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Extracellular Galectin-3 Induces Accelerated Oligodendroglial Differentiation Through Changes in Signaling Pathways and Cytoskeleton Dynamics

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Abstract

Galectin-3 (Gal-3) is a chimeric protein structurally composed of unusual tandem repeats of proline and short glycine-rich segments fused onto a carbohydrate recognition domain. Our studies have previously demonstrated that Gal-3 drives oligodendrocyte (OLG) differentiation to control myelin integrity and function. The cytoskeleton plays a key role in OLG maturation: the initial stage of OLG process extension requires dynamic actin filament assembly, while subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins which are dependent on myelin basic protein (MPB) expression. In this context, the aim of the present work was to elucidate the mechanism by which recombinant Gal-3 (rGal-3) induces OLG maturation, giving special attention to the actin cytoskeleton. Our results show that rGal-3 induced early actin filament assembly accompanied by Erk signaling deactivation, which led to a decrease in the number of platelet-derived growth factor receptor α (PDGFRα)+ cells concomitantly with an increase in the number of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase)+ cells at 1 day of treatment (TD1), and Akt signaling activation at TD1 and TD3. Strikingly, rGal-3 induced an accelerated shift from polymerized to depolymerized actin between TD3 and TD5, accompanied by a significant increase in MBP, gelsolin, Rac1, Rac1-GTP, and β-catenin expression at TD5. These results were strongly supported by assays using Erk 1/2 and Akt inhibitors, indicating that both pathways are key to rGal-3-mediated effects. Erk 1/2 inhibition in control-treated cells resembled an rGal-3 like state characterized by an increase in MBP, β-catenin, and gelsolin expression. In contrast, Akt inhibition in rGal-3-treated cells reduced MBP, β-catenin, and gelsolin expression, indicating a blockade of rGal-3 effects. Taken together, these results indicate that rGal-3 accelerates OLG maturation by modulating signaling pathways and protein expression which lead to changes in actin cytoskeleton dynamics.

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Acknowledgements

We thank Leandro N. Marziali and Teresa Elola for their assistance in rGal-3 production and purification; and to Victoria S.B. Wies Mancini for her help in primary OLG cultures. This study was supported by grants from the Argentine Agency for Promotion of Science and Technology (PICT 2012-0282; PICT 2014-3116), and the University of Buenos Aires (UBACYT-20020130100305BA).

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  1. Department of Biological Chemistry, School of Pharmacy and Biochemistry, Institute of Chemistry and Biological Physicochemistry (IQUIFIB), University of Buenos Aires and National Research Council (CONICET), Junín 956, C1113, Buenos Aires, Argentina

    Laura Thomas & Laura Andrea Pasquini

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Correspondence to Laura Andrea Pasquini.

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Highlights

Gal-3 drives early OLG process outgrowth by fostering actin cytoskeleton assembly and a decrease in Erk activation.

Gal-3 regulates OLG maturation by inducing Akt activation and MBP expression, which promote gelsolin release and actin cytoskeleton disassembly.

Electronic Supplementary Material

Fig. S1

rGal-3 purification. a Workflow. Immunoblot and Coomassie blue staining showing the presence or absence of rGal-3 in the different steps of rGal-3 purification. b Lactose affinity chromatography and c polymyxin affinity chromatography. SN before chromatography (SN BC); SN after chromatography (SN PC); Wash (W); eluted fraction (EF). d Chromatographic profiles from column blank and sample after HPLC-MS/MS using LCQ-Duo (ESI-IT, Thermofisher). e Peptides identified using Sequest and Mascot software. f Proteins identified. (GIF 419 kb)

High Resolution Image (TIF 25194 kb)

Fig. S2

a Viability of OLG treated with rGal-3: TD0 OPC were treated with different rGal-3 concentrations for 24 h and later MTT assayed. Graph shows absorbance at 570 nm quantification for each condition. Comparisons were performed using two-way ANOVA followed by Bonferroni post hoc test (* and #P < 0.05; ** and ##P < 0.01; ***P < 0.001; * between control and 50 μg/ml; # between 20 μm/ml and 50 μg/ml). b Viability of OLG treated with signaling pathway inhibitors or sucrose: TD1, TD3, and TD5 OLG treated with or without LY, UO, lactose, or sucrose and later MTT assayed. Graph shows absorbance at 570 nm for each condition. Comparisons were performed using two-way ANOVA followed by Bonferroni post hoc test. c Western blot analysis of OLG treated with signaling pathway inhibitors or sucrose: representative immunoblot for β-catenin, pAkt, tAkt, pErk1/2, tErk1/2, gelsolin, MBP, and GAPDH of TD1, TD3, and TD5 OLG treated with sucrose and with or without rGal-3. Graphs show immunoblot quantification for band intensities normalized to GAPDH as fold increases over TD1 control Comparisons were performed using two-way ANOVA followed by Bonferroni post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001; * regarding control, ♦ regarding control-suc). (GIF 158 kb)

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Thomas, L., Pasquini, L.A. Extracellular Galectin-3 Induces Accelerated Oligodendroglial Differentiation Through Changes in Signaling Pathways and Cytoskeleton Dynamics. Mol Neurobiol 56, 336–349 (2019). https://doi.org/10.1007/s12035-018-1089-6

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