Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons.

Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol-anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI-anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes. The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI-PLC) was similar for all the GPI-anchored molecules, which could mean that cell-specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line. Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N-CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.     

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.     

GPI-anchored human placental alkaline phosphatase has a nonpolarized distribution on the cell surface of mouse cerebellar granule neurons in vitro

The glycosyl phosphatidylinositol (GPI) lipid anchor, which directs GPI-anchored proteins to the apical cell surface in certain polarized epithelial cell types, has been proposed to act as an axonal protein targeting signal in neurons. However, as several GPI-anchored proteins have been found on both the axonal and somatodendritic cell-surface domains of a variety of neuronal cell types, the role of the GPI anchor in protein localization to the axon remains unclear. To begin to address the role of the GPI anchor in neuronal protein localization, we used a replication-incompetent retroviral vector to express a model GPI-anchored protein, human placental alkaline phosphatase (hPLAP), in early postnatal mouse cerebellar granule neurons developing in vitro. Purified granule neurons were cultured in large mitotically active cellular reaggregates to allow retroviral infection of undifferentiated, proliferating granule neuron precursors. To more easily visualize hPLAP localization during the sequence of differentiation of single postmitotic granule neurons, reaggregates were dissociated following infection, plated as high-density monolayers, and maintained for 1-9 days under serum-free culture conditions. As we previously demonstrated for uninfected granule neurons developing in monolayer culture, hPLAP-expressing granule neurons likewise developed in vitro through a series of discrete temporal stages highly similar to those observed in situ. hPLAP-expressing granule neurons first extended either a single neurite or two axonal processes, and subsequently attained a mature, well-polarized morphology consisting of multiple short dendrites and one or two axons that extended up to 3 mm across the culture substratum. hPLAP was expressed uniformly on the entire cell surface at each stage of granule neuron differentiation. Thus, it appears that the GPI anchor is not sufficient to confer axonal localization to an exogenous GPI-anchored protein expressed in a well-polarized primary neuronal cell type in vitro; other signals, such as those present in the extracellular domain of these proteins, may be necessary for the polarized targeting or retention of axon-specific GPI-anchored proteins.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions

Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.     

Antibodies to the L1 adhesion molecule inhibit Schwann cell ensheathment of neurons in vitro

To investigate whether neural adhesion molecules are involved in neuron- induced Schwann cell differentiation, cocultures of pure dorsal root ganglion neurons, and Schwann cells were maintained in the presence of antibodies to evaluate possible perturbing effects. Several parameters characteristic of differentiating Schwann cells were studied, such as transition of spindle-shaped to flattened, i.e., more epithelioid morphology, association with neuronal cell bodies, ensheathment of neurites, production of basal lamina and collagen fibrils, and expression of the myelin associated glycoprotein (MAG). A complete ablation of Schwann cell differentiation in all features studied was seen with antibodies to the neural adhesion molecule L1. Antibodies to N-CAM did not reduce the association of Schwann cells with neurites but abolished the interdigitation of Schwann cell processes into neurite bundles, while leaving the other parameters studied unaffected. Fab fragments of antibodies to J1, MAG, and mouse liver membranes did not interfere with the manifestation of any of these parameters. None of the antibodies changed incorporation of [3H]thymidine into Schwann cells.     

Cell adhesion mechanisms in gangliogenesis studied in avian embryo and in a model system

Neural crest cells undergo rapid changes in their cell-to-cell and cell-to-extracellular matrix adhesion during the ontogeny of the peripheral nervous system. The mechanisms of adhesion have been analyzed to assess the respective roles played by the cell adhesion molecules (CAMs) and the differentiated junctions. Crest cells which lose their terminal bar junctions after emigration from the neural tube contain only very few gap junctions during gangliogenesis. The calcium-dependent cell adhesion molecules, L-CAM, disappear from the neural crest and never reappear in crest cell derivatives. In contrast, the number of calcium-independent cell adhesion molecules, N-CAM, diminishes transiently during the migratory phase. In vitro, N-CAM is expressed de novo either just before or at the onset of aggregation into autonomic ganglion rudiments, whereas it is delayed in the dorsal root ganglion cells. In vitro, N-CAM mediates the calcium-independent aggregation mechanism; the rate of aggregation is, however, similar whether crest cells are derived from well-spread cultures or from two- and three-dimensional clusters. Crest cells also exhibit a calcium-dependent mechanism of adhesion controlled by molecules differing from N-CAM but which may codistribute on many different cell types during embryogenesis. These two classes of cell adhesion molecules are present on the surface of neural precursors prior to their differentiation into neurons and glial cells.     

Neural cell adhesion molecule expression is regulated by Schwann cell- neuron interactions in culture

To investigate the cellular and molecular signals underlying regulation of cell adhesion molecule expression, the influence of interactions between dorsal root ganglion neurons and Schwann cells on their expression of L1 and N-CAM was quantitated by immunogold electronmicroscopy. The numbers of antibody binding sites on cell surfaces of neurons and glia were compared between pure populations and co-cultures. After 3 d of co-culture, expression of L1 was reduced by 91% on Schwann cells and 36% on neurons, with expression in pure cultures being taken as 100%. N-CAM expression was unchanged on neurons and reduced by 43% on Schwann cells. Within 3 d after removal of neurons from Schwann cell-neuron co-cultures by immunocytolysis, expression of L1 and N-CAM on Schwann cell surfaces increased by 69 and 84%, respectively. Cell surface antigens recognized by an antibody to mouse liver membranes were unchanged in co-cultures. Furthermore, in co-cultures of neurons and sciatic nerve fibroblasts neither of the three antibodies detected any changes in expression of antigens when pure and co-cultures were compared. These observations suggest that adhesion molecules are not only involved in neuron-Schwann cell recognition and neurite outgrowth on Schwann cells (Seilheimer, B., and M. Schachner. 1988. J. Cell Biol. 107: 341-351), but that cell interactions, in turn, modulate the extent of adhesion molecule expression.     

Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development.

Using a monoclonal antibody that recognizes specifically a high polysialylated form of N-CAM (high PSA N-CAM), the temporal and spatial expression of this molecule was studied in developing spinal cord and neural crest derivatives of mouse truncal region. Temporal expression was analyzed on immunoblots of spinal cord and dorsal root ganglia (DRGs) extracts microdissected at different developmental stages. Analysis of the ratio of high PSA N-CAM to total N-CAM indicated that sialylation and desialylation are independently regulated from the expression of polypeptide chains of N-CAM. Motoneurons, dorsal root ganglia cells and commissural neurons present a homogeneous distribution of high PSA N-CAMs on both their cell bodies and their neurites. Sialylation of N-CAM can occur in neurons after their aggregation in peripheral ganglia as demonstrated for dorsal root ganglia at E12. Furthermore, peripheral ganglia express different levels of high PSA N-CAM. With in vitro models using mouse neural crest cells, we found that expression of high PSA N-CAM was restricted to cells presenting an early neuronal phenotype, suggesting a common regulation for the expression of high PSA N-CAM molecules, neurofilament proteins and sodium channels. Using perturbation experiments with endoneuraminidase, we confirmed that high PSA N-CAM molecules are involved in fasciculation and neuritic growth when neurons derived from neural crest grow on collagen substrata. However, we demonstrated that these two parameters do not appear to depend on high PSA N-CAM molecules when cells were grown on a fibronectin substratum, indicating the existence of a hierarchy among adhesion molecules.     

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM)

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

Differentiation state determines neural effects on microvascular endothelial cells

Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells.     

A neuronal cell surface heparan sulfate proteoglycan is required for dorsal root ganglion neuron stimulation of Schwann cell proliferation

Axons of dorsal root ganglion neurons express on their surfaces one or more proteins which are mitogenic for Schwann cells (Salzer, J., R. P. Bunge, and L. Glaser, 1980, J. Cell Biol., 84:767-778). Incubation of co-cultures of dorsal root ganglion neurons and Schwann cells with 4- methylumbelliferyl-beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, decreases the mitogenic response of the Schwann cell by over 95%. The effect of the beta-D-xyloside has been localized to the neurons; pretreatment of neurons but not of Schwann cells with the inhibitor causes a marked reduction of the mitogenic response. In addition, Schwann cells treated with beta-D-xyloside are still mitogenically responsive to soluble Schwann cell mitogens (cholera toxin and glial growth factor). Neurons treated with heparitinase and membrane vesicles prepared from heparitinase-treated neurons show diminished mitogenicity for Schwann cells, while other proteoglycan lyases have no effect. We conclude that a cell surface heparan sulfate proteoglycan is a component of the Schwann cell mitogen present on the surface of dorsal root ganglion neurons.     

Highly Basic 30- and 32-Kilodalton Proteins Associated with Synapse Formation on Polylysine-Coated Beads in Enriched Neuronal Cell Cultures

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.     

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.     

Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.