Receptor tyrosine phosphatase-delta is a homophilic, neurite-promoting cell adhesion molecular for CNS neurons

Appropriate regulation of tyrosine phosphorylation is essential for axon growth and guidance; evidence from invertebrates indicates that receptor-type tyrosine phosphatases (RPTPs) are required for correct axon growth during CNS development. One vertebrate RPTP, PTP-delta, is highly expressed in brain and has a cell adhesion molecule-like extracellular domain (ECD) comprising three immunoglobulin repeats and eight fibronectin type III repeats. Using fluorescent beads (Covaspheres) coated with the PTP-delta ECD, as well as insect cells expressing PTP-delta on their surfaces, we show that PTP-delta is a homophilic cell adhesion molecule. A variety of chick neurons adhere strongly to an Fc fusion protein containing the PTP-delta ECD. Additionally, substrate-bound PTP-delta ECD fusion protein strongly promotes neurite outgrowth from forebrain neurons; this effect is separable from its effect on adhesion. Our results indicate that PTP-delta is a neurite-promoting cell adhesion molecule for CNS neurons.     

Different Domains of the F3 Neuronal Adhesion Molecule are Involved in Adhesion and Neurite Outgrowth Promotion

The mouse F3 cell surface protein is preferentially expressed on axons of subpopulations of neurons and is anchored to the membrane by a glycosyl-phosphatidylinositol group. It consists of six immunoglobulin-like domains and four fibronectin type III homologous repeats, and can be found both in membrane-anchored and soluble forms. We have previously established that F3 fulfils the operational criteria of a cell adhesion molecule when anchored to the plasma membrane and that its soluble form stimulates neurite initiation and neurite outgrowth. To further characterize F3-mediated adhesion and to investigate whether adhesion and neurite outgrowth promoting activities are displayed by different parts of the molecule, we (i) selected F3 transfected CHO cells expressing increasing levels of F3 at their surface and (ii) prepared transfectants expressing an F3 molecule with its fibronectin type III repeats deleted. We show that the F3 molecule mediates divalent-cation-independent, temperature-dependent binding. The levels of aggregation of F3 transfectants are proportional to the level of F3 expression. Transfectants expressing F3 deleted of the fibronectin type Ill repeats lose their adhesive properties; conversely, cells expressing wild-type F3 and treated with collagenase, specifically removing the immunoglobulin-like domains, are still able to aggregate. Therefore, in this model adhesion site(s) mapped to the fibronectin type III repeats. By contrast, transfectants expressing deleted F3, as well as the soluble forms of this F3 deleted molecule, were able to stimulate neurite outgrowth of sensory neurons similarly to wild-type F3. Our data indicate that F3 is a multifunctional molecule and that adhesion and neurite outgrowth promoting properties are expressed by distinct and independent domains.     

Cell adhesion and neurite outgrowth are promoted by neurofascin NF155 and inhibited by NF186

Neurofascin (NF) is a neural cell adhesion molecule in the L1-family containing six Ig domains and multiple fibronectin type III (FnIII) repeats in its extracellular region. NF has many splicing variants and two of these are exemplars that have different cellular patterns of expression during development. NF186, which is expressed on neurons, contains an unusual mucin-like region and NF155, which is expressed on glia, contains a unique FnIII repeat with an RGD motif. Analysis of Fc fusion proteins representing different extracellular regions of NF indicate that NF186 inhibits cell adhesion and neurite outgrowth, and the inhibition is associated with the region containing the mucin-like domain. NF155 promotes neural cell adhesion and neurite outgrowth, and the RGD motif in its third FnIII repeat is critical for cell spreading and neurite outgrowth. The results suggest that different splicing variants of NF expressed on neurons and glia play distinct roles during neural development.     

Single chain Fv antibodies against neural cell adhesion molecule L1 trigger L1 functions in cultured neurons

The neural cell adhesion molecule L1 plays important roles in cell adhesion, neuronal migration, neurite outgrowth, fasciculation and pathfinding, neuronal survival, and synaptic plasticity. Many of these functions have been identified and characterized by using antibodies. Because of the need for reproducible and functionally active antibodies, we have generated two single-chain variable fragment antibodies against mouse L1 from a human synthetic phage display library. The complementarity determining region 3 of the variable heavy chains of the two antibodies differed in length and sequence. Both antibodies recognized mouse, but not human L1, by enzyme-linked immunosorbent assay, Western blot, and immunofluorescence staining of cultured neurons. Epitope mapping showed reactivity with the fibronectin type III repeats 1-2 of mouse L1. The antibodies stimulated neurite outgrowth from cerebellar dorsal, root ganglion and motor neurons when offered in substrate-coated form in a dose-dependent manner with maximal effects at approximately 32 nM. Furthermore, substrate-coated antibodies enhanced survival of cerebellar neurons. Peptides comprising 8 and 11 amino acids derived from the complementarity determining region 3 of the variable heavy chains of the two single-chain variable fragment antibodies also promoted neurite outgrowth. The combined observations indicate that single-chain variable fragment antibodies against L1 and peptides derived from their binding domains can mimic some beneficial functions of homophilically binding L1 in vitro and may thus serve to trigger these functions in vivo.     

Distinct Effects of Recombinant Tenascin-R Domains in Neuronal Cell Functions and Identification of the Domain Interacting with the Neuronal Recognition Molecule F3/11

We have identified distinct domains of the rat extracellular matrix glycoprotein tenascin-R using recombinant fragments of the molecule that confer neuronal cell functions. In short-term adhesion assays (0.5 h), cerebellar neurons adhered best to the fragment representing the fibrinogen knob (FG), but also the fibronectin type Ill (FN) repeats 1-2 and 6-8. FG, FN1-2 and FN3-5 were the most repellent fragments for neuronal cell bodies. Neurites and growth cones were strongly repelled from areas coated with fragments containing the cysteine-rich stretch and the EGF-like domains (EGF-L), FN1-2, FN3-5 and FG. Polarization of morphology of hippocampal neurons was exclusively associated with FG, while EGF-L prevented neurite outgrowth altogether. The binding site of the neuronal receptor for tenascin-R, the immunoglobulin superfamily adhesion molecule F3/11, was localized to EGF-L. The combined observations show distinct, but also overlapping functions for the different tenascin-R domains. They further suggest the existence of multiple neuronal tenascin-R receptors which influence the response of neurons to their extracellular matrix environment.     

Distinct effects of recombinant tenascin-C domains on neuronal cell adhesion,growth cone guidance,and neuronal polarity

Using a set of recombinantly expressed proteins, distinct domains of the mouse extracellular matrix glycoprotein tenascin-C, hereafter called tenascin, have been identified to confer adhesion, anti-adhesion, and changes in morphology of neuronal cells. In short-term adhesion assays (1 hr), cerebellar and hippocampal neurons adhered to several domains, encompassing the fibronectin type III-like (FN III) repeats 1–2 and 6–8, as well as to the alternatively spliced FN III repeats and to tenascin itself. Although no short-term adhesion to the EGF repeats containing fragment could be detected under the conditions used, it was anti-adhesive for neuronal cell bodies and repellent for growth cone advance and neuritogenesis. FN III repeats 3–5 were repellent only for growth cones but not for neuronal cell bodies. Neurite outgrowth promoting activities at early stages and induction of a polarized neuronal morphology at later stages of differentiation were associated with the EGF repeats and the FN III repeats 6–8. These observations suggest differential effects of particular domains of the tenascin molecule on distinct cellular compartments, i.e., cell body, axon and dendrite, and existence of multiple neuronal receptors with distinct intracellular signaling features. © 1996 Wiley-Liss, Inc.     

AMIGO and friends: an emerging family of brain-enriched, neuronal growth modulating, type I transmembrane proteins with leucine-rich repeats (LRR) and cell adhesion molecule motifs

Leucine-rich repeats (LRR) are protein-protein interaction domains (20-29 amino acid residues in length) found in proteins with diverse structure and functions. We note here an emerging group of central nervous system-enriched, type I surface proteins with an ectodomain containing LRR repeats and motifs found in cell adhesion molecules. Members of this group include the Amphoterin-induced gene and ORF-1 (AMIGO-1), LRR and Ig domain containing Nogo Receptor interacting protein I (LINGO-1) and the netrin-G1 ligand NGL-1. The above proteins carry, in addition to the LRR repeats, an immunoglobin (Ig)-like segment in their ectodomain. Two other related families of molecules, the NLRRs and the FLRTs, have in addition, a fibronectin type III repeat. The LRR domain distinguishes these molecules from the more extensively studied Ig-like family of cell adhesion molecules, and the transmembrane domain differentiate them from the family of secreted extracellular proteoglycans with LRRs. Functionally, many members of this group of proteins could modulate neurite outgrowth of neurons, at least in vitro. LINGO-1, initially discovered as a component of the Nogo-66 receptor complex which inhibits neurite growth, also regulates oligodendrocyte differentiation and myelination. Current knowledge and recent findings pertaining to the functions of this interesting group of proteins in the nervous system are discussed.     

Identification of neural cell adhesion molecule L1‐derived neuritogenic ligands of the fibroblast growth factor receptor

The neural cell adhesion molecule L1 plays an important role in axon growth, neuronal survival, and synaptic plasticity. We recently demonstrated that the L1 fibronectin type III (FN3) modules interact directly with the fibroblast growth factor (FGF) receptor (FGFR). Sequence alignment of individual L1 FN3 modules with various FGFs suggested that four sequence motifs located in the third and fifth L1 FN3 modules might be involved in interactions with FGFR. The present study found that corresponding synthetic peptides, termed elcamins 1, 2, 3, and 4, bind and activate FGFR in the absence of FGF1. Conversely, in the presence of FGF1, elcamins inhibited receptor phosphorylation, indicating that the peptides are FGFR partial agonists. Elcamins 1, 3, and 4 dose dependently induced neurite outgrowth in cultured primary cerebellar neurons. The neuritogenic effect of elcamins was dependent on FGFR activation, insofar as the effect was abolished by the receptor inhibition. Thus, the identified peptides act as L1 mimetics with regard to activation of FGFR and induction of neurite outgrowth. © 2009 Wiley‐Liss, Inc.     

Signalling pathways underlying neural cell adhesion molecule-mediated survival of dopaminergic neurons

Stimulation of the neural cell adhesion molecule (NCAM) by homophilic interactions is known to lead to neurite outgrowth as well as to neuronal survival. Whereas a complex network of signalling molecules is known to be of importance to NCAM-mediated neurite extension, only limited information is available regarding signalling underlying NCAM-mediated neuroprotection. Here, we present data suggesting a difference in the signalling events required for survival of rat dopaminergic neurons as compared with neurite outgrowth from the same cell type. Whereas Fyn, fibroblast growth factor receptor, mitogen-activated protein and ERK kinase, protein kinase A and protein kinase C are required for both responses to NCAM-induced signalling, phospholipase C and Ca(2+)-calmodulin-dependent kinase II are only necessary for the neurite outgrowth response, but dispensable for neuroprotection.     

NCAM expression induces neurogenesis in vivo

Neural cell adhesion molecule (NCAM) plays an important role during neural development and in the adult brain, whereby most functions of NCAM have been ascribed to its unique polysialic acid (PSA) modification. Recently we presented evidence suggesting that expression of NCAM in vivo interferes with the maintenance of forebrain neuronal stem cells. We here aimed at investigating the fate of cells generated from NCAM-overexpressing stem cells in postnatal mouse brain and at elucidating the functional domains of NCAM mediating this effect. We show that ectopic expression of the NCAM140 isoform in radial glia and type C cells induces an increase in cell proliferation and consequently the presence of additional neuronal type A cells in the rostral migratory stream. A mutant NCAM protein comprising only fibronectin type III repeats and immunoglobulin-like domain 5 was sufficient to induce this effect. Furthermore, we show that the neurogenic effect is independent of PSA, as transgenic NCAM is not polysialylated in radial glia and type C cells. These results suggest that heterophilic interactions of NCAM with other components of the cell membrane must be involved.     

Characterization of BASP1-mediated neurite outgrowth

The brain acid-soluble protein BASP1 (CAP-23, NAP-22) belongs to the family of growth-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite outgrowth. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite outgrowth in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite outgrowth was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the fibroblast growth factor receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite outgrowth. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite outgrowth to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite outgrowth. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite outgrowth, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite outgrowth. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite outgrowth and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.     

Identification of an ectodomain within the LAR protein tyrosine phosphatase receptor that binds homophilically and activates signalling pathways promoting neurite outgrowth

Elucidation of mechanisms by which receptor protein tyrosine phosphatases (PTPs) regulate neurite outgrowth will require characterization of ligand-receptor interactions and identification of ligand-induced signalling components mediating neurite outgrowth. The first identified ligand of the leucocyte common antigen-related (LAR) receptor PTP consists of a 99-residue ectodomain isoform, termed LARFN5C, which undergoes homophilic binding to LAR and promotes neurite outgrowth. We employed peptide mapping of LARFN5C to identify an active neurite-promoting domain of LAR. A peptide mimetic consisting of 37 residues (L59) and corresponding to the fifth LAR fibronectin type III (FNIII) domain prevented LARFN5C homophilic binding, demonstrated homophilic binding to itself and promoted neurite outgrowth of mouse E16-17 hippocampal neurons and of dorsal root ganglia explants. Response to L59 was partially lost when using neurons derived from LAR-deficient (-/-) mice or neurons treated with LAR siRNA, consistent with homophilic interaction of L59 with LAR. L59 neurite-promoting activity was decreased in the presence of inhibitors of Src, Trk, PLCgamma, PKC, PI3K and MAPK. L59 activated Src (a known substrate of LAR), FAK and TrkB and also activated downstream signalling intermediates including PKC, ERK, AKT and CREB. BDNF augmented the maximal neurite-promoting activity of L59, a finding consistent with the presence of shared and distinct signalling pathways activated by L59 with BDNF and L59 with TrkB. These studies are the first to identify an ectodomain of LAR (located within the fifth FNIII domain) capable of promoting neurite outgrowth and point to novel approaches for promotion of neurite outgrowth.     

Defasciculation of neurites is mediated by tenascin-R and its neuronal receptor F3/11

Fasciculation and defasciculation of axons are major morphogenetic events in the formation of neuronal pathways during development. We have identified the extracellular matrix glycoprotein tenascin-R (TN-R) and its neuronal receptor, the immunoglobulin superfamily recognition molecule F3, as promoters of neurite defasciculation in cerebellar explant cultures. Perturbation of the interaction between these two molecules using both antibodies and an antisense oligonucleotide resulted in increased neurite fasciculation. The domains involved in defasciculation were identified as the N-terminal region of TN-R containing the cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats and the immunoglobulin-like domains of F3. Fasciculation induced by antibodies and the antisense oligonucleotide could be reverted by a phorbol ester activator of protein kinase C, whereas the protein kinase inhibitor staurosporine increased fasciculation. Our observations indicate that defasciculated neurite outgrowth does not only depend on the reduction of the expression of fasciculation enhancing adhesion molecules, such as L1 and the neural cell adhesion molecule (NCAM), but also on recognition molecules that actively induce defasciculation by triggering second messenger systems. J. Neurosci. Res. 52:390–404, 1998. © 1998 Wiley-Liss, Inc.     

Accelerated Differentiation in Response to Retinoic Acid After Retrovirally Mediated Gene Transfer of GAP-43 into Mouse Neuroblastoma Cells

Although substantial evidence exists for the involvement of growth-associated protein-43 (GAP-43) in neuronal development and regeneration, the precise role of this protein in neurite outgrowth is currently debated. To investigate the role of GAP-43 in the initiation of neurite outgrowth, we transfected a full-length cDNA coding for GAP-43 into a mouse neuroblastoma cell line (Neuro-2a) which can be differentiated to a neuronal phenotype using retinoic acid (RA). We show that the consequent overexpression of GAP-43 results in a change in the basic morphology of these cells, but is not in itself sufficient to induce the extension of neurites. However, overexpression of GAP-43 results in a marked acceleration of neurite formation in response to RA. We propose that while GAP-43 does not trigger the initiation of neurite extension, its expression is rate-limiting for neurite outgrowth in response to differentiation agents such as RA.     

Fibronectin type III (FN3) modules of the neuronal cell adhesion molecule L1 interact directly with the fibroblast growth factor (FGF) receptor

The neuronal cell adhesion molecule (CAM) L1 promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). The present study demonstrates a direct interaction between L1 fibronectin type III (FN3) modules I-V and FGFR1 immunoglobulin (Ig) modules II and III by surface plasmon resonance analysis. Binding of L1 to FGFR1 was enhanced by adenosine 5'-triphosphate (ATP), adenylylmethylenediphosphonate (AMP-PCP), and guanosine-5'-triphosphate (GTP), but not adenosine monophosphate (AMP). The L1-FN3 modules were capable of activating FGFR1, reflected by receptor phosphorylation, and this resulted in the induction of differentiation of primary neurons, reflected by neurite outgrowth. Furthermore, ATP modulated L1-induced neuronal differentiation and FGFR1 phosphorylation through regulation of the L1-FGFR1 interaction.     

Physical and functional cooperation of neural cell adhesion molecule and ��1-integrin in neurite outgrowth induction

Neural cell adhesion molecule (NCAM) and ??1-integrin are both involved in cell differentiation, with changes in the expression of these two molecules correlating with changes in the malignancy of tumor cells. There is a known functional correlation between NCAM and ??1-integrin in adhesion and also neurite outgrowth in tumor cells. In the present study, we used immunostaining and immunoprecipitation studies to demonstrate that isoform 120 of NCAM associates physically as well as functionally with ??1-integrin in the induction of neurite outgrowth in SH-SY5Y-human neuroblastoma cells. The interaction between these two molecules is mandatory for neurite outgrowth. NCAM blockage completely inhibits the effects of ??1-integrin on neurite outgrowth. These findings further our understanding of the interactions between NCAMs and integrins in malignancy.     

Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide library

The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion and signal transduction. By screening a combinatorial library of synthetic peptides with NCAM purified from postnatal day 10 rat brains, we identified a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth induced specifically by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathway similar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from intracellular calcium stores in addition to calcium influx from extracellular sources. By single-cell calcium imaging we further demonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system.     

Central and peripheral neurite outgrowth differs in preference for heparin-binding versus integrin-binding sequences.

Neurons of the CNS and PNS differ in their response to fibronectin (FN) and proteolytic fragments of FN. The 33 kDa C-terminal cell and heparin-binding fragment of FN, in particular, is a strong promoter of CNS neurite outgrowth. To define further the neurite-promoting activity of the 33 kDa fragment, and to investigate further the differences between PNS and CNS responses to FN and the 33 kDa fragment, we contrasted neurite outgrowth by CNS and PNS neurons on three synthetic peptides representing sequences from this fragment of FN: two heparin-binding peptides, FN-C/H I and FN-C/H II (McCarthy et al., 1990), and an integrin-binding peptide, CS1 (Humphries et al., 1987). Spinal cord (SC) neurons, from the CNS, differed from dorsal root ganglion (DRG) neurons, from the PNS, with respect to substratum preference for heparin-binding versus integrin-binding peptides. SC neurite outgrowth was greatest on the heparin-binding peptide FN-C/H II, while DRG neurite outgrowth was greatest on the a4 beta 1 integrin-binding peptide CS1. To test whether the difference in substratum preference was due to differences in the molecular mechanism by which SC and DRG neurons interact with the 33 kDa fragment of FN, anti-beta 1 integrin antibodies and/or soluble heparin were added to the cultures as potential inhibitors of integrin-mediated or proteoglycan-mediated interactions with FN. SC neurite outgrowth was much more sensitive to the effect of heparin than anti-beta 1 integrin, indicating SC neurite outgrowth may involve predominantly a heparin-sensitive mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

NCAM-fibronectin-type-III-domain substrata with and without a six-amino-acid-long proline-rich insert increase the dendritic and axonal arborization of spinal motoneurons

The neural cell adhesion molecule (NCAM) is a modulator of neurite outgrowth in vitro and in vivo. To see if single or tandem extracellular NCAM domains can influence neurite outgrowth, motoneurons from embryonic rat spinal cord were cultured on several NCAM fusion protein substrata. Motoneurons growing on either of two fusion proteins comprising the combined two fibronectin type III homology domains of NCAM with or without a six-amino-acid-long, proline-rich insert (F3I,II+ and F3I,II, respectively) usually developed three or more neurites per cell. Motoneurons grown on NCAM-immunoglobulin domain I (IgI), by contrast, developed many unipolar and bipolar cells, a situation also seen when motoneurons were cultured on control substrata. The neuritic trees of motoneurons grown on F3I,II and F3I,II+ appeared broader and rounder than motoneurons cultured on either control or IgI substrata, and the spreading indices of motoneurons grown on F3I,II and F3I,II+ were significantly lower than when the other substrata were used. Neither of the NCAM-F3 fusion proteins stimulated the outgrowth of single neurites. By contrast, IgI substratum was able to stimulate neurite outgrowth over control substrata. Both NCAM-F3 substrata induced branches in axons and dendrites, whereas IgI substratum did not affect neurite branching significantly. These data indicated that neurite outgrowth and neurite branching on the chosen substrata were not closely linked to each other. Furthermore, the branching characteristics of motoneuron neurites potentially depend on their differentiation states and, possibly, on the conformation of the two NCAM-F3 domains. J. Neurosci. Res. 48:112–121, 1997. © 1997 Wiley-Liss, Inc.