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• W2041126270 abstract "II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid β-galactosidase (β-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in β-gal knockout (β-gal−/−) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and β-gal−/− mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice.Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders. II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid β-galactosidase (β-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in β-gal knockout (β-gal−/−) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and β-gal−/− mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders. ErrataJournal of Lipid ResearchVol. 46Issue 6PreviewIn the article “Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice” by Kasperzyk et al., published in the April 2004 issue of the Journal of Lipid Research (Volume 46, pages 744–751), the affiliations should read as follows: Full-Text PDF Open Access GM1 gangliosidosis is an incurable glycosphingolipid (GSL) lysosomal storage disease caused by a genetic deficiency in lysosomal acid β-galactosidase (β-gal) and leads to the storage of II3NeuAc-GgOse4Cer (GM1) and its asialo derivative (GA1) in the central nervous system (CNS) (1Suzuki Y. Sakuraba H. Oshima A. β-Galactosidase deficiency (β-galactosidosis): GM1 gangliosidosis and Morquio B disease.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, Inc., New York1995: 2785-2823Google Scholar). Age of onset ranges from infancy to adulthood, and excessive CNS ganglioside accumulation produces progressive neurodegeneration and brain dysfunction in humans. A β-gal-deficient mouse model of GM1 gangliosidosis was generated using homologous recombination and embryonic stem cell technology (2Hahn C.N. del Pilar Martin M. Schroder M. Vanier M.T. Hara Y. Suzuki K. Suzuki K. d'Azzo A.A. Generalized CNS disease and massive GM1-ganglioside accumulation in mice defective in lysosomal acid beta-galactosidase.Hum. Mol. Genet. 1997; 6: 205-211Crossref PubMed Scopus (134) Google Scholar). We recently showed that these mice accumulate GM1 and GA1 in the brain as early as postnatal day 5 (p-5), thus mimicking the neurochemical features of the infantile disease form (3Kasperzyk J.L. El-Abbadi M.M. Hauser E.C. D'Azzo A. Platt F.M. Seyfried T.N.N. N-Butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis.J. Neurochem. 2004; 89: 645-653Crossref PubMed Scopus (70) Google Scholar). In contrast to infantile-onset patients, in whom ganglioside accumulation leads to behavioral and developmental abnormalities within the first few years of life, β-gal-deficient mice are phenotypically indistinguishable from normal mice until adult ages (2Hahn C.N. del Pilar Martin M. Schroder M. Vanier M.T. Hara Y. Suzuki K. Suzuki K. d'Azzo A.A. Generalized CNS disease and massive GM1-ganglioside accumulation in mice defective in lysosomal acid beta-galactosidase.Hum. Mol. Genet. 1997; 6: 205-211Crossref PubMed Scopus (134) Google Scholar). Most therapies for ganglioside storage diseases focus on augmenting lysosomal enzyme levels and involve bone marrow transplantation, enzyme replacement, stem cell therapy, gene therapy, and chemical chaperone therapy (4Dobrenis K. Cell-mediated delivery systems.in: Platt F.M. Walkley S.U. Lysosomal Disorders of the Brain. Oxford University Press, New York2004: 339-380Crossref Google Scholar, 5Lacorazza H.D. Flax J.D. Snyder E.Y. Jendoubi M. Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells.Nat. Med. 1996; 2: 424-429Crossref PubMed Scopus (196) Google Scholar, 6Norflus F. Tifft C.J. McDonald M.P. Goldstein G. Crawley J.N. Hoffmann A. Sandhoff K. Suzuki K. Proia R.L.L. Bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in Sandhoff disease mice.J. Clin. Invest. 1998; 101: 1881-1888Crossref PubMed Scopus (133) Google Scholar, 7Matsuda J. Suzuki O. Oshima A. Yamamoto Y. Noguchi A. Takimoto K. Itoh M. Matsuzaki Y. Yasuda Y. Ogawa S. Sakata Y. Nanba E. Higaki K. Ogawa Y. Tominaga L. Ohno K. Iwasaki H. Watanabe H. Brady R.O. Suzuki Y.Y. Chemical chaperone therapy for brain pathology in GM1-gangliosidosis.Proc. Natl. Acad. Sci. USA. 2003; 100: 15912-15917Crossref PubMed Scopus (251) Google Scholar, 8Schiffmann R. Brady R.O. New prospects for the treatment of lysosomal storage diseases.Drugs. 2002; 62: 733-742Crossref PubMed Scopus (91) Google Scholar, 9Takaura N. Yagi T. Maeda M. Nanba E. Oshima A. Suzuki Y. Yamano T. Tanaka A.A. Attenuation of ganglioside GM1 accumulation in the brain of GM1 gangliosidosis mice by neonatal intravenous gene transfer.Gene Ther. 2003; 10: 1487-1493Crossref PubMed Scopus (32) Google Scholar, 10Tropak M.B. Reid S.P. Guiral M. Withers S.G. Mahuran D. Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff patients.J. Biol. Chem. 2004; 279: 13478-13487Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 11Brady R.O. Enzyme replacement therapy: conception, chaos and culmination.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 915-919Crossref PubMed Scopus (63) Google Scholar). These therapeutic approaches, however, have difficulty reducing GSL storage products throughout the CNS. Alternatively, substrate reduction therapy, originally proposed by Radin (12Radin N.S. Inhibitors and stimulators of glucocerebroside metabolism.Prog. Clin. Biol. Res. 1982; 95: 357-383PubMed Google Scholar) and others (13Platt F.M. Butters T.D. Inhibition of substrate synthesis: a pharmacological approach for glycosphingolipid storage disease therapy.in: Platt F.M. Walkley S.U. Lysosomal Disorders of the Brain. Oxford University Press, New York2004: 381-408Crossref Google Scholar, 14Tifft C.J. Proia R.L. Stemming the tide: glycosphingolipid synthesis inhibitors as therapy for storage diseases.Glycobiology. 2000; 10: 1249-1258Crossref PubMed Scopus (43) Google Scholar, 15Butters T.D. Mellor H.R. Narita K. Dwek R.A. Platt F.M.M. Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 927-945Crossref PubMed Scopus (61) Google Scholar, 16Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells.J. Biol. Chem. 1999; 274: 1140-1146Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 17Andersson U. Smith D. Jeyakumar M. Butters T.D. Borja M.C. Dwek R.A. Platt F.M.M. Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease.Neurobiol. Dis. 2004; 16: 506-515Crossref PubMed Scopus (74) Google Scholar), aims to reduce GSL synthesis to counterbalance the impaired rate of catabolism, thus reducing GSL accumulation and disease progression. Platt and colleagues (15Butters T.D. Mellor H.R. Narita K. Dwek R.A. Platt F.M.M. Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 927-945Crossref PubMed Scopus (61) Google Scholar, 18Vunnam R.R. Radin N.S. Analogs of ceramide that inhibit glucocerebroside synthetase in mouse brain.Chem. Phys. Lipids. 1980; 26: 265-278Crossref PubMed Scopus (119) Google Scholar, 19Butters T.D. Dwek R.A. Platt F.M. Therapeutic applications of imino sugars in lysosomal storage disorders.Curr. Top. Med. Chem. 2003; 3: 561-574Crossref PubMed Scopus (162) Google Scholar, 20Platt F.M. Jeyakumar M. Andersson U. Heare T. Dwek R.A. Butters T.D.D. Substrate reduction therapy in mouse models of the glycosphingolipidoses.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 947-954Crossref PubMed Scopus (50) Google Scholar, 21Platt F.M. Walkley S.U. Lysosomal Disorders of the Brain. Oxford University Press, New York2004Crossref Google Scholar) used this therapy to treat adult-onset ganglioside storage disease in mice more recently. We recently provided evidence that substrate reduction therapy using N-butyldeoxygalactonojirimycin (N B-DGJ) could significantly reduce total brain ganglioside and GM1 content in neonatal normal and β-gal-deficient mutant mice (3Kasperzyk J.L. El-Abbadi M.M. Hauser E.C. D'Azzo A. Platt F.M. Seyfried T.N.N. N-Butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis.J. Neurochem. 2004; 89: 645-653Crossref PubMed Scopus (70) Google Scholar). The imino sugars N-butyldeoxynojirimycin (N B-DNJ) and N B-DGJ are competitive inhibitors of the ceramide-specific glucosyltransferase that catalyzes the first step in GLS biosynthesis (15Butters T.D. Mellor H.R. Narita K. Dwek R.A. Platt F.M.M. Small-molecule therapeutics for the treatment of glycolipid lysosomal storage disorders.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2003; 358: 927-945Crossref PubMed Scopus (61) Google Scholar). Substrate reduction therapy using N B-DNJ and N B-DGJ have successfully reduced brain ganglioside content, delayed symptom onset, and increased survival in adult mouse models of the GM2 gangliosidoses (Tay-Sachs disease and Sandhoff disease) (13Platt F.M. Butters T.D. Inhibition of substrate synthesis: a pharmacological approach for glycosphingolipid storage disease therapy.in: Platt F.M. Walkley S.U. Lysosomal Disorders of the Brain. Oxford University Press, New York2004: 381-408Crossref Google Scholar, 17Andersson U. Smith D. Jeyakumar M. Butters T.D. Borja M.C. Dwek R.A. Platt F.M.M. Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease.Neurobiol. Dis. 2004; 16: 506-515Crossref PubMed Scopus (74) Google Scholar, 22Platt F.M. Neises G.R. Reinkensmeier G. Townsend M.J. Perry V.H. Proia R.L. Winchester B. Dwek R.A. Butters T.D.D. Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin.Science. 1997; 276: 428-431Crossref PubMed Scopus (333) Google Scholar). N B-DNJ is also approved for the treatment of type 1 Gaucher disease (glucosylceramide storage) and is under trial evaluation for late-onset Tay-Sachs disease (23Cox T.M. Aerts J.M. Andria G. Beck M. Belmatoug N. Bembi B. Chertkoff R. Dahl S. Vom Elstein D. Erikson A. Giralt M. Heitner R. Hollak C. Hrebicek M. Lewis S. Mehta A. Pastores G.M. Rolfs A. Miranda M.C. Zimran A.A. The role of the iminosugar N-butyldeoxynojirimycin (miglustat) in the management of type I (non-neuronopathic) Gaucher disease: a position statement.J. Inherit. Metab. Dis. 2003; 26: 513-526Crossref PubMed Scopus (210) Google Scholar, 24Lachmann R.H. Miglustat. Oxford GlycoSciences/Actelion.Curr. Opin. Invest. Drugs. 2003; 4: 472-479PubMed Google Scholar, 25Kolodny E.H. Neudorfer O. Gianutsos J. Zaroff C. Barnett N. Zeng B. Raghavan S. Torres P. Pastores G.G. Late-onset Tay-Sachs disease: natural history and treatment with OGT 918.J. Neurochem. 2004; 90: 54Google Scholar). N B-DGJ, however, may be more suitable for treating neonates than N B-DNJ, because digestive abnormalities and weight loss do not occur with N B-DGJ treatment (3Kasperzyk J.L. El-Abbadi M.M. Hauser E.C. D'Azzo A. Platt F.M. Seyfried T.N.N. N-Butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis.J. Neurochem. 2004; 89: 645-653Crossref PubMed Scopus (70) Google Scholar, 17Andersson U. Smith D. Jeyakumar M. Butters T.D. Borja M.C. Dwek R.A. Platt F.M.M. Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease.Neurobiol. Dis. 2004; 16: 506-515Crossref PubMed Scopus (74) Google Scholar, 26Andersson U. Butters T.D. Dwek R.A. Platt F.M. N-Butyldeoxygalactonojirimycin: a more selective inhibitor of glycosphingolipid biosynthesis than N-butyldeoxynojirimycin, in vitro and in vivo.Biochem. Pharmacol. 2000; 59: 821-829Crossref PubMed Scopus (135) Google Scholar). Furthermore, we previously showed that N B-DGJ could reduce GSL and ganglioside biosynthesis by 90% in normal mouse embryos without impairing viability, growth, or morphogenesis (27Brigande J.V. Platt F.M. Seyfried T.N. Inhibition of glycosphingolipid biosynthesis does not impair growth or morphogenesis of the postimplantation mouse embryo.J. Neurochem. 1998; 70: 871-882Crossref PubMed Scopus (25) Google Scholar). Our studies in embryonic and neonatal mice suggest that N B-DGJ may be an effective early intervention therapy for the gangliosidoses. In this study, we investigated for the first time the effects of N B-DGJ in normal C57BL/6J (B6) mice and β-gal-deficient (β-gal−/−) mutant mice from p-9 to p-15. This is a period of most active cerebellar development and CNS myelinogenesis in the mouse involving the proliferation and migration of granule cells, synaptogenesis, and the rapid accumulation of cerebroside, a myelin-enriched GSL (28Muse E.D. Jurevics H. Toews A.D. Matsushima G.K. Morell P.P. Parameters related to lipid metabolism as markers of myelination in mouse brain.J. Neurochem. 2001; 76: 77-86Crossref PubMed Scopus (89) Google Scholar, 29Seyfried T.N. Miyazawa N. Yu R.K. Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant.J. Neurochem. 1983; 41: 491-505Crossref PubMed Scopus (60) Google Scholar, 30Seyfried T.N. Yu R.K. Heterosis for brain myelin content in mice.Biochem. Genet. 1980; 18: 1229-1238Crossref PubMed Scopus (31) Google Scholar, 31Matthieu J.M. Widmer S. Herschkowitz N. Biochemical changes in mouse brain composition during myelination.Brain Res. 1973; 55: 391-402Crossref PubMed Scopus (63) Google Scholar) Also, this developmental period is comparable to late-stage embryonic and early development in humans (32Verbitskaya L.B. Some aspects of ontophylogenesis of the cerebellum.in: Llinas R. Neurobiology of Cerebellar Evolution and Development. Institute for Biomedical Research AMA/ERF, Chicago1969: 859-874Google Scholar, 33Morell P. Quarles R.H. Myelin formation, structure and biochemistry.in: Siegel G.J. Agranoff B.W. Albers R.W. Fisher S.K. Uhler M.D.D. Basic Neurochemistry. Lippincott-Raven, New York1999: 69-93Google Scholar). Our results show that N B-DGJ significantly reduces cerebrum-brainstem (C-BS) and cerebellar total ganglioside and GM1 content during this critical developmental period. Moreover, no adverse effects of the drug treatment were observed in the normal or mutant mice for cerebellar growth and morphology or brain cerebroside content. The B6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). B6/129Sv mice, heterozygous for the GM1 gene (β-gal +/−), were obtained from Dr. Alessandra d'Azzo (Saint Jude Children's Research Hospital, Memphis, TN). The B6 mice were used as normal controls because this genetic background largely composed that of the knockout mice. The knockout mice were derived by homologous recombination and recombinant stem cell technology as previously described (2Hahn C.N. del Pilar Martin M. Schroder M. Vanier M.T. Hara Y. Suzuki K. Suzuki K. d'Azzo A.A. Generalized CNS disease and massive GM1-ganglioside accumulation in mice defective in lysosomal acid beta-galactosidase.Hum. Mol. Genet. 1997; 6: 205-211Crossref PubMed Scopus (134) Google Scholar). Homozygous β-gal−/− mouse pups were derived from crossing β-gal−/− adults, thus ensuring that control and drug-treated β-gal−/− pups were compared within the same litter. Likewise, control and drug-treated normal B6 pups were compared within the same litter. Genotypes were determined postmortem by measuring β-gal-specific activity in tail tissue using a modification of the Galjaard procedure (34Galjaard H. Genetic Metabolic Disease: Diagnosis and Prenatal Analysis. Elsevier Science Publishers, Amsterdam1980Google Scholar, 35Hauser E.C. Kasperzyk J.L. d'Azzo A. Seyfried T.N. Inheritance of lysosomal acid b-galactosidase activity and gangliosides in crosses of DBA/2J and knockout mice.Biochem. Genet. 2004; 42: 241-257Crossref PubMed Scopus (31) Google Scholar). All mice were propagated at the Boston College Animal Facility and were housed in plastic cages with filter tops containing Sani-Chip bedding (P. J. Murphy Forest Products Corp., Montville, NJ). The room was maintained at 22°C on a 12 h light/dark cycle. Food (Prolab RMH 3000; PMI LabDiet, Richmond, IN) and water were provided ad libitum. Cotton nesting pads were provided to nursing females for the duration of the experiment. All animal experiments were carried out with ethical committee approval in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee. N B-DGJ was purchased from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). N B-DGJ solutions were prepared in sterile saline to yield a final concentration of ∼0.2 mg/μl. Postnatal B6 and β-gal−/− mice were injected daily intraperitoneally from p-9 to p-15 with either vehicle (0.9% saline) or N B-DGJ at 600 mg/kg body weight. Injections were performed using a Hamilton syringe (26 gauge, point style 2, 0.5 inch needle length), and volumes ranged from ∼10 to 25 μl/mouse. Body weights were measured before cervical dislocation ∼4 h after the final injection on p-15. Cerebellum was separated from the C-BS, and the tissues were frozen on dry ice to obtain wet weights. Two or three cerebella were pooled for each B6 sample, but each β-gal−/− cerebellum as well as each C-BS was analyzed separately. The samples were frozen at −80°C and lyophilized to determine water content. Additional mice were killed by CO2 asphyxiation for histology. The brains of these mice were exposed after midline resection along the sutures of the skull and were immersed in ∼25 ml of Bouin's fixative (LabChem, Inc., Pittsburgh, PA) for 3 days at room temperature. The brains were then removed from the skull, placed in diluted Bouin's fixative [1:10 with deionized H2O (dH2O)], and stored at 4°C. Total lipids were isolated and purified from the lyophilized brain tissue using modifications of previously described procedures (3Kasperzyk J.L. El-Abbadi M.M. Hauser E.C. D'Azzo A. Platt F.M. Seyfried T.N.N. N-Butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis.J. Neurochem. 2004; 89: 645-653Crossref PubMed Scopus (70) Google Scholar, 35Hauser E.C. Kasperzyk J.L. d'Azzo A. Seyfried T.N. Inheritance of lysosomal acid b-galactosidase activity and gangliosides in crosses of DBA/2J and knockout mice.Biochem. Genet. 2004; 42: 241-257Crossref PubMed Scopus (31) Google Scholar, 36Seyfried T.N. Glaser G.H. Yu R.K. Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures.J. Neurochem. 1978; 31: 21-27Crossref PubMed Scopus (104) Google Scholar). Briefly, total lipids were extracted with 5 ml of CHCl3 and CH3OH (1:1, v/v) and 0.5 ml of dH2O. The solution was placed on a magnetic stirrer at room temperature for at least 8 h and then centrifuged for 10 min at 1,200 g. The supernatant was removed and the pellet was washed with 2 ml of CHCl3/CH3OH (1:1, v/v). The combined supernatants were converted to a CHCl3/CH3OH/dH2O ratio of 30:60:8 (solvent A) by adding 2.5 ml of CHCl3, 8.5 ml of CH3OH, and 1.6 ml of dH2O. Neutral and acidic lipids were separated using DEAE-Sephadex (A-25; Pharmacia Biotech, Uppsala, Sweden) column chromatography as previously described (37Macala L.J. Yu R.K. Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry.J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar). DEAE-Sephadex was prepared in bulk by washing the resin three times with solvent B (CHCl3/CH3OH/0.8 M Na acetate, 30:60:8, v/v), equilibrating in solvent B overnight, followed by washing three times with solvent A until neutral. The total lipid extract, suspended in solvent A, was then applied to a DEAE-Sephadex column (1.2 ml bed volume) that had been equilibrated previously with solvent A. The column was washed twice with 20 ml of solvent A, and the entire neutral lipid fraction, consisting of the initial eluent plus washes, was collected. This fraction contained the following lipids: cholesterol, phosphatidylcholine, phosphatidylethanolamine, plasmalogens, ceramide, sphingomyelin, cerebrosides, and GA1. Next, acidic lipids were eluted from the column with 30 ml of solvent B. This fraction contained the gangliosides and other less hydrophilic acidic lipids. The acidic lipid fraction containing gangliosides was dried by rotary evaporation and transferred to a 15 ml graduated glass conical tube using CHCl3/CH3OH (1:1, v/v) and adjusted to 7 ml using the same solvent. Water (2.6 ml) was added and the mixture was inverted, vortexed, and centrifuged for ∼10 min at 1,200 g to partition gangliosides into the upper phase (36Seyfried T.N. Glaser G.H. Yu R.K. Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures.J. Neurochem. 1978; 31: 21-27Crossref PubMed Scopus (104) Google Scholar, 38Folch J. Lees M. Sloane-Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The upper aqueous phase was removed and the lower organic phase was washed once with 4.5 ml of the Folch pure solvent upper phase (CHCl3/CH3OH/dH2O, 3:48:47, v/v). The combined upper phases, containing the gangliosides, were adjusted to 11 ml using the pure solvent upper phase. An aliquot of the ganglioside fraction (Folch upper phase) was evaporated under a stream of nitrogen and analyzed for sialic acid content using a modified resorcinol method (39Svennerholm L. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method.Biochim. Biophys. Acta. 1957; 24: 604-611Crossref PubMed Scopus (2165) Google Scholar, 40Miettinen T. Takki-Luukkainen I.T. Use of butyl acetate in the determination of sialic acid.Acta Chem. Scand. A. 1959; 13: 856-858Crossref Google Scholar, 41Suzuki K. A simple and accurate micromethod for quantitative determination of ganglioside patterns.Life Sci. 1964; 3: 1227-1233Crossref PubMed Scopus (217) Google Scholar). N-acetylneuraminic acid (Sigma, St. Louis, MO) was used as an external standard. Samples were dissolved in 1 ml of resorcinol reagent/dH2O (1:1, v/v), boiled for 15 min, and then cooled in an ice bath. Butyl acetate/1-butanol (1.5 ml; 85:15, v/v) was then added, and the samples were vortexed and centrifuged at 1,200 g. The violet supernatant was removed and analyzed at 580 nm in the Shimadzu UV-1601 ultraviolet-visible spectrophotometer (Shimadzu, Kyoto, Japan). After removing the aliquots for the resorcinol assay, the ganglioside fraction was evaporated under a stream of nitrogen and treated with mild base (1 ml of 0.5 M NaOH) in a shaking water bath at 37°C for 1.5 h. Base and salts were separated from the gangliosides using a modification of a previously described method (42Williams M.A. McCluer R.H. The use of Sep-Pak C18 cartridges during the isolation of gangliosides.J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar). The sample was applied to a C18 reverse-phase Bond Elute column (Varian, Harbor City, CA) that was equilibrated with 5 ml each of CHCl3/CH3OH (1:1, v/v), CHCl3, and 0.1 M NaCl. The column was washed with 25 ml of dH2O to remove salts. Gangliosides were eluted from the column with 2 ml of CHCl3 followed by 4 ml of CHCl3/CH3OH (1:1, v/v) and stored at 4°C. Neutral lipids were dried by rotary evaporation and resuspended in 10 ml of CHCl3/CH3OH (2:1, v/v). To further purify GA1, a 4 ml aliquot of the neutral lipid fraction was evaporated under a stream of nitrogen, base treated, and Folch partitioned as described previously and above (3Kasperzyk J.L. El-Abbadi M.M. Hauser E.C. D'Azzo A. Platt F.M. Seyfried T.N.N. N-Butyldeoxygalactonojirimycin reduces neonatal brain ganglioside content in a mouse model of GM1 gangliosidosis.J. Neurochem. 2004; 89: 645-653Crossref PubMed Scopus (70) Google Scholar). The Folch lower phase containing GA1 was evaporated under a stream of nitrogen and resuspended in 10 ml of CHCl3/CH3OH (2:1, v/v). All lipids were analyzed qualitatively by high-performance thin-layer chromatography (HPTLC) according to previously described methods (36Seyfried T.N. Glaser G.H. Yu R.K. Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures.J. Neurochem. 1978; 31: 21-27Crossref PubMed Scopus (104) Google Scholar, 37Macala L.J. Yu R.K. Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry.J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar, 43Ando S. Chang N.C. Yu R.K. High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.Anal. Biochem. 1978; 89: 437-450Crossref PubMed Scopus (362) Google Scholar). Lipids were spotted on 10 × 20 cm Silica gel 60 HPTLC plates (E. Merck, Darmstadt, Germany) using a Camag Linomat III auto-TLC spotter (Camag Scientific, Inc., Wilmington, NC). The amount of lipid per lane was equivalent to 1.5 μg of total sialic acid for gangliosides and either 0.4 or 0.3 mg of tissue dry weight for GA1 or neutral lipids, respectively. To enhance precision, an internal standard (oleoyl alcohol) was added to the neutral lipid standards and samples as previously described (37Macala L.J. Yu R.K. Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry.J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar). Purified lipid standards were either purchased from Matreya, Inc. (Pleasant Gap, PA), or Sigma or were a gift from Dr. Robert Yu (Medical College of Georgia, Augusta, GA). For gangliosides and GA1, the HPTLC plates were developed by a single ascending run with CHCl3/CH3OH/dH2O (55:45:10, v/v for gangliosides; 65:35:8, v/v for GA1) containing 0.02% CaCl2·2H2O. The plates were sprayed with either the resorcinol-HCl reagent or the orcinol-H2SO4 reagent and heated at 95°C for 30 min to visualize gangliosides or GA1, respectively (39Svennerholm L. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method.Biochim. Biophys. Acta. 1957; 24: 604-611Crossref PubMed Scopus (2165) Google Scholar, 44Bruckner J. Estimation of monosaccharides by the orcinol-sulphuric acid reaction.Biochem. J. 1955; 60: 200-205Crossref PubMed Scopus (108) Google Scholar). For neutral lipids, the plates were developed to a height of 4.5 cm with chloroform-methanol-acetic acid-formic acid-water (35:15:6:2:1, v/v), dried for ∼20 min, and developed to the top with hexanes-diisopropyl ether-acetic acid (65:35:2, v/v) as previously described (37Macala L.J. Yu R.K. Ando S. Analysis of brain lipids by high performance thin-layer chromatography and densitometry.J. Lipid Res. 1983; 24: 1243-1250Abstract Full Text PDF PubMed Google Scholar, 45Seyfried T.N. Bernard D. Mayeda F. Macala L. Yu R.K. Genetic analysis of cerebellar lipids in mice susceptible to audiogenic seizures.Exp. Neurol. 1984; 84: 590-595Crossref PubMed Scopus (12) Google Scholar). Neu" @default.
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• W2041126270 title "Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice" @default.
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