Null Mutation of the Murine ATP7B (Wilson Disease) Gene Results in Intracellular Copper Accumulation and Late-Onset Hepatic Nodular Transformation

Abstract

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.

Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper

“…Atp7b Ϫ/Ϫ mice demonstrate intracellular copper accumulation, low serum oxidase activity, and increased copper excretion in the urine and liver pathology, similar to Wilson's disease patients (12,32). Transcriptome analysis of the Atp7b Ϫ/Ϫ mouse liver revealed copper-induced alterations of lipid metabolism and cholesterol homeostasis, which are also observed in Wilson's disease patients (33).…”

“…Furthermore, the pathways affected by toxic concentrations of copper may be unique from those affected by physiological levels. In the present study, transcriptomes were generated using HepG2 cells exposed to four concentrations of copper (100, 200, 400, and 600 M) for four time periods (4,8,12, and 24 h). These conditions were selected based on our previous cytotoxicity results (64) and copper levels reported from human case studies.…”

Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson's disease

“…18 Animals were housed in the Laboratory Animal Facility of the Colket Translational Research Building at The Children's Hospital of Philadelphia (CHOP). Mice were maintained in sterilized plastic micro isolator cages and given sterilized standard laboratory chow and tap water ad libitum.…”

“…Mice were genotyped at 3 weeks of age and Atp7b null mice identified according to the original protocol. 18 Heterozygous animals were mated for 12 --18 h to achieve accurate time-dated pregnancies and the day of removal of male mice was considered E0. All experimental protocols were approved by The Institutional Animal Care and Use Committee at Children's Hospital of Philadelphia, and followed guidelines set forth in the 'Guide for Care and Use of Laboratory Animals' by the National Institutes of Health.…”

“…These mice were generated by inserting early termination codons into the second exon of the Atp7b gene, which resulted in a truncated mRNA and complete loss of the ATP7B protein. 18 An advantage of these animals is the availability of morphological and biochemical data for the entire course of disease progression. 19 --22 By 6 weeks, copper accumulates in the livers of Atp7b À/À mice.…”