Rapid Turnover of Cortical NCAM1 Regulates Synaptic Reorganization after Peripheral Nerve Injury

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Neural cell adhesion molecule (NCAM) as a quantitative marker in synaptic remodeling

The neural cell adhesion molecule (NCAM) participates in adhesion and neuritic outgrowth during nervous system development. In the adult brain, NCAM is considered to be involved in neuronal sprouting and synaptic remodeling. the NCAM concentration of brain tissue has proved to be a useful marker of these processes, especially when viewed in comparison with the concentration of a marker of mature synapses, e.g. D3-protein (SNAP-25) or synaptophysin. The present review focusses on studies of adult brain in which NCAM concentration estimates and NCAM/D3 ratios have been used to evaluate the rate of synaptic remodeling in brain damage and degenerative diseases.Special issue dedicated to Dr. Robert Balázs.     

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.     

Effects of Melatonin on Behavioral Reactions and on the Expression of NCAM in Rats

We studied the effects of i.p. injection of melatonin in pharmacotherapeutic doses and of constant illumination (a melatonin synthesis-suppressing factor) on the behavior of rats in the open-field test and on the content of the main isoforms of neural cell adhesion molecule (NCAM) in the hippocampus, cerebellum, and neocortex of these animals. In the studied brain structures of the rats kept under conditions preventing the melatonin synthesis, we observed suppression of the behavioral activity of animals and a decrease in the expression of the NCAM180 isoform. In rats injected with 10 mg/kg melatonin, changes in the behavioral activity were insignificant. In the hippocampus and neocortex of rats of this group, the NCAM180 content increased. Our experiments showed that melatonin can be involved in the control of balance of the expression of different NCAM isoforms. Such a balance is a crucial factor determining plastic rearrangements of the synaptic contacts.     

Astrogliosis in the hippocampus and cortex and cognitive deficits in rats with streptozotocin-induced diabetes: Effects of melatonin

We examined, using a Western blot technique, the contents and compositions of a specific neuronal protein, NCAM, and of an astrocyte marker, GFAP, in the hippocampus and cortex of rats with streptozotocin (STZ)-induced diabetes and compared these indices with those in control (intact) animals and STZ-diabetic rats treated with melatonin. Behavioral cognitive indices manifested in the passive avoidance test (PAT) and Morris water maze (MWM) learning performance were also estimated in the above groups of animals. As was found, STZ-diabetic rats demonstrated clear cognitive deficits according to the values of the retention latency in the PAT and time of reaching the escape platform in the MWM performance. In these animals, the GFAP content was elevated, and the amount of degraded products of this protein increased, as compared with the control. Simultaneously, considerable down-regulation of the NCAM expression and modifications of NCAM isoform composition were found in diabetic animals. In addition, significantly increased levels of lipid peroxidation (according to the amounts of malondialdehyde + 4-hydroxyalkenals) were measured in the cortex and hippocampus of rats with stable diabetic hyperglycemia. All the above-mentioned shifts were significantly smoothed or even nearly completely compensated in the case of treatment of STZ-diabetic rats with melatonin (10 mg/kg per day). The role of diabetes-related changes in the amount and composition of specific neural and glial proteins in the development of cognitive deficits, the involvement of oxidative stress in the mechanisms of the respective shifts, and possible mechanisms of the neuroprotective effect of melatonin with respect to diabetes-related pathological biochemical and behavioral shifts are discussed. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 105–111, March–April, 2008.     

Induction of distinct phenotypes in clonal and variant GH4 pituitary cells

Summary GH cells are a widely used cell strain for the investigation of mechanisms regulating hormone release and synthesis. This report identifies two inducible phenotypes of the GH₄ clone (epithelioid and motile) which may extend studies of this well-characterized cell line to different stages of pituitary cell development. GH₄C₁ cells treated in suspension with epidermal growth factor plus tetradecanoylphorbol acetate aggregate to form large epithelioid colonies with extensive cell-to-cell and cell-to-substratum adhesion. These cells cease replicating within 48 h, increase 50% in cell volume, and synthesize 40-fold more prolactin. A GH₄C₁ variant with enhanced substratum adhesion and little or no cell-to-cell adhesion (GH₄S₁), responds differently to this treatment. These cells cease replicating immediately, show increased cell separation, develop leading lamellae, and display locomotory activity. Each phenotype coexists in mixed cultures of GH₄C₁ and GH₄S₁ cells. This indicates that the different inducible response of the variant does not result from autocrine secretion. A molecular basis for cell-to-cell adhesion in GH₄ cells was investigated. GH₄C₁, but not the variant cells, express a 180 kDa immunoreactive protein indistinguishable from an isoform of the neural cell adhesion molecule. Therefore the absence of cell-to-cell adhesion and inability to develop extensive cell-to-cell adhesion characteristic of the epithelioid phenotype may result from altered expression of the neural cell adhesion molecule. These findings are important because they have defined an in vitro approach to investigate genetic and cellular changes associated with the development and progression of pituitary cell phenotype. This study was initiated in the laboratory of Dr. A. H. Tashjian, Jr., under the support of grant DK11011 from the National Institutes of Health, Bethesda, MD. The completion of this study was supported by the Medical University of South Carolina Biomedical Support Grant of 1987–1988 and the American Cancer Society grant 1N-175.     

Identification and characterization of a novel neural cell adhesion molecule (NCAM)-associated protein from quail myoblasts: relationship to myotube formation and induction of neurite-like protrusions

Abstract We identified a novel neural cell adhesion molecule (NCAM)-associated protein, myo genesis-related and N CAM- a ssociated p rotein (MYONAP), the expression of which increases during the formation of myotubes in quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells). MYONAP shares homology with PL48 in human cytotrophoblasts and KIAA0386 in human brain. Excess expression of MYONAP in presumptive QM-RSV myoblasts induced long protrusions like neurites in cooperation with microtubules. Suppression of MYONAP by antisense cDNA prevented myotubes from forming in spite of the expression of myogenin, creatine kinase, and myosin, and rendered myoblast membranes resistant to fusion. Yeast two-hybrid screening showed that MYONAP interacted with NCAM specifically. Deletion of the NCAM-associated domain resulted in a loss of the function that induces neurite-like protrusions to form and disturbed the elongation of microtubules. The results suggested that MYONAP influenced the functions of microtubules and was involved in the formation of myotubes via its interaction with NCAM.     

Deficits of olfactory interneurons in polysialyltransferase‐ and NCAM‐deficient mice

The neurogenic niche of the anterior subventricular zone (SVZ) persistently generates neuroblasts, which migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into granule and periglomerular cells. Loss of the neural cell adhesion molecule NCAM or its post‐translational modification polysialic acid (polySia) impairs migration causing accumulations of cells in the proximal RMS and decreased OB volume. Polysialylation of NCAM is implemented by two polysialyltransferases, ST8SIA2 and ST8SIA4, with overlapping functions. Here, we used mice with Ncam1 and polysialyltransferase deletions to analyze how partial or complete loss of polySia synthesis or a combined loss of polySia and NCAM affects the RMS and the interneuron composition in the OB. Numerous calretinin (CR)‐positive cells were detected dispersed around the RMS in Ncam1 knockout, St8sia2, St8sia4 double‐knockout, and St8sia2, St8sia4, Ncam1 triple‐knockout mice, as well as in St8sia2 ^?/? but not in St8sia4 ^?/? mice. These changes were not reflected by reductions of CR‐positive cells in the granule or glomerular layer of the OB. Instead, calbindin‐positive periglomerular interneurons were strongly reduced in all polySia‐NCAM negative mice and slightly attenuated in St8sia2 ^?/? as well as in the St8sia4 ^?/? mice, which were devoid of ectopic CR‐positive cells along the RMS. Consistent with the early developmental generation of calbindin‐ as compared with CR‐positive OB interneurons, this phenotype was fully developed at postnatal day 5. Together, these results demonstrate that the early development of calbindin‐positive periglomerular interneurons depends on the presentation of polySia on NCAM and requires the activity of both polysialyltransferases. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 421–433, 2016     

Effect of Melatonin on Cognitive Ability of Rats and Expression of NCAM in the Brain Structures in Streptozotocin-Induced Diabetes

We studied a protective effect of a course injections of melatonin on cognitive deficiency in rats with streptozotocin-induced diabetes (STZD). The mean time necessary for the fulfillment of the Morris' water test in animals with STZD after 7 days of testing was three times greater than the corresponding index in the control group. Rats with STZD, which were injected with 10 mg/kg melatonin daily for 21 days after introduction of STZ, demonstrated a significantly lower level of cognitive deficiency ((in these rats the mean time necessary for the test fulfillment was only 48% greater than that in the control animals). In rats with STZD, substantial changes in the content of NCAM isoforms in the brain structures (significant decreases in the NCAM180 content in the hippocampus, neocortex, and cerebellum, and in that of NCAM140 in the cerebellum) were observed. Course injections of melatonin into the rats with STZD promoted significant normalization of the composition of NCAM isoforms in the structures under study. The data obtained indicate that control of expression of separate NCAM isoforms can be one of the mechanisms through which melatonin prevents the development of cognitive deficiency in diabetic animals.     

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant > 100 μM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.     

Induction of filopodia-like protrusions by transmembrane agrin: Role of agrin glycosaminoglycan chains and Rho-family GTPases

Filopodia sense the extracellular environment and direct movement in many cell types, including neurons. Recent reports suggest that the transmembrane form of the widely expressed proteoglycan agrin (TM-agrin) regulates formation and stability of neuronal filopodia. In order to elucidate the mechanism by which TM-agrin regulates filopodia, we investigated the role of agrin's glycosaminoglycan (GAG) chains in the induction of filopodia formation by TM-agrin over-expression in hippocampal neurons, and in the induction of filopodia-like processes in COS7 cells. Deletion of the GAG chains of TM-agrin sharply reduced formation of filopodia-like branched retraction fibers (BRFs) in COS7 cells, with deletion of the heparan sulfate GAG chains being most effective, and eliminated filopodia induction in hippocampal neurons. GAG chain deletion also reduced the activation of Cdc42 and Rac1 resulting from TM-agrin over-expression. Moreover, dominant-negative Cdc42 and Rac1 inhibited BRF formation. Lastly, over-expression of TM-agrin increased the adhesiveness of COS7 cells and this increase was reduced by deletion of the GAG chains. Our results suggest that TM-agrin regulates actin-based protrusions in large part through interaction of its GAG chains with extracellular or transmembrane proteins, leading to the activation of Cdc42 and Rac1.     

Impairment of neuropsychological behaviors in ganglioside GM3-knockout mice

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. Although its expression is tightly controlled during early embryo development and postnatal development and maturation in the brain, the physiological role of ganglioside GM3 in the regulation of neuronal functions has not been elucidated. In the present study, we examined motor activity, cognitive and emotional behaviors, and drug administration in juvenile GM3-knockout (GM3-KO) mice. GM3-KO male and female mice showed hyperactivity in the motor activity test, Y-maze test, and elevated plus maze test. In the Y-maze test, there was significantly less spontaneous alternation behavior in GM3-KO male mice than in wild-type mice. In the elevated plus maze test, the amount of time spent on the open arms by GM3-KO male mice was significantly higher than that of sex-matched wild-type mice. In contrast, there was no significant difference between GM3-KO and wild-type female mice in these tests. Thus, juvenile GM3-KO mice show gender-specific phenotypes resembling attention-deficit hyperactivity disorder (ADHD), namely hyperactivity, reduced attention, and increased impulsive behaviors. However, administration of methylphenidate hydrochloride (MPH) did not ameliorate hyperactivity in either male or female GM3-KO mice. Although these data demonstrate the involvement of ganglioside GM3 in ADHD and the ineffectiveness of MPH, the first-choice psychostimulant for ADHD medication, our studies indicate that juvenile GM3-KO mice are a useful tool for neuropsychological studies.     

On the mechanisms of neuroprotection by creatine and phosphocreatine

Creatine and phosphocreatine were evaluated for their ability to prevent death of cultured striatal and hippocampal neurons exposed to either glutamate or 3-nitropropionic acid (3NP) and to inhibit the mitochondrial permeability transition in CNS mitochondria. Phosphocreatine (PCr), and to a lesser extent creatine (Cr), but not (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801), dose-dependently ameliorated 3NP toxicity when applied simultaneously with the 3NP in Mg2+-free media. Pre-treatment of PCr for 2 or 5 days and Cr for 5 days protected against glutamate excitotoxicity equivalent to that achieved by MK801 post-treatment. The combination of PCr or Cr pre-treatment and MK801 post-treatment did not provide additional protection, indicating that both prevented the toxicity attributable to secondary glutamate release. To determine if Cr or PCr directly inhibited the permeability transition, mitochondrial swelling and depolarization were assayed in isolated, purified brain mitochondria. PCr reduced the amount of swelling induced by calcium by 20%. Cr decreased mitochondrial swelling when inhibitors of creatine kinase octamer-dimer transition were present. However, in brain mitochondria prepared from rats fed a diet supplemented with 2% creatine for 2 weeks, the extent of calcium-induced mitochondrial swelling was not altered. Thus, the neuroprotective properties of PCr and Cr may reflect enhancement of cytoplasmic high-energy phosphates but not permeability transition inhibition.     

NCAM regulates the proliferation,apoptosis, autophagy,EMT, and migration of human melanoma cells via the Src/Akt/mTOR/cofilin signaling pathway

The neural cell adhesion molecule (NCAM) plays critical roles in multiple cellular processes in neural cells, mesenchymal stem cells, and various cancer cells. However, the effect and mechanism of NCAM in human melanoma cells are still unclear. In this study, we found that NCAM regulated the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells by determining the biological behavior of NCAM knockdown A375 and M102 human melanoma cells. Further studies revealed that NCAM knockdown impaired the organization of actin cytoskeleton and reduced the phosphorylation of cofilin, an actin-cleaving protein. When cells were transfected with cofilin S3A (dephosphorylated cofilin), biological behavior similar to that of NCAM knockdown cells was observed. Research on the underlying molecular mechanism showed that NCAM knockdown suppressed activation of the Src/Akt/mTOR pathway. Specific inhibitors of Src and PI3K/Akt were employed to further verify the relationship between Src/Akt/mTOR signaling and cofilin, and the results showed that the phosphorylation level of cofilin decreased following inhibition of the Src/Akt/mTOR pathway. These results indicated that NCAM may regulate the proliferation, apoptosis, autophagy, migration, and epithelial-to-mesenchymal transition of human melanoma cells via the Src/Akt/mTOR/cofilin pathway-mediated dynamics of actin cytoskeleton.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28 , 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time a functional interaction between NCAM and EGFR in mammalian cells and investigates the molecular mechanisms underlying this interaction. First, NCAM and EGFR are shown to play opposite roles in neurite outgrowth regulation in cerebellar granular neurons. The data presented indicate that negative regulation of EGFR is one of the mechanisms underlying the neuritogenic effect of NCAM. Second, it is demonstrated that expression of the NCAM-180 isoform induces EGFR down-regulation in transfected cells and promotes EGFR down-regulation induced by EGF stimulation. It is demonstrated that the mechanism underlying this NCAM-180-induced EGFR down-regulation involves increased EGFR ubiquitination and lysosomal EGFR degradation. Furthermore, NCAM-180-mediated EGFR down-regulation requires NCAM homophilic binding and interactions of the cytoplasmic domain of NCAM-180 with intracellular interaction partners, but does not require NCAM-mediated fibroblast growth factor receptor activation.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

MicroRNA-29a suppresses the growth,migration, and invasion of lung adenocarcinoma cells by targeting carcinoembryonic antigen-related cell adhesion molecule 6

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an important regulator of cell adhesion, invasion, and metastasis. The aim of this study was to evaluate the functional roles of CEACAM6 in lung adenocarcinoma and to identify miRNAs that inhibit the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. CEACAM6 expression is associated with poor prognosis of patients with lung adenocarcinoma, and CEACAM6 has important functional roles in controlling the growth, migration, and invasion of lung adenocarcinoma cells in vitro and in vivo. Furthermore, miR-29a can suppress the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. Therefore, miR-29a/CEACAM6 axis represents a potential therapeutic target for treatment of lung adenocarcinoma.