Fragile X syndrome results from the transcriptional silencing of a gene, Fmr1 , that codes for an mRNA-binding protein (fragile X mental retardation protein, FMRP) present in neuronal dendrites. FMRP can act as a translational suppressor, and its own translation in dendrites is regulated by group I metabotropic glutamate receptors (mGluRs). Multiple lines of evidence suggest that mGluR-induced translation is exaggerated in Fragile X syndrome because of a lack of translational inhibition normally provided by FMRP. We characterized the role of FMRP in the regulation of mRNA granules, which sediment as a heavy peak after polysomes on sucrose gradients. In WT mouse brain, FMRP distributed with polysomes and granules. EM and biochemical analyses suggested that the granule fraction itself contained clusters of polysomes. In Fmr1 knockout brain, we observed a significant decrease in the amount of mRNA granules relative to WT mice. This difference appeared to be due to a role of FMRP in regulating the activation of granules during mGluR-induced translation; in vivo administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine increased granule content in Fmr1 knockout mouse brain to levels comparable with those seen in WT brain. In accord with a role of mGluR5 in the regulation of ongoing translation in vivo , we observed that the phosphorylation of several initiation factors in response to application of the mGluR1/5 agonist S -3,5-dihydroxyphenylglycine in vitro was blocked by methyl-6-(phenylethynyl)pyridine. Together, these data suggest that although large, polysome-containing granules can form in the absence of FMRP, their use in response to mGluR-induced translation is exaggerated.

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.

We previously demonstrated that the neural cell adhesion molecule (N-CAM) inhibited the proliferation of cultured rat hippocampal progenitor cells and increased the number of neurons generated. We demonstrate here that the continued presence of fibroblast growth factor 2 along with N-CAM or brain-derived neurotrophic factor over 12 days of culture greatly increased the number of both progenitors and neurons. These progenitor-derived neurons expressed neurotransmitters, neurotransmitter receptors, and synaptic proteins in vitro consistent with those expressed in the mature hippocampus. Progenitor cells cultured on microelectrode plates formed elaborate neural networks that exhibited spontaneously generated action potentials after 21 days. This activity was observed only in cultures grown in the presence of fibroblast growth factor 2 and either N-CAM or brain-derived neurotrophic factor. Analysis of neuronal activity after various pharmacological treatments indicated that the networks formed functional GABAergic and glutamatergic synapses. We conclude that mitogenic growth factors can synergize with N-CAM or neurotrophins to generate spontaneously active neural networks from neural progenitors.

Homophilic binding of the neural cell adhesion molecule (N-CAM) mediates the calcium-independent cell-cell adhesion that is involved in neuronal development. Two hypotheses have been advanced for the mechanism of homophilic binding. Cell-based experiments have implicated each of the five extracellular immunoglobulin (Ig) domains of N-CAM in the homophilic adhesion interaction, and have predicted that the third domain (Ig III) self-associates. The alternative hypothesis is based on solution observations, which implicate a specific antiparallel interaction between the first two Ig domains (Ig I and Ig II). In order to test these hypotheses, we have determined a high-resolution solution structure of recombinant Ig III (sequence derived from chicken N-CAM) and examined the aggregation behavior of isolated Ig domains in solution. The structure shows that Ig III adopts a canonical Ig fold, in which the β strands ABED and A′GFCC′ form two β sheets that are linked by a disulfide bond. In contrast to the demonstrated aggregation of Ig III on solid supports, we were unable to demonstrate self-association of Ig III under any of a variety of solution conditions. The structure shows that the surface of Ig III is dominated by two large acidic patches, which may explain our failure to observe self-association in solution. To evaluate the involvement of the Ig I-Ig II interaction in cell-cell adhesion, we designed a point mutation in Ig I (F19S) that proved sufficient to abrogate the Ig I-Ig II interaction seen in solution. However, the introduction of this mutation into full-length N-CAM expressed in COS-7 cells failed to affect N-CAM-mediated cell-cell adhesion. The inability to observe Ig III self-association in solution, combined with the failure of the F19S mutation to affect N-CAM-mediated cell-cell adhesion, suggests that, although solution studies can give important insights into the structures of individual domains, the interactions observed in solution between the domains may not be representative of the interactions that occur on the cell surface.

The neural cell adhesion molecule, N-CAM, is expressed on the surface of astrocytes and neurons, and N-CAM homophilic binding has been shown to alter gene expression in both of these cell types. To determine mechanisms by which N-CAM regulates gene expression, we have analyzed DNA binding of and transcriptional activation by NF-kappa B after N-CAM binding to the cell surface. Addition of purified N-CAM, the recombinant third immunoglobulin domain of N-CAM, or N-CAM antibodies either to neonatal rat forebrain astrocytes or to cerebellar granule neurons increased NF-kappa B/DNA binding activity in nuclear extracts as measured by electrophoretic mobility shift assays. Analysis using supershifting antibodies indicated that, in both cell types, p50 and p65 but not p52, c-Rel, or Rel B were contained in the NF-kappa B-binding complex. NF-kappa B-mediated transcription was increased after N-CAM binding to astrocytes and neurons as demonstrated by the activation of two different luciferase reporter constructs containing NF-kappa B-binding sites. N-CAM binding also resulted in degradation of I kappa B-alpha protein followed by an increase in the levels of I kappa B-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappa B binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappa B, however, did not influence the previously reported ability of N-CAM to inhibit astrocyte proliferation. These observations together support the hypothesis that N-CAM binding activates multiple pathways leading to changes in gene expression in both astrocytes and neurons.

Cell adhesion molecules (CAMs) play important roles during the development of the nervous system. On the basis of our previous observations that binding of the neural CAM (N-CAM) inhibits astrocyte proliferation and alters gene expression, we hypothesized that N-CAM may influence the balance between the proliferation and the differentiation of neural progenitor cells. Rat and mouse hippocampal progenitor cells were cultured and showed dependence on basic FGF for proliferation, immunoreactivity for nestin, the presence of limited numbers of differentiated cells, and the ability to generate glial cells and neurons under different culture conditions. Addition of soluble N-CAM reduced cell proliferation in a dose-dependent manner with no evidence of apoptosis. The inhibition of proliferation by N-CAM was accompanied by an induction of differentiation to the neuronal lineage, as indicated by a twofold increase in the percentage of microtubule-associated protein 2-positive cells even in the presence of mitogenic growth factors. Experiments using hippocampal cells from N-CAM knock-out mice indicated that N-CAM on the cell surface is not required for these effects, suggesting the existence of heterophilic signaling. These results support a role for N-CAM and N-CAM ligands in the inhibition of proliferation and the induction of neural differentiation of hippocampal neural progenitor cells.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.

The neural cell adhesion molecule N-CAM is expressed at key sites during embryonic development and mediates homophilic adhesion between cells both in the embryo and in the adult. N-CAM is expressed in multiple forms and two of the major isoforms differ in their cytoplasmic domains, one (ld form) having an insert of 261 amino acids that is missing in the other (sd form). N-CAM has been previously shown to be palmitoylated, but the sites of acylation have not been localized. We show here that the cytoplasmic domain of the N-CAM became palmitoylated after transfection of a cDNA encoding N-CAM into COS-7 cells, and that this acylation occurs on the four closely spaced cysteines in the cytoplasmic domain of N-CAM. Moreover, when a cDNA encoding only the cytoplasmic domain was transfected into cells, the protein was palmitoylated and associated with membranes even though it lacked a membrane spanning segment. Site directed mutagenesis of the four cysteine residues to serines at positions 5, 11, 16, and 22 in the cytoplasmic domain (723, 729, 734, and 740 in the native protein) eliminated both the palmitoylation and association with the membrane fraction. Mutagenesis of the cysteines individually, in pairs, and in groups of three indicated that C5 is not acylated with either palmitate or oleate, but the other three cysteines are acylated to different extents. Cytoplasmic domains with single cysteine mutations localized primarily in the membrane fraction, while those with three mutations were found primarily in the cytoplasm. Proteins containing two mutated cysteines were found in both the cytoplasm and the membrane fraction with C11 and C16 having the most influence on the distribution in accord with their higher level of acylation. Mutation of the cysteines did not affect the ability of full-length N-CAM to promote aggregation when transfected into COS-7 cells. Based on these results we suggest that the primary role of palmitoylation is to provide a second anchor in the plasma membrane to direct the protein to discrete membrane microdomains or to organize the cytoplasmic region for interaction with factors that affect signaling events resulting from N-CAM mediated adhesion.

The neural cell adhesion molecule (N-CAM) inhibits astrocyte proliferation in vitro and in vivo , and this effect is partially reversed by the glucocorticoid antagonist RU-486. The present studies have tested the hypothesis that N-CAM-mediated inhibition of astrocyte proliferation is caused by homophilic binding and involves the activation of glucocorticoid receptors. It was observed that all N-CAM Ig domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other N-CAM-expressing cells also was inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings support the hypothesis that it is binding of soluble N-CAM to N-CAM on the astrocyte surface that leads to decreased proliferation. Signaling pathways stimulated by growth factors include activation of mitogen-activated protein (MAP) kinase. Addition of N-CAM inhibited MAP kinase activity induced by basic fibroblast growth factor in astrocytes. In accord with previous findings that RU-486 could partially prevent the proliferative effects of N-CAM, inhibition of MAP kinase activity by N-CAM was reversed by RU-486. The ability of N-CAM to inhibit astrocyte proliferation was unaffected, however, by agents that block the ability of N-CAM to promote neurite outgrowth. Together, these findings indicate that homophilic N-CAM binding leads to inhibition of astrocyte proliferation via a pathway involving the glucocorticoid receptor and that the ability of N-CAM to influence astrocyte proliferation and neurite outgrowth involves different signal pathways.

The neural cell adhesion molecule (N-CAM) mediates homophilic binding between a variety of cell types including neurons, neurons and glia, and neurons and muscle cells. The mechanism by which N-CAM on one cell interacts with N-CAM on another, however, is unknown. Attempts to identify which of the five immunoglobulin-like domains (Ig I-V) and the two fibronectin type III repeats (FnIII 1-2) in the extracellular region of N-CAM are involved in this process have led to ambiguous results. We have generated soluble recombinant proteins corresponding to each of the individual immunoglobulin domains and the combined FnIII 1-2 and prepared polyclonal antibodies specific for each. The purified proteins and antibodies were used in aggregation experiments with fluorescent microspheres and chicken embryo brain cells to determine possible contributions of each domain to homophilic adhesion. The recombinant domains were tested for their ability to bind to purified native N-CAM, to bind to each other, and to inhibit the aggregation of N-CAM on microspheres and the aggregation of neuronal cells. Each of the immunoglobulin domains bound to N-CAM, and in solution all of the immunoglobulin domains inhibited the aggregation of N-CAM-coated microspheres. Soluble Ig II, Ig III, and Ig IV inhibited neuronal aggregation; antibodies against whole N-CAM, the Ig III domain, and the Ig I domain all strongly inhibited neuronal aggregation, as well as the aggregation of N-CAM-coated microspheres. Of all the domains, the third immunoglobulin domain alone demonstrated the ability to self-aggregate, whereas Ig I bound to Ig V and Ig II bound to Ig IV. The combined FnIII 1-2 exhibited a slight ability to self-aggregate but did not bind to any of the immunoglobulin-like domains. These results suggest that N-CAM-N-CAM binding involves all five immunoglobulin domains and prompt the hypothesis that in homophilic cell-cell binding mediated by N-CAM these domains may interact pairwise in an antiparallel orientation.

After a penetrating lesion in the central nervous system, astrocytes enlarge, divide, and participate in creating an environment that adversely affects neuronal regeneration. We have recently shown that the neural cell adhesion molecule (N-CAM) partially inhibits the division of early postnatal rat astrocytes in vitro. In the present study, we demonstrate that addition of N-CAM, the third immunoglobulin-like domain of N-CAM, or a synthetic decapeptide corresponding to a putative homophilic binding site in N-CAM partially inhibits astrocyte proliferation after a stab lesion in the adult rat brain. Animals were lesioned in the cerebral cortex, hippocampus, or striatum with a Hamilton syringe and needle at defined stereotaxic positions. On one side, the lesions were concomitantly infused with N-CAM or with one of the N-CAM-related molecules. As a control, a peptide of the same composition as the N-CAM decapeptide but of random sequence was infused on the contralateral side of the brain. We consistently found that the population of dividing astrocytes was significantly smaller on the side in which N-CAM or one of the N-CAM-related molecules was infused than on the opposite side. The inhibition was greatest in the cortical lesion sites (approximately 50%) and was less pronounced in the hippocampus (approximately 25%) and striatum (approximately 20%). Two weeks after the lesion, the cerebral cortical sites infused with N-CAM continued to exhibit a significantly smaller population of dividing astrocytes than the sites on the opposite side. When N-CAM and basic fibroblast growth factor, which is known to stimulate astrocyte division in vitro, were coinfused into cortical lesion sites, astrocyte proliferation was still inhibited. These results suggest the hypothesis that, by reducing glial proliferation, N-CAM or its peptides may help create an environment that is more suitable for neuronal regeneration.

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Many significant advances have been made recently in our understanding of the structure and function of cell adhesion molecules (CAMs). The most provocative, however, are those that indicate that CAM-mediated adhesion may lead to changes in gene expression and those that suggest that the expression of CAM genes may be regulated by the products of Hox and related genes.

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.