Adhesion molecule L1 inhibition increases infarct size in cerebral ischemia-reperfusion without change in blood-brain barrier disruption.

OBJECTIVE: Neural cell adhesion molecule L1CAM (L1) is involved in neuroprotection. To investigate a possible neuroprotective effect of L1 during ischemia, we determined whether blocking L1 with an antagonistic antibody would worsen the outcome of focal cerebral ischemia-reperfusion and increase blood-brain barrier (BBB) disruption. METHODS: Transient middle cerebral artery occlusion (MCAO) was performed in anesthetized rats. Five µg of antagonistic mouse IgG monoclonal L1 antibody 324 or non-immune control mouse IgG was applied on the ischemic-reperfused cortex during one hour of MCAO and two hours of reperfusion. At two hours of reperfusion, BBB permeability, size of infarct using tetrazolium staining, number of TUNEL-labeled apoptotic cells, and immunohistochemistry for expression of PTEN and p53 were studied. RESULTS: The antagonistic L1 antibody 324 increased the percentage of cortical infarct area (+36%), but did not affect BBB permeability in the ischemic-reperfused cortex. The antagonistic L1 antibody increased number of apoptotic neurons and p53 expression, but decreased PTEN expression. CONCLUSION: Functional antagonism of L1 increases infarct size by increasing numbers of apoptotic neurons without affecting BBB permeability during the early stage of cerebral ischemia-reperfusion. Our data suggest that L1 affects primarily the brain parenchyma rather than BBB during early stages of cerebral ischemia-reperfusion and that endogenous brain L1 may be neuroprotective.

Bilirubin inhibits lipid raft dependent functions of L1 cell adhesion molecule in rat pup cerebellar granule neurons.

BACKGROUND: The mechanism of bilirubin neurotoxicity is poorly understood. We hypothesize that bilirubin inhibits the function of lipid rafts (LR), microdomains of the plasma membrane critical for signal transduction. To test this hypothesis, we measured the effect of free bilirubin (Bf) between 7.6 and 122.5 nM on LR-dependent functions of L1 cell adhesion molecule (L1). METHODS: Cerebellar granule neurons (CGN) were plated on poly-L-lysine overnight, and neurite length was determined after 1 h treatment with L1 alone or L1 and bilirubin. L1 activation of ERK1/2 was measured in CGN in the presence or absence of bilirubin. The effect of bilirubin on L1 distribution in LR was quantitated, and the localization of bilirubin to LR was determined. RESULTS: The addition of bilirubin to CGN treated with L1 significantly decreased neurite length compared to L1 alone. L1 activation of ERK1/2 was inhibited by bilirubin. Bilirubin redistributed L1 into LR. Bilirubin was associated only with LR-containing fractions of a sucrose density gradient. CONCLUSION: Bf significantly inhibits LR-dependent functions of L1 and are found only associated with LR, suggesting one mechanism by which bilirubin may exert neurotoxicity is through the dysfunction of protein-LR interactions. IMPACT: This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin. This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.

L1CAM as an E-selectin Ligand in Colon Cancer.

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.

The cell adhesion molecule L1 interacts with nuclear proteins via its intracellular domain.

Proteolytic cleavage of the cell adhesion molecule L1 (L1) in brain tissue and in cultured cerebellar neurons results in the generation and nuclear import of a 30 kDa fragment comprising most of L1's C-terminal, intracellular domain. In search of molecules that interact with this domain, we performed affinity chromatography with the recombinant intracellular L1 domain and a nuclear extract from mouse brains, and identified potential nuclear L1 binding partners involved in transcriptional regulation, RNA processing and transport, DNA repair, chromatin remodeling, and nucleocytoplasmic transport. By co-immunoprecipitation and enzyme-linked immunosorbent assay using recombinant proteins, we verified the direct interaction between L1 and the nuclear binding partners non-POU domain containing octamer-binding protein and splicing factor proline/glutamine-rich. The proximity ligation assay confirmed this close interaction in cultures of cerebellar granule cells. Our findings suggest that L1 fragments regulate multiple nuclear functions in the nervous system. We discuss possible physiological and pathological roles of these interactions in regulation of chromatin structure, gene expression, RNA processing, and DNA repair.

Axonal Growth of Midbrain Dopamine Neurons is Modulated by the Cell Adhesion Molecule ALCAM Through Trans-Heterophilic Interactions with L1cam, Chl1, and Semaphorins.

The growth of axons corresponding to different neuronal subtypes is governed by unique expression profiles of molecules on the growth cone. These molecules respond to extracellular cues either locally though cell adhesion interactions or over long distances through diffusible gradients. Here, we report that that the cell adhesion molecule ALCAM (CD166) can act as an extracellular substrate to selectively promote the growth of murine midbrain dopamine (mDA) neuron axons through a trans-heterophilic interaction with mDA-bound adhesion molecules. In mixed-sex primary midbrain cultures, the growth-promoting effect of ALCAM was abolished by neutralizing antibodies for components of the Semaphorin receptor complex Nrp1, Chl1, or L1cam. The ALCAM substrate was also found to modulate the response of mDA neurites to soluble semaphorins in a context-specific manner by abolishing the growth-promoting effect of Sema3A but inducing a branching response in the presence of Sema3C. These findings identify a previously unrecognized guidance mechanism whereby cell adhesion molecules act in trans to modulate the response of axonal growth cones to soluble gradients to selectively orchestrate the growth and guidance of mDA neurons.SIGNIFICANCE STATEMENT The mechanisms governing the axonal connectivity of midbrain dopamine (mDA) neurons during neural development have remained rather poorly understood relative to other model systems for axonal growth and guidance. Here, we report a series of novel interactions between proteins previously not identified in the context of mDA neuronal growth. Significantly, the results suggest a previously unrecognized mechanism involving the convergence in signaling between local, adhesion and long-distance, soluble cues. A better understanding of the molecules and mechanisms involved in establishment of the mDA system is important as a part of ongoing efforts to understand the consequence of conditions that may result from aberrant connectivity and also for cell replacement strategies for Parkinson's disease.

L1CAM induces perineural invasion of pancreas cancer cells by upregulation of metalloproteinase expression.

Pancreas cancer cells have a tendency to invade along nerves. Such cancerous nerve invasion (CNI) is associated with poor outcome; however, the exact mechanism that drives cancer cells to disseminate along nerves is unknown. Immunohistochemical analysis of human pancreatic ductal adenocarcinoma (PDAC) specimens showed overexpression of the L1 cell adhesion molecule (L1CAM) in cancer cells and in adjacent Schwann cells (SC) in invaded nerves. By modeling the neural microenvironment, we found that L1CAM secreted from SCs acts as a strong chemoattractant to cancer cells, through activation of MAP kinase signaling. L1CAM also upregulated expression of metalloproteinase-2 (MMP-2) and MMP-9 by PDAC cells, through STAT3 activation. Using a transgenic Pdx-1-Cre/KrasG12D /p53R172H (KPC) mouse model, we show that treatment with anti-L1CAM Ab significantly reduces CNI in vivo. We provide evidence of a paracrine response between SCs and cancer cells in the neural niche, which promotes cancer invasion via L1CAM secretion.

Gene CG15630 (fipi) is involved in regulation of the interpulse interval in Drosophila courtship song.

To study the central pattern generators functioning, previously we identified genes, whose neurospecific knockdowns led to deviations in the courtship song of Drosophila melanogaster males. Reduced expression of the gene CG15630 caused a decrease in the interpulse interval. To investigate the role of CG15630, which we have called here fipi (factor of interpulse interval), in the courtship song production, at first, we have characterized fipi transcripts and protein (FIPI) in the mutant flies carrying P insertion and deletions in this gene and in flies with its RNAi knockdown. FIPI is homologous to the mammalian NCAM2 protein, an important factor of neuronal development in the olfactory system. In this study, we have revealed that local fipi knockdown in the antennal olfactory sensory neurons (OR67d and IR84a), which are responsible for reception of chemosignals modulating courtship behavior, alters the interpulse interval in the opposite directions. Thus, a proper fipi expression seems to be necessary for perception of sexual chemosignals, and the effect of fipi knockdown on IPI value depends on the type of chemoreceptor neurons affected.

Cell Recognition Molecule L1 Regulates Cell Surface Glycosylation to Modulate Cell Survival and Migration.

Background: Cell recognition molecule L1 (L1) plays an important role in cancer cell differentiation, proliferation, migration and survival, but its mechanism remains unclear. Methodology/Principal: Our previous study has demonstrated that L1 enhanced cell survival and migration in neural cells by regulating cell surface glycosylation. In the present study, we show that L1 affected cell migration and survival in CHO (Chinese hamster ovary) cell line by modulation of sialylation and fucosylation at the cell surface via the PI3K (phosphoinositide 3-kinase) and Erk (extracellularsignal-regulated kinase) signaling pathways. Flow cytometry analysis indicated that L1 modulated cell surface sialylation and fucosylation in CHO cells. Activated L1 upregulated the protein expressions of ST6Gal1 (ß-galactoside α-2,6-sialyltransferase 1) and FUT9 (Fucosyltransferase 9) in CHO cells. Furthermore, activated L1 promoted CHO cells migration and survival as shown by transwell assay and MTT assay. Inhibitors of sialylation and fucosylation blocked L1-induced cell migration and survival, while decreasing FUT9 and ST6Gal1 expressions via the PI3K-dependent and Erk-dependent signaling pathways. Conclusion : L1 modulated cell migration and survival by regulation of cell surface sialylation and fucosylation via the PI3K-dependent and Erk-dependent signaling pathways.

Inhibition of N1-Src kinase by a specific SH3 peptide ligand reveals a role for N1-Src in neurite elongation by L1-CAM.

In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src-/- mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src.

Role of L1CAM in the Regulation of the Canonical Wnt Pathway and Class I MAGE Genes.

Molecule L1CAM is specific for nerve cells and tumors of various localizations. The expression of L1CAM is significantly higher in melanoma in comparison with benign nevi and correlates with the progress of melanoma and transition from radial to vertical growth. Monoclonal antibodies to L1CAM effectively and specifically attenuate melanoma growth, though stimulates the epithelial-mesenchymal transition. shRNA-mediated knock-down of L1CAM showed the involvement of L1CAM in regulation of activity of the canonical Wnt pathway and expression of genes of class I melanoma-associated antigens (MAGE).

Enzymatic Depletion of the Polysialic Acid Moiety Associated with the Neural Cell Adhesion Molecule Inhibits Antidepressant Efficacy.

Antidepressant drugs are too often ineffective, the exact mechanism of efficacy is still ambiguous, and there has been a paucity of novel targets for pharmacotherapy. In an attempt to understand the pathogenesis of depression and subsequently develop more efficacious antidepressant drugs, multiple theories have been proposed, including the modulation of neurotransmission, the upregulation of neurogenesis and neurotrophic factors, normalizing hypothalamic-pituitary-adrenal reactivity, and the reduction of neuroinflammation; all of which have supporting lines of evidence. Therefore, an ideal molecular target for novel pharmaceutical intervention would function at the confluence of these theories. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) functions broadly, serving to mediate synaptic plasticity, neurogenesis, neurotrophic factor signaling, and inflammatory signaling throughout the brain; all of which are associated with the pathophysiology and treatment of depression. Moreover, the expression of PSA-NCAM is reduced by depression, and conversely enhanced by antidepressant treatment, particularly within the hippocampus. Here we demonstrate that selectively cleaving the polysialic acid moiety, using the bacteriophage-derived enzyme endoneuraminidase N, completely inhibits the antidepressant efficacy of the selective-serotonin reuptake inhibitor fluoxetine (FLX) in a chronic unpredictable stress model of depression. We also observe a corresponding attenuation of FLX-induced hippocampal neuroplasticity, including decreased hippocampal neurogenesis, synaptic density, and neural activation. These data indicate that PSA-NCAM-mediated neuroplasticity is necessary for antidepressant action; therefore PSA-NCAM represents an interesting, and novel, target for pharmacotherapy.

The role of L1cam in murine corticogenesis, and the pathogenesis of hydrocephalus.

L1cam (L1), one of the cell adhesion molecules belonging to the immunoglobulin superfamily, plays critical roles in neuronal migration, axon growth, guidance, fasciculation, and synaptic plasticity in the central as well as the peripheral nervous system. A number of X-linked forms of mental retardation have been associated with mutations in the L1 gene, including X-linked hydrocephalus in humans. Although model mice with different sites of L1 mutation have been studied, the pathogenetic mechanisms of hydrocephalus and mental retardation still remain unsolved. We herein present an overview of the function of L1 in the central nervous system and describe a human case of L1 mutation and knock-in mice that showed deleted sixth immunoglobulin of L1. Finally, we present experimental evidence showing that L1 is involved in murine neocortical histogenesis and propose a hypothetical mechanism of L1-linked hydrocephalus, with reference to corticogenesis.

Neurite outgrowth induced by NGF or L1CAM via activation of the TrkA receptor is sustained also by the exocytosis of enlargeosomes.

NGF binding to its protein kinase receptor TrkA is known to induce neurite outgrowth and neural cell differentiation. The plasma membrane expansion, necessary for the process, was shown to be contributed by the VAMP7-dependent exocytosis of endocytic vesicles. Working with wild-type PC12 (wtPC12), a cell model widely used to investigate NGF-induced neurite outgrowth, we found that a few hours of treatment with the neurotrophin (and to a lower extent with basic FGF and EGF) induces the appearance of enlargeosome vesicles competent for VAMP4-dependent exocytosis abundant in high REST-PC12 clones. Both the neurite length assay and the immunocytochemistry of enlargeosomes exocytosis revealed that activation of TrkA is induced not only by NGF, but also by the L1 adhesion protein, L1CAM, whose soluble construct binds the receptor with submicromolar affinity. In the intact wtPC12, the L1CAM construct induced autophosphorylation and internalization of TrkA followed by the activation of the PI3K, MEK, and PKCγ signaling cascades, analogous to the responses induced by NGF. Down-regulation of either VAMP7 or VAMP4 revealed the coparticipation of the two corresponding vesicles to the outgrowth responses induced by NGF and L1CAM. Finally, mixing experiments of wtPC12 cells rich in TrkA with high REST PC12 cells transfected with L1CAM documented the transactivation of the receptor by the adhesion protein surface-exposed in adjacent cells. In view of the known inhomogeneous surface distribution of both L1CAM and TrkA in various neural cells including neurons, their transcellular binding could be restricted to discrete sites, governing local signaling events distinct from those induced by soluble messengers.

Choline partially prevents the impact of ethanol on the lipid raft dependent functions of l1 cell adhesion molecule.

BACKGROUND: Fetal alcohol spectrum disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol (EtOH) teratogenicity is the disruption of the functions of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of EtOH on neurobehavior. We sought to determine whether choline could prevent the effect of EtOH on L1 function using a simple experimental system. METHODS: Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution, and neurite outgrowth were measured in the presence or absence of EtOH. RESULTS: Choline significantly reduced the effect of EtOH on L1 signaling, the distribution of L1 in lipid rafts and L1-mediated neurite outgrowth. However, choline supplemented EtOH-exposed cultures remained significantly different than controls. CONCLUSIONS: Choline pretreatment of CGN significantly reduces the disruption of L1 function by EtOH, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline.

Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization.

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

Chlorhexidine inhibits L1 cell adhesion molecule-mediated neurite outgrowth in vitro.

BACKGROUND: Chlorhexidine is a skin disinfectant that reduces skin and mucous membrane bacterial colonization and inhibits organism growth. Despite numerous studies assessing chlorhexidine safety in term infants, residual concerns have limited its use in hospitalized neonates, especially low-birth-weight preterm infants. The aim of this study was to assess the potential neurotoxicity of chlorhexidine on the developing central nervous system using a well-established in vitro model of neurite outgrowth that includes laminin and L1 cell adhesion molecule (L1) as neurite outgrowth-promoting substrates. METHODS: Cerebellar granule neurons are plated on poly L-lysine, L1, or laminin. Chlorhexidine, hexachlorophene, or their excipients are added to the media. Neurons are grown for 24 h, fixed, and neurite length is measured. RESULTS: Chlorhexidine significantly reduced the length of neurites grown on L1 but not on laminin. Chlorhexidine concentrations as low as 125 ng/ml statistically significantly reduced neurite length on L1. Hexachlorophene did not affect neurite length. CONCLUSION: Chlorhexidine at concentrations detected in the blood following topical applications in preterm infants specifically inhibited L1-mediated neurite outgrowth of cerebellar granule neurons. It is now vital to determine whether the blood-brain barrier is permeable to chlorhexidine in preterm infants.

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. Cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. L1 stimulation triggers sumoylation and cleavage of L1, thus generating the L1-70 fragment (1) which is cleaved by cathepsin E (2) yielding the L1-30 fragment that is imported to the nucleus (3), may bind to DNA and/or nuclear proteins (4), to regulate diverse cellular functions.

The dendritic spines of interneurons are dynamic structures influenced by PSA-NCAM expression.

Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation.

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.