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.     

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.     

Glycolipids: Essential regulator of neuro-inflammation,metabolism and gliomagenesis

Gene knockout mice of glycosyltransferases have clearly showed roles of their products in the bodies, while there are examples where phenotype of knockout was much less severe than expected probably due to functional redundancy. The most striking novel finding obtained from ganglioside-deficient mice was that progressive inflammatory reaction took place, leading to neurodegeneration. In particular, dysfunction of complement-regulatory proteins due to deteriorated architecture of lipid rafts seemed to be essential mechanisms for the inflammation. Furthermore, roles of gangliosides in neurons were demonstrated by neuron-specific transgenic of B4galnt1 with genetic background of B4galnt1 deficiency. From study of gene knockout mice of St8sia1, new roles of b-series gangliosides in leptin secretion from adipocytes, and roles of a-series gangliosides in leptin receptor, ObR in hypothalamus were demonstrated, leading to apparent intact balance of energy. Essential roles of b-series gangliosides in malignant properties of gliomas were also shown, suggesting their roles in the regulation of inflammation and proliferation in nervous tissues. How to apply these findings for the control of newly discovered patients with ganglioside deficiency remains to be investigated. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Attenuation of interleukin 2 signal in the spleen cells of complex ganglioside-lacking mice.

T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor alpha, beta, and gamma were almost equivalent, and up-regulation of alpha chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.     

ABCA2 deficiency results in abnormal sphingolipid metabolism in mouse brain

ABCA2, a member of the ATP-binding cassette (ABC) transporter family, is localized mainly to late endosome/lysosomes of oligodendrocytes in brain, but the physiological role and function of ABCA2 are unknown. In this study, we generated mutant mice (ABCA2-null) by targeting the abca2 gene. ABCA2-null mice exhibited a phenotype including lower pregnancy rate and body weight, shorter latency period on the balance beam, and sensitization to environmental stress compared with wild type mice but no abnormality in the cytoarchitectonic and compact myelin structure or oligodendroglial differentiation. Lipid analysis of brain from 11 days to 64 weeks of age revealed significant accumulation of gangliosides along with reduced sphingomyelin (SM) from 4 weeks to 64 weeks of age and accumulation of cerebrosides and sulfatides at 64 weeks of age in ABCA2-null mice compared with wild type mice. In addition, a significant accumulation of the major ganglioside GM1 and reduced SM was detected in the myelin fraction of ABCA2-null brain. Comparison of ABCA2-null and wild type mice revealed weak ABCA2 immunoreactivity in some large pyramidal cells of wild type brain. These results suggest that ABCA2 is involved in the intracellular metabolism of sphingolipids in the brain, particularly SM and gangliosides in oligodendrocytes and certain neurons.     

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.     

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.     

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.     

Intracranial V. cholerae sialidase protects against excitotoxic neurodegeneration

Converging evidence shows that GD3 ganglioside is a critical effector in a number of apoptotic pathways, and GM1 ganglioside has neuroprotective and noötropic properties. Targeted deletion of GD3 synthase (GD3S) eliminates GD3 and increases GM1 levels. Primary neurons from GD3S−/− mice are resistant to neurotoxicity induced by amyloid-β or hyperhomocysteinemia, and when GD3S is eliminated in the APP/PSEN1 double-transgenic model of Alzheimer''s disease the plaque-associated oxidative stress and inflammatory response are absent. To date, no small-molecule inhibitor of GD3S exists. In the present study we used sialidase from Vibrio cholerae (VCS) to produce a brain ganglioside profile that approximates that of GD3S deletion. VCS hydrolyzes GD1a and complex b-series gangliosides to GM1, and the apoptogenic GD3 is degraded. VCS was infused by osmotic minipump into the dorsal third ventricle in mice over a 4-week period. Sensorimotor behaviors, anxiety, and cognition were unaffected in VCS-treated mice. To determine whether VCS was neuroprotective in vivo, we injected kainic acid on the 25th day of infusion to induce status epilepticus. Kainic acid induced a robust lesion of the CA3 hippocampal subfield in aCSF-treated controls. In contrast, all hippocampal regions in VCS-treated mice were largely intact. VCS did not protect against seizures. These results demonstrate that strategic degradation of complex gangliosides and GD3 can be used to achieve neuroprotection without adversely affecting behavior.     

Genetic polymorphism of rat liver gangliosides

Liver ganglioside patterns of eight rat strains were classified according to two phenotypes: SHR type, characterized by predominance of b-series gangliosides (GD1b, GT1b, GQ1b), and DA type, characterized by predominance of a-series gangliosides (GM1, GD1a). Comparison of ganglioside pattern expressed in the liver of F1 hybrids and backcross F2 hybrids indicated that SHR type is controlled by a single autosomal-dominant gene which probably determines the expression of sialytransferase 2 activity for synthesis of GD3 from GM3.     

Life without gangliosides

Gangliosides are sialic acid containing glycosphingolipids that are present on all plasma cell membranes. Although they represent the major sialoconjugates in the central nervous system, their precise functions remain obscure. We have generated a series of knockout mice with gene disruptions in the ganglioside biosynthetic pathway. Individually these mice lack subsets of gangliosides allowing investigations into their specific functions. By breeding mice to contain multiple mutations in the biosynthetic pathway we have now produced mice without neuronal gangliosides. These mice display an extremely severe phenotype and neuronal pathology highlighting the essential function of gangliosides in the central nervous system.     

HNK-1 carbohydrate synthesis in retinoic acid-induced P19 cells

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK-1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC-binding protein, SBP-1 has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long-term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non-neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP-1 with the GC molecules. The results indicated that in vivo SBP-1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.     

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.     

Csx/Nkx2-5 is required for homeostasis and survival of cardiac myocytes in the adult heart

Csx/Nkx2-5, which is essential for cardiac development of the embryo, is abundantly expressed in the adult heart. We here examined the role of Csx/Nkx2-5 in the adult heart using two kinds of transgenic mice. Transgenic mice that overexpress a dominant negative mutant of Csx/Nkx2-5 (DN-TG mice) showed degeneration of cardiac myocytes and impairment of cardiac function. Doxorubicin induced more marked cardiac dysfunction in DN-TG mice and less in transgenic mice that overexpress wild type Csx/Nkx2-5 (WT-TG mice) compared with non-transgenic mice. Doxorubicin induced cardiomyocyte apoptosis, and the number of apoptotic cardiomyocytes was high in the order of DN-TG mice, non-transgenic mice, and WT-TG mice. Overexpression of the dominant negative mutant of Csx/Nkx2-5 induced apoptosis in cultured cardiomyocytes, while expression of wild type Csx/Nkx2-5 protected cardiomyocytes from doxorubicin-induced apoptotic death. These results suggest that Csx/Nkx2-5 plays a critical role in maintaining highly differentiated cardiac phenotype and in protecting the heart from stresses including doxorubicin.     

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.     

Beta1,4-N-acetylgalactosaminyltransferase--GM2/GD2 synthase: a key enzyme to control the synthesis of brain-enriched complex gangliosides

Beta1,4-N-acetylgalactosaminyltransferase (GM2/GD2 synthase) is a key enzyme which catalyzes the conversion of GM3, GD3 and lactosylceramide (LacCer) to GM2, GD2 and asialo-GM2 (GA2), respectively. This step is critical for the synthesis of all complex gangliosides enriched in the nervous system of vertebrates. Following the cloning of cDNAs encoding GM2/GD2 synthase by an expression cloning approach, substantial evidence for the roles of complex gangliosides have been obtained. Above all, knock-out mice lacking all complex gangliosides revealed important roles of complex gangliosides in vivo, i.e., in the maintenance and repair of nervous tissues, in the intact differentiation of spermatocytes via the transport of testosterone, and in the regulation of interleukin-2 receptor complex. Molecular mechanisms for these functions of complex gangliosides in vivo remain to be clarified.     

Neuronal Ganglioside Increases Dependent on the Neuron-Glia Interaction in Primary Culture

Dissociated neuronal cells from rat embryonic hemispheres were cultivated on astroglial layers. The increase in ganglioside content of the cocultures was more rapid than that of neuronal cultures seeded on polylysine surfaces for the first 24 h, and the extent of the increase was greater 7 days after inoculation, probably because of interaction between the preformed astroglial layers and the neuronal cells in vitro. The promoted expression of the a-pathway gangliosides, GM1 and GD1a, was recognized by TLC and the increase in GM1 was immunologically ascertained. The incorporation of 3H-labeled N-acetyl-D-mannosamine into GD3 and b-series gangliosides was elevated for the first 24 h. However, cocultures in which there was no contact between neuronal cells and the astroglial sheet showed no appreciable increase in incorporation. Thus, cell surface changes were induced at the membrane glycolipid level in the neuronal cells by contact with astroglial layers. The synthesis and expression of neuronal gangliosides are discussed in relation to the onset of neuron--glia interaction.     

The R116C mutation in alpha A-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis.

alphaA-crystallin is a small heat-shock protein expressed preferentially in the lens and is detected during the early stages of lens development. Recent work indicates that the expression of alphaA-crystallin enhances lens epithelial cell growth and resistance to stress conditions. Mutation of the arginine 116 residue to cysteine (R116C) in alphaA-crystallin has been associated with congenital cataracts in humans. However, the physiological consequences of this mutation have not been analyzed in lens epithelial cells. In the present study, we expressed wild type or R116C alphaA-crystallin in the human lens epithelial cell line HLE B-3. Immunofluorescence and confocal microscopy indicated that both wild type and R116C alphaA-crystallin were distributed mainly in the cytoplasm of lens epithelial cells. Size-exclusion chromatography indicated that the size of the alphaA-crystallin aggregate in lens epithelial cells increased from 500 to 600 kDa for the wild type protein to >2 MDa in the R116C mutant. When cells were exposed to physiological levels of UVA radiation, wild type alphaA-crystallin protected cells from apoptotic death as shown by annexin labeling and flow cytometric analysis, whereas the R116C mutant had a 4- to 10-fold lower protective ability. UVA-irradiated cells expressing the wild type protein had very low TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining, whereas cells expressing R116C mutant had a high level of TUNEL staining. F-actin was protected in UVA-treated cells expressing the wild type alphaA-crystallin but was either clumped around the apoptotic cells or was absent in apoptotic cells in cultures expressing the R116C mutant. Structural changes caused by the R116C mutation could be responsible for the reduced ability of the mutant to protect cells from stress. Our study shows that comparing the stress-induced apoptotic cell death is an effective way to compare the protective abilities of wild type and mutant alphaA-crystallin. We propose that the diminished protective ability of the R116C mutant in lens epithelial cells may contribute to the pathogenesis of cataract.     

Pharmacological correction of the apoptosis level in cortical neurons of ageing HER2/neu transgenic mice

Neurodegenerative changes and neuronal death underlie ageing of the nervous system. We investigated the mechanisms of apoptosis in sensorimotor cortical neurons of HER2/neu transgenic mice during ageing, as well as the functional changes in the cortex and the involvement of exogenous neurometabolites (cytoflavin, piracetam) in the regulation of neuronal death and locomotor and psycho-emotional status in mice. The level of apoptosis and expression of the apoptotic protein markers (TUNEL, immunohistochemistry, Western blotting) were detected in HER2/neu transgenic mice versus wild type mice (FBV strain). In ageing wild type mice, the basal activity decreases while the anxiety level increases correlating with the high level of neuronal apoptosis. We revealed specific behavioral features of HER2/neu transgenic mice—their low basal activity which remains intact during ageing. Previously, we have shown that in this mouse strain the level of apoptosis is low, with no age-related dynamics, due to the suppression primarily of the p53-dependent pathway by HER2 (tyrosine kinase receptor) overexpression. Here we show that cytoflavin and piracetam have a pronounced neuroprotective effect, preserving and restoring the nervous system functions (improving locomotion and psychological status) in both mouse strains. The effect of the tested neurometabolites on neuronal apoptosis is ambiguous. In case of low-level apoptosis, there occurs its moderate stimulation in HER2/neu transgenic mice that are characterized by a high level of carcinogenesis (via the extrinsic and p53-dependent pathways with caspase-3 activation) which probably prevents tumor development. By contrast, in aged wild-type mice there is a marked decrease in the level of age-related apoptosis (via the stimulation of antiapoptotic protein Mcl-1 expression) supposed to prevent neurodegeneration.