NCAM Proteins Research Articles

The dynamic TRPV2 ion channel proximity proteome reveals functional links of calcium flux with cellular adhesion factors NCAM and L1CAM in neurite outgrowth

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2’s physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation.Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2’s calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

Effects of Resistance Aerobic Training on the Expression of SEMA3A and NCAM Genes and Proteins in the Soleus Muscle of Male Rats in Different Age Groups

Background Exercise and physical activity improve damage and destructive changes in the peripheral nervous system. Objective This study aims to investigate the effects of resistance aerobic training on the expression of SEMA3A and NCAM genes and proteins of soleus muscle in male rats of different age groups. Methods A total of 30 male Wistar rats in three groups of 2-week-old (infant) (n=10), 6-week-old (young) (n=10), and 96-week-old (elderly) (n=10) were divided into two groups of training and control. The training program included 6 days of resistance and aerobic training one day in between. The rats were euthanized and the soleus muscle was extracted 48 h after the last training session. A fluorescent microscope was used to measure SEMA3A and NCAM proteins in muscle fibers. Gene expressions were measured by real-time polymerase chain reaction. Results The results showed that SEMA3A gene expression was not significantly different between exercise and control groups in all three age groups; however, SEMA3A protein expression was significantly reduced in infant and young exercise groups (P≤0.001). Also, NCAM gene expression was not significantly different between training and control groups in all three age groups; while there was a significant decrease in NCAM protein expression in young and old training groups compared to the control group (P≤0.001). Conclusion The results showed that resistance aerobic training can inhibit the process of peripheral nerve damage in infant and young rats, as well as the growth and strength of the neuromuscular junction in young and elderly rats.

Evaluation of Morphological Changes in Muscle Fibers and Neuronal Terminals of Motor Neurons in Male Rats from Childhood to Old Age

Introduction: The aim of this study was to investigate the effect of time and age on morphological changes in male rats. Methods: For conducting this study, 15 male Wistar rats in three age groups of 2 weeks (150-100 g), 6 weeks (250-220 g) and 96 weeks (320-280 g) were kept in Pasargad Tissue and Gene Laboratory in Tehran City for six weeks and 48 hours later they were dissected and the soleus muscles was extracted. Cross-sectional area and number of muscle fibers were measured by light microscopy. Immunohistochemical method was used to measure sema3a and NCAM proteins of muscle fibers. To determine the differences between the groups, one-way analysis of variance with Tukey post hoc test was used at the significant level of p≤0.05. Statistical analysis was performed by Prism5 software. Results: The results showed that there was a significant difference in the cross section of muscle fibers between the three age groups: children and young people (p=0.01), children and the elderly (p=0.049), and finally young people and the elderly (p=0.049). There was also a significant decrease in the number of muscle fibers in the age group of elderly compared to children and elderly to young people (p≤0.001). Moreover, sema3a protein expression increased significantly over time between the age groups of children and young (p≤0.048), children and elderly (p=0.0001) and finally young and elderly (p=0.0001). NCAM protein expression significantly reduced in three age groups: children and young people (p=0.048), children and controls (p=0.0001), and young and elderly people (p=0.001). Conclusion: The results showed that with increasing growth from childhood to youth, hypertrophy increases and decreases in old age, in addition the process of hyperplasia will decrease with age. In addition to proteins involved in axonal growth due to growth decrease and neurodegenerative proteins also increase.

Effective Conformational Sampling in Explicit Solvent with Gaussian Biased Accelerated Molecular Dynamics.

In this Article, a user-friendly Gaussian biased accelerated molecular dynamics (GbAMD) method is presented that uses a sum of Gaussians of potential energies as the biased force to accelerate the conformational sampling. The easy parameter setting of GbAMD is demonstrated in a variety of simulation tests for the conformational transitions of proteins with various complexity including the folding of Trpcage, GB1p, and HP35 peptides as well as the functional conformational changes of nCaM and HIV-1 PR proteins. Additionally, the ability of GbAMD in conformational sampling and free-energy evaluation is quantitatively assessed through the comparison of GbAMD simulations on the folding of α-helical Trpcage and β-hairpin GB1p with the accompanying standard dual boost AMD and conventional MD (cMD) simulations. While GbAMD can fold both peptides into their native structures repeatedly in individual trajectories, AMD can only fold Trpcage and cMD fails the folding in both cases. As a result, only GbAMD can quantitatively measure the properties of the equilibrium conformational ensemble of protein folding consistent with experimental data. Also notable is that the structural properties of the indispensable unfolded and transition states in the folding pathways of Trpcage and GB1p characterized by GbAMD simulations are in great agreement with previous simulations on the two peptides. In summary, GbAMD has an effective conformational sampling ability that provides a convenient and effective access for simulating the structural dynamics of biomolecular systems.

Ascending midbrain dopaminergic axons require descending GAD65 axon fascicles for normal pathfinding.

The Nigrostriatal pathway (NSP) is formed by dopaminergic axons that project from the ventral midbrain to the dorsolateral striatum as part of the medial forebrain bundle. Previous studies have implicated chemotropic proteins in the formation of the NSP during development but little is known of the role of substrate-anchored signals in this process. We observed in mouse and rat embryos that midbrain dopaminergic axons ascend in close apposition to descending GAD65-positive axon bundles throughout their trajectory to the striatum. To test whether such interaction is important for dopaminergic axon pathfinding, we analyzed transgenic mouse embryos in which the GAD65 axon bundle was reduced by the conditional expression of the diphtheria toxin. In these embryos we observed dopaminergic misprojection into the hypothalamic region and abnormal projection in the striatum. In addition, analysis of Robo1/2 and Slit1/2 knockout embryos revealed that the previously described dopaminergic misprojection in these embryos is accompanied by severe alterations in the GAD65 axon scaffold. Additional studies with cultured dopaminergic neurons and whole embryos suggest that NCAM and Robo proteins are involved in the interaction of GAD65 and dopaminergic axons. These results indicate that the fasciculation between descending GAD65 axon bundles and ascending dopaminergic axons is required for the stereotypical NSP formation during brain development and that known guidance cues may determine this projection indirectly by instructing the pathfinding of the axons that are part of the GAD65 axon scaffold.

Abstract 33: Implementing CAD cells quantify the relationships between Ig-CAM-mediated adhesion, force transduction, and actin dynamics

Abstract Several neuro-degenerative diseases and motor defects have been linked to malfunctions in the motility of neurons. This project is focused on the determining whether the roles of the transmembrane proteins NCAM and L1-CAM can be studied using the CAD cell line as a model. CAD cells are a cancer cell line that come from mice and can be stimulated to grow as either neurons or glial cells. In addition, this cell line has been shown to be an easily transfected line. In order to determine usefulness of this cell line several techniques were used. Western blots were implemented in order to determine if the NCAM and/or L1-CAM and proteins known to be involved in their signaling pathways were present in the cell line. Western blots were then confirmed by immunocytochemistry staining. Next, the CAD cell line was transfected with photoactivateable actin. Finally, immuno beads were attached to the membranes of these CAD cells and then holographic optical tweezers were used to determine whether or not thsee ligands coupled to the retrograde actin flow in the cells. The combination of these experiments should provide the evidence for whether or not the CAD cells would be a feasible model to study the nature of the NCAM and L1-CAM proteins and their relationship to the movement of actin in the cytoskeleton of neurons. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 33. doi:1538-7445.AM2012-33

Impact of maternal separation on neural cell adhesion molecules expression in dopaminergic brain regions of juvenile, adolescent and adult rats

Stressful experiences in the early stages of life can influence brain development and maturation, and they can also increase the risk for some psychiatric disorders; however, the specific mechanisms of this effect are still poorly understood. Neural cell adhesion molecules (NCAM 120, 140, 180kDa) are known to play an important role in normal brain development and synaptic plasticity. Therefore, we decided to investigate whether maternal separation (MS) in rats, a paradigm which models an early life stress, has any impact on the expression of NCAM proteins in the juvenile, adolescent and adult brains of both male and female rats. Specifically, we focused our efforts on the brain regions associated with dopaminergic neurotransmission. In juvenile rats, MS decreased the levels of NCAM-140 in the substantia nigra (SN) of females and NCAM-180 in the ventral tegmental area of males. During adolescence, a reduction in NCAM-180 levels in the SN and medial prefrontal cortex (mPFC) of MS females was revealed. Finally, in adulthood, a decrease in NCAM-180 expression was observed in the mPFC of MS males. The results that we obtained indicate that early life stress can affect maturation and NCAM-driven plasticity in dopaminergic brain areas at different stages of ontogenesis and with a sex-specific manner.

Inhibition of neuronal cell–cell adhesion measured by the microscopic aggregation assay and impedance sensing

Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron–neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron–neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control of spatial arrangement of cells in culture. In the literature N-CAM, L1 and N-cadherin proteins are pointed out as main regulators of neuronal adhesion. In this study, these three main cell adhesion molecules were used to inhibit neuron-to-neuron adhesion and aggregation. Both soluble extracellular domains and antigen antibodies were added to these adhesion molecules. They were investigated for their blocking ability in neuronal cultures. First, in a 96 h aggregation assay on a low-adhesive substrate, the effect of inhibition of the three proteins on aggregation of cortical neurons was investigated optically. Both L1 antibody and L1 protein had no effect on the degree of aggregation. An N-cadherin antibody however was shown to be effective in aggregation inhibition at concentrations of 1 and 3 µg ml−1. Up to 96 h no aggregation occurred. A similar effect was achieved by the N-cadherin protein, although less distinct. N-CAM blocking revealed no inhibition of aggregation. Second, results from IS corresponded to those of the aggregation assays. In these experiments neuron–neuron adhesion was also inhibited by blocking N-CAM L1 and N-cadherin. Cortical neurons were cultured in small wells containing circular 100 µm diameter gold electrodes, so small changes in cell–cell interactions in monolayers of neurons could be monitored by IS. Impedances of neuron-covered electrodes were significantly lower in the presence of the N-cadherin antibody and protein at concentrations of 1, 3 and 10 µg ml−1, indicating a less profound binding between adjacent neurons. Results from the aggregation assays and impedance measurements demonstrate the applicability of blocking cell adhesion molecules for inhibition of cell–cell adhesion and aggregation.

Repeated risperidone treatment increases the expression of NCAM and PSA-NCAM protein in the rat medial prefrontal cortex

The present study investigates whether the anti-schizophrenic drug risperidone may evoke changes in the expression of NCAM/PSA-NCAM proteins, an indispensable element in the remodeling of synaptic arrangements, in the medial prefrontal cortex (mPFC). Rats were treated with risperidone (0.2 mg/kg, i.p.) either once or repeatedly (once a day, for 21 days). The expression of NCAM and PSA-NCAM proteins was analyzed via western blot and immunohistochemistry at intervals of 3 h and 3, 6, and 9 days after the single or the last risperidone dose. Repeated (but not acute) administration of risperidone was found to increase the expression of NCAM-180, NCAM-140 and PSA-NCAM proteins at 3 or 6 days after treatment. PSA-NCAM immunoreactivity was found in cell bodies, perisomatic-like sites, and in the neuropil of the mPFC. Neither single nor repeated risperidone administration changed the number of PSA-NCAM neurons in the mPFC. In contrast, the repeated risperidone treatment increased the number of PSA-NCAM perisomatic-like sites and the length density of PSA-NCAM positive neuropil at 3 days after the last injection. The data obtained indicate that risperidone, given repeatedly, may promote the remodeling of the structure of presumably GABA-ergic interneurons and that it may evoke the rearrangement of the synaptic contact in the mPFC.

A Novel α-Helix in the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Is Critical for N-Glycan Polysialylation

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

Analysis of a new homozygous deletion in the tumor suppressor region at 3p12.3 reveals two novel intronic noncoding RNA genes

Homozygous deletions or loss of heterozygosity (LOH) at human chromosome band 3p12 are consistent features of lung and other malignancies, suggesting the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene has been cloned thus far from the overlapping region deleted in lung and breast cancer cell lines U2020, NCI H2198, and HCC38. It is DUTT1 (Deleted in U Twenty Twenty), also known as ROBO1, FLJ21882, and SAX3, according to HUGO. DUTT1, the human ortholog of the fly gene ROBO, has homology with NCAM proteins. Extensive analyses of DUTT1 in lung cancer have not revealed any mutations, suggesting that another gene(s) at this location could be of importance in lung cancer initiation and progression. Here, we report the discovery of a new, small, homozygous deletion in the small cell lung cancer (SCLC) cell line GLC20, nested in the overlapping, critical region. The deletion was delineated using several polymorphic markers and three overlapping P1 phage clones. Fiber-FISH experiments revealed the deletion was approximately 130 kb. Comparative genomic sequence analysis uncovered short sequence elements highly conserved among mammalian genomes and the chicken genome. The discovery of two EST clusters within the deleted region led to the isolation of two noncoding RNA (ncRNA) genes. These were subsequently found differentially expressed in various tumors when compared to their normal tissues. The ncRNA and other highly conserved sequence elements in the deleted region may represent miRNA targets of importance in cancer initiation or progression.

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. We have studied the functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, our findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM.

Immunohistochemistry and Electron Microscopy for Diagnosis of Neuroendocrine Tumors

This review article describes various neuroendocrine (NE) markers used for the diagnosis of NE neoplasms. These NE markers include synaptophysin, chromogranin A, N-CAM, and SNARE proteins (SNAP −25). EM demonstration of secretory granules is also emphasized in the definitive diagnosis of NE tumors. Three representative cases are included in which immunohistochemistry and electron microscopy are applied in diagnostic workup.

Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules

Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1–10 μg/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1α, IL-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1α and IL-1β, and IL-6, IL-8 and TNF-α as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria.

C-erbB2/neuTransfection Induces Gap Junctional Communication Incompetence in Glial Cells

Astrocytes form functional networks that participate in active signaling in which external stimuli are generated and amplified in many of the same ways as in neurons. Gap junctions between astrocytes offer the structural avenue by which the electrical and metabolic signals are propagated from one cell to another. Little is known about the trafficking, assembly, and degradation mechanisms of the major astrocytic gap junction protein connexin43. We have studied a glial cell line transfected with the C-erbB2/neu oncogene (neu+), finding severe interruption of gap junctional communication after stable transfection. Evidence from Western blotting and phosphorylation studies showed that the processing of connexin43 to its higher phosphorylated isoforms is disturbed. Confocal laser imaging indicates that the major deficit in the neu+ cells is attributable to a lack in plaque assembly of connexin43. Because the neu+ cells also lack N-CAM proteins and because work from others has indicated a close relationship between communication competence and constitutive CAM expression, our data suggest that expression of C-erbB2/neu oncogene alters cell-cell association via CAM proteins, which thereby affects gap junction plaque assembly and appropriate phosphorylation of connexin43.

Negative regulation of the P0 gene in Schwann cells: suppression of P0 mRNA and protein induction in cultured Schwann cells by FGF2 and TGF beta 1, TGF beta 2 and TGF beta 3.

During the development of peripheral nerves, Schwann cells are induced to form myelin sheaths round the larger axons. This process involves a complex series of events and the nature of the molecular signals that regulate and control myelin formation in Schwann cells is not well understood. Our previous experiments on rat Schwann cells in vitro, using serum-free defined medium, showed that a myelin-related protein phenotype could be induced in early postnatal Schwann cells in culture by elevation of intracellular cyclic AMP levels in the absence of growth factors, conditions under which the cells are not dividing. Cells with this phenotype expressed the major myelin glycoprotein P0 and expression of p75 NGF receptor, N-CAM, GFAP and A5E3 proteins was down-regulated. These changes are all characteristics associated with myelination in vivo. In contrast, when cyclic AMP levels were elevated in the presence of serum, suppression of cyclic AMP-induced differentiation resulted and DNA synthesis was induced. In this paper, we have used this model system and extended our analysis to explore the relationship between defined growth factors and suppression of myelination. We have used pure recombinant growth factors normally present in peripheral nerves, i.e. FGF1 and FGF2 and TGF beta 1, TGF beta 2, and TGF beta 3 and shown that, like serum, they can strongly suppress the forskolin-mediated induction of the P0 gene, both at the level of mRNA and protein synthesis. For both growth factor families, the suppression of P0 gene expression is dose-dependent and takes place in serum-starved cells that are mitotically quiescent. In the case of FGF2, however, even more complete suppression is obtained when the cells are simultaneously allowed to enter the cell cycle by inclusion of high concentrations of insulin in the culture medium. The present results raise the possibility that, in addition to the positive axonal signals that are usually envisaged to control the onset of myelination, growth factors present in the nerve may exert negative regulatory signals during development and thus help control the time of onset and the rate of myelination in peripheral nerves.

Transcriptional activation of the NCAM gene during myogenic differentiation

Earlier work has shown that in vitro myogenesis of muscle cells is accompanied by important changes in the expression of NCAM mRNAs and proteins, consisting mainly in an upregulation of the smaller transcripts and the 120- to 125-kD NCAM isoforms they encode. By measuring run-on transcription rates in nuclei isolated from C2 myoblasts and myotubes, we show in this report that transcriptional control can account for the increase in NCAM mRNA levels during myogenesis. Hence, the quantitative changes in overall abundance of NCAM transcripts and the concomitant qualitative changes in the mRNA pattern are regulated by independent mechanisms. Moreover, as transcriptional activity was stimulated to similar extents in the 5′ and 3′ region of the gene, we can exclude premature transcription termination as a mechanism involved in the preferential synthesis of shorter transcripts. Further support for transcriptional regulation of the NCAM gene is provided by experiments showing increased NCAM promoter activity in differentiating cells. As this increase was observed with 4.5 kb but not with 426 bp of upstream sequence, it seems to involve one or more upstream regulatory elements.

The three size classes of mouse NCAM proteins arise from a single gene by a combination of alternative splicing and use of different polyadenylation sites

The three size classes of mouse NCAM proteins arise from a single gene by a combination of alternative splicing and use of different polyadenylation sites