Wilson Disease Model Research Articles

The role of mTOR signaling pathway in regulating autophagy in liver injury of TX mice with Wilson’s disease

Wilson disease (WD), known as hepatolenticular degeneration (HLD), is a treatable autosomal recessive disorder of copper metabolism. Because copper deposits in the liver first, the liver is not only the original defective organ but also the most affected organ. The liver injury is also one of the main causes of death throughout the course of the disease. Therefore, the treatment of liver injury is the main task of WD treatment, which is of great significance to improve the prognosis of patients. Autophagy is a process that promotes cell survival through degradation, recycling, and absorption in order to maintain the normal physiological function of cells, while excessive autophagy can aggravate cell death. In view of the abnormal damage of liver cells in patients with WD, which may be related to the change of autophagy level, in this study, we established an animal model of WD through toxic milk (TX) mice, observed the change of autophagy level in the liver, and observed the change of liver damage in mice after treatment with autophagy inhibitors. It was found that the mTOR signaling pathway was activated and autophagy was inhibited in Wilson mouse liver. After treatment with rapamycin, the autophagy level of mice liver was upregulated, and the copper content of mice liver was reduced, and the damage was alleviated. TX mouse hepatocytes were isolated, after using siRNA to interfere with mTOR expression, the copper accumulation was significantly reduced, which was the same with RAPA treatment. The results showed that in TX mice, the damage caused by copper accumulation in the liver may be related to the decrease of autophagy level caused by the activation of the mTOR signaling pathway. Our findings suggested that RAPA or the use of siRNA targeting mTOR may have potential applications in the treatment of Wilson’s disease.

MtDNA depletion-like syndrome in Wilson disease.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, with its main pathology attributed to copper-mediated oxidative damage. The limited therapeutic effect of copper chelators and the early occurrence of mitochondrial deficits, however, undermine the prevalence of this mechanism. We characterized mitochondrial DNA copy number and mutations as well as bioenergetic deficits in blood from patients with WD and in livers of tx-j mice, a mouse model of hepatic copper accumulation. In vitro experiments with hepatocytes treated with CuSO4 were conducted to validate in vivo studies. Here, for the first time, we characterized the bioenergetic deficits in WD as consistent with a mitochondrial DNA depletion-like syndrome. This is evidenced by enriched DNA synthesis/replication pathways in serum metabolomics and decreased mitochondrial DNA copy number in blood of WD patients as well as decreased mitochondrial DNA copy number, increased citrate synthase activity, and selective Complex IV deficit in livers of the tx-j mouse model of WD. Tx-j mice treated with the copper chelator penicillamine, methyl donor choline or both ameliorated mitochondrial DNA damage but further decreased mitochondrial DNA copy number. Experiments with copper-loaded HepG2 cells validated the concept of a direct copper-mitochondrial DNA interaction. This study underlines the relevance of targeting the copper-mitochondrial DNA pool in the treatment of WD separate from the established copper-induced oxidative stress-mediated damage.

Accurate Measurement of Copper Overload in an Experimental Model of Wilson Disease by Laser Ablation Inductively Coupled Plasma Mass Spectrometry.

Wilson disease is a rare inherited autosomal recessive disorder. As a consequence of genetic alterations in the ATP7B gene, copper begins to accumulate in the body, particularly in the liver and brain. Affected persons are prone to develop liver cancer and severe psychiatric and neurological symptoms. Clinically, the development of corneal Kayser-Fleischer rings and low ceruloplasmin concentrations (<20 mg/dL) are indicative of Wilson disease. However, the detection of elevated hepatic copper content (>250 µg/g dry weight) alone is still considered as the best but not exclusive diagnostic test for Wilson disease. Presently, specific copper stains (e.g., rhodanine) or indirect staining for copper-associated proteins (e.g., orcein) are widely used to histochemically visualize hepatic copper deposits. However, these procedures only detect lysosomal copper, while cytosolic copper is not detectable. Similarly, elemental analysis in scanning electron microscope with energy dispersive X-ray analysis (EDX) often leads to false negative results and inconsistencies. Here, we tested the diagnostic potential of laser ablation inductively-coupled mass spectrometry (LA-ICP-MS) that allows quantitative analysis of multiple elements. Comparative studies were performed in wild type and the Atp7b null mouse model. We propose LA-ICP-MS as a versatile and powerful method for the accurate determination of hepatic copper in people with Wilson disease with high spatial resolution.

Application of 9.4T MRI in Wilson Disease Model TX Mice With Quantitative Susceptibility Mapping to Assess Copper Distribution.

In the current study, we used 9.4-tesla magnetic resonance imaging (9.4T MRI) and inductively coupled plasma mass spectrometry (ICP-MS) to investigate the distribution of copper in the brain samples of a murine model of Wilson’s disease (WD) following penicillamine (PCA) treatment. We also evaluated if the distribution of copper in the brain samples of mice was correlated with behavioral symptoms. Results from the behavioral experiments showed that 7 days of PCA treatment decreased the total distance traveled in the open field and the number of rearing and climbing instances among the toxic milk (TX) mice as compared with model group. We also observed that the open arm ratio in the elevated plus-maze (EPM) was reduced, escape latency in the Barnes maze test was increased, and avoidance in the open field was enhanced in TX mice following 14 days of PCA treatment as compared with those in untreated TX mice. We found that PCA treatment for 21–28 days improved the cognitive abilities, exploratory behavior, and movement behavior of TX mice. The PCA-treated mice also exhibited varying degrees of magnetic susceptibilities in the cortex, corpus striatum, hippocampus, and amygdaloid nucleus across the treatment period. Low copper concentrations were found in all of the analyzed brain regions of PCA-treated mice after 21–28 days as compared with the model group (P < 0.05). However, copper concentrations were increased in the primary motor cortex and cerebellum at 7 days post-PCA treatment as compared with those in the model group (P < 0.05). After 14 days of PCA treatment, the copper concentrations in the sensorimotor cortex, corpus striatum, hippocampus, and amygdaloid nucleus were higher than those detected without treatment. The results from a Pearson’s correlation analysis revealed that there was a significant (P < 0.05) correlation between copper concentrations and magnetic susceptibility in all of the brain regions that were analyzed. Therefore, our results indicate that copper concentration and magnetic susceptibility are associated with alterations in mood-related behavior, recognition memory, and movement behaviors in TX mice that are treated with PCA. The redistribution of copper in the TX mouse brain during PCA treatment may aggravate changes in behavioral performance.

Gandouling Tablets Inhibit Excessive Mitophagy in Toxic Milk (TX) Model Mouse of Wilson Disease via Pink1/Parkin Pathway.

Objective Gandouling (GDL) tablet is a Chinese patent medicine approved by the National Medical Product Administration, which is used to treat Wilson disease (WD) in China. In this study, we aimed to investigate the effects of GDL on mitophagy in the hippocampus in the toxic milk (TX) mouse model of WD. Methods Mice were randomly divided into the following four groups: control, Wilson (model group), D-penicillamine (DPA), and GDL groups. The animal behaviors were evaluated by the water maze experiment, traction test, and pole test. Transmission electron microscopy was used for the detection of mitochondrion structure. An enzyme-linked immunosorbent assay (ELISA) was performed for the analysis of the changes in liver function. Colocalization of mitophagy-related proteins was detected by fluorescence microscopy. Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR) were conducted for the detection of protein expression and mRNA levels, respectively. Results Significant reduction in neurological impairments was observed in the WD model group. All of these results were significantly reversed by GDL intervention. Compared with the levels in the Wilson group, the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), and albumin (ALB) changed obviously. Colocalization between mitophagy-related proteins pink1, parkin, and mitochondria was changed significantly. The mitophagy-related mRNA (pink1, parkin, and LC3II) and protein expression levels (pink1, parkin, and the rate of LC3II/LC3I) were decreased significantly, while p62 was remarkably increased after GDL intervention. Conclusion Our findings indicated that the neuroprotective mechanism of GDL may occur via the inhibition of excessive mitophagy through the regulation of the pink1/parkin pathway in the TX mouse brain of WD.

Dysregulated Choline, Methionine, and Aromatic Amino Acid Metabolism in Patients with Wilson Disease: Exploratory Metabolomic Profiling and Implications for Hepatic and Neurologic Phenotypes

Wilson disease (WD) is a genetic copper overload condition characterized by hepatic and neuropsychiatric symptoms with a not well-understood pathogenesis. Dysregulated methionine cycle is reported in animal models of WD, though not verified in humans. Choline is essential for lipid and methionine metabolism. Defects in neurotransmitters as acetylcholine, and biogenic amines are reported in WD; however, less is known about their circulating precursors. We aimed to study choline, methionine, aromatic amino acids, and phospholipids in serum of WD subjects. Hydrophilic interaction chromatography-quadrupole time-of-flight mass spectrometry was employed to profile serum of WD subjects categorized as hepatic, neurologic, and pre-clinical. Hepatic transcript levels of genes related to choline and methionine metabolism were verified in the Jackson Laboratory toxic milk mouse model of WD (tx-j). Compared to healthy subjects, choline, methionine, ornithine, proline, phenylalanine, tyrosine, and histidine were significantly elevated in WD, with marked alterations in phosphatidylcholines and reductions in sphingosine-1-phosphate, sphingomyelins, and acylcarnitines. In tx-j mice, choline, methionine, and phosphatidylcholine were similarly dysregulated. Elevated choline is a hallmark dysregulation in WD interconnected with alterations in methionine and phospholipid metabolism, which are relevant to hepatic steatosis. The elevated phenylalanine, tyrosine, and histidine carry implications for neurologic manifestations and are worth further investigation.

Laser Ablation Inductively Coupled Plasma Spectrometry: Metal Imaging in Experimental and Clinical Wilson Disease

Wilson disease is an inherited disorder caused by mutations in the ATP7B gene resulting in copper metabolism disturbances. As a consequence, copper accumulates in different organs with most common presentation in liver and brain. Chelating agents that nonspecifically chelate copper, and promote its urinary excretion, or zinc salts interfering with the absorption of copper from the gastrointestinal tract, are current medications. Also gene therapy, restoring ATP7B gene function or trials with bis-choline tetrathiomolybdate (WTX101) removing excess copper from intracellular hepatic copper stores and increasing biliary copper excretion, is promising in reducing body’s copper content. Therapy efficacy is mostly evaluated by testing for evidence of liver disease and neurological symptoms, hepatic synthetic functions, indices of copper metabolisms, urinary copper excretions, or direct copper measurements. However, several studies conducted in patients or Wilson disease models have shown that not only the absolute concentration of copper, but also its spatial distribution within the diseased tissue is relevant for disease severity and outcome. Here we discuss laser ablation inductively coupled plasma spectrometry imaging as a novel method for accurate determination of trace element concentrations with high diagnostic sensitivity, spatial resolution, specificity, and quantification ability in experimental and clinical Wilson disease specimens.

Unique Aspects of Copper Binding Components in the Blood Plasma of Dogs

It is well known that dogs suffer from at least two unusual traits related to Cu metabolism, which are (a) an inability of their serum albumin to bind Cu tightly, due to no histidine in the N terminal binding site; and (b) 10–20‐times higher liver Cu concentrations accompanied by a tendency to develop Cu overload.[1,2] We have identified and investigated two additional peculiarities in dogs related to components in their blood plasma. One has to do with ceruloplasmin (Cp), a 132 kDa glycoprotein, which has functions ranging from delivery of Cu to cells and scavenging reactive oxygen species, to oxidation of Fe(II) and various amines. It is also the main Cu binding component of the blood plasma. Although liver Cu concentrations are extremely high compared to other species, total Cu levels in plasma are normally much lower than those in human and rats (200–400 ng/ml versus about 1200). We found that when the plasma of Labrador retrievers was fractionated in large pore size exclusion chromatography (SEC), the main Cu peak eluted much earlier than is the case with plasma from other mammals. In Superdex 200 FPLC equilibrated with 20 mM phosphate, pH 7, the main Cu peak eluted near the void volume (Mr ~ 700 kDa) as compared to that of humans mice, rats and pigs (Mr~150 kDa). The same was the case for plasma from other dog breeds. Western blotting determined that Cp protein eluted in parallel with the main Cu peak and was of normal size in SDS‐PAGE. Enzyme activities associated with Cp (ferroxidase and p‐phenylenediamine oxidase) also eluted in parallel. This indicated that canine Cp was either bound to some other protein(s) or that it was aggregating. Lactoferrin, which has been reported to bind to Cp in milk, was not detected. Canine plasma proteins were further fractionated on Sephacryl S300, which gives a better separation of large components. Stained SDS‐PAGE gels of fractions from such columns did not show any bands that eluted in parallel with those of Cp. This is consistent with the concept that Cp was not binding another protein but aggregating. To further examine that possibility, fractionation was carried out in buffers of higher ionic strength. Isotonic phosphate‐buffered saline did not alter the elution of canine Cp in Superdex 200. However with 300 mM phosphate (pH 6.8), canine Cp eluted in the same way as the Cp of other mammals, indicating it had dissociated. The other canine peculiarity investigated relates to the tendency towards Cu toxicosis. Plasma from Labrador retrievers with mutations in the Cu transporter ATP7B and high liver copper concentrations had markedly increased levels of a small Cu carrier in their plasma and urine compared with the wild type, as is the case with Wilson disease model mice.[3] Our results indicate that increased levels of a small Cu carrier may be helpful in diagnosis of canine Cu overload, and suggest that canine Cp is circulating as a multimeric protein.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Gene Therapy Approach to Improve Copper Metabolism and Prevent Liver Damage in a Mouse Model of Wilson Disease.

Wilson disease (WD), an autosomal recessive disease caused by mutations in a copper-transporting P-type ATPase (Atp7b), causes severe liver damage. This disease is currently treated with the lifelong use of copper chelation therapy, which has side effects and does not fix copper metabolism. Here, we thoroughly characterized a mouse model of WD, the toxic milk mouse, and used the model to test a gene therapy approach for treating WD. WD mice accumulated copper in the liver from birth; severe copper accumulation and concurrent liver disease were evident by 2 months of age. Intravenously administering an adeno-associated viral (AAV) 8 vector expressing a codon-optimized version of the human ATP7B transgene into 2-month-old WD mice significantly decreased liver copper levels compared with age-matched, uninjected, WD mice. We also observed a significant dose-dependent decrease in liver disease. Male mice injected with 1011 genome copies of AAV8 vector showed only mild histopathological findings with a complete lack of liver fibrosis. Therefore, we conclude that administering gene therapy at the early stages of disease onset is a promising approach for reducing liver damage and correcting copper metabolism in WD.

Epigenetic changes of the thioredoxin system in the tx-j mouse model and in patients with Wilson disease.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, leading to copper accumulation in the liver and brain. Excess copper inhibits S-adenosyl-L-homocysteine hydrolase, leading to variable WD phenotypes from widespread alterations in DNA methylation and gene expression. Previously, we demonstrated that maternal choline supplementation in the Jackson toxic milk (tx-j) mouse model of WD corrected higher thioredoxin 1 (TNX1) transcript levels in fetal liver. Here, we investigated the effect of maternal choline supplementation on genome-wide DNA methylation patterns in tx-j fetal liver by whole-genome bisulfite sequencing (WGBS). Tx-j Atp7b genotype-dependent differences in DNA methylation were corrected by choline for genes including, but not exclusive to, oxidative stress pathways. To examine phenotypic effects of postnatal choline supplementation, tx-j mice were randomized to one of six treatment groups: with or without maternal and/or continued choline supplementation, and with or without copper chelation with penicillamine (PCA) treatment. Hepatic transcript levels of TXN1 and peroxiredoxin 1 (Prdx1) were significantly higher in mice receiving maternal and continued choline with or without PCA treatment compared to untreated mice. A WGBS comparison of human WD liver and tx-j mouse liver demonstrated a significant overlap of differentially methylated genes associated with ATP7B deficiency. Further, eight genes in the thioredoxin (TXN) pathway were differentially methylated in human WD liver samples. In summary, Atp7b deficiency and choline supplementation have a genome-wide impact, including on TXN system-related genes, in tx-j mice. These findings could explain the variability of WD phenotype and suggest new complementary treatment options for WD.

Brain copper storage after genetic long-term correction in a mouse model of Wilson disease.

Wilson disease is a rare autosomal recessive condition caused by mutations in the copper-transporting ATPase ATP7B gene (OMIM: 606882) provoking loss of function and resulting in variable hepatic and neurologic symptoms. Currently, the treatment of Wilson disease focuses on achieving a negative copper balance either with chelators (e.g., d-penicillamine, trientine, and tetrathiomolybdate) or zinc, which reduces copper absorption, or a combination thereof.1 However, these lifelong treatment regimens often cause side effects and do not restore normal copper metabolism. The authors thank Astrid Kuppers (Zentralinstitut fur Engineering, Elektronik und Analytik, Forschungszentrum Julich, Julich, Germany) for excellent technical assistance in LA-ICP-MS measurements. They acknowledge Drs Nick Weber and Bernard Benichou for critical comments and careful review and Dr. Andreas Matusch (Division of Molecular Neuroimaging, Institute of Neuroscience and Medicine-2, Forschungszentrum Julich, Germany) for help in preparing murine brains.

Metabolic disposition of WTX101 (bis-choline tetrathiomolybdate) in a rat model of Wilson disease

1. WTX101 (bis-choline tetrathiomolybdate) is an investigational copper (Cu)-protein-binding agent developed for the treatment of Wilson disease (WD), a rare genetic disorder caused by mutations in the ATP7B Cu-transporter and resulting in toxic Cu accumulation.2. Mass balance of a single intravenous WTX101 dose, measured as molybdenum (Mo), was assessed over 168 h in control (Long Evans Agouti [LEA]) and Long-Evans Cinnamon (LEC) rats, a WD model.3. In LEC rats, Mo was partially excreted (up to 45%); 29% by renal clearance, and faecal clearance, still ongoing at 168 h, accounted for 16%. In contrast, in LEA rats, Mo was almost fully excreted (∼87%); 79% was renally cleared with only 7% faecal excretion.4. In LEC rats, the proportion of faecal to renal Mo excretion was enhanced (4:6) compared to controls (1:9).5. Substantially more Mo was found in LEC liver and kidney compared with LEA tissues, in line with tissue Cu distribution.6. These findings are consistent with the WTX101 mechanism of action: in the WD model, excess Cu is removed from hepatic metallothionein and retained within the stable tetrathiomolybdate-Cu-albumin tripartite complex, preventing tetrathiomolybdate degradation and resulting in less urinary elimination and greater faecal excretion than in controls.

Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.

Lipid and copper metabolism in humans with wilson disease: Enigmatic relationship.

Potential conflict of interest: Nothing to report. To the Editor: We read with interest the article by Hamilton et al. published in hepatology, an experimental study highlighting the role of nuclear receptors in a mouse model of Wilson disease (WD).1 Their previous study demonstrated that lipid metabolism was inhibited in response to hepatic copper accumulation in the WD mouse model,2 and this expanded study showed that up‐regulation of the nuclear receptor by liver X receptor agonist administration can increase serum lipid values to normal despite significant hepatic copper overload. These findings seem very convincing and throw light on WD pathogenesis. The only human study describing attenuation in lipid metabolism in WD suggests low cholesterol levels, but the study does not provide correlation of lipids with hepatic copper.3 We had opportunity to retrospectively study serum lipid profiles, liver function tests, and hepatic copper in our WD children (n = 26) seen at King's College Hospital from 2005 to 2015. WD diagnosis was established with ATP7B mutations in all. Serum total cholesterol (TC) and triglyceride (TG) data before starting treatment were collected. Hepatic copper was measured in four explant livers of WD/acute liver failure children and 22 core liver biopsy tissues from WD/non–acute liver failure children. The median age at diagnosis was 10.86 (1.64‐17.26) years, and 62% were female. The median hepatic copper was high (513 [7‐4225] μg/g dry weight liver). Interestingly, the median TC and TG in our study were normal, with values of 4.45 (1.7‐6.3) and 1.3 (0.5‐3.2) mmol/L. Indeed, the median body mass index that might affect the lipid profile level was also normal (19.8 [13.7‐40.7] kg/m2). We further assessed the correlation between serum lipid profiles with hepatic copper and liver function tests. There was no correlation between lipid profiles (TC and TG) and hepatic copper (Fig. 1). TG has no correlation with other parameters, while TC significantly correlates with total bilirubin (r = –0.621, P = 0.001) and alanine aminotransferase (r = 0.422, P = 0.028).Figure 1: The correlation between lipid profiles and hepatic copper content.Our data suggest that low cholesterol in hepatic WD patients might relate to severity of liver disease. Our data on correlation of hepatic copper with cholesterol in children with liver disease are not in keeping with the observation in the animal model. However, we were not able to perform nuclear receptor studies in our patient population, but our finding may be of relevance before applying the data from animal studies to humans.

A microbial peptide to rescue severe and fulminant Wilson disease?

Wilson disease is characterized by massive copper overload caused by a mutation of the liver-specific copper-transporting ATPase, ATP7B. Presently, liver transplantation is the only treatment available to patients with advanced or acute liver disease. In this paper, the authors describe the therapeutic effect of methanobactin, a potent bacterial copper-binding protein, in a rat model of Wilson disease, the Atp7b-/- rat. Their results show a marked improvement of clinical, biochemical and ultrastructural abnormalities. They propose that methanobactin is a candidate drug for Wilson disease patients with severe liver disease.

Methanobactin reverses acute liver failure in a rat model of Wilson disease.

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

Reply

Potential conflict of interest: Nothing to report. Reply: We would like to thank you for your interest in our article. We agree that our article, and the work of others,1 has shed a new light on the role of nuclear receptor signaling, and especially liver X receptor/retinoid X receptor (LXR/RXR) activation as a relatively early response to copper misbalance. We propose that drug‐mediated improvement of liver disease in the Wilson disease (WD) mice involves activation of LXR/RXR function not only in hepatocytes, but also in nonparenchymal liver cells (NPCs). Our findings suggest that copper overload negatively regulates LXR and RXR. The mechanisms underlying this regulation by copper remain to be fully elucidated. However, the implication that NPCs are involved in the pathogenesis of WD is an exciting new finding. Investigation of the cross‐talk between hepatocytes and stellate cells/Kupffer cells in this animal model may have ramifications for all fibrotic liver diseases. Although T0901317 was first reported to be a specific LXR agonist,4 we agree that the drug may target other nuclear receptors that form heterodimers with RXR. In fact, it could be that the pleotropic effect of the drug accounts for beneficial anti‐inflammatory and antifibrotic effects that we observe in WD mice. Identifying all targets of T0901317 in the WD model is an important future goal. Future modifications of the drug and/or careful evaluation of dosage/time of treatment are also likely to be necessary to offset the potential untoward effects (such as hepatic steatosis). Nonetheless, modulation of LXR activity appears to have promise as a novel therapy for WD.

Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B−/− (Wilson disease) mice

Wilson disease (WD) is a hepatoneurological disorder caused by mutations in the copper‐transporter, ATP7B. Copper accumulation in the liver is a hallmark of WD. Current therapy is based on copper chelation, which decreases the manifestations of liver disease, but often worsens neurological symptoms. We demonstrate that in Atp7b−/− mice, an animal model of WD, liver function can be significantly improved without copper chelation. Analysis of transcriptional and metabolic changes in samples from WD patients and Atp7b−/− mice identified dysregulation of nuclear receptors (NRs), especially the liver X receptor (LXR)/retinoid X receptor heterodimer, as an important event in WD pathogenesis. Treating Atp7b−/− mice with the LXR agonist, T0901317, ameliorated disease manifestations despite significant copper overload. Genetic markers of liver fibrosis and inflammatory cytokines were significantly decreased, lipid profiles normalized, and liver function and histology were improved. Conclusions: The results demonstrate the major role of an altered NR function in the pathogenesis of WD and suggest that modulation of NR activity should be explored as a supplementary approach to improving liver function in WD. (Hepatology 2016;63:1828‐1841)

Early Application of Auxiliary Partial Orthotopic Liver Transplantation in Murine Model of Wilson Disease.

Liver transplantation (LT) is the only option of treatment for Wilson disease (WD) when chelation therapy fails, but it is limited due to the shortage of donor. Auxiliary partial orthotopic LT (APOLT) has been performed successfully in end-stage WD patients, which expands the donor pool. Atp7bmice were used as experimental model of WD. Eight- and 20-week-old mice were used as different timepoints to perform APOLT. Serum copper, tissue copper, serum ceruloplasmin (CP), and liver histological examination were observed after operation. Hepatic and serum copper levels in Atp7b mice decreased after APOLT, and copper metabolism disorder of WD mice was relieved at both early and late stages. The progression of pathology in the native liver was delayed only when transplantation was performed at an early stage. Auxiliary partial orthotopic LT can significantly improve copper metabolism disorder in the Atp7b mice, and early transplantation may prevent the disease progression.

Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy

Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.