Genome-wide Responses to Mitochondrial DysfunctionV⃞

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1154Q/2Q mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1154Q/2Q mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as preventing oxidative stress-induced DNA / RNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.

From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans.

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.

A systematic review of evidence for silver nanoparticle-induced mitochondrial toxicity

© The Royal Society of Chemistry 2016.Silver nanoparticles (AgNPs) are extensively used for their antibacterial properties in a diverse set of applications, ranging from the treatment of municipal wastewater to infection control in hospitals. However, the properties of AgNPs that render them conducive to bactericidal use in commerce may influence their potential toxicity to non-bacterial organisms. Based on the physiological and phylogenetic similarities between bacteria and mitochondria within eukaryotic cells, mitochondria are a likely intracellular target of AgNP toxicity. Mitochondria-specific outcomes of AgNP exposures have been identified in multiple cell types, including (but not limited to) loss of membrane potential, inhibition of enzymes involved in oxidative phosphorylation, and changes in calcium sequestration. However, the biological significance of mitochondrial toxicity due to AgNP exposure is currently incompletely understood. This review examines the existing evidence of mitochondrial toxicity induced by AgNP exposure, with discussions of the role of the physicochemical properties of the nanoparticles themselves in mitochondrial toxicity. The impacts of potentially differential cell- and tissue-specific significance of AgNP-induced mitochondrial dysfunction are also discussed.

Study of insulin-like growth factor signaling in mitochondrial function

Insulin-like Growth Factor-1 (IGF-1) signalling promotes cell growth and is associated with cancer progression, including metastasis, epithelial-mesenchymal transition (EMT), and resistance to therapy. Mitochondria play an essential role in cancer cell metabolism and accumulating evidence demonstrates that dysfunctional mitochondria associated with release of mitochondrial reactive oxygen species (ROS) can influence cancer cell phenotype and invasive potential. We previously isolated a mitochondrial UTP carrier (PNC1/SLC25A33) whose expression is regulated by IGF-1, and which is essential for mitochondrial maintenance. PNC1 suppression in cancer cells results in mitochondrial dysfunction and acquisition of a profound ROS-dependent invasive (EMT) phenotype. Moreover, over-expression of PNC1 in cancer cells that exhibit an EMT phenotype is sufficient to suppress mitochondrial ROS production and reverse the invasive phenotype. This led us to investigate the IGF-1-mitochondrial signalling axis in cancer cells. We found that IGF-1 signalling supports increased mitochondrial mass and Oxphos potential through a PI3K dependant pathway. Acute inhibition of IGF-1R activity with a tyrosine kinase inhibitor results in dysfunctional mitochondria and cell death. We also observed an adaptive response to IGF-1R inhibition upon prolonged exposure to the kinase inhibitor, where increased expression of the EGF receptor can compensate for loss of mitochondrial mass through activation of PI3K/mTOR signalling. However, these cells exhibit impaired mitochondrial biogenesis and mitophagy. We conclude that the IGF-1 is required for mitochondrial maintenance and biogenesis in cancer cells, and that pharmacological inhibition of this pathway may induce mitochondrial dysfunction and may render the cells more sensitive to glycolysis-targeted drugs.

Role of mitochondrial antioxidant defense systems in fatty acid β-oxidation defects

Mitochondrial fatty acid oxidation (FAO) plays a pivotal role in energy homeostasis, namely during periods of fasting or metabolic stress. FAO defects are a group of inherited metabolic disorders that encompass at least twelve distinct enzyme or transporter deficiencies, and can present with a wide range of clinical symptoms with various degrees of severity. Besides recent advances, many doubts still remain on the degree and characteristics of mitochondrial dysfunction on FAOD and its contribution to the clinical phenotype.

Identification of telomere dysfunction in Friedreich ataxia.

Background: Friedreich ataxia (FRDA) is a progressive inherited neurodegenerative disorder caused by mutation of the FXN gene, resulting in decreased frataxin expression, mitochondrial dysfunction and oxidative stress. A recent study has identified shorter telomeres in FRDA patient leukocytes as a possible disease biomarker. Results: Here we aimed to investigate both telomere structure and function in FRDA cells. Our results confirmed telomere shortening in FRDA patient leukocytes and identified similar telomere shortening in FRDA patient autopsy cerebellar tissues. However, FRDA fibroblasts showed significantly longer telomeres at early passage, occurring in the absence of telomerase activity, but with activation of an alternative lengthening of telomeres (ALT)-like mechanism. These cells also showed accelerated telomere shortening as population doubling increases. Furthermore, telomere dysfunction-induced foci (TIF) analysis revealed that FRDA fibroblasts have dysfunctional telomeres. Conclusions: Our finding of dysfunctional telomeres in FRDA cells provides further insight into FRDA molecular disease mechanisms, which may have implications for future FRDA therapy.

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Intake of trans Fatty Acids Causes Nonalcoholic Steatohepatitis and Reduces Adipose Tissue Fat Content

We investigated the effects of dietary trans fatty acids, PUFA, and SEA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SEA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPAR alpha, PPAR gamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SEA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1 c and PPAR gamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPAR alpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome. J. Nutr. 140: 1127-1132, 2010.

Timing-dependent protection of hypertonic saline solution administration in experimental liver ischemia/reperfusion injury

Background. Diving liver ischemia, the decrease in mitochondrial energy causes cellular damage that is aggravated after reperfusion. This injury can trigger a systemic inflammatory syndrome, also producing remote organ damage. Several substances have been employed to decrease this inflammatory response during liver transplantation, liver resections, and hypovolemic shock. The aim of this study was to evaluate the effects of hypertonic saline solution and the best timing of administration to prevent organ injury during experimental liver ischemia/reperfusion. Methods. Rats underwent 1 hr of warm liver ischemia followed by reperfusion. Eighty-four rats Were allocated into 6 groups: sham group, control of ischemia group) (C), pre-ischemia treated NaCl 0.9% (ISS) and NaCl 7.5% (HTS) groups, pre-repefusion ISS, and HTS groups. Blood and tissue samples were collected 4 hr after reperfusion. Results. HTS showed beneficial effects in prevention of live ischemia/reperfusion injury. HTS groups developed increases in AST and ALT levels that were significantly less than ISS groups; however, the HTS pre-reperfusion group showed levels significantly less than the HTS pre-ischemia group. No differences in IL-6 and IL-10 levels, were observed. A significant decrease in mitochondrial dysfunction as well as hepatic edema was observed in the HTS pre-reperfusion group. Pulmonary vascular permeability Was significantly less in the pre-reperfusion HTS group compared to the ISS group. No differences in myeloperoxidase activity were observed. The liver histologic score was significantly less in the pre-reperfusion HTS group compared to the pre-ischemia I-ITS group. Conclusion. HTS ameliorated local and systemic injuries in experimental liver ischemia/reperfusion. Infusion of HTS in the pre-reperfusion period may be an important adjunct to accomplish the best results. (Surgery 2010;147:415-23.)

Anti-Inflammatory Effects of Peritoneal Lavage in Acute Pancreatitis

Objectives: Intraperitoneal administration of trypsin stimulates the production of cytokines from peritoneal macrophages. Removing the pancreatitis-associated ascitic fluid from the peritoneal cavity may decrease the systemic inflammatory response in acute pancreatitis (AP). We investigated the effect of peritoneal lavage on the systemic inflammatory response in severe AP. Methods: Acute pancreatitis was induced in Wistar rats by 5% taurocholate intraductal injection. Peritoneal lavage was performed for 4 hours after onset of AP. At 4 hours after induction of AP, serum samples were assayed for amylase and inflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-10). Expression of pancreatic cyclooxygenase-2 and inducible nitric oxide synthase, liver mitochondrial function, and pulmonary myeloperoxidase activities were determined. Results: Peritoneal lavage after AP led to a decrease in serum levels of tumor necrosis factor alpha and IL-6 and an increase in IL-10. In the pancreas, this treatment reduced cyclooxygenase-2 and inducible nitric oxide synthase expression. Liver mitochondrial dysfunction was also reduced. There were no differences on serum amylase levels and pulmonary myeloperoxidase between groups with AP. Conclusions: Peritoneal lavage has a systemic anti-inflammatory effect in severe AP and may be able to decrease the severity of severe AP.