Susumu Ando

High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species

Abstract Improved resolution of complex brain ganglioside mixtures was achieved by high-performance thin-layer chromatography. The percentage distribution of individual gangliosides was then determined by direct densitometric seanning, employing a transmittance mode, of the resorcinol-positive spots on the plate. As little as 90 pmol (29 ng) of lipid-bound sialic acid could be detected with a good signal-to-noise ratio. A linear detector response was observed up to 3.0 μg of lipid-bound sialic acid. The brain white matter ganglioside patterns of eight animal species, including human, chimpanzee, monkey, chicken, bovine, sheep, and pig, were examined in detail. In addition, human brain gray matter, rat cerebral, rat brain gray matter, and rat cerebellar ganglioside patterns were also studied. Ganglioside G M4 (G 7 ) was found to be one of the major components in primate and chicken brain white matter, but it represented only a minor ganglioside in other species. Other major gangliosides in all brain samples studied were G M1 , G D1a , G D1b , and G T1b . G M1 was more abundant in white matter than in gray matter. G T1a , a recently discovered ganglioside species, was found in all species examined, but was most abundant in the rat cerebellum. The latter source also contained high proportions of G T1b and G Q1b .

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid‐containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c‐series gangliosides such as GT3 and GQ1c), Chol‐1α (GT1aα and GQ1bα), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage‐specific embryonic antigen‐1 (Lewis x) glycolipids, and human natural killer‐1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12–E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a‐series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage‐specific embryonic antigen‐1 were expressed in embryonic brains. Human natural killer‐1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol‐1α, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up‐regulation of complex a‐ and b‐series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.

Structures of some new complex gangliosides of fish brain.

Three novel trisialogangliosides of fish brain, GT3, GT2 and GT1c, have been isolated in their intact forms and their structures characterized. The discovery of these ganglioside species provides essential links for a new possible biosynthetic pathway leading to the major tetrasialoganglioside, GQ1c, of the fish brain.

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain.

The accumulation of Aβ (amyloid β-protein) is one of the major pathological hallmarks in AD (Alzheimers disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aβ. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aβ was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aβ.

Brain gangliosides of a transgenic mouse model of Alzheimer's disease with deficiency in GD3-synthase: expression of elevated levels of a cholinergic-specific ganglioside, GT1aα

In order to examine the potential involvement of gangliosides in AD (Alzheimers disease), we compared the ganglioside compositions of the brains of a double-transgenic (Tg) mouse model [APP (amyloid precursor protein)/PSEN1 (presenilin)] of AD and a triple mutant mouse model with an additional deletion of the GD3S (GD3-synthase) gene (APP/PSEN1/GD3S−/−). These animals were chosen since it was previously reported that APP/PSEN1/GD3S−/− triple-mutant mice performed as well as WT (wild-type) control and GD3S−/− mice on a number of reference memory tasks. Cholinergic neuron-specific gangliosides, such as GT1aα and GQ1bα, were elevated in the brains of double-Tg mice (APP/PSEN1), as compared with those of WT mice. Remarkably, in the triple mutant mouse brains (APP/PSEN1/GD3S−/−), the concentration of GT1aα was elevated and as expected there was no expression of GQ1bα. On the other hand, the level of c-series gangliosides, including GT3, was significantly reduced in the double-Tg mouse brain as compared with the WT. Thus, the disruption of the gene of a specific ganglioside-synthase, GD3S, altered the expression of cholinergic neuron-specific gangliosides. Our data thus suggest the intriguing possibility that the elevated cholinergic-specific ganglioside, GT1aα, in the triple mutant mouse brains (APP/PSEN1/GD3S−/−) may contribute to the memory retention in these mice.

Conversion of deoxycorticosterone to 3-keto-4-etienic acid, catalyzed by a partially purified preparation from bovine adrenocortical mitochondria

Bovine adrenocortical mitochondria were sonicated and subjected to extraction with sodium cholate. The extract contained not only cytochrome P-450 activities, but also an activity which catalyzed the conversion of deoxycorticosterone to an unknown steroid (designated X). The latter activity was concentrated by (NH4)2SO4 fractionation in the presence of sodium cholate, and separated from P-450 by taking advantage of their different solubilities in phosphate buffer without sodium cholate. The specific activity of the partially purified enzyme fraction was 70 times higher than that of sonicated mitochondria. The conversion of deoxycorticosterone to steroid X required NAD or NADP. The conversion rate was dependent on the concentration of deoxycorticosterone. The major product, steroid X, was isolated from the reaction mixture by means of silicic acid and Iatrobeads column chromatography. The steroid was characterized as 3-keto-4-etienic acid (3-oxoandrost-4-ene-17beta-carboxylic acid). This result suggests that an enzyme system for the conversion of deoxycorticosterone to 3-keto-4-etienic acid exists in adrenocortical mitochondria.

A Novel Disialoganglioside (GD1α) with N-Acetylneuraminyl (α2 → 6)-N-Acetylgalactosamine Linkage in Rat Ascites Hepatoma Cells

Gangliosides in plasma membranes have been assumed to be involved in cellular recognition sites such as receptors of bacterial toxins (van Heyningen et al., 1971; Cuatrecasas, 1973), viruses (Haywood, 1974; Holmgren et al., 1980), hormones (Mullin et al., 1976) and chemical mediators (Woolley and Gommi, 1965). On the other hand, some special gangliosides have been proposed to be regulators of cell growth or differentiation (Bremer et al., 1984; Tsuji et al., 1983). Furthermore, alteration of gangliosides associated with oncogenic transformation are well known phenomena (Hakomori, 1984). Since sialic acid of gangliosides plays a key role in these cellular reactions, number of sialic acids, manner of linkage and location of sialyl residues in the back bone structure of gangliosides are thought to be critical for the function of the cells. Sialic acids in ganglioside are attached to the internal or terminal galactose of ganglio-N-tetraose. In the biosynthesis of ganglio series gangliosides, sialic acid is transferred to the galactose moiety of lactosyl-ceramide, then N-acetylgalactosamine and galactose are transferred in stepwise fashion to form GMla. However, in rat ascites hepatoma cells, a biosynthetic pathway of ganglioside via asialogangliosides has been demonstrated by our structural and metabolic studies (Hirabayashi et al., 1978; Taki et al., 1979a). In the tumor cells, N-acetylgalactosamine is preferentially transferred to lactosylceramide and followed by a transfer of galactose to form gangliotetraosylceramide. Sialic acid is then transferred to the terminal galactose resulting in the formation of GMlb. From the series of study on glycolipids in the tumor cells, two major gangliosides, mono- and disialo-, were isolated.