Orie Tajima

Targeted Disruption of Gb3/CD77 Synthase Gene Resulted in the Complete Deletion of Globo-series Glycosphingolipids and Loss of Sensitivity to Verotoxins

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 μg of VT-2 or 1.0 μg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

Gangliosides play pivotal roles in the regulation of complement systems and in the maintenance of integrity in nerve tissues

Gangliosides are considered to be essential in the maintenance and repair of nervous tissues; however, the mechanisms for neurodegeneration caused by ganglioside defects are unknown. We examined gene expression profiles in double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes and showed that the majority of complement genes and their receptors were up-regulated in cerebellum in DKO mice. Inflammatory reactions were demonstrated in those tissues by measuring up-regulated inflammatory cytokines, indicating the presence of complement activation and inflammation as reported in Alzheimers disease. Immunoblotting of fractionated membrane extracts by sucrose density gradient revealed that complement-regulatory molecules such as decay-accelerating factor and CD59 were dispersed from glycolipid-enriched microdomain/rafts in DKO cerebellum. Immunohistostaining of these molecules showed disordered membrane localization. These results suggested that dysfunction of complement-regulatory molecules may be due to abnormal glycolipid-enriched microdomain/rafts that triggered complement activation, subsequent inflammation, and neurodegeneration in DKO mice. Generation of the triple KO mice lacking complement activity in addition to the two glycosyltransferases suggested that complement activation is involved in the inflammatory reactions and neurodegeneration caused by the ganglioside deficiency.

Ganglioside GD3 Enhances Adhesion Signals and Augments Malignant Properties of Melanoma Cells by Recruiting Integrins to Glycolipid-enriched Microdomains

Ganglioside GD3 is widely expressed in human malignant melanoma cell lines and tumors. Previously, we reported that GD3+ cells show stronger tyrosine phosphorylation of focal adhesion kinase (FAK), p130Cas, and paxillin when treated with fetal calf serum than GD3− cells. In this study, we analyzed the changes in the signals mediated by the interaction between integrins and extracellular matrices (ECM) to clarify how GD3 enhances cell signals in the vicinity of the cell membrane. An adhesion assay with a real time cell electronic sensing system revealed that GD3+ cells had stronger adhesion to all extracellular matrices examined. In particular, GD3+ cells attached more strongly to collagen type I and type IV than controls. Correspondingly, they showed stronger tyrosine phosphorylation of FAK and paxillin during adhesion to collagen type I. In the floating pattern of detergent extracts, a high level of integrin β1 was found in glycolipid-enriched microdomain (GEM)/rafts in GD3+ cells before adhesion, whereas a smaller amount of integrin β1 was detected in the GEM/rafts of controls. Some phosphorylated forms of FAK as well as total FAK were found in GEM/rafts during cell adhesion only in GD3+ cells. Another signal consisting of integrin-linked kinase/Akt was also activated during adhesion more strongly in GD3+ cells than in controls. In double stained GD3+ cells, GD3 and integrin β1 co-localized at the focal adhesion with a punctate pattern. All these results suggested that integrins assembled and formed a cluster in GEM/rafts, leading to the enhanced signaling and malignant properties under GD3 expression.

Gangliosides are essential in the protection of inflammation and neurodegeneration via maintenance of lipid rafts: elucidation by a series of ganglioside-deficient mutant mice.

J. Neurochem. (2011) 116, 926–935.

Sensory nerve-dominant nerve degeneration and remodeling in the mutant mice lacking complex gangliosides

Gangliosides, sialic acid-containing glycosphingo-lipids, are enriched in the mammalian nervous system. Since mutant mice with disrupted beta1,4-N-acetylgalactosaminyl-transferase (GM2/GD2 synthase) were generated, there have been several studies on the pathology of the mutant mice, i.e. mild functional disorders and Wallerian degeneration in the peripheral nervous system. To further analyze the chronological alteration in the mutant mice, we examined the peripheral and CNS mainly with morphological approaches, such as electron microscopy and immunohistochemistry. Accordingly with the sensory dysfunction, neural degeneration, glial proliferation and synaptic remodeling in the dorsal horn of the spinal cord were found in adult mice. Thick astrocytic processes with densely packed glial filaments were extended among the neuropils and around blood vessels. Morphological changes in the synaptic vesicles and modes of synaptic contacts with central terminals were detected, suggesting synaptic remodeling following the degeneration. These results suggest that complex gangliosides are essential in the maintenance of integrity in architecture and function of the nervous system, lack of which results in neural degeneration in a sensory nerve-dominant manner.

Reduced motor and sensory functions and emotional response in GM3-only mice: emergence from early stage of life and exacerbation with aging.

Sialic acid-containing glycosphingolipids (gangliosides) have been believed to play a role in the regulation and protection of nervous tissues. To clarify their function in the nervous system in vivo, double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes were generated and abnormal behaviors were analyzed. Mutant mice exhibited reduced weight and a round shape of the whole brain that progressively emerged with aging, and displayed motor dysfunction in the footprint, traction, open-field, and 24h locomotion activity tests. Sensory functions were also reduced in the von Frey and hot plate tests and greatly reduced in the acoustic startle response test. For emotional behavior, fear response was clearly decreased. Numerous neuronal dysfunctions were found even in younger mutant mice examined at 10-23 weeks after birth, which were exacerbated with aging. These results suggest that a lack of gangliosides other than GM3 induces severe neuronal degeneration in the early stage of life, and that the expression of complex gangliosides is essential to maintain the integrity of the nervous system throughout life.

Membrane sialidase NEU3 is highly expressed in human melanoma cells promoting cell growth with minimal changes in the composition of gangliosides

NEU3 is a membrane sialidase specific for gangliosides. Its increased expression and implication in some cancers have been reported. Here, we analyzed NEU3 expression in malignant melanoma cell lines and its roles in the cancer phenotypes. Quantitative RT‐PCR revealed that high levels of the NEU3 gene were expressed at almost equivalent levels with those in colon cancers. To examine the effects of overexpression of NEU3, NEU3 cDNA‐transfectant cells were established using a melanoma cell line SK‐MEL‐28 and its mutant N1 lacking GD3. SK‐MEL‐28 sublines overexpressing both the NEU3 gene and NEU3 enzyme activity showed no changes in both cell growth and ganglioside expression, while N1 cells showed a mild increase in cell proliferation with increased phosphorylation of the EGF receptor and neo‐synthesis of Gb3 after NEU3 transfection. In contrast, NEU3 silencing resulted in a definite reduction in cell growth in a melanoma line MeWo, while ganglioside patterns underwent minimal changes. Phosphorylation levels of ERK1/2 with serum stimulation decreased in the NEU3‐silenced cells. All these results suggest that NEU3 is highly expressed to enhance malignant phenotypes including apoptosis inhibition in malignant melanomas. (Cancer Sci 2011; 102: 2139–2149)

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21.

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9‐O‐acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography‐immunostaining, two‐dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9‐O‐ acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O‐acetyl ganglioside synthase were not up‐regulated. Tis21 which had been reported to be a 9‐O‐acetylation inducer was partially down‐regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9‐O‐acetyl‐GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9‐O‐acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild‐type brain might be able to synthesize very low levels of 9‐O‐acetyl GD3. Increased 9‐O‐acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.

Impaired hypoglossal nerve regeneration in mutant mice lacking complex gangliosides: down-regulation of neurotrophic factors and receptors as possible mechanisms.

Gangliosides, sialic acid-containing glycosphingolipids, have been considered to play roles as neurotrophic factors. Exogenous gangliosides added to the culture medium of neuronal cells or injected in artificially injured sites of nerve tissues actually showed neurotrophic factor-like effects such as neurite extension and alleviation of nerve tissue deterioration. In this study, neuroregeneration in the mutant mice lacking complex gangliosides was examined. To determine whether the nervous system maintains regenerative activity in the long-term absence of complex gangliosides, we analyzed hypoglossal nerve regeneration after axotomy in the mutant mice of GM2/GD2 synthase. These mice exhibited marked impairment of regenerative activity both in the number of surviving neurons and in the number of peroxidase-positive neurons. Moreover, reduced levels of gene expression of neurotrophic factors and their receptors including CNTF, p75 NTR, TrkB, and others in hypoglossal neurons were observed in real-time reverse transcription-polymerase chain reaction combined with laser capture microdissection, suggesting that these molecules are, at least partly, involved in the regeneration of lesioned nerves and that their expression levels are precisely controlled in the presence of intact expression of complex gangliosides.

Dysfunction of muscarinic acetylcholine receptors as a substantial basis for progressive neurological deterioration in GM3-only mice

To examine the effects of deletion of gangliosides on the nervous system by avoiding masking effects with the remaining structures, we established double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase genes, i.e., GM3-only mice. They showed progressive sensory and motor neuron deficits with aging. We further examined higher order neurological functions, and found progressive dysfunction of motor coordination with rota-rod test and marked deterioration in memory and learning with eight-arm radial maze test in the DKO mice. The results of oxotremorine treatment suggested that they undergo strong suppression of muscarinic type acetylcholine receptors (mAChRs) functions, and that the damage in the GM3-only mice is due to a mAChR receptor deficit. On the other hand, expression levels of mRNAs of mAChRs were generally up-regulated, suggesting compensatory increase of expression due to reduced functions. Since central mAChRs are involved in the regulation of cognitive, behavioral, sensory, motor, and autonomic functions, we investigated changes in the expressions levels of subtypes of the mAChR genes in various regions of brain tissues. M1 and M4 receptors were conspicuously up-regulated in cortex and striatum in the DKO, suggesting that suppressed functions of mAChRs are responsible for the altered neurological features, in particular for deteriorated memory and learning, observed in the behavioral analyses. Thus, dysfunction of mAChRs might be a substantial basis for the progressive neurological deterioration in DKO mice.