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.     

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

Sensitivity of neurite outgrowth to microfilament disruption varies with adhesion molecule substrate

Interactions between the cytoskeleton and cell adhesion molecules are presumed responsible for neurite extension. We have examined the role of microfilaments in neurite outgrowth on the cell adhesion molecules L1, P84, N-CAM, and on laminin. Cerebellar neurons growing on each substrate exhibited differing growth cone morphologies and rates of neurite extension. Growth of neurites in the presence of cytochalasin B (CB) was not inhibited on substrates of L1 or P84 but was markedly inhibited on N-CAM. Neurons on laminin were initially unable to extend neurites in the presence of CB but recovered this ability within 9 h. These studies suggest that neurite outgrowth mediated by different cell adhesion molecules proceeds via involvement of distinct cytoskeletal interactions. © 1993 John Wiley & Sons, Inc.     

Cell adhesion molecules in the early developing mouse retina: Retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in intitiation and establishment of the optic pathway. © 1994 John Wiley & Sons, Inc.     

N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro

Retinal ganglion neurons extend axons that grow along astroglial cell surfaces in the developing optic pathway. To identify the molecules that may mediate axon extension in vivo, antibodies to neuronal cell surface proteins were tested for their effects on neurite outgrowth by embryonic chick retinal neurons cultured on astrocyte monolayers. Neurite outgrowth by retinal neurons from embryonic day 7 (E7) and E11 chick embryos depended on the function of a calcium-dependent cell adhesion molecule (N-cadherin) and beta 1-class integrin extracellular matrix receptors. The inhibitory effects of either antibody on process extension could not be accounted for by a reduction in the attachment of neurons to astrocytes. The role of a third cell adhesion molecule, NCAM, changed during development. Anti-NCAM had no detectable inhibitory effects on neurite outgrowth by E7 retinal neurons. In contrast, E11 retinal neurite outgrowth was strongly dependent on NCAM function. Thus, N-cadherin, integrins, and NCAM are likely to regulate axon extension in the optic pathway, and their relative importance varies with developmental age.     

Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes.

Triggering neural cell adhesion molecules of the immunoglobulin superfamily with specific ligands or antibodies inhibited the phosphorylation of tryosyl residues in a subpopulation of alpha- and beta-tubulin associated with membranes from a subcellular fraction of nerve growth cones from fetal rat brain. Preincubation of these membranes with purified extracellular fragments of L1, N-CAM, or myelin-associated glycoprotein, or with antibodies directed against the extracellular domains of L1 or N-CAM, inhibited pp60c-src-dependent phosphorylation of tubulin in an endogenous membrane kinase reaction. Other proteins that affect neurite outgrowth (fibronectin, laminin, antibodies against N-cadherin) had no effect. The results suggest that cell adhesion molecules transduce cell surface events to intracellular signals by modulating the activity of protein tyrosine kinases or phosphatases in axonal membranes to influence cytoskeletal dynamics at the growth cone.     

Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons.

Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes. The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line. Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Glycosylphosphatidylinositol anchored recognition molecules that function in axonal fasciculation, growth and guidance in the nervous system.

A large number of glycoproteins in the central nervous system are attached to the cell membrane via covalent linkage to glycosylphosphatidylinositol (GPI). Many of them, including the drosophila fasciclin 1 as well as the mammalian glycoproteins Thy-1, TAG1, N-CAM and F11,F3, contactin are members of the immunoglobulin gene superfamily. These and other GPI-linked molecules have been implicated in key developmental events including selective axonal fasciculation and highly specific growth to and innervation of target tissues. In model systems fasciclin 1, TAG1 and N-CAM have been shown to be capable of mediating cell-cell adhesion via a homophilic binding mechanism confirming their operational classification as cell adhesion molecules (CAMs). However, of these molecules, only N-CAM has been shown to mediate a complex response (neurite outgrowth) via a homophilic binding mechanism. Whether the other molecules in this family mediate biological responses by binding to themselves and/or other molecules remains to be determined. Studies on N-CAM provide an ideal model system for understanding the function of GPI anchors since alternative splicing of the NCAM gene generates both lipid-linked and transmembrane N-CAM isoforms. Recent studies have shown that neurons can recognise and respond (by increased neurite outgrowth) to both lipid-linked and transmembrane N-CAM isoforms expressed on the surface of non-neuronal cells following transfection with appropriate cDNAs. The major determinant of neuronal responsiveness was the level of N-CAM expression rather than the isoform type. Neurite outgrowth in response to transfected N-CAM is mediated by transmembrane N-CAM isoforms expressed by neurons and this involves the activation of classical second messenger pathways in the neurons. One possibility is that GPI anchors are utilised when a cell has simply to provide recognition or positional information to a second cell whereas transmembrane molecules might be required for cells that actively respond to such information. The hypothesis is compatible with all the known information on N-CAM expression and function and may be extended to other adhesive events.     

Neurite Outgrowth Stimulated by L1 Requires Calcium Influx into Neurons but is Not Associated with Changes in Steady State Levels of Calcium in Growth Cones

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N- type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth

N-cadherin is the predominant mediator of calcium-dependent adhesion in the nervous system (Takeichi, M. 1988. Development (Camb.). 102: 639-655). Investigations using antibodies to block N-cadherin function (Bixby, J.L., R.L. Pratt, J. Lilien, and L.F. Reichardt. 1987. Proc. Natl. Acad. Sci. USA. 84:2555-2569; Bixby, J.L., J. Lilien, and L.F. Reichardt. 1988. J. Cell Biol. 107:353-362; Tomaselli, K.J., K.N. Neugebauer, J.L. Bixby, J. Lilien, and L.F. Reichardt. 1988. Neuron. 1:33-43) or transfection of the N-cadherin gene into heterologous cell lines (Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi. 1988. Nature (Lond.). 334:62-64) have provided evidence that N-cadherin, alone or in combination with other molecules, can participate in the induction of neurite extension. We have developed an affinity purification procedure for the isolation of whole N-cadherin from chick brain and have used the isolated protein as a substrate for neurite outgrowth. N-cadherin promotes the rapid extension of neurites from chick ciliary ganglion neurons, which extend few or no neurites on adhesive but noninducing substrates such as polylysine, tissue culture plastic, and collagens. N-cadherin is extremely potent, more so than the L1 adhesion molecule, and comparable to the extracellular matrix protein laminin. Compared to laminin, however. N-cadherin promotes outgrowth from ciliary ganglion neurons extremely rapidly and with a distinct morphology. These results provide a direct demonstration that N-cadherin is sufficient to induce neurite outgrowth when substrate bound and suggest that the mechanism(s) involved may differ from that induced by laminin.     

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.     

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.     

Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N-cadherin, fused to the Fc region of human IgG (NCAD-Fc) supports calcium-dependent cell adhesion and promotes neurite outgrowth following affinity-capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD-Fc stimulated neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant-negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD-Fc. In cells transfected with full-length N-cadherin and the FGF receptor, antibody-clustering of N-cadherin resulted in a co-clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N-cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N-cadherin and the FGF receptor co-clustering in cells provides a basis for the neurite outgrowth response stimulated by N-cadherin being dependent on FGF receptor function.     

Neutralization of Schwann Cell-Secreted VEGF Is Protective to In Vitro and In Vivo Experimental Diabetic Neuropathy

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus.

Monolayers of control 3T3 fibroblasts and 3T3 cells expressing transfected NCAM or N-cadherin have been used as a culture substratum for rat hippocampal neurons. Both NCAM and N-cadherin are expressed in the hippocampus through embryonic day 17 (E17) to postnatal day 4 (PND4); however, whereas E17 neurons responded to transfected NCAM by extending considerably longer neurites, PND4 neurons responded very poorly. The converse was true for responsiveness to N-cadherin. These data demonstrate a switch in neuronal responsiveness to NCAM and N-cadherin in the developing hippocampus. NCAM-dependent neurite outgrowth from E17 neurons was largely dependent on the presence of alpha 2-8-linked polysialic acid (PSA) on neuronal NCAM. NCAM-dependent neurite outgrowth could be fully inhibited by pertussis toxin or a combination of L- and N-type calcium channel antagonists thus providing direct evidence concerning the nature of the second messenger pathway activated in primary neurons by cell adhesion molecules (CAMs).