Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.     

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.     

Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.     

Synergistic activity of fibronectin and fibroblast growth factor receptors on neuronal adhesion and neurite extension through extracellular signal-regulated kinase pathway

Fibroblast growth factor (FGF) is an important modulator of cell growth and differentiation of various cells including neuron. Cells need to adhere specifically to cellular and extracellular components of their environment to carry out diverse physiological functions. Here, we examined whether fibronectin (FN) and FGF can cooperate for neuronal adhesion and neurite outgrowth. Using recombinant FN peptide (FNIII9-10), we found that FNIII9-10-mediated adhesion promotes the effect of FGF1 on neurite outgrowth of PC12 cells, while FGF1 enhances the FNIII9-10-mediated neuronal adhesion of PC12 cells. This collaboration of FNIII9-10 and FGF1 was the result of the sustained activation of extracellular signal-regulated kinase (ERK)-type MAP kinase. Finally, the synergistic activity of FGF1 and FN was inhibited by PD98059, an MEK inhibitor. Taken together, these findings indicate that FN-mediated signaling can collaborate with FGFRs signaling for neurite outgrowth through selective activation of ERK-type MAP kinase in PC12 cells, and suggest that FN and FGF act in concert to regulate cell differentiation in the nervous system.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.     

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60src

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60^src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60^src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60^src.     

Effects of ethanol and NAP on cerebellar expression of the neural cell adhesion molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10(-12) M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression.     

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.     

Alcohol inhibits cell-cell adhesion mediated by human L1 [published erratum appears in J Cell Biol 1996 Jun;133(5):1139-40]

Mental retardation, hydrocephalus, and agenesis of the corpus callosum are observed both in fetal alcohol syndrome (FAS) and in children with mutations in the gene for the cell adhesion molecule L1. We studied the effects of ethanol on cell-cell adhesion in mouse fibroblasts transfected with human L1. L1-transfected fibroblasts exhibited increased cell-cell adhesion compared with wild-type or vector- transfected controls. Ethanol potently and completely inhibited L1- mediated adhesion both in transfected L cells and NIH/3T3 cells. Half- maximal inhibition was observed at 7 mM ethanol, a concentration achieved in blood and brain after ingesting one alcoholic beverage. In contrast, ethanol did not inhibit the adhesion of fibroblasts transfected with vector alone or with N-CAM-140. L1-mediated cell-cell adhesion was inhibited with increasing potency by n-propanol and n- butanol, but was not inhibited at all by n-alcohols of 5 to 8 carbons, acetaldehyde, or acetate, suggesting that ethanol interacts directly with a small hydrophobic pocket within L1. Phenylalanine, teratogenic anticonvulsants, and high concentrations of glucose did not inhibit L1- mediated cell-cell adhesion. Ethanol also inhibited potently the heterotypic adhesion of rat cerebellar granule cells to a monolayer of L1-transfected NIH/3T3 cells, but had no effect on their adhesion to N- CAM-140 or vector-transfected NIH/3T3 cells. Because L1 plays a role in both neural development and learning, ethanol inhibition of L1-mediated cell-cell interactions could contribute to FAS and ethanol-associated memory disorders.     

Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth

Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on G(s) and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.     

Substrate-bound beta-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures

Accumulation of the beta-amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism of Abeta toxicity remains unclear. Abeta can bind to the extracellular matrix, a structure that regulates adhesive events such as neurite outgrowth and synaptogenesis. The binding of Abeta to the extracellular matrix suggests that Abeta may disrupt cell-substrate interactions. Therefore, the effect of substrate-bound Abeta on the growth of isolated chick sympathetic and mouse cortical neurons was examined. Abeta1-40 and Abeta1-42 had dose-dependent effects on cell morphology. When tissue culture plates were coated with 0.1-10 ng/well Abeta, neurite outgrowth increased. Higher amounts of Abeta peptides (> or =3 microg/well) inhibited outgrowth. The inhibitory effect was related to aggregation of the peptide, as preincubation of Abeta1-40 for 24 h at 37 degrees C (a process known to increase amyloid fibril formation) was necessary for inhibition of neurite outgrowth. Abeta29-42, but not Abeta1-28, also inhibited neurite outgrowth at high concentrations, demonstrating that the inhibitory domain is located within the hydrophobic C-terminal region. Abeta1-40, Abeta1-42, and Abeta29-42 also inhibited cell-substrate adhesion, indicating that the effect on neurite outgrowth may have been due to inhibition of cell adhesion. The results suggest that accumulation of Abeta may disrupt cell-adhesion mechanisms in vivo.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.     

PTPmu regulates N-cadherin-dependent neurite outgrowth

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule-like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPmu is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPmu promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPmu was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPmu and N-cadherin, the expression level and enzymatic activity of PTPmu were perturbed in retinal explant cultures. Downregulation of PTPmu expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPmu significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPmu specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPmu plays a dual role in the regulation of neurite outgrowth.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin

Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.     

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.     

Identification of a Homophilic Binding Site in Immunoglobulin-Like Domain 2 of the Cell Adhesion Molecule L1

Abstract: The cell adhesion molecule L1 plays an important role in neural development, and mutations in human L1 have been implicated in X-linked hydrocephalus and related neurological diseases. We have previously demonstrated that recombinant proteins containing the second immunoglobulin-like domain (Ig2) of L1 contain both homophilic binding and neuritogenic activities. In this report, the involvement of L1 Ig2 in cell-cell adhesion and neuritogenesis was further evaluated in cell transfection studies. Transfectants expressing intact L1 were capable of undergoing L1-dependent self-aggregation and promoting neurite outgrowth from neural retinal cells. However, both activities were abolished in transfectants expressing L1Δ2, a mutant L1 with Ig2 deleted. In competition experiments, the wild-type Ig2 fusion protein inhibited L1-dependent cell aggregation, whereas an Ig2 fusion protein containing the hydrocephalus mutation R184Q did not. Oligopeptides flanking Arg¹⁸⁴ were therefore synthesized and assayed for their effects on L1-mediated cell-cell binding and neuritogenesis. The peptide L1-A, spanning the residues His¹⁷⁸ and Gly¹⁹¹, inhibited both L1- and Ig2 fusion protein-mediated homophilic binding. When neural retinal cells were cultured on substrate-coated Ig2 fusion protein, peptide L1-A also abolished L1-dependent neurite outgrowth. Substitutions of several charged residues and hydrophobic residues with alanine in peptide analogues led to the loss of inhibitory effects, suggesting that multiple amino acids might be involved in L1-L1 binding. Taken together, these results identify an L1 homophilic binding site within the sequence HIKQDERVTMGQNG of Ig2 and demonstrate the requirement of L1 homophilic binding in the promotion of neurite outgrowth.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

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.