Transgenic Study of ALS-Linked CCS Mutations

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种由大脑和脊髓运动神经元变性引起的进行性麻痹疾病。大多数ALS病例是散发的，但约有5-10%是家族性的。家族性肌萎缩侧索硬化症的13个基因位点已经建立，9个基因的突变已经确定。在这些基因中，Cu， zn -超氧化物歧化酶基因（SOD1）的突变约占家族性ALS的20%，并且代表了ALS最普遍的已知原因。动物模型的研究，特别是SOD1转基因小鼠模型的研究，为了解ALS的发病机制提供了有价值的数据。众所周知，一些同源基因的基因突变会产生类似的疾病表型，如早老素1和早老素2的同源基因突变会导致阿尔茨海默病。由于SOD1的铜伴侣蛋白（CCS）与SOD1具有高度的同源性，并且在ALS转基因小鼠模型中，野生型CCS （wtCCS）的过表达显著增强了SOD1G93A的ALS相关毒性，因此我们测试了CCS基因的突变是否与ALS的一个子集相关。我们分析了201例家族性ALS和338例散发性ALS患者中CCS基因的整个编码序列，发现了两个突变。在3例ALS患者的家系中发现了CCSG222R突变，在1例散发性ALS病例中发现了CCSR71W突变。在1118名对照受试者中未发现这两种突变。生化分析表明，这两个与ALS相关的CCS突变体在蛋白质折叠/再折叠方面表现出显著缺陷，并显著增加了形成纤维聚集体的倾向。目前尚不确定这两种CCS突变是否会导致ALS，尽管我们的遗传和生化数据表明这些CCS突变可能通过纤维聚集体的形成导致ALS。由于ALS是一种迟发性致死性疾病，平均生存期仅为3年，因此很难收集ALS大家族来验证CCS突变是否具有致病性。测试这种可能性的一种直接和经济有效的方法是利用这些突变开发和表征转基因小鼠。如果转基因小鼠表现出als样表型和病理，则CCS可能是一种新的als致病基因。对这些小鼠模型的进一步研究将进一步揭示ALS的致病机制。考虑到当前的挑战和机遇，我们建议使用人类CCS转基因基因：野生型CCS （wtCCS）、CCSR71W和CCSG222R来开发和表征转基因小鼠模型。wtCCS小鼠模型将作为CCSR71W和CCSG222R小鼠的对照进行表征。该项目的前景是，我们不仅可以证明CCS是一种新的ALS致病基因，而且可以为ALS研究界的进一步研究提供新的资源。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of motor neurons in the brain and spinal cord. Most ALS cases are sporadic, but approximately 5-10% are familial. Thirteen genetic loci for familial ALS have been established and mutations in nine genes have been identified. Among these genes, mutations in the Cu, Zn-superoxide dismutase gene (SOD1) account for approximately 20% of familial ALS, and represent the most prevalent known cause of ALS. Studies of animal models, especially the SOD1 transgenic mouse models, have provided valuable data for understanding the pathogenesis of ALS. It is well recognized that genetic mutations in some homologous genes produce similar disease phenotypes, such as mutations in the homologous genes presenilin 1 and 2 cause Alzheimer disease. Because the copper chaperone for SOD1 (CCS) shares high homology with SOD1 and overexpression of wild-type CCS (wtCCS) significantly enhances the ALS-associated toxicity of SOD1G93A in transgenic mouse model of ALS, we tested if mutations in the CCS gene are associated with a subset of ALS. We analyzed the entire coding sequence of the CCS gene in a large population with ALS, including 201 familial ALS and 338 sporadic ALS cases, and found two mutations. A CCSG222R mutation was identified in a pedigree with three patients affected with ALS and a CCSR71W mutation was found in a sporadic ALS case. These two mutations were not found in 1,118 control subjects. Biochemical analysis demonstrated that these two ALS- associated CCS mutants exhibit remarkable defects in protein folding/refolding and significantly increased propensity to form fibrillar aggregates. Currently, it is not certain if these two CCS mutations cause ALS, even though our genetic and biochemical data suggest that these CCS mutations may cause ALS, possibly through fibrillar aggregate formation. Because ALS is a late-onset and fatal disease, with an average survival of only three years, it is rather difficult to collect large ALS families to verify if mutations in the CCS are pathogenic. One direct and cost-effective way to test this possibility is to develop and characterize transgenic mice using these mutations. If the transgenic mice develop ALS-like phenotype and pathology, then we may conclude that the CCS is a novel ALS-causing gene. Further studies of these mouse models should shed additional light on the pathogenic mechanisms of ALS. In consideration of the current challenges and opportunities, we propose to develop and characterize transgenic mouse models using human CCS transgenes: wild-type CCS (wtCCS), CCSR71W and CCSG222R. The wtCCS mouse model will be used as a control for the CCSR71W and CCSG222R mice in characterizations. The promise of this project is that we may not only prove that the CCS is a novel ALS-causing gene, but also provide novel resources for further studies in ALS research community. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by degeneration of motor neurons in the brain and spinal cord. The etiology and pathogenesis of ALS are largely unknown. Mutations in the Cu, Zn- superoxide dismutase gene (SOD1) account for approximately 20% of familial ALS, and represent the most prevalent known cause of ALS. Mutations in the SOD1 homolog and functional partner, copper chaperone for SOD1 (CCS), have been identified in ALS patients. However, it remains to be determined if these CCS mutations are causative of ALS. This project is designed to test if CCS is a novel ALS-causing gene, using a transgenic approach.