Prevention of neuronal cell death by neural adhesion molecules L1 and CHL1

The effects of L1-Fc and CHL1-Fc fusion proteins on neuronal survival were investigated. Cerebellar granule neurons of mouse and hippocampal neurons of rat embryo undergo apoptosis when cultured in serum-free medium. Treatment with chimeric proteins containing the extracellular domains of the neural adhesion molecules L1 or CHL1 fused to the Fc region of human immunoglobulin significantly enhanced the survival of neurons. Compared to the control, the percentage of surviving neurons increased about 60% and 45% with L1 and CHL1 fusion proteins, respectively. A fusion protein containing the extracellular domain of NCAM had no effect on survival. The L1 and CHL1 fusion proteins were effective both in soluble form or when offered as a substrate, with the maximal effect at about 1 microg/mL. To explore the intracellular events related to the neuronal survival effects of L1-Fc fusion protein, Bcl-2 and c-Jun expression were analyzed by Western blotting. The level of Bcl-2 in cerebellar granule neurons was increased by treatment with L1-Fc at both 1 and 5 days of culture. The level of c-Jun was not significantly affected at the early time point and was reduced by L1-Fc fusion protein after long-term culture. The results demonstrate that the neural adhesion molecule L1 and its relative CHL1 are potential neuronal survival factors for neurons of the central nervous system. Bcl-2 may serve as one of the intracellular mediators of the neuronal survival effects of L1.     

Two alcohol binding residues interact across a domain interface of the L1 neural cell adhesion molecule and regulate cell adhesion

Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371–375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.     

Long noncoding RNA CHL1-AS1 promotes cell proliferation and migration by sponging miR-6076 to regulate CHL1 expression in endometrial cancer

Endometrial cancer (EC) is deemed to be the most typical gynecologic malignant tumor. Despite the incidence of EC being lower in Asia than that in western countries, substantial increased incidence has been observed in the past few decades in Asia. Although various molecular testing methods and genomic science have developed, the overall prognosis is still disappointing. LncRNAs have been found to influence the progression of various cancers. CHL1-AS1 has been found to be upregulated in ovarian endometriosis, nevertheless, the molecular mechanism and biological function of CHL1-AS1 in EC have not been explored. In our exploration, both CHL1-AS1 and CHL1 were upregulated in EC cells. Knockdown of CHL1-AS1 or CHL1 inhibited cell proliferation and migration in EC. Furthermore, microRNA-6076 (miR-6076) could bind with CHL1-AS1 or CHL1, and regulate the expression of CHL1. Finally, absence of miR-6076 or overexpression of CHL1 can partially rescue the effect of CHL1-AS1 knockdown or miR-6076 upregulation on cell proliferation and migration, respectively. All in all, our research was the first endeavor to study the underlying mechanism of CHL1-AS1 in EC and confirmed that CHL1-AS1 regulated EC progression via targeting the miR-6076/CHL1 axis, offering new insight into treating EC.     

Loss of cell adhesion molecule CHL1 improves homeostatic adaptation and survival in hypoxic stress

Close homologue of L1 (CHL1) is a transmembrane cell adhesion molecule that is critical for brain development and for the maintenance of neural circuits in adults. Recent studies revealed that CHL1 has diverse roles and is involved in the regulation of recovery after spinal cord injury. CHL1 expression was downregulated in the cerebral cortex, hypothalamus, and brain stem after the induction of acute hypoxia (AH). In the current study, we sought to address the role of CHL1 in regulating homeostasis responses to hypoxia using CHL1-knockout (CHL1^−/−) mice. We found that, compared with wild-type littermates, CHL1^−/− mice showed a dramatically lower mortality rate and an augmented ventilatory response after they were subjected to AH. Immunofluorescence staining revealed that CHL1 was expressed in the carotid body (CB), the key oxygen sensor in rodents, and CHL1 expression level in the CB as assayed by western blot was decreased after hypoxic exposure. The number of glomus cells and the expression of tyrosine hydroxylase (a marker for glomus cells) in the CB of CHL1^−/− mice appeared to be increased compared with CHL1^+/+ mice. In addition, in the ex vivo CB preparation, hypoxia induced a significantly greater afferent nerve discharge in CHL1^−/− mice compared with CHL1^+/+ mice. Furthermore, the arterial blood pressure and plasma catecholamine levels of CHL1^−/− mice were also significantly higher than those of CHL1^+/+ mice. Our findings first demonstrate that CHL1 is a novel intrinsic factor that is involved in CB function and in the ventilatory response to AH.     

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.     

Overlapping functions of the cell adhesion molecules Nr-CAM and L1 in cerebellar granule cell development

The structurally related cell adhesion molecules L1 and Nr-CAM have overlapping expression patterns in cerebellar granule cells. Here we analyzed their involvement in granule cell development using mutant mice. Nr-CAM-deficient cerebellar granule cells failed to extend neurites in vitro on contactin, a known ligand for Nr-CAM expressed in the cerebellum, confirming that these mice are functionally null for Nr-CAM. In vivo, Nr-CAM-null cerebella did not exhibit obvious histological defects, although a mild size reduction of several lobes was observed, most notably lobes IV and V in the vermis. Mice deficient for both L1 and Nr-CAM exhibited severe cerebellar folial defects and a reduction in the thickness of the inner granule cell layer. Additionally, anti-L1 antibodies specifically disrupted survival and maintenance of Nr-CAM-deficient granule cells in cerebellar cultures treated with antibodies. The combined results indicate that Nr-CAM and L1 play a role in cerebellar granule cell development, and suggest that closely related molecules in the L1 family have overlapping functions.     

The immunoglobulin superfamily of neuronal cell adhesion molecules: Lessons from animal models and correlation with human disease

Neuronal cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in the formation of neural circuits at different levels: cell migration, axonal and dendritic targeting as well as synapse formation. Furthermore, in perinatal and adult life, neuronal IgCAMs are required for the formation and maintenance of specialized axonal membrane domains, synaptic plasticity and neurogenesis. Mutations in the corresponding human genes have been correlated to several human neuronal disorders. Perturbing neuronal IgCAMs in animal models provides powerful means to understand the molecular and cellular basis of such human disorders. In this review, we concentrate on the NCAM, L1 and contactin subfamilies of neuronal IgCAMs summarizing recent functional studies from model systems and highlighting their links to disease pathogenesis.     

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.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

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.     

mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment

Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.     

Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain

To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca²⁺, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca²⁺ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface-bound-L1 as a prominent signal-transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca²⁺. © 1995 John Wiley & Sons, Inc.     

The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i‐AAA protease YME1L

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring‐like structures and locally specify the protein–lipid composition in a variety of cellular membranes. Stomatin‐like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i‐AAA protease YME1L, which we term the SPY complex (for SLP2–PARL–YME1L). Association with SLP2 in the SPY complex regulates PARL‐mediated processing of PTEN‐induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress‐activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1‐mediated processing of the dynamin‐like GTPase OPA1 allowing stress‐induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.     

Stromal cell derived factor-1 acutely promotes neural progenitor cell proliferation in vitro by a mechanism involving the ERK1/2 and PI-3K signal pathways

Stromal cell derived factor-1 (SDF-1), a member of the chemotactic cytokine family, has attracted attention in recent years. It participates in diverse processes such as the regulation of neuronal migration and activation of CD4+ T cells; it is also a co-receptor for human immunodeficiency virus-1 (HIV-1). Here, we show that the proliferation of neural progenitor cells dissociated from rat cortex and cultured in vitro with basic fibroblast growth factor (bFGF) is stimulated by SDF-1. PD98059 and wortmannin, which are, respectively, specific inhibitors of the extracellular regulated kinase1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI-3K) signal pathways, markedly attenuate this stimulation of proliferation. These findings indicate that SDF-1 acutely promotes the proliferation of NPCs in vitro involving the ERK1/2 and PI-3 kinase pathways, suggesting that it plays a basic role in the development of neural progenitors.     

The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.