The goal of this research is to clarify the nature of ganglioside abnormalities in mutant mouse embryos. Gangliosides are a family of sialic acid-containing glycosphingolipids that are enriched in the central nervous system (CNS) and are thought to play an important role in neuronal differentiation. The tW1 mouse mutation is located within the T/t complex on chromosome 17 and causes embryonic lethality from failed neuronal differentiation within the neural tube. Recent findings indicate the tW1/tW1 embryos express reductions of gangliosides in the """"""""b"""""""" metabolic pathway (GD3, GD1b, GT1b, and GQ1b) and elevations of ganglioside in the """"""""a"""""""" metabolic pathway (GM3, GM2, GM1, and GD1a). This shift in ganglioside distribution strongly suggests that the mutants have a partial deficiency in the sialyltransferase activity that catalyzes the synthesis of GD3 from GM3: a key enzyme in the synthesis of the complex gangliosides. Moreover, gangliosides of the """"""""b"""""""" pathway have been implicated in neuronal differentiation and neuritogenesis. This hypothesis will be tested through a developmental analysis of ganglioside composition and metabolism in normal and tW1/tW1 mutants. The gangliosides will be studied from embryonic day 13 (E-13) to E-17 in whole embryos and in embryonic CNS tissues. The composition of neutral glycolipids will also be examined. Total ganglioside content will be analyzed using gas-liquid chromatography and the individual ganglioside species will be analyzed using high performance thin-layer chromatography and densitometry. A specific objective will be to determine if the ganglioside abnormalities are a primary or secondary effect of the tW1 mutation. This will be approached through in vivo and in vitro studies of ganglioside biosynthesis in the +/+ and tW1/tW1 embryos and in the phenotypically normal +/tW1 heterozygotes. The sialyltransferase activities that catalyze the synthesis of GM3, GD3, GD1a, and GQ1b will be analyzed in enzyme enriched membrane preparations from the +/+, +/tW1, and tW1/tW1 embryos. The N- acetylgalactosaminyltransferase activity that catalyzes the synthesis of GM2 from GM3 (a key enzyme in the synthesis of """"""""a"""""""" pathway gangliosides) will also be analyzed in the normal and mutant embryos. The activity of GM1 beta-galactosidase will also be studied using GM1 as a natural substrate in embryos of inbred DBA/2 and C57BL/6 mice and their F1 hybrids. Because DBA mice have elevated levels of GM1 at both embryonic and juvenile ages, they may express a mild form of GM1 gangliosidosis and serve as an important animal model for this disorder. Since this is the first study of ganglioside abnormalities associated with inherited embryonic lethality and failed CNS development in mammals, new insight can be obtained on ganglioside function and on the genetic regulation of ganglioside metabolism.
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