Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV- 1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin- 1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.     

Binding between the neural cell adhesion molecules axonin-1 and Nr- CAM/Bravo is involved in neuron-glia interaction

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.     

A homologue of the axonally secreted protein axonin-1 is an integral membrane protein of nerve fiber tracts involved in neurite fasciculation

Axonin-1 is a glycoprotein that is released from axons of cultured neurons (Stoeckli, E. T., P. F. Lemkin, T. B. Kuhn, M. A. Ruegg, M. Heller, and P. Sonderegger. 1989. Eur. J. Biochem. 180:249-258). It has recently been purified from the ocular vitreous fluid of the chicken embryo (Ruegg, M. A., E. T. Stoeckli, T. B. Kuhn, M. Heller, R. Zuellig, and P. Sonderegger. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:55-63). Immunohistochemistry localized axonin-1 prevalently in developing nerve fiber tracts. The presence of anti-axonin-1 Fab fragments during axon growth in vitro resulted in antibody binding to the axonal surfaces and in a marked perturbation of the fasciculation pattern. Hence, a fraction of axonin-1 is associated with axonal membranes and, by operational criteria, qualifies as a cell adhesion molecule. The major proportion of membrane-associated axonin-1 co-solubilized with the integral membrane proteins. By physico-chemical, immunological, and protein-chemical criteria, the integral membrane form was found to be highly similar to soluble axonin-1. In common with a number of other cell adhesion molecules, both soluble and membrane-bound axonin-1 express the L2/HNK-1 and the L3 epitopes. Radioactive pulse-chase and double-labeling experiments revealed that the released form was not derived from the membrane-bound form by shedding from the membrane surface, but directly secreted from an intracellular pool. Due to its high degree of similarity to the membrane-associated form and the presence of the L2/HNK-1 and L3 epitopes, reported to be ligands in adhesive cell interactions, adhesive properties are postulated for secreted axonin-1. As a soluble adhesive protein, it may function as a regulator of cell adhesion around its most likely site of secretion, the growth cone.     

Expression and function of cell adhesion molecules during neural crest migration

Neural crest cells are highly migratory cells that give rise to many derivatives including peripheral ganglia, craniofacial structures and melanocytes. Neural crest cells migrate along defined pathways to their target sites, interacting with each other and their environment as they migrate. Cell adhesion molecules are critical during this process. In this review we discuss the expression and function of cell adhesion molecules during the process of neural crest migration, in particular cadherins, integrins, members of the immunoglobulin superfamily of cell adhesion molecules, and the proteolytic enzymes that cleave these cell adhesion molecules. The expression and function of these cell adhesion molecules and proteases are compared across neural crest emigrating from different axial levels, and across different species of vertebrates.     

The L2/HNK-1 carbohydrate of neural cell adhesion molecules is involved in cell interactions

We investigated whether the L2/HNK-1 carbohydrate epitope, expressed by two unusual glycolipids and several neural adhesion molecules, including L1, neural cell adhesion molecule, J1, and the myelin-associated glycoprotein, is involved in adhesion. Monoclonal L2 antibodies, the L2/HNK-1-reactive, sulfate-3-glucuronyl residue carrying glycolipids (L2 glycolipid) and a tetrasaccharide derived from the L2 glycolipid (L2 tetrasaccharide) were added to microexplant cultures of early postnatal mouse cerebellum, and cell migration and process extension were monitored. On the substrate poly-D-lysine, Fab fragments of L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes and migration of cell bodies, but only L2 glycolipid and L2 tetrasaccharide reduced neurite outgrowth. On laminin, L2 antibodies, L2 glycolipid, and L2 tetrasaccharide inhibited outgrowth of astrocytic processes. Additionally, L2 glycolipid and L2 tetrasaccharide inhibited cell migration and neurite outgrowth. Several negatively charged glycolipids, lipids, and saccharides were tested for control and found to have no effect on outgrowth patterns, except for sulfatide and heparin, which modified outgrowth patterns in a similar fashion as L2 glycolipid and L2 tetrasaccharide. On astrocytes none of the tested compounds interfered with explant outgrowth. In short-term adhesion assays L2 glycolipid, sulfatide, and heparin inhibited adhesion of neural cells to laminin. L2 glycolipid and sulfatide interfered with neuron to astrocyte and astrocyte to astrocyte adhesion, but not with neuron-neuron adhesion. The most straightforward interpretation of these observations is that the L2/HNK-1 carbohydrate and the sulfated carbohydrates, sulfatide and heparin, act as ligands in cell adhesion.     

Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.

The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.     

Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1

The effects of L1-Fc and CHL1-Fc fusion proteins on neuronal survival were investigated. Cerebellar granule neurons of mouse and hippocampal neurons of rat embryo undergo apoptosis when cultured in serum-free medium. Treatment with chimeric proteins containing the extracellular domains of the neural adhesion molecules L1 or CHL1 fused to the Fc region of human immunoglobulin significantly enhanced the survival of neurons. Compared to the control, the percentage of surviving neurons increased about 60% and 45% with L1 and CHL1 fusion proteins, respectively. A fusion protein containing the extracellular domain of NCAM had no effect on survival. The L1 and CHL1 fusion proteins were effective both in soluble form or when offered as a substrate, with the maximal effect at about 1 microg/mL. To explore the intracellular events related to the neuronal survival effects of L1-Fc fusion protein, Bcl-2 and c-Jun expression were analyzed by Western blotting. The level of Bcl-2 in cerebellar granule neurons was increased by treatment with L1-Fc at both 1 and 5 days of culture. The level of c-Jun was not significantly affected at the early time point and was reduced by L1-Fc fusion protein after long-term culture. The results demonstrate that the neural adhesion molecule L1 and its relative CHL1 are potential neuronal survival factors for neurons of the central nervous system. Bcl-2 may serve as one of the intracellular mediators of the neuronal survival effects of L1.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

The crystal structure of the ligand binding module of axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1(Ig1-4) is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.     

Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development

Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.     

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments, to provide an overview of progress in this rapidly-developing area of cell and developmental biology.     

Ganglioside modulation of neural cell adhesion molecule and N-cadherin- dependent neurite outgrowth

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.     

The neuronal chondroitin sulfate proteoglycan neurocan binds to the neural cell adhesion molecules Ng-CAM/L1/NILE and N-CAM, and inhibits neuronal adhesion and neurite outgrowth

We have previously shown that aggregation of microbeads coated with N- CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I- neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.     

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.     

Semaphorin-1a acts in concert with the cell adhesion molecules fasciclin II and connectin to regulate axon fasciculation in Drosophila

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. Here, by manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, we provide further evidence that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons.     

Proteolytic cleavage of the neural cell adhesion molecule by ADAM17/TACE is involved in neurite outgrowth

The transmembrane and multidomain neural cell adhesion molecule (NCAM) plays important functional roles in the developing and adult nervous system. NCAM is proteolytically processed and appears in soluble forms in the cerebrospinal fluid and in serum under normal and pathological conditions. In this report, we present evidence that the metalloprotease a disintegrin and a metalloprotease (ADAM)17/tumour necrosis factor alpha converting enzyme (TACE) cleaves the polysialylated as well as the non-polysialylated transmembrane isoforms of NCAM, whereas the glycophosphatidylinositol-linked isoform of NCAM is not proteolytically cleaved. A truncated, enzymatically inactive mutant of TACE did not result in release of the NCAM110 cleavage product. Proteolytic cleavage was enhanced by a calmodulin-specific inhibitor and the actin-destabilizing agents cytochalasin D and latrunculin B. In contrast, the microtubule-stabilizing agent colchicine or microtubule-destabilizing agent paclitaxel did not affect the release of the 110-kDa fragment of NCAM. Neurite outgrowth from cerebellar microexplants was inhibited in the presence of the metalloprotease inhibitor GM 6001 on substrate-coated NCAM, but not on poly-l-lysine. Upon transfection of hippocampal neurones with an enzymatically inactive mutant of TACE, NCAM-stimulated neurite outgrowth was inhibited without affecting neurite outgrowth on poly-l-lysine, showing that proteolytic processing of NCAM by the metalloprotease TACE is involved in NCAM-mediated neurite outgrowth.     

Functional cooperation between the neural adhesion molecules L1 and N- CAM is carbohydrate dependent

The neural cell adhesion molecules L1 and N-CAM have been suggested to interact functionally by formation of a complex between the two molecules (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990. J. Cell Biol. 110:193-208). To determine the molecular mechanisms underlying this functional cooperation, we have studied the contribution of carbohydrates to the association of the two molecules at the cell surface. Aggregation or adhesion between L1- and N-CAM-positive neuroblastoma N2A cells was reduced when the synthesis of complex and/or hybrid glycans was modified by castanospermine. Fab fragments of polyclonal antibodies to L1 inhibited aggregation and adhesion of castanospermine-treated cells almost completely, whereas untreated cells were inhibited by approximately 50%. Fab fragments of polyclonal antibodies to N-CAM did not interfere with the interaction between castanospermine-treated cells, whereas they inhibited aggregation or adhesion of untreated cells by approximately 50%. These findings indicate that cell interactions depending both on L1 and N-CAM ("assisted homophilic" binding) can be reduced to an L1-dominated interaction ("homophilic binding"). Treatment of cells with the carbohydrate synthesis inhibitor swainsonine did not modify cell aggregation in the absence or presence of antibodies compared with untreated cells, indicating that castanospermine-sensitive, but swainsonine-insensitive glycans are involved. To investigate whether the appropriate carbohydrate composition is required for an association of L1 and N-CAM in the surface membrane (cis-interaction) or between L1 on one side and L1 and N-CAM on the other side of interacting partner cells (trans-interaction), an L1-positive lymphoid tumor cell line was coaggregated with and adhered to neuroblastoma cells in the various combinations of castanospermine-treated and untreated cells. The results show that it is the cis-interaction between L1 and N-CAM that depends on the appropriate carbohydrate structures.     

神经细胞粘附分子结构特征和生理功能

神经细胞粘附分子是一类调节细胞与细胞、细胞与细胞外基质间粘附作用的膜表面糖蛋白,主要有NCAM-180、NCAM-140、NCAM-120三种形式,多与PSA结合在一起。在神经系统中,NCAM的表达具有时间和空间特异性,最主要的作用为调节神经系统的可塑性,这种作用可能是通过PSA-NCAM对AMPA的调节作用,主要是通过调节蛋白激酶的表达和细胞内Ca^2 浓度来实现的。