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.     

Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4)

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.     

Distinct subpopulations of sensory afferents require F11 or axonin-1 for growth to their target layers within the spinal cord of the chick.

Dorsal root ganglion neurons project axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality. Using an in vivo approach, we demonstrate an involvement of the two immunoglobulin superfamily cell adhesion molecules axonin-1/TAG-1 and F11/F3/contactin in subpopulation-specific sensory axon guidance. Proprioceptive neurons, which establish connections with motoneurons in the ventral horn, depend on F11 interactions. Nociceptive fibers, which target to layers in the dorsal horn, require axonin-1 for pathfinding. In vitro NgCAM and NrCAM were shown to bind to both axonin-1 and F11. However, despite this fact and despite their ubiquitous expression in the spinal cord, NgCAM and NrCAM are selective binding partners for axonin-1 and F11 in sensory axon guidance. Whereas nociceptive pathfinding depends on NgCAM and axonin-1, proprioceptive fibers require NrCAM and F11.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

A cytomechanical investigation of neurite growth on different culture surfaces

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.     

Hodgkin's cell lectin, a lymphocyte adhesion molecule and mitogen

We have previously shown a novel galactose/N-acetylgalactosamine specific lectin activity (Hodgkin's disease (HD) lectin) on the surface of cultured HD cells (lines L428, its variants, and line L540) to mediate lymphocyte adhesion. We here demonstrate that both surface membrane-bound and secreted HD lectin activities participate in the activation of agglutinated lymphocytes. Among known adhesion molecules expressed by the HD cells, only the intercellular adhesion molecule-1 (ICAM-1) contributed to this activation as an alternative PBL binding site. As yet we have not identified the cellular ligand(s) for the HD lectin on the lymphocyte surface. Pretreatment of lymphocytes with mAb to the accessory molecules CD2, CD3, CD4, CD8, CD11b, or CD11c did not interfere with their response to HD cells. mAb to CD11a (LFA-1), the alleged ligand of ICAM-1, inhibited the ICAM-1 but not the HD lectin-mediated lymphocyte stimulation. Although lymphocyte binding could proceed via either pathway, lymphocyte activation always depended upon factors secreted by the HD cells, one of which we identified as a soluble form of the HD lectin based on its shared properties with the membrane-bound form including immunologic cross-recognition and carbohydrate-binding specificity. Although HD cell-conditioned medium alone stimulated lymphocytes, HD cell plasma membranes could compensate for low concentrations of this medium. In addition, resting lymphocytes, normally unresponsive, were triggered into DNA synthesis by growth medium when cocultured with HD cell membranes. The unique functions of the surface-expressed HD lectin and its soluble counterpart as lymphocyte adhesion molecule and mitogen might be physiologically relevant to the severe immunodeficiencies occurring in patients with HD.     

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.     

Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons

The effects of glycosaminoglycans (GAG) on cell-to-substratum adhesion and neurite elongation were examined in primary cultures of fetal rat hippocampal neurons using tissue culture dishes coated with GAGs coupled to dipalmitoylphosphatidylethanolamine (PE), a novel probe for biological functions of GAGs. Both chondroitin sulfate conjugate to PE (CS-PE) and hyaluronic acid conjugate to PE (HA-PE) promoted neurite elongation from neurons in a dose-dependent manner when immobilized onto polylysine-coated dishes at various concentrations up to 1.0 microg/ml. The coating of CS-PE or HA-PE at a concentration higher than 1.0 microg/ml resulted in failure of neurite extension and adhesion of neurons to the substrata. In contrast, heparin conjugate to PE (HP-PE) did not exert any effects on neurite elongation or on cell attachment at these concentrations. These findings suggest that GAGs serve as a modulator for neurite elongation during neuronal network formation in the developing central nervous system.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.     

Neurite outgrowth from hippocampal neurons is promoted by choroid plexus ependymal cells <Emphasis Type="Italic">in vitro</Emphasis>

Choroid plexus ependymal cells (CPECs) were known to promote axonal growth when choroid plexus is grafted into the adult rat spinal cord. The present study was carried out to examine whether CPECs promote axonal outgrowth from neurons derived from the CNS in vitro. Hippocampal neurons were cocultured on CPEC monolayers. After 24 h, neurite extension was evaluated using various parameters in comparison with cultures grown on poly-L-lysine (PLL)-coated plates and cocultures grown on astrocyte monolayers. The primary neurite length and total neurite length were longest in the cocultures with CPECs. The number of primary neurites and the number of branches were larger in the cultures with CPECs than in the cultures on PLL-coated plates, but almost the same as in the cocultures with astrocytes. Next, we examined whether the neurite extension-promoting effect occurring within 24 h is due primarily to contact with the CPECs or to factors secreted by CPECs into the culture medium. The CPEC monolayers were killed by ethanol fixation, and neurons cultured on them. The neurons extended long neurites with elaborate branching, as in the case of cocultures grown on living CPECs. On the other hand, CPEC-conditioned medium exhibited less promoting effect on neurite outgrowth from hippocampal neurons. These results indicate that CPECs have a capacity to promote neurite outgrowth from CNS neurons in vitro, and that surface plasma membrane-bound components of CPECs strongly contribute to the enhancement of neurite outgrowth in the present coculture system.