CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

神经系统疾病的化学病理学

• 批准号: 6040889
• 负责人: KUNIHIKO SUZUKI
• 金额: $ 31.02万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6040889
• 关键词: apoptosis; beta galactosidase; congenital nervous system disorder; cytotoxicity; disease /disorder model; embryonic stem cell; fibroblasts; gangliosidosis GM1; gene targeting; genetically modified animals; genotype; glycosylation; inborn lysosomal enzyme disorder; laboratory mouse; model design /development; molecular pathology; mutant; neurochemistry; point mutation; protein structure function; sphingolipids; tissue /cell culture

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.

两个主要的项目被提议继续两年多我毕生对神经遗传疾病的研究兴趣。他的第一个项目涉及溶酶体β -半乳糖苷酶两种小鼠突变体的杂交实验，扭曲小鼠（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 -神经节脂病模型）。这个已经在进行的实验产生了一个完全出乎意料和矛盾的结果，除了预期的大量乳糖神经酰胺的积累。这些观察结果与关于常染色体隐性遗传病的传统观点相反，因此必须对其进行随访，以便最终阐明潜在的遗传和代谢机制。这些矛盾的发现的本质是，具有额外的酸性-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-半乳糖苷酶基因正常补体的抽搐小鼠，而具有单一功能辅助-半乳糖苷酶基因的抽搐小鼠具有最严重的疾病，并伴有任何其他基因型中未见的额外神经元病变。还应注意的是，我们只比较同一实验中双载体交配的后代，以排除小鼠遗传背景的变化。提出对半乳糖神经酰胺、神经节苷脂及其相关化合物，包括精神碱（半乳糖-鞘氨醇）进行详细的分析研究，为今后的研究奠定坚实的基础。双缺陷小鼠中GM1-神经节苷脂的积累以某种方式抵消psychosine的凋亡效应的可能性将在培养的胚胎小鼠成纤维细胞中进行测试。第二个主要项目是产生鞘脂激活蛋白A和D （sap, saposin A， D）分别发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整个身体中的重要性进行最终测试。我们已经利用Cre- loxP系统生成了必要的靶向载体，在ES细胞液前体基因的液A和D结构域引入点突变。引入的点突变是：(1)去除糖基化位点，(2)去除六个半胱氨酸残基中的一个。这些点突变在人类sap B和C中已知，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加工。