Form Of NCAM Research Articles

Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

Perineuronal nets (PNNs) are implicated in closure of critical periods of synaptic plasticity in the brain, but the molecular mechanisms by which PNNs regulate synapse development are obscure. A receptor complex of NCAM and EphA3 mediates postnatal remodeling of inhibitory perisomatic synapses of GABAergic interneurons onto pyramidal cells in the mouse frontal cortex necessary for excitatory/inhibitory balance. Here it is shown that enzymatic removal of PNN glycosaminoglycan chains decreased the density of GABAergic perisomatic synapses in mouse organotypic cortical slice cultures. Neurocan, a key component of PNNs, was expressed in postnatal frontal cortex in apposition to perisomatic synapses of parvalbumin-positive interneurons. Polysialylated NCAM (PSA-NCAM), which is required for ephrin-dependent synapse remodeling, bound less efficiently to neurocan than mature, non-PSA-NCAM. Neurocan bound the non-polysialylated form of NCAM at the EphA3 binding site within the immunoglobulin-2 domain. Neurocan inhibited NCAM/EphA3 association, membrane clustering of NCAM/EphA3 in cortical interneuron axons, EphA3 kinase activation, and ephrin-A5-induced growth cone collapse. These studies delineate a novel mechanism wherein neurocan inhibits NCAM/EphA3 signaling and axonal repulsion, which may terminate postnatal remodeling of interneuron axons to stabilize perisomatic synapses in vivo.

Neural cell adhesion molecule and its polysialic acid moiety exhibit opposing and linked effects on neuropathic hyperalgesia

Spinal lamina II, where nociceptive C-fibers terminate, expresses high amounts of the polysialylated form of neural cell adhesion molecule (PSA-NCAM). While enzymatic removal of the PSA moiety from NCAM did not affect normal sensitivity to thermal stimuli, it exacerbated nerve injury-induced neuropathic hyperalgesia. The genetic removal of the NCAM core protein also did not alter thermal sensitivity. However in the presence of a peripheral nerve injury, NCAM-null mutants exhibited a complete suppression of thermal hyperalgesia. This strong NCAM mutant phenotype appears to involve the long form of NCAM's cytoplasmic domain, in that it is duplicated by selective genetic deletion of the NCAM-180 isoform. PSA appears therefore to provide a mechanism for modulation of chronic sensory overload, by means of attenuation of the activity of the NCAM-180 isoform, which reduces nociceptive transmission.

Podocalyxin Is a Novel Polysialylated Neural Adhesion Protein with Multiple Roles in Neural Development and Synapse Formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro

Schwann cells, the myelin-forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl-transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase-N stripping of PSA. PSA-positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX-transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co-cultures. Thus, migratory properties of PNS myelin-forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.

Blockade of N-methyl- d-aspartate receptors by phencyclidine causes the loss of corticostriatal neurons

Perinatal administration of the N-methyl- d-aspartate (NMDA) receptor antagonist phencyclidine (PCP) has been reported to produce regionally selective apoptotic cell death in the frontal cortex. The development of certain behavioral abnormalities following PCP treatment suggested that extracortical regions such as the striatum also could be affected. In this study, perinatal PCP treatment caused a marked reduction in striatal, but not hippocampal, staining for polysialic acid–neural cell adhesion molecule (PSA-NCAM), an NMDA-regulated molecule important in synaptogenesis. In order to isolate striatal influences to the cortex, this investigation was continued in vitro using corticostriatal slices. For these experiments we cultured coronal corticostriatal slices from postnatal day 7 rats. After 4 days in vitro, PCP was added for 48 h and then washed out for 24 h before harvesting the tissue. Similar to what was observed in vivo, we found that PCP treatment results in a marked reduction in striatal staining for PSA-NCAM. No change was observed in the mature form of NCAM. In striatal synaptoneurosomes, immunoblot analysis confirmed that the levels of PSA-NCAM and synaptophysin, a molecule often used as a marker of synaptogenesis, were substantially down-regulated by PCP. These effects were prevented by M40403, a superoxide dismutase mimetic that also prevented the PCP-induced terminal dUTP nick-end labeling of DNA fragments that was observed selectively in the cortex. These data suggest that PCP causes cell death by apoptosis selectively in the cortex, but not in the striatum, following either in vivo treatment of perinatal rat pups or in vitro treatment of corticostriatal slices. Further, cortical apoptosis induced by PCP negatively impacts striatal synaptogenesis, a process important in normal neural development. This deficit is probably caused by a reduction in corticostriatal neurotransmission. It is possible that the dysregulation of striatal synaptogenesis contributes to the behavioral abnormalities observed following perinatal PCP administration in vivo.

Non-granule PSA-NCAM immunoreactive neurons in the rat hippocampus

The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) continues to be expressed in the adult hippocampus, mainly in a subset of neurons located in the innermost portion of the granule cell layer. PSA-NCAM immunoreactive neurons have also been described outside this layer in humans, where they are severely reduced in schizophrenic brains. Given this important clinical implication, we were interested in finding whether similar neurons existed in the adult rat hippocampus and to characterize their distribution, morphology and phenotype. PSA-NCAM immunocytochemistry reveals labeled neurons in the subiculum, fimbria, alveus, hilus, and stratum oriens, lucidum and radiatum of CA3 and CA1. They are mainly distributed in the ventral hippocampus, and have polygonal or fusiform somata with multipolar or bipolar morphology. These neurons show long straight dendrites, which reach several strata and even enter the fimbria and the alveus. These dendrites are often varicose, appear devoid of excrescences and apparently do not show spines. Most of these neurons display GABA immunoreactivity and further analysis has shown that a subpopulation expresses calretinin, but not somatostatin, neuropeptide Y, parvalbumin, calbindin or NADPH diaphorase. Our study demonstrates that there is an important subpopulation of PSA-NCAM immunoreactive neurons, many of which can be considered interneurons, outside the rat granule cell layer, probably homologous to those described in the human hippocampus. The presence of the polysialylated form of NCAM in these neurons could indicate that they are undergoing continuous remodeling during adulthood and may have an important role in hippocampal structural plasticity.

Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection.

There is a molecular mimicry between the polysialic acid polysaccharide of bacterial pathogens causing sepsis and meningitis, and the carbohydrate units of the neural cell adhesion molecule NCAM. We investigated whether bacteriophage mutants with catalytically disabled endosialidase, which bind but do not cleave polysialic acid, could recognise and bind to bacterial and eukaryotic polysialic acid. In nitrocellulose dot blot assay the mutant bacteriophages, but not the wild-type phages, remained specifically bound to polysialic acid-containing bacteria including Escherichia coli K1 and K92, group B meningococci, Mannheimia (Pasteurella) haemolytica A2, and Moraxella nonliquefaciens. A minimum binding requirement was determined to be 10 sialyl residues in the polysialic acid chain. In Western blots the mutant phages specifically bound to the embryonic polysialylated form of NCAM, but not to the adult less sialylated form of the molecule. The mutant phages together with secondary anti-phage antibodies were subsequently successfully used in fluorescence microscopy of cultured cells and light microscopy of paraffin-embedded tissue sections as a probe for the eukaryotic polysialic acid. Thus, mutant bacteriophages of meningitis causing bacteria bind to and detect the molecularly mimicked polysialic acid of the neural cell adhesion molecule in host tissues.

Revisiting the function of PSA-NCAM in the nervous system.

Since its first description the polysialylated form of NCAM (PSA-NCAM) is thought to be a major regulator of cell-cell interactions in the nervous system. Over the past few years many crucial questions have been answered concerning PSA biosynthesis and function. Among these are the identification and cloning of the key enzymes that are responsible for its synthesis and the fact that expression of PSA is not restricted to developmental stages but maintained in the adult nervous system. In the adult, PSA has been shown to be not only a marker of structural plasticity but seems to be a major player in these processes. Originally suggested to be a purely anti-adhesive factor, modulating cell-cell interactions in general and by this allowing plasticity, there is now increasing evidence that this might not be the whole story. Instead, it appears possible that PSA-NCAM interacts with secreted signaling molecules and by this fulfills a more instructive function in brain plasticity.

Cellular signaling by neural cell adhesion molecules of the immunoglobulin superfamily.

Neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult. In addition to their adhesive properties, binding of CAMs can affect intracellular signaling. Their ability to influence developmental events, including cell migration, proliferation, and differentiation can therefore result both from their adhesive as well as their signaling properties. This review focuses on the two CAMs for which the most information is known, the neural CAM, N-CAM, and L1. N-CAM was the first CAM to be characterized and, therefore, has been studied extensively. The binding of N-CAM to cells leads to a number of signaling events, some of which result in changes in gene expression. Interest in L1 derives from the fact that mutations in its gene lead to human genetic diseases including mental retardation. Much is known about modifications of the L1 cytoplasmic domain and its interaction with cytoskeletal molecules. The study of CAM signaling mechanisms has been assay-dependent rather than molecule-dependent, with particular emphasis on assays of neurite outgrowth and gene expression, an emphasis that is maintained throughout the review. The signals generated following CAM binding that lead to alterations in cell morphology and gene expression have been linked directly in only a few cases. We also review information on other CAMs, giving special consideration to those that are anchored in the membrane by a phospholipid anchor. These proteins, including a form of N-CAM, are presumed to be localized in lipid rafts, membrane substructures that include distinctive subsets of cytoplasmic signaling molecules such as members of the src-family of nonreceptor protein tyrosine kinases. In the end, these studies may reveal that what CAMs do after they bind cells together may have as profound consequences for the cells as the adhesive interactions themselves. This area will therefore remain a rich ground for future studies.

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain

During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment. J. Comp. Neurol. 420:171–181, 2000. © 2000 Wiley-Liss, Inc.

Enzymatic removal of polysialic acid from neural cell adhesion molecule perturbs the migration route of luteinizing hormone-releasing hormone neurons in the developing chick forebrain.

During development in the chick embryo, luteinizing hormone-releasing hormone (LHRH) neurons migrate along the olfactory nerve from the olfactory epithelium to the forebrain. At embryonic day 5.5 (E5.5) to E6.0, the majority of LHRH neurons begin to enter the medial forebrain and then course dorsocaudally along the forebrain substance just beneath the pia matter in association with the somatostatin (SST)-positive fibers, which branch medially from the SST-positive olfactory nerve. By E6.5, the neurons and SST-positive medial branch of the olfactory nerve have proceeded toward the septal area. Intense immunoreactivity for the polysialylated form of neural cell adhesion molecule (PSA-NCAM) on both the LHRH neurons and the SST-positive fibers during this period suggests that this less adhesive form of NCAM is involved in the migratory process. This possibility was examined by using a polysialic acid (PSA)-specific endoneuraminidase. PSA removal did not alter the behavior or appearance of the SST-positive olfactory fibers within the migration pathway. However, it induced a significant deviation of migrating LHRH neurons from the regular path in the forebrain. The effect of PSA removal is more likely to involve changes in the interaction of the migrating neurons with a subset of the SST-positive olfactory fibers and/or other elements in the forebrain rather than an alteration in the pattern of their axonal substrate. On the basis of these results, it is suggested that PSA contributes to the specific pattern of LHRH neuronal migration in the forebrain by limiting interaction of these LHRH neurons with their surrounding environment.

Emergence of oligodendrocytes from human neural spheres.

To study the development of human oligodendrocyte precursors (OP), we expanded human embryonic brain-derived neural precursors into spheres with basic fibroblast growth factor (FGF2). Over 90% of the cells in the expanded spheres were precursors coexpressing nestin and the polysialylated (PSA) form of NCAM. The remaining cells were mostly astrocytes and neuronal cells located at the periphery of the floating spheres. When spheres were allowed to adhere on fibronectin-coated substrate in the absence of FGF2, neural precursors migrated in the outgrowth and often formed chains of cells expressing high levels of PSA-NCAM. Many migrating cells also expressed beta-3 tubulin while only scattered elongated cells radiating from the spheres were GFAP+ astrocytes. Spindle-shaped cells not associated with the chains were labeled for the PDGF-alpha receptor and often coexpressed MAP2 neuronal isoforms. Neuronal cells in the outgrowth rapidly established a rich neuritic network where OP expressing O4 and DM20/proteolipid antigens appeared. T3 treatment of neural spheres increased the rate of OP formation and the complexity of their shape. Thus, the generation of human oligodendrocytes from neural precursors is tightly correlated with growth of neuronal processes and enhanced by hormonal signals.

Supportive Role of Cellular Bridge of Neurons Expressing a Highly Polysialylated Form of NCAM (NCAM-H) at the Initial Stage of Migration of LHRH Neurons

The early stage of cell migration from the olfactory placode to the forebrain was studied immunohistochemically in chick embryos to investigate the nature of early migrated cells and the role of these cells in the sebsequent migration of luteinizing hormone releasing hormone (LHRH) neurons. The initial cells migrating from the olfactory placode stained strongly positive for the highly polysialylated neural cell adhesion molecule (NCAM-H), but were negative for LHRH. These migrating NCAM-H immunoreactive cells were observed on embryonic day (ED) 2.5 as a bulge with a few cells from the base of the placode. By ED 3, they formed a wide cellular strand and developed into a cellular bridge between the olfactory placode and the ventro-rostral surface of the forebrain. These migrating cells are neurons because they stained positive for growth associated protein-43 (GAP-43), microtubule associated protein (MAP) 2, and MAP 5. Some of these cells seemed to migrate caudally along the ventro-lateral surface of the forebrain. LHRH-immunoreactive cells were not detected in the olfactory epithelium until after ED 3.5, which was one day after formation of the cellular bridge. Then, LHRH-immunoreactive cells appeared and began to cross the cellular bridge. The outgrowth of the olfactory nerve axon bundles from the olfactory epithelium was detected at around the same time when LHRH-immunoreactive cells first appeared. These olfactory nerve axons expressed NCAM-H, GAP-43, and MAP 5, as assessed by immunochemistry. After bundle formation, the olfactory nerve appeared to provide the migratory routes for LHRH neurons. These results suggest that the cellular bridge formed by the NCAM-H-immunoreactive neurons plays an important supportive role for LHRH neurons at the initial stage of their migration in chick embryos.

Network of tangential pathways for neuronal migration in adult mammalian brain.

Cells in the brains of adult mammals continue to proliferate in the subventricular zone (SVZ) throughout the lateral wall of the lateral ventricle. Here we show, using whole mount dissections of this wall from adult mice, that the SVZ is organized as an extensive network of chains of neuronal precursors. These chains are immunopositive to the polysialylated form of NCAM, a molecule present at sites of plasticity, and TuJ1, an early neuronal marker. The majority of the chains are oriented along the rostrocaudal axis and many join the rostral migratory stream that terminates in the olfactory bulb. Using focal microinjections of DII and transplantation of SVZ cells carrying a neuron-specific reporter gene, we demonstrate that cells originating at different rostrocaudal levels of the SVZ migrate rostrally and reach the olfactory bulb where they differentiate into neurons. Our results reveal an extensive network of pathways for the tangential chain migration of neuronal precursors throughout the lateral wall of the lateral ventricle in the adult mammalian brain.

Expression of cell adhesion molecules and common acute lymphoblastic leukaemia antigen in hepatoblastoma.

Hepatoblastoma is an embryonal tumour of the liver, which often contains tissue components with multidirectional differentiation. The occurrence of cell surface antigens in this tumour has not been studied systematically, and we therefore investigated 20 hepatoblastomas for the expression of common acute lymphoblastic leukaemia antigen (CALLA) and cell adhesion molecules (CAMs) in their different tissue components. Epithelial tumour cells of fetal differentiation contained E-cadherin. This protein did not occur in tumour areas with embryonal or mesenchymal differentiation. In contrast, immature embryonal and anaplastic cells expressed CALLA and the hyaluronate receptor (HCAM, CD44). Both fetal and embryonal areas stained irregularly positive for ICAM-1, which, in contrast, was not present on anaplastic cells. Immature fibrous tissue did not contain any of these molecules except for ICAM-1. However, some cells adjacent to, or enclosed in, osteoid were positive for HCAM and NCAM. Like small undifferentiated hepatoblastoma cells, primitive mesenchymal spindle-shaped cells also expressed CALLA, HCAM, and the polysialylated embryonic form of NCAM strongly. This last is not present on other epithelial or mesenchymal tumour cells. Hepatoblastoma cells of varying differentiation express distinct patterns of CAMs and CALLA. Our results give further insight into their histogenesis and cellular interactions and may explain their variable ability for invasive growth and formation of metastases.

Embryonic form of N-CAM and development of the rat corticospinal tract; immuno-electron microscopical localization during spinal white matter ingrowth

The neural cell adhesion molecule N-CAM has been proposed to function in the guidance of outgrowing axons in the peripheral and central nervous system. Light microscopic observations have shown that the embryonic form of N-CAM (200–230 kDa) is present in the ventralmost part of the dorsal funiculus during corticospinal tract (CST) ingrowth in the first postnatal week (Joosten, Dev. Brain Res., 78 (1994) 226–236). Here, the subcellular localization of the embryonic form of N-CAM (E-NCAM) is determined by pre-embedding staining on vibratone sections and by postembedding immunogold-labelling on Epon embedded spinal cord sections. The electron microscopical observations indicate that E-NCAM is present on the outer membrane of CST growth cones as well as other unmyelinated axons which are present in the ventralmost part of the dorsal funiculus. Furthermore, E-NCAM is localized in an irregular patchy way on the outer side of the axonal membrane of small unmyelinated, later arriving CST axons. From these results it may be deduced that E-NCAM is involved in CST tract formation through guidance of outgrowing pioneer CST growth cones along other unmyelinated axons and through mediation of axon fasciculation of later arriving CST axons.

Expression of a NK cell-restricted epitope on decidual large granular lymphocytes.

Decidual large granular lymphocytes (DLGL) are the most abundant lymphoid cell type found in the first trimester maternal decidua. The function of DLGL remains controversial, although freshly isolated DLGL have been shown to exert a weak NK activity. We report here the phenotypic characterization of two DLGL subpopulations by immunofluorescence, using mAb against CD56, PEN5 as well as adhesion molecules potentially involved in cell-cell contact between DLGL and trophoblasts. DLGL are CD56bright and express the CD2, CD11a, CD18, CD38 and CD50 molecules, dimly the CD54 molecule, and poorly the CD102 and CD69 molecules. A strong expression of the polysialylated form of N-CAM (or CD56) was also observed on the surface of DLGL. Finally, 40% of CD56bright DLGL cells express the PEN5 epitope, which is selectively expressed on CD56dim cells NK in the peripheral blood. No other phenotypic difference was detected between the CD56brightPEN5+ and the CD56brightPEN5- DLGL populations. Our results show that DLGL are heterogeneous and suggest that the CD56brightPEN5+ DLGL subset belongs to the classical NK cell lineage.

Expression of adhesion molecules by endovascular trophoblast and decidual endothelial cells: Implications for vascular invasion during implantation

Summary During the process of implantation, maternal spiral arteries within the decidua are invaded by trophoblast cells that adhere to and migrate along the luminal surface of the vascular endothelial cells. This phenomenon resembles the events that occur during the migration of neutrophils into an acute inflammatory site, therefore it is possible that similar mechanisms are involved. Indeed, previous observations have shown that endovascular trophoblast expresses the blood group-related antigen sialyl Le x . In this study, we show, by immunohistology, the expression of both E- and P-selectin by vascular endothelial cells only in the decidua basalis and not in decidua parietalis. In contrast, ICAM-1 is expressed by all vascular endothelium throughout the decidua. Expression of VCAM-1 is variable at the implantation site, and is not expressed by vascular endothelial cells in decidua parietalis. Interestingly, we demonstrate the strong expression of a polysialylated form of NCAM by endovascular trophoblast. Our data suggests that vascular invasion by trophoblast is regulated by the expression of appropriate adhesion molecules which permit interaction between endovascular trophoblast and decidual endothelial cells.

Requirement of polysialic acid for the migration of the O-2A glial progenitor cell from neurohypophyseal explants

While the capacity of O-2A oligodendrocyte progenitors to migrate in cell culture and during in vivo myelin formation is well documented, little is known about factors that regulate the motility of these cells. Here, we report on an in vitro model that allowed us to evaluate the contribution of alpha 2-8 linked polysialic acid (PSA) to O-2A cell motility. Using explant cultures of newborn rat neurohypophysis, we observed that individual glial fibrillary acidic protein (GFAP)-positive cells rapidly disperse from the explants, and that cells of the O-2A lineage predominate in the migratory cell pool. Presumed O-2A progenitor cells had a round or bipolar morphology and presented both A2B5 and GFAP immunoreactivity. When cultured in medium containing 10% fetal calf serum, these cells differentiated into stellate-shaped, A2B5/GFAP-positive type 2 astrocytes. In serum-free medium most of them developed into O4/galactocerebroside-positive oligodendrocytes. O-2A lineage cells were found only in a specific developmental period extending from embryonic day 21 to postnatal day 3. A monoclonal antibody, which recognizes the alpha 2-8 linked PSA, characteristic of the embryonic form of NCAM, revealed immunoreactivity on the surface of O-2A progenitor cells, whereas mature oligodendrocytes, type 2, type 1 astrocytes as well as flat GFAP-negative cells were negative. Treatment of the explants with endoneuraminidase purified from phage K1, which specifically removes PSA from the surface of the cell, resulted in a complete blockade of the dispersion of O-2A lineage population from the explant. The effects of the enzymatic treatment were both selective and reversible: migration of GFAP-negative fibroblast-like cells that are normally PSA negative was not influenced, and upon removal of the enzyme, cells of the O-2A lineage were readily detectable in the migrating population. These results provide direct evidence that alpha 2-8 linked PSA contribute to the motility of O-2A, glial progenitor cells.

In vitro differentiation of E‐N‐CAM expressing rat neural precursor cells isolated by FACS during prenatal development

Most fetal rat brain cells expressing the embryonal, highly sialylated form of the cell adhesion molecule N-CAM (E-N-CAM) are precursor cells, as judged from the absence of marker molecules specific for mature neural cell types. However, the detection of E-N-CAM+ cells in frozen sections does not provide information on the lineage-specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E-N-CAM+ cells were isolated at different times of brain development by fluorescence-activated cell sorting (FACS), using a monoclonal antibody (Mab RB21-7) which specifically recognizes polysialic acid (PSA) residues on E-N-CAM. Double-immunofluorescence analyses showed that the majority of E-N-CAM+ cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21-7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E-N-CAM+ astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E-N-CAM to mature forms of N-CAM was never observed in neurons during cultivation, E-N-CAM+ cells of the astrocyte lineage switched to N-CAM soon after the onset of GFAP expression. A lineage-specific transition of E-N-CAM to mature N-CAM expression is, therefore, suggested for these astrocytic progenitor cells during rat brain development.