Neuroinflammatory and behavioural changes in the Atp7B mutant mouse model of Wilson's disease

Wilson's disease (WD) is caused by mutations in the copper transporting ATPase 7B (Atp7b). Patients present with liver pathology or behavioural disturbances. Studies on rodent models for WD so far mainly focussed on liver, not brain. The effect of knockout of atp7b on sensori-motor and cognitive behaviour, as well as neuronal number, inflammatory markers, copper and synaptic proteins in brain were studied in so-called toxic milk mice. Copper accumulated in striatum and hippocampus of toxic milk mice, but not in cerebral cortex. Inflammatory markers were increased in striatum and corpus callosum, but not in cerebral cortex and hippocampus, whereas neuronal numbers were unchanged. Toxic milk mice were mildly impaired in the rotarod and cylinder test and unable to acquire spatial memory in the Morris water maze. Despite the latter observation only synaptophysin of a number of synaptic proteins, was altered in the hippocampus of toxic milk mice. In addition to disturbances in neuronal signalling by increased brain copper, inflammation and inflammatory signalling from the periphery to the brain might add to the behavioural disturbances in the toxic milk mice. These mice can be used to evaluate therapeutic strategies to alleviate behavioural disturbances and cerebral pathology observed in WD.     

Liver Ischemia and Ischemia–Reperfusion Induces and Trafficks the Multi-specific Metal Transporter Atp7b to Bile Duct Canaliculi: Possible Preferential Transport of Iron into Bile

Both Atp7b (Wilson disease gene) and Atp7a (Menkes disease gene) have been reported to be trafficked by copper. Atp7b is trafficked to the bile duct canaliculi and Atp7a to the plasma membrane. Whether or not liver ischemia or ischemia–reperfusion modulates Atp7b expression and trafficking has not been reported. In this study, we report for the first time that the multi-specific metal transporter Atp7b is significantly induced and trafficked by both liver ischemia alone and liver ischemia–reperfusion, as judged by immunohistochemistry and Western blot analyses. Although hepatocytes also stained for Atp7b, localized intense staining of Atp7b was found on bile duct canaliculi. Inductive coupled plasma-mass spectrometry analysis of bile copper, iron, zinc, and manganese found a corresponding significant increase in biliary iron. In our attempt to determine if the increased biliary iron transport observed may be a result of altered bile flow, lysosomal trafficking, or glutathione biliary transport, we measured bile flow, bile acid phosphatase activity, and glutathione content. No significant difference was found in bile flow, bile acid phosphatase activity, and glutathione, between control livers and livers subjected to ischemia–reperfusion. Thus, we conclude that liver ischemia and ischemia–reperfusion induction and trafficking Atp7b to the bile duct canaliculi may contribute to preferential iron transport into bile.     

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b^−/− mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu²⁺/H₂O₂/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b^−/− mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b^−/− mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b^−/− mice in contrast to control with increasing age. Hepatocytes from elder Atp7b^−/−mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.     

Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.     

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid.To study the function of gastric parietal cells during gastric epithelium homeostasis,we generated a transgenie mouse line,namely,Atp4b-Cre,in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H+-,K+-ATPase gene(Atp4b).In order to test the tissue distribution and excision activity of Cre recombinase in vivo,the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles(Smad4Co/Co).Multiple-tissue PCR of Atp4b-Cre;Smad4Co/+mice revealed that the recombination only happened in the stomach.As indicated by LacZ staining,ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells.These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.     

Urinary copper elevation in a mouse model of Wilson's disease is a regulated process to specifically decrease the hepatic copper load

Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson's disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b(-/-) mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b(-/-) livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr1(-/-) knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD.     

Heterozygous <Emphasis Type="BoldItalic">tx</Emphasis> mice have an increased sensitivity to copper loading: Implications for Wilson’s disease carriers

Wilson’s disease carriers constitute 1% of the human population. It is unknown whether Wilson’s disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson's disease mouse model – the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4–7, 8–11, 12–15 or 16–20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney – an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilson’s disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.     

The copper chaperone Atox1 in canine copper toxicosis in Bedlington terriers

Copper toxicosis, resulting in liver disease, commonly occurs in Bedlington terriers. This recessively inherited disorder, similar in many respects to Wilson disease, is of particular interest because the canine Atp7b gene, homologous to ATP7B defective in Wilson disease, is not responsible for canine copper toxicosis as has been expected. Atox1, a copper chaperone delivering copper to Atp7b, therefore became a potential candidate. We cloned canine Atox1, which shows conserved motifs of the copper-binding domain (MTCXXC) and of the lysine-rich region (KTGK), and showed 88, 80, and 41% amino acid sequence identity with the orthologous mouse, human, and yeast proteins. No gross deletions of Atox1 could be identified in the affected Bedlington terriers by Southern blot analysis of genomic DNA. The canine Atox1 gene spans about 4 kb, with a 204-bp open reading frame cDNA contained within two exons. Sequence analysis of the coding regions, including intron/exon boundaries, showed no mutations in Atox1 from genomic DNA of an affected dog. We have also identified an apparently nontranscribed canine Atox1 pseudogene, with 12 sequence changes and no intron. Mapping of Atox1 and a marker closely linked to the canine copper toxicosis locus indicated lack of synteny. Atox1 is therefore excluded as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene must be responsible for this copper storage disease in dogs and also suggesting the possibility of a similar gene responsible for a copper storage disease in humans.     

Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.     

Exploration of the copper-related compensatory response in the Belgrade rat model of genetic iron deficiency

The Menkes copper ATPase (Atp7a) and metallothionein (Mt1a) are induced in the duodenum of iron-deficient rats, and serum and hepatic copper levels increase. Induction of a multi-copper ferroxidase (ceruloplasmin; Cp) has also been documented. These findings hint at an important role for Cu during iron deficiency. The intestinal divalent metal transporter 1 (Dmt1) is also induced during iron deficiency. The hypothesis that Dmt1 is involved in the copper-related compensatory response during iron deficiency was tested, utilizing a mutant Dmt1 rat model, namely the Belgrade (b/b) rat. Data from b/b rats were compared with phenotypically normal, heterozygous +/b rats. Intestinal Atp7a and Dmt1 expression was increased in b/b rats, whereas Mt1a expression was unchanged. Serum and liver copper levels did not increase in the Belgrades nor did Cp protein or activity. The lack of fully functional Dmt1 may thus partially blunt the compensatory response to iron deficiency by 1) decreasing copper levels in enterocytes, as exemplified by a lack of Mt1a induction and a lesser induction of Atp7a, 2) abolishing the frequently described increase in liver and serum copper, and 3) attenuating the documented increase in Cp expression and activity.     

Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964-G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic Atp7b gene expression does not change with iron deficiency, suggesting that liver copper excretion is not altered. We have developed polyclonal antibodies against rat ATP7A, and we demonstrate the specificity of the immunogenic reaction. We show that the ATP7A protein is present on apical domains of duodenal enterocytes in control rats and on brush-border and basolateral membrane domains in iron-deprived rats. This localization is surprising, as previous in vitro studies have suggested that ATP7A traffics between the trans-Golgi network and the basolateral membrane. We further demonstrate that ATP7A protein levels are dramatically increased in brush-border and basolateral membrane vesicles isolated from iron-deficient rats. Other experiments show that iron refeeding partially corrects the hematological abnormalities seen in iron-deficient rats but that it does not ameliorate ATP7A protein induction, suggesting that Atp7a does not respond to intracellular iron levels. We conclude that ATP7A is involved in copper loading observed during iron deficiency and that increased intestinal copper transport is of physiological relevance, as copper plays important roles in overall body iron homeostasis.     

Silencing of the Menkes copper-transporting ATPase (Atp7a) gene increases cyclin D1 protein expression and impairs proliferation of rat intestinal epithelial (IEC-6) cells

The Menkes copper-transporting ATPase (Atp7a) has dual roles in mammalian enterocytes: pumping copper into the trans-Golgi network (to support cuproenzyme synthesis) and across the basolateral membrane (to deliver dietary copper to the blood). Atp7a is strongly induced in the rodent duodenum during iron deprivation, suggesting that copper influences iron homeostasis. To investigate this possibility, Atp7a was silenced in rat intestinal epithelial (IEC-6) cells. Irrespective of its influence on iron homeostasis, an unexpected observation was made in the Atp7a knockdown (KD) cells: the cells grew slower (∼40% fewer cells at 96 h) and were larger than negative-control shRNA-transfected cells. Lack of Atp7a activity thus perturbed cell cycle control. To elucidate a possible molecular mechanism, expression of two important cell cycle control proteins was assessed. Cyclin D1 (CD1) protein expression increased in Atp7a KD cells whereas proliferating-cell nuclear antigen (PCNA) expression was unaltered. Increased CD1 expression is consistent with impaired cell cycle progression. Expression of additional cell proliferation marker genes (p21 and Ki67) was also investigated; p21 expression increased, whereas Ki67 decreased, both consistent with diminished cell growth. Further experiments were designed to determine whether increased cellular copper content was the trigger for the altered growth phenotype of the Atp7a KD cells. Copper loading, however, did not influence the expression patterns of CD1, p21 or Ki67. Overall, these findings demonstrate that Atp7a is required for normal proliferation of IEC-6 cells. How Atp7a influences cell growth is unclear, but the underlying mechanism could relate to its roles in intracellular copper distribution or cuproenzyme synthesis.     

Hepatic copper-transporting ATPase ATP7B: function and inactivation at the molecular and cellular level

Copper-transporting ATPase ATP7B (Wilson disease protein) is a member of the P-type ATPase family with characteristic domain structure and distinct ATP-binding site. ATP7B plays a central role in the regulation of copper homeostasis in the liver by delivering copper to the secretory pathway and mediating export of excess copper into the bile. The dual function of ATP7B in hepatocytes is coupled with copper-dependent intracellular relocalization of the transporter. The final destination of ATP7B in hepatocytes during the copper-induced trafficking process is still under debate. We show the results of immunocytochemistry experiments in polarized HepG2 cells that support the model in which elevated copper induces trafficking of ATP7B to sub-apical vesicles, and transiently to the canalicular membrane. In Atp7b ^-/- mice, an animal model of Wilson disease, both copper delivery to the trans-Golgi network and copper export into the bile are disrupted despite large accumulation of copper in the cytosol. We review the biochemical and physiological changes associated with Atp7b inactivation in mouse liver and discuss the pleiotropic consequences of the common Wilson disease mutation, His1069Gln.     

Absence of linkage between radiosensitivity and the predisposing atp7b gene mutation for heritable hepatitis in the LEC rat

The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F(1) animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F(1) x LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.