Myelin-associated glycoprotein (MAG) binds to the nerve cell surface and inhibits nerve regeneration. The nerve cell surface ligand(s) for MAG are not established, although sialic acid-bearing glycans have been implicated. We identify the nerve cell surface gangliosides GD1a and GT1b as specific functional ligands for MAG-mediated inhibition of neurite outgrowth from primary rat cerebellar granule neurons. MAG-mediated neurite outgrowth inhibition is attenuated by (i) neuraminidase treatment of the neurons; (ii) blocking neuronal ganglioside biosynthesis; (iii) genetically modifying the terminal structures of nerve cell surface gangliosides; and (iv) adding highly specific IgG-class antiganglioside mAbs. Furthermore, neurite outgrowth inhibition is mimicked by highly multivalent clustering of GD1a or GT1b by using precomplexed antiganglioside Abs. These data implicate the nerve cell surface gangliosides GD1a and GT1b as functional MAG ligands and suggest that the first step in MAG inhibition is multivalent ganglioside clustering.
SUMMARY Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here we show that FMRP regulates the translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is ubiquitously expressed, NOS1 protein is transiently co-expressed with FMRP during early synaptogenesis in layer- and region-specific subpopulations of pyramidal neurons. These include mid-fetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca’s area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases but not FMRP-deficient mice. Thus, alterations in FMRP post-transcriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.
Antibodies targeting major gangliosides that are broadly distributed in the nervous system are sometimes associated with clinical symptoms that imply selective nerve damage. For example, anti-GD1a antibodies are associated with acute motor axonal neuropathy (AMAN), a form of Guillain-Barré syndrome that selectively affects motor nerves, despite reports that GD1a is present in human axons and myelin and is not expressed differentially in motor versus sensory roots. We used a series of high-affinity monoclonal antibodies (mAbs) against the major nervous system gangliosides GM1, GD1a, GD1b and GT1b to test whether any of them bind motor or sensory fibres differentially in rodent and human peripheral nerves. The following observations were made. (i) Some of the anti-GD1a antibodies preferentially stained motor fibres, supporting the association of human anti-GD1a antibodies with predominant motor neuropathies such as AMAN. (ii) A GD1b antibody preferentially stained the large dorsal root ganglion (DRG) neurones, in keeping with the proposed role of human anti-GD1b antibodies in sensory ataxic neuropathies. (iii) Two mAbs with broad structural cross-reactivity bound to both gangliosides and peripheral nerve proteins. (iv) Myelin was poorly stained; all clones stained axons nearly exclusively. Our findings suggest that anti-ganglioside antibody fine specificity as well as differences in ganglioside accessibility in axons and myelin influence the selectivity of injury to different fibre systems and cell types in human autoimmune neuropathies.
Gangliosides-sialylated glycosphingolipids-are the major glycoconjugates of nerve cells. The same four structures-GM1, GD1a, GD1b and GT1b-comprise the great majority of gangliosides in mammalian brains. They share a common tetrasaccharide core (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'Cer) with one or two sialic acids on the internal galactose and zero (GM1 and GD1b) or one (GD1a and GT1b) α2-3-linked sialic acid on the terminal galactose. Whereas the genes responsible for the sialylation of the internal galactose are known, those responsible for terminal sialylation have not been established in vivo. We report that St3gal2 and St3gal3 are responsible for nearly all the terminal sialylation of brain gangliosides in the mouse. When brain ganglioside expression was analyzed in adult St3gal1-, St3gal2-, St3gal3- and St3gal4-null mice, only St3gal2-null mice differed significantly from wild type, expressing half the normal amount of GD1a and GT1b. St3gal1/2-double-null mice were no different than St3gal2-single-null mice; however, St3gal2/3-double-null mice were >95% depleted in gangliosides GD1a and GT1b. Total ganglioside expression (lipid-bound sialic acid) in the brains of St3gal2/3-double-null mice was equivalent to that in wild-type mice, whereas total protein sialylation was reduced by half. St3gal2/3-double-null mice were small, weak and short lived. They were half the weight of wild-type mice at weaning and displayed early hindlimb dysreflexia. We conclude that the St3gal2 and St3gal3 gene products (ST3Gal-II and ST3Gal-III sialyltransferases) are largely responsible for ganglioside terminal α2-3 sialylation in the brain, synthesizing the major brain gangliosides GD1a and GT1b.
Gangliosides - sialic acid-bearing glycolipids - are major cell surface determinants on neurons and axons. The same four closely related structures, GM1, GD1a, GD1b and GT1b, comprise the majority of total brain gangliosides in mammals and birds. Gangliosides regulate the activities of proteins in the membranes in which they reside, and also act as cell-cell recognition receptors. Understanding the functions of major brain gangliosides requires knowledge of their tissue distribution, which has been accomplished in the past using biochemical and immunohistochemical methods. Armed with new knowledge about the stability and accessibility of gangliosides in tissues and new IgG-class specific monoclonal antibodies, we investigated the detailed tissue distribution of gangliosides in the adult mouse brain. Gangliosides GD1b and GT1b are widely expressed in gray and white matter. In contrast, GM1 is predominately found in white matter and GD1a is specifically expressed in certain brain nuclei/tracts. These findings are considered in relationship to the hypothesis that gangliosides GD1a and GT1b act as receptors for an important axon-myelin recognition protein, myelin-associated glycoprotein (MAG). Mediating axon-myelin interactions is but one potential function of the major brain gangliosides, and more detailed knowledge of their distribution may help direct future functional studies.
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