Targeting of regulated necrosis in kidney disease.

The term acute tubular necrosis was thought to represent a misnomer derived from morphological studies of human necropsies and necrosis was thought to represent an unregulated passive form of cell death which was not amenable to therapeutic manipulation. Recent advances have improved our understanding of cell death in acute kidney injury. First, apoptosis results in cell loss, but does not trigger an inflammatory response. However, clumsy attempts at interfering with apoptosis (e.g. certain caspase inhibitors) may trigger necrosis and, thus, inflammation-mediated kidney injury. Second, and most revolutionary, the concept of regulated necrosis emerged. Several modalities of regulated necrosis were described, such as necroptosis, ferroptosis, pyroptosis and mitochondria permeability transition regulated necrosis. Similar to apoptosis, regulated necrosis is modulated by specific molecules that behave as therapeutic targets. Contrary to apoptosis, regulated necrosis may be extremely pro-inflammatory and, importantly for kidney transplantation, immunogenic. Furthermore, regulated necrosis may trigger synchronized necrosis, in which all cells within a given tubule die in a synchronized manner. We now review the different modalities of regulated necrosis, the evidence for a role in diverse forms of kidney injury and the new opportunities for therapeutic intervention.

Valproic acid-induced hepatotoxicity in Alpers syndrome is associated with mitochondrial permeability transition pore opening-dependent apoptotic sensitivity in an induced pluripotent stem cell model.

UNLABELLED: Valproic acid (VPA) is widely used to treat epilepsy, migraine, chronic headache, bipolar disorder, and as adjuvant chemotherapy, but potentially causes idiosyncratic liver injury. Alpers-Huttenlocher syndrome (AHS), a neurometabolic disorder caused by mutations in mitochondrial DNA polymerase gamma (POLG), is associated with an increased risk of developing fatal VPA hepatotoxicity. However, the mechanistic link of this clinical mystery remains unknown. Here, fibroblasts from 2 AHS patients were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (AHS iPSCs-Hep). Both AHS iPSCs-Hep are more sensitive to VPA-induced mitochondrial-dependent apoptosis than controls, showing more activated caspase-9 and cytochrome c release. Strikingly, levels of both soluble and oligomeric optic atrophy 1, which together keep cristae junctions tight, are reduced in AHS iPSCs-Hep. Furthermore, POLG mutation cells show reduced POLG expression, mitochondrial DNA (mtDNA) amount, mitochondrial adenosine triphosphate production, as well as abnormal mitochondrial ultrastructure after differentiation to hepatocyte-like cells. Superoxide flashes, spontaneous bursts of superoxide generation, caused by opening of the mitochondrial permeability transition pore (mPTP), occur more frequently in AHS iPSCs-Hep. Moreover, the mPTP inhibitor, cyclosporine A, rescues VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. This result suggests that targeting mPTP opening could be an effective method to prevent hepatotoxicity by VPA in AHS patients. In addition, carnitine or N-acetylcysteine, which has been used in the treatment of VPA-induced hepatotoxicity, is able to rescue VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. CONCLUSION: AHS iPSCs-Hep are more sensitive to the VPA-induced mitochondrial-dependent apoptotic pathway, and this effect is mediated by mPTP opening. Toxicity models in genetic diseases using iPSCs enable the evaluation of drugs for therapeutic targets.

Myocardial reperfusion injury: looking beyond primary PCI.

Coronary heart disease (CHD) is the leading cause of death and disability in Europe. For patients presenting with an acute ST-segment elevation myocardial infarction (STEMI), timely myocardial reperfusion using either thrombolytic therapy or primary percutaneous coronary intervention (PPCI) is the most effective therapy for limiting myocardial infarct (MI) size, preserving left-ventricular systolic function and reducing the onset of heart failure. Despite this, the morbidity and mortality of STEMI patients remain significant, and novel therapeutic interventions are required to improve clinical outcomes in this patient group. Paradoxically, the process of myocardial reperfusion can itself induce cardiomyocyte death-a phenomenon which has been termed 'myocardial reperfusion injury' (RI), the irreversible consequences of which include microvascular obstruction and myocardial infarction. Unfortunately, there is currently no effective therapy for preventing myocardial RI in STEMI patients making it an important residual target for cardioprotection. Previous attempts to translate cardioprotective therapies (antioxidants, calcium-channel blockers, and anti-inflammatory agents) for reducing RI into the clinic, have been unsuccessful. An improved understanding of the pathophysiological mechanisms underlying RI has resulted in the identification of several promising mechanical (ischaemic post-conditioning, remote ischaemic pre-conditioning, therapeutic hypothermia, and hyperoxaemia), and pharmacological (atrial natriuretic peptide, cyclosporin-A, and exenatide) therapeutic strategies, for preventing myocardial RI, many of which have shown promise in initial proof-of-principle clinical studies. In this article, we review the pathophysiology underlying myocardial RI, and highlight the potential therapeutic interventions which may be used in the future to prevent RI and improve clinical outcomes in patients with CHD.

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition.

Treatment with the omega-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the omega-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca2+-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of omega-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca2+-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca2+ retention capacity and decreased Ca2+-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca2+-induced MPTP opening.

Exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and tetraethyl lead affects lung mitochondria bioenergetics.

Environmental pollutants such as TCDD and tetraethyl lead are extremely toxic and related with pulmonary disease development. Lung mitochondria are primary cellular targets for dioxins exposure-induced toxicity. TCDD showed a delay in the repolarization after a phosphorylative cycle and a decrease on state 3 respiration, suggesting alterations at the phosphorylative system level. The ATPase activity showed no differences between control and lung mitochondria incubated with TCDD, implying alterations in other components of the phosphorylative system. Tetraethyl lead also showed a delay in the repolarization after a phosphorylative cycle and a decrease on RCR. These data suggest that lung mitochondria incubated with TCDD and tetraethyl lead showed impaired mitochondrial function, reflecting the loss of oxidative phosphorylation capacity.

A novel intermembrane space-targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding.

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step-specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.

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.

Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.

Pre-synaptic nerve terminals (synaptosomes) require ATP for neurotransmitter exocytosis and recovery and for ionic homeostasis, and are consequently abundantly furnished with mitochondria. Pre-synaptic mitochondrial dysfunction is implicated in a variety of neurodegenerative disorders, although there is no precise definition of the term 'dysfunction'. In this study, we test the hypothesis that partial restriction of electron transport through Complexes I and II in synaptosomes to mimic possible defects associated with Parkinson's and Huntington's diseases respectively, sensitizes individual terminals to mitochondrial depolarization under conditions of enhanced proton current utilization, even though these stresses are within the respiratory capacity of the synaptosomes when averaged over the entire population. We combine two novel techniques, firstly using a modification of a plate-based respiration and glycolysis assay that requires only microgram quantities of synaptosomal protein, and secondly developing an improved method for fluorescent imaging and statistical analysis of single synaptosomes. Conditions are defined for optimal substrate supply to the in situ mitochondria within mouse cerebrocortical synaptosomes, and the energetic demands of ion cycling and action-potential firing at the plasma membrane are additionally determined.

Vagal nerve stimulation prevents reperfusion injury through inhibition of opening of mitochondrial permeability transition pore independent of the bradycardiac effect.

BACKGROUND: In spite of recent advances in coronary interventional therapy, reperfusion injury is still considered to be a major problem in patients undergoing surgical procedures, such as bypass grafting. Here we demonstrate a novel therapeutic strategy against ischemia-reperfusion injury: vagally mediated prevention of reperfusion-induced opening of mitochondrial permeability transition pore. METHODS: We investigated the effects of efferent vagal stimulation on myocardial reperfusion injury with ex vivo and in vitro rat models. In the ex vivo model the hearts were perfused with intact vagal innervation, which allowed us to study the effects of the vagal nerve on the heart without other systemic effects. RESULTS: Compared with sham stimulation, vagal stimulation exerted a marked anti-infarct effect irrespective of the heart rate (34% +/- 6% vs 85% +/- 9% at a heart rate of 300 beats/min, 37% +/- 4% vs 43% +/- 5% at a heart rate of 250 beats/min, and 39% +/- 4% vs 88% +/- 7% at a heart rate of 350 beats/min) after a 30-minute period of global ischemia, activated cell-survival Akt cascade, prevented downregulation of the antiapoptotic protein Bcl-2, and suppressed cytochrome-c release and caspase-3 activation. Furthermore, vagal stimulation-treated hearts exhibited a significant improvement in left ventricular developed pressure (78 +/- 5 vs 45 +/- 8 mm Hg) and a significant attenuation in an incremental change in left ventricular end-diastolic pressure during reperfusion. These beneficial effects of vagal stimulation were abolished by a permeability transition pore opener, atractyloside. In the in vitro study with primary-cultured cardiomyocytes, acetylcholine prevented a reoxygenation-induced collapse in mitochondrial transmembrane potential through inhibition of permeability transition pore opening. CONCLUSION: Vagal stimulation would be a potential adjuvant therapy for the rescue of ischemic myocardium from reperfusion injury, and the protective effects are independent of its bradycardiac effects.

The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.

The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy.

In tumours, polyamines and amine oxidases increase as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H2O2 and aldehydes produced by the reaction. Increasing the incubation temperature from 37 to 42 degrees C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. Since the tumour cells release endogenous substrates of BSAO, the administration of spermine is not required. Combination with hyperthermia improves the cytocidal effect of polyamines oxidation products. Our findings show that multidrug resistant (MDR) cells are more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity which induces the generation of intracellular ROS prior to the onset of mitochondrial permeability transition (MPT). It makes this new approach attractive, since the development of MDR is one of the major problems of conventional cancer therapy.

A mutation inactivating the mitochondrial inner membrane folate transporter creates a glycine requirement for survival of chinese hamster cells.

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.