Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase

Abstract

Recent studies in Saccharomyces cerevisiae suggest that the delivery of copper to Cu/Zn superoxide dismutase (SOD1) is mediated by a cytosolic protein termed the copper chaperone for superoxide dismutase (CCS). To determine the role of CCS in mammalian copper homeostasis, we generated mice with targeted disruption of CCS alleles (CCS(-/-) mice). Although CCS(-/-) mice are viable and possess normal levels of SOD1 protein, they reveal marked reductions in SOD1 activity when compared with control littermates. Metabolic labeling with (64)Cu demonstrated that the reduction of SOD1 activity in CCS(-/-) mice is the direct result of impaired Cu incorporation into SOD1 and that this effect was specific because no abnormalities were observed in Cu uptake, distribution, or incorporation into other cuproenzymes. Consistent with this loss of SOD1 activity, CCS(-/-) mice showed increased sensitivity to paraquat and reduced female fertility, phenotypes that are characteristic of SOD1-deficient mice. These results demonstrate the essential role of any mammalian copper chaperone and have important implications for the development of novel therapeutic strategies in familial amyotrophic lateral sclerosis.

Figure 1

Targeted disruption of the CCS gene by homologous recombination. (a) Maps of the wild-type CCS allele, the targeting vector, and the targeted CCS locus. Exons 1 to 5 of the CCS gene are denoted by black boxes. The targeting vector shows the replacement of exons 1and 2 and flanking genomic sequences including portions of the promoter by the neomycin gene (neo) and the HSV thymidine kinase gene (tk). Lines below indicate expected sizes from a Southern blot for EcoRI-digested fragments detected with a 5′-probe (black bar) from targeted and endogenous CCS alleles. B, BamHI; E, EcoRI; H, HindIII; S, SalI; X, XhoI. Arrows denote the sites within the targeted and wild-type CCS locus from which PCR primers were chosen for genotyping. (b) Analysis of genomic DNA from ES cells (lanes 1 and 2) and from progeny of CCS^+/− crosses (lanes 3–5). Genotypes for the CCS targeted allele and the EcoRI fragments detected for endogenous (9.5 kb) and targeted (7.2 kb) CCS alleles with the 5′ probe are indicated. (c) PCR analysis of DNA extracted from tail clips. By using primers indicated in a, the 0.4-kb or 0.6-kb fragment is specific to the endogenous or targeted CCS allele respectively; wild-type (lane 4), heterozygous (lanes 1, 2, 3, and 5), and homozygous (lanes 6 and 7) CCS knockout mice are indicated. (d) Protein extracts (20 μg) from various tissues of wild-type (lanes 1, 4, 7, 10, and 13), heterozygous (lanes 2, 5, 8, 11, and 14), and homozygous (lanes 3, 6, 9, 12, and 15) CCS knockout mice were immunoblotted by using antisera specific for CCS, SOD1, and α-tubulin. Bound antibodies were detected by using an enhanced chemiluminescent detection method.

Figure 2

Diminished SOD1 enzymatic activity in CCS knockout mice. (a) SOD1 activity assay gel of 25 or 50 μg protein extracts, respectively, from brain, spinal cord, muscle, and liver or from lung, heart, and kidney of wild-type (lanes 2, 5, 8, 11, 14, 17, and 20), heterozygous (lanes 3, 6, 9, 12, 15, 18, and 21), and homozygous (lanes 4, 7, 10, 13, 16, 19, and 22) CCS knockout mice. Arrow denote the position of mouse SOD1. Purified human erythrocyte SOD1 is shown in lane 1. (b) SOD1 activity determined from tissue extracts indicated by using a cytochrome c/xanthine oxidase method. The averages of SOD1 activities +/− standard deviations from three mice for each CCS genotype are shown. “Wild-type” values, as previously reported (21), are included for comparison.

Figure 3

Copper incorporation into SOD1 is impaired in CCS knockout mice. Protein extracts from various tissues of wild-type, heterozygous, and homozygous CCS knockout mice and homozygous SOD1 knockout mice were obtained. (a) SDS/PAGE gel of liver (50 μg), kidney (75 μg), brain and spinal cord (100 μg), immunoblotted with antisera to SOD1. (b) SOD1 gel activity assay on same samples as a. (c) ⁶⁴Cu incorporation into SOD1, 100 μg of protein lysate for each tissue. (d) Analysis of serum samples from wild-type, heterozygous, and homozygous CCS knockout mice and homozygous ceruloplasmin knockout mice. (Top to Bottom) Immunoblot with antisera to human ceruloplasmin, ⁶⁴Cu incorporation to ceruloplasmin, and ceruloplasmin activity gel assay.

Figure 4

Decreased copper incorporation into SOD1 is detected in fibroblasts derived from CCS^−/− mice. Primary fibroblast cultures were isolated from wild-type, heterozygous, and homozygous CCS knockout mice. (a) Protein lysates (50 μg) immunoblotted with antisera to SOD1. (b) SOD1 gel activity assay of 50 μg protein extract. (c) Protein lysate (50 μg) of ⁶⁴Cu-labeled fibroblasts.

Figure 5

Increased sensitivity to paraquat in CCS^−/− mice. Cumulative probability of survival of age-matched CCS^+/+ and CCS^−/− mice of both genders after exposure of paraquat at a dose of (a) 15 (P < 0.025) or (b) 25 (P < 0.005) mg/kg body weight.

Figure 6

Abnormal development of follicles in CCS^−/− mice. Histological analysis of ovaries from age-matched (a) CCS^+/+ or (b and c) CCS^−/− mice. Several corpora lutea (CL) and follicles in different stages of development are observed in control mice in a. In contrast, instead of corpora lutea, abnormally developed follicles are frequently seen in ovaries of CCS^−/− mice (arrows in b and c). Bar = 40 μm in a and 10 μm in c.