Agenesis of the corpus callosum in Nogo receptor deficient mice.

The corpus callosum (CC) is the largest fiber tract in the mammalian brain, linking the bilateral cerebral hemispheres. CC development depends on the proper balance of axon growth cone attractive and repellent cues leading axons to the midline and then directing them to the contralateral hemisphere. Imbalance of these cues results in CC agenesis or dysgenesis. Nogo receptors (NgR1, NgR2, and NgR3) are growth cone directive molecules known for inhibiting axon regeneration after injury. We report that mice lacking Nogo receptors (NgR123-null mice) display complete CC agenesis due to axon misdirection evidenced by ectopic axons including cortical Probst bundles. Because glia and glial-derived growth cone repellent factors (especially the diffusible factor Slit2) are required for CC development, their distribution was studied. Compared with wild-type mice, NgR123-null mice had a sharp increase in the glial marker glial fibrillary acidic protein (GFAP) and in Slit2 at the glial wedge and indusium griseum, midline structures required for CC formation. NgR123-null mice displayed reduced motor coordination and hyperactivity. These data are consistent with the hypotheses that Nogo receptors are membrane-bound growth cone repellent factors required for migration of axons across the midline at the CC, and that their absence results directly or indirectly in midline gliosis, increased Slit2, and complete CC agenesis. J. Comp. Neurol. 525:291-301, 2017. © 2016 Wiley Periodicals, Inc.

Sialylation regulates brain structure and function.

Every cell expresses a molecularly diverse surface glycan coat (glycocalyx) comprising its interface with its cellular environment. In vertebrates, the terminal sugars of the glycocalyx are often sialic acids, 9-carbon backbone anionic sugars implicated in intermolecular and intercellular interactions. The vertebrate brain is particularly enriched in sialic acid-containing glycolipids termed gangliosides. Human congenital disorders of ganglioside biosynthesis result in paraplegia, epilepsy, and intellectual disability. To better understand sialoglycan functions in the nervous system, we studied brain anatomy, histology, biochemistry, and behavior in mice with engineered mutations in St3gal2 and St3gal3, sialyltransferase genes responsible for terminal sialylation of gangliosides and some glycoproteins. St3gal2/3 double-null mice displayed dysmyelination marked by a 40% reduction in major myelin proteins, 30% fewer myelinated axons, a 33% decrease in myelin thickness, and molecular disruptions at nodes of Ranvier. In part, these changes may be due to dysregulation of ganglioside-mediated oligodendroglial precursor cell proliferation. Neuronal markers were also reduced up to 40%, and hippocampal neurons had smaller dendritic arbors. Young adult St3gal2/3 double-null mice displayed impaired motor coordination, disturbed gait, and profound cognitive disability. Comparisons among sialyltransferase mutant mice provide insights into the functional roles of brain gangliosides and sialoglycoproteins consistent with related human congenital disorders.

Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells.

BACKGROUND: Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid-binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. OBJECTIVE: The histologic distribution, expression, and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. METHODS: Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by means of siglec blotting and isolation by means of siglec capture. RESULTS: Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, and connective tissue); both were significantly upregulated in patients with chronic rhinosinusitis. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B (MUC5B) under inflammatory control through the nuclear factor κB pathway, and MUC5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. CONCLUSION: Inflammation results in upregulation of immune-inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands are upregulated through the nuclear factor κB pathway, resulting in their enhanced expression on MUC5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation.

Ganglioside regulation of AMPA receptor trafficking.

Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10(-4)), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

Mice deficient in the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) exhibit enhanced allergic eosinophilic airway inflammation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec)-F is a proapoptotic receptor on mouse eosinophils, but little is known about its natural tissue ligand. OBJECTIVE: We previously reported that the St3gal3 gene product α2,3 sialyltransferase (ST3Gal-III) is required for constitutive Siglec-F lung ligand synthesis. We therefore hypothesized that attenuation of ST3Gal-III will decrease Siglec-F ligand levels and enhance allergic eosinophilic airway inflammation. METHODS: C57BL/6 wild-type mice and St3gal3 heterozygous or homozygous deficient (St3gal3(+/-) and St3gal3(-/-)) mice were used. Eosinophilic airway inflammation was induced through sensitization to ovalbumin (OVA) and repeated airway OVA challenge. Siglec-F human IgG1 fusion protein (Siglec-F-Fc) was used to detect Siglec-F ligands. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for inflammation, as well as various cytokines and chemokines. Serum was analyzed for allergen-specific immunoglobulin levels. RESULTS: Western blotting with Siglec-F-Fc detected approximately 500-kDa and approximately 200-kDa candidate Siglec-F ligands that were less abundant in St3gal3(+/-) lung extracts and nearly absent in St3gal3(-/-) lung extracts. After OVA sensitization and challenge, Siglec-F ligands were increased in wild-type mouse lungs but less so in St3gal3 mutants, whereas peribronchial and BALF eosinophil numbers were greater in the mutants, with the following rank order: St3gal3(-/-) ≥ St3gal3(+/-) > wild-type mice. Levels of various cytokines and chemokines in BALF were not significantly different among these 3 types of mice, although OVA-specific serum IgG1 levels were increased in St3gal3(-/-) mice. CONCLUSIONS: After OVA sensitization and challenge, St3gal3(+/-) and St3gal3(-/-) mice have more intense allergic eosinophilic airway inflammation and less sialylated Siglec-F ligands in their airways. One possible explanation for these findings is that levels of sialylated airway ligands for Siglec-F might be diminished in mice with attenuated levels of ST3Gal-III, resulting in a reduction in a natural proapoptotic pathway for controlling airway eosinophilia.