Abstract
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.
Highlights
In a previous study, we cloned a full-length cDNA of rat ABCA2, and showed that ABCA2 is expressed at high levels in brain white matter regions and is localized mainly to late endosome/lysosomes in oligodendrocytes [14, 15]
We demonstrated that ABCA2 is not localized to astrocytes, microglias, or adult oligodendroglial progenitors, suggesting a role of ABCA2 in lipid transport linked to myelination processes in maturated myelinating oligodendrocytes [16]
We investigated the contents of glycosphingolipids an increase of cerebrosides and sulfatides only at 64 weeks such as galactosylceramides, sulfatides, and gangliosides, suggesting that ABCA2 is involved in the which are abundant in brain compared with other tissues. metabolism of SM and glycosphingolipids primarily in the gan
Summary
Generation of ABCA2-null Mice—The abca gene fragment was isolated from a 129Sv mouse -bacteriophage genomic library (Stratagene). Chromatographic Separations—For separation of each ganglioside, aliquots of the purified gangliosides were developed on silica gel 60 high-performance thin-layer chromatography plates (Merck, 10 ϫ 20 cm) with appropriate standards, using chloroform:methanol:0.2% CaCl2 in water (55:45:10, v/v). For the separation of cerebrosides, each phospholipid, and sulfatides, aliquots of total lipids were developed on silica gel 60 thinlayer chromatography plates (Merck, 20 ϫ 20 cm) using twodimensional solvent systems; the first and the second chromatographic runs were performed with chloroform:methanol: M ammonia (65:30:4, v/v) and with chloroform:methanol:acetic acid:water (170:25:25:4, v/v) at 90° to the original direction [26]. For measurement of the amount of sphingoid base in cerebrosides and sulfatides, the lipids were extracted from silica gel and hydrolyzed by aqueous methanolic HCl reagent (concentrated hydrochloric acid:water:methanol (8.6:9.4:82) v/v) for 18 h at 70 °C and quantified colorimetrically by the methyl orange method using N-palmitoyl-D-sphingosine for calibration [28]. Several correctly targeted ES cell clones were isolated and injected into blastocysts, which developed germ line transmission
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