Polysialic acid controls NCAM-induced differentiation of neuronal precursors into calretinin-positive olfactory bulb interneurons

Understanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis is polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule NCAM. In the present study, we investigated the role of polySia in differentiation of neuronal precursors isolated from the subventricular zone of early postnatal mice. Removal of polySia promoted neurite induction and selectively enhanced maturation into a calretinin-positive phenotype. Expression of calbindin and Pax6, indicative for other lineages of olfactory bulb interneurons, were not affected. A decrease in the number of TUNEL-positive cells indicated that cell survival was slightly improved by removing polySia. Time lapse imaging revealed the absence of chain migration and low cell motility, in the presence and absence of polySia. The changes in survival and differentiation, therefore, could be dissected from the well-known function of polySia as a promoter of precursor migration. The differentiation response was mimicked by exposure of cells to soluble or substrate-bound NCAM and prevented by the C3d-peptide, a synthetic ligand blocking NCAM interactions. Moreover, a higher degree of differentiation was observed in cultures from polysialyltransferase-depleted mice and after NCAM exposure of precursors from NCAM-knockout mice demonstrating that the NCAM function is mediated via heterophilic binding partners. In conclusion, these data reveal that polySia controls instructive NCAM signals, which direct the differentiation of subventricular zone-derived precursors towards the calretinin-positive phenotype of olfactory bulb interneurons.     

Neural cell adhesion molecule induces intracellular signaling via multiple mechanisms of Ca2+ homeostasis

The neural cell adhesion molecule (NCAM) plays a pivotal role in the development of the nervous system, promoting neuronal differentiation via homophilic (NCAM-NCAM) as well as heterophilic (NCAM-fibroblast growth factor receptor [FGFR]) interactions. NCAM-induced intracellular signaling has been shown to affect and be dependent on the cytoplasmic Ca2+ concentration ([Ca2+]i). However, the molecular basis of this remains unclear. In this study, we determined [Ca2+]i regulating mechanisms involved in intracellular signaling induced by NCAM. To mimic the effect of homophilic NCAM interaction on [Ca2+]i in vitro, we used a peptide derived from a homophilic binding site of NCAM, termed P2, which triggers signaling cascades similar to those activated by NCAM-NCAM interaction. We found that P2 increased [Ca2+]i in primary hippocampal neurons. This effect depended on two signaling pathways. The first pathway was associated with activation of FGFR, phospholipase Cgamma, and production of diacylglycerol, and the second pathway involved Src-family kinases. Moreover, NCAM-mediated Ca2+ entry required activation of nonselective cation and T-type voltage-gated Ca2+ channels. These channels, together with the Src-family kinases, were also involved in neuritogenesis induced by physiological, homophilic NCAM interactions. Thus, unanticipated mechanisms of Ca2+ homeostasis are shown to be activated by NCAM and to contribute to neuronal differentiation.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

Binding properties of detergent-solubilized NCAM

An assay has been designed for the identification of NCAM-binding proteins present in an NP-40 detergent extract of brain membranes. This method, which is capable of analyzing both heterophilic and homophilic interactions, uses species-specific antibodies against NCAM in combination with radioiodination, so that after unlabeled chicken and iodinated frog brain membrane proteins were allowed to interact, the chicken NCAM could be specifically isolated by immunoaffinity adsorption. The radiolabeled frog proteins coisolated with chicken NCAM were then characterized by one- and two-dimensional gel electrophoresis in combination with immunoblotting. The only detectable NCAM-binding proteins were identified as the 140- and 180-kD forms of NCAM. The presence and absence of polysialic acid on NCAM did not change the amount or nature of the frog proteins immunopurified under these conditions. As an alternative for detecting heterophilic ligands, a simplified immunoprecipitation method was employed using either iodine or sulfate radiolabels. Again under these conditions only NCAM was detected. These results are consistent with the hypothesis that the major binding protein for NCAM is NCAM itself, and suggest that differences in polysialic acid content do not directly alter the properties of NCAM's homophilic binding site.     

Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.     

Zippers Make Signals: NCAM-mediated Molecular Interactions and Signal Transduction

The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell–cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitroand in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.Special issue dedicated to Lawrence F. Eng.     

Polysialic acid profiles of mice expressing variant allelic combinations of the polysialyltransferases ST8SiaII and ST8SiaIV

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate approximately 90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.     

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.     

Polysialic acid directs tumor cell growth by controlling heterophilic neural cell adhesion molecule interactions

Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), promotes neural plasticity and tumor malignancy, but its mode of action is controversial. Here we establish that PSA controls tumor cell growth and differentiation by interfering with NCAM signaling at cell-cell contacts. Interactions between cells with different PSA and NCAM expression profiles were initiated by enzymatic removal of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the cell surface led to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these effects. Combinatorial transinteraction studies with cells and membranes with different PSA and NCAM phenotypes revealed that heterophilic NCAM binding mimics the cellular responses to PSA removal. In conclusion, our data demonstrate that PSA masks heterophilic NCAM signals, having a direct impact on tumor cell growth. This provides a mechanism for how PSA may promote the genesis and progression of highly aggressive PSA-NCAM-positive tumors.     

Retinoic acid evoked-differentiation of neuroblastoma cells predominates over growth factor stimulation: an automated image capture and quantitation approach to neuritogenesis.

To facilitate the characterization of compounds that have positive growth factor mimetic effects on neuritogenesis, we have implemented a high-throughput functional assay which measures, in a multiparametric manner, the proliferation and differentiation characteristics of cells in a microtiter plate. Conditions were established using chronic incubation of SH-SY5Y human neuroblastoma cells with retinoic acid (RA) and/or nerve growth factor (NGF) in which discernible alterations in proliferation, growth, and differentiation of cells were induced. SH-SY5Y cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on Cellomics ArrayScanII was utilized to quantify the effects of these treatments on cell characteristics. NGF and retinoic acid were found to increase multiple parameters of SH-SY5Y differentiation, including an increased proportion of cells having neurites and increased extent of branching. However, marked differences in the effects of these compounds on SH-SY5Y growth and differentiation were also detected: whereas NGF increased cell number, RA treatment decreased cell number, and RA but not NGF caused significant elongation of neurites. This study quantifies and characterizes the effects of differentiating and proliferating agents on a human-derived neuroblastoma cell line. The high-content, rapid-throughput nature of this assay makes it ideal for functional identification and characterization of compounds regulating cell behavior.     

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.     

Polysialic acid and mucin type o-glycans on the neural cell adhesion molecule differentially regulate myoblast fusion

Polysialic acid attached to the neural cell adhesion molecule (NCAM) is thought to play a critical role in development. NCAM in muscle tissue contains a muscle-specific domain (MSD) to which mucin type O-glycans are attached. In the present study, using the C2C12 myoblast system, we show that NCAM containing MSD is increasingly expressed on the cell surface as myotubes form. Polysialic acid is primarily attached to N-glycans of NCAM, and polysialylated NCAM is expressed on the outer surface of myotube bundles. By transfecting cDNAs encoding wild type and mutant forms of NCAM, we found that NCAM containing MSD facilitates myoblast fusion, and this effect is diminished by mutating O-glycosylation sites at MSD. By contrast, forced expression of polysialic acid in early differentiation stages reduces myotube formation and delays the expression of NCAM containing the MSD domain. Strikingly, inhibition of polysialic acid synthesis by antisense DNA approach induced differentiation in both human rhabdomyosarcoma cells, which overexpress polysialic acid, and C2C12 cells. These results indicate that polysialic acid and mucin type O-glycans on NCAM differentially regulate myoblast fusion, playing critical roles in muscle development.     

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.     

Up-regulation of embryonic NCAM in an EC cell line by retinoic acid

The impact of retinoic acid (RA) on the expression of the neural cell adhesion molecules (NCAMs) and their developmentally regulated polysialic acid (PSA) moiety was studied in embryonal carcinoma (EC) cell lines. These cell lines are known to be capable of RA-induced differentiation into neurons (murine P19 cells) or parietal endoderm (murine F9 cells), respectively. Monoclonal antibodies were employed to monitor expression of NCAM and PSA. F9 and P19 cells were both found to express NCAM but only P19 cells carried the highly polysialylated "embryonic form" of NCAM (E-NCAM). The amount of NCAM in aggregated P19 cells but not in F9 cells was dramatically increased upon treatment with RA. Since NCAMs play an important role in cell interactions during embryogenesis it is tempting to speculate that the regulative impact of RA on NCAMs is related to its morphogenic property.