Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.     

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.     

神经细胞粘附分子结构特征和生理功能

神经细胞粘附分子是一类调节细胞与细胞、细胞与细胞外基质间粘附作用的膜表面糖蛋白,主要有NCAM-180、NCAM-140、NCAM-120三种形式,多与PSA结合在一起。在神经系统中,NCAM的表达具有时间和空间特异性,最主要的作用为调节神经系统的可塑性,这种作用可能是通过PSA-NCAM对AMPA的调节作用,主要是通过调节蛋白激酶的表达和细胞内Ca^2 浓度来实现的。     

Profilin1 is required for glial cell adhesion and radial migration of cerebellar granule neurons

Cerebellar granule neurons (CGNs) exploit Bergmann glia (BG) fibres for radial migration, and cell-cell contacts have a pivotal role in this process. Nevertheless, little is known about the mechanisms that control CGN-BG interaction. Here we demonstrate that the actin-binding protein profilin1 is essential for CGN-glial cell adhesion and radial migration. Profilin1 ablation from mouse brains leads to a cerebellar hypoplasia, aberrant organization of cerebellar cortex layers and ectopic CGNs. Conversely, neuronal progenitor proliferation, tangential migration of neurons and BG morphology appear to be independent of profilin1. Our mouse data and the mapping of developmental neuropathies to the chromosomal region of PFN1 suggest a similar function for profilin1 in humans.     

Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination

The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function‐triggering L1 antibody leads to cathepsin E‐mediated generation of a sumoylated 30 kDa L1 fragment (L1‐30) and to import of L1‐30 into the nucleus. Mutation of the sumoylation site at K₁₁₇₂ or the cathepsin E cleavage site at E₁₁₆₇ abolishes generation of L1‐30, while mutation of the nuclear localization signal at K₁₁₄₇ prevents nuclear import of L1‐30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1‐30 and inhibits L1‐induced migration of cerebellar neurons and Schwann cells as well as L1‐dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1‐stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non‐mutated L1. In addition, L1‐stimulated migration of HEK293 cells expressing non‐mutated L1 is also abolished upon knock‐down of cathepsin E expression and enhanced by over‐expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1‐30 regulate neuronal and Schwann cell migration as well as myelination.

     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.     

IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over‐expression of LAMP or OBCAM increased synaptic number, while the over‐expression of Kilon reduced synaptic number and Ntm had no effects. The double over‐expression of Kilon‐Ntm, Kilon‐OBCAM, LAMP‐Ntm, and Ntm‐OBCAM decreased synaptic number and that of Kilon‐LAMP and LAMP‐OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons. Copyright © 2009 John Wiley & Sons, Ltd.     

Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display

The recognition molecule L1 plays important functional roles in the nervous system and in non-neural tissues. Since antibodies to L1 are of prime importance to study its functional properties, we have generated affinity matured human single chain variable fragment (scFv) antibodies against mouse L1 by introducing random mutations in the complementarity determining regions (CDRs) of a previously isolated scFv antibody heavy chain (CDR1 and CDR2) and light chain (CDR3). After biopanning the mutant library, a clone (5F7) that gave the strongest ELISA signal was expressed, purified, and characterized. The dissociation constant of 5F7 (2.86 x 10(-8)M) was decreased 60-fold compared to the wild type clone G6 (1.72 x 10(-6)M). 5F7 detected L1 by Western blot analysis in mouse brain homogenates and recognized L1 in L1 transfected cells and cryosections from mouse retina and optic nerve by immunofluorescence. Bivalent 5F7 scFv antibody (5F7-Cys) was also generated and showed a dissociation constant of 5.22 x 10(-9)M that is 5.5-fold lower than that of monomeric 5F7 antibody. The bivalent affinity matured L1 scFv antibody thus showed stronger binding by a factor of 310 compared to the wild type clone. This antibody should be useful in various biological assays.     

Brain development in mice lacking L1-L1 homophilic adhesion

A new mouse line has been produced in which the sixth Ig domain of the L1 cell adhesion molecule has been deleted. Despite the rather large deletion, L1 expression is preserved at normal levels. In vitro experiments showed that L1-L1 homophilic binding was lost, along with L1-alpha5beta1 integrin binding. However, L1-neurocan and L1-neuropilin binding were preserved and sema3a responses were intact. Surprisingly, many of the axon guidance defects present in the L1 knockout mice, such as abnormal corticospinal tract and corpus callosum, were not observed. Nonetheless, when backcrossed on the C57BL/6 strain, a severe hydrocephalus was observed and after several generations, became an embryonic lethal. These results imply that L1 binding to L1, TAG-1, or F3, and L1-alpha5beta1 integrin binding are not essential for normal development of a variety of axon pathways, and suggest that L1-L1 homophilic binding is important in the production of X-linked hydrocephalus.     

Ontogeny of the cell adhesion molecule L1 in the cerebellum of weaver and reeler mutant mice

The ontogeny of cell adhesion molecule L1 in cerebellum was quantitatively assessed in weaver and reeler mutant mice and in heterozygous litter-mate controls. In the latter the concentration and the amount of L1 both increased from the first postnatal week to become maximum at the second. In contrast, in the weaver and reeler neurologic mutant mice, L1 decreased steadily. The L1 concentration and the amount of L1 was lower in the cerebellum of homozygous mutant mice than in litter-mate controls. The findings are consistent with L1 being a component of axonal plasma membranes. However, no evidence was found of any direct effect of thewv andrl phenotypes on L1 expression.     

细胞粘附分子群相互作用网络模型研究

通过相互作用网络可视化软件Cytoscape对整联蛋白粘合体156种成份及相互间的690个反应实现网络可视化,并通过网络分析软件network analyser得到该网络节点间的平均连接并不复杂是一个不可分割的整体网络,网络较稳定,信息传递很快等。直接可视化的网络通过节点的形状改变视图有助于我们在原始数据的基础上更好的认识这一复杂的作用网络的相关信息。     

Ethanol inhibits L1 cell adhesion molecule tyrosine phosphorylation and dephosphorylation and activation of pp60src

Fetal alcohol syndrome is a leading cause of mental retardation. The neuropathology found in patients with fetal alcohol syndrome overlaps with those with mutations in the gene for cell adhesion molecule (L1). We have previously shown that L1-mediated neurite outgrowth and L1 activation of extracellular receptor kinases 1/2 are inhibited at low concentrations of ethanol. One possible mechanism for this effect is through disruption of a tyrosine-based sorting signal, Y(1176)RSLE, on the cytoplasmic domain of L1. Our goal was to determine if ethanol inhibited the sorting signal or its phosphorylation state. Using cerebellar granule neurons and dorsal root ganglion neurons, we found that ethanol had no effect on L1 distribution to the growth cone or its ability to be expressed on the cell surface as determined by confocal microscopy. In cerebellar granule neurons, clustering of L1 resulted in increased dephosphorylation of Y(1176), increased L1 tyrosine phosphorylation, and an increase in the activation of pp60^src as measured by immunoblot. All changes were inhibited by 25 mM ethanol. Using PP2 to inhibit pp60^src activation resulted in inhibition of increases in L1 tyrosine and extracellular receptor kinases 1/2 phosphorylation, and Y(1176) dephosphorylation. We conclude that ethanol disrupts L1 trafficking/signaling following its expression on the surface of the growth cone, and prior to its activation of pp60^src.     

Immunocytological characterization of the expression of cell adhesion molecule L1 during early innervation of mouse otocysts

Summary Doubts exist as to whether afferent nerve fibers exert a neurotrophic effect on the differentiation of sensory cells in the developing vestibular neuroepithelium. To determine whether innervation of hair cells precedes their differentiation, we have used the L1 adhesion molecule as a marker for axons. The detection of L1 on afferent axons in the otic vesicle of mouse embryos on gestation day 11 shows that nerve fibers penetrate the neuroepithelium before the sensory cells differentiate. L1-immunoreactivity of nerve endings also reveals the considerable fiber ramification on gestation days 14 and 15, i.e., corresponding to the first stages of sensory cell differentiation. The expression of L1 at successive stages of nerve fiber growth in the neuroepithelium, such as fasciculation and ramification, is not consistent with the previous role proposed for L1 as a fascicule-promoting factor and raises the possibility that other mechanisms are involved in L1 mediaded adhesion.     

Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival.

This study investigated the function of the adhesion molecule L1 in unmyelinated fibers of the peripheral nervous system (PNS) by analysis of L1- deficient mice. We demonstrate that L1 is present on axons and Schwann cells of sensory unmyelinated fibers, but only on Schwann cells of sympathetic unmyelinated fibers. In L1-deficient sensory nerves, Schwann cells formed but failed to retain normal axonal ensheathment. L1-deficient mice had reduced sensory function and loss of unmyelinated axons, while sympathetic unmyelinated axons appeared normal. In nerve transplant studies, loss of axonal-L1, but not Schwann cell-L1, reproduced the L1-deficient phenotype. These data establish that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells, stabilize the polarization of Schwann cell surface membranes, and mediate a trophic effect that assures axonal survival.     

The calcium‐sensing receptor and integrins modulate cerebellar granule cell precursor differentiation and migration

In the developing cerebellum granule cell precursors (GCPs) proliferate in the external granule cell layer before differentiating and migrating to the inner granule cell layer. Aberrant GCP proliferation leads to medulloblastoma, the most prevalent form of childhood brain cancer. Here, we demonstrate that the calcium‐sensing receptor (CaSR), a homodimeric G‐protein coupled receptor, functions in conjunction with cell adhesion proteins, the integrins, to enhance GCP migration and cell homing by promoting GCP differentiation. During the second postnatal week a robust peak in CaSR expression was observed in GCPs; reciprocal immunoprecipitation experiments conducted during this period established that the CaSR and β1 integrins are present together in a macromolecular protein complex. Analysis of cell‐surface proteins demonstrated that activation of the CaSR by positive allosteric modulators promoted plasma membrane expression of β1 integrins via ERK2 and AKT phosphorylation and resulted in increased GCP migration toward an extracellular matrix protein. The results of in vivo experiments whereby CaSR modulators were injected i.c.v. revealed that CaSR activation promoted radial migration of GCPs by enhancing GCP differentiation, and conversely, a CaSR inhibitor disrupted GCP differentiation and promoted GCP proliferation. Our results demonstrate that an ion‐sensing G‐protein coupled receptor acts to promote neuronal differentiation and homing during cerebellar maturation. These findings together with those of others also suggest that CaSR/integrin complexes act to transduce extracellular calcium signals into cellular movement, and may function in this capacity as a universal cell migration/homing complex in the developing brain. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 375–389, 2016