GANGLIOSIDE STUDIES

本研究的目的是阐明神经节苷脂的性质 突变小鼠胚胎的异常。 神经节苷脂是唾液酸的一个家族， 富含中枢神经系统的含酸鞘糖脂 中枢神经系统（CNS），并被认为在神经元 分化 tW 1小鼠突变位于T/t复合物内 在17号染色体上， 神经管内的分化。 最近的研究结果表明， tW 1/tW 1胚胎在“B”代谢中表达神经节苷脂减少， 途径（GD 3，GD 1b，GT 1b和GQ 1b）和神经节苷脂升高 “a”代谢途径（GM 3、GM 2、GM 1和GD 1a）。 神经节苷脂的这种变化 分布强有力地表明，突变体具有部分缺陷， 唾液酸转移酶催化GD 3的合成 GM 3：复合神经节苷脂合成的关键酶。 此外，委员会认为， “B”途径的神经节苷脂与神经元的 分化和轴突发生。 这一假设将通过 神经节苷脂组成和代谢的发育分析， 正常和tW 1/tW 1突变体。 神经节苷脂将从 胚胎第13天（E-13）至E-17（整个胚胎和胚胎CNS） 组织中 还将检查中性糖脂的组成。 将使用气液色谱法分析总神经节苷脂含量 并且将使用高浓度的神经节苷脂分析单个神经节苷脂种类。 高效薄层色谱法和光密度测定法。 特定 目的是确定神经节苷脂异常是否是 tW 1突变的主要或次要影响。 这将被处理 通过在+/+中神经节苷脂生物合成的体内和体外研究， 和tW 1/tW 1胚胎以及表型正常+/tW 1杂合子。 催化GM 3、GD 3、 将在酶富集膜制备物中分析GD 1a和GQ 1b +/+、+/tW 1和tW 1/tW 1胚胎。 N- 乙酰氨基半乳糖转移酶活性，催化 从GM 3（“a”途径神经节苷脂合成中的关键酶）得到GM 2 也将在正常和突变胚胎中进行分析。 的活性 GM 1 β-半乳糖苷酶也将使用GM 1作为天然酶进行研究。 在近交系DBA/2和C57 BL/6小鼠及其F1杂种的胚胎中的底物。 因为DBA小鼠在胚胎期和幼年期的GM 1水平都很高， 年龄，他们可能表达一种轻度形式的GM 1神经节苷脂沉积症，并作为一种免疫反应。 这种疾病的重要动物模型。 因为这是第一次研究 神经节苷脂异常与遗传性胚胎致死相关， 哺乳动物中枢神经系统发育失败，可以获得新的见解 神经节苷脂的功能及其基因调控 新陈代谢.

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.