CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

两个主要项目被提议继续两年多我的终身研究兴趣在神经遗传性疾病。他的第一个项目涉及两种溶酶体β-半乳糖苷酶突变小鼠之间的杂交育种实验，扭转小鼠（半乳糖神经酰胺酶缺乏症，人类克拉伯病模型）和我们最近产生的酸性β-半乳糖苷酶敲除小鼠（GM-神经节苷脂沉积症模型）。除了预期的乳糖神经酰胺的大量积累之外，这个已经在进行中的实验还产生了完全出乎意料和矛盾的结果。这些观察结果与关于常染色体隐性遗传疾病的传统观点完全相反，因此必须对其进行随访，以最终澄清潜在的遗传和代谢机制。矛盾的发现的本质是，额外的酸性β-半乳糖苷酶缺乏症的抽搐小鼠具有迄今为止最温和的表型，其次是具有两个酸性β-半乳糖苷酶基因的正常补体的抽搐小鼠，并且具有单一功能性辅助β-半乳糖苷酶基因的抽搐小鼠具有最严重的疾病，具有在任何其他基因型中未观察到的额外的神经元病变。还应该注意的是，我们在同一实验中只比较了双携带者交配的后代，以排除小鼠遗传背景的变化。详细的分析研究相关的半乳糖神经酰胺，神经节苷脂和相关化合物，包括psychosine（半乳糖鞘氨醇）的建议，奠定了坚实的基础，为今后的研究。 将在培养的胚胎小鼠成纤维细胞中测试在双重缺陷小鼠中GM 1-神经节苷脂的积累以某种方式抵消精神病肽的凋亡作用的可能性。第二个主要项目是产生分别在鞘脂激活蛋白A和D（sap、saposin A、D）中具有点突变的小鼠突变体。尽管在体外和细胞培养中进行了大量的研究，我们仍然需要对这些激活蛋白在整个身体中的必要性进行最终的测试。我们已经产生了必要的靶向载体，其使用Cre-loxP系统在ES细胞sap前体基因的sap A和D结构域中引入点突变。要引入的点突变是（1）消除糖基化位点，和（2）消除六个半胱氨酸残基之一。已知这些点突变存在于人的sap B和C中，由于相应激活蛋白的缺乏而引起临床疾病，而不影响其它结构域的加工。

Two major projects are proposed to continue for two more years my life- long research interest in the neurogenetic disorders.. He first project concerns a cross-breeding experiment between two mouse mutants of lysosomal beta-galactosidases, the twister mouse (galactosylceramidase deficiency, model of human Krabbe disease) and acid beta-galactosidase knockout mouse we recently generated (model of GM-gangliosidosis). This already ongoing experiment has yielded a totally unexpected and paradoxical results in addition to the anticipated massive accumulation of lactosylceramide. The observations are so contrary to the conventional wisdom concerning autosomal recessive disorders that they must be followed up in order to eventually clarify the underlying genetic and metabolic mechanism. The essence of the paradoxical findings is that twitcher mice with additional acid beta-galactosidase deficiency have by far the mildest phenotype, followed by twitcher mice with the normal complement of two acid beta-galactosidase genes and that the twitcher mice with a single functional aid beta-galactosidase gene have the most severe disease with additional neuronal lesions not seen in any other genotypes. It should also be noted that we compare only offspring of double-carrier mating in the same experiment in order to exclude variations in the genetic background of the mice. Detailed analytical studies related to galactosylceramide, ganglioside and related compounds, including psychosine (galactosyl-sphingosine) are proposed to lay a solid ground for future studies. The possibility that accumulation that accumulation of GM1- ganglioside in the doubly deficient mice somehow counteracts the apoptotic effect of psychosine will be tested in cultured embryonic mouse fibroblasts. The second major project is to generate mouse mutants with point mutations in the sphingolipid activator proteins A and D (sap, saposin A, D) respectively. Despite may studies in vitro and cell cultures, we still need the ultimate test for the essentiality of these activator proteins in the whole body. We have already generated the necessary targeting vectors which introduce point mutations in the sap A and D domains of the ES cell sap precursor gene with the use of the Cre- loxP system. The point mutations to be introduced are (1) to abolish the glycosylation site, and (2) to abolish one of the six cysteine residues. These point mutations are known in sap B and C in humans causing clinical diseases due to deficiency of the respective activator proteins without affecting processing of other domains.