Normal Cortical Myelination in Galectin-4-Deficient Mice.

Myelin, critical for the correct function of the nervous system, is organized in different patterns that can include long non-myelinated axonal segments. How myelin patterning is regulated remains unexplained. The carbohydrate-binding protein galectin-4 (Gal-4) influences oligodendrocyte differentiation in vitro and is associated with non-myelinable axon segments (NMS) in cultured neurons. In consequence, Gal-4 has been proposed as a myelin patterning regulator, although no in vivo studies have corroborated this hypothesis. We used Gal-4-deficient mice (Lgals4-KO) to study the role of Gal-4 in cortical myelination in vivo. We show that cultured neurons of Lgals4-KO mice form NMS that are regulated as in control neurons. In addition, oligodendrocyte/myelin markers expression measured by biochemical and immunochemical means, and cortical myelin microstructure studied by in-depth image analysis appear unaltered in these animals. Consistently, myelin displays an essentially normal function assessed by in vivo electrophysiology and locomotion analyses. In conclusion, cortical myelin of Lgals4-KO mice does not show any significant defect in composition, organization or function, pointing to a negligible role of Gal-4 in myelination in vivo or, as discussed, to unknown mechanisms that compensate its absence.

How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins.

BACKGROUND: Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. METHODS: Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. RESULTS: Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. CONCLUSIONS: Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. GENERAL SIGNIFICANCE: Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.

Neurons define non-myelinated axon segments by the regulation of galectin-4-containing axon membrane domains.

The mechanism underlying selective myelination of axons versus dendrites or neuronal somata relies on the expression of somatodendritic membrane myelination inhibitors (i.e. JAM2). However, axons still present long unmyelinated segments proposed to contribute to axonal plasticity and higher order brain functions. Why these segments remain unmyelinated is still an unresolved issue. The bifunctional lectin galectin-4 (Gal-4) organizes the transport of axon glycoproteins by binding to N-acetyllactosamine (LacNac) termini of N-glycans. We have shown that Gal-4 is sorted to segmental domains (G4Ds) along the axon surface, reminiscent of these long unmyelinated axon segments in cortical neurons. We report here that oligodendrocytes (OLGs) do not deposit myelin on Gal-4 covered surfaces or myelinate axonal G4Ds. In addition, Gal-4 interacts and co-localizes in G4Ds with contactin-1, a marker of another type of non-myelinated segments, the nodes of Ranvier. Neither Gal-4 expression nor G4D dimensions are affected by myelin extracts or myelinating OLGs, but are reduced with neuron maturation. As in vitro, Gal-4 is consistently segregated from myelinated structures in the brain. Our data shape the novel concept that neurons establish axon membrane domains expressing Gal-4, the first inhibitor of myelination identified in axons, whose regulated boundaries delineate myelination-incompetent axon segments along development.

The sugar code in neuronal physiology.

Carbohydrate-related interactions are necessary for the correct development and function of the nervous system. As we illustrate with several examples, those interactions are controlled by carbohydrate-modifying enzymes and by carbohydrate-binding proteins that regulate a plethora of complex axonal processes. Among others, glycan-related proteins as sialidase Neu3 or galectins-1, -3, and -4 play central roles in the determination of axonal fate, axon growth, guidance and regeneration, as well as in polarized axonal glycoprotein transport. In addition, myelination is also highly dependent on glycans, and the stabilization of myelin architecture requires the interaction of the myelin-associated glycoprotein (siglec-4) with gangliosides in the axonal membrane. The roles of glycans in neuroscience are far from being completely understood, though the cases presented here underscore the importance and potential of carbohydrates to establish with precision key molecular mechanisms of the physiology of the nervous system. New specific applications in diagnosis as well as the definition of new molecular targets to treat neurological diseases related to lectins and/or glycans are envisioned in the future.

Axon glycoprotein routing in nerve polarity, function, and repair.

Nervous system function relies on the capacity of neurons to organize specialized domains for impulse reception or transmission. Such a polarized architecture relies on highly discriminatory and efficient mechanisms for the transport and targeting of required molecules to their functional positions. Glycans play a central role in polarized traffic based on their extraordinary capacity to encrypt bio-information. Glycan-based interactions exquisitely regulate cargo selection, trafficking, and targeting to the axon membrane. This generates segregated functional domains, where basal nerve processes such as axon growth, synaptic activity, or myelination take place. Deciphering the details of the glycan structures and carbohydrate-binding molecules that underlie these mechanisms improves our knowledge of nerve physiology and defines novel specific approaches for neurological treatments.

Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering.

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.

Phosphorylation of adhesion- and growth-regulatory human galectin-3 leads to the induction of axonal branching by local membrane L1 and ERM redistribution.

Serine phosphorylation of the beta-galactoside-binding protein galectin-3 (Gal-3) impacts nuclear localization but has unknown consequences for extracellular activities. Herein, we reveal that the phosphorylated form of galectin-3 (pGal-3), adsorbed to substratum surfaces or to heparan sulphate proteoglycans, is instrumental in promoting axon branching in cultured hippocampal neurons by local actin destabilization. pGal-3 interacts with neural cell adhesion molecule L1, and enhances L1 association with Thy-1-rich membrane microdomains. Concomitantly, membrane-actin linker proteins ezrin-radixin-moesin (ERM) are recruited to the same membrane site via interaction with the intracellular domain of L1. We propose that the local regulation of the L1-ERM-actin pathway, at the level of the plasma membrane, underlies pGal-3-induced axon branching, and that galectin phosphorylation in situ could act as a molecular switch for the axon response to Gal-3.