| Neural cell adhension molecule L1 itself is one of many carbohydrate-carrying molecules in the nervous system, where it is widely expressed and involved in many aspects of neural development, regeneration and synaptic plasticity in the adult. Various human genetic disorders with prominent nervous system defects are caused by mutations in L1. Cell recognition molecules at the cell surface are not only donors, but also acceptors of carbohydrates. Glycosyl- ation of proteins and lipids is a prime example of a cellular process that is not under the direct control of the genome. This contributes to the functional diversity required to generate extensive phenotypes from a limited genotype. Glycosylation is a crucial postor co- translational modification of more than 50% of all eukaryotic proteins according to database analyses . It is affected by a multitude of factors, such as cellular metabolism and the rate of cell growth. Accumulated evidence indicates that glycan structures play important roles in various contexts, including differentiation, development, fertilization, inflammation, and cell–cell recognition.1.Glycosylation patterns on the cell surface of L1+/y and L1-/y neurons with and without treatment with anti-L1 antibodies:(1)Neurons isolated from L1+/y (L1wt) and L1-/y (L1ko) mice were subjected to flow cytometry analysis using a panel of carbohydrate surface markers, including lectins and antibodies against carbohydrates. The quantitative result showed that the expression of carbohydrates recognized by SNA, MAA, UEAI, and DSL as well as L5 antibody was significantly decreased in the neurons of L1-/y mice versus L1+/y mice. (2)After treatment with anti-L1 antibodies, neurons isolated from L1+/y (L1wt-L1Ab) and L1-/y (L1ko-L1Ab) mice were assayed by flow cytometry using a panel of carbohydrate surface markers, including lectins and antibodies against carbohydrates. The quantitative result showed that the expression of carbohydrates recognized by SNA, MAA, UEAI, DSL, and L5 antibody was significantly increased in L1wt-L1Ab neurons versus neurons treated with rat IgG only ("L1wt"). The quantitative result showed that the expression of carbohydrates recognized by SNA, MAA, UEAI, DSL, and L5 antibody was no significant different in L1ko-L1Ab neurons compared to L12/y neurons treated with rat IgG only ("L1ko").2.The mRNA expression of ST6Gal1 and FUT9 is modulated by L1.(1)cDNA microarray analysis was performed to investigate the expression of genes involved in the synthesis and hydrolysis of the carbohydrates recognized by SNA, MAA, UEAI lectins and L5 antibodies by comparing between L1 antibody-treated neurons and rat IgG-treated neurons ("neurons-L1Ab"versus"neurons"group); and neurons isolated from the cerebellum of L1-/y mice versus L1+/y mice ("L1ko"versus"L1wt"group). Two microarrays per group were finished. The expression of fucosyltransferase 9 (FUT9) was elevated in the"neurons-L1Ab"versus"neurons"group (1.414-fold) and decreased in the L1ko neurons versus L1wt neurons group (0.218-fold), and b-galactoside-a2,6- sialyltransferase (ST6Gal1) expression was elevated in the"neurons-L1Ab"compared to the"neurons"group (1.249-fold) and decreased in the L1ko neurons versus L1wt neurons group (0.574-fold).(2)To verify the results obtained by cDNA microarray analysis, quantitative RT-real-time PCR was performed. The expression of ST6Gal1 and FUT9 mRNAs was significantly downregulated in neurons isolated from L1-/y (L1ko) versus L1+/y (L1wt) mice and upregulated in the L1-Fc-treated ("L1wt-L1") versus Fc-treated ("L1wt") group of L1+/y neurons. No significant differences were observed in L1-Fc-treated ("L1ko-L1") versus Fc-treated ("L1ko") L1-/y neurons.3.Activation of L1 promotes neurite outgrowth, cell migration and survival. (1)Cerebellar granule neurons isolated from cerebella of 6- to 8-day-old L1+/y (L1wt) and L1-/y (L1ko) mice were seeded on coverslips coated with PLL. Bright field micrographs of cerebellar neurons isolated from L1+/y (L1wt) and L1-/y (L1ko) mice and cultured with different concentrations of L1 antibodies (L1Ab: 50, 100, and 200 mg/ml). The length of the longest neurite and the total length of all neuritis per cell were measured. Notably, L1 antibodies promoted neurite outgrowth of neurons isolated from L1+/y mice 1.5–2-fold, but not outgrowth of L12/y neurons, both in terms of longest neurite and of total neurite lengths.(2)To verify the role of L1 in cell migration, the undersurface of transwell membranes was coated with the L1 antibody. In the control group, the undersurface of transwell membranes was coated with rat IgG. Relative numbers of cells transmigrating through the membrane were determined by staining cells on the undersurface of the transwell membrane followed by cell lysis and measurement of the absorbance value. Neuronal migration inducedby L1 antibody was significantly increased compared with neuronal migration induced by rat IgG in the neurons isolated from L1+/y (L1wt) mice, but not L1-/y (L1ko) mice. (3)To investigate the role of L1 in cell survival, MTT analysis was performed. After anti-L1 antibody (L1Ab) treatment (white bars), cell survival was significan- tly enhanced in the neurons isolated from L1+/y (L1wt) mice, but not L12/y (L1ko) mice. Rat IgG treatment was used as control.4.ShRNA-mediated knockdown of FUT9 and ST6Gal1 blocks L1-induced neurite outgrowth, cell migration and survival.(1)Neurons were dissected from the cerebellum of 6-to 8-day-old mice. Neurons were co-transfected with GFP plasmid plus shRNA of FUT9 or ST6Gal1 shRNA by electroporation. GFP plasmid plus empty vector transfection was used as control. Immunocytochemistry for FUT9 or ST6Gal1 (red) in transfected neurons (green) indicated the knockdown efficiency: FUT9, 86.35%; ST6Gal1, 85.47%. So we can think FUT9 and ST6Gal1 of green cells is knock down. (2)Longest neurite length and total neurite length were measured. ShRNA-mediated knockdown of FUT9 and ST6Gal1 blocks L1-induced neurite outgrowth.(3)Cell migration assay was performed in L1antibody-treated neurons (Neurons-L1Ab) co-transfect- ed with GFP plasmid plus empty vector (control) or shRNA of FUT9 or ST6Gal1. ShRNA-mediated knockdown of FUT9 and ST6Gal1 blocks L1-induced cell migration.(4)MTT analysis of L1antibody-treated neurons (Neurons-L1Ab) was performed after cotransfection with GFP plasmid plus empty vector or shRNA of FUT9 or ST6Gal1. ShRNA-mediated knockdown of FUT9 and ST6Gal1 blocks L1-induced survival.5.L1 influences FUT9 and ST6Gal1 mRNA levels, neurite outgrowth, cell migration and cell survival via various intracellular signaling mediators. (1)Cerebellar granule neurons isolated from the cerebella of wild type mice (6- to 8-day-old) were seeded on coverslips coated with PLL and PLL plus L1 antibodies (neurons-L1Ab). After 1 hour in culture, inhibitors of PLCγ(U73122, 10.5 mM), CDC25 phosphatase (CDC25 inhibitor II, 1.05 mM), PI3K (LY294002, 16.5 mM), Erk (Erk activation inhibitor I, 50 mM) or PKA (KT5720, 280 nM) were added into the culture medium and the cells were cultured for a further 24 hours. Levels of FUT9 mRNA and ST6Gal1 mRNA were quantified by RT-real-time PCR. In neurons treated with L1 antibody, the phospholipase Cγ(PLCγ) inhibitor U73122, the PKA inhibitor KT5720 and the Erk inhibitor reduced both FUT9 and ST6Gal1 mRNA levels, with U73122 and Erk inhibitors having the strongest effects.(2)Neurons were seeded on coverslips coated with PLL plus L1 antibodies (L1Ab). Rat IgG coating was used as control. After treatment with the inhibitors of signal transduction pathways, longest neurite length and total neurite length were measured. PLCγ, Cdc25, PI3K, Erk and PKA inhibitors all decreased L1 antibody-induced neurite outgrowth in cerebellar neurons, with the strongest effect being produced by the PLCγinhibitor.(3)Neurons were seeded on coverslips coated with PLL plus L1 antibodies (neurons-L1Ab). After treatment with the inhibitors of signal transduction pathways, cell survival was quantified by MTT assay. PLCγ, PI3K and Erk inhibitors all reduced survival in L1 antibody-treated neurons, whereas the Cdc25 and PKA inhibitors had no effect on survival.(4)Neurons were cotransfected with GFP plasmid plus empty vector (control) or shRNA for PLCγand seeded on coverslips coated with L1 antibody (L1Ab), rat IgG coating was used as control. ShRNA-mediated knockdown of PLCγblocks L1- induced neurite outgrowth, cell migration and survival.In our study, a PLCγ-dependent pathway contributes to the L1- induced increases in expression of the sialyltransferase and fucosyltransferase and to the L1-mediated neurite outgrowth and cell survival.
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