The axonally secreted cell adhesion molecule, axonin-1. Primary structure, immunoglobulin-like and fibronectin-type-III-like domains and glycosyl-phosphatidylinositol anchorage.

Axonin-1 is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM L1(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-1. Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-1 fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type III repeats. Axonin-1 exhibits 75% amino acid identity with the AxCAM TAG-1 of the rat, suggesting that it is the chicken homologue of TAG-1. Like TAG-1, axonin-1 is glycosyl-PtdIns-anchored to the neuronal membrane; in contrast to TAG-1, it does not exhibit an Arg-Gly-Asp sequence.     

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.     

The axonally secreted protein axonin-1 is a potent substratum for neurite growth

Axonin-1 is a neuronal glycoprotein occurring both as a membrane-bound and a secreted form. Membrane-bound axonin-1 is predominantly located in membranes of developing nerve fiber tracts and has recently been characterized as a cell adhesion molecule; the soluble form is secreted from axons and accumulates in the cerebrospinal fluid and the vitreous fluid of the eye. In the present study, we addressed the question as to whether secreted axonin-1 was released in a functionally competent form and we found that it strongly promotes neurite outgrowth when presented to neurons as an immobilized substratum. Neurite lengths elaborated by embryonic dorsal root ganglia neurons on axonin-1 were similar to those on the established neurite-promoting substrata L1 and laminin. Fab fragments of axonin-1 antibodies completely inhibited neurite growth on axonin-1, but not on other substrata. In soluble form, axonin-1 had an anti-adhesive effect, as revealed by perturbation of neurite fasciculation. In view of their structural similarity, we conclude that secreted and membrane-bound axonin-1 interact with the same growth-promoting neuritic receptor. The fact that secreted axonin-1 is functionally active, together with our previous findings that it is secreted from an internal cellular pool, suggests a functional dualism between membrane-bound and secreted axonin-1 at the site of secretion, which is most likely the growth cone. The secretion of adhesion molecules could represent a powerful and rapidly acting regulatory element of growth cone-neurite interactions in the control of neurite elongation, pathway selection, and possibly target recognition.     

Neurite Fasciculation Mediated by Complexes of Axonin-1 and Ng Cell Adhesion Molecule

Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1–NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM–NgCAM interaction could be established simultaneously with the axonin-1–NgCAM interaction. In contrast, the axonin-1–NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-1₂/NgCAM₂ tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-1₂/NgCAM₂ complexes and, thus, neurite fasciculation in DRG neurons.     

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.     

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.     

Nr-CAM promotes neurite outgrowth from peripheral ganglia by a mechanism involving axonin-1 as a neuronal receptor.

Nr-CAM is a neuronal cell adhesion molecule (CAM) belonging to the immunoglobulin superfamily that has been implicated as a ligand for another CAM, axonin-1, in guidance of commissural axons across the floor plate in the spinal cord. Nr-CAM also serves as a neuronal receptor for several other cell surface molecules, but its role as a ligand in neurite outgrowth is poorly understood. We studied this problem using a chimeric Fc-fusion protein of the extracellular region of Nr-CAM (Nr-Fc) and investigated potential neuronal receptors in the developing peripheral nervous system. A recombinant Nr-CAM-Fc fusion protein, containing all six Ig domains and the first two fibronectin type III repeats of the extracellular region of Nr-CAM, retains cellular and molecular binding activities of the native protein. Injection of Nr-Fc into the central canal of the developing chick spinal cord in ovo resulted in guidance errors for commissural axons in the vicinity of the floor plate. This effect is similar to that resulting from treatment with antibodies against axonin-1, confirming that axonin-1/Nr-CAM interactions are important for guidance of commissural axons through a spatially and temporally restricted Nr-CAM positive domain in the ventral spinal cord. When tested as a substrate, Nr-Fc induced robust neurite outgrowth from dorsal root ganglion and sympathetic ganglion neurons, but it was not effective for tectal and forebrain neurons. The peripheral but not the central neurons expressed high levels of axonin-1 both in vitro and in vivo. Moreover, antibodies against axonin-1 inhibited Nr-Fc-induced neurite outgrowth, indicating that axonin-1 is a neuronal receptor for Nr-CAM on these peripheral ganglion neurons. The results demonstrate a role for Nr-CAM as a ligand in axon growth by a mechanism involving axonin-1 as a neuronal receptor and suggest that dynamic changes in Nr-CAM expression can modulate axonal growth and guidance during development.     

Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites

Neurofascin and NrCAM are two axon-associated transmembrane glycoproteins belonging to the L1 subgroup of the Ig superfamily. In this study, we have analyzed the interaction of both proteins using neurite outgrowth and binding assays. A neurofascin-Fc chimera was found to stimulate the outgrowth of tectal cells when immobilized on an inert surface but not as a soluble form using polylysine as substrate. Antibody blocking experiments demonstrate that neurite extension on immobilized neurofascin is mediated by NrCAM on the axonal surface. Under the reverse experimental conditions where NrCAM induces neurite extension, F11, and not neurofascin, serves as axonal receptor. Binding studies using transfected COS7 cells and immunoprecipitations reveal a direct interaction between neurofascin and NrCAM. This binding activity was mapped to the Ig domains within neurofascin. The neurofascin-NrCAM binding can be modulated by alternative splicing of specific stretches within neurofascin. These studies indicate that heterophilic interactions between Ig-like proteins implicated in axonal extension underlie a regulation by the neuron.     

Purification of axonin-1, a protein that is secreted from axons during neurogenesis.

Using selective metabolic labelling in a compartmental cell culture system two proteins, denoted axonin-1 and axonin-2, were found to be secreted by axons of dorsal root ganglia neurons from chicken embryos. Based on its characteristic coordinates and spot morphology in two-dimensional gel electrophoresis, axonin-1 was detected in the cerebrospinal fluid and the vitreous fluid, axonin-1 was purified 476-fold to homogeneity by a four-step chromatographic procedure. The identity of the purified protein as axonin-1 was confirmed by immunological methods. Axonin-1 is a glycoprotein that subdivides into at least 16 immunologically similar isoelectric variants; their molecular weight range extends from 132 to 140 kd and their pI range from 5.3 to 6.2. In the vitreous fluid of the embryo, axonin-1 could first be detected on the embryonic day 5 and highest concentrations were measured during the second half of embryonic life; in the vitreous fluid of the adult chicken, concentrations were approximately 20 times lower. The early onset of secretion and the time course of expression suggest a role for axonin-1 in the development of the 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.     

Intracellular signaling is changed after clustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation

Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association.     

The role of cell adhesion molecules in neurite outgrowth on Müller cells

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.     

The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against α_vβ₃ integrin, but not an anti-β₁ antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the α_vβ₃ integrin on neuronal cells.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.     

Autonomous and non-autonomous regulation of mammalian neurite development by Notch1 and Delta1

BACKGROUND: On the basis of experiments suggesting that Notch and Delta have a role in axonal development in Drosophila neurons, we studied the ability of components of the Notch signaling pathway to modulate neurite formation in mammalian neuroblastoma cells in vitro. RESULTS: We observed that N2a neuroblastoma cells expressing an activated form of Notch, Notch1(IC), produced shorter neurites compared with controls, whereas N2a cell lines expressing a dominant-negative Notch1 or a dominant-negative Delta1 construct extended longer neurites with a greater number of primary neurites. We then compared the effects on neurites of contacting Delta1 on another cell and of overexpression of Delta1 in the neurite-extending cell itself. We found that N2a cells co-cultured with Delta1-expressing quail cells produced fewer and shorter neuritic processes. On the other hand, high levels of Delta1 expressed in the N2a cells themselves stimulated neurite extension, increased numbers of primary neurites and induced expression of Jagged1 and Notch1. CONCLUSIONS: These studies show that Notch signals can antagonize neurite outgrowth and that repressing endogenous Notch signals enhances neurite outgrowth in neuroblastoma cells. Notch signals therefore act as regulators of neuritic extension in neuroblastoma cells. The response of neuritic processes to Delta1 expressed in the neurite was opposite to that to Delta1 contacted on another cell, however. These results suggest a model in which developing neurons determine their extent of process outgrowth on the basis of the opposing influences on Notch signals of ligands contacted on another cell and ligands expressed in the same cell.     

Mouse acetylcholinesterase enhances neurite outgrowth of rat R28 cells through interaction with laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert 'non-classical', morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1.     

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.