. Embryonic Ng-CAM migrates primarily as a single component of Mr 135,000; its apparent Mr shifted to 127,000 after neuraminidase treatment. In contrast, the embryonic form of N-CAM migrates on NaDodSO4/polyacrylamide gels in the apparent Mr range of 200,000-250,000; after neuraminidase treatment, N-CAM migrates as two components of Mr 170,000 and Mr 140,000. Although both Ng-CAM and N-CAM have calcium-independent binding mechanisms, immunologically based cell adhesion assays suggested that they have different specificities in mediating cell adhesion. Whereas 0.25 micrograms of Ng-CAM partially neutralized the ability of 0.5 mg of polyspecific antineural Fab' fragments to inhibit the heterotypic binding of neuronal membrane vesicles to glial cells and larger amounts of Ng-CAM completely neutralized this inhibition, 20 micrograms of N-CAM had no neutralization activity in this assay. Reciprocally, 0.25 micrograms of N-CAM partially neutralized the ability of 0.5 mg of the same Fab' fragments to inhibit the direct homotypic aggregation of neuronal cells, but 20 micrograms of Ng-CAM had no detectable activity. Although peptide maps of the two cell adhesion molecules differed considerably and despite the differences in binding specificity of these molecules, two independently derived monoclonal antibodies were found to crossreact with both Ng-CAM and N-CAM. Therefore, these different neuronal cell adhesion molecules with distinct binding specificities share at least one antigenic determinant, raising the possibility that they arose from a common evolutionary precursor.

Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis.

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50-fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron-glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.

This study represents a global survey of the times of the first appearance of the neuron-glia cell adhesion molecule (Ng-CAM) in various regions and on particular cells of the chick embryonic nervous system. Ng-CAM, originally characterized by means of an in vitro binding assay between glial cells and brain membrane vesicles, first appears in development at the surface of early postmitotic neurons. By 3 d in the chick embryo, the first neurons detected by antibodies to Ng-CAM are located in the ventral neural tube; these precursors of motor neurons emit well-stained fibers to the periphery. To identify locations of appearance of Ng-CAM in the peripheral nervous system (PNS), we used a monoclonal antibody called NC-1 that is specific for neural crest cells in early embryos to show the presence of numerous crest cells in the neuritic outgrowth from the neural tube; neither these crest cells nor those in ganglion rudiments bound anti-Ng-CAM antibodies. The earliest neurons in the PNS stained by anti-Ng-CAM appeared by 4 d of development in the cranial ganglia. At later stages and progressively, all the neurons and neurities of the PNS were found to contain Ng-CAM both in vitro and in vivo. Many central nervous system (CNS) neurons also showed Ng-CAM at these later stages, but in the CNS, the molecule was mostly associated with neuronal processes (mainly axons) rather than with cell bodies; this regional distribution at the neuronal cell surface is an example of polarity modulation. In contrast to the neural cell adhesion molecule and the liver cell adhesion molecule, both of which are found very early in derivatives of more than one germ layer, Ng-CAM is expressed only on neurons of the CNS and the PNS during the later epoch of development concerned with neural histogenesis. Ng-CAM is thus a specific differentiation product of neuroectoderm. Ng-CAM was found on developing neurons at approximately the same time that neurofilaments first appear, times at which glial cells are still undergoing differentiation from neuroepithelial precursors. The present findings and those of previous studies suggest that together the neural cell adhesion molecule and Ng-CAM mediate specific cellular interactions during the formation of neuronal networks by means of modulation events that govern their prevalence and polarity on neuronal cell surfaces.

The sequential appearance of the extracellular matrix (ECM) protein, cytotactin, was examined during development of the chicken embryo by immunohistochemical techniques. Although cytotactin was identified as a molecule that mediates glia-neuron interactions, preliminary immunohistochemical localization of the molecule suggested that it was an ECM protein with a widespread but nonetheless more restricted distribution than either fibronectin or laminin. In the present study, it was found that cytotactin is first present in the gastrulating chicken embryo. It appears later in the basement membrane of the developing neural tube and notochord in a temporal sequence beginning in the cephalic regions and proceeding caudally. Between 2 and 3 d of development, the molecule is present at high levels in the early neural crest pathways (surrounding the neural tube and somites) but, in contrast to fibronectin and laminin, is not found in the lateral plate mesoderm or ectoderm. At later times, cytotactin is expressed extensively in the central nervous system, in lesser amounts in the peripheral nervous system, and in a number of nonneural sites, most prominently in all smooth muscles and in basement membranes of lung and kidney. Cytotactin appears in adult tissues with distributions that are similar to those seen in embryonic tissues. The findings raise the possibility that certain ECM proteins contribute to pattern formation in embryogenesis as a result of their restricted expression in a spatiotemporally regulated fashion at some sites but not at others.

Immunocytochemical methods were used to show that Ng-CAM (the neuron-glia cell adhesion molecule), N-CAM (the neural cell adhesion molecule), and the extracellular matrix protein cytotactin are highly concentrated at nodes of Ranvier of the adult chicken and mouse. In contrast, unmyelinated axonal fibers were uniformly stained by specific antibodies to both CAMs but not by antibodies to cytotactin. Ultrastructural immunogold techniques indicated that both N-CAM and Ng-CAM were enriched in the nodal axoplasm and axolemma of myelinated fibers as well as within the nodal regions of the myelinating Schwann cell. At embryonic day 14, before myelination had occurred, small-caliber fibers of chick embryos showed periodic coincident accumulations of the two CAMs but not of cytotactin, with faint labeling in the axonal regions between accumulations. Cytotactin was found on Schwann cells and in connective tissue. By embryonic day 18, nodal accumulations of CAMs were first observed in a few medium- and large-caliber fibers. Immunoblot analyses indicated that embryonic to adult conversion of N-CAM and a progressive decrease in the amount of Ng-CAM and N-CAM occurred while nodes were forming. Sciatic nerves of mouse mutants with defects in cell interactions showed abnormalities in the distribution patterns and amount of Ng-CAM, N-CAM, and cytotactin that were consistent with the known morphological nodal disorders. In trembler (+/Tr), intense staining for both CAMs appeared all along the fibers and the amounts of N-CAM in the sciatic nerve were found to be increased. In mice with motor endplate disease (med/med), Ng-CAM and N-CAM, but not cytotactin, were localized in the widened nodes. Both trembler and med/med Schwann cells stained intensely for cytotactin, in contrast to normal Schwann cells which stained only slightly. All of these findings are consistent with the hypothesis that surface modulation of neuronal CAMs mediated by signals shared between neurons and glia may be necessary for establishing and maintaining the nodes of Ranvier.

An extracellular matrix protein, cytotactin, with widespread tissue distribution has been identified, isolated, and partially characterized. Cytotactin mediates glia-neuron adhesion in vitro, but unlike Ng-CAM, the neuron-glia cell-adhesion molecule, it is absent from neurons. Cytotactin was isolated from 14-day embryonic chicken brains as structurally related polypeptides of Mr 220,000, 200,000, and 190,000. These polypeptides were efficiently extracted in the absence of detergent and appeared to be disulfide-linked into higher polymers. Immunofluorescence staining with specific antibodies indicated that cytotactin is found in extracellular spaces and in basement membranes of a variety of non-neural tissues including smooth muscle, lung, and kidney. In the cerebellum, it appears on glial end-feet, on Bergmann glial fibers, and in extracellular spaces. The molecule is synthesized by glia and cells from smooth muscle, lung, and kidney. It is found at the surface of glia in culture in a cell-associated fibrillar pattern. A survey of the times and sites of its appearance during embryogenesis is consistent with the hypothesis that cytotactin is a cell-substrate adhesion molecule that may mediate cell migration in a site-restricted fashion.