Neuritogenic and survival-promoting effects of the P2 peptide derived from a homophilic binding site in the neural cell adhesion molecule

The neural cell adhesion molecule (NCAM) plays a pivotal role in neural development, regeneration, and plasticity. NCAM mediates adhesion and subsequent signal transduction through NCAM-NCAM binding. Recently, a peptide ligand termed P2 corresponding to a 12-amino-acid sequence in the FG loop of the second Ig domain of NCAM was shown to mimic NCAM homophilic binding as reflected by induction of neurite outgrowth in hippocampal neurons. We demonstrate here that in concentrations between 0.1 and 10 microM, P2 also induced neuritogenesis in primary dopaminergic and cerebellar neurons. Furthermore, it enhanced the survival rate of cerebellar neurons although not of mesencephalic dopaminergic neurons. Moreover, our data indicate that the protective effect of P2 in cerebellar neurons was due to an inhibition of the apoptotic process, in that caspase-3 activity and the level of DNA fragmentation were lowered by P2. Finally, treatment of neurons with P2 resulted in phosphorylation of the ser/thr kinase Akt. Thus, a small peptide mimicking homophilic NCAM interaction is capable of inducing differentiation as reflected by neurite outgrowth in several neuronal cell types and inhibiting apoptosis in cerebellar granule neurons.     

Phosphorylation of serine 774 of the neural cell adhesion molecule is necessary for cyclic adenosine monophosphate response element binding protein activation and neurite outgrowth

The neural cell adhesion molecule (NCAM) plays a fundamental role during development and regeneration. NCAM is expressed in three major isoforms, two of them with intracellular domains of different length and one without any intracellular domain. The cytoplasmic domain of NCAM contains, depending on the isoform, up to 49 phosphorylation sites, and it has been demonstrated previously by phosphoproteomic analysis that NCAM is phosphorylated on serine 774. However, the impact of NCAM phosphorylation is unclear. Here we have analyzed the phosphorylation of serine 774 in more detail and found that phosphorylation of this site is crucial for NCAM-mediated signal transduction. A serine-to-alanine exchange at position 774 (NCAM140-S774A) resulted in decreased activation of the cAMP response element binding protein (CREB) after NCAM stimulation and, as a consequence, in decreased neurite outgrowth of NCAM140-S774A-transfected B35 neuroblastoma cells.     

Cytoplasmic domain of NCAM 180 reduces NCAM-mediated neurite outgrowth

The neural cell adhesion molecule (NCAM) is one of the best-characterized cell adhesion molecules of the immunoglobulin superfamily. In the nervous system, NCAM is involved in cell migration, axon fasciculation and in neurite outgrowth. Neurite outgrowth is mediated by homophilic NCAM-NCAM interactions. Alternative splicing generates three major isoforms of NCAM differing in their intracellular portion. Two of them, NCAM 180 and NCAM 140, are transmembrane proteins with large intracellular domains. The present study is concerned with novel details of the intracellular domains of NCAM 140 and NCAM 180. We expressed these NCAM isoforms consisting only of the transmembrane and intracellular domains (without extracellular domains) in PC12 cells and elaborated their function in neurite outgrowth assays. Our data demonstrate that membrane-associated NCAM 180 interferes with neurite outgrowth, whereas membrane-associated NCAM 140 promotes neurite outgrowth.     

Neural cell adhesion molecule function is regulated by metalloproteinase-mediated ectodomain release

The neural cell adhesion molecule (NCAM) is involved in development of the nervous system, in brain plasticity associated with learning and memory, and in neuronal regeneration. NCAM regulates these processes by influencing cell adhesion, cell migration, and neurite outgrowth. NCAM activates intracellular signaling upon homophilic NCAM binding, and this is a prerequisite for NCAM-stimulated neurite outgrowth. NCAM is synthesized in three main membrane-bound isoforms, NCAM-120, NCAM-140, and NCAM-180. Soluble forms of NCAM in blood and cerebrospinal fluid have also been found, although the functional significance of these forms remains unclear. In this report, we demonstrate that NCAM can be released from primary hippocampal neurons in culture. The release was enhanced by application of ATP and inhibited by the metalloproteinase inhibitor BB-3103. ATP also induced metalloproteinase-dependent release of all three major NCAM isoforms from NCAM-transfected fibroblastoid L-cells. In this model system, the extracellular ATP-binding site of NCAM was shown not to be necessary for ATP-induced NCAM release. Furthermore, inhibition of serine, cysteine, and aspartic proteinases could not prevent ATP-induced down-regulation of NCAM in L-cells, suggesting that NCAM is cleaved directly by a metalloproteinase. Aggregation of hippocampal neurons in culture was increased in the presence of the metalloproteinase inhibitor GM 6001, consistent with a metalloproteinase-dependent shedding of NCAM occurring in these cells. Moreover, NCAM-dependent neurite outgrowth was significantly reduced by application of GM 6001. Taken together, these results suggest that membrane-bound NCAM can be cleaved extracellularly by a metalloproteinase and that metalloproteinase-dependent shedding of NCAM regulates NCAM-mediated neurite outgrowth.     

Extracellular adenosine triphosphate affects neural cell adhesion molecule (NCAM)-mediated cell adhesion and neurite outgrowth.

The neural cell adhesion molecule (NCAM) plays an important role in synaptic plasticity in embryonic and adult brain. Recently, it has been demonstrated that NCAM is capable of binding and hydrolyzing extracellular ATP. The purpose of the present study was to evaluate the role of extracellular ATP in NCAM-mediated cellular adhesion and neurite outgrowth. We here show that extracellularly added adenosine triphosphate (ATP) and its structural analogues, adenosine-5'-O-(3-thiothiophosphate), beta, gamma-methylenadenosine-5'-triphosphate, beta, gamma-imidoadenosine-5-triphosphate, and UTP, in varying degrees inhibited aggregation of hippocampal neurons. Rat glial BT4Cn cells are unable to aggregate when grown on agar but acquire this capacity when transfected with NCAM. However, addition of extracellular ATP to NCAM-transfected BT4Cn cells inhibited aggregation. Furthermore, neurite outgrowth from hippocampal neurons in cultures allowing NCAM-homophilic interactions was inhibited by addition of extracellular nucleotides. These findings indicate that NCAM-mediated adhesion may be modulated by extracellular ATP. Moreover, extracellularly added ATP stimulated neurite outgrowth from hippocampal neurons under conditions non-permissive for NCAM-homophilic interactions, and neurite outgrowth stimulated by extracellular ATP could be inhibited by a synthetic peptide corresponding to the so-called cell adhesion molecule homology domain (CHD) of the fibroblast growth factor receptor (FGFR) and by FGFR antibodies binding to this domain. Antibodies against the fibronectin type-III homology modules of NCAM, in which a putative site for ATP binding and hydrolysis is located, also abolished the neurite outgrowth-promoting effect of ATP. The non-hydrolyzable analogues of ATP all strongly inhibited neurite outgrowth. Our results indicate that extracellular ATP may be involved in synaptic plasticity through a modulation of NCAM-mediated adhesion and neurite outgrowth.     

NCAM-induced intracellular signaling revisited

The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, synaptic plasticity, and regeneration. NCAM works as "smart glue" that not only mediates cell-cell adhesion but also induces activation of a complex network of intracellular signaling cascades on homophilic or heterophilic binding. Stimulation of NCAM by homophilic interactions induces neuronal differentiation through activation of a number of signaling molecules, including the fibroblast growth factor receptor, non-receptor kinases Fyn and focal adhesion kinase, growth-associated protein-43, the mitogen-activated protein kinase pathway, intracellular Ca(2+), and protein kinases A, C, and G. This review presents and discusses the current knowledge in the area of NCAM signaling with a focus on the events involved in NCAM-mediated neurite outgrowth.     

VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth

Interactions between the neural cell adhesion molecule (NCAM) with NCAM-expressing neurons (trans-interaction) stimulate outgrowth of neurites. The extent of NCAM-triggered neurite outgrowth depends on the presence of 10 amino acids derived from the variable alternatively spliced exon (VASE or π-exon) in the fourth immunoglobulin-like domain of NCAM (Ig4): NCAM with VASE reduces and without VASE enhances neurite outgrowth in cis- or trans-interaction. We have investigated the role of VASE in neurite outgrowth by characterizing the receptors at the cell surface of cultured cerebellar neurons. Results from experiments with L1 and NCAM antibodies and with cerebellar neurons derived from wild-type or NCAM-deficient mice show that substrate-coated Ig4 with VASE (Ig4+) or without VASE (Ig4−) stimulates neurite outgrowth by a trans-interaction with L1 and that Ig4− promotes neurite outgrowth more strongly than Ig4+ by a transinteraction with NCAM. J. Neurosci. Res. 50:62–68, 1997. © 1997 Wiley-Liss, Inc.     

Signal functions of NCAM

The neuronal protein of cell adhesion belongs to the immunoglobulin superfamily of cell adhesion proteins. It consists of an extracellular domain providing homo-and heterophilic interactions with surrounding molecules that are located on the cell surface or are components of the extracellular matrix, a transmembrane part, and intracellular domains (NCAM140 and NCAM180). In addition to its role in cell adhesion, NCAMs act as a signal receptor molecule. Adhesion and initiation of signal cascades induced by binding to the NCAM extracellular domains occur interdependently but influence each other. The homo-and heterophilic binding of NCAM can activate a number of intracellular signal cascades resulting in neurite growth, axone guidance, axone myelinization, and formation of nerve fibers. It has been established that the intracellular signal is initiated by the interactions between NCAMs and fibroblast growth factor receptors (FGFR), non-receptor tyrosine kinases (Fyn and FAK), glia-derived neurotrophic factor (GDNF), ATP, prion proteins, and several other molecules. The review discusses possible mechanisms of functioning of these signal cascades.     

Cyclic guanosine monophosphate signalling pathway plays a role in neural cell adhesion molecule-mediated neurite outgrowth and survival

The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, regeneration, and synaptic plasticity associated with learning and memory consolidation. Homophilic binding of NCAM leads to neurite extension and neuroprotection in various types of primary neurons through activation of a complex network of signalling cascades, including fibroblast growth factor receptor, Src-family kinases, the mitogen-activated protein kinase pathway, protein kinase C, phosphatidylinositol-3 kinase, and an increase in intracellular Ca(2+). Here we present data indicating an involvement of cyclic GMP in NCAM-mediated neurite outgrowth in both hippocampal and dopaminergic neurons and in NCAM-mediated neuroprotection of dopaminergic neurons. In addition, evidence is presented suggesting that NCAM mediates activation of cGMP via synthesis of nitric oxide (NO) by NO synthase (NOS) and activation of soluble guanylyl cyclase by NO, leading to an increased synthesis of cGMP and activation by cGMP of protein kinase G.     

Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule

Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development.     

A soluble version of the receptor-like protein tyrosine phosphatase kappa stimulates neurite outgrowth via a Grb2/MEK1-dependent signaling cascade.

Receptor-like protein tyrosine phosphatase kappa (RPTPkappa) is expressed in the nervous system in a manner consistent with a role in axonal growth and guidance. The extracellular domain of RPTPkappa shares structural features with cell adhesion molecules and can support homophilic adhesion. In the present study we produced a soluble Fc-chimeric protein containing the full extracellular domain of RPTPkappa. Following affinity capture, the RPTPkappa-Fc was shown to promote the aggregation of Covasphere beads, confirming its homophilic binding activity. When added to cultures of cerebellar neurons as a soluble molecule, the RPTPkappa chimera stimulated neurite outgrowth. The neurite outgrowth response was substantially inhibited by a cell-permeable peptide inhibitor of Grb2 and by PD 098059, a drug that has been used to inhibit MEK1 activation in a wide range of cell types. These results demonstrate that RPTPkappa can stimulate neurite outgrowth and provide evidence that this might involve the coupling of Grb2 to a MAPK signal transduction cascade.     

Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon

The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM ± VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM ? VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM ± VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM ? VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM ± VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM ? VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM ? VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a © Wiley-Liss, Inc. significant factor in the reduced axonation capabilities of mature neurons. © 1993 Wiley-Liss, Inc.     

The fibroblast growth factor receptor (FGFR) agonist FGF1 and the neural cell adhesion molecule‐derived peptide FGL activate FGFR substrate 2α differently

Activation of fibroblast growth factor (FGF) receptors (FGFRs) both by FGFs and by the neural cell adhesion molecule (NCAM) is crucial in the development and function of the nervous system. We found that FGFR substrate 2α (FRS2α), Src homologous and collagen A (ShcA), and phospholipase‐Cγ (PLCγ) were all required for neurite outgrowth from cerebellar granule neurons (CGNs) induced by FGF1 and FGL (an NCAM‐derived peptide agonist of FGFR1). Like FGF1, FGL induced tyrosine phosphorylation of FGFR1, FRS2α, ShcA, and PLCγ in a time‐ and dose‐dependent manner. However, the activation of FRS2α by FGL was significantly lower than the activation by FGF1, indicating a differential signaling profile induced by NCAM compared with the cognate growth factor. © 2010 Wiley‐Liss, Inc.     

The neural cell adhesion molecule in synaptic plasticity and ageing

By mediating cell adhesion and signal transduction, the neural cell adhesion molecule (NCAM) regulates neurite outgrowth, fasciculation and target recognition in the developing nervous system. In addition, a number of studies suggest an important role for the NCAM in regeneration and learning in the adult nervous system. NCAM-deficient mice are impaired in spatial learning. Moreover, by interfering with normal NCAM function by intracranial injections of NCAM-antibodies, long-term potentiation (LTP) in rat hippocampal slices and learning in rats and chicks have been inhibited. In the vertebrate nervous system, NCAM is the dominant carrier of polysialic acid (PSA), an unusual carbohydrate consisting of long homopolymers of sialic acid. The PSA-NCAM expression decreases markedly during development. However, an upregulation of polysialic acid (PSA) in restricted brain areas including the hippocampus has been observed following learning. Moreover, enzymatic removal of PSA results in impaired LTP and learning. In muscle, the PSA-NCAM expression is upregulated following denervation. This response is weakened in aging rats. The expression of NCAM and PSA have been shown to be regulated by neuronal activity suggesting that the NCAM may promote structural remodelling in an activity dependent manner associated with learning and regeneration.     

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.     

A peptide derived from a trans‐homophilic binding site in neural cell adhesion molecule induces neurite outgrowth and neuronal survival

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N‐terminal immunoglobulin (Ig)‐like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis‐interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans‐interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding. © 2010 Wiley‐Liss, Inc.     

NCAM mimetic peptides

The neural cell adhesion molecule (NCAM) plays an important role in neuronal differentiation and synaptic plasticity, making it an attractive target for the development of drugs for the treatment of neurodegenerative disorders. NCAM binds to itself (homophilic binding) and to a series of counter-receptors, including the fibroblast growth factor receptor (FGFR), other adhesion molecules, and various extracellular matrix components (heterophilic binding). By means of combinatorial chemistry and based on the unraveling of the structure of NCAM, it has been possible to develop a number of peptides that mimic NCAM homophilic binding. These peptides interfere with cell adhesion and promote differentiation and cell survival. Recently, a peptide mimicking the heterophilic binding to FGFR has also been identified. It binds and activates the receptor, thereby modulating neurite extension and synaptic plasticity.     

The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans.

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.     

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.