Different response of the knockout mice lacking b-series gangliosides against botulinum and tetanus toxins

We assessed the response in knockout mice lacking the b-series (G(D2), G(D1b), G(T1b) and G(Q1b)) gangliosides against Clostridium botulinum (types A, B and E) and tetani toxins. We found that botulinum toxins were fully toxic, while tetanus toxin was much less toxic in the knockout mice. Combining the present results with our previous finding that tetanus toxin and botulinum types A and B toxins showed essentially no toxic activity in the knockout mice lacking both the a-series and b-series gangliosides (complex gangliosides), we concluded that the b-series gangliosides is the major essential substance for tetanus toxin, while b-series gangliosides may be not the essential substance for botulinum toxins, at the initial step during the intoxication process in mouse.     

b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve.

The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain alpha-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas.     

Regulatory Mechanisms of Nervous Systems with Glycosphingolipids

A number of studies have suggested functions of sialic acid-containing glycosphingolipids (gangliosides) in the nervous system. However, results of analyses of the mutant mice lacking gangliosides suggested that they play crucial roles in the maintenance of integrity and repair of the nervous tissues. Furthermore, results of double knockout mice lacking all gangliosides except GM3 (GM3-only mice) suggested that deficiency of gangliosides induced complement activation and inflammation, leading to neurodegeneration. Generation of triple knockout mice by mating GM3-only mice and C3-deficient mice verified the involvement of complement systems in the inflammation and neurodegeneration. For the mechanisms of the complement activation, functional disorders of complement-regulatory proteins such as CD55 and CD59, which belong to GPI-anchored proteins, should be main factors. These results suggested that normal composition of gangliosides is essential for the maintenance of lipid rafts. Therefore, it was suggested that regulation of the complement systems and suppression of the inflammation should be important for the treatment of neurodegeneration, having common aspects with other neurodegenerative diseases such as Alzheimer disease.     

Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 - A quantitative Golgi cox study

Gangliosides are characteristic plasma membrane constituents of vertebrate brain used as milestones of neuronal development. As neuronal morphology is a good indicator of neuronal differentiation, we analyzed how lack of the ganglioside biosynthetic gene Galgt1 whose product is critical for production of four major adult mammalian brain complex gangliosides (GM1, GD1a, GD1b and GT1b) affects neuronal maturation in vivo. To define maturation of cortical neurons in mice lacking B4galnt1 we performed a morphological analysis of Golgi-Cox impregnated pyramidal neurons in primary motor cortex and granular cells of dentate gyrus in 3, 21 and 150 days old B4galnt1-null and wild type mice. Quantitative analysis of basal dendritic tree on layer III pyramidal neurons in the motor cortex showed very immature dendritic picture in both mice at postnatal day 3. At postnatal day 21 both mice reached adult values in dendritic length, complexity and spine density. No quantitative differences were found between B4galnt1-null and wild type mice in pyramidal cells of motor cortex or granular cells of dentate gyrus at any examined age. In addition, the general structural and neuronal organization of all brain structures, qualitatively observed on Nissl and Golgi-Cox, were similar Our results demonstrate that neurons can develop normal dendritic complexity and length without presence of complex gangliosides in vivo. Therefore, behavioral differences observed in B4galnt1-null mice may be attributed to functional rather than morphological level of dendrites and spines of cortical pyramidal neurons.     

Expression of the Blood-Group-Related Gene B4galnt2 Alters Susceptibility to Salmonella Infection

Glycans play important roles in host-microbe interactions. Tissue-specific expression patterns of the blood group glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) are variable in wild mouse populations, and loss of B4galnt2 expression is associated with altered intestinal microbiota. We hypothesized that variation in B4galnt2 expression alters susceptibility to intestinal pathogens. To test this, we challenged mice genetically engineered to express different B4galnt2 tissue-specific patterns with a Salmonella Typhimurium infection model. We found B4galnt2 intestinal expression was strongly associated with bacterial community composition and increased Salmonella susceptibility as evidenced by increased intestinal inflammatory cytokines and infiltrating immune cells. Fecal transfer experiments demonstrated a crucial role of the B4galnt2-dependent microbiota in conferring susceptibility to intestinal inflammation, while epithelial B4galnt2 expression facilitated epithelial invasion of S. Typhimurium. These data support a critical role for B4galnt2 in gastrointestinal infections. We speculate that B4galnt2-specific differences in host susceptibility to intestinal pathogens underlie the strong signatures of balancing selection observed at the B4galnt2 locus in wild mouse populations.     

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.     

Immunohistochemical and biochemical analyses of GD3, GT1b,and GQ1b gangliosides during neural differentiation of P19 EC cells

In an earlier study, we showed that expressions of GD3, GT1b, and GQ1b gangliosides in P19 embryonic carcinoma (EC) cells were enhanced during their neural differentiation induced by retinoic acid. We now further demonstrated that this increase of the b-series gangliosides is due to an increase in their corresponding synthases (sialyltransferase-II, -IV, and -V) in the Golgi. Of the three gangliosides studied, GQ1b appeared to be the best candidate for monitoring such differentiation process. We also used fluorescence-labeled monoclonal antibodies and confocal fluorescence microscopy to obtain direct visual information about the relationship of gangliosides and neural specific proteins in neuron development. Again, GQ1b is the most interesting as it localizes with synaptophysin and neural cell adhesion molecules (NCAMs) on synaptic boutons or dendritic spines in RA-induced neurons (R/N). This suggests that GQ1b could be used as a marker for synapse formation during construction of the neural network.     

Neuronal PTP1B regulates body weight, adiposity and leptin action

Obesity is a major health problem and a risk factor for type 2 diabetes. Leptin, an adipocyte-secreted hormone, acts on the hypothalamus to inhibit food intake and increase energy expenditure. Most obese individuals develop hyperleptinemia and leptin resistance, limiting the therapeutic efficacy of exogenously administered leptin. Mice lacking the tyrosine phosphatase PTP1B are protected from diet-induced obesity and are hypersensitive to leptin, but the site and mechanism for these effects remain controversial. We generated tissue-specific PTP1B knockout (Ptpn1(-/-)) mice. Neuronal Ptpn1(-/-) mice have reduced weight and adiposity, and increased activity and energy expenditure. In contrast, adipose PTP1B deficiency increases body weight, whereas PTP1B deletion in muscle or liver does not affect weight. Neuronal Ptpn1(-/-) mice are hypersensitive to leptin, despite paradoxically elevated leptin levels, and show improved glucose homeostasis. Thus, PTP1B regulates body mass and adiposity primarily through actions in the brain. Furthermore, neuronal PTP1B regulates adipocyte leptin production and probably is essential for the development of leptin resistance.     

Structure and biosynthesis of glycoproteins. Preface

We used the knockout mice lacking gangliosides and evaluated their response to tetanus and botulinum toxins. We found that tetanus toxin and botulinum type A or B toxin was less toxic in the knockout mice. We conclude that the toxins bind to the gangliosides on the synapses in the initial step of intoxication prior to penetration of the toxins into the neural cells.     

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.     

Specific ganglioside changes in extraneural tissues of adult rats with hypothyroidism

Adults rats with hypothyroidism were prepared by administration of 6-propyl-2-thiouracil (PTU) or methimazole, and the tissues were examined for their gangliosides through methods including glycolipid-overlay techniques. Normal thyroid tissue contained GM3, GD3, and GD1a as the major gangliosides, with GM1, GD1b, GT1b, and GQ1b in lesser amounts. The goitrous tissue of PTU-induced hypothyroid rats had higher concentrations of GM1 and GD1a with a concomitant decrease of GM3. The amount of GT3 in thyroid tissue was increased in hypothyroid animals. While normal liver tissue had a complex ganglioside pattern with a- and b-series gangliosides, the PTU-induced hypothyroid tissue showed a simpler ganglioside profile that consisted mainly of a-series gangliosides with almost undetectable amounts of b-series gangliosides. The expression of c-series gangliosides was suppressed in the hypothyroid liver tissue. Heart tissue had higher contents of GM3 and GT3 than control. No apparent change was observed in the compositions of major and c-series gangliosides in other extraneural tissues (i.e., kidney, lung, spleen, thymus, pancreas, testis, skeletal muscle, and eye lenses), and neural tissues (i.e., cerebrum and cerebellum) from PTU-induced hypothyroid rats. The ganglioside changes of thyroid, liver, and heart tissues were reproduced in corresponding tissues of methimazole-induced hypothyroid rats. These results suggest that hypothyroid conditions affect the biosynthesis and expression of gangliosides in specific tissue and cell types.     

Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation

Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.     

Essential Roles of Gangliosides in the Formation and Maintenance of Membrane Microdomains in Brain Tissues

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.     

AMPKα2 knockout enhances tumour inflammation through exacerbated liver injury and energy deprivation‐associated AMPKα1 activation

Tissue damage and its associated‐inflammation act as tumour initiators or propagators. AMP‐activated protein kinase (AMPK) is activated by environmental or nutritional stress factors, such as hypoxia, glucose deprivation, and other cell injury factors, to regulate cell energy balance and differentiation. We previously have reported that AMPKα2 deficiency resulted in the energy deprivation in tumour‐bearing liver and the enhanced‐hepatocyte death. In this study, AMPKα2 knockout mice and the liver metastasis model of colon cancer cells were used to address the role of AMPKα isoforms in tumour inflammation. First, we found that the AMPKα2 deficiency exacerbated the liver injury and recruitment of macrophages. Meanwhile, although compensatory expression of AMPKα1 was not significant after AMPKα2 knockout, AMPKα1 phosphorylation was elevated in remnant liver in AMPKα2 knockout mice, which was positively associated with the enhanced energy deprivation in the AMPKα2 deficient mice. Furthermore, the activated AMPKα1 in macrophage contributed to its polarizing to tumour‐associated phenotype. Thus, the enhanced tumour‐associated inflammation and activation of AMPKα1 in the AMPKα2 deficient mice may exacerbate the tumour development by affecting the tumour inflammatory microenvironment. Our study suggests that the two isoforms of AMPKα, AMPKα1 and AMPKα2 play different roles in controlling tumour development.     

Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice

The effects of high-fat feeding on the development of obesity were evaluated in intercellular adhesion molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat diet for < or =50 days. Serum and tissues were collected at baseline and after 1, 11, and 50 days on the diet. After 11 days on the diet, ICAM-1-deficient, but not B6, mice developed fatty livers and showed a significant increase in inguinal fat pad weight. At day 50, ICAM-1-deficient mice weighed less, and their adiposity index and circulating leptin levels were significantly lower than those of B6 controls. To better understand the early differential response to the diet, liver gene expression was analyzed at three time points by use of Affymetrix GeneChips. In both strains, a similar pattern of gene expression was detected in response to the high-fat diet. However, sterol regulatory element-binding protein-1, apolipoprotein A4, and adipsin mRNAs were significantly induced in ICAM-1-deficient livers, suggesting that these genes and their associated pathways may be involved in the acute diet response observed in the knockout mice.     

SOCS-1 deficiency does not prevent diet-induced insulin resistance

Obesity is associated with inflammation and increased expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit cytokine and insulin signaling. Thus, reducing SOCS expression could prevent the development of obesity-induced insulin resistance. Using SOCS-1 knockout mice, we investigated the contribution of SOCS-1 in the development of insulin resistance induced by a high-fat diet (HFD). SOCS-1 knockout mice on HFD gained 70% more weight, displayed a 2.3-fold increase in epididymal fat pads mass and increased hepatic lipid content. This was accompanied by increased mRNA expression of leptin and the macrophage marker CD68 in white adipose tissue and of SREBP1c and FAS in liver. HFD also induced hyperglycemia in SOCS-1 deficient mice with impairment of glucose and insulin tolerance tests. Thus, despite the role of SOCS proteins in obesity-related insulin resistance, SOCS-1 deficiency alone is not able to prevent insulin resistance induced by a diet rich in fat.     

Long-term balancing selection at the blood group-related gene B4galnt2 in the genus Mus (Rodentia; Muridae)

Recent surveys of the human genome have highlighted the significance of balancing selection in relation to understanding the evolutionary origins of disease-associated variation. Cis-regulatory variation at the blood group-related glycosyltransferase B4galnt2 is associated with a phenotype in mice that closely resembles a common human bleeding disorder, von Willebrand disease. In this study, we have performed a survey of the 5' flanking region of the B4galnt2 gene in several Mus musculus subspecies and Mus spretus. Our results reveal a clear pattern of trans-species polymorphism and indicate that allele classes conferring alternative tissue-specific expression patterns have been maintained for >2.8 My in the genus Mus. Furthermore, analysis of B4galnt2 expression patterns revealed the presence of an additional functional class of alleles, supporting a role for gastrointestinal phenotypes in the long-term maintenance of expression variation at this gene.     

Lactoferrin Deficiency Promotes Colitis-Associated Colorectal Dysplasia in Mice

Nonresolving inflammatory processes affect all stages of carcinogenesis. Lactoferrin, a member of the transferrin family, is involved in the innate immune response and anti-inflammatory, anti-microbial, and anti-tumor activities. We previously found that lactoferrin is significantly down-regulated in specimens of nasopharyngeal carcinoma (NPC) and negatively associated with tumor progression, metastasis, and prognosis of patients with NPC. Additionally, lactoferrin expression levels are decreased in colorectal cancer as compared with normal tissue. Lactoferrin levels are also increased in the various phases of inflammation and dysplasia in an azoxymethane–dextran sulfate sodium (AOM-DSS) model of colitis-associated colon cancer (CAC). We thus hypothesized that the anti-inflammatory function of lactoferrin may contribute to its anti-tumor activity. Here we generated a new Lactoferrin knockout mouse model in which the mice are fertile, develop normally, and display no gross morphological abnormalities. We then challenged these mice with chemically induced intestinal inflammation to investigate the role of lactoferrin in inflammation and cancer development. Lactoferrin knockout mice demonstrated a great susceptibility to inflammation-induced colorectal dysplasia, and this characteristic may be related to inhibition of NF-κB and AKT/mTOR signaling as well as regulation of cell apoptosis and proliferation. Our results suggest that the protective roles of lactoferrin in colorectal mucosal immunity and inflammation-related malignant transformation, along with a deficiency in certain components of the innate immune system, may lead to serious consequences under conditions of inflammatory insult.