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.