Transgenic Study of ALS-Linked CCS Mutations

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种由大脑和脊髓运动神经元退化引起的进行性瘫痪疾病。大多数肌萎缩侧索硬化症病例是散发性的，但大约5-10%是家族性的。家族性肌萎缩侧索硬化症的13个遗传位点已被建立，9个基因的突变已被鉴定。在这些基因中，铜锌超氧化物歧化酶基因(SOD1)突变约占家族性ALS的20%，是ALS最常见的已知病因。动物模型的研究，特别是SOD1转基因小鼠模型的研究，为理解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相似的疾病表型，例如同源基因早老素1和2的突变会导致阿尔茨海默病。由于SOD1G93A的铜伴侣基因(CCS)与SOD1有很高的同源性，并且野生型CCS(WtCCS)的过表达显著增强了SOD1G93A在ALS转基因小鼠模型中的毒性，我们检验了CCS基因的突变是否与ALS的一个子集相关。我们对包括201例家族性ALS和338例散发性ALS在内的ALS患者进行了CCS基因的全序列分析，发现了两个突变。在3例ALS患者家系中发现CCSG222R突变，在1例散发ALS患者中发现CCSR71W突变。在1118名对照组受试者中没有发现这两种突变。生化分析表明，这两个与肌萎缩侧索硬化症相关的CCS突变体在蛋白质折叠/重折叠方面表现出明显的缺陷，并显著增加了形成纤维状聚集体的倾向。目前，尚不确定这两个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形成导致ALS。由于ALS是一种起病晚、致命性强的疾病，平均生存期只有三年，因此很难收集大型ALS家系来验证CCS突变是否具有致病性。测试这种可能性的一种直接且经济有效的方法是开发和鉴定使用这些突变的转基因小鼠。如果转基因小鼠出现了类似ALS的表型和病理，那么我们可以得出结论，CCS是一个新的ALS致病基因。对这些小鼠模型的进一步研究将进一步阐明ALS的致病机制。考虑到当前的挑战和机遇，我们建议建立和鉴定利用人CCS转基因基因：野生型CCS(WtCCS)、CCSR71W和CCSG222R的转基因小鼠模型。WtCCS小鼠模型将作为CCSR71W和CCSG222R小鼠的对照。该项目的前景是，我们不仅可以证明CCS是一个新的ALS致病基因，而且还可以为ALS研究界的进一步研究提供新的资源。 公共卫生相关性：肌萎缩侧索硬化症(ALS)是一种由大脑和脊髓运动神经元变性引起的致命性瘫痪疾病。肌萎缩侧索硬化症的病因和发病机制尚不清楚。铜锌超氧化物歧化酶基因(SOD1)突变约占家族性ALS的20%，是ALS最常见的致病原因。在肌萎缩侧索硬化症患者中，已经发现了SOD1同源基因及其功能伴侣铜伴侣(CCS)的突变。然而，这些CCS突变是否是ALS的原因仍有待确定。该项目旨在使用转基因方法来测试CCS是否是一种新的导致ALS的基因。

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.