Analysis of in vivo function of glycosphingolipids with glycosylation mutant mice

在这个项目中，我们分析了糖在神经节苷脂，含唾液酸的酸性鞘糖脂中的作用，以及它们的机制，使用基因敲除小鼠的糖基转移酶负责合成它们。分析的突变小鼠是缺乏复合神经节苷脂的GM 2/GD 2合酶敲除小鼠、缺乏b系列神经节苷脂的GD 3合酶敲除小鼠、通过使上述两种突变体交配产生的双敲除小鼠和负责乳糖神经酰胺（大多数神经节苷脂的前体）合成的β4-半乳糖基转移酶6敲除小鼠。在缺乏复合神经节苷脂的无效突变小鼠中，还观察到周围神经和脊髓随增龄而变性和破坏，以及小脑的明显萎缩和变性。另一方面，在缺乏b系列神经节苷脂的GD 3合酶的无效突变小鼠中，未检测到明显的异常表型。然而，只有雄性突变体在行为检查中表现出异常的神经功能。在由上述两种突变体产生的双敲除突变体中，甚至在幼龄小鼠中也发现了明确的神经元变性;并且在出生后12周时在面部和颈部出现了难治性皮肤病变。基于周围神经变性，疼痛感觉功能降低可能会导致对伤口部位的频繁重复抓挠，并可能引发皮肤病变。虽然我们已经尝试使用DNA阵列或cDNA减法鉴定在无效突变小鼠和野生型小鼠之间显示出表达水平的大差异的基因，但是还没有鉴定出绝对重要的基因。通过从组织的特定位点提取RNA或用最低水平的RNA进行表达分析，我们将在不久的将来鉴定参与退化和再生的基因，并阐明这些基因产物的原始分子功能。

In this project, we have analyzed the roles of carbohydrates in gangliosides, sialic acid-containing acidic glycosphingolipids, and their mechanisms using gene knock-out mice of glycosyltransferases responsible for the synthesis of them. The mutant mice analyzed are GM2/GD2 synthase knock-out mice lacking complex gangliosides, GD3 synthase knock-out mice lacking b-series gangliosides, double knock-out mice generated by mating the two mutants described above, and β4-galactosyltransferase 6 knock-out mice responsible for the synthesis of lactosylceramide, a precursor of the majority of gangliosides. In the null mutant mice lacking complex gangliosides, degeneration and destruction peripheral nerves and spinal cords with aging, and marked atrophy and degeneration of cerebellum were also observed. On the other hand, in the null mutant mice of GD3 synthase lacking b-series gangliosides, no apparent abnormal phenotypes were detected. However, only male mutants showed abnormal neurological function in the behavioral examination. In the double knock-out mutants generated from the two mutants described above, definite neuronal degeneration was found even in the young mice ; and refractory skin lesions appeared at faces and necks at 12 weeks after birth. Based on the peripheral nerve degeneration, lowered function of pain sensation might induce frequent repeated scratching toward the wound site, and might trigger the skin lesions. Although we have tried to identify genes which show big differences in the expression levels between the null mutant and wild type mice using a DNA array or cDNA subtraction, no definitely important genes have not yet identified. With RNA extraction from specific sites of tissues or expression analysis with minimal levels of RNA, we will identify the genes involved in the degeneration and regeneration, and clarify the original molecular function of those gene products in the near future.