Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Rat neural antigen-2 (RAN-2): A cell surface antigen on astrocytes, ependymal cells, Müller cells and lepto-meninges defined by a monoclonal antibody

We have immunized mice with enriched populations of cultured rat astrocytes and fused their spleen cells with NS-1 myeloma cells to generate antibody-secreting hybridomas. We have isolated two stable hybridoma clones which secrete monoclonal IgG₂ antibodies that react with the surface of the great majority of rat astrocytes in culture. We have studied one of these antibodies in indirect immunofluorescence assays and show that it binds to the surface of rat ependymal cells, retinal Müller cells and leptomeningeal cells as well as to astrocytes, but not to cultured neurones, oligodendrocytes, Schwann cells, microglia or various non-neural cells. The antigen defined by this monoclonal antibody is protease-sensitive and rat-specific and we have called it rat neural antigen-2 (Ran-2).We also show that isolated rat ependymal cells and cultured rat Müller cells do not express other neural cell-type-specific markers, such as tetanus toxin receptors, rat neural antigen-1 (Ran-1), galactocerebroside or glial fibrillary acidic protein (GFAP). Nor do these cells express cell surface Fc receptors for IgG, phagocytose latex beads or make detectable amounts of the Thy-1 or fibronectin glycoproteins.     

Transferrin gene expression and secretion by rat brain cells in vitro

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (Tf) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Tf mRNA levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.     

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers. COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.     

Concurrent isolation and characterization of oligodendrocytes, microglia and astrocytes from adult human spinal cord

A cellular preparation of highly enriched oligodendrocytes was obtained from adult human spinal cord by Percoll gradient centrifugation followed by either differential adhesion or fluorescence-activated cell sorting after immunostaining with an antibody against galactocerebroside (Ol). The adherent and O1-negative cell fractions were 96% microglia. The non-adherent and O1-positive fractions were 96% positive for the oligodendrocyte markers O4 and O1, 0–2% positive for glial fibrillary acidic protein, and were devoid of neuronal or microglial markers. If the oligodendrocyte fraction was co-cultured with purified dissociated rat dorsal root ganglion neurons, the oligodendrocytes adhered to the axons and their numbers increased over a 4 week period. However, myelin sheaths were not produced around axons in these cultures. In contrast, if the oligodendrocyte cell fraction was grown alone in culture for 3 weeks, the number of oligodendrocytes decreased and a layer of astrocytes developed underneath the oligodendrocytes. The oligodendrocytes could be eliminated from these cultures by subsequent passaging, thus producing cultures of pure astrocytes. The astrocytes accumulated both K⁺ and glutamate with kinetic properties similar to those reported for rodent astrocytes. We suggest that these astrocytes arose in part from an O4/O1-positive precursor which did not initially express glial fibrillary acidic protein. These results define a relatively simple method by which highly enriched populations of oligodendrocytes, astrocytes and microglia can be obtained from adult human spinal cord.     

Uptake of [3H]GABA by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study

Uptake of [3H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [3H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galactocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [3H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by beta-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [3H]beta-alanine, which labeled only astrocytes. Oligodendroglial [3H]GABA uptake was Na+-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

Oligodendrocyte-derived J1-160/180 extracellular matrix glycoproteins are adhesive or repulsive depending on the partner cell type and time of interaction

We have studied the functional involvement of J1-160 and J1-180 in the interaction between oligodendrocytes and neurons, astrocytes, or L cells in short- and long-term adhesion assays using monoclonal antibodies directed against topographically distinct epitopes on the molecules. Whereas antibodies to mouse liver membranes and monoclonal antibody 597 do not interfere with neuron-oligodendrocyte or astrocyte-oligodendrocyte adhesion after 30 min of coculture, antibodies 596, 619, and 620 interfere with astrocyte to oligodendrocyte and neuron to oligodendrocyte adhesion. The adhesion of L cells to oligodendrocytes is not affected by the antibodies. When neurons or astrocytes are cultured on oligodendrocytes for more than 30 min, monoclonal antibody 619 continues to reduce adhesion of astrocytes to oligodendrocytes after 1 and 2 h. However, during this time period the antibody affects neuron to oligodendrocyte adhesion in a different manner. It does not interfere with adhesion of neurons to oligodendrocytes at 1 h and enhances the adhesion of neurons to oligodendrocytes after 2 h of coculture. After 6 and 24 h of coculture, antibody 619 does not affect the adhesion of neurons or astrocytes to oligodendrocytes, suggesting that other adhesive mechanisms are predominant at later times of interaction. At all times studied, neurons and astrocytes adhered well to the oligodendrocytes. To study the influence of the J1 molecules on neuronal interactions in the absence of other oligodendrocyte-derived cell surface components, purified J1-160 was coated as a substrate and neuron attachment was measured as a function of time. Two hours after plating neurons adhered well to J1-160, as they did to laminin, while cell detachment was subsequently observed from J1-160, but not from laminin. These results implicate J1-160 and J1-180 in a recognition process between oligodendrocytes and neurons or astrocytes, but not fibroblasts. This recognition process appears to merge into adhesion or stabilization of cell contacts for astrocytes and destabilization of cell interactions or repulsion for neurons. It is likely that these two opposite effects in cell behavior elicited by the J1 molecules result from differential intracellular responses to a cell surface trigger possibly mediated by different cell surface receptors and/or different consequences in intracellular signaling networks.     

A cell surface protein of astrocytes, Ran-2, distinguishes non-myelin-forming Schwann cells from myelin-forming Schwann cells

Defining the molecular phenotype of adult glial cells in the peripheral nervous system in situ forms a good basis for subsequent studies on the development of these cells, and for determining the role of neurons in the attainment and maintenance of the mature glial phenotype. We report here the characterization of the glial surface antigen, Ran-2, and describe its distribution in the peripheral and central nervous system of adult rats. Immunoprecipitation of the antigen from cultured astrocytes with monoclonal Ran-2, antibodies, showed that Ran-2 is a protein with an apparent molecular weight of 140,000 daltons. In immunofluorescence studies of teased nerve preparations, Ran-2 was found on the surface of non-myelin-forming Schwann cells in all nerves surveyed, i.e. the sciatic, dorsal and ventral roots, cervical sympathetic trunk and the brachial plexus. In contrast, it was not detected immunohistochemically on myelin-forming Schwann cells. The antigen was also absent, or present at very low levels, on the satellite cells of dorsal root sensory ganglia, although, as we reported previously, it was present on the glial cells of the enteric nervous system. The Ran-2 antigen was also associated with perineurial cells. In short-term cell cultures of the sciatic nerve and cervical sympathetic trunk from 19-day-old rats, Ran-2 could be localized on the surface of individual viable Schwann cells. In the central nervous system, the antigen was present on astrocytes in sections of the optic nerve.     

Cultured human and rat oligodendrocytes and rat Schwann cells do not have immune response gene associated antigen (Ia) on their surface

In order to determine if oligodendrocytes or Schwann cells had surface immune response gene associated antigen (Ia), we studied the binding of: (a) mouse monoclonal antibodies to rat Ia, to cultures of rat oligodendrocytes and Schwann cells; and, (b) mouse monoclonal antibodies to human Ia, to cultures of human oligodendrocytes employing radioimmunoassay and indirect immunofluorescence. Cells were identified using phenotypic markers; rabbit anti-galactocerebroside (GalC) for oligodendrocytes; rabbit anti-GalC and rabbit anti-Schwann cell for Schwann cells; rabbit anti-glial fibrillary acidic protein for astrocytes; rabbit anti-fibronectin for fibro-blasts and leptomeningeal cells, and the capacity to ingest latex particles for macrophage-microglia. Ia could not be detected on the surface of oligodendrocytes, Schwann cells, astrocytes, fibroblasts, or leptomeningeal cells. A small number of latex ingesting cells bound anti-Ia even after blocking of surface Fc receptors.     

Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro

To study the interaction of neurons with CNS glial cells, dissociated sympathetic or sensory ganglion cells or fetal retinal cells were plated onto cultures of dissociated optic nerve glial cells of young rats. Whereas astrocytes favored neuron adhesion and neurite outgrowth, oligodendrocytes differed markedly in their properties as neuronal substrates. Immature (O4+, A2B5+, GalC-) oligodendrocytes were frequently contacted by neurons and neurites. In contrast, differentiated oligodendrocytes (O4+, A2B5-, GalC+) represented a nonpermissive substrate for neuronal adhesion and neurite growth. When neuroblastoma cells or 3T3 fibroblasts were plated into optic nerve glial cultures, the same differences were observed; differentiated oligodendrocytes were nonpermissive for cell adhesion, neurite growth, or fibroblast spreading. These nonpermissive oligodendrocytes were characterized by a radial, highly branched process network, often contained myelin basic protein, and may, therefore, correspond to cells actively involved in the production of myelin-like membranes. Isolated myelin from adult rat spinal cord was adsorbed to polylysine-coated culture dishes and tested as a substrate for peripheral neurons, neuroblastoma cells, or 3T3 cells. Again, cell attachment, neurite outgrowth, and fibroblast spreading was strongly impaired. General physicochemical properties of myelin were not responsible for this effect, since myelin from rat sciatic nerves favored neuron adhesion and neurite growth as well as spreading of 3T3 cells. These results show that differentiated oligodendrocytes express nonpermissive substrate properties, which may be of importance in CNS development or regeneration.     

Type 1 astrocytes inhibit myelination by adult rat oligodendrocytes in vitro

We have determined the effect of Type 1 astrocytes on the myelination of dorsal root ganglion cell axons by oligodendrocytes obtained from adult animals. Experiments were initiated by the addition of oligodendrocytes [purified either by density gradient centrifugation and treatment in culture with 5-fluorodeoxyuridine (FdU) or by fluorescence-activated cell sorting after immunostaining with antigalactocerebroside antibody] to cultures of purified neurons. In control conditions, the added oligodendrocytes proliferate and, after 4 weeks, accomplish substantial myelination of the sensory axons. Type 1 astrocytes (purified from cultures of dissociated newborn rat cerebral cortex by vigorous shaking to remove less adherent cells) or fibroblasts (purified from cultures of cranial periosteum by serial replating) were added to some of these cultures after the oligodendrocytes had attached and started to proliferate. We observed that the added Type 1 astrocytes, but not the added fibroblasts, strongly inhibited myelination and caused decreased oligodendrocyte proliferation or survival. These effects of added Type 1 astrocytes were reproduced with Type 1 astrocyte-conditioned medium. We conclude that Type 1 astrocytes can release soluble factors that inhibit oligodendrocyte myelination.     

Uptake [H]GABA by oligodendrocytes in dissociated brain cell culture: A combined autoradiographic and immunocytochemical study

Uptake of [³H]GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography. Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers. In 12 days in vitro (DIV) cultures [³H]GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes. This was confirmed by complement-dependent antibody-mediated cytotoxicity against galatocerebroside-positive oligodendrocytes. There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes. Heavily labeled astrocytes were only occasionally observed. Oligodendrocytes accumulated [³H]GABA more rapidly than astrocytes but slower than neurons. Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes. The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by β-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake. Oligodendrocytes did not accumulate [³H]β-alanine, which labeled only astrocytes. Oligodendroglial [³H]GABA uptake was Na⁺-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA. The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.     

Tyrosine phosphorylated proteins decrease during differentiation of neuronal and glial cells.

Tyrosine kinases have been implicated in the development of the nervous system. To investigate their role, immunoblotting with phosphotyrosine antibodies was used to identify substrates of tyrosine kinases during glial and neuronal differentiation in the rat and mouse. Fourteen prominent phosphotyrosine-containing proteins were detected in oligodendrocyte-type 2 astrocyte (O2A) progenitor cells. When O2A cells differentiated into type 2 astrocytes, a phosphotyrosine-modified protein of 74 kilodaltons (kDa) decreased 15-fold in abundance, and phosphotyrosine-containing proteins of 36-40 kDa declined. When O2A cells differentiated into oligodendrocytes, a prominent 71-kDa phosphotyrosine-modified protein became undetectable. During retinoic acid-induced neuronal differentiation of the mouse embryonal carcinoma cell line P19S101A1 (P19), an 80-kDa phosphotyrosine-containing protein decreased from high levels in the undifferentiated state to undetectable levels after 96 h in aggregation. Retinoic acid treatment also caused a rapid decrease in levels of a 96-kDa phosphotyrosine-containing protein. Cell-cell contact occurring as a result of aggregation resulted in decreases in 130- and 90-kDa phosphotyrosine-containing proteins in both retinoic acid-induced and control cultures. Cultured rat central nervous system cerebral cortical neurons and peripheral nervous system dorsal root ganglion neurons exhibited prominent phosphotyrosine-modified proteins of 90 and 46 kDa the same sizes as those in P19 neurons. The phosphotyrosine-containing proteins involved in the retinoic acid-induced differentiation of P19 cells to neurons were different from those altered in the glial differentiation of O2A cells to astrocytes or oligodendrocytes, indicating that the tyrosine kinase substrates modified during nervous system differentiation may be cell-type-specific.     

Cell-specific expression of neutral glycosphingolipids in vertebrate brain: immunochemical localization of 3-O-acetyl-sphingosine-series glycolipid(s) in myelin and oligodendrocytes

The tissue- and cell-specific expression of three neutral glycosphingolipids, gangliotetraosylceramide (GA1), gangliopentaosylceramide (GalNAc-GA1), and the novel 3-O-acetyl-sphingosine-series glycolipid (FMC-5), were examined with monospecific polyclonal antibodies. Immunohistochemical studies of rodent brain cross-sections indicated that both GA1 and FMC-5 antibodies stained myelin. In contrast, GalNAc-GA1 antibody distinctly stained neurons in cerebral cortex, but only partially delineated Purkinje cells and other neurons in cerebellum. Preliminary studies of mixed glial cultures suggested the following: 1) both FMC-5 and GA1 antibodies stained oligodendrocytes and oligo progenitors, and 2) GalNAc-GA1 antibody did not stain any cells in the culture. Because the GalNAc-GA1 was associated with neurons, we examined the immunoreactivity of GalNAc-GA1 antibody in primary neuronal cultures. Further studies using primary cultures of rat brain oligodendrocytes, and dissociated cerebellar neuronal cultures indicated that both GA1 and FMC-5 are specifically expressed by oligodendrocytes, whereas GalNAc-GA1 is primarily localized in interneurons and to some extent in Purkinje neurons.     

Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.     

Human and simian glial cells infected by human T-lymphotropic virus type I in culture

Although human T-lymphotropic virus type I (HTLV-I) has been implicated in the etiology of tropical spastic paraparesis (TSP) and HTLV-I associated myelopathy (HAM), the direct infectivity of the virus against constituent cells in the central nervous system remains undetermined. To investigate the neurotropism of HTLV-I, we exposed cultured human and simian glial cells to HTLV-I. Primary mixed glial cell cultures of astrocytes and oligodendrocytes were obtained from adult human and cynomolgus monkey (Macaca fascicularis) brains by an enzyme digestion-Percoll gradient method. After two weeks in vitro, the cells were co-cultured with irradiated MT-2 cells, an HTLV-I-producing T-cell line. Cultures were double stained with antibodies against cell-type specific markers and anti-HTLV-I p19 (gag) monoclonal antibody. The HTLV-I antigen was demonstrated in small numbers of glial fibrillary acidic protein-positive cells (astrocytes) and galactocerebroside-positive cells (oligodendrocytes) in both the human and simian cultures. Electron microscopy demonstrated the presence of type C virus-like particles in the cytoplasm of astrocytes. These results indicate that HTLV-I is capable of infecting human and simian glial cells in vitro.     

LM and EM immunolocalization of the gap junctional protein connexin 43 in rat brain

A site-specific antibody against the principal gap junctional protein in heart (connexin 43) was used to determine immunohistochemically the cellular localization of this protein in rat brain. Structures labelled with the antibody included gap junctional membranes between glial, ependymal, pial and arachnoid cells as well as cytoplasmic membranes and intracellular organelles in close proximity to junctions between these various cell types. No labelling was detected within cell bodies of oligodendrocytes and neurons and no labelled neuronal gap junctions were found. The results suggest that connexin 43 is one of the major gap junctional proteins utilized for junctional coupling between astrocytes and between cells lining the surfaces of the brain.     

Ran-2, a glial lineage marker,is a GPI-anchored form of ceruloplasmin

Cell interactions in the nervous system are frequently mediated by surface proteins that are attached to the membrane by a glycosyl phosphatidylinositol (GPI) anchor. In this study, we have characterized the expression of such proteins on glial cells. We have detected a major GPI-anchored protein on astrocytes and Schwann cells, with a molecular weight of 140 kD. When Schwann cells were treated with forskolin to promote a myelinating phenotype, expression of this 140-kD protein dramatically decreased, whereas another GPI-anchored protein of 80 kD was strongly induced; expression of other integral membrane proteins were likewise dramatically altered. The size and pattern of expression of the 140-kD protein suggested that it might correspond to the Ran-2 antigen, a glial lineage marker. This notion was confirmed by immunoprecipitating this 140-kD protein with the Ran-2 monoclonal antibody. The Ran-2 antigen is expressed over the entire Schwann cell surface in a punctate fashion; it is removed by phosphatidylinositol phospholipase C treatment, thereby confirming that it is GPI-anchored. When Schwann cells are cocultured with neurons, the Ran-2 antigen initially concentrates at sites of Schwann cell contact with neurons, suggesting that it may play a role in early Schwann cell–neuron interactions; it is then downregulated. Protein sequencing of the Ran-2 antigen immunopurified from rat brain membranes showed complete identity over two extended segments with the copper binding protein ceruloplasmin. These findings indicate that astrocytes and Schwann cells express a novel GPI-anchored form of ceruloplasmin and suggest that this GPI form plays a role in axonal–glial interactions. J. Neurosci. Res. 54:147–157, 1998. © 1998 Wiley-Liss, Inc.     

Injuring neurons induces neuronal differentiation in a population of hippocampal precursor cells in culture

A novel population of hippocampal precursor cells (HPCs) that can be induced to differentiate into astrocytes and oligodendrocytes can be derived from hippocampal cultures grown in serum-free media. The HPCs are PDGF-responsive, do not proliferate with bFGF, and grow as sheets of cells rather than gathering into neurospheres. The HPCs share many markers (A2B5, GD3, poly-sialylated neuronal common adhesion molecule (PSA-NCAM), and NG2) with oligodendrocyte precursor cells (OPCs). The HPCs do not express markers for mature neurons, astrocytes, or oligodendrocytes. Like OPCs, the HPCs differentiate into glial fibrillary acidic protein (GFAP)+ astrocytes and GalC+ oligodendrocytes with the addition of bone morphogenetic protein-4 (BMP-4) and triiodothyronine (T3), respectively. They do not differentiate into neurons with the addition or withdrawal of basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), or retinoic acid (RA). These HPCs can be stimulated to differentiate into neuron-like cells by the induction of neuronal injury or cell death in nearby cultured neurons or by conditioned medium from injured neuronal cultures. Under these conditions, HPCs grow larger, develop more extensive dendritic processes, become microtubule-associated protein-2-immunoreactive, express large voltage-dependent sodium currents, and form synaptic connections. The conversion of endogenous pluripotent precursor cells into neurons in response to local brain injury may be an important component of central nervous system homeostasis.