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Kinuko Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Dramatic phenotypic improvement during pregnancy in a genetic leukodystrophy: estrogen appears to be a critical factor
Human molecular genetics, 2001Co-Authors: Junko Matsuda, Kinuko Suzuki, Marie T. Vanier, Yuko Saito, Kunihiko SuzukiAbstract:Globoid cell leukodystrophy is one of the classical genetic leukodystrophies in humans. The typical infantile disease in man (Krabbe disease) is caused by deficiency of lysosomal Galactosylceramidase. We recently generated a new mouse model of a late-onset, chronic form of the disease by inactivating saposin A, the essential activator of Galactosylceramidase. The phenotypic features of saposin A(-/-) mice are qualitatively identical but milder than those of twitcher mice, which is caused by genetic Galactosylceramidase deficiency. During intercrossing of saposin A(-/-) mice, we observed that affected females that are continually pregnant showed greatly improved neurological symptoms compared to affected females that do not experience pregnancy, or affected males. The pathological hallmark of globoid cell leukodystrophy, demyelination with infiltration of globoid cells, largely disappeared. The immune-related gene expression (MCP-1, TNF-alpha) was significantly down-regulated in the brain of pregnant saposin A(-/-) mice. In addition, we found intense expression of the estrogen receptors (ER alpha and ER beta) on the globoid cells, activated astrocytes and microglia in the demyelinating area of saposin A(-/-) mice. When saposin A(-/-) mice were subcutaneously implanted with time-release 17 beta-estradiol (E2) pellets from 30 to 90 days, the pathology was vastly improved. These findings suggest that a higher level of estrogen during pregnancy is one of the important factors in the protective effect of pregnancy. While we should be cautious in extrapolating these observations in the mouse to human disease, the phenomenon is spectacularly dramatic and estrogen administration might be worth a consideration as a supplementary treatment for some chronic genetic leukodystrophies.
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Paradoxical influence of acid β-galactosidase gene dosage on phenotype of the twitcher mouse (genetic Galactosylceramidase deficiency)
Human molecular genetics, 2000Co-Authors: Jun Tohyama, Kinuko Suzuki, Junko Matsuda, Marie T. Vanier, Takanori Ezoe, Kunihiko SuzukiAbstract:We have cross-bred twitcher mice (Galactosylceramidase deficiency) and acid beta-galactosidase knockout mice (G(M1) gangliosidosis) and found that the acid beta-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid beta-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid beta-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that G(M1) gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc(-/-), bgal(+/-) mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc(-/-), bgal(+/-) mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc(-/-), bgal(+/-) mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc(-/-), bgal(+/-) mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid beta-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic Galactosylceramidase deficiency.
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Suppressed UDP-galactose: ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain.
Journal of Neuroscience Research, 1998Co-Authors: Masako Taniike, Kunihiko Suzuki, Jill Marcus, Toshinori Nishigaki, Nobuya Fujita, Brian Popko, Kinuko SuzukiAbstract:The developmental changes in expression of steady-state mRNA that encode proteins that are important for myelination (myelin basic protein, myelin-associated glycoprotein, proteolipid protein, UDP-galactose: ceramide galactosyltransferase) and glial fibrillary acidic protein were investigated in the brain of the twitcher mouse, a model of human globoid cell leukodystrophy. This disease is caused by a mutation in the gene encoding the lysosomal enzyme, Galactosylceramidase, which catalyzes the degradation of the myelin lipid galactosylceramide. Before postnatal day (PND) 20, the levels of myelin protein mRNA were similar in twitcher and normal mice. With progression of demyelination after PND 25-30, myelin protein mRNA levels gradually decreased. The period of maximum expression of the myelin protein genes in twitcher mice was, however, similar to that of normal control mice. mRNA levels for the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase which is responsible for catalyzing the final step in galactosylceramide synthesis, was exceptionally down-regulated from the early stages of the disease. The increase of glial fibrillary acidic protein (GFAP) mRNA levels preceded morphological evidence of demyelination.
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Impairment of protein kinase C activity in twitcher Schwann cells in vitro
Brain Research, 1996Co-Authors: H Yamada, Parthena M. Martin, Kinuko SuzukiAbstract:Abstract Twitcher (twi/twi) is a murine model of globoid cell leukodystrophy in humans caused by a genetic deficiency in activity of Galactosylceramidase. Our previous study demonstrated that the rate of Schwann cell proliferation in twi/twi was considerably lower than that of the control ( + / + ) in vitro. We hypothesize that the lower mitotic rate in twi/twi results from the metabolic perturbation of Schwann cells caused by an accumulation of the toxic metabolite of Galactosylceramidase, psychosine, a potent inhibitor of protein kinase C (PKC). Mouse Schwann cells are known to be stimulated to divide by growth factors in media containing fetal bovine serum. The stimulation by glial growth factor (GGF) or platelet-derived growth factor-BB (PDGF-BB) is thought to be through the PKC pathway, but not by the basic fibroblast growth factor (bFGF) or transforming growth factor-β (TGF-/3 ). Thus, we tested responses of twi/twi and +/+ Schwann cells to these growth factors. Schwann cells were isolated from the dorsal root ganglia at 30 days of age and the experiments were carried out at 21 days in vitro. In media containing PDGF-BB or bovine pituitary extract (BPE), the mitotic rate of twi/twi Schwann cells was 76% or 69% of the + / + value, respectively, while significant differences were detected between twi/twi and + / + in cultures containing TGF-β or bFGF. When phorbol 12,13-dibutyrylate, a specific activator of PKC, was added to the media containing PDGF-BB or BPE, the mitotic rate of twi/twi Schwann cells improved up to 90%v of + / + cells. Staurosporine, an inhibitor of PKC, suppressed the proliferation of both twi/twi and + / + Schwann cells. However, proliferation of twi/twi Schwann cells was suppressed by one-tenth of the concentration required for + / + Schwann cells. These results are consistent with an accumulation of psychosine, an inhibitor of PKC, and suggest that the signal transduction system through PKC is impaired in the twi/twi Schwann cells.
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Murine model of genetic demyelinating disease: The twitcher mouse
Microscopy research and technique, 1995Co-Authors: Kinuko Suzuki, Masako TaniikeAbstract:Twitcher mouse is an authentic murine model of human genetic demyelinating disease, globoid cell leukodystrophy (GLD), or Krabbe disease. Since its discovery at the Jackson Laboratory (Bar Harbor, ME) this model has been used extensively for the morphological, biochemical-enzymatic studies to clarify pathogenesis and also for therapeutic manipulation of genetic demyelinating disease in humans. As a result of these studies, now we know that (1) GLD is caused by a deficiency of lysosomal enzyme Galactosylceramidase, and a toxic metabolite, psychosine, accumulates in the tissue, including the nervous system, damaging myelin forming cells and resulting in secondary demyelination; (2) morphological features of demyelination and associated cellular reactions in demyelination in this mutant are similar to those seen in autoimmune or toxic demyelination; and (3) with enzyme supplementation provided by bone marrow transplantation, remyelination occurs to some extent in demyelinated fibers in both central and peripheral nervous systems of twitcher mouse.
Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris.
Acta crystallographica. Section F Structural biology and crystallization communications, 2006Co-Authors: Robert Schultz-heienbrok, Konrad Sandhoff, Natascha Remmel, R Klingenstein, Maksim Rossocha, Wolfram Saenger, Timm MaierAbstract:The amphiphilic saposin proteins (A, B, C and D) act at the lipid-water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and beta-Galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.
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Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris.
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Robert Schultz-heienbrok, Konrad Sandhoff, Natascha Remmel, R Klingenstein, Maksim Rossocha, Wolfram Saenger, Timm MaierAbstract:The amphiphilic saposin proteins (A, B, C and D) act at the lipid–water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and β-Galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 A. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins.
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Hydrolysis of lactosylceramide by human Galactosylceramidase and GM1‐β‐galactosidase in a detergent‐free system and its stimulation by sphingolipid activator proteins, sap‐B and sap‐C Activator proteins stimulate lactosylceramide hydrolysis
FEBS Journal, 1994Co-Authors: Alexandra Zschoche, Werner Fürst, Günter Schwarzmann, Konrad SandhoffAbstract:Two exo-β-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-β-galactosidase (EC 3.2.1.23) and Galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-β-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by Galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-β-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by Galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-β-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1-β-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by Galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.
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hydrolysis of lactosylceramide by human Galactosylceramidase and gm1 β galactosidase in a detergent free system and its stimulation by sphingolipid activator proteins sap b and sap c activator proteins stimulate lactosylceramide hydrolysis
FEBS Journal, 1994Co-Authors: Alexandra Zschoche, Werner Fürst, Günter Schwarzmann, Konrad SandhoffAbstract:Two exo-β-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-β-galactosidase (EC 3.2.1.23) and Galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-β-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by Galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-β-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by Galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-β-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1-β-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by Galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.
Alice Luddi - One of the best experts on this subject based on the ideXlab platform.
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Impaired spermatogenesis in the twitcher mouse: A morphological evaluation from the seminiferous tubules to epididymal transit
Systems biology in reproductive medicine, 2017Co-Authors: Alice Luddi, Martina Gori, Elvira Costantino-ceccarini, Giuseppe Belmonte, Laura Crifasi, Camilla Marrocco, Paola PiomboniAbstract:Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa. In this developmental process the interactions between different cell types are finely regulated, hence any disruption in these relationships leads to male infertility. The twitcher mouse, the murine model of Krabbe disease, is characterized by deficiency of Galactosylceramidase, an enzyme also involved in the metabolism of the galactosyl-alkyl-acyl-glycerol, the precursor of sulfogalactosyl-alkyl-acyl-glycerol, the most abundant glycolipid in spermatozoa. Twitcher mice are sterile due to alterations of spermatogenesis resulting in the production of spermatozoa with abnormally swollen acrosomes and bent flagella, mainly at the midpiece-principal piece junction. The current study employs light, fluorescence, and electron microscopy to examine the defective spermiogenesis leading to the morphological abnormalities of mature sperm. This study reveals that alterations in germ cell development can be initially detected at the stage VIII and IX of spermatogenesis. The disrupted spermatogenetic process leads to a reduced number of elongating spermatids and spermatozoa in these mutant animals. Electron microscopy analysis demonstrates major acrosomal and chromatin condensation defects in the mutants. In addition, in twitcher mice, the epididymal architecture is impaired, with stereocilia of caput and corpus broken, detached and completely spread out into the lumen. These findings indicate that seminolipid expression is crucial for proper development of spermatocytes and spermatids and for their normal differentiation into mature spermatozoa. ABBREVIATIONS GALC: Galactosylceramidase; GalAAG: galactosyl-alkyl-acyl-glycerol; SGalAAG: sulfogalactosylalkylacylglycerol; PND: postnatal day; PAS: periodic acid-Schiff stain; TEM: transmission electron microscopy; SEM: scanning electron microscopy; PFA: paraformaldheyde.
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Morphological and molecular characterisation of Twitcher mouse spermatogenesis: an update
Reproduction fertility and development, 2015Co-Authors: Erica Puggioni, Paola Piomboni, Laura Governini, Martina Gori, Elvira Costantino-ceccarini, Giuseppe Belmonte, Alice LuddiAbstract:Spermatogenesis is a complex developmental program in which interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis. The Twitcher mouse is a model of Krabbe disease, characterised by the deficiency of Galactosylceramidase, the enzyme that hydrolyses galactosylceramide and galactosylsphingosine. Galactosyl-alkyl-acyl-glycerol, a precursor of seminolipid, the most abundant glycolipid in spermatozoa, is also a substrate for Galactosylceramidase. Altered sphingolipid metabolism has been suggested to be the cause of the morphological abnormalities reported previously in the spermatogenesis of Twitcher. However, given the frequency of infertility associated with neurological impairment, we hypothesised that an unbalanced hormonal profile could contribute to male infertility in this mutant. In order to clarify this issue, we investigated potential variations in the expression of hormones and hormone receptors involved in the regulation of spermatogenesis. Our data show that, in the brain of Twitcher mouse, gonadotrophin-releasing hormone (GnRH), LH and FSH gene expression is decreased, whereas expression of androgen receptor (AR) and inhibin βA (INHβA) is increased. The changes in gene expression for the LH and FSH receptors and AR in the testes support the hypothesis that altered sphingolipid metabolism is not the only cause of Twitcher infertility.
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New players in the infertility of a mouse model of lysosomal storage disease: the hypothalamus-pituitary-gonadal axis.
Frontiers in endocrinology, 2014Co-Authors: Paola Piomboni, Laura Governini, Martina Gori, Erica Puggioni, Elvira Costantino-ceccarini, Alice LuddiAbstract:Mammalian spermatogenesis is a complex hormone-dependent developmental program where interactions between different cell types are finely regulated. Mouse models in which any of the sperm maturation steps are perturbed provide major insights into the molecular control of spermatogenesis.The Twitcher mouse is a model for the Krabbe disease, characterized by the deficiency of Galactosylceramidase, a lysosomal enzyme that hydrolyzes the terminal galactose from galactosylceramide, a typical component of the myelin membrane. In addition, Galactosylceramidase catalyzes the hydrolysis of the terminal galactose from galactosyl-alkyl-acyl-glycerol, precursor of seminolipids, specifically expressed on the membrane of germ cells. Previous data reported by our group demonstrated that glycolipids play an important role in sperm maturation and differentiation. Moreover, we hypothesized that the severe impairment of the Central Nervous System that affects the Twitcher mouse could interfere with the hypothalamus-pituitary-gonadal axis function, contributing to infertility. To highlight this hypothesis we have determined, at molecular level, the potential variation in expression pattern of brain hormones involved in spermatogenesis regulation.
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Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy.
Experimental cell research, 2004Co-Authors: Alice Luddi, Michelina Strazza, M. Carbone, Elena Moretti, Elvira Costantino-ceccariniAbstract:The classical recessive mouse mutant, "the twitcher," is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme Galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.
Alexandra Zschoche - One of the best experts on this subject based on the ideXlab platform.
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Hydrolysis of lactosylceramide by human Galactosylceramidase and GM1‐β‐galactosidase in a detergent‐free system and its stimulation by sphingolipid activator proteins, sap‐B and sap‐C Activator proteins stimulate lactosylceramide hydrolysis
FEBS Journal, 1994Co-Authors: Alexandra Zschoche, Werner Fürst, Günter Schwarzmann, Konrad SandhoffAbstract:Two exo-β-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-β-galactosidase (EC 3.2.1.23) and Galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-β-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by Galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-β-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by Galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-β-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1-β-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by Galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.
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hydrolysis of lactosylceramide by human Galactosylceramidase and gm1 β galactosidase in a detergent free system and its stimulation by sphingolipid activator proteins sap b and sap c activator proteins stimulate lactosylceramide hydrolysis
FEBS Journal, 1994Co-Authors: Alexandra Zschoche, Werner Fürst, Günter Schwarzmann, Konrad SandhoffAbstract:Two exo-β-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-β-galactosidase (EC 3.2.1.23) and Galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-β-galactosidase, sap-C preferentially stimulated lactosylceramide hydrolysis by Galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C2>C4>C6>C8>C10>C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-β-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by Galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-β-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. Acta 481, 561–572]. GM1-β-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by Galactosylceramidase proceeds almost exclusively on liposomal surfaces: both activators, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.
Kunihiko Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Models of Krabbe Disease
Myelin Biology and Disorders, 2004Co-Authors: Kunihiko SuzukiAbstract:Publisher Summary Globoid cell leukodystrophy, GLD is classical form of human Krabbe disease. It is caused by genetic deficiency of Galactosylceramidase activity. Galactosylceramidase cDNA is cloned and disease-causing mutations have been identified in mouse, West Highland and Cairn terriers, and the Rhesus monkey. These models have been extensively utilized for studies of the pathogenesis and treatment of GLD. This chapter emphasizes on the mouse models and refers to other models, as appropriate. It discusses the twitcher mutant, its clinical manifestations, pathology, and biochemistry. In addition to the naturally occurring twitcher mutant, a new experimental model of GLD because of genetic Galactosylceramidase deficiency and an entirely new, genetically distinct mouse model of GLD are also described. They provide varying advantages and disadvantages as animal models because of their different sizes and life spans. It is noted that GLD identified by its unique neuropathology has also been described in other mammalian species and some other strain of dogs.
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Globoid cell leukodystrophy (Krabbe's disease): update.
Journal of child neurology, 2003Co-Authors: Kunihiko SuzukiAbstract:The classic globoid cell leukodystrophy (Krabbe's disease) is caused by genetic defects in a lysosomal enzyme, Galactosylceramidase. It is one of the two classic genetic leukodystrophies, together with metachromatic leukodystrophy. The mode of inheritance is autosomal recessive. Typically, the disease occurs among infants and takes a rapidly fatal course, but rarer late-onset forms also exist. Clinical manifestations are exclusively neurologic with prominent white-matter signs. The pathology is unique, consisting of a rapid and nearly complete disappearance of myelin and myelin-forming cells--the oligodendrocytes in the central nervous system and the Schwann cells in the peripheral nervous system, reactive astroytic gliosis, and infiltration of the unique and often multinucleated macrophages ("globoid cells") that contain strongly periodic acid-Schiff (PAS)-positive materials. A normally insignificant but highly cytotoxic metabolite, galactosylsphingosine (psychosine), is also a substrate of Galactosylceramidase and is considered to play a critical role in the pathogenesis. The Galactosylceramidase gene has been cloned, and a large number of disease-causing mutations have been identified. Equivalent genetic Galactosylceramidase deficiency occurs in several mammalian species, such as mouse, dog, and monkey. Recently, deficiency of one of the sphingolipid activator proteins, saposin A, was demonstrated to cause a late-onset, slowly progressive globoid cell leukodystrophy at least in the mouse, with all of the phenotypic consequences of impaired degradation of Galactosylceramidase substrates. Human globoid cell leukodystrophy owing to saposin A deficiency might be anticipated and should be suspected in human patients with a late-onset leukodystrophy with normal Galactosylceramidase activity when other possibilities are also excluded. The only serious attempt at treating human patients is bone marrow transplantation, which can provide significant alleviation of symptoms, particularly in those patients with later-onset, more slowly progressive globoid cell leukodystrophy.
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Dramatic phenotypic improvement during pregnancy in a genetic leukodystrophy: estrogen appears to be a critical factor
Human molecular genetics, 2001Co-Authors: Junko Matsuda, Kinuko Suzuki, Marie T. Vanier, Yuko Saito, Kunihiko SuzukiAbstract:Globoid cell leukodystrophy is one of the classical genetic leukodystrophies in humans. The typical infantile disease in man (Krabbe disease) is caused by deficiency of lysosomal Galactosylceramidase. We recently generated a new mouse model of a late-onset, chronic form of the disease by inactivating saposin A, the essential activator of Galactosylceramidase. The phenotypic features of saposin A(-/-) mice are qualitatively identical but milder than those of twitcher mice, which is caused by genetic Galactosylceramidase deficiency. During intercrossing of saposin A(-/-) mice, we observed that affected females that are continually pregnant showed greatly improved neurological symptoms compared to affected females that do not experience pregnancy, or affected males. The pathological hallmark of globoid cell leukodystrophy, demyelination with infiltration of globoid cells, largely disappeared. The immune-related gene expression (MCP-1, TNF-alpha) was significantly down-regulated in the brain of pregnant saposin A(-/-) mice. In addition, we found intense expression of the estrogen receptors (ER alpha and ER beta) on the globoid cells, activated astrocytes and microglia in the demyelinating area of saposin A(-/-) mice. When saposin A(-/-) mice were subcutaneously implanted with time-release 17 beta-estradiol (E2) pellets from 30 to 90 days, the pathology was vastly improved. These findings suggest that a higher level of estrogen during pregnancy is one of the important factors in the protective effect of pregnancy. While we should be cautious in extrapolating these observations in the mouse to human disease, the phenomenon is spectacularly dramatic and estrogen administration might be worth a consideration as a supplementary treatment for some chronic genetic leukodystrophies.
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Psychosine Is as Potent an Inducer of Cell Death as C6-Ceramide in Cultured Fibroblasts and in MOCH-1 Cells
Neurochemical Research, 2001Co-Authors: Jun Tohyama, Junko Matsuda, Kunihiko SuzukiAbstract:Cytotoxic capacity of psychosine (galactosylsphingosine) was evaluated in comparison with C6-ceramide in cultured fibroblasts and the glia-derived MOCH-1 cells that have characteristics of myelinating cells (1). Psychosine caused cytotoxic cell death and DNA fragmentation at concentrations similar to C6-ceramide and MOCH-1 cells were substantially more sensitive to their cytotoxic effects than fibroblasts. In this system, pretreatment with GM1-ganglioside failed to protect the cells from the deleterious effects of these compounds. These findings are consistent with the hypothesis that psychosine is the cytotoxic metabolite that causes apoptotic death of the oligodendrocyte in globoid cell leukodystrophy (Krabbe disease). They further suggest that the protective capacity of GM1-ganglioside is unlikely to be the explanation for the paradoxical improvement of the phenotype of globoid cell leukodystrophy in the mouse simultaneously deficient in two lysosomal β-galactosidases, Galactosylceramidase and GM1-ganglioside β-galactosidase.
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Paradoxical influence of acid β-galactosidase gene dosage on phenotype of the twitcher mouse (genetic Galactosylceramidase deficiency)
Human molecular genetics, 2000Co-Authors: Jun Tohyama, Kinuko Suzuki, Junko Matsuda, Marie T. Vanier, Takanori Ezoe, Kunihiko SuzukiAbstract:We have cross-bred twitcher mice (Galactosylceramidase deficiency) and acid beta-galactosidase knockout mice (G(M1) gangliosidosis) and found that the acid beta-galactosidase gene dosage exerts an unexpected and paradoxical influence on the twitcher phenotype. Twitcher mice with an additional complete deficiency of acid beta-galactosidase have the mildest phenotype with the longest lifespan and nearly rescued CNS pathology. In contrast, twitcher mice with a single functional acid beta-galactosidase gene have the most severe disease with the shortest lifespan, despite the fact that G(M1) gangliosidosis carrier mice with an otherwise normal genetic background are phenotypically normal. A significant proportion of these galc(-/-), bgal(+/-) mice clinically develop additional extreme hyper-reactivity and generalized seizures not seen in any other genotypes. Consistent with the clinical seizures, widespread neuronal degeneration is present in the galc(-/-), bgal(+/-) mice, most prominently in the CA3 region of the hippocampus. The double knockout mice show a massive accumulation of lactosylceramide in all tissues. The brain inexplicably contains only a half-normal amount of galactosylceramide, which may account for the mild clinical and pathological phenotype. On the other hand, brain psychosine level is increased in all twitcher mice, but galc(-/-), bgal(+/-) mice show a significantly higher level than other genotypes. The reduced galactosylceramide in the brain of the double knockout mice and the significantly higher psychosine in the brain of the galc(-/-), bgal(+/-) mice cannot readily be explained from the genotypes of these mice. These observations are contrary to the expected outcome of Mendelian autosomal recessive single gene disorders and may also be interpreted as that the acid beta-galactosidase gene functions as a modifier gene for the phenotypic expression of genetic Galactosylceramidase deficiency.
