VASE exon expression alters NCAM-mediated cell-cell interactions

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30-bp VASE exon in the fourth immunoglobulin-like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell-cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM ± the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self-aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co-aggregation assay: cells expressing NCAM — VASE formed aggregates which tended to exclude cells expressing NCAM + VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells. © 1994 Wiley-Liss, Inc.     

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.     

Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells

A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared to cells transfected with expression-vector alone or untransfected cells. However, when injected subcutaneously into nude mice, both NCAM expressing cells and control cells produced invasive tumors. Nude mice injected with NCAM positive cells developed tumors with slower growth rates as compared to those induced by NCAM negative cells. This implies that NCAM may not only be involved in adhesive and motile behaviour of glioma cells, but also in their growth regulation.     

The TINS Lecture. Understanding the roles of Otx1 and Otx2 in the control of brain morphogenesis

The murine homologs of the orthodenticle (otd) gene of Drosophila, Otx1 and Otx2, have an important role in brain morphogenesis.Analysis of Otx1 and Otx2 null mice reveals that Otx1 is required primarily for corticogenesis and sense-organ development,while Otx2 is necessary for specification and maintenance of anterior neural plate as well as for proper gastrulation. Cross-phylum recoveries of Otx1 abnormalities by Drosophila otd, and vice versa, indicate that genetic functions required in mammalian-brain development evolved in a primitive ancestor of flies and mice. Knock-in mouse models in which Otx2 was replaced with Otx1, and vice versa, provide evidence that the existence of Otx1-/- and Otx2-/- divergent phenotypes largely reflects differences in expression patterns rather than in the biochemical activity of OTX1 and OTX2. In evolutionary terms, some of these findings lead us to hypothesize a fascinating and crucial role for Otx genes that contributes to the genetic program required for the specification of the development of the vertebrate head.     

Rapid and efficient electroporation-based gene transfer into primary dissociated neurons

Non-viral gene transfer into neurons has proved to be a formidable task. Here, we describe an electroporation-based method that allows efficient and reliable DNA transfer into dissociated neural cells before they are plated and cultured. In hippocampal neural cells derived from either neonatal mouse or embryonic chicken brains, a high transfection rate was already observed 5 h after transfection, and reached 40-80% in 24 h, as monitored by expression of enhanced green fluorescent protein (eGFP). The level of eGFP expression per cell depended on the amount of DNA used in a gene transfer experiment. The survival and neuritic length of transfected cells resembled that of non-electroporated cells. The transfected neurons showed normal immunostaining for endogenous synaptic protein synaptophysin and the neural cell adhesion molecule (NCAM). Furthermore, efficient gene transfer of the NCAM isoform NCAM140 and eGFP-tagged NCAM140 could be achieved, allowing visualization of NCAM140 expression. Also, a glycosylphosphatidylinositol-anchored eGFP could be efficiently expressed, highlighting lipid rafts without altering electrophysiological properties of transfected neurons. When neurons transfected with green and red fluorescent proteins were cocultured, fine details of their interactions could be revealed in time-lapse experiments. Thus, the method provides a useful tool for elucidation of genes involved in different neuronal functions, including neurite outgrowth, synaptogenesis and synaptic transmission.     

Low-level lead exposure attenuates the expression of three major isoforms of neural cell adhesion molecule

Toxic lead (Pb) exposure poses serious risks to human health, especially to children at developmental stages, even at low exposure levels. Neural cell adhesion molecule (NCAM) is considered to be a potential early target in the neurotoxicity of Pb due to its role in cell adhesion, neuronal migration, synaptic plasticity, and learning and memory. However, the effect of low-level Pb exposure on the specific expression of NCAM isoforms has not been reported. In the present study, we found that Pb could concentration-dependently (1-100 nM) inhibit the expression of three major NCAM isoforms (NCAM-180, -140, and -120) in primary cultured hippocampal neurons. Furthermore, it was verified that levels of all three major isoforms of NCAM were reduced by Pb exposure in human embryonic kidney (HEK)-293 cells transiently transfected with NCAM-120, -140, or -180 isoform cDNA constructs. In addition, low-level Pb exposure delayed the neurite outgrowth and reduced the survival rate of cultured hippocampal neurons at different time-points. Together, our results demonstrate that developmental low-level Pb exposure can attenuate the expression of all three major NCAM isoforms, which may contribute to the observed Pb-mediated neurotoxicity.     

Expression of the neural cell adhesion molecule (NCAM) and polysialic acid during taste bud degeneration and regeneration

Taste receptor cells are replaced throughout life, accompanied by continuing synaptogenesis between newly formed taste cells and first-order gustatory fibers. The neural cell adhesion molecule (NCAM) is expressed by a subset of taste cells in adult rodents and appears on gustatory nerve fibers during development prior to differentiation of the taste buds. We employed antibodies against the extracellular domain of the NCAM polypeptide (mAb 3F4) and against polysialic acid (PSA) residues found on embryonic forms of NCAM (mAb 5A5) to investigate the relationship between the expression of these molecules and the innervation of taste buds in adult rats. In unoperated rats, anti-NCAM recognized a subset of cells within the vallate taste buds and also the fibers of the glossopharyngeal (IXth) nerve, including those innervating the gustatory epithelium. Taste bud cells did not express PSA but mAb 5A5 immunoreactivity was observed on some fibers of the IXth nerve, including a few that entered the taste buds. Bilateral crush of the IXth nerve resulted in the loss of NCAM expression from the gustatory epithelium within 8 days. As IXth nerve fibers reinnervated the epithelium, NCAM expression was seen first in the nerve, followed by increased expression in the epithelium as the taste cells differentiated from their precursors. PSA expression by fibers of the IXth nerve did not return to normal until well after the regeneration of the vallate taste buds. The present results demonstrate that taste cell expression of NCAM is dependent upon innervation by the IXth nerve and that NCAM expression appears in the nerve prior to its expression in the differentiating epithelium during regeneration. The occurrence of a similar temporal sequence in the developing taste system suggests that NCAM could play a role in cell-cell interactions that are important for the differentiation of the taste epithelium. Ongoing taste cell turnover and synaptogenesis between IXth nerve fibers and newly differentiating taste cells also requires recognition and adhesion, in which NCAM could play a role. © 1994 Wiley-Liss, Inc.     

The modulations of NCAM polysialylation state that follow transient global ischemia are brief on neurons but enduring on glia

To investigate the role of polysialylated neural cell adhesion molecule (NCAM PSA)-mediated plasticity after injury, we examined the temporal and spatial expression of NCAM PSA immunoreactivity in the medial temporal lobe following global ischemia. Male Mongolian gerbils were subjected to bilateral common carotid artery occlusion for 5 min and killed at increasing times post-occlusion. The well-characterized delayed CAl pyramidal cell death was observed 5-7 days post-occlusion. At post-occlusion days 1-2 there was a small but significant increase of NCAM PSA-positive hippocampal granule cells followed by an equally significant decrease at post-occlusion day 5. In contrast, a substantial increase in glial PSA expression was observed in all hippocampal regions at 1-7 days post-occlusion that was associated generally with stellate astroglia and specifically with the radial processes of glia traversing the granule cell layer of the dentate gyrus. Administration of the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-ben-zo(F)quinoxaline significantly blocked the ischemia-induced modulation of neuronal and glial NCAM PSA expression. Astroglial NCAM polysialylation became attenuated by 35 days post-occlusion except in the CAI area of cell death. The temporal and regional pattern of polysialylated NCAM expression in the ischemic gerbil hippocampus implicates this neuroplastic marker in mechanisms of neurotrophic-dependent repair/remodeling that ensue following transient interruption of blood flow.     

Different expression of adhesion molecules on stromal cells and endothelial cells of capillary hemangioblastoma

Cell-cell and cell-matrix interactions are essential for many basic functions, including differentiation and development. In pathological conditions such as inflammation and tumorigenesis adhesive events also play a major role. Cellular adhesion is mediated by specific molecules expressed by both normal and neoplastic tissues. Capillary hemangioblastoma is a tumor of controversial origin, characterized by two major components, vacuolated stromal cells and a capillary network. In order to shed light on the differentiation of the stromal cells and the interactions between the two major components of hemangioblastoma we studied the expression of several adhesion molecules by immunocytochemistry. The endothelium-associated adhesion molecules (ICAM-1, ICAM-2, VCAM-1, PECAM-1 and ELAM-1) were expressed by endothelial cells within the tumors, but not by stromal cells. In contrast, the stromal cells showed strong neuronal cell adhesion molecule (NCAM/CD56) expression, further distinguishing them from endothelial cells. In addition, the stromal cells expressed CD44, which is of interest, as this membrane protein is linked to ezrin, a cytoskeleton-associated protein also expressed by stromal cells. We conclude that the stromal cells and endothelial cells of capillary hemangioblastoma exhibit quite divergent expression patterns of adhesion molecules. The NCAM expression in stromal cells suggests neuroectodermal or mesenchymal differentiation of this tumor. In addition, the NCAM expression could contribute to the sometimes problematic differential diagnosis between capillary hemangioblastoma and metastatic renal cell carcinoma of the central nervous system. Received: 17 December 1995 / Revised, accepted: 13 May 1996     

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.     

Differential expression of cell adhesion molecules (CAM), neural CAM and epithelial cadherin in ependymomas and choroid plexus tumors

A series of frozen specimens of 18 ependymomas and 7 choroid plexus tumors were examined for their expression of cell adhesion molecules, such as neural cell adhesion molecule (NCAM), its polysialylated isoforms (PSA NCAM), and epithelial (E-) cadherin, and of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP) and cytokeratin, using various monoclonal and polyclonal antibodies. Normal choroid plexus and ependyma were taken as controls. Anti-E-cadherin immunoreactivity was observed on the basolateral part of most adult choroid plexus and benign choroid plexus papilloma cells. However, a small number of atypical papillomas and carcinoma cells showed anti- E-cadherin immunoreactivity throughout their cell surface membrane. NCAM were not expressed by adult choroid plexus and benign papilloma cells. Only a few cells expressed NCAM and PSA NCAM in developing choroid plexus, atypical papillomas and carcinomas. Cytokeratin expression was always observed in choroid plexus and their tumors; GFAP expression was variable from case to case. In contrast, ependymal cells and their tumors never expressed E-cadherin but strongly expressed NCAM. PSA NCAM was found in ependymomas exhibiting anaplastic features. All ependymomas strongly expressed GFAP and a few demonstrated slight expression of cytokeratin. These data suggest that, besides GFAP and cytokeratin, NCAM and E-cadherin are of potential diagnostic value in distinguishing choroid plexus tumors from ependymomas. E-cadherin and NCAM may play a role in the functional organization of normal choroid plexus and ependyma, respectively. In particular, incomplete or irregular anti-E-cadherin expression in choroid plexus tumors and PSA NCAM immunoreativity in ependymomas and choroid plexus tumors correlates with the emergence of anaplastic histological features.     

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.     

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.     

Expression of neural cell adhesion molecule (NCAM) characterizes a subpopulation of type 1 astrocytes in human optic nerve head

The human optic nerve contains a heterogeneous population of astrocytes. In situ, a specialized subpopulation of astrocytes was distinguished in the adult optic nerve head by expression of neural cell adhesion molecule (NCAM). To further study the biology of astrocytes, we have developed and characterized cells grown from explanted optic nerve heads and myelinated optic nerves as in vitro model systems. Second or third passage cells were processed for immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP) and cell surface epitopes: CD56/NCAM, HNK-1/NCAM, A2B5, and O4. Synthesis and gene expression of NCAM were characterized by Western blot analysis and RNase protection assay. Cells grown from myelinated optic nerves expressing GFAP, but not NCAM or A2B5, were identified as type 1A astrocytes, and cells expressing GFAP and A2B5, but not NCAM, were identified as type 2 astrocytes. Cells grown from explanted optic nerve head expressing GFAP, NCAM, and O4 were identified as type 1B astrocytes. Expression of NCAM by type 1B astrocytes may provide these cells with adhesion properties that allow them specialized responses in their microenvironment. Astrocytes from the lamina cribrosa may form a functional barrier to prevent myelination of the retina. In glaucoma, these astrocytes may be exposed to stresses due to fluctuation in intraocular pressure and therefore participate in the optic nerve changes associated with glaucomatous optic neuropathy. GLIA 20:262–273, 1997. © 1997 Wiley-Liss, Inc.     

Neural cell adhesion molecule differentially interacts with isoforms of the fibroblast growth factor receptor

The fibroblast growth factor receptor (FGFR) can be activated through direct interactions with various fibroblast growth factors or through a number of cell adhesion molecules, including the neural cell adhesion molecule (NCAM). We produced recombinant proteins comprising the ligand-binding immunoglobulin-like modules 2 and 3 of FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4, and found that all FGFR isoforms, except for FGFR4, interacted with NCAM. The binding affinity of NCAM-FGFR interactions was considerably higher for splice variant 'b' than for splice variant 'c'. We suggest that the expression pattern of various FGFR isoforms determines the cell context-specific effects of NCAM signaling through FGFR.