ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson's Disease Rats.

BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

Copper impairs the intestinal barrier integrity in Wilson disease.

In Wilson disease (WD), liver copper (Cu) excess, caused by mutations in the ATPase Cu transporting beta (ATP7B), has been extensively studied. In contrast, in the gastrointestinal tract, responsible for dietary Cu uptake, ATP7B malfunction is poorly explored. We therefore investigated gut biopsies from WD patients and compared intestines from two rodent WD models and from human ATP7B knock-out intestinal cells to their respective wild-type controls. We observed gastrointestinal (GI) inflammation in patients, rats and mice lacking ATP7B. Mitochondrial alterations and increased intestinal leakage were observed in WD rats, Atp7b-/- mice and human ATP7B KO Caco-2 cells. Proteome analyses of intestinal WD homogenates revealed profound alterations of energy and lipid metabolism. The intestinal damage in WD animals and human ATP7B KO cells did not correlate with absolute Cu elevations, but likely reflects intracellular Cu mislocalization. Importantly, Cu depletion by the high-affinity Cu chelator methanobactin (MB) restored enterocyte mitochondria, epithelial integrity, and resolved gut inflammation in WD rats and human WD enterocytes, plausibly via autophagy-related mechanisms. Thus, we report here before largely unrecognized intestinal damage in WD, occurring early on and comprising metabolic and structural tissue damage, mitochondrial dysfunction, and compromised intestinal barrier integrity and inflammation, that can be resolved by high-affinity Cu chelation treatment.

Farnesoid X receptor activation by bile acids suppresses lipid peroxidation and ferroptosis.

Ferroptosis is a regulated cell death modality that occurs upon iron-dependent lipid peroxidation. Recent research has identified many regulators that induce or inhibit ferroptosis; yet, many regulatory processes and networks remain to be elucidated. In this study, we performed a chemical genetics screen using small molecules with known mode of action and identified two agonists of the nuclear receptor Farnesoid X Receptor (FXR) that suppress ferroptosis, but not apoptosis or necroptosis. We demonstrate that in liver cells with high FXR levels, knockout or inhibition of FXR sensitized cells to ferroptotic cell death, whereas activation of FXR by bile acids inhibited ferroptosis. Furthermore, FXR inhibited ferroptosis in ex vivo mouse hepatocytes and human hepatocytes differentiated from induced pluripotent stem cells. Activation of FXR significantly reduced lipid peroxidation by upregulating the ferroptosis gatekeepers GPX4, FSP1, PPARα, SCD1, and ACSL3. Together, we report that FXR coordinates the expression of ferroptosis-inhibitory regulators to reduce lipid peroxidation, thereby acting as a guardian of ferroptosis.

Bis-choline tetrathiomolybdate prevents copper-induced blood-brain barrier damage.

In Wilson disease, excessive copper accumulates in patients' livers and may, upon serum leakage, severely affect the brain according to current viewpoints. Present remedies aim at avoiding copper toxicity by chelation, for example, by D-penicillamine (DPA) or bis-choline tetrathiomolybdate (ALXN1840), the latter with a very high copper affinity. Hence, ALXN1840 may potentially avoid neurological deterioration that frequently occurs upon DPA treatment. As the etiology of such worsening is unclear, we reasoned that copper loosely bound to albumin, that is, mimicking a potential liver copper leakage into blood, may damage cells that constitute the blood-brain barrier, which was found to be the case in an in vitro model using primary porcine brain capillary endothelial cells. Such blood-brain barrier damage was avoided by ALXN1840, plausibly due to firm protein embedding of the chelator bound copper, but not by DPA. Mitochondrial protection was observed, a prerequisite for blood-brain barrier integrity. Thus, high-affinity copper chelators may minimize such deterioration in the treatment of neurologic Wilson disease.

A High-Calorie Diet Aggravates Mitochondrial Dysfunction and Triggers Severe Liver Damage in Wilson Disease Rats.

BACKGROUND & AIMS: In Wilson disease, ATP7B mutations impair copper excretion into bile. Hepatic copper accumulation may induce mild to moderate chronic liver damage or even acute liver failure. Etiologic factors for this heterogeneous phenotype remain enigmatic. Liver steatosis is a frequent finding in Wilson disease patients, suggesting that impaired copper homeostasis is linked with liver steatosis. Hepatic mitochondrial function is affected negatively both by copper overload and steatosis. Therefore, we addressed the question of whether a steatosis-promoting high-calorie diet aggravates liver damage in Wilson disease via amplified mitochondrial damage. METHODS: Control Atp7b+/- and Wilson disease Atp7b-/- rats were fed either a high-calorie diet (HCD) or a normal diet. Copper chelation using the high-affinity peptide methanobactin was used in HCD-fed Atp7b-/- rats to test for therapeutic reversal of mitochondrial copper damage. RESULTS: In comparison with a normal diet, HCD feeding of Atp7b-/- rats resulted in a markedly earlier onset of clinically apparent hepatic injury. Strongly increased mitochondrial copper accumulation was observed in HCD-fed Atp7b-/- rats, correlating with severe liver injury. Mitochondria presented with massive structural damage, increased H2O2 emergence, and dysfunctional adenosine triphosphate production. Hepatocellular injury presumably was augmented as a result of oxidative stress. Reduction of mitochondrial copper by methanobactin significantly reduced mitochondrial impairment and ameliorated liver damage. CONCLUSIONS: A high-calorie diet severely aggravates hepatic mitochondrial and hepatocellular damage in Wilson disease rats, causing an earlier onset of the disease and enhanced disease progression.

The exceptional sensitivity of brain mitochondria to copper.

Wilson disease (WD) is characterized by a disrupted copper homeostasis resulting in dramatically increased copper levels, mainly in liver and brain. While copper damage to mitochondria is an established feature in WD livers, much less is known about such detrimental copper effects in other organs. We therefore assessed the mitochondrial sensitivity to copper in a tissue specific manner, namely of isolated rat liver, kidney, heart, and brain mitochondria. Brain mitochondria presented with exceptional copper sensitivity, as evidenced by a comparatively early membrane potential loss, profound structural changes already at low copper dose, and a dose-dependent reduced capacity to produce ATP. This sensitivity was likely due to a copper-dependent attack on free protein thiols and due to a decreased copper reactive defense system, as further evidenced in neuroblastoma SHSY5Y cells. In contrast, an increased production of reactive oxygen species was found to be a late-stage event, only occurring in destroyed mitochondria. We therefore propose mitochondrial protein thiols as major targets of mitochondrial copper toxicity.

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

In Wilson disease (WD), functional loss of ATPase copper-transporting ß (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.