Rescue of mitochondrial import failure by intercellular organellar transfer.

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cell's energy supplies - particularly troublesome for energy-demanding cells of nervous tissue and muscle. Unsurprisingly then, import breakdown is implicated in disease. Here, we explore the consequences of import failure in mammalian cells; wherein, blocking the import machinery impacts mitochondrial ultra-structure and dynamics, but, surprisingly, does not affect import. Our data are consistent with a response involving intercellular mitochondrial transport via tunnelling nanotubes to import healthy mitochondria and jettison those with blocked import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial dysfunction.

The MitoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import Within Mammalian Cells.

Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase-providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes.

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintenance of mitochondrial fitness.

Maintenance of complex I and its supercomplexes by NDUF-11 is essential for mitochondrial structure, function and health.

Mitochondrial supercomplexes form around a conserved core of monomeric complex I and dimeric complex III; wherein a subunit of the former, NDUFA11, is conspicuously situated at the interface. We identified nduf-11 (B0491.5) as encoding the Caenorhabditis elegans homologue of NDUFA11. Animals homozygous for a CRISPR-Cas9-generated knockout allele of nduf-11 arrested at the second larval (L2) development stage. Reducing (but not eliminating) expression using RNAi allowed development to adulthood, enabling characterisation of the consequences: destabilisation of complex I and its supercomplexes and perturbation of respiratory function. The loss of NADH dehydrogenase activity was compensated by enhanced complex II activity, with the potential for detrimental reactive oxygen species (ROS) production. Cryo-electron tomography highlighted aberrant morphology of cristae and widening of both cristae junctions and the intermembrane space. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development presumably arises due to its involvement in complex I and supercomplex maintenance. This highlights the importance of respiratory complex integrity for health and the potential for its perturbation to cause mitochondrial disease. This article has an associated First Person interview with Amber Knapp-Wilson, joint first author of the paper.

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration.

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease.

Hexokinase II dissociation alone cannot account for changes in heart mitochondrial function, morphology and sensitivity to permeability transition pore opening following ischemia.

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes.

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.

Early Cardiac Mitochondrial Molecular and Functional Responses to Acute Anthracycline Treatment in Wistar Rats.

Doxorubicin (DOX) is an anticancer drug widely used to treat human and nonhuman tumors but the late and persistent cardio-toxicity reduces the therapeutic utility of the drug. The full mechanism(s) of DOX-induced acute, subchronic and delayed toxicity, which has a preponderant mitochondrial component, remains unclear; therefore, it is clinically relevant to identify early markers to identify patients who are predisposed to DOX-related cardiovascular toxicity. To address this, Wistar rats (16 weeks old) were treated with a single DOX dose (20 mg/kg, i.p.); then, mRNA, protein levels and functional analysis of mitochondrial endpoints were assessed 24 h later in the heart, liver, and kidney. Using an exploratory data analysis, we observed cardiac-specific alterations after DOX treatment for mitochondrial complexes III, IV, and preferentially for complex I. Conversely, the same analysis revealed complex II alterations are associated with DOX response in the liver and kidney. Interestingly, H2O2 production by the mitochondrial respiratory chain as well as loss of calcium-loading capacity, markers of subchronic toxicity, were not reliable indicators of acute DOX cardiotoxicity in this animal model. By using sequential principal component analysis and feature correlation analysis, we demonstrated for the first time alterations in sets of transcripts and proteins, but not functional measurements, that might serve as potential early acute markers of cardiac-specific mitochondrial toxicity, contributing to explain the trajectory of DOX cardiac toxicity and to develop novel interventions to minimize DOX cardiac liabilities.

The role of succinate and ROS in reperfusion injury - A critical appraisal.

We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca2+]. However, IP only attenuates [Ca2+] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1.

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

Cardiac cytochrome c and cardiolipin depletion during anthracycline-induced chronic depression of mitochondrial function.

AIMS: It is still unclear why anthracycline treatment results in a cardiac-specific myopathy. We investigated whether selective doxorubicin (DOX) cardiotoxicity involving mitochondrial degeneration is explained by different respiratory complexes reserves between tissues by comparing and contrasting treatment effects in heart vs liver and kidney. Alternatively, we have also explored if the degeneration is due to alterations of mitochondrial thresholds to incompatible states. METHODS AND RESULTS: Heart, liver and kidney mitochondria were isolated from male Wistar rats weekly injected with DOX during 7weeks. Global flux and isolated step curves were obtained for Complex I, III, IV, as well as for the adenine nucleotide translocator. We show treatment-related alterations in global flux curve for Complex III in all analyzed tissues and in Complex IV activity curve solely in heart. However, all mitochondrial threshold curves remained unchanged after treatment in the analyzed tissues. No treatment-related differences were detected on transcript or protein analysis of selected respiratory complexes subunits. However, a specific loss of cytochrome c and cardiolipin was measured in heart, but not in other organs, mitochondria from DOX-treated animals. CONCLUSIONS: Contrary to our hypothesis, impaired mitochondrial respiration could not be explained by intrinsic differences in respiratory complexes reserves among tissues or, by alterations in mitochondrial thresholds after treatment. Instead, we propose that loss of cytochrome c and cardiolipin are responsible for the depressed mitochondrial respiration observed after chronic DOX treatment. Moreover, cardiac cytochrome c and cardiolipin depletion decreases metabolic network buffering, hindering cardiac ability to respond to increased workload, accelerating cardiac aging.

The role of hexokinase in cardioprotection - mechanism and potential for translation.

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose-6-phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre-ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B-cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target.

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death.

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.

Dioxin-induced acute cardiac mitochondrial oxidative damage and increased activity of ATP-sensitive potassium channels in Wistar rats.

The environmental dioxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a Group 1 human carcinogen and teratogenic agent. We hypothesize that TCDD-induced oxidative stress may also interfere with mitochondrial ATP-sensitive potassium channels (mitoKATP), which are known to regulate and to be regulated by mitochondrial redox state. We investigated the effects of an acute treatment of male Wistar rats with TCDD (50 µg/kg i.p.) and measured the regulation of cardiac mitoKATP. While the function of cardiac mitochondria was slightly depressed, mitoKATP activity was 52% higher in animals treated with TCDD. The same effects were not observed in liver mitochondria isolated from the same animals. Our data also shows that regulation of mitochondrial ROS production by mitoKATP activity is different in both groups. To our knowledge, this is the first report to show that TCDD increases mitoKATP activity in the heart, which may counteract the increased oxidative stress caused by the dioxin during acute exposure.

Edelfosine and perifosine disrupt hepatic mitochondrial oxidative phosphorylation and induce the permeability transition.

Edelfosine and perifosine are alkylphospholipids that have been intensively studied as potential antitumor agents. Apoptotic cell death caused by these two compounds is mediated, at least in part, through mitochondria. Additionally, previous works demonstrated that edelfosine induces changes in mitochondrial membrane permeability that are somehow reduced by using cyclosporin A. Therefore, the objective of the present study was not only to confirm mitochondrial permeability transition but also identify direct effects of both ether lipids on mitochondrial hepatic fractions, namely on mitochondrial oxidative phosphorylation and generation of hydrogen peroxide (H(2)O(2)) through the respiratory chain. Results show that edelfosine and perifosine inhibit mitochondrial respiration and decrease transmembrane electric potential. However, despite these effects, edelfosine and perifosine were still able to induce mitochondrial permeability transition in non-energized mitochondria. Interestingly, edelfosine decreased H(2)O(2) production through the respiratory chain. In conclusion, the present work demonstrates previously unknown alterations of mitochondrial physiology directly induced by edelfosine and perifosine. The study is relevant in the understanding of mitochondrial-target effects of both compounds, as well as to acknowledge possible toxic responses in non-tumor organs.

Mitochondrionopathy phenotype in doxorubicin-treated Wistar rats depends on treatment protocol and is cardiac-specific.

Although doxorubicin (DOX) is a very effective antineoplastic agent, its clinical use is limited by a dose-dependent, persistent and cumulative cardiotoxicity, whose mechanism remains to be elucidated. Previous works in animal models have failed to use a multi-organ approach to demonstrate that DOX-associated toxicity is selective to the cardiac tissue. In this context, the present work aims to investigate in vivo DOX cardiac, hepatic and renal toxicity in the same animal model, with special relevance on alterations of mitochondrial bioenergetics. To this end, male Wistar rats were sub-chronically (7 wks, 2 mg/Kg) or acutely (20 mg/Kg) treated with DOX and sacrificed one week or 24 hours after the last injection, respectively. Alterations of mitochondrial bioenergetics showed treatment-dependent differences between tissues. No alterations were observed for cardiac mitochondria in the acute model but decreased ADP-stimulated respiration was detected in the sub-chronic treatment. In the acute treatment model, ADP-stimulated respiration was increased in liver and decreased in kidney mitochondria. Aconitase activity, a marker of oxidative stress, was decreased in renal mitochondria in the acute and in heart in the sub-chronic model. Interestingly, alterations of cardiac mitochondrial bioenergetics co-existed with an absence of echocardiograph, histopathological or ultra-structural alterations. Besides, no plasma markers of cardiac injury were found in any of the time points studied. The results confirm that alterations of mitochondrial function, which are more evident in the heart, are an early marker of DOX-induced toxicity, existing even in the absence of cardiac functional alterations.

Drug-induced cardiac mitochondrial toxicity and protection: from doxorubicin to carvedilol.

Mitochondria have long been involved in several cellular processes beyond its role in energy production. The importance of this organelle for cardiac tissue homeostasis has been greatly investigated and its impairment can lead to cell death and consequent organ failure. Several compounds have been described in the literature as having direct effects on cardiac mitochondria which can provide a mechanistic explanation for their toxicological or pharmacological effects. The present review describes one classic example of drug-induced cardiac mitochondrial toxicity and another case of drug-induced mitochondrial protection. For the former, we present the case for doxorubicin, an anticancer agent whose treatment is associated with a cumulative and dose-dependent cardiomyopathy with a mitochondrial etiology. Following this, we present the case of carvedilol, a ß-blocker with intrinsic antioxidant activity, which has been described to protect cardiac mitochondria from oxidative injury. The final part of the review integrates information from the previous chapters, demonstrating how carvedilol can contribute to reduce doxorubicin toxicity on cardiac mitochondria. The two referred examples result in important take-home messages: a) drug-induced cardiac mitochondrial dysfunction is an important contributor for drug-associated organ failure, b) protection of mitochondrial function is involved in the beneficial impact of some clinically-used drugs and c) a more accurate prediction of toxic vs. beneficial effects should be an important component of drug development by the pharmaceutical industry.

Sub-chronic administration of doxorubicin to Wistar rats results in oxidative stress and unaltered apoptotic signaling in the lung.

Despite the vast published data on cardiac toxicity, there is still little work done regarding the toxicity of the antineoplastic agent Doxorubicin (DOX) in the lung. The aim of the present work was to determine if DOX causes alterations in selected apoptotic proteins and oxidative stress in the lung, in a similar manner to what occurs in the heart. For that purpose, lungs from Wistar-Han rats sub-chronically treated with vehicle or DOX for seven weeks were collected and analyzed concerning several proteins involved in mitochondrial permeabilization and apoptotic pathways, including p53, Bax and Bcl-2 and different oxidative stress markers. After sub-chronic DOX treatment, no alterations in lung proteins involved in mitochondrial membrane permeabilization or caspase 3 and 9-like activities were found. Nevertheless, an increase in malondialdehyde levels and a decrease in the lung concentration of vitamin E were detected, despite no alterations in reduced and oxidized glutathione. The results obtained indicate for the first time that lungs from DOX-treated rats appear to be susceptible to increased lipid peroxidation, which can explain some cases of DOX-induced lung toxicity.

Can drug safety be predicted and animal experiments reduced by using isolated mitochondrial fractions?

Mitochondrial toxicity has resulted in the withdrawal of several drugs from the market. One particular example is nefazodone, an anti-depressant withdrawn in the USA due to hepatoxicity caused by drug-induced mitochondrial dysfunction. Drug development and safety testing can involve the use of large numbers of laboratory animals, which, without a decisive pre-screening for mitochondrial toxicity, are often unable to pre-empt higher mortality rates in some patient groups. The use of isolated mitochondria as a screening tool for drug safety can decrease the number of laboratory animals used in pre-clinical studies, thus improving animal welfare and healthcare outcomes and costs. Novel techniques involving high-throughput methods can be used to investigate whether a molecule is a mitochondrial toxicant. Moreover, these screens are mechanistically-based, since the effects of the drug on oxidative phosphorylation, calcium homeostasis and mitochondrial genetics can be assessed. This review is intended to demonstrate that isolated mitochondrial fractions are suitable for predicting drug and general chemical safety in toxicological screenings, thus contributing to the refinement and reduction of animal use in laboratory research.