Down-regulation of polysialic acid is required for efficient myelin formation

Oligodendrocyte precursor cells modify the neural cell adhesion molecule (NCAM) by the attachment of polysialic acid (PSA). Upon further differentiation into mature myelinating oligodendrocytes, however, oligodendrocyte precursor cells down-regulate PSA synthesis. In order to address the question of whether this down-regulation is a necessary prerequisite for the myelination process, transgenic mice expressing the polysialyltransferase ST8SiaIV under the control of the proteolipid protein promoter were generated. In these mice, postnatal down-regulation of PSA in oligodendrocytes was abolished. Most NCAM-120, the characteristic NCAM isoform in oligodendrocytes, carried PSA in the transgenic mice at all stages of postnatal development. Polysialylated NCAM-120 partially co-localized with myelin basic protein and was present in purified myelin. The permanent expression of PSA-NCAM in oligodendrocytes led to a reduced myelin content in the forebrains of transgenic mice during the period of active myelination and in the adult animal. In situ hybridizations indicated a significant decrease in the number of mature oligodendrocytes in the forebrain. Thus, down-regulation of PSA during oligodendrocyte differentiation is a prerequisite for efficient myelination by mature oligodendrocytes. Furthermore, myelin of transgenic mice exhibited structural abnormalities like redundant myelin and axonal degeneration, indicating that the down-regulation of PSA is also necessary for myelin maintenance.     

Expression of the neural cell adhesion molecule NCAM in endocrine cells

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.     

Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule.

The rodent neural cell adhesion molecule (NCAM) consists of three glycoproteins with Mr of 180,000, 140,000 and 120,000. The Mr 120,000 protein (NCAM-120) has been shown to exist in membrane-bound and soluble forms but the nature of its membrane association and release has remained obscure. We show here that phosphatidylinositol-specific phospholipase C (PI-PLC), but not a phospholipase C of different specificity, releases a substantial proportion of NCAM-120 from brain membranes and solubilizes almost quantitatively NCAM-120 present at the surface of C6 astroglial cells. The PI-PLC effect was highly selective since only one other protein species was detectably released from C6 cells. These results suggest that NCAM-120 is held in the membrane by covalently bound phosphatidylinositol or a closely related lipid in a way similar to several other surface proteins from eukaryotic cells. The presence of NCAM in a form which can be released from the cell surface by a highly selective mechanism raises additional possibilities for modulation and control of cell--cell adhesion.     

Role of GPI-anchored NCAM-120 in rabies virus infection

Although the neural cell adhesion molecule (NCAM) -140 and -180 have been shown to serve as a receptor for rabies virus (RV), it was not known whether the other major isoform of NCAM, GPI-anchored NCAM-120 functions as RV receptor. In this study, we have established HEp-2 cells stably expressing NCAM-120 or NCAM-140, and their susceptibilities to RV infection were compared. The results demonstrated that NCAM-120 served as virus attachment protein; however, the cells expressing NCAM-120 did not support efficient RV replication. Furthermore, the level of IFN-ss mRNA was apparently elevated in NCAM-120 expressing cells but not in NCAM-140 expressing cells, suggesting that GPI-anchored NCAM-120 suppressed RV replication via induction of IFN-ss even though NCAM-120 was able to promote virus penetration into the cells.     

Cultured microvascular endothelial cells derived from the bovine corpus luteum possess NCAM-140

Previously, five phenotypically different, stable types of microvascular endothelial cells (MVE) were isolated from the bovine corpus and cultured successfully. We found that three out of these five types of MVE express the neural cell adhesion molecule (NCAM). As shown by immunocytochemistry, weak NCAM immunoreactivity occurred mainly in the perinuclear area of cell type 1. Monolayers of types 2 and 5 revealed heavy NCAM immunoreactivity, which was localized predominantly at the lateral cell surface outlining the contact zones of adjacent cells. In contrast, cell types 3 and 4 were not NCAM immunoreactive. Western blot analyses substantiated these results: While cell type 1 showed a weak immunoreactive band, cell types 2 and 5 displayed strong NCAM-immunoreactive bands of a molecular weight of approximately 140 kDa (NCAM-140), which was absent in cell types 3 and 4. These results reveal for the first time that NCAM can be expressed by cultured MVE and may serve in mediating endothelial cell contacts. Since luteal cells also express NCAM-140, this adhesion molecule could in addition be involved in the interactions of luteal cells with MVE.     

Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth

The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.     

Embryonic Neural Cell Adhesion Molecule in Cerebrospinal Fluid of Younger Children: Age-Dependent Decrease During the First Year

Poly-alpha-2,8-N-acetylneuraminic acid (poly-alpha-2,8-NeuAc) is developmentally expressed in neural tissue of higher animals, where it is covalently attached to the neural cell adhesion molecule (NCAM), a large integral membrane glycoprotein mediating cell-cell adhesion during neuronal development. NCAM exists in several molecular forms, of which only embryonic NCAM carries lengthy chains (n greater than 5) of poly-alpha-2,8-NeuAc. Chemically identical poly-alpha-2,8-NeuAc of bacterial origin is an important virulence factor in infections caused by Neisseria meningitidis group B and Escherichia coli K1, the predominant pathogens of bacterial meningitis. A quantitative enzyme-linked immunoassay was developed using monoclonal antibody (MAb) 735, an MAb specifically recognizing poly-alpha-2,8-NeuAc, and applied to CSF specimens from younger children. Poly-alpha-2,8-NeuAc contents were within the range of 20-0.2 micrograms/ml, decreasing from day 1 to day 300. Immunoprecipitation, immunoblot with a rabbit anti-mouse NCAM serum recognizing the protein part of human NCAM by cross-reactivity, affinity enrichment using immobilized MAb 735, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that poly-alpha-2,8-NeuAc in CSF is bound to human NCAM, probably NCAM-120.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.     

Distribution of NCAM-180 and polysialic acid in the developing tectum mesencephali of the frog Discoglossus pictus and the salamander Pleurodeles waltl

The 180 kDa component of the neural cell adhesion molecule (NCAM-180), total NCAM (NCAM-total) and the polysialic acid modification of NCAM (PSA) show similar temporal and spatial regulation in the developing tecta of Pleurodeles waltl (salamander) and Discoglossus pictus (frog). Whereas NCAM-total is found throughout the tectal tissue on neurons and glia, NCAM-180 is only found on nonproliferating neurons and in fiber layers. PSA is expressed by a subset of NCAM-180-positive cells. Western blots show that there is little polysialylated NCAM-140 in the developing amphibian tectum. Regions unstained for PSA and NCAM-180 correspond precisely to the growth zones of the tectum. NCAM-180 and PSA are not present in tecta of early larvae. Staining intensity is strongest at midlarval stages for both antigens. At metamorphosis, PSA is strongly downregulated, whereas NCAM-180 is downregulated in juvenile animals. Both antigens are still present in fiber layers of adult animals. In dissociated tissue culture of the frog tectum, NCAM-180 is not present on astrocytes, but on neuronal cells. Expression is enhanced at cell contact sites, suggesting that NCAM-180 is involved in cell contact stabilization. This study shows that general features of temporal and spatial regulation of NCAM isoforms and PSA are highly conserved in frog and salamander tecta, despite large differences in the rate of cell migration and the degree of lamination in these homologous brain regions.     

Adult human brain expresses four different molecular forms of neural cell adhesion molecules

Using antibodies to rat neural cell adhesion molecules (NCAM), we analyzed the NCAM of adult human brain. Various regions of the brain were analyzed quantitatively by Western blot. Grey matter showed four bands of NCAM with apparent molecular weights of 180,000, 170,000, 140,000 and 120,000. White matter showed one major band with an apparent M_r of 120,000 and a minor band of 180,000. Cerebellar grey matter contained mainly 170,000, 140,000 and 120,000, white cerebellar white matter had only 180,000 and 120,000 M₁ NCAMS. Spinal cord showed mainly 120,000 M_r NCAM. Deglycosylation using N-glycanase resulted in 170,000, 160,000, 130,000 and 110,000 M_r proteins, suggesting that the four forms of human NCAM are derived from individual polypeptides. The presence of 170,000 M₁ NCAM is unique to human brain.     

Neural cell adhesion molecule (NCAM-/-) null mice show impaired sensitization of the startle response

The neural cell adhesion molecule (NCAM) plays important roles in development of the nervous system and in synaptic plasticity and memory formation in the adult. The present study sought to further investigate the role of NCAM in learning by testing habituation and footshock sensitization learning of the startle response (SR) in NCAM null mutant (NCAM-/-) and wildtype littermate (NCAM+/+) mice. Whereas habituation is a form of non-associative learning, footshock sensitization is induced by rapid contextual fear conditioning. Habituation was tested by repetitive presentation of acoustic and tactile startle stimuli. Although NCAM-/- mice showed differences in sensitivity in both stimulus modalities, habituation learning was intact in NCAM-/- mice, suggesting that NCAM does not play a role in the mechanisms underlying synaptic plasticity in the startle pathway. Footshock sensitization was elicited by presentation of electric footshocks between two series of acoustic stimuli. In contrast to habituation, footshock sensitization learning was attenuated in NCAM-/- mice: the acoustic SR increase after the footshocks was lower in the mutant than in wildtype mice, indicating that NCAM plays an important role in the relevant brain areas, such as amygdala and/or the hippocampus.     

Multistratified expression of polysialic acid and its relationship to VAChT-containing neurons in the inner plexiform layer of adult rat retina.

We investigated the localization of polysialic acid (PSA), neural cell adhesion molecule (NCAM), and vesicular acetylcholine transporter (VAChT) in adult rat retina by using immunofluorescence with a confocal laser scanning microscope. Western blot analysis showed a typical broad smear of PSA and isoforms of NCAM (120, 140, and 180 kD). PSA immunofluorescence revealed multistratification in the inner plexiform layer (IPL). Dual immunostaining for PSA and NCAM exhibited the selective co-expression of PSA and NCAM on Müller cells. Moreover, dual immunolabeling for PSA and VAChT completely separated the five strata in the IPL. Strata 1, 3, and 5 were immunoreactive for PSA and Strata 2 and 4 for VAChT. These results suggest the possibility that PSA molecules on Müller cells are spatially related to ON and OFF retinal channels in the IPL.     

Expression level of neural cell adhesion molecule (NCAM) inversely correlates with the ability of neuroblastoma cells to adhere to endothelium in vitro

The precise function of cell adhesion molecules in the hematogenous phase of neuroblastoma metastasis is poorly understood. The aim of this study was to investigate whether neural cell adhesion molecule (NCAM) modulates neuroblastoma cell (NB) adhesion and transendothelial penetration in a coculture model. Our data, assessed on 11 NB cell lines, demonstrate an inverse correlation between NCAM expression and NB cell adhesion. Transfection of the NB cell line UKF-NB-4 with a cDNA encoding the human NCAM-140 kD isoform enhanced NCAM expression and the amount of tumor cell aggregates, reduced the amount of single tumor cells, and diminished initial NB cell adhesion to an endothelial cell monolayer. Treatment of UKF-NB-4 with NCAM antisense oligonucleotides reduced NCAM surface level, increased the number of single tumor cells, and induced up-regulation of NB cell adhesion to endothelium. Modulation of NCAM expression had no effect on transendothelial penetration. Fluorescence analysis revealed a down-regulation of NCAM in single tumor cells, prior to NB adhesion. The data support the view that low levels of NCAM are necessary for NB cells to leave a tumor cell aggregate and adhere to endothelial cells.     

Identification of novel mRNAs expressed in oligodendrocytes

To identify new proteins, which are expressed in oligodendrocytes and which may have a functional role in myelination, a rat oligodendrocyte cDNA library was screened using differential and subtractive screening techniques. Ten clones that have elevated levels of expression in brain were isolated. Two of these clones were characterized further and one clone, pC26.H2, was found to be closely related to mouse stearoyl-CoA desaturase 2 (SCD2), which catalyzes the synthesis of unsaturated fatty acid. From Northern blot and in situ hybridization studies, SCD2 mRNA was expressed primarily in brain with lower levels found in lung and spleen. In brain sections, SCD2 mRNA was found primarily in oligodendrocytes, although mRNA was detected at a low level in neurons, in particular in Purkinje cells in the cerebellum. Northern blot analysis of the other clone, p973.HB, indicated that it was expressed more selectively in brain. In mixed glial cultures oligodendrocytes were the only cells that expressed this mRNA, whereas in brain, neurons expressed this mRNA at a higher level than in oligodendrocytes. This clone is being actively pursued because of its unique expression exclusively in oligodendrocytes and neurons.Special issue dedicated to Dr. Marjorie B. Lees.