Neural Cell Adhesion Molecule Protein Research Articles

Swimming alters some proteins of skeletal muscle tissue in rats with Alzheimer-like phenotype

ObjectivesExercise training plays a significant role in preventing the destruction of central nerve neurons and muscle atrophy. The purpose of the present study was to investigate the effect of a period of swimming training on the expression of Neural cell adhesion molecule (NCAM), Semaphorin 3A (SEMA3A), and Profilin-1 (PFN1) proteins in the gastrocnemius muscle of Alzheimer-like phenotype rats. Methods & materials32 Wistar males were (6 weeks of age) divided into four groups: Healthy Control (HC), Alzheimer-like phenotype's Control (AC), Healthy Training (HT), and Alzheimer-like phenotype's Training (AT). Alzheimer-like phenotypes were induced by beta-amyloid injection in the hippocampus. The training program consisted of 20 swimming sessions. Gastrocnemius muscle was removed after the intervention, and NCAM, SEMA3A, and PFN1 proteins were measured by the immunohistoflorescent method. ResultsThe results showed that SEMA3A was increased (p = 0.001), and NCAM (p = 0.001), and PFN1 (p = 0.001) were decreased in AC compared to the HC group. Also, the results showed that NCAM (p = 0.001) and Pfn1 (p = 0.002) increased in the HT group compared to HC, and the NCAM (p = 0.001) and Pfn1 (p = 0.002) in AT group compared to AC (p = 0.001) increased significantly, while SEMA3A was reduced in the HT group compared to HC (p = 0.001) and AT group compared to AC (p = 0.001) ConclusionSwimming effectively improves axon regeneration and neuronal formation in motor neurons and, therefore, can be an effective intervention to prevent and control the complications of Alzheimer-like phenotype.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, −4, and −5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, −4, and −5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and −4 or TRPC1 and −5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, −4, and −5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.

Molecular Interactions of the Polysialytransferase Domain (PSTD) in ST8Sia IV with CMP-Sialic Acid and Polysialic Acid Required for Polysialylation of the Neural Cell Adhesion Molecule Proteins: An NMR Study.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

The Role of Forced and Voluntary Training on Accumulation of Neural Cell Adhesion Molecule and Polysialic Acid in Muscle of Mice with Experimental Autoimmune Encephalomyelitis.

It has been suggested that depletion of adhesion molecules is one of the factors associated with or possibly responsible for multiple sclerosis (MS) progression. The aim of this study was to investigate the effect of forced and voluntary training before and after induction of experimental autoimmune encephalomyelitis (EAE) on accumulation of neural cell adhesion molecule (NCAM) and polysialic acid (PSA) in neuromuscular junction denervation in plantaris and soleus muscles in C57BL/6 female mice. A total of 40 female C57BL/6 mice, 10-week-old, were randomly divided into four groups, including induced control groups without EAE induction, induced EAE without training, and forced and voluntary training groups. Myelin oligodendrocyte glycoprotein peptide 35–55 (300 μg in saline; MOG 35–55; KJ Ross-Petersen ApS, Denmark) was injected subcutaneously at the base of the tail of each mouse. Clinical assessment of EAE was performed daily using a 15-point scoring system following immunization. Training groups performed the swimming program for 30 min/day, 5 times/week, for 4 weeks. Mice had access to a treadmill for one hour per day, 5times/week, for 4 weeks in individual cage. The mice were scarified, and the plantaris and soleus muscles were then isolated for investigation of proteins expression using IHC. An analysis of the preventive exercise (before) and recovery exercise (after) of the EAE was performed. Images of the stained sections were taken using a fluorescent microscope. Quantitative image analysis was performed using ImageJ software package. The obtained data from the mean percentage expression of PSA and NCAM in pre- and post-soleus and plantaris muscles showed that the highest and lowest expression levels of PSA and NCAM belonged to control and swim EAE (SE) groups, respectively. The low expression levels of PSA and NCAM were detected in rat with MS without intervention. In conclusion, the relationship between increasing levels of NCAM and PSA protein expression and voluntary and compulsory activity were detectable both in pre and post-soleus and plantaris. However, voluntary activity resulted in more expression levels of NCAM and PSA than that of compulsory. In conclusion, since it has been suggested that depletion of NCAM is one of the factors associated with or possibly responsible for MS progression, these findings show exercise MS progression may be reduced by increasing expression of exercise-related adhesion molecule such as NCAM and PSA (a glycan modification of the NCAM).

MiR-125b promotes tau phosphorylation by targeting the neural cell adhesion molecule in neuropathological progression.

MicroRNAs, small noncoding RNAs, not only regulate gene expression at the post-transcriptional level in a variety of physiological processes but also accompany the initiation and progression of a vast number of diseases, including dementia. While miR-125b has been shown to be aberrantly expressed in some dementia patients, its role in the pathological process remains ambiguous. Presenilin-1/2 conditional double knockout mice exhibit a range of symptoms, including impaired cognition and memory, increased tau phosphorylation, neuroinflammation, and apoptosis, and are therefore regarded as a useful dementia model. In the prefrontal cortices of double knockout mice, miR-125b was found to be abnormally increased in an age-dependent manner. We further verified the neural cell adhesion molecule (NCAM) as an miR-125b target using the dual luciferase reporter assay. The NCAM protein level was decreased when miR-125b was overexpressed (OE) in neuronal growth factor-induced differentiated PC12cells, which further inhibited the neuronal growth factor-induced phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at the Ser9 site and ultimately increased the GSK3β activity and tau phosphorylation. Moreover, on serum deprivation, high GSK3β activity in differentiated miR-125b-OE PC12cells induced increased caspase-3 activation. Finally, adeno-associated virus-mediated miR-125b overexpression in the prefrontal cortexes of wild-type C57B/L6 mice resulted in decreased dendritic spine density. In addition, similar to the invitro data, elevated GSK3β activity and hyperphosphorylation of the tau protein were confirmed. Taken together, our findings reveal a direct regulation of miR-125b on NCAM, which leads to further effects on downstream GSK3β activity and tau phosphorylation and may contribute to the generation of neurofibrillary tangles in neuropathological progression.

Effects of Angelica Extract on Schwann Cell Proliferation and Expressions of Related Proteins

The present study investigated the effects of Angelica extract (AE) on Schwann cell proliferation and expressions of related proteins, including brain derived neurotrophic factor (BDNF), neural cell adhesion molecule (NCAM), and proliferating cell nuclear antigen (PCNA). Proliferation activity and cell cycles of SCs were evaluated by MTT assay and flow cytometry methods, respectively, after 12 h treatment of AE at different concentrations (62.5, 125, 250, 1000, 2000, 4000, and 8000 mg/L). SCs were treated by 500, 1000, and 2000 mg/L AE for 24 h or 48 h; the related genes mRNA and proteins expressions in SCs were detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit. At the concentration range of 125–2000 mg/L, the SC proliferation was induced by AE in a dose-dependent manner, especially 1000 and 2000 mg/L; cells in drug-treated groups showed the most increase. Cells counts were ascended significantly in (G2/M + S) phase compared to control group. BDNF, NCAM, and PCNA protein expressions significantly increased at drug-treated groups. Relative genes mRNA expressions levels were also significantly higher compared to control group. The results indicated that AE facilitated SC proliferation and related genes and proteins expressions, which provided a basic guideline for nerve injury repair in clinic.

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis.

21q21 deletion involving NCAM2: Report of 3 cases with neurodevelopmental disorders

Here we report three patients affected with neurodevelopmental disorders and harbouring 21q21 deletions involving NCAM2 gene. NCAM (Neural Cell Adhesion Molecule) proteins are involved in axonal migration, synaptic formation and plasticity. Poor axonal growth and fasciculation is observed in animal models deficient for NCAM2. Moreover, this gene has been proposed as a candidate for autism, based on genome-wide association studies. In this report, we provide a comprehensive molecular and phenotypical characterisation of three deletion cases giving additional clues for the involvement of NCAM2 in neurodevelopment.

Partial reduction in neural cell adhesion molecule (NCAM) in heterozygous mice induces depression-related behaviour without cognitive impairment

The neural cell adhesion molecule (NCAM) plays an important role in brain plasticity. Using mice deficient in all isoforms of NCAM we have previously demonstrated that constitutive deficiency in the NCAM gene (NCAM−/−) resulted in cognitive impairment, anhedonic behaviour and a reduced ability to cope with stress. This was accompanied by reduced basal phosphorylation of the fibroblast growth factor receptor 1 (FGFR1) and reduced phosphorylation of calcium-calmodulin kinase (CaMK) II and IV and cAMP response element binding protein (CREB). The present study was aimed to investigate how partial deficiency in NCAM in mice (NCAM+/−) affected phenotype. We found that NCAM+/− mice showed a longer period of immobility in the tail suspension test, increased latency to feed in the novelty-suppressed feeding test and reduced preference for sucrose in sucrose preference test. Both NCAM+/− and NCAM−/− mice showed reduced extinction of contextual fear. In contrast to NCAM−/− mice, NCAM+/− mice did not demonstrate memory impairment in either object recognition or contextual fear conditioning tests. Levels of phosphorylated FGFR1 in the hippocampus and prefrontal/frontal cortex of NCAM+/− mice were partially reduced and no changes in the phosphorylation of CaMKII, CaMKIV or CREB in the hippocampus were found. We conclude that a constitutive partial reduction in NCAM proteins results in a behavioural phenotype related to depression without impairment in cognitive functions, also affecting the level of FGFR1 phosphorylation without major alterations in CaMKII and CaMKIV intracellular signalling. Partial reduction in FGFR1 phosphorylation might explain the observed behavioural phenotype in NCAM+/− mice.

Neural cell–cell and cell–substrate adhesion through N-cadherin, N-CAM and L1

In this study neural (N)-cadherin, neural cell adhesion molecule (N-CAM) and L1 proteins and their antibody equivalents were covalently immobilized on a polyethylene-imine (PEI)-coated glass surface to form neuron-adhesive coatings. Impedance sensing and (supplementary) image analysis were used to monitor the effects of these CAMs. Immobilization of high concentrations of both N-cadherin protein and antibody led to good adhesion of neurons to the modified surface, better than surfaces treated with 30.0 and 100.0 µg ml−1 N-CAM protein and antibody. L1 antibody and protein coating revealed no significant effect on neuronal cell–substrate adhesion. In a second series of combinatorial experiments, we used the same antibodies and proteins as medium-additives to inhibit cell–cell adhesion between neurons. Adhesion of neurons cultured on N-cadherin protein or antibody-modified surfaces was lowered by the addition of a soluble N-cadherin protein and antibody to the culturing medium, accelerating neuronal aggregation. The presence of a soluble N-CAM antibody or protein had no effect on the adhesion of neuronal cells on a N-cadherin protein-modified surface. On a N-cadherin antibody-coated surface, the addition of a soluble N-CAM protein led to cell death of neurons after 48 h, while a N-CAM antibody had no effect. In the presence of a soluble N-cadherin protein and antibody the aggregation of neurons was inhibited, both on N-CAM protein and N-CAM antibody-modified surfaces. Neurons cultured on immobilized antibodies were less affected by the addition of soluble CAM blockers than neurons cultured on immobilized proteins, indicating that antibody–protein bonds are more stable compared to protein–protein bonds.

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro(510)-Tyr(511)-Ser(512) (PYS) and Gln(516)-Val(517)-Gln(518) (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro(510)-Tyr(511) eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.

Identification of NCAM that interacts with the PHE-CoV spike protein

BackgroundThe spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies.ResultsWe identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection.ConclusionsA novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

5-HT 1A receptors mediate detrimental effects of cocaine on long-term potentiation and expression of polysialylated neural cell adhesion molecule protein in rat dentate gyrus

The present study investigated the involvement of 5-HT 1A receptors in the inhibitory effect of single administration of cocaine (COC, 15 mg/kg i.p.) on the induction of long-term potentiation (LTP) in slices of rat dentate gyrus (DG), prepared 30 min and 2 days after COC administration. These effects of COC were blocked by an antagonist of 5-HT 1A receptors, WAY 100635 (0.4 mg/kg i.p.), which had been administered 20 min before COC. The detrimental effect of COC on LTP in slices prepared 30 min after COC administration could be prevented by blocking glucocorticoid receptors (GRs) using mifepristone (RU 38486, 10 mg/kg s.c. given 1 h before COC), similar as in slices obtained 2 days after COC as reported previously [ Maćkowiak et al. (2008) Eur J Neurosci 27:2928—2937]. After a single administration of an agonist of 5-HT 1A receptors, 8-OH-DPAT, (0.5 mg/kg i.p.), the level of LTP in slices prepared 2 days later was significantly decreased resembling the effect of COC. This effect of 8-OH-DPAT was antagonized by WAY 100635 (0.4 mg/kg i.p.), administered 20 min before 8-OH-DPAT and by RU 38486, given 1 h before 8-OH-DPAT. COC-induced inhibition of LTP could be blocked by the inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), SL 327 (50 mg/kg i.p.), administered 1 h before COC, but not by the inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), LY 294002 (80 mg/kg i.p.). COC-induced reduction in the number of polysialylated neural cell adhesion molecule (PSA-NCAM)—positive neurons in rat dentate gyrus could also be prevented by WAY 100635, given 20 min before COC. These data indicate that the indirect 5-HT 1A receptor activation by a single COC administration and subsequent stimulation of extracellular signal-regulated kinases (ERK 1/2) signaling pathway result in a decrease of the potential for long-term increase in synaptic efficacy in rat DG lasting at least two but less than 7 days, most likely via activation of the hypothalamic-pituitary-adrenal (HPA) axis.

NCAM expression induces neurogenesis in vivo

Neural cell adhesion molecule (NCAM) plays an important role during neural development and in the adult brain, whereby most functions of NCAM have been ascribed to its unique polysialic acid (PSA) modification. Recently we presented evidence suggesting that expression of NCAM in vivo interferes with the maintenance of forebrain neuronal stem cells. We here aimed at investigating the fate of cells generated from NCAM-overexpressing stem cells in postnatal mouse brain and at elucidating the functional domains of NCAM mediating this effect. We show that ectopic expression of the NCAM140 isoform in radial glia and type C cells induces an increase in cell proliferation and consequently the presence of additional neuronal type A cells in the rostral migratory stream. A mutant NCAM protein comprising only fibronectin type III repeats and immunoglobulin-like domain 5 was sufficient to induce this effect. Furthermore, we show that the neurogenic effect is independent of PSA, as transgenic NCAM is not polysialylated in radial glia and type C cells. These results suggest that heterophilic interactions of NCAM with other components of the cell membrane must be involved.

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.

IgIII (270–280)-fragment-like H 2N-DDSDEEN-COOH peptide modulates N-CAM expression via Ca 2+-dependent ERK signaling during “in vitro neurogenesis”

The two major isoforms (180 kDa and 140 kDa) of the neural cell adhesion molecule (N-CAM) are crucially involved in neurogenesis and brain repair via activation of the mitogen-activated protein kinase (MAPK) cascade. Modification by glycosylation, and homophilic and heterophilic interactions regulate the function of N-CAM, but little is known about the interplay of these processes. In the neuron-like PC12 cell line, extracellular small acidic peptides have been shown to modulate the expression of N-CAM mRNA and protein and regulate its translocation to the plasma membrane. Among these peptides, a synthetic Ig-III-like short sequence (H 2N-DDSDEEN-COOH), designated sSP, was particularly potent. In this study, we analyzed the cross-talk between nerve growth factor (NGF) and extracellular sSP in native and N-CAM-transfected PC12 cells to determine if these systems interact to modulate transduction pathways and regulate early steps of neurogenesis in vitro. Our results indicate that sSP accelerated the phosphorylation of extracellular regulated kinase-1 (ERK1) and -2 (ERK2) and promoted plasma membrane translocation of 180 kDa N-CAM. By stabilizing cell–cell contacts and promoting cell cluster formation, these events, which were mediated via a significant increase in intracellular Ca 2+, regulated some of the early stages of the NGF-induced differentiation process.

WITHDRAWN: Dendritic Spine and Synapse Morphological Alterations Induced by a Neural Cell Adhesion Molecule (NCAM) Mimetic.

The neural cell adhesion molecule (NCAM) is a glycoprotein expressed on the surface of neurons and glial cells. It plays a key role in morphogenesis of the nervous system, regeneration of damaged neural tissue and synaptic plasticity. The extracellular domain of NCAM engages in homophilic interactions (NCAM binding to NCAM) and in heterophilic interactions between NCAM and other proteins such as the fibroblast growth factor (FGF) receptor. It promotes synaptogenesis and activity-dependent remodelling of synapses but less is know of its influence on synaptic and dendritic morphology. Recently, quantitative electron microscopy and 3-dimensional reconstruction (3-D) of ultrathin serial sections has been used to examine the morphology of synapses and dendritic spines in the hippocampus of rats treated with a neural cell adhesion molecule-derived fibroblast growth factor receptor agonist, FGL-peptide (an NCAM mimetic). These data show clearly that the FGL peptide has marked influences on both spine and synaptic form.

Polysialylation Increases Lateral Diffusion of Neural Cell Adhesion Molecule in the Cell Membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.

Valproic acid inhibits adhesion of vincristine- and cisplatin-resistant neuroblastoma tumour cells to endothelium

Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-, doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA), a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-2CDDP, UKF-NB-3VCR, UKF-NB-3CDDP, UKF-NB-6VCR, UKF-NB-6CDDP) to an endothelial cell monolayer, accompanied by downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins were enhanced in UKF-NB-3VCR and UKF-NB-3CDDP, compared to the drug naïve controls. p73 was diminished, whereas the p73 isoform deltaNp73 was upregulated in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid blocked adhesion of UKF-NB-3VCR and UKF-NB-3CDDP, but not of UKF-NB-3DOX, and induced the upregulation of NCAM surface expression, NCAM protein content and NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73 in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive processes.

Cimetidine induces apoptosis of human salivary gland tumor cells

It has been reported that cimetidine, a histamine type-2 receptor (H2R) antagonist, inhibits the growth of glandular tumors such as colorectal cancer. However, its effects against salivary gland tumors are still unknown. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express the neural cell adhesion molecule (NCAM) and also that HSG cell proliferation could be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. In the present study, we investigated the effects of cimetidine via the expression of NCAM on tumor growth and perineural/neural invasion in salivary gland tumor cells. Expression of both NCAM mRNA and protein was found to decrease in a dose-dependent manner upon treatment with cimetidine for 24 h. The MTT assay and confocal laser microscopy clearly showed that HSG cells underwent apoptosis after treatment with cimetidine. Activation of caspases 3, 7, 8 and 9 was observed in HSG cells after cimetidine treatment, thus confirming that the apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with cimetidine. We also found that the cimetidine-mediated down-regulation of NCAM expression in HSG cells did not occur via blocking of the histamine receptor, even though H2R expression was observed on HSG cells, as two other H2R antagonists, famotidine and ranitidine, did not show similar effects. We demonstrated for the first time that cimetidine can induce significant apoptosis of salivary gland tumor cells, which express NCAM, at least in part by down-regulation of NCAM expression on the cells. These findings suggest that the growth, development and perineural/neural invasion of salivary gland tumor cells can be blocked by cimetidine administration through down-regulation of NCAM expression, as well as induction of apoptosis.