TRANSGENIC C ELEGANS AS AMYLOID DISEASE MODEL

描述：P.I.提议使用线虫线虫作为模型 研究阿尔茨海默病背后的病理生理学的系统。在他的 初步结果他已经表达了一段人类淀粉样前体 一种蛋白质(APP)，称为UNC-54/b1-42，存在于蠕虫的躯体肌肉系统中。 APP的细胞外沉积很可能是潜在的原因之一 阿尔茨海默氏症在人类中的作用，尽管尚不清楚它们是如何导致 广泛性神经元死亡是这种疾病的典型表现。应用程序的表达方式 片段b1-42导致肌肉系统的细胞内沉积，尽管 未见细胞外分泌物。然而，中枢神经系统中的液泡 在其他突变中，这种突变是细胞死亡的标志。这项建议 需要进行以下四组实验： 1.现有APP表达株系的表型分析 更详细：将使用TM和特定标记物来确定 出现了中枢神经系统空泡的细胞；怀疑是围绕着 神经元。其他实验解决了病理生理联系。 B-肽表达与空泡形成之间的关系。细胞内钙水平 将通过注射荧光色素Fura-2进行监测。B1-42的表达 动物将在不同的离子条件下饲养，以测试其影响 这些离子对液泡形成的影响。将应用钠通道阻滞剂 以测试钠通道是否参与液泡的形成。一个数字 已知的在正常神经元中抑制空泡形成的突变 细胞死亡将被测试其抑制空泡的能力。 表达B-肽的动物。 2.产生新的品系，其中其他b1-42变体和不同 调控元件将被用来实现在其他组织中的表达。在……里面 特别是，将使用mec-7、unc-4和unc-119基因的启动子 这应该会推动神经元亚群的表达。达到分泌的目的 在b1-42中，将进行几项实验。它将是第一个 确定了肽的太小尺寸是否导致了 将该肽与绿色荧光蛋白(GFP)融合，从而 增加了它的大小。可替换地，在 UNC-54/b1-42结构可能有故障；信号序列成功 有导致分泌的另一种多肽，转甲状腺素，将被使用。 最后，线虫的一个基因bli-4，编码一种前蛋白酶，被路由到 分泌途径将与b肽融合，以确保其分泌。 几种新的结构，其中b肽的选定残基是 在体外改变将转化为蠕虫以测试是否增加或 表型下降所致。P.I.是由脊椎动物细胞引导的 培养研究表明，相应的残基变化 以一定的方式对APP押金的毒性进行修改。 3.怀疑可改变沉积的其他人类蛋白的共表达 APP与b1-42多肽联合应用。初步结果显示， B1-42和野生型反式甲状腺素的共同表达减少了 转基因蠕虫中的淀粉样沉积。两者之间的互动 这一结果所暗示的蛋白质将通过共表达一种 在体外试验中不相互作用的转甲状腺素的突变形式 用b型多肽。在另一项实验中，编码载脂蛋白E的基因 根据临床和体外数据，它促进了 淀粉样蛋白，将与b1-42共表达，以测试相互作用是否可以 在蠕虫系统中复制。 4.线虫致病表型修饰基因的遗传筛选 提出了B1-42表达式。建议使用三个屏幕： A.表达b1-42的动物将被EMS处理；F2将被染色 用组织学染色刚果红来显示是否增加或 存款的数量已经出现了下降。感兴趣的动物会 被回收，并重新测试其后代是否有蚀变沉积。动物与 改变的沉淀物将通过酶联免疫吸附试验检测是否表达水平 B1-42没有改变，因为只有影响基因的突变 对矿床形成能力感兴趣。 B.筛选麻痹表型的抑制因子：观察到 表达B-肽的动物患瘫痪，可能是由 肌肉内淀粉样物沉积。一种获得突变的EMS诱变方法 提出了影响麻痹表型的基因。C.屏幕用于 液泡表型的抑制因子。因为b1-42中的液泡 有表情的动物很少被观察到，可能是因为 变性细胞的碎片化，ced-2突变体允许死亡 要保留的单元格将被引入UNC-54/b1-42背景。

DESCRIPTION: The P.I. proposes to use the nematode C. elegans as a model system to study the pathophysiology underlying Alzheimer's disease. In his preliminary results he has expressed a fragment of human amyloid precursor protein (APP), called unc-54/b1-42, in the somatic musculature of the worm. Extracellular deposits of APP are most likely one of the underlying causes of Alzheimer's disease in humans, although it is unclear how they cause the widespread neuronal death typical of this disease. Expression of the APP fragment b1-42 leads to intracellular deposits in the musculature, although no extracellular secretion could be noted. However, vacuoles in the CNS developed which in other mutations are sign of cell death. The proposal entails the following four sets of experiments: 1. Analysis of the phenotype of the existing APP expressing lines in greater detail: TEM and specific markers will be used to establish in which cells of the CNS vacuoles appear; suspected are glia cells surrounding the neurons. Other experiments address the pathophysiological connection between b-peptide expression and vacuole formation. Intracellular Ca levels will be monitored by injection of the fluorochrome fura-2. b1-42 expressing animals will be reared in different ionic conditions to test the influence of these ions on vacuole formation. Sodium-channel blockers will be applied to test whether sodium channels are involved in vacuole formation. A number of mutations known to suppress vacuole formation during regular neuronal cell death will be tested as to their ability to also suppress vacuoles in b-peptide expressing animals. 2. Generation of novel lines in which other b1-42 variants and different regulatory elements will be used to achieve expression in other tissues. In particular, promoters of the mec-7, unc-4 and unc-119 genes will be used which should drive expression in subsets of neurons. To achieve secretion of the b1-42, several experiments will be carried out. It will be first established whether the too small size of the peptide is responsible, by fusing the peptide with green fluorescent protein (GFP) and thereby increasing its size. Alternatively, the signal sequence used in the unc-54/b1-42 construct may be faulty; signal sequences which successfully have lead to secretion of another peptide, transthyretin, will be used. Finally, a C. elegans gene, bli-4, encoding a pro-protease routed to the secretory pathway will be fused with b-peptide to ensure its secretion. Several new constructs in which selected residues of the b peptide are changed in vitro will be transformed into worms to test for increase or decrease of the phenotype caused. The P.I. is guided by vertebrate cell culture studies in which the corresponding residue changes were shown to modify the toxicity of the APP deposits in a certain way. 3. Coexpression of other human proteins suspected to modify the deposition of APP together with b1-42 peptide. Preliminary results had shown that co-expression of b1-42 and wild-type transthyretin reduces the number of amyloid-like deposits in transgenic worms. The interaction between the two proteins suggested by this result will be further tested by coexpressing a mutant form of transthyretin which in an in vitro assay does not interact with b-peptide. In another experiment, the gene encoding apolipoprotein E which according to clinical and in vitro data enhances the formation of amyloid, will be co-expressed with b1-42 to test whether the interaction can be replicated in the worm system. 4. Genetic screen for C. elegans genes modifying the phenotype caused by b1-42 expression is proposed. Three screens are proposed: A. b1-42 expressing animals will be treated with EMS; the F2 will be stained with the histological stain Congo Red to visualize whether increase or decrease in the amount of deposits has occurred. Animals of interest will be recovered and their progeny retested for altered deposits. Animals with altered deposits will be tested by ELISA whether the level of expression of b1-42 has not changed, since only mutations in genes which affect the ability of deposit formation are of interest. B. Screen for suppressors of the paralysis phenotype: it was observed that b-peptide expressing animals suffer from paralysis, possibly caused by intramuscular amyloid deposits. An EMS mutagenesis to obtain mutations in genes which affect the paralysis phenotype is proposed. C. Screen for suppressors of the vacuole phenotype. Since the vacuoles in b1-42 expressing animals are only infrequently observed, possibly due to the fragmentation of the degenerating cell, the ced-2 mutant which allows dead cells to persist will be introduced into the unc-54/b1-42 background.