CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

两个主要项目被提出，让我对神经遗传性疾病的毕生研究兴趣再延续两年。他的第一个项目涉及两个溶酶体β-半乳糖苷酶突变小鼠之间的杂交实验，即TWISTER小鼠(半乳糖基神经酰胺酶缺乏症，人类Krabbe病模型)和我们最近培育的酸性β-半乳糖苷酶基因敲除小鼠(GM-神经节苷脂缺乏模型)。这项已经在进行的实验除了预期乳糖基神经酰胺的大量积累外，还产生了完全意想不到和自相矛盾的结果。这些观察结果与关于常染色体隐性遗传病的传统看法截然相反，因此必须对其进行追踪，以便最终弄清潜在的遗传和代谢机制。这些矛盾发现的实质是，到目前为止，缺乏额外酸性β-半乳糖苷酶的Twitcher小鼠具有最轻微的表型，其次是具有两个酸性β-半乳糖苷酶基因正常补充的Twitcher小鼠，具有单一功能辅助β-半乳糖苷酶基因的Twitcher小鼠具有最严重的疾病，其额外的神经元损伤在任何其他基因类型中都没有见过。还应该注意的是，我们只比较了同一实验中双携带者交配的后代，以排除小鼠遗传背景的变异。建议对半乳糖神经酰胺、神经节苷脂和相关化合物进行详细的分析研究，包括心磷脂(半乳糖-鞘氨醇)，为今后的研究奠定坚实的基础。GM1神经节苷脂在双缺陷小鼠体内的积累以某种方式抵消了精神病碱的凋亡效应，这一可能性将在培养的小鼠胚胎成纤维细胞中进行测试。第二个主要项目是分别在鞘脂激活蛋白A和D(SAP、皂苷A、D)上产生点突变的小鼠突变体。尽管在体外和细胞培养中进行了许多研究，但我们仍然需要对这些激活蛋白在全身的重要性进行最终测试。我们已经利用Cre-loxP系统构建了在ES细胞sap前体基因的sap A和D结构域引入点突变的必要的靶向载体。将要引入的点突变是(1)取消糖基化位点，(2)取消六个半胱氨酸残基中的一个。这些点突变已知在人类的汁液B和C中，由于各自激活蛋白的缺乏而导致临床疾病，而不影响其他结构域的处理。

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.