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Abstract Background The permeabilization of mitochondrial membranes is a decisive event in apoptosis or necrosis culminating in cell death. One fundamental mechanism by which such permeabilization events occur is the calcium-induced mitochondrial permeability transition. Upon Ca2+-uptake into mitochondria an increase in inner membrane permeability occurs by a yet unclear mechanism. This leads to a net water influx in the mitochondrial matrix, mitochondrial swelling, and finally the rupture of the outer membrane. Although already described more than thirty years ago, many unsolved questions surround this important biological phenomenon. Importantly, theoretical modeling of the mitochondrial permeability transition has only started recently and the existing mathematical models fail to characterize the swelling process throughout the whole time range. Results We propose here a new mathematical approach to the mitochondrial permeability transition introducing a specific delay equation and resulting in an optimized representation of mitochondrial swelling. Our new model is in accordance with the experimentally determined course of volume increase throughout the whole swelling process, including its initial lag phase as well as its termination. From this new model biological consequences can be deduced, such as the confirmation of a positive feedback of mitochondrial swelling which linearly depends on the Ca2+-concentration, or a negative exponential dependence of the average swelling time on the Ca2+-concentration. Finally, our model can show an initial shrinking phase of mitochondria, which is often observed experimentally before the actual swelling starts. Conclusions We present a model of the mitochondrial swelling kinetics. This model may be adapted and extended to diverse other inducing/inhibiting conditions or to mitochondria from other biological sources and thus may benefit a better understanding of the mitochondrial permeability transition.

Introduction/aims: Swim training and regulation of copper metabolism result in clinical benefits in amyotrophic lateral sclerosis (ALS) mice. Therefore, the study aimed to determine whether swim training improves copper metabolism by modifying copper metabolism in the skeletal muscles of ALS mice., Methods: SOD1G93A mice (n = 6 per group) were used as the ALS model, and wild-type B6SJL (WT) mice as controls (n = 6). Mice with ALS were analyzed before the onset of ALS (ALS BEFORE), at baseline ALS (first disease symptoms, trained and untrained, ALS ONSET), and at the end of ALS (last stage disease, trained and untrained, ALS TERMINAL). Copper concentrations and the level of copper metabolism proteins in the skeletal muscles of the lower leg were determined., Results: ALS disease caused a reduction in the copper concentration in ALS TERMINAL untrained mice compared with the ALS BEFORE (10.43 ± 1.81 and 38.67 ± 11.50 μg/mg, respectively, p = .0213). The copper chaperon for SOD1 protein, which supplies copper to SOD1, and ATPase7a protein (copper exporter), increased at the terminal stage of disease by 57% (p = .0021) and 34% (p = .0372), while the CTR1 protein (copper importer) decreased by 45% (p = .002). Swim training moderately affected the copper concentration and the concentrations of proteins responsible for copper metabolism in skeletal muscles., Discussion: The results show disturbances in skeletal muscle copper metabolism associated with ALS progression, which is moderately affected by swim training. From a clinical point of view, exercise in water for ALS patients should be an essential element of rehabilitation for maintaining quality of life., (© 2024 The Author(s). Muscle & Nerve published by Wiley Periodicals LLC.)

Higher eukaryotes' life is impossible without copper redox activity and, literally, every breath we take biochemically demonstrates this. However, this dependence comes at a considerable price to ensure target-oriented copper action. Thereto its uptake, distribution but also excretion are executed by specialized proteins with high affinity for the transition metal. Consequently, malfunction of copper enzymes/transporters, as is the case in hereditary Wilson disease that affects the intracellular copper transporter ATP7B, comes with serious cellular damage. One hallmark of this disease is the progressive copper accumulation, primarily in liver but also brain that becomes deadly if left untreated. Such excess copper toxicity may also result from accidental ingestion or attempted suicide. Recent research has shed new light into the cell-toxic mechanisms and primarily affected intracellular targets and processes of such excess copper that may even be exploited with respect to cancer therapy. Moreover, new therapies are currently under development to fight against deadly toxic copper., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest HZ is scientific consultant for ArborMed Co. Ltd. GK is supported by the Ligue contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR) – Projets blancs; AMMICa US23/CNRS UMS3655; Association pour la recherche sur le cancer (ARC); Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen; European Joint Programme on Rare Diseases (EJPRD); European Research CouncilAdvanced Investigator Award (ERC-2021-ADG, ICD-Cancer, Grant No. 101052444), European Union Horizon 2020 Projects Oncobiome, Prevalung (grant No. 101095604) and Crimson; Institut National du Cancer (INCa); Institut Universitaire de France; LabEx Immuno-Oncology ANR-18-IDEX-0001; a Cancer Research ASPIRE Award from the Mark Foundation; the RHUs Immunolife and LUCA-pi; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM). This study contributes to the IdEx Université de Paris ANR-18-IDEX-0001. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union, the European Research Council or any other granting authority. Neither the European Union nor any other granting authority can be held responsible for them., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Essential transition metals have key roles in oxygen transport, neurotransmitter synthesis, nucleic acid repair, cellular structure maintenance and stability, oxidative phosphorylation, and metabolism. The balance between metal deficiency and excess is typically ensured by several extracellular and intracellular mechanisms involved in uptake, distribution, and excretion. However, provoked by either intrinsic or extrinsic factors, excess iron, zinc, copper, or manganese can lead to cellular damage upon chronic or acute exposure, frequently attributed to oxidative stress. Intracellularly, mitochondria are the organelles that require the tightest control concerning reactive oxygen species production, which inevitably leaves them to be one of the most vulnerable targets of metal toxicity. Current therapies to counteract metal overload are focused on chelators, which often cause secondary effects decreasing patients' quality of life. New therapeutic options based on synthetic or natural antioxidants have proven positive effects against metal intoxication. In this review, we briefly address the cellular metabolism of transition metals, consequences of their overload, and current therapies, followed by their potential role in inducing oxidative stress and remedies thereof.

Lagunamide A is a biologically active natural product with a yet unidentified molecular mode of action. Cellular studies revealed that lagunamide A is a potent inhibitor of cancer cell proliferation, promotes apoptosis and causes mitochondrial dysfunction. To decipher the cellular mechanism responsible for these effects, we utilized thermal protein profiling (TPP) and identified EYA3 as a stabilized protein in cells upon lagunamide A treatment. EYA3, involved in the DNA damage repair process, was functionally investigated via siRNA based knockdown studies and corresponding effects of lagunamide A on DNA repair were confirmed. Furthermore, we showed that lagunamide A sensitized tumor cells to treatment with the drug doxorubicin highlighting a putative therapeutic strategy., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.

Swim training has increased the life span of the transgenic animal model of amyotrophic lateral sclerosis (ALS). Conversely, the progress of the disease is associated with the impairment of iron metabolism and insulin signaling. We used transgenic hmSOD1 G93A (ALS model) and non-transgenic mice in the present study. The study was performed on the muscles taken from trained (ONSET and TERMINAL) and untrained animals at three stages of the disease: BEFORE, ONSET, and TERMINAL. In order to study the molecular mechanism of changes in iron metabolism, we used SH-SY5Y and C2C12 cell lines expression vector pcDNA3.1 and transiently transfected with specific siRNAs. The progress of ALS resulted in decreased P-Akt/Akt ratio, which is associated with increased proteins responsible for iron storage ferritin L, ferritin H, PCBP1, and skeletal muscle iron at ONSET. Conversely, proteins responsible for iron export- TAU significantly decrease. The training partially reverses changes in proteins responsible for iron metabolism. AKT silencing in the SH-SY5Y cell line decreased PCBP2 and ferroportin and increased ferritin L, H, PCBP1, TAU, transferrin receptor 1, and APP. Moreover, silencing APP led to an increase in ferritin L and H. Our data suggest that swim training in the mice ALS model is associated with significant changes in iron metabolism related to AKT activity. Down-regulation of AKT mainly upregulates proteins involved in iron import and storage but decreases proteins involved in iron export., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023. Published by Elsevier B.V.)

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., (© 2023. The Author(s).)

Labile copper(II) ions (Cu 2+ ) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu 2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu 2+ subset. This study introduces a fluorometric high throughput assay using the novel Cu 2+ binding fluoresceine-peptide sensor FP4 (Kd of the Cu 2+ -FP4-complex 0.38 pM) to determine labile Cu 2+ in human and rat serum. Using 96 human serum samples, labile Cu 2+ was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu 2+ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu 2+ in the sera of 19 patients with WD showed a significant decrease in labile Cu 2+ following copper chelation therapy, suggesting that labile Cu 2+ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress., (© 2023. Springer Nature Limited.)

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., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Selenium homeostasis depends on hepatic biosynthesis of selenoprotein P (SELENOP) and SELENOP-mediated transport from the liver to e.g. the brain. In addition, the liver maintains copper homeostasis. Selenium and copper metabolism are inversely regulated, as increasing copper and decreasing selenium levels are observed in blood during aging and inflammation. Here we show that copper treatment increased intracellular selenium and SELENOP in hepatocytes and decreased extracellular SELENOP levels. Hepatic accumulation of copper is a characteristic of Wilson's disease. Accordingly, SELENOP levels were low in serum of Wilson's disease patients and Wilson's rats. Mechanistically, drugs targeting protein transport in the Golgi complex mimicked some of the effects observed, indicating a disrupting effect of excessive copper on intracellular SELENOP transport resulting in its accumulation in the late Golgi. Our data suggest that hepatic copper levels determine SELENOP release from the liver and may affect selenium transport to peripheral organs such as the brain., (© 2023. The Author(s).)

Mutations in the HFE / Hfe gene cause Hereditary Hemochromatosis (HH), a highly prevalent genetic disorder characterized by elevated iron deposition in multiple tissues. HFE acts in hepatocytes to control hepcidin expression, whereas HFE actions in myeloid cells are required for cell-autonomous and systemic iron regulation in aged mice. To address the role of HFE specifically in liver-resident macrophages, we generated mice with a selective Hfe deficiency in Kupffer cells ( Hfe Clec4fCre ). The analysis of the major iron parameters in this novel Hfe Clec4fCre mouse model led us to the conclusion that HFE actions in Kupffer cells are largely dispensable for cellular, hepatic and systemic iron homeostasis.

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X-Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids., (open access.)

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP + ) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C 10 -TPP + ). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing., Methods: In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference., Results: ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below., Conclusion: Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)