The Polysialylated Neural Cell Adhesion Molecule Promotes Neurogenesis in vitro

A characteristic feature of neurogenic sites in the postnatal brain is the expression of the polysialylated forms of the neural cell adhesion molecule (PSA-NCAM). To investigate the role of PSA-NCAM in generation of neuronal populations, we developed an in vitro model where neurogenesis occurs in primary cortical cultures following serum withdrawal. We show that removal or inactivation of the PSA tail of NCAM in these cultures leads to a significant decrease in the number of newly generated neurons. Similarly, cultures prepared from NCAM knock-out mice exhibit a significantly reduced neurogenesis. Pulse-chase experiments using the proliferation marker BrdU reveal that the lack of PSA does not affect the mitotic rate of neural progenitors but rather, it reduces the early survival of newly generated neurons. These results suggest that, in addition to its role in the migration of neuronal progenitors, PSA-NCAM is required for the adequate survival of these cells.     

Chronic Low-Level Lead Exposure Impairs Embryonic to Adult Conversion of the Neural Cell Adhesion Molecule

The neural cell adhesion molecule (N-CAM) is a complex of surface glycoproteins that are developmentally regulated and believed to be intimately involved in the orderly structuring of the CNS. Here the effect of chronic low-level lead exposure on their expression in the postnatally developing cerebellum is described. Rat pups were chronically exposed to lead via their dam's drinking water which contained either 200 or 400 mg PbCl2/L from time of birth. Pup postnatal blood lead levels ranged between 10 and 20 micrograms/dl until day 16 after which they became elevated to 40 micrograms/dl on day 20. During this period the developmental sialylation state of N-CAM, which is believed to regulate cell-cell interaction, fibre outgrowth, and synapse formation, was monitored by rocket immunoelectrophoresis. In control animals the expected desialylation of N-CAM was found to occur at times coincident with postnatal synaptogenesis. In contrast, desialylation in animals chronically exposed to lead was found to be significantly impaired when blood lead levels exceeded 20 micrograms/dl. This could not be attributed to lead-induced undernutrition or alterations in immunoprecipitate formation. These observations could account for the neurobehavioural deficits that are known to be induced at similar blood lead levels, and the potential contribution of impaired N-CAM desialylation to synaptic elaboration is discussed.     

Neural Cell Adhesion Molecule (N-CAM) Is Required for Cell Type Segregation and Normal Ultrastructure in Pancreatic Islets

Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells have established that cellular cohesiveness, mediated by cell adhesion molecules, is important in determining the organization of cells within tissue and organs. We have employed N-CAM-deficient mice to determine whether N-CAM plays a functional role in the proper segregation of cells during the development of islets of Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing α cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected reduction of cell–cell adhesion in N-CAM-deficient mice, a significant increase in the clustering of cadherins, F-actin, and cell–cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei and Na⁺/K⁺-ATPase indicate that islet cell polarity is also affected. Finally, degranulation of β cells suggests that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type segregation during organogenesis. Possible mechanisms underlying this phenomenon may include changes in cadherin-mediated adhesion and cell polarity.     

S-Adenosylmethionine Decarboxylase Levels in Rat Retina During Postnatal Development

Abstract: S -Adenosylmethionine decarboxylase from rat retina is similar to that isolated from other rat tissues with regard to kinetic parameters. pH optimum, putrescine requirement, and sensitivity to spermine. The enzymic activity increases during the first 7 days of postnatal life but decreases until the 20th day. After this period AdoMet decarboxylase activity increases, to reach the highest values at the 90th day. This behavior suggests that such enzymic activity is responsible for spermidine and spermine levels in rat retina and that a high content of retinal spermine might have a role in the photoreceptor outer segment renewal.     

Heterogeneity of Soluble Neural Cell Adhesion Molecule

Soluble neural cell adhesion molecule (NCAM) from rat brain neuronal cell culture media consists predominantly of a polypeptide of Mr approximately 115,000. Minor amounts of a polypeptide of Mr approximately 180,000 and two inconsistently appearing components of Mr 160,000 and 145,000 are also observed. The Mr 115,000 component is derived from the neuronal membrane NCAM components NCAM-A of Mr 190,000, NCAM-B of Mr 140,000, or both. Thus, as a part of the catabolism of membrane NCAM-A plus -B, a minor fraction is posttranslationally cleaved and recovered in the media as discernible soluble NCAM polypeptides. The half-life of membrane NCAM-A plus -B is less than 24 h. Astrocyte culture media contains a predominant soluble NCAM component of Mr 120,000 derived from membrane-associated NCAM-C. A close comparison of deglycosylated soluble NCAM from astrocyte and neuronal cultures showed a small but consistent difference in Mr, a result suggesting that different NCAM polypeptides are released from the membrane of neurons and astrocytes. In contrast to the Mr 115,000-120,000 NCAM polypeptides, the Mr 180,000 polypeptide from neuronal culture media does not seem to be derived from membrane-attached NCAM and may therefore represent a secreted NCAM isoform.     

Localization of the Extracellular Matrix Protein SC1 Coincides with Synaptogenesis During Rat Postnatal Development

SC1 is an extracellular matrix protein that belongs to the SPARC family of matricellular molecules. This anti-adhesive protein localizes to synapses in the adult rat brain and has been postulated to modulate synapse shape. In this study, increased levels of SC1 were detected from postnatal days 10–20, with a peak at postnatal day 15, a period of intense synaptogenesis. During this time, increased colocalization of SC1 with the synaptic marker synaptophysin was observed in synapse-rich regions of the cerebellum and the cerebral cortex. These findings indicate that the pattern of SC1 localization coincided with synaptogenesis during rat postnatal development.     

Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin

The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).     

神经细胞粘连分子和多聚唾液酸在神经发育和再生中的作用

神经系统的形成依赖于细胞间的互相粘连。本文综述了神经细胞粘连分子（ＮＣＡＭ）及其多聚唾液酸（ＰＳＡ）组份对神经发育和再生的作用。ＮＣＡＭ的基本功能是介导细胞粘连,ＰＳＡ则由于其特殊的分子结构而降低细胞间的粘连。研究表明,鸡胚的发育过程中,ＰＳＡ含量在三个关键时期表达的高低决定了运动神经元能否准确地识别和支配肌肉。成年大鼠周围神经损伤后,肌肉内ＮＣＡＭ含量的高低决定于该肌肉的神经支配状况。成年大鼠脑内,切断内嗅皮层与海马的神经联系,发现齿回外分子层ＰＳＡ含量显著增加,并至少可持续６０天。已有的研究资料提示在去神经靶区域ＰＳＡ的重新表达可能有利于移植神经元轴突的生长并与宿主重建突触联系。     

Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane-attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell-intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane-integrated phosphorylated L1–30. Phase separation experiments with Triton X-114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane-anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1-containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.     

Elasticity and Rupture of a Multi-Domain Neural Cell Adhesion Molecule Complex

The neural cell adhesion molecule (NCAM) plays an important role in nervous system development. NCAM forms a complex between its terminal domains Ig1 and Ig2. When NCAM of cell A and of cell B connect to each other through complexes Ig12(A)/Ig12(B), the relative mobility of cells A and B and membrane tension exerts a force on the Ig12(A)/Ig12(B) complex. In this study, we investigated the response of the complex to force, using steered molecular dynamics. Starting from the structure of the complex from the Ig1-Ig2-Ig3 fragment, we first demonstrated that the complex, which differs in dimensions from a previous structure from the Ig1-Ig2 fragment in the crystal environment, assumes the same extension when equilibrated in solvent. We then showed that, when the Ig12(A)/Ig12(B) complex is pulled apart with forces 30-70 pN, it exhibits elastic behavior (with a spring constant of ∼0.03 N/m) because of the relative reorientation of domains Ig1 and Ig2. At higher forces, the complex ruptures; i.e., Ig12(A) and Ig12(B) separate. The interfacial interactions between Ig12(A) and Ig12(B), monitored throughout elastic extension and rupture, identify E16, F19, K98, and L175 as key side chains stabilizing the complex.     

Casein Kinase II Phosphorylates the Neural Cell Adhesion Molecule L1

Abstract: L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144–1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173–1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177–1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser^1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser^1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.     

Embryonic Neural Cell Adhesion Molecule in Cerebrospinal Fluid of Younger Children: Age-Dependent Decrease During the First Year

Poly-alpha-2,8-N-acetylneuraminic acid (poly-alpha-2,8-NeuAc) is developmentally expressed in neural tissue of higher animals, where it is covalently attached to the neural cell adhesion molecule (NCAM), a large integral membrane glycoprotein mediating cell-cell adhesion during neuronal development. NCAM exists in several molecular forms, of which only embryonic NCAM carries lengthy chains (n greater than 5) of poly-alpha-2,8-NeuAc. Chemically identical poly-alpha-2,8-NeuAc of bacterial origin is an important virulence factor in infections caused by Neisseria meningitidis group B and Escherichia coli K1, the predominant pathogens of bacterial meningitis. A quantitative enzyme-linked immunoassay was developed using monoclonal antibody (MAb) 735, an MAb specifically recognizing poly-alpha-2,8-NeuAc, and applied to CSF specimens from younger children. Poly-alpha-2,8-NeuAc contents were within the range of 20-0.2 micrograms/ml, decreasing from day 1 to day 300. Immunoprecipitation, immunoblot with a rabbit anti-mouse NCAM serum recognizing the protein part of human NCAM by cross-reactivity, affinity enrichment using immobilized MAb 735, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that poly-alpha-2,8-NeuAc in CSF is bound to human NCAM, probably NCAM-120.     

Nefiracetam (DM-9384) Preserves Hippocampal Neural Cell Adhesion Molecule-Mediated Memory Consolidation Processes During Scopolamine Disruption of Passive Avoidance Training in the Rat

Abstract: Scopolamine (0.15 mg/kg), a muscarinic antagonist, when administered during training or at a discrete 6-h posttraining time point, is demonstrated to inhibit the recall of a step-down passive avoidance response when tested at 24 and 48 h after task acquisition. Nefiracetam (3 mg/ kg), a piracetam-related nootropic, when given with scopolamine during training tended to improve task recall, and this effect was more pronounced when given at the 6-h posttraining time. Co-administration of nefiracetam with scopolamine was not necessary to achieve the antiamnesic action, as nefiracetam given during training significantly improved the memory deficits produced by scopolamine at the 6-h posttraining time. The paradigm-specific increase in hippocampal neural cell adhesion molecule sialylation, which is observed during consolidation of a passive avoidance response, was attenuated by the presence of scopolamine during training and at the 6-h posttraining time, and this effect was reversed by co-administration of nefiracetam, albeit in a paradigm-independent manner. These results suggest nefiracetam exerts a neurotrophic action that protects memory consolidation from drug inter- ventive insults.     

Structure and Function of the Neural Cell Adhesion Molecule (NCAM)

Based on published data and on our own experimental findings, we analyze key aspects of the structure and functions of the neural cell adhesion molecule (NCAM). We conclude that identification of NCAM mimetics opens up novel prospects for elucidation of the role of NCAM in neural differentiation and plasticity and, therefore, for practical development of new tools useful for the treatment of neurodegenerative disorders.     

Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder

Abstract: Neural cell adhesion molecule (N-CAM) is involved in cell-cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N-CAM in the CSF. Soluble human N-CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N-CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N-CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N-CAM immunoreactive proteins, primarily the 120-kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N-CAM concentrations. It is possible that the 120-kDa N-CAM band present in the CSF is derived from CNS cells as a secreted soluble N-CAM isoform. Our results suggest the possibility of latent state-related disturbances in N-CAM cellular function, i.e., residue from a previous episode, or abnormal N-CAM turnover in the CNS of patients with mood disorder.     

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, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (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 Pro⁵¹⁰-Tyr⁵¹¹ 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.     

Postnatal Development and Isolation of Peroxisomes from Brain

We analyzed the postnatal peroxisome development in rat brain by measuring the enzyme activities of catalase and acyl-CoA oxidase and beta-oxidation of [1-14C]lignoceric acid. These enzyme activities were higher between 10 and 16 days of postnatal life and then decreased. We developed and compared two different methods for isolation of enriched peroxisomes from 10-day-old rat brain by using a combination of differential and density gradient centrifugation techniques. Peroxisomes in Percoll (self-generating gradient) banded at a density of 1.036 +/- 0.012 g/ml and in Nycodenz continuous gradient at 1.125 +/- 0.014 g/ml. Acyl-CoA oxidase, D-amino acid oxidase, L-pipecolic acid oxidase, and dihydroxyacetone phosphate acyltransferase activities and activities for the oxidation of very long chain fatty acid (lignoceric acid) were almost exclusively associated with catalase activity (a marker enzyme for peroxisomes) in the gradient. The postnatal increase in peroxisomal activity with the onset of myelination and the presence of enzyme for the biosynthesis of plasmalogens and oxidation of very long chain fatty acid (both predominant constituents of myelin) suggest that brain peroxisomes may play an important role in the assembly and turnover of myelin.     

大鼠和家兔生后发育各阶段比目鱼肌纤维的比较

为研究大鼠与家兔骨骼肌各类型肌纤维的数量和二维分布以及生后发育对其影响,取生后2d和2、4、6、8、10周龄(体重10g和32、95、190、280、320g)大鼠及生后2d和2、4、8、12、16、20、24周龄(体重100g和220、400、750、1200、1600、2100、2500g)家兔的比目鱼肌做琥珀酸脱氢酶染色。实验结果表明,大鼠和家兔比目鱼肌纤维被分成Ⅰ型(SO),ⅡX型(FO)和ⅡA型(FOG)3型。使用图像分析系统分析每型肌纤维在生后发育各阶段的相关变化,大鼠和家兔比目鱼肌中：Ⅰ型纤维分布于整块肌肉,其数量随着生后发育而增加。幼体ⅡX型纤维分布在整块肌肉中,其数量随生后发育而减少;ⅡA型分布在肌肉中深层,数量几乎无变化;至成体时只有少量的ⅡX和ⅡA分布在肌表层。整个发育期间未见ⅡB型纤维。ⅡA型纤维直径最大,Ⅰ型中等,而ⅡX型最小。家兔3型肌纤维的平均横切面积比大鼠的大。这些结果表明大鼠和家兔后肢肌各种类型肌纤维的数量比例和分布随生长过程发生改变。     

Unique Intracellular Trafficking Processes Associated With Neural Cell Adhesion Molecule and Its Intracellular Signaling

Abstract

Homophilic binding of the neural cell adhesion molecule (NCAM) results in intracellular signaling, which also involves heterophilic engagement of coreceptors such as the fibroblast growth factor receptor (FGFR) and receptor protein tyrosine phosphatase-α (RPTPα). NCAM's own cellular dynamic itinerary includes endocytosis and recycling to the plasma membrane. Recent works suggest that NCAM could influence the trafficking of other receptor molecules that it associates with, particularly the FGFR. Furthermore, it was demonstrated that NCAM could undergo proteolytic processing upon activation. A processed fragment of NCAM, together with an N-terminal fragment of focal adhesion kinase (FAK), is translocated into the nucleus. Here, the authors discuss these rather unique (though not without precedence and analogues) receptor trafficking activities that are associated with NCAM and NCAM signaling.