The changing role of NCAM as a neurite outgrowth-promoting molecule during development.
Explore the source record for details and available documents.
Aging↗
Biomedical subjects
P Doherty
Publications and source records attributed to P Doherty.
Ganglioside modulation of neural cell adhesion molecule and N-cadherin-dependent neurite outgrowth.
We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.
3T3 Cells↗
Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth.
We present evidence that direct activation of neuronal second messenger pathways in PC12 cells by opening voltage-dependent calcium channels mimics cell adhesion molecule (CAM)-induced differentiation of these cells. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin in the presence of KCl or a calcium channel agonist Bay K 8644. Both potassium depolarization and agonist-induced activation of calcium channels promoted substantial neurite outgrowth from PC12 cells cultured on control 3T3 monolayers and increased neurite outgrowth from those cultured on N-cadherin-expressing 3T3 monolayers. The potassium-induced response could be inhibited by L- and N-type calcium channel antagonists and by kinase inhibitor K-252b but was unaffected by pertussis toxin. In contrast activators of protein kinase C did not stimulate neurite outgrowth, and the neurite outgrowth response induced by activation of protein kinase A was not inhibited by calcium channel antagonists or pertussis toxin. These studies support the postulate that CAM-induced neuronal differentiation involves a specific transmembrane signaling pathway and suggest that activation of this pathway after CAM binding may be more important for the neurite outgrowth response than CAM-dependent adhesion per se.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1.
We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.
3T3 Cells↗
'Is N-CAM a 'non'-cell adhesion molecule?'--Reply.
Explore the source record for details and available documents.
Animals↗
Second messengers underlying cell-contact-dependent axonal growth stimulated by transfected N-CAM, N-cadherin, or L1.
Explore the source record for details and available documents.
Animals↗
Alternative splicing of the cytoplasmic domain of neural cell adhesion molecule alters its ability to act as a substrate for neurite outgrowth.
A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.
Animals↗
Use of the neural cell adhesion molecule VASE exon by neurons is associated with a specific down-regulation of neural cell adhesion molecule-dependent neurite outgrowth in the developing cerebellum and hippocampus.
The development of the CNS is associated with an increasing use of the 30-bp variable alternative, spliced exon (VASE) in neural cell adhesion molecule (NCAM). We have assessed the relative usage of VASE by reverse transcriptase-linked polymerase chain reaction in the developing cerebellum and hippocampus at times when neurons isolated from these tissues can respond to substrate-associated NCAM by increased axonal growth and also at later developmental stages, when they are no longer responsive to substrate-associated NCAM. Neurons isolated from the developing cerebellum at postnatal day 6 respond to NCAM with increased neurite growth. NCAM transcripts from these cells were found to have negligible levels of VASE usage. In contrast, neurons that are isolated at later stages of development (postnatal days 8, 10, and 11) and do not respond to NCAM were found to synthesise a much higher proportion of NCAM transcripts containing VASE. In the hippocampus, embryonic day 18 neurons, which are responsive to NCAM, express low levels of VASE, whereas postnatal days 4 and 5 neurons, which are not responsive to NCAM, have a greater proportion of transcripts containing VASE. Thus, the level of NCAM VASE exon usage by neurons appears to be a good indicator of the ability of these cells to respond to non-VASE-containing NCAM (expressed in a cellular substratum) by increased neurite outgrowth.
Alternative Splicing↗
A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus.
Monolayers of control 3T3 fibroblasts and 3T3 cells expressing transfected NCAM or N-cadherin have been used as a culture substratum for rat hippocampal neurons. Both NCAM and N-cadherin are expressed in the hippocampus through embryonic day 17 (E17) to postnatal day 4 (PND4); however, whereas E17 neurons responded to transfected NCAM by extending considerably longer neurites, PND4 neurons responded very poorly. The converse was true for responsiveness to N-cadherin. These data demonstrate a switch in neuronal responsiveness to NCAM and N-cadherin in the developing hippocampus. NCAM-dependent neurite outgrowth from E17 neurons was largely dependent on the presence of alpha 2-8-linked polysialic acid (PSA) on neuronal NCAM. NCAM-dependent neurite outgrowth could be fully inhibited by pertussis toxin or a combination of L- and N-type calcium channel antagonists thus providing direct evidence concerning the nature of the second messenger pathway activated in primary neurons by cell adhesion molecules (CAMs).
3T3 Cells↗
Morphoregulatory activities of NCAM and N-cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca2+ channels.
We present evidence that the morphoregulatory activities of neural cell adhesion molecule (NCAM) and N-cadherin involve activation of intracellular second messenger pathways. PC12 cells were cultured on monolayers of control 3T3 cells or 3T3 cells expressing transfected N-cadherin or NCAM. NCAM and N-cadherin directly induced a transcription-independent change in the morphology of PC12 cells from an adrenal to neuronal phenotype and also specifically increased Thy-1, but not L1/NILE or low affinity NGF receptor, immunoreactivity. The morphological response was more rapid and, in the case of N-cadherin, more substantial than that induced by NGF. It could be fully inhibited by pertussis toxin and a combination of L- and N-type Ca2+ channel antagonists, but not by broad-specificity kinase inhibitors. It was blocked, however, by the kinase inhibitor K-252b. These studies suggest that cell adhesion molecules directly alter cell phenotype and provide direct evidence for transmembrane signaling mediating both the morphological and biochemical responses induced by NCAM and N-cadherin.
3T3 Cells↗
Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barré syndrome.
In a retrospective study, we have analysed sera from a well-characterised Guillain-Barré syndrome (GBS) patient group for antibodies that react with gangliosides. Of 95 GBS patients and 85 control patients analysed, we found that 14 (15%) of GBS patients but only one control patient had antibodies that react with the gangliosides GM1 and/or GD1b but not GM2, GD1a and GT1b using a sensitive enzyme-linked immunosorbent assay (ELISA). This pattern of reactivity suggests binding to the carbohydrate structure Gal(beta 1-3)GalNAc which is shared between some glycolipids and glycoproteins. Similar antibodies have been found previously in a subpopulation of patients with lower motor neuron disease. In the present study, the predominant immunoglobulin class of these anti-glycoconjugate antibodies was IgG rather than IgM. A correlation was found between the presence of these antibodies and prognosis in terms of disability at 3 and 12 months after presentation. Patients with anti-glycoconjugate antibodies also had a higher incidence of previous Campylobacter infections than the rest of the patient group, although the significance of this remains to be determined.
Antibodies↗
Glycosylphosphatidylinositol anchored recognition molecules that function in axonal fasciculation, growth and guidance in the nervous system.
A large number of glycoproteins in the central nervous system are attached to the cell membrane via covalent linkage to glycosylphosphatidylinositol (GPI). Many of them, including the drosophila fasciclin 1 as well as the mammalian glycoproteins Thy-1, TAG1, N-CAM and F11,F3, contactin are members of the immunoglobulin gene superfamily. These and other GPI-linked molecules have been implicated in key developmental events including selective axonal fasciculation and highly specific growth to and innervation of target tissues. In model systems fasciclin 1, TAG1 and N-CAM have been shown to be capable of mediating cell-cell adhesion via a homophilic binding mechanism confirming their operational classification as cell adhesion molecules (CAMs). However, of these molecules, only N-CAM has been shown to mediate a complex response (neurite outgrowth) via a homophilic binding mechanism. Whether the other molecules in this family mediate biological responses by binding to themselves and/or other molecules remains to be determined. Studies on N-CAM provide an ideal model system for understanding the function of GPI anchors since alternative splicing of the NCAM gene generates both lipid-linked and transmembrane N-CAM isoforms. Recent studies have shown that neurons can recognise and respond (by increased neurite outgrowth) to both lipid-linked and transmembrane N-CAM isoforms expressed on the surface of non-neuronal cells following transfection with appropriate cDNAs. The major determinant of neuronal responsiveness was the level of N-CAM expression rather than the isoform type. Neurite outgrowth in response to transfected N-CAM is mediated by transmembrane N-CAM isoforms expressed by neurons and this involves the activation of classical second messenger pathways in the neurons. One possibility is that GPI anchors are utilised when a cell has simply to provide recognition or positional information to a second cell whereas transmembrane molecules might be required for cells that actively respond to such information. The hypothesis is compatible with all the known information on N-CAM expression and function and may be extended to other adhesive events.
Amino Acid Sequence↗
Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs.
Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.
Animals↗
Campylobacter neuropathy.
Explore the source record for details and available documents.
Campylobacter Infections↗
The contrasting roles of N-CAM and N-cadherin as neurite outgrowth-promoting molecules.
The neural cell adhesion molecule (N-CAM) is a prominent member of the immunoglobulin gene superfamily of recognition molecules. It operates in a calcium-independent manner to promote cell-cell adhesion. Alternative splicing of a single gene generates more than twenty N-CAM isoforms and these can be further modified by the differential addition of complex N- and O-linked carbohydrates. In contrast, N-cadherin is a major calcium-dependent adhesion molecule in the brain; it is not a member of the immunoglobulin gene superfamily and, as far as we know, exists as a single gene product with no evidence of differential post-translational modification. Both molecules are believed to operate through a homophilic binding mechanism and both are expressed at key developmental times in a number of tissues including the brain. Antibody perturbation experiments suggest that both of the above cell adhesion molecules (CAMs) can support neurite outgrowth over complex cellular substrata such as astrocytes and Schwann cells. In the present review we discuss the use of a molecular genetic approach to study the neurite outgrowth-promoting activity of these molecules. Using this approach we have found that both CAMs are potent inducers of neurite outgrowth from a variety of neurons. However, whereas a critical value of N-CAM expression is required for increased neurite outgrowth, with small increases above this value having substantial effects, N-cadherin promotes neurite outgrowth in a highly linear manner. In addition, whereas N-CAM promotes chick retinal ganglion cell (RGC) neurite outgrowth at E6 but not E11, N-cadherin does so throughout this developmental period.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Long-term effect of mexiletine on left ventricular function and relation to suppression of ventricular arrhythmia.
The effects of oral mexiletine on left ventricular (LV) ejection fraction (EF) and ventricular arrhythmias--and a possible relation between these effects--were evaluated during 3 months of therapy in 29 patients with chronic ventricular premature complexes (VPCs) and a moderately reduced to normal LVEF by 24-hour Holter monitoring and by radionuclide ventriculography at rest and during maximum tolerable exercise testing. After an average titration period of 13 days, a mean daily mexiletine dose of 739 mg was maintained throughout the treatment. At the end of titration and after 3 months of treatment, patients with a baseline LVEF less than or equal to 40% (group 2) responded with a median reduction of the hourly VPC rate by 90 and 81%, respectively, compared with 79 and 72% in those with a baseline LVEF greater than 40% (group 1). Couplets and runs of ventricular tachycardia were almost completely suppressed in nearly all patients. A single patient had a proarrhythmic increase in VPCs during treatment. Compared with baseline, there were no significant changes in resting or exercise LVEF after 1 or 3 months of treatment in either of the 2 groups of patients. No correlation was found between treatment-induced changes in arrhythmia frequency and in resting EF. No symptoms of congestive heart failure developed. The study confirms that long-term use of mexiletine is efficacious and relatively free of cardiac depressant effects even in patients with diminished LV function.
Aged↗
A threshold effect of the major isoforms of NCAM on neurite outgrowth.
Interactions between recognition molecules on the surface of neuronal growth cones and guidance cues present in the local cellular environment are thought to account for the growth of neurites in the highly stereospecific manner that contributes to correct target cell innervation. In vitro assays have been used to identify candidate molecular components of this system, either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. The role of the neural cell adhesion molecule (NCAM) in pathway finding is not fully understood. Some immunological studies support a positive role; others do not, and it has been reported that purified NCAM does not support neurite outgrowth. We have previously shown that an arbitrary biochemical index of neurite outgrowth, the relative level of immunoreactive neurofilament protein, is increased when human and rat dorsal root ganglion neurons are cultured on monolayers of cells expressing transfected human NCAM. But, the complexity of growth precluded a simple morphological analysis and we did not determine the 'dose-response' relationship between NCAM expression and neuronal response. Here, we report on the morphology of rat cerebellar neurons cultured on monolayers of 3T3 cells transfected with complementary DNAs encoding all of the main NCAM isoforms found in cells such as astrocytes, Schwann cells and skeletal muscle. The data indicate that both transmembrane and glycosyl-phosphatidylinositol linked NCAM isoforms are potent substrates for neurite extension. A critical threshold value of NCAM expression is required for increased neurite outgrowth. Above this threshold, small increases in NCAM induce substantial increases in neurite outgrowth.
Animals↗
Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid.
We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.
Animals↗