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.

Subzero project: comparing trace element profiles of enriched mitochondria fractions from frozen and fresh liver tissue.

From organs to subcellular organelles, trace element (TE) homeostasis is fundamental for many physiological processes. While often overlooked in early stages, manifested TE disbalance can have severe health consequences, particularly in the context of aging or pathological conditions. Monitoring TE concentrations at the mitochondrial level could identify organelle-specific imbalances, contributing to targeted diagnostics and a healthier aging process. However, mitochondria isolation from frozen tissue is challenging, as it poses the risk of TE losses from the organelles due to cryodamage, but would significantly ease routine laboratory work. To address this, a novel method to isolate an enriched mitochondria fraction (EMF) from frozen tissue was adapted from already established protocols. Validation of manganese (Mn), iron (Fe), and copper (Cu) quantification via inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) showed sufficiently low quantification limits for EMF TE analysis. Successful mitochondrial enrichment from frozen liver samples was confirmed via immunoblots and transmission electron microscopy (TEM) revealed sufficient structural integrity of the EMFs. No significant differences in EMF TEs between frozen and fresh tissue were evident for Mn and Cu and only slight decreases in EMF Fe. Consequently, EMF TEs were highly comparable for isolates from both tissue states. In application, this method effectively detected dietary differences in EMF Fe of a murine feeding study and identified the disease status in a Wilson disease rat model based on drastically increased EMF Cu. In summary, the present method is suitable for future applications, facilitating sample storage and high-throughput analyses of mitochondrial TEs.

OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

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.

Lysosomal TRPML1 regulates mitochondrial function in hepatocellular carcinoma cells.

Liver cancers, including hepatocellular carcinoma (HCC), are the second leading cause of cancer death worldwide, and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 (also known as MCOLN1) has gained focus in cancer research because it represents an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for overactivation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis and proteomic alterations. Furthermore, we analyzed mitochondrial function in detail by performing confocal and transmission electron microscopy combined with SeahorseTM and Oroboros® functional analysis. We report that TRPML1 overactivation mediated by a novel, isoform-selective small-molecule activator induces apoptosis by impairing mitochondrial function in a Ca2+-dependent manner. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy.

IL-6-induced FOXO1 activity determines the dynamics of metabolism in CD8 T cells cross-primed by liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) are liver-resident antigen (cross)-presenting cells that generate memory CD8 T cells, but metabolic properties of LSECs and LSEC-primed CD8 T cells remain understudied. Here, we report that high-level mitochondrial respiration and constitutive low-level glycolysis support LSEC scavenger and sentinel functions. LSECs fail to increase glycolysis and co-stimulation after TLR4 activation, indicating absence of metabolic and functional maturation compared with immunogenic dendritic cells. LSEC-primed CD8 T cells show a transient burst of oxidative phosphorylation and glycolysis. Mechanistically, co-stimulatory IL-6 signaling ensures high FOXO1 expression in LSEC-primed CD8 T cells, curtails metabolic activity associated with T cell activation, and is indispensable for T cell functionality after re-activation. Thus, distinct immunometabolic features characterize non-immunogenic LSECs compared with immunogenic dendritic cells and LSEC-primed CD8 T cells with memory features compared with effector CD8 T cells. This reveals local features of metabolism and function of T cells in the liver.

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.

Mitochondrial Impairment by MitoBloCK-6 Inhibits Liver Cancer Cell Proliferation.

Augmenter of liver regeneration (ALR) is a critical multi-isoform protein with its longer isoform, located in the mitochondrial intermembrane space, being part of the mitochondrial disulfide relay system (DRS). Upregulation of ALR was observed in multiple forms of cancer, among them hepatocellular carcinoma (HCC). To shed light into ALR function in HCC, we used MitoBloCK-6 to pharmacologically inhibit ALR, resulting in profound mitochondrial impairment and cancer cell proliferation deficits. These effects were mostly reversed by supplementation with bioavailable hemin b, linking ALR function to mitochondrial iron homeostasis. Since many tumor cells are known for their increased iron demand and since increased iron levels in cancer are associated with poor clinical outcome, these results help to further advance the intricate relation between iron and mitochondrial homeostasis in liver cancer.

Isolation and Electron Microscopic Analysis of Liver Cancer Cell Mitochondria.

Isolation of mitochondria is a crucial method for examining molecular details of this organelle's manifold functions. Historically, mitochondrial isolations required large amounts of sample material which impeded their isolation from cultured cells. We have therefore developed a method allowing for controlled and reproducible isolation of intact and functional mitochondria from diverse cell types in culture. Here we provide a methodological update of this approach together with a protocol for the subsequent analysis of such isolated mitochondria by electron microscopy. Combining the isolation procedure with this powerful imaging method can reveal ultrastructural mitochondrial peculiarities in disease settings that might not be evident in intact cells and allows for assessment of mitochondrial membrane integrity and sample purity.

Adipocyte hypertrophy parallels alterations of mitochondrial status in a cell model for adipose tissue dysfunction in obesity.

AIMS: Adipocyte hypertrophy is the main cause of obesity. A deeper understanding of the molecular mechanisms regulating adipocyte dysfunction may help to plan strategies to treat/prevent obesity and its metabolic complications. Here, we investigated in vitro the molecular alterations associated with early adipocyte hypertrophy, focusing on mitochondrial dysfunction. MAIN METHODS: As model of adipocyte hypertrophy, we employed 3T3-L1 preadipocytes firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids. As a function of differentiation and hypertrophy, we assessed triglyceride content, lipid droplet size, radical homeostasis by spectrophotometry and microscopy, as well as the expression of PPARγ, adiponectin and metallothioneins. Mitochondrial status was investigated by electron microscopy, oxygraph 2 k (O2K) high-resolution respirometry, fluorimetry and western blot. KEY FINDINGS: Compared to mature adipocytes, hypertrophic adipocytes showed increased triglyceride accumulation and lipid peroxidation, larger or unique lipid droplet, up-regulated expression of PPARγ, adiponectin and metallothioneins. At mitochondrial level, early-hypertrophic adipocytes exhibited: (i) impaired mitochondrial oxygen consumption with parallel reduction in the mitochondrial complexes; (ii) no changes in citrate synthase and HSP60 expression, and in the inner mitochondrial membrane polarization; (iii) no stimulation of mitochondrial fatty acid oxidation. Our findings indicate that the content, integrity, and catabolic activity of mitochondria were rather unchanged in early hypertrophic adipocytes, while oxygen consumption and oxidant production were altered. SIGNIFICANCE: In the model of early adipocyte hypertrophy exacerbated oxidative stress and impaired mitochondrial respiration were observed, likely depending on reduction in the mitochondrial complexes, without changes in mitochondrial mass and integrity.

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.

Mitochondrial adaptation in steatotic mice.

Western lifestyle-associated malnutrition causes steatosis that may progress to liver inflammation and mitochondrial dysfunction has been suggested as a key factor in promoting this disease. Here we have molecularly, biochemically and biophysically analyzed mitochondria from steatotic wild type and immune-compromised mice fed a Western diet (WD) - enriched in saturated fatty acids (SFAs). WD-mitochondria demonstrated lipidomic changes, a decreased mitochondrial ATP production capacity and a significant sensitivity to calcium. These changes preceded hepatocyte damage and were not associated with enhanced ROS production. Thus, WD-mitochondria do not promote steatohepatitis per se, but demonstrate bioenergetic deficits and increased sensitivity to stress signals.

Data on chow, liver tissue and mitochondrial fatty acid compositions as well as mitochondrial proteome changes after feeding mice a western diet for 6-24 weeks.

The data presented in this article describe the fatty acid composition of chow, liver tissue and isolated liver mitochondria from mice fed for 6-24 weeks with a high caloric western diet (WD) in comparison to control diet (normal diet, ND). The fatty acid composition was measured via gas chromatography flame ionization detection (GC-FID). Moreover, WD-induced mitochondrial protein changes are presented in this work and were analyzed by mass spectrometry (LC-MS/MS). For further interpretation and discussion of the presented data please refer to the research article entitled "Mitochondrial adaptation in steatotic mice" (Einer et al., 2017) [1].

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.

A protocol for the parallel isolation of intact mitochondria from rat liver, kidney, heart, and brain.

Mitochondria are key organelles for cellular energy production and cell death decisions. Consequently, a plethora of conditions which are toxic to cells are known to directly attack these organelles. However, mitochondria originating from different tissues differ in their sensitivity to toxic insults. Thus, in order to predict the potential organ-specific toxicity of a given drug or pathological condition at the mitochondrial level, test settings are needed that directly compare the responses and vulnerabilities of mitochondria from different organs. As a prerequisite for such test strategies, we provide here a robust, prompt, and easy-to-follow step-by-step protocol to simultaneously isolate functional and intact mitochondria from rat liver, kidney, heart, and brain. This isolation procedure ensures mitochondrial preparations of comparable purity and reproducible quantities which can be subsequently analyzed for organ-specific mitochondrial toxicity.

Isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses.

We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66-74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66-74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities.

Why to compare absolute numbers of mitochondria.

Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.

A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies.

Mitochondrial dysfunctions decisively contribute to the progression of human diseases, implying that functional tests of isolated mitochondria may furnish conclusive information for diagnosis and therapy. Classical mitochondrial isolation methods, however, lack precisely adjustable settings for cell rupture, which is the most critical step in this procedure, and this complicates subsequent analyses. Here, we present an efficient method to isolate functionally active, intact mitochondria from cultured or primary cells and minute tissue samples in a rapid, highly reproducible manner.

TCDD induces the expression of insulin-like growth factor binding protein 4 in 5L rat hepatoma cells: a cautionary tale of the use of this cell line in studies on dioxin toxicity.

Previous quantitative proteomic studies on the actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells, a cell model frequently used for investigating the mechanisms of TCDD toxicity, had indicated that dioxin exposure reduced the abundance of numerous proteins which are regulated at the level of protein synthesis initiation. In the present study, we have analysed the mechanism mediating this inhibition. TCDD treatment of the cells largely prevented the activation of eukaryotic translation initiation factor 4E-binding protein 1, a regulator of translation initiation and substrate of the mammalian target of rapamycin (mTOR). By "working upwards" from mTOR, we observed that TCDD inhibited endogenous and IGF-I-induced AKT and ERK activation by interfering with tyrosine phosphorylation of insulin receptor substrate 1. This inhibition was mediated by a TCDD-induced secreted factor which was identified as insulin-like growth factor binding protein 4 (IGFBP-4). The induction of IGFBP-4 protein was dependent on a functional aryl hydrocarbon receptor and was preceded by a rapid increase in the level of IGFBP-4 mRNA indicating that IGFBP-4 is a previously unknown transcriptional target of TCDD in 5L cells. IGFBP-4 was not induced by TCDD in the parental cell line of 5L cells, Fao, and in various closely related rat hepatoma cell lines as well as in other unrelated cell types. Analysis of 5L cell chromosomes by multicolour spectral karyotyping (SKY) revealed that the cells carry several hitherto uncharacterised chromosomal translocations. The observations suggest that in 5L cells the Igfbp-4 gene may have got under the control of a promoter containing dioxin responsive element(s) leading to the induction of IGFBP-4 by TCDD. These findings emphasise a particular caution when interpreting and extrapolating results on the action mechanisms of TCDD obtained in studies using 5L cells as a model system.