Miclxin, a Novel MIC60 Inhibitor, Induces Apoptosis via Mitochondrial Stress in ß-Catenin Mutant Tumor Cells.

The Wnt signaling pathway regulates diverse cellular processes. ß-Catenin is one of the major components of this pathway, in which it plays a main role. Although it has been established that ß-catenin is mutated in a wide variety of tumors, there are currently no effective therapeutic agents that target ß-catenin. In this study, we searched for the compound that targets mutant ß-catenin and found DS37262926 (miclxin). Miclxin exhibited ß-catenin-dependent apoptosis in ß-catenin-mutated HCT116 cells and isogenic HCT116 (CTNNB1 Δ45/-) cells; however, this effect was not observed in isogenic HCT116 (CTNNB1 +/-) cells. Using miclxin-immobilized beads, MIC60, one of the major components of the mitochondrial contact site and cristae organizing system (MICOS) complex, was identified as a target protein of miclxin. We revealed that MIC60 dysfunction caused by miclxin induced a mitochondrial stress response in a mutant ß-catenin-dependent manner. Activation of the mitochondrial stress response was responsible for the downregulation of Bcl-2, leading to severe loss of mitochondrial membrane potential and subsequent apoptosis-inducing factor-dependent apoptosis. Our findings suggest that targeting MIC60 is a potential strategy with which tumor cells can be killed through induction of severe mitochondrial damage in a mutant ß-catenin-dependent manner.

Targeting putative components of the mitochondrial permeability transition pore for novel therapeutics.

Few discoveries have influenced drug discovery programs more than the finding that mitochondrial membranes undergo swings in permeability in response to cellular perturbations. The conductor of these permeability changes is the aptly named mitochondrial permeability transition pore which, although not yet precisely defined, is comprised of several integral proteins that differentially act to regulate the flux of ions, proteins and metabolic byproducts during the course of cellular physiological functions but also pathophysiological insults. Pursuit of the pore's exact identity remains a topic of keen interest, but decades of research have unearthed provocative functions for the integral proteins leading to their evaluation to develop novel therapeutics for a wide range of clinical indications. Chief amongst these targeted, integral proteins have been the Voltage Dependent Anion Channel (VDAC) and the F1FO ATP synthase. Research associated with the roles and ligands of VDAC has been extensive and we will expand upon 3 examples of ligand:VDAC interactions for consideration of drug discovery projects: Tubulin:VDAC1, Hexokinase I/II:VDAC1 and olesoxime:VDAC1. The discoveries that cyclosporine blocks mitochondrial permeability transition via binding to cyclophilin D, and that cyclophilin D is an important component of F1FO ATP synthase, has heightened interest in the F1FO ATP synthase as a focal point for drug discovery, and we will discuss 2 plausible campaigns associated with disease indications. To date no drug has emerged from prospective targeting these integral proteins; however, continued exploration such as the approaches suggested in this Commentary will increase the likelihood of providing important therapeutics for severely unmet medical needs.

The mitochondrial carrier Citrin plays a role in regulating cellular energy during carcinogenesis.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

NURR1 inhibition reduces hypoxia-mediated cardiomyocyte necrosis via blocking Mst1-JNK-mPTP pathway.

Context: Although many studies have investigated the molecular mechanisms underlying hypoxia-related cardiomyocyte damage, the role of necrosis in cardiomyocyte death.Objective: The aim of our study is to explore the pathological role of nuclear receptor related 1 protein (NURR1) in regulating cardiomyocyte viability under hypoxia stress.Materials and methods: Cardiomyocyte was treated with hypoxia and siRNA against NURR1 was transfected into cardiomyocyte. Pathway agonist was used to activate the Mst1-JNK-mPTP pathway in cardiomyocyte.Results: In our study, the expression of NURR1 was rapidly increased in cardiomyocyte transfected with NURR1. Knockout of NURR1 could promote cardiomyocyte survival, reduce cell death and repress inflammation response. Mechanistically, NURR1 upregulation was associated with an activation of Mst1-JNK pathway and the latter promoted the mPTP opening in cardiomyocyte. Excessive mPTP opening was followed by cardiomyocyte necrosis and this effect could be reversed by NURR1 deletion. Besides, re-activation of Mst1-JNK pathway could abolish the protective effects of NURR1 deletion on cardiomyocytes, as evidenced by increased cell survival and decreased necrosis. Besides, re-activation of Mst1-JNK pathway also abolished NURR1 deletion-mediated mPTP opening.Conclusions: Hypoxia-mediated cardiomyocyte death is associated with NURR1 upregulation which contributes to the activation Mst1-JNK-mPTP pathways.

Second-Generation Inhibitors of the Mitochondrial Permeability Transition Pore with Improved Plasma Stability.

Excessive mitochondrial matrix Ca2+ and oxidative stress leads to the opening of a high-conductance channel of the inner mitochondrial membrane referred to as the mitochondrial permeability transition pore (mtPTP). Because mtPTP opening can lead to cell death under diverse pathophysiological conditions, inhibitors of mtPTP are potential therapeutics for various human diseases. High throughput screening efforts led to the identification of a 3-carboxamide-5-phenol-isoxazole compounds as mtPTP inhibitors. While they showed nanomolar potency against mtPTP, they exhibited poor plasma stability, precluding their use in in vivo studies. Herein, we describe a series of structurally related analogues in which the core isoxazole was replaced with a triazole, which resulted in an improvement in plasma stability. These analogues were readily generated using the copper-catalyzed "click chemistry". One analogue, N-(5-chloro-2-methylphenyl)-1-(4-fluoro-3-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxamide (TR001), was efficacious in a zebrafish model of muscular dystrophy that results from mtPTP dysfunction whereas the isoxazole isostere had minimal effect.

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

Slc25a36 modulates pluripotency of mouse embryonic stem cells by regulating mitochondrial function and glutathione level.

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening.

Necrosis is a key factor in myocardial injury during cardiac pathological processes, such as myocardial infarction (MI), ischemia/reperfusion (I/R) injury and heart failure. Increasing evidence suggests that several aspects of necrosis are programmed and tightly regulated, so targeting the necrosis process has become a new trend for myocardial protection. Multiple cellular signaling pathways have been implicated in necrotic cell death, such as the death receptor-mediated extrinsic and mitochondrial intrinsic pathways. However, the precise mechanisms underlying myocardial necrosis remain unclear. In this study, we showed that apoptosis repressor with caspase recruitment domain (ARC) participated in the mitochondrial intrinsic pathway and inhibited myocardial necrosis by preventing the opening of the mitochondrial permeability transition pore (mPTP). ARC attenuated necrotic cell death triggered by exposure to 500 µM hydrogen peroxide (H2O2) in the cardiomyocyte cell line H9c2. In mice, ARC ameliorated myocardial necrosis, reduced the myocardial infarct size and improved long-term heart function during I/R injury. Mechanistically, it has been shown that the inhibition of necrosis by ARC was dependent on its mitochondrial localization and that ARC prevented the opening of mPTP by targeting CypD, the main regulator of mPTP. In addition, ARC expression was negatively regulated by the transcription factor p53 at the transcriptional level during the necrosis process. These findings identified the novel role of ARC in myocardial necrosis and delineated the p53-ARC-CypD/mPTP necrosis pathway during ischemia- and oxidative stress-induced myocardial damage, which can provide a new strategy for cardiac protection.

Hypoxia induces lactate secretion and glycolytic efflux by downregulating mitochondrial pyruvate carrier levels in human umbilical vein endothelial cells.

The mitochondrial pyruvate carrier (MPC) complex, located on the inner mitochondrial membrane, transports pyruvate to the mitochondrial matrix for oxidative phosphorylation. Previous studies have shown that the MPC complex is a key regulator of glycolysis in tumor cells. The present study evaluated the role of the MPC under hypoxic conditions in human umbilical vein endothelial cells, which rely on glycolysis for energy generation. It was indicated that hypoxia led to an increase in lactate secretion and a decrease in MPC1 and MPC2 levels, which were upregulated following re­oxygenation. In addition, the knockdown of MPC1 or treatment with the MPC inhibitor UK5099 increased the levels of glycolytic enzymes, HK2, PFKFB3, and LDHA, promoting glycolysis and lactate secretion. Taken together, the present data revealed that hypoxia can induce lactate secretion and glycolytic efflux by downregulating MPC levels.

CypD-mPTP axis regulates mitochondrial functions contributing to osteogenic dysfunction of MC3T3-E1 cells in inflammation.

Bone is a dynamic organ, the bone-forming osteoblasts and bone-resorbing osteoclasts form the physiological basis of bone remodeling process. During pathological process of numerous inflammatory diseases, these two aspects are uncoupled and the balance is usually tipped in favor of bone destruction. Evidence suggests that the inflammatory destruction of bone is mainly attributed to oxidative stress and is closely related to mitochondrial dysfunction. The mechanisms underlying osteogenic dysfunction in inflammation still need further investigation. Reactive oxygen species (ROS) is associated with mitochondrial dysfunction and cellular damage. Here, we reported an unexplored role of cyclophilin D (CypD), the major modulator of mitochondrial permeability transition pore (mPTP), and the CypD-mPTP axis in inflammation-induced mitochondrial dysfunction and bone damage. And the protective effects of knocking down CypD by siRNA interference or the addition of cyclosporin A (CsA), an inhibitor of CypD, were evidenced by rescued mitochondrial function and osteogenic function of osteoblast under tumor necrosis factor-α (TNF-α) treatment. These findings provide new insights into the role of CypD-mPTP-dependent mitochondrial pathway in the inflammatory bone injury. The protective effect of CsA or other moleculars affecting the mPTP formation may hold promise as a potential novel therapeutic strategy for inflammation-induced bone damage via mitochondrial pathways.

Discovery of a new mitochondria permeability transition pore (mPTP) inhibitor based on gallic acid.

Pharmacological interventions targeting mitochondria present several barriers for a complete efficacy. Therefore, a new mitochondriotropic antioxidant (AntiOxBEN3) based on the dietary antioxidant gallic acid was developed. AntiOxBEN3 accumulated several thousand-fold inside isolated rat liver mitochondria, without causing disruption of the oxidative phosphorylation apparatus, as seen by the unchanged respiratory control ratio, phosphorylation efficiency, and transmembrane electric potential. AntiOxBEN3 showed also limited toxicity on human hepatocarcinoma cells. Moreover, AntiOxBEN3 presented robust iron-chelation and antioxidant properties in both isolated liver mitochondria and cultured rat and human cell lines. Along with its low toxicity profile and high antioxidant activity, AntiOxBEN3 strongly inhibited the calcium-dependent mitochondrial permeability transition pore (mPTP) opening. From our data, AntiOxBEN3 can be considered as a lead compound for the development of a new class of mPTP inhibitors and be used as mPTP de-sensitiser for basic research or clinical applications or emerge as a therapeutic application in mitochondria dysfunction-related disorders.

Lactate dehydrogenase activity drives hair follicle stem cell activation.

Although normally dormant, hair follicle stem cells (HFSCs) quickly become activated to divide during a new hair cycle. The quiescence of HFSCs is known to be regulated by a number of intrinsic and extrinsic mechanisms. Here we provide several lines of evidence to demonstrate that HFSCs utilize glycolytic metabolism and produce significantly more lactate than other cells in the epidermis. Furthermore, lactate generation appears to be critical for the activation of HFSCs as deletion of lactate dehydrogenase (Ldha) prevented their activation. Conversely, genetically promoting lactate production in HFSCs through mitochondrial pyruvate carrier 1 (Mpc1) deletion accelerated their activation and the hair cycle. Finally, we identify small molecules that increase lactate production by stimulating Myc levels or inhibiting Mpc1 carrier activity and can topically induce the hair cycle. These data suggest that HFSCs maintain a metabolic state that allows them to remain dormant and yet quickly respond to appropriate proliferative stimuli.

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

Development of a Mitochondriotropic Antioxidant Based on Caffeic Acid: Proof of Concept on Cellular and Mitochondrial Oxidative Stress Models.

Targeting mitochondrial oxidative stress is an effective therapeutic strategy. In this context, a rational design of mitochondriotropic antioxidants (compounds 22-27) based on a dietary antioxidant (caffeic acid) was performed. Jointly named as AntiOxCINs, these molecules take advantage of the known ability of the triphenylphosphonium cation to target active molecules to mitochondria. The study was guided by structure-activity-toxicity-property relationships, and we demonstrate in this work that the novel AntiOxCINs act as mitochondriotropic antioxidants. In general, AntiOxCINs derivatives prevented lipid peroxidation and acted as inhibitors of the mitochondrial permeability transition pore. AntiOxCINs toxicity profile was found to be dependent on the structural modifications performed on the dietary antioxidant. On the basis of mitochondrial and cytotoxicity/antioxidant cellular data, compound 25 emerged as a potential candidate for the development of a drug candidate with therapeutic application in mitochondrial oxidative stress-related diseases. Compound 25 increased GSH intracellular levels and showed no toxicity on mitochondrial morphology and function.

Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion.

Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signalling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion.

Protection of PC12 cells from cocaine-induced cell death by inhibiting mitochondrial permeability transition.

Cocaine abuse induces brain injury and neurodegeneration by a mechanism that has not yet been fully elucidated. Mitochondria play a key role in cell death processes, notably through the opening of the permeability transition pore (PTP). In this work, we examined the involvement of the PTP in cocaine-induced toxicity in PC12 cell lines. We used two different PTP inhibitors -i.e. cyclosporin A (CsA) and metformin-to assess their ability to counteract the cocaine induced effects. We first observed that a 48 h exposure to cocaine strongly sensitized cells to calcium overload, as measured by the calcium retention capacity. CsA and metformin significantly decreased the cocaine-induced PTP opening sensitization. We next showed by confocal microscopy that cocaine induced a permanent PTP opening in intact living cells, a phenomenon characterized by the collapse of the mitochondrial membrane potential and the relocation of the NAD(P)H from the mitochondrial matrix to the cytosol. As expected, a cocaine-induced PTP opening was prevented by PTP inhibitors. Finally, a flow cytometry analysis revealed that cocaine induced cell death while CsA and metformin promoted cell survival. Our results demonstrate that cocaine induces PC12 cell death through a mechanism involving permanent PTP opening.

2',3'-Cyclic nucleotide 3'-phosphodiesterase as a messenger of protection of the mitochondrial function during melatonin treatment in aging.

The process of aging is considered to be tightly related to mitochondrial dysfunction. One of the causes of aging is an increased sensitivity to the induction of mitochondrial permeability transition pore (mPTP) opening in the inner membrane of mitochondria. Melatonin, a natural antioxidant, is a hormone produced by the pineal gland. The role of melatonin whose level decreases with aging is well understood. In the present study, we demonstrated that long-term treatment of aged rats with melatonin improved the functional state of mitochondria; thus, the Ca2+ capacity was enhanced and mitochondrial swelling was deaccelerated in mitochondria. Melatonin prevented mPTP and impaired the release of cytochrome c and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from mitochondria of both young and aged rats. Our data suggest that melatonin retains СNPase inside mitochondria, thereby providing the protection of the protein against deleterious effects of 2',3'-cAMP in aging.

Pioglitazone inhibits mitochondrial pyruvate metabolism and glucose production in hepatocytes.

Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14 C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies.