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Biomedical subjects

G Rougon

Publications and source records attributed to G Rougon.

At least 19 recordsLinked to original sources
PubMed1999

The interaction between F3 immunoglobulin domains and protein tyrosine phosphatases zeta/beta triggers bidirectional signalling between neurons and glial cells.

F3, a mouse glycosyl-phosphatidylinositol anchored molecule of the immunoglobulin superfamily, is known to influence axonal growth and fasciculation via multiple interactions of its modular immunoglobulin-like domains. We prepared an Fc chimeric molecule (F3IgFc) to identify molecules interacting with these domains and characterize the functional impact of the interactions. We affinity-isolated tenascin-C and isoforms of the proteoglycan-type protein tyrosine phosphatases zeta/beta (PTPzeta/RPTPbeta) from extracts of developing mouse brain. We showed that both PTPzeta/RPTPbeta and tenascin-C can bind directly to F3, possibly in an exclusive manner, with the highest affinity for the F3-PTPzeta/RPTPbeta interaction. We observed a strong binding of F3IgFc-coated fluorospheres to astrocytes in neural primary cultures and to C6 astrocytoma cells, and demonstrated, in antibody perturbation experiments, that F3-Ig binding on astrocytes depends on its interaction with PTPzeta/RPTPbeta. We also found by confocal analysis that tenascin-C and PTPzeta/RPTPbeta were colocalized on astrocytes which suggests a complex interplay of interactions between PTPzeta/RPTPbeta, tenascin-C and F3. We showed that the interaction between PTPzeta/RPTPbeta and F3-Ig-like domains can trigger bidirectional signalling. C6 glia-expressed PTPzeta/RPTPbeta stimulated neurite outgrowth by cortical and cerebellar neurons, whereas preclustered F3IgFc specifically modified the distribution of phosphotyrosine labelling in these glial cells. Both effects could be prevented and/or mimicked by anti-F3 and anti-6B4PG antibodies. These results identify F3 and PTPzeta/RPTPbeta as potential mediators of a reciprocal exchange of information between glia and neurons.

Animals↗
PubMed1999

Adrenalectomy increases neurogenesis but not PSA-NCAM expression in aged dentate gyrus.

Ageing is accompanied by a decline in neurogenesis and in polysialylated isoforms of neural cell adhesion molecule (PSA-NCAM) expression within the hippocampus and by elevated basal levels of circulating corticosterone. In a companion study, we demonstrated that suppression of corticosterone by adrenalectomy increased neurogenesis and PSA-NCAM expression in the dentate gyrus of adult rats. Here we show that adrenalectomy increased neurogenesis in this structure in old rats, as measured by the incorporation of 5-bromo-2'-deoxyuridine in neuronal progenitors. This effect was prevented by corticosterone replacement. In contrast, PSA-NCAM expression remained unchanged in comparison with controls. Thus, in the aged brain, stem cells are still present and able to enter the cell cycle. This may point to ways of protecting or treating age-related cognitive impairments.

Adrenal Glands↗
PubMed1998

Expression of L1 and PSA during sprouting and regeneration in the adult hippocampal formation.

The objective of the present study was to evaluate the expression of polysialic acid (PSA) and the cell adhesion molecule L1 during axonal regeneration and sprouting after injury to the adult rat brain. All animals received a complete lesion of the fimbria-fornix (FF). Grafts of nerve growth factor (NGF)- or beta-galactosidase (betaGal)-producing fibroblasts were placed in the FF lesion cavity and induced septohippocampal cholinergic regeneration or sympathetic tyrosine hydroxylase (TH)-positive sprouting, respectively. Cholinergic regeneration was evaluated from 2 to 8 weeks following grafting of NGF-producing fibroblasts in the FF lesion cavity. In the graft area, choline acetyltransferase (ChAT)-positive fibers expressed L1 and PSA. Once cholinergic axons reached the hippocampal formation (HF), they no longer expressed L1 or PSA. Eight weeks after a lesion of the FF and transplantation of betaGal-producing fibroblasts, TH-positive fibers sprouted in the denervated HF and expressed L1 but not PSA. At the zone of reactive gliosis, PSA but not L1 expression was increased following a lesion of the FF and transplantation of NGF- or betaGal-producing fibroblasts. In animals that received a graft of NGF-producing fibroblasts in the FF lesion cavity, numerous ChAT-positive axons were observed along these areas rich in PSA and reactive astrocytes. Taken together, these results suggest that the expression of PSA and L1 is upregulated on regenerating cholinergic axons during axonal elongation and downregulated upon target innervation. In contrast, TH-positive fibers that sprout in the denervated HF express and maintain their expression of L1. Finally, the expression of PSA in the area of reactive gliosis may contribute to a permissive environment for axonal regrowth.

Age Factors↗
PubMed1998

A functional interaction between the neuronal adhesion molecules TAG-1 and F3 modulates neurite outgrowth and fasciculation of cerebellar granule cells.

F3 and TAG-1 are two closely related adhesion glycoproteins of the Ig superfamily that are both expressed by the axons of cerebellar granule cells. In an in vitro system in which cerebellar granule cells were cultured on monolayers of transfected Chinese hamster ovary (CHO) cells, we show that F3 and TAG-1 interact functionally. F3 transfectants have been shown to inhibit outgrowth and induce fasciculation of granule cell neurites. By contrast TAG-1 transfectants have no effect on these events. However, when TAG-1 is coexpressed with F3, the inhibitory effect of F3 is blocked. Two possible mechanisms may account for this functional interaction: (1) either TAG-1 and F3 compete for the same neuronal receptor, and in favor of this we observed that binding sites for microspheres conjugated with F3 and TAG-1 are colocalized on the granule cell growth cones, (2) or alternatively, F3 and TAG-1 associate in a multimolecular complex after their binding to independent receptors. Extensive co-clustering of F3 with TAG-1 can in fact be achieved by anti-TAG-1 antibody-mediated cross-linking in double-transfected CHO cells. Moreover, F3 coimmunoprecipitates with TAG-1 in Triton X-100-insoluble microdomains purified from newborn brain. These data strongly suggest that F3 and TAG-1 may associate under physiological conditions to modulate neurite outgrowth and fasciculation of the cerebellar granule cells.

Animals↗
PubMed1998

Growth and fate of PSA-NCAM+ precursors of the postnatal brain.

Oligodendrocyte-type 2 astrocyte (O-2A) lineage cells are derived from multipotential stem cells of the developing CNS. Precursors of O-2A progenitors express the polysialylated (PSA) form of the neural cell adhesion molecule (NCAM) and are detected in neonatal rat brain glial cultures. It is unclear how such PSA-NCAM+ "pre-progenitors" are related to neural stem cells and whether they still have the potential to differentiate along several neural lineages. Here we isolated PSA-NCAM+ pre-progenitor cells from glial cultures by immunopanning and found that most of these cells expressed nestin and PDGF-receptor-alpha but not O-2A antigens. PSA-NCAM+ cells synthesized transcripts for fibroblast growth factor (FGF) receptors 1, 2, and 3 and responded to FGF2 by survival and proliferation, growing into large clusters resembling neural spheres. FGF2-induced proliferation of PSA-NCAM+ pre-progenitors was significantly enhanced by thyroid hormone (T3), which on its own did not increase cell survival or mitosis. After adhesion and withdrawal of the mitogen, spheres generated mostly oligodendrocytes and astrocytes but very rarely neurons. PSA-NCAM immunopanned cells grown in epidermal growth factor (EGF) also adopted a mostly glial fate after differentiation. In contrast, PSA-NCAM-negative cells and striatal neonatal stem cells, grown in EGF or FGF2, generated the three CNS cell types. Like neural stem cells, PSA-negative cells generated more oligodendrocytes and fewer neurons when expanded in FGF2 and T3. Thus emergence of PSA-NCAM at the surface of neonatal brain precursors coincides with their restriction to a glial fate. T3 modulates these events by enhancing PSA-NCAM+ pre-progenitor growth in FGF2 and favoring an oligodendrocyte fate.

Animals↗
PubMed1998

Regulated expression of the cell adhesion glycoprotein F3 in adult hypothalamic magnocellular neurons.

F3, a glycoprotein of the immunoglobulin superfamily implicated in axonal growth, occurs in oxytocin (OT)-secreting and vasopressin (AVP)-secreting neurons of the adult hypothalamo-neurohypophysial system (HNS) whose axons undergo morphological changes in response to stimulation. Immunocytochemistry and immunoblot analysis showed that during basal conditions of HNS secretion, there are higher levels of this glycosylphosphatidyl inositol-anchored protein in the neurohypophysis, where their axons terminate, than in the hypothalamic nuclei containing their somata. Physiological stimulation (lactation, osmotic challenge) reversed this pattern and resulted in upregulation of F3 expression, paralleling that of OT and AVP under these conditions. In situ hybridization revealed that F3 expression in the hypothalamus is restricted to its magnocellular neurons and demonstrated a more than threefold increase in F3 mRNA levels in response to stimulation. Confocal and electron microscopy localized F3 in secretory granules in all neuronal compartments, a localization confirmed by detection of F3 immunoreactivity in granule-enriched fractions obtained by sucrose density gradient fractionation of rat neurohypophyses. F3 was not visible on any cell surface in the magnocellular nuclei. In contrast, in the neurohypophysis, it was present not only in secretory granules but also on the surface of axon terminals and glia and in extracellular spaces. Taken together, our observations reveal that the cell adhesion glycoprotein F3 is colocalized with neurohypophysial peptides in secretory granules. It follows, therefore, the regulated pathway of secretion in HNS neurons to be released by exocytosis at their axon terminals in the neurohypophysis, where it may intervene in activity-dependent structural axonal plasticity.

Animals↗
PubMed1998

Defasciculation of neurites is mediated by tenascin-R and its neuronal receptor F3/11.

Fasciculation and defasciculation of axons are major morphogenetic events in the formation of neuronal pathways during development. We have identified the extracellular matrix glycoprotein tenascin-R (TN-R) and its neuronal receptor, the immunoglobulin superfamily recognition molecule F3, as promoters of neurite defasciculation in cerebellar explant cultures. Perturbation of the interaction between these two molecules using both antibodies and an antisense oligonucleotide resulted in increased neurite fasciculation. The domains involved in defasciculation were identified as the N-terminal region of TN-R containing the cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats and the immunoglobulin-like domains of F3. Fasciculation induced by antibodies and the antisense oligonucleotide could be reverted by a phorbol ester activator of protein kinase C, whereas the protein kinase inhibitor staurosporine increased fasciculation. Our observations indicate that defasciculated neurite outgrowth does not only depend on the reduction of the expression of fasciculation enhancing adhesion molecules, such as L1 and the neural cell adhesion molecule (NCAM), but also on recognition molecules that actively induce defasciculation by triggering second messenger systems.

Animals↗
PubMed1998

Complex regulation of the expression of the polysialylated form of the neuronal cell adhesion molecule by glucocorticoids in the rat hippocampus.

The gyrus dentatus is one of the few areas of the brain that continues to produce neurons after birth. The newborn cells differentiate into granule cells which project axons to their postsynaptic targets. This step is accompanied by the transient expression of the polysialylated isoforms of neuronal cell adhesion molecules (PSA-NCAM) by the developing neurons. Glucocorticoid hormones have been shown to inhibit neurogenesis. We noted a functional correlation between PSA-NCAM expression and glucocorticoid action after manipulation of corticosterone levels in the adrenalectomized rat. Adrenalectomy increased neurogenesis, evaluated from the incorporation of 5-bromo-2'-deoxyuridine in neuronal precursors, as well as PSA-NCAM expression. The increase in PSA-NCAM-immunoreactive (IR) cells in the gyrus dentatus, evidenced 72 h following adrenalectomy, persisted for at least a month. It was accompanied by enhanced dendritic arborization of PSA-NCAM-IR cells in the gyrus dentatus and by an increase in number of PSA-NCAM-IR fibres in the CA3 subfield. Neurogenesis was normalized by restitution of diurnal or nocturnal levels of corticosterone, whereas normalization of PSA-NCAM expression was only observed after simulation of the complete circadian fluctuation of the hormone. Our findings reveal the complex action of corticosterone in modulating the expression of PSA-NCAM in the gyrus dentatus of the hippocampal formation. They also highlight the importance of corticosterone fluctuations in the control of neurogenesis and plasticity in this structure.

Adrenalectomy↗
PubMed1998

Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner.

Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.

Amino Acid Sequence↗
PubMed1998

[Polysialylated NCAM in CSF, a marker for invasive medulloblastoma].

We described a double-site enzyme-linked immunosorbent assay (ELISA) to measure polysialic acid neural cell adhesion molecule (PSA-NCAM) level in CSF. Immunocapture of PSA-bearing molecules is first effected by means of a monoclonal antibody (anti-MenB), directed against sialic acid polymers and adsorbed into plastic wells. Linked PSA-NCAM is then revealed by means of a second antibody, directed against an aminoacid sequence of NCAM and labelled with peroxydase. The lowest amount of PSA-NCAM detectable was estimated to be 0.11 microgram/l. This value was considered as the threshold for positivity. PSA-NCAM level was measured using this method in CSF from 29 patients with medulloblastoma. CSF had been collected at different times following tumor excision and stored at--80 degrees C. At the same times, cytological examination in CSF (medulloblastoma metastatic cells) and craniospinal imaging (tomographic scan or MRI) had been performed. PSA-NCAM was never detected in control CSF. For patients in remission, beyond the post-operative period of 1 or 2 months, 18 on 21 exhibited a PSA-NCAM level below the threshold value. For refractory patients, so classified according to the positivity of cytology and/or imaging, whatever the time after the tumor excision, PSA-NCAM was always positive (23/23), while either cytology or imaging were positive less frequently (16/23 for both). For relapses, PSA-NCAM was more frequently positive (6/7) than cytology and imaging (1/7 and 5/7, respectively). We concluded that PSA-NCAM positivity in CSF may be a reliable marker to detect the invasive or metastatic feature of medulloblastoma.

Biomarkers, Tumor↗
PubMed1997

Isolation of a tenascin-R binding protein from mouse brain membranes. A phosphacan-related chondroitin sulfate proteoglycan.

We have isolated a chondroitin sulfate proteoglycan from mouse brain by affinity chromatography with a fragment of the extracellular matrix glycoprotein tenascin-R (TN-R) that comprises the amino-terminal cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats. The isolated chondroitin sulfate proteoglycan has a molecular mass of 500-600 kDa and carries the HNK-1 carbohydrate epitope. Treatment with chondroitinase ABC reveals a major band of approximately 400 kDa and two minor bands at 200 and 150 kDa. Immunoblot analysis relates the molecule to phosphacan but not to the chondroitin sulfate proteoglycans neurocan and versican. Binding of the phosphacan-related molecule to the epidermal growth factor-like repeats of TN-R is Ca2+-dependent. Co-localization of the molecule with TN-R in the retina and optic nerve by immunocytochemistry suggests a functional relationship between the two molecules in vivo. Inhibition of neurite outgrowth from hippocampal neurons by the phosphacan-related molecule in vitro is neutralized by TN-R when coated as a uniform substrate. Furthermore, the phosphacan-related molecule neutralizes growth cone repulsion induced by TN-R coated as a sharp substrate boundary with or without prior treatment with chondroitinase ABC. These observations indicate that TN-R can interact with a phosphacan-related molecule and thereby modulate its inhibitory influence on neuritogenesis.

Animals↗
PubMed1997

Regulation of the polysialylated form of the neural cell adhesion molecule in the developing striatum: effects of cortical lesions.

Early in development, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed by growth cones, neuronal processes, and neuronal cell bodies. In rat striatum, PSA-NCAM expression becomes progressively restricted to pre- and postsynaptic membranes and is undetectable by postnatal day 25 (P25), i.e., after corticostriatal synaptogenesis. This study examined the effects of cortical lesions performed on P14, when the corticostriatal projection is already primarily unilateral and cortical inputs have not yet formed asymmetric synapses on striatal neurons. Rats were killed on P25, and PSA-NCAM expression was examined by immunoblotting and immunohistochemistry with light and electron microscopy. In contrast to the case in controls, PSA-NCAM expression was maintained in the striatum of lesioned pups. Ultrastructural studies showed that PSA-NCAM was present 1) in growth cone-like structures and neuronal processes and 2) in striatal neurons. Together with the presence of growth cones, the observation that the number of asymmetric synapses was unchanged in the denervated striatum suggests that axonal sprouting occurred in response to the lesion. This was confirmed by axonal labeling in the denervated striatum after injection of Fluoro-Ruby in the contralateral cortex. The data indicate that P14 cortical lesions affect PSA-NCAM expression in the developing striatum 1) by inducing a robust axonal plasticity resulting in the presence of immature presynaptic elements that contain PSA-NCAM and 2) by delaying the loss of PSA-NCAM expression in striatal neurons, suggesting that the lesion affects the time course of striatal maturation.

Aging↗
PubMed1997

Signaling events following the interaction of the neuronal adhesion molecule F3 with the N-terminal domain of tenascin-R.

Interaction between the extracellular matrix protein tenascin-R and the neuronal adhesion molecule F3 might be involved in the formation of neuronal networks. In this study, the fragment of tenascin-R comprising epithelial growth factor (EGF)-like repeats and the cysteine-rich NH2 terminal stretch (EGF-L), known to be inhibitory for growing neurites and repellent for growth cones, was used to investigate the signaling events following the F3/EGF-L interaction. We addressed this question using an in vitro test with F3-transfected Chinese hamster ovary (CHO) cells that allowed us to measure the kinetics, magnitude and specificity of the repellent effect resulting from the specific F3/EGF-L interaction. We showed that the repellent effect was counteracted by addition of the serine/threonine kinase and -phosphatase modulators (staurosporine, okadaic acid and H7) but not by modulators of tyrosine kinase or -phosphatases. This result indicates that the intracellular signals activated by the repellent effect involve a serine/threonine kinase pathway. Furthermore, the repellent effect of the EGF-L fragment for growth cones of cultured cerebellar neurons was also abolished by the identical modulators of serine/threonine kinase and -phosphatases. The inhibition of neurite outgrowth from hippocampal neurons by EGF-L was abolished in the presence of the serine threonine-kinase inhibitor H7. These results strongly suggest that the F3/tenascin-R interaction through EGF-L involves an intracellular activation of serine/ threonine kinase(s) in all F3-expressing cells tested.

Animals↗
PubMed1997

Decrease in highly polysialylated neuronal cell adhesion molecules and in spatial learning during ageing are not correlated.

Age-dependent spatial memory impairments have been related to a decline in hippocampal plasticity. Highly polysialylated neuronal cell adhesion molecules (PSA-NCAM) show a strong expression during adulthood within regions associated with neuroplastic events. Furthermore, NCAM molecules have been proposed to mediate neuronal plasticity during learning and memory. The aim of the present study was to examine the effect of ageing on the expression of PSA-NCAM within the hippocampus. To investigate whether age-dependent changes in expression of PSA-NCAM were accentuated in aged rats with learning impairment, animals were in a first step assessed for their cognitive abilities using a Morris water maze. Seven-month-old and 24-month-old-rats were tested for their performance in the Morris water maze. The animals were sacrificed and brain sections were processed for PSA-NCAM immunohistochemistry. Ageing was accompanied by an overall decrease in PSA-NCAM-immunoreactivity (-IR) within the forebrain, presenting a important decrease of the number of PSA-NCAM-IR perikarya within the hippocampus. These results were confirmed by Western blot analysis. No difference in PSA-NCAM immunoreactivity was observed in aged rats with or without spatial learning impairment. It is concluded that although changes in PSA-NCAM accompanied the decrease in cognitive abilities, our data did not evidence a causal relationship between these two parameters.

Aging↗
PubMed1997

Cell biology of polysialic acid.

The unusual carbohydrate polysialic acid (PSA), attached uniquely to neural cell adhesion molecule (NCAM) through a developmentally regulated process, modulates neural cell interactions. Major advances in the past two years have increased our understanding of PSA biosynthesis and regulation. Of particular interest is the cloning of the genes encoding polysialyltransferases (PSTs) and the finding that a single enzyme is able to confer polysialylation to NCAM. The electrical activity of neurons and transmembrane signalling are probably major players in controlling both PSA biosynthesis and its expression at the cell surface. A direct causal relationship between PSA expression and activity-induced synaptic plasticity has been reported.

Animals↗
PubMed1997

Polysialylated neural cell adhesion is involved in target-induced morphological differentiation of arcuate dopaminergic neurons.

We have previously shown that the morphological and biochemical maturation of developing rat hypothalamic dopaminergic neurons is accelerated when they are cocultivated with pituitary intermediate lobe cells, one of their targets. Only two subsets of hypothalamic dopaminergic neurons (arcuate, A12, and periventricular, A14, nuclei) may project to the pars intermedia. In order to determine whether the two populations are equally responsive to coculture conditions, we microdissected the hypothalamus of 17-day-old rat fetuses in two fragments containing cell bodies from the A12 and from the A14 regions, prepared neuronal cultures from both portions and incubated them separately with intermediate lobe cells. The presence of intermediate lobe cells increased tyrosine hydroxylase levels in both dopaminergic neuron subsets, but morphological differentiation was accelerated in dopaminergic neurons originating in the arcuate nucleus only. We then investigated whether physical contact between developing arcuate neurons and their target cells was a prerequisite of the morphological effect by interposing a semipermeable membrane between cultivated neurons and intermediate lobe cells in transwell culture dishes. The morphological effect was no longer observed under transwell coculture conditions, pointing to the involvement of membrane-bound molecules. Accordingly, the stimulating effect of coculture on arcuate dopaminergic neurons was completely abolished by the removal of polysialic acid on neural cell adhesion molecules by endoneuraminidase N treatment. Thus, maturation of A12 and A14 dopaminergic neurons exhibits differential susceptibility to intermediate lobe target cells, and polysialylated-NCAM is required for the contact-dependent effect.

Animals↗
PubMed1997

Expression of PSA-N-CAM in human neuroblastoma cells induced to neuronal differentiation by retinoic acid.

The neural cell adhesion molecule (N-CAM) plays a significant role in the development of the nervous system. Three different isoforms of the molecule have been described, with molecular masses of 180, 140 and 120 kDa, whose differential expression in neurons seems to be related to their state of differentiation. We took advantage of the use of the human neuroblastoma cell line LAN-5, which can be differentiated in vitro by retinoic acid (RA) into neuronal cells, for studying the expression of N-CAM isoforms, and their polysialic acid (PSA) content, at the protein and mRNA levels. Anti-N-CAM polyclonal antibodies recognizing all the N-CAM isoforms and a monoclonal antibody recognizing PSA were used in Western blot experiments with extracts from undifferentiated and RA-differentiated cells. We found that undifferentiated cells express very little of the 180 kDa N-CAM isoform and a large amount of the 140 kDa isoform. A 4-fold increase in the expression of the 180 kDa N-CAM isoform was obtained when LAN-5 cells were differentiated by RA for 8 days, whereas a 1.8-fold increase in the expression of the 140 kDa N-CAM isoform was observed upon differentiation. Similarly, the levels of the 7.4 kb mRNA coding for N-CAM 180 kDa, determined by Northern blot analysis, were barely detectable in undifferentiated cells, and showed a 3.8-fold increase upon differentiation. By contrast, only a 1.3-fold increase in the 6.7 kb mRNA, coding for the 140 kDa N-CAM isoform, was observed. N-CAM was always found in its polysialylated form in both undifferentiated and RA-differentiated cells. This indicates that, in LAN-5 cells, the expression and activity of the polysialytransferase enzyme precedes the acquisition of a neuronal phenotype.

Antibody Specificity↗