Consequences of copper accumulation in the livers of the Atp7b-/- (Wilson disease gene) knockout mice

Abstract

Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene, ATP7B, has been identified, but the molecular events leading to Wilson disease remain poorly understood. Here, we demonstrate that genetically engineered Atp7b-/- mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of Atp7b-/- livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with Atp7b inactivation.

Figure 1

Copper incorporation into ceruloplasmin is disrupted by Atp7b inactivation. A: Total amount of ceruloplasmin in the serum analyzed under reducing and denaturing conditions. B: Detection of holo- and apo-ceruloplasmin in the same samples after separation under nonreducing, nondenaturing conditions. In both cases, the equal amounts of total serum protein from four different 6-week-old WT and KO mice were analyzed. C: Oxidase activity in serum of 6-week-old WT and KO mice. D: Daily urinary copper excretion in 6-week-old WT and KO mice (n = 5 to 7; *statistical significance for KO versus WT mice, P < 0.01).

Figure 2

Macroscopic and microscopic changes in the Atp7b^−/− livers. A: Control liver (left) and two Atp7b^−/− livers (middle and right) illustrating different degrees of pathological changes at 20 weeks. B: H&E staining at 6 weeks. Left: Unremarkable histology. Right: Enlarged hepatocytes, containing pleomorphic nuclei, foci of single cell necrosis, and mild lobular inflammation. C: Electron microscopy of Atp7b^−/− liver at 6 weeks. Left: Mitochondria have abnormal shape and size, as in human WD. Right: A dilated bile canaliculus and microsteatosis of a hepatocyte. Original magnifications: ×200 (B); ×10,000 (C, left); ×3000 (C, right).

Figure 3

Extensive pathology, subsequent liver regeneration, and development of cholangiocarcinoma. A: Inflammation and necrosis of markedly enlarged hepatocytes with huge and deformed nuclei. B: Focal bile duct dilation and diffuse lobular chronic inflammation. C: Electron microscopy of a markedly enlarged nucleus with pseudoinclusions, lipid inclusions, and fragmented nucleoli. D: The dysplastic changes in hepatocytes are revealed by cytoplasmic accumulation of α-fetoprotein (brown). The central nodule of regenerating hepatocytes is unstained. E: Restoration of normal parenchyma (left) and tumor development (right). F: Focal extensive atypical proliferation of bile ducts and acute inflammation. G: Region of normal parenchyma contains an unremarkable portal tract with intact bile duct. H: Electron microscopy of a completely normal hepatocyte from the regenerating portions of the liver. I and J: Examples of marked complexity and irregularity of the tubular structures and significant pleomorphism of the epithelium, with the loss of usual basal nuclear polarity, anisonucleosis, and occasional mitoses. Original magnifications: ×200 (A, D, G, I); ×125 (B); ×2000 (C); ×100 (E); ×500 (F); ×1200 (H); ×360 (J).

Figure 4

Elevated copper accumulates mostly in the cytosol and binds to small molecular weight protein(s). A: Distribution of copper between membrane and soluble fractions isolated from the liver homogenates of Atp7b^−/− mice at 6 and 20 weeks. B: Size-exclusion chromatography of a soluble fraction (6-week-old mice). The line indicates a protein elution profile, the bars indicate the concentration of copper in the fractions. The arrows point to the position of the molecular weight markers.

Figure 5

Distribution of copper in subcellular fractions of control (WT) and Atp7b^−/− (KO) hepatocytes. A: Copper concentration in the total membrane fraction from Atp7b^−/− (black bars) and WT (white bars) livers at 6 and 20 weeks. B: Relative copper distribution in subcellular fractions enriched in nuclei (500 g), mitochondria (3000 g), endoplasmic reticulum, Golgi, lysosome (15,000 g), plasma membranes (125,000 g). The total amount of copper in fractions was taken as 100%.

Figure 6

Accumulation of copper in the nuclei is associated with early changes in nuclear structure and function. A: Analysis of the purity of nuclear fraction using organelle markers. T, total homogenate; P, postnuclear supernatant; M, crude mitochondria; N, nuclei; Na/K ATPase α 1, marker for plasma membrane; cytochrome c, marker for mitochondria; histone H2B, marker for nuclei. B: Copper concentration in the purified nuclear fractions. C: H&E staining of liver sections from control and Atp7b^−/− mice. All results are for 6-week-old animals. Original magnifications, ×600.

Figure 7

Detection of proliferation marker Ki-67 using immunostaining with Mib1 antibody in the Atp7b^−/− livers at different stages of the disease. A: Six weeks: increased hepatocellular DNA synthesis (black nuclear staining) before detectable hepatocellular injury. B: Twelve weeks: many positive hepatocytes at time of maximal injury. C: Forty-four weeks: normal-appearing hepatocytes with minimal DNA synthesis. D: Forty-four weeks: the proliferating bile duct epithelium is strongly positive. E: Fifty-six weeks: minimal hepatocyte staining distant from the cholangitis. F: Fifty-six weeks: increased DNA synthesis in the vicinity of the proliferating bile ducts. Original magnifications, ×200.