Enzymatic Removal Of Polysialic Acid Research Articles

Enzymatic removal of polysialic acid from neural cell adhesion molecule interrupts gonadotropin releasing hormone (GnRH) neuron–glial remodeling

There is abundant evidence to prove that the astrocytes are highly dynamic cell type in CNS and under physiological conditions such as reproduction, these cells display a remarkable structural plasticity especially at the level of their distal processes ensheathing the gonadotropin releasing hormone (GnRH) axon terminals. The morphology of GnRH axon terminals and astrocytes in the median eminence region of hypothalamus show activity dependent structural plasticity during different phases of estrous cycle. In the current study, we have assessed the functional contribution of ∞-2,8-linked polysialic acid (PSA) on neural cell adhesion molecule (PSA-NCAM) in this neuronal–glial plasticity using both in vitro and in vivo model systems. In vivo experiments were carried out after stereotaxic injection of endoneuraminidase enzyme (endo-N) near median eminence region of hypothalamus to specifically remove PSA residues on NCAM followed by localization of GnRH, PSA-NCAM and glial fibrillary acidic protein (GFAP) by immunostaining. Using in vitro model, structural remodeling of GnV-3 cells, (a conditionally immortalized GnRH cell line) co-cultured with primary astrocytes was studied after treating the cells with endo-N. Marked morphological changes were observed in GnRH axon terminals in proestrous phase rats and control GnV-3 cells as compared to endo-N treatment i.e. after removal of PSA. The specificity of endo-N treatment was also confirmed by studying the expression of PSA-NCAM by Western blotting in cultures treated with and without endo-N. Removal of PSA from surfaces with endo-N prevented stimulation associated remodeling of GnRH axon terminals as well as their associated glial cells under both in vivo and in vitro conditions. The current data confirms the permissive role of PSA to promote dynamic remodeling of GnRH axon terminals and their associated glia during reproductive cycle in rats.

NCAM and polysialyltransferase profiles match dopaminergic marker gene expression but polysialic acid is dispensable for development of the midbrain dopamine system

The modification of the neural cell adhesion molecule (NCAM) with polysialic acid plays a pivotal role in the developing nervous system. Here we studied the expression and function of polysialic acid during development of the mesencephalic dopaminergic system of mice. Using immunohistochemistry, polysialic acid was detected on nestin-positive radial glia processes and on cell somata in the pial zone of the midbrain at embryonic day E11.5 and E14.5. As studied by quantitative real-time RT-PCR, mRNA profiles of NCAM and the polysialyltransferases, ST8SiaII and ST8SiaIV, matched the course of tyrosine hydroxylase, dopamine transporter, nur-related factor 1, and paired-like homeodomain transcription factor 3 expression, which were used as marker genes of dopaminergic development. Asking for a possible role of polysialylation during formation of the dopaminergic system, mice lacking polysialic acid because of ablation of both St8siaII and St8siaIV were analyzed at selected time points by tyrosine hydroxylase immunohistochemistry and by real-time RT-PCR of dopaminergic markers. Surprisingly, no differences between wild-type and mutant mice could be detected. Likewise, enzymatic removal of polysialic acid from cultured neurons of the ventral embryonic midbrain had no effect on the expression of dopaminergic marker genes. We conclude that despite its abundance polysialic acid is dispensable for the formation of the mesencephalic dopaminergic system.

NCAM in Long-Term Potentiation and Learning

The neural cell adhesion molecule (NCAM) is important in regulating neuronal development and plasticity by modulating cell adhesion and signal transduction. Studies using NCAM antibodies have demonstrated a role of NCAM in hippocampal long-term potentiation and in animal learning models. These findings have been supported by the observation that NCAM-deficient mice show altered LTP as well as learning deficits. A particular carbohydrate modification of NCAM, polysialic acid (PSA), has been shown to be important for synaptic plasticity, as demonstrated by impaired LTP and learning in mice deficient in polysialyltransferase enzymes necessary for adding PSA to the NCAM protein as well as in wildtype mice after enzymatic removal of PSA. It is suggested that NCAM and its PSA moiety are involved in synaptic remodeling in LTP and learning, possibly by modulating signal transduction pathways as well as by mediating and permitting adhesive changes in an activity-dependent manner.

Polysialylated NCAM Represses E-Cadherin-Mediated Cell-Cell Adhesion in Pancreatic Tumor Cells

Inhibition of cell-cell adhesion between epithelial cells represents an early step during tumor metastasis. Down-regulation or perturbation of E-cadherin-mediated adherens junctions is an essential requirement in this process. The interaction between polysialylated neural cell adhesion molecule (PSA-NCAM) and the E-cadherin adhesion complex was studied by coimmunoprecipitation assays. The presence of PSA-NCAM was correlated with tumor invasion by using cell-cell aggregation and cell migration assays. The importance of polysialic acid (PSA) in the interaction of NCAM with E-cadherin and inhibition of cell-cell adhesion was confirmed by enzymatic removal of PSA from NCAM and down-regulation of PSA-transferases by siRNA. Expression of oncogenic K-Ras(V12) in pancreatic carcinoma cells resulted in induction of PSA-NCAM expression and reduced E-cadherin-mediated cellular adhesion. The association of PSA-NCAM with the E-cadherin adhesion complex correlated with decreased cell-cell aggregation and elevated cell migration of pancreatic carcinoma cells. Enzymatic removal of PSA from NCAM or reduction of polysialyltransferase expression led to reduced association between NCAM and E-cadherin and subsequently increased E-cadherin-mediated cell-cell aggregation and reduced cell migration. Our data suggest the induction of PSA-NCAM by oncogenic K-Ras as a novel molecular mechanism by which E-cadherin-mediated cellular adhesion is reduced and dissemination of tumor cells is facilitated.

Polysialyltransferase expression is linked to neuronal migration in the developing and adult zebrafish

Modulation of cell-cell adhesion is crucial for regulating neuronal migration and maintenance of structural plasticity in the embryonic and mature brain. Such modulation can be obtained by the enzymatic attachment of polysialic acid (PSA) to the neural cell adhesion molecule (NCAM) by means of the polysialyltransferases STX and PST. Thus, differential expression of STX and PST is likely to be responsible for varying functions of PSA-NCAM during neuronal differentiation, maintenance, plasticity, and regeneration. We have isolated the zebrafish homologues of STX (St8sia2) and PST (St8sia4) and demonstrate that their expression in the embryonic and adult nervous system is often confined to regions of neuronal migration. Moreover, in the adult cerebellum, the complementary expression pattern of both polysialyltransferases suggests a function in regulating cerebellar neuronal plasticity. Enzymatic removal of PSA in the embryonic cerebellum results in impaired neuronal migration, suggesting that PSA-NCAM is a key regulator of motility for cerebellar neuronal progenitors.

Neural Cell Adhesion Molecule-associated Polysialic Acid Inhibits NR2B-containing N-Methyl-d-aspartate Receptors and Prevents Glutamate-induced Cell Death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.

Polysialylated Neural Cell Adhesion Molecule Promotes Remodeling and Formation of Hippocampal Synapses

Expression of the neural cell adhesion molecule (NCAM) has been shown to promote long-term potentiation (LTP) and stabilization of synapses during early synaptogenesis. Here, we searched for the mechanisms of synaptogenic activity of NCAM, focusing on the role of polysialic acid (PSA), an unusual carbohydrate preferentially associated with NCAM. We show that enzymatic removal of PSA with endoneuraminidase-N (endo-N) abolished preferential formation of synapses on NCAM-expressing cells in heterogenotypic cocultures of wild-type and NCAM-deficient hippocampal neurons. Transfection of NCAM-deficient neurons with either of three major NCAM isoforms (different in intracellular domains but identical in extracellular domains and carrying PSA) stimulated preferential synapse formation on NCAM isoform-expressing neurons. Enzymatic removal of heparan sulfates from cultured neurons and a mutation in the heparin-binding domain of NCAM diminished synaptogenic activity of neuronally expressed PSA-NCAM, suggesting that interaction of NCAM with heparan sulfate proteoglycans mediates this activity. PSA-NCAM-driven synaptogenesis was also blocked by antagonists to fibroblast growth factor receptor and NMDA subtype of glutamate receptors but not by blockers of non-NMDA glutamate receptors and voltage-dependent Na+ channels. Enzymatic removal of PSA and heparan sulfates also blocked the increase in the number of perforated spine synapses associated with NMDA receptor-dependent LTP in the CA1 region of organotypic hippocampal cultures. Thus, neuronal PSA-NCAM in complex with heparan sulfate proteoglycans promotes synaptogenesis and activity-dependent remodeling of synapses.

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.

Differential Effects of Unnatural Sialic Acids on the Polysialylation of the Neural Cell Adhesion Molecule and Neuronal Behavior

In this study we have examined how unnatural sialic acids can alter polysialic acid expression and influence the adhesive properties of the neural cell adhesion molecule (NCAM). Unnatural sialic acids are generated by metabolic conversion of synthetic N-acyl mannosamines and are typically incorporated into cell-surface glycoconjugates. However, N-butanoylmannosamine and N-pentanoylmannosamine are effective inhibitors of polysialic acid (PSA) synthesis in stably transfected HeLa cells expressing NCAM and the polysialyltransferase STX. These cells were used as substrates to examine the effect of inhibiting PSA synthesis on the development of neurons derived from the chick dorsal root ganglion. N-butanoylmannosamine blocked polysialylation of NCAM and significantly reduced neurite outgrowth comparable with enzymatic removal of PSA by endoneuraminidases. As a result, neurite outgrowth was similar to that observed for non-polysialylated NCAM. In contrast, previous studies have shown that N-propanoyl sialic acid (SiaProp), generated from N-propanoylmannosamine, is readily accepted by polysialyltransferases and permits the extension of poly(SiaProp) on NCAM. Despite being immunologically distinct, poly(SiaProp) can promote neurite outgrowth similarly to natural polysialic acid. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM.

The Polysialic Acid Moiety of the Neural Cell Adhesion Molecule Is Involved in Intraretinal Guidance of Retinal Ganglion Cell Axons

We have characterized the antigen recognized by mab10, a monoclonal antibody that has been shown to modify outgrowth of thalamic and cortical axons in vitro, and investigated the influence of this antibody on axonal growth in the chicken retina in vivo. Immunopurification, peptide sequencing, and biochemical characterization proved the epitope recognized by mab10 to be polysialic acid (PSA), associated with the neural cell adhesion molecule (NCAM). Intravitreal injections of antibody-secreting hybridoma cells were combined with whole-mount studies using the fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Pathfinding at the optic fissure was affected, resulting in a failure of axons to exit into the nerve. Misprojections also occurred in more peripheral areas of the retina; however, axons eventually oriented toward the center. Similar projection errors were observed after enzymatic removal of PSA by injecting endoneuraminidase N (endo N). Quantitative measurements of the optic nerve diameter as well as the width of the optic fiber layer confirmed that many axons failed to leave the retina and grew back in the optic fiber layer of the retina. Our findings suggest that NCAM-linked PSA is involved in guiding ganglion cell axons in the retina and at the optic fissure.

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

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

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

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

The Pattern of Avian Intramuscular Nerve Branching Is Determined by the Innervating Motoneuron and Its Level of Polysialic Acid

Most skeletal muscles are composed of a heterogeneous population of fast and slow muscle fibers that are selectively innervated during development by fast and slow motoneurons, respectively. It is well recognized that, in both birds and mammals, fast and slow motoneurons have substantially different intramuscular branching patterns, a difference critical for proper motor function. However, the cellular mechanisms regulating these differences in motoneuron branching are unknown. In a previous study, we showed that the fast and slow pattern of intramuscular branching, in a chick muscle containing distinct fast and slow muscle regions, was remarkably similar to normal when formed by foreign motoneurons. Whether this was attributable to some property of the innervating "fast" or "slow" motoneurons or to some property of the developing fast-slow muscle fibers was not determined. To distinguish between these two possibilities, we performed chick-quail hindlimb chimeras to force slow chick plantaris motoneurons to innervate a fast quail plantaris muscle. The pattern of intramuscular nerve branching in the fast plantaris of these chimeras closely resembled the slow branching pattern normally observed in chick slow plantaris muscles. Enzymatic removal of polysialic acid (PSA) from nerve and muscle during normal quail plantaris development dramatically changed the normal fast pattern to more closely resemble a slow pattern. In contrast, removal of PSA from chick plantaris motoneurons and muscle fibers had little effect on the pattern of nerve branching. Together, these results indicate that the pattern of intramuscular nerve branching is determined by the level of PSA on the innervating motoneurons.

Role of polysialic acid on outgrowth of rat olfactory receptor neurons

Towards elucidating the role of polysialic acid (PSA) in developing olfactory neuron of the rat, we injected neuraminidase (endo-N) into the olfactory nerve pathway under whole embryo culture, then employed immunohistochemistry to (i) detect expression of highly sialylated neural cell adhesion molecules (NCAM-H) and (ii) identify olfactory neurons via anti-microtubule-associated protein 1B (MAP1B) antibody. Olfactory axonal outgrowth from basal lamina occurred at the 31-somite stage and reached the olfactory bulb primordium at the 42-somite stage, being coincident with the timing and expression of NCAM-H immunoreactivity. Enzymatic removal of PSA by endo-N remarkably affected developmental processes of axonal outgrowth, extension, and pathfinding, i.e. individual axons appeared to have become stuck in the mesenchyme. Results indicate that PSA is critically involved with anti-adhesion cues associated with individual axonal growth during olfactory system development.

N-CAM mutation inhibits tangential neuronal migration and is phenocopied by enzymatic removal of polysialic acid

The mutation of N-CAM in mice produces a phenotype dominated by an undersized olfactory bulb and accumulation of precursors in the subependymal layer. We demonstrate here that this defect can be duplicated by injection of an enzyme that specifically destroys the polysialic acid (PSA) moiety associated with N-CAM. Studies of BrdU-labeled and pyknotic cells suggest that this defect reflects a decrease in the rostral migration of olfactory precursors and not a change in the proliferation or rate of death of these cells. In addition to their ectopic location, these cells had fewer growth cone-like processes oriented along the migration route. In contrast to tangential movement, radial migration of granule cells in the olfactory bulb was not affected by loss of PSA. These results support the proposed role for PSA in cell translocation, discriminate between different mechanisms of cell migration, and provide insight as to the nature of the N-CAM mutant phenotype.

Neural cell adhesion molecule regulates cell contact-mediated changes in choline acetyltransferase activity of embryonic chick sympathetic neurons.

E10 chick sympathetic ganglion cells display a cell contact-dependent rise in choline acetyltransferase (ChAT) specific activity over the first several days in culture. This effect can be mimicked by addition of crude membrane fractions prepared from E10 retina and adult chicken brain, but not by those from E10 brain. The effects of both cell-cell and membrane-cell contact are inhibited by the addition of anti-NCAM Fab fragments. The membranes capable of increasing ChAT and those which are ineffective all contain NCAM, however their relative levels of NCAM polysialic acid differ. Whereas membranes with high polysialic acid NCAM are ineffective, selective enzymatic removal of polysialic acid renders them capable of producing an increase in ChAT. The inhibition of NCAM-mediated adhesion produced by Fab fragments can be compensated for by addition of wheat germ agglutinin, but only with membranes whose NCAM has low levels of polysialic acid. Taken together, these data suggest that NCAM can regulate cell contact-mediated increases in ChAT activity. We propose that NCAM-mediated adhesion promotes contact between cell membranes to allow the transmission of an otherwise NCAM-independent signal. In addition, NCAM's polysialic acid moiety appears to influence the ability of cells to transmit this signal, even in the presence of an alternative adhesion mechanism.

General practice audit of asthma in childhood.

It has been suggested that asthma is underdiagnosed in children. We studied the records of 52 children with asthma in our group practice. Although asthma had been positively diagnosed in roughly 10% of the children aged 18 months to 11 years, to make the diagnosis required an average of 16 to 20 consultations for respiratory problems. According to the symptoms recorded before the diagnosis was made, only three quarters of the children had presented with wheeze and roughly a third had had difficulty in sleeping. We think that the doctor's time would be better spent teaching patients about their asthma than by repeatedly treating non-existent respiratory infection.