Intracellular signaling is changed after clustering of the neural cell adhesion molecules axonin-1 and NgCAM during neurite fasciculation

Neural cell adhesion molecules of the immunoglobulin/fibronectin type III family on axons have been implicated in promotion of neurite outgrowth, fasciculation, and the mediation of specific cell adhesion. The present study demonstrates that two of these molecules on dorsal root ganglion neurons are associated with distinct protein kinases, axonin-1 with the src-related nonreceptor tyrosine kinase fyn and NgCAM with a casein kinase II-related activity and a serine/ threonine kinase related to S6 kinase. When neurites grew without contacts involving axonin-1 and NgCAM, strong fyn kinase activity was associated with axonin-1, whereas the NgCAM-associated kinase activities were low. Clustering of axonin-1 with NgCAM induced by the formation of cell-cell contacts correlated with a reduction of the axonin-1-associated fyn activity and an increased phosphorylation of NgCAM by the associated casein kinase II-related activity. Thus, axonin-1 and NgCAM trigger distinctive intracellular signals during in vitro differentiation depending on their state of association.     

The Arg-Gly-Asp motif in the cell adhesion molecule L1 promotes neurite outgrowth via interaction with the alphavbeta3 integrin

This review focuses on the mechanisms of control of heart glycolysis under conditions of normal and reduced oxygen supply. The kinetic properties and the biochemical characteristics of control steps (glucose transporters, hexokinase, glycogen phosphorylase and phosphofructokinases) in the heart are reviewed in the light of recent findings and are considered together to explain the control of glycolysis by substrate supply and availability, energy demand, oxygen deprivation and hormones. The role of fructose 2,6-bisphosphate in the control of glycolysis is analysed in detail. This regulator participates in the stimulation of heart glycolysis in response to glucose, workload, insulin and adrenaline, and it decreases the glycolytic flux when alternative fuels are oxidized. Fructose 2,6-bisphosphate integrates information from various metabolic and signalling pathways and acts as a glycolytic signal. Moreover, a hierarchy in the control of glycolysis occurs and is evidenced in the presence of adrenaline or cyclic AMP, which relieve the inhibition of glycolysis by alternative fuels and stimulate fatty acid oxidation. Insulin and glucose also stimulate glycolysis, but inhibit fatty acid oxidation. The mechanisms of control underlying this fuel selection are discussed. Finally, the study of the metabolic adaptation of glucose metabolism to oxygen deprivation revealed the implication of nitric oxide and cyclic GMP in the control of heart glucose metabolism.     

Cerebellar granule cell differentiation in mutant and X-irradiated rodents revealed by the neural adhesion molecule TAG-1

In the external granular layer of the cerebellum, the granule cell precursors express the transient axonal glycoprotein TAG-1, a molecule involved in adhesion and neurite outgrowth. Granule cells express TAG-1 transiently, just as they extend neurites before migrating over the radial glia. The present study aims to investigate whether the expression pattern of TAG-1 is altered when granule cells develop abnormally. We studied in vivo models in which Purkinje and/or granule cell defects occur during postnatal development. These include the cerebellar mutant mice staggerer and lurcher as well as rats irradiated during postnatal development. Neither alterations in Purkinje cell differentiation nor the related granule cell loss in the mouse mutants impairs the ability of the surviving granule cell precursors to express TAG-1. Also, early granule cell loss in the X-irradiated rats do not disturb the TAG-1 expression phase in the patches of surviving granule cell precursors. Ectopic granule cells found in the adult cerebellum of X-irradiated rats do not bear the molecule, although they are located in the most superficial part of the molecular layer, occupied by the immunopositive cells a few days earlier. Thus, TAG-1 marks a very precise stage of granule cell differentiation, and the inward migration process itself is not required for the cessation of the expression. We postulate that TAG-1 may be involved in local differentiation steps restricted to the deep external granular layer such as parallel migratory routes or synchrony of axonal growth.     

Depolarization of cerebellar granule cells increases phosphorylation of rabphilin-3A

Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using 32P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 +/- 13%) (mean +/- SEM) on depolarization of the cells with K+ (56 mM) in the presence of external Ca2+ (1 mM). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 +/- 21%). Inhibitors of Ca2+/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca2+-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K+ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 +/- 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca2+-activated K+ depolarization.     

Molecular cloning and functional expression of gicerin, a novel cell adhesion molecule that binds to neurite outgrowth factor

Gicerin is an integral membrane glycoprotein of about 82 kd that is transiently expressed in the developing CNS. Gicerin was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family of extracellular matrix proteins. By isolating and sequencing a gicerin cDNA, we have found that this protein is a novel member of the immunoglobulin superfamily. The deduced protein (584 amino acids) consists of five immunoglobulin-like loop structures in an extracellular domain, a single transmembrane region, and a short cytoplasmic tail. Cells transfected stably with gicerin cDNA adhered to NOF and aggregated with each other, indicating that gicerin exhibits both heterophilic and homophilic adhesion activities.     

Enhancement of neurite outgrowth by the soluble form of human L1 (neural cell adhesion molecule)

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.     

Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.     

Contribution of neurotrophin-3 to the neuropeptide Y-induced increase in neurite outgrowth of rat dorsal root ganglion cells

Recent studies show that neuropeptide Y acts indirectly, via release of a neurotrophic factor(s) from the spinal cord, to increase the neurite outgrowth of dissociated adult rat dorsal root ganglion cells. This study examines further the neuropeptide Y-induced increase in neurite outgrowth. To characterize the factor(s) mediating the neuropeptide Y-induced increase in neurite outgrowth, we have examined whether antisera to either nerve growth factor or neurotrophin-3 influence the neuropeptide Y-induced increase in neurite outgrowth. Spinal cord slices were incubated with media alone or in combination with 10 nM neuropeptide Y for 2 h at 37 degrees C. The supernatant of spinal cord incubated with neuropeptide Y significantly enhanced the neurite outgrowth of normal dorsal root ganglion cells. Antiserum against nerve growth factor had no effect on the trophic actions of the supernatant. Antiserum against neurotrophin-3, however, significantly attenuated the increase in neurite outgrowth. Consistent with this finding, neurotrophin-3 also increased the percentage of cells with neurites. Transganglionic labelling of A-fibres with choleragenoid-horseradish peroxidase in animals treated intrathecally with neurotrophin-3 for 14 days via an osmotic pump showed that the area of choleragenoid-horseradish peroxidase label expanded into lamina II. In comparison, saline-treated animals had no label in lamina II. In addition, neurotrophin-3-treated animals also had a significant decrease in mechanical nociceptive threshold. The results suggest that neuropeptide Y acts via neurotrophin-3 to mediate an increase in neurite outgrowth of dorsal root ganglion cells. These results have important implications for the mechanisms underlying neuropathic pain.     

Soluble factors from rat basophilic leukemia (RBL-2H3) cells stimulated cooperatively the neurite outgrowth of PC12 cells

Culture media from rat basophilic leukemia cells (RBL-2H3) induced the neurite outgrowth of rat pheochromocytoma PC12 cells, a model system for neuronal differentiation. The extension of the neurite outgrowth was dependent on the culture time of RBL-2H3 cells in the DMEM medium. The DMEM medium conditioned by RBL-2H3 cells for 48 h induced neurite outgrowth of PC12 cells significantly. The neurite extension was much higher than that by medium containing 1 ng/ml nerve growth factor (NGF) but was rather lower than that by medium containing 10 or 50 ng/ml NGF. The neurite extension by 50 ng/ml NGF was completely suppressed by excess anti-NGF antibody (1-1.5 microg/ml), while the extension by culture medium conditioned by RBL-2H3 cells for 48 h was not completely suppressed in the presence of the same amount of anti-NGF antibody. The neurite extension by the culture medium of RBL-2H3 cells was also suppressed by anti-interleukin (IL)-6 antibody (1 microg/ml), although IL-6 itself (20 units) could scarcely induce the neurite outgrowth of PC12 cells. This suggests that IL-6 in the culture medium of RBL-2H3 cells could be effective in inducing the neurite extension in cooperation with NGF. In the presence of an excess of both anti-NGF and anti-IL-6 antibodies, the culture medium of RBL-2H3 cells induced the neurite extension of PC12 cells. This suggests that the action of the various factors from RBL-2H3 cells may be synergistic as far as the neurite outgrowth of PC12 cells is concerned.     

Functional cooperation of beta1-integrins and members of the Ig superfamily in neurite outgrowth induction

Neurite outgrowth is a central aspect of the ontogenetic formation of neural networks and is regulated by distinct groups of cell surface molecules. One protein involved in neurite elongation and fasciculation is the neural Ig superfamily member F11/contactin. We have shown previously that F11 promotes neurite extension of chick tectal neurons by interaction with the tectal receptor NrCAM, a member of the L1 subgroup of the Ig superfamily. By contrast, it does not induce outgrowth of retinal neurons despite the fact that these cells also express NrCAM, suggesting that in retinal cells the F11-NrCAM interaction alone is not sufficient to induce neurite extension. In this report we present a novel image analysis procedure to quantify neurite outgrowth and use it to demonstrate that F11 enhances the fibronectin-induced outgrowth response of embryonic retinal neurons. We reveal that NrCAM is the neuronal receptor mediating the enhanced outgrowth of retinal neurons, whereas the related F11-binding molecule NgCAM is not involved. Furthermore, we provide evidence that a beta1-integrin may represent the fibronectin-dependent receptor that cooperates indirectly with the F11-NrCAM pathway. Our results support the concept of a combinatorial labeling of cells in nervous system histogenesis by different classes of cell surface proteins, in particular by integrins and molecules of the Ig superfamily.     

Expression of the immunoglobulin superfamily cell adhesion molecule F3 by oligodendrocyte-lineage cells

We have analysed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins by oligodendrocyte-lineage cells. Biosynthetic labeling of mouse oligodendroglial primary cultures and an oligodendroglial precursor cell line demonstrated that these cells synthesise a variety of different GPI-anchored proteins. GPI-anchored proteins were isolated as a bulk preparation from the precursor cell line, and the individual proteins separated by 2D gel electrophoresis and analysed by microsequencing after tryptic digestion of the separated components. One of the most prominent GPI-anchored proteins synthesised by the cell line was identified as the cell adhesion molecule F3, previously thought to be exclusively expressed by neurons. Western blotting and immunoprecipitation with several polyclonal sera confirmed the expression of F3 by oligodendrocyte-lineage cells and demonstrated the presence of F3 in myelin. Double staining with a panel of oligodendrocyte-specific antibodies and anti-F3 antibodies of cerebellar cultures, as well as oligodendrocytes isolated by panning, showed a colocalization of F3 with oligodendrocyte markers. Oligodendrocyte F3 is shown to be susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage, similar to neuronal F3. Northern blots demonstrated that the oligodendroglial F3 mRNA is the same size as the neuronal message; however, no F3 mRNA could be detected in cortical astrocytes and an astrocytic cell line. Thus, in addition to the expression by neurons, the cell-type specificity of F3 expression must be extended to oligodendroglial cells, underscoring the importance of this Ig superfamily member in the nervous system.     

Endothelin-1 increases the levels of mRNA and protein of muscarinic acetylcholine receptors and c-fos mRNA in cerebellar granule cells

Endothelin-1 (ET-1) induced a time- and dose-dependent increase in the levels of mRNA of m2- and m3-muscarinic acetylcholine receptors (mAChRs) in cultured cerebellar granule cells. The levels of immunoprecipitable m3-mAChR protein and total mAChR binding sites were also increased by ET-1 treatment. The up-regulation of m2- and m3-mAChR was blocked by phorbol ester pretreatment to inhibit ET-1-stimulated phosphoinositide hydrolysis and was preceded by an increase in c-fos mRNA levels. Treatments that prevented ET-1-induced c-fos mRNA increase also abolished the subsequent m2- and m3-mAChR mRNA up-regulation, suggesting that c-Fos protein is involved in the ET-1-induced mAChR expression.     

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

Cross talk between adhesion molecules: control of N-cadherin activity by intracellular signals elicited by beta1 and beta3 integrins in migrating neural crest cells

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin-mediated intercellular contacts. In the present study we provide evidence that, in these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to beta1 and beta3 integrins caused rapid N-cadherin-mediated cell clustering. Restoration of stable intercellular contacts resulted essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulated into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. In addition, agents that cause elevation of intracellular Ca2+ after entry across the plasma membrane were potent inhibitors of cell aggregation and reduced the N-cadherin- mediated junctions in the cells. Finally, elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompanied the restoration of intercellular contacts. These results indicate that, in migrating neural crest cells, beta1 and beta3 integrins are at the origin of a cascade of signaling events that involve transmembrane Ca2+ fluxes, followed by activation of phosphatases and kinases, and that ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell-cell and cell-substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis.     

The effect of benomyl on neurite outgrowth in mouse NB2A and human SH-SY5Y neuroblastoma cells in vitro

The commercial fungicide methyl 1-[(butylamino) carbonyl]-1H-benzimidazol-2-ylcarbamate (benomyl) is teratogenic in rats. Its mode of action is believed to be related to its ability to inhibit the polymerization of brain tubulin. In this study its effects were studied in cultured neuronal cells during differentiation and neurite outgrowth. Mouse NB2a and human SH-SY5Y neuroblastoma cells were induced to differentiate by addition of dibutyryl cyclic AMP and at the same time were exposed to various concentrations of benomyl. Benomyl significantly inhibited neurite outgrowth in both cell lines at concentrations of 10(-8) M and above with IC50 values of 5.9 x 10(-7) M and 1.0 x 10(-6) M in the NB2a and SH-SY5Y cells respectively. The results show that benomyl inhibits neuronal cell differentiation at concentrations likely to be achieved during the development of fetal abnormalities in rats in vivo.     

Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-alpha-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-alpha for 24 hr ICAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-alpha treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 microM). The level of HO activity in endothelial cells exposed to heme or TNF-alpha was increased from 24.7 +/- 5.7 pmol bilirubin/mg protein/min in control to 70.0 +/- 9.5 and 36.7 +/- 3.1 pmol bilirubin/mg protein/min in heme- and TNF-alpha-stimulated cells, respectively. These results suggest that upregulation of ICAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.     

Modulation of expression of glucose transporters GLUT3 and GLUT1 by potassium and N-methyl-D-aspartate in cultured cerebellar granule neurons

Depolarization is known to stimulate neuronal oxidative metabolism. As glucose is the primary fuel for oxidative metabolism in the brain, the entry of glucose into neural cells is a potential control point for any regulatory events in brain metabolism. Therefore, the effects of depolarizing stimuli, high K+ and N-methyl-D-aspartate (NMDA), were examined on the functional expression of glucose transporter isoforms GLUT1 and GLUT3 in primary cultured cerebellar granule neurons. Higher levels of glucose transport activity were observed in neurons cultured in 25 mM KCl (K25) compared to those in 5 and 15 mM KCl (K5 and K15). The elevated glucose transport activity correlated with increased levels of GLUT3 protein and, to a lesser extent, GLUT1. Both GLUT3 and GLUT1 were regulated at the level of mRNA expression. Addition of NMDA to K5 and K15 cultures increased both glucose uptake and GLUT3 protein levels, with smaller changes in GLUT1. NMDA effects were not additive with K25 effects. All these changes were observed only with chronic exposure of neurons to high K+ or NMDA; no acute effects on glucose uptake or transporter expression were found. Thus, chronic depolarization of primary cerebellar granule neurons acts as a stimulus for the expression of the neuronal GLUT3 glucose transporter isoform.     

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.