Analyzing the Therapeutic Efficacy of Bis-Choline-Tetrathiomolybdate in the Atp7b−/− Copper Overload Mouse Model

Bis-choline-tetrathiomolybdate, introduced as WTX101 (now known as ALXN1840), is a first-in-class copper-protein-binding agent for oral therapy of Wilson’s disease. In contrast to other decoppering agents such as trientine or D-penicillamine it acts by forming a tripartite complex with copper and albumin, thereby detoxifying excess liver and blood copper through biliary excretion. Preclinical animal experimentation with this drug was typically done with the alternative ammonium salt of tetrathiomolybdate, which is expected to have identical properties in terms of copper binding. Here, we comparatively analyzed the therapeutic efficacy of ALXN1840, D-penicillamine and trientine in lowering hepatic copper content in Atp7b−/− mouse. Liver specimens were subjected to laser ablation inductively conductively plasma mass spectrometry and electron microscopic analysis. We found that ALXN1840 caused a massive increase of hepatic copper and molybdenum during early stages of therapy. Prolonged treatment with ALXN1840 reduced hepatic copper to an extent that was similar to that observed after administration of D-penicillamine and trientine. Electron microscopic analysis showed a significant increase of lysosomal electron-dense particles in the liver confirming the proposed excretory pathway of ALXN1840. Ultrastructural analysis of mice treated with dosages comparable to the bis-choline-tetrathiomolybdate dosage used in an ongoing phase III trial in Wilson’s disease patients, as well as D-penicillamine and trientine, did not show relevant mitochondrial damage. In contrast, a high dose of ALXN1840 applied for four weeks triggered dramatic structural changes in mitochondria, which were notably characterized by the formation of holes with variable sizes. Although these experimental results may not be applicable to patients with Wilson’s disease, the data suggests that ALXN1840 should be administered at low concentrations to prevent mitochondrial dysfunction and overload of hepatic excretory pathways.

Copper restoration by liver Ceruloplasmin ablation ameliorates NAFLD via SCO1-AMPK-LKB1 complex

Copper is an essential nutrient and a co-factor of numerous enzymes governing a wide range of intracellular processes. Copper deficiency has emerged to be associated with various lipid metabolism diseases, including non-alcoholic fatty liver disease (NAFLD). However, the molecular mechanisms of how copper regulates lipid metabolism and is sensed remain elusive. Here, we reveal that copper elevation caused by hepatic ceruloplasmin (CP) ablation enhances lipid catabolism by promoting the assembly of copper-load SCO1/AMPK complex. We report that overnutrition-mediated CP elevation results in hepatic copper loss, and that liver-specific CP ablation counteracts this reduction in copper levels and ameliorates NAFLD in mice. Mechanistically, SCO1 constitutively interacts with LKB1 even in the absence of copper, and copper-loaded SCO1 directly tethers LKB1 to AMPK, thereby activating AMPK and consequently promoting mitochondrial biogenesis and fatty acid oxidation in hepatocytes. Therefore, this study reveals an unexpected role for AMPK to sense copper alteration via SCO1 and uncovers a previously unidentified mechanism by which copper, as a signaling molecule, improves hepatic lipid catabolism, and indicates that targeting copper-AMPK signaling pathway ameliorates NAFLD development by modulating AMPK activity.

Diagnosis of Wilson Disease and Its Phenotypes by Using Artificial Intelligence

WD is caused by ATP7B variants disrupting copper efflux resulting in excessive copper accumulation mainly in liver and brain. The diagnosis of WD is challenged by its variable clinical course, onset, morbidity, and ATP7B variant type. Currently it is diagnosed by a combination of clinical symptoms/signs, aberrant copper metabolism parameters (e.g., low ceruloplasmin serum levels and high urinary and hepatic copper concentrations), and genetic evidence of ATP7B mutations when available. As early diagnosis and treatment are key to favorable outcomes, it is critical to identify subjects before the onset of overtly detrimental clinical manifestations. To this end, we sought to improve WD diagnosis using artificial neural network algorithms (part of artificial intelligence) by integrating available clinical and molecular parameters. Surprisingly, WD diagnosis was based on plasma levels of glutamate, asparagine, taurine, and Fischer’s ratio. As these amino acids are linked to the urea–Krebs’ cycles, our study not only underscores the central role of hepatic mitochondria in WD pathology but also that most WD patients have underlying hepatic dysfunction. Our study provides novel evidence that artificial intelligence utilized for integrated analysis for WD may result in earlier diagnosis and mechanistically relevant treatments for patients with WD.

Dietary Iron Intake in Excess of Requirements Impairs Intestinal Copper Absorption in Sprague Dawley Rat Dams, Causing Copper Deficiency in Suckling Pups

Physiologically relevant iron-copper interactions have been frequently documented. For example, excess enteral iron inhibits copper absorption in laboratory rodents and humans. Whether this also occurs during pregnancy and lactation, when iron supplementation is frequently recommended, is, however, unknown. Here, the hypothesis that high dietary iron will perturb copper homeostasis in pregnant and lactating dams and their pups was tested. We utilized a rat model of iron-deficiency/iron supplementation during pregnancy and lactation to assess this possibility. Rat dams were fed low-iron diets early in pregnancy, and then switched to one of 5 diets with normal (1×) to high iron (20×) until pups were 14 days old. Subsequently, copper and iron homeostasis, and intestinal copper absorption (by oral, intragastric gavage with 64Cu), were assessed. Copper depletion/deficiency occurred in the dams and pups as dietary iron increased, as evidenced by decrements in plasma ceruloplasmin (Cp) and superoxide dismutase 1 (SOD1) activity, depletion of hepatic copper, and liver iron loading. Intestinal copper transport and tissue 64Cu accumulation were lower in dams consuming excess iron, and tissue 64Cu was also low in suckling pups. In some cases, physiological disturbances were noted when dietary iron was only ~3-fold in excess, while for others, effects were observed when dietary iron was 10–20-fold in excess. Excess enteral iron thus antagonizes the absorption of dietary copper, causing copper depletion in dams and their suckling pups. Low milk copper is a likely explanation for copper depletion in the pups, but experimental proof of this awaits future experimentation.

COMMD1 Exemplifies the Power of Inbred Dogs to Dissect Genetic Causes of Rare Copper-Related Disorders

Wilson’s Disease is a rare autosomal recessive disorder in humans, often presenting with hepatic copper overload. Finding the genetic cause of a rare disease, especially if it is related to food constituents like the trace element copper, is a Herculean task. This review describes examples of how the unique population structure of in-bred dog strains led to the discovery of a novel gene and two modifier genes involved in inherited copper toxicosis. COMMD1, after the discovery in 2002, was shown to be a highly promiscuous protein involved in copper transport, protein trafficking/degradation, regulation of virus replication, and inflammation. Mutations in the ATP7A and ATP7B proteins in Labrador retrievers and Dobermann dogs resulted in a wide variation in hepatic copper levels in these breeds. To our knowledge, numerous dog breeds with inherited copper toxicosis of unknown genetic origin exist. Therefore, the possibility that men’s best friend will provide new leads in rare copper storage diseases seems realistic.

Wilson’s Disease Presenting in Late Adult Life

Wilson’s disease (WD) is an autosomal recessive disease affecting the copper metabolism resulting in various clinical presentations. Diagnosis includes the presence of low serum copper and ceruloplasmin concentrations, increased urinary copper excretion, and/or increased hepatic copper concentrations. Yet, genetic testing remains diagnostic. Management includes copper chelating agents and liver transplant in advance cases. We report a case of WD presenting with liver function impairment in late adult life and started on treatment. Therefore, early diagnosis and treatment of WD can prevent related complications.