N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Metabolic Features of Brain Function with Relevance to Clinical Features of Alzheimer and Parkinson Diseases.

Brain metabolism is comprised in Alzheimer's disease (AD) and Parkinson's disease (PD). Since the brain primarily relies on metabolism of glucose, ketone bodies, and amino acids, aspects of these metabolic processes in these disorders-and particularly how these altered metabolic processes are related to oxidative and/or nitrosative stress and the resulting damaged targets-are reviewed in this paper. Greater understanding of the decreased functions in brain metabolism in AD and PD is posited to lead to potentially important therapeutic strategies to address both of these disorders, which cause relatively long-lasting decreased quality of life in patients.

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

The Crowded Crosstalk between Cancer Cells and Stromal Microenvironment in Gynecological Malignancies: Biological Pathways and Therapeutic Implication.

The tumor microenvironment plays a pillar role in the progression and the distance dissemination of cancer cells in the main malignancies affecting women-epithelial ovarian cancer, endometrial cancer and cervical cancer. Their milieu acquires specific properties thanks to intense crosstalk between stromal and cancer cells, leading to a vicious circle. Fibroblasts, pericytes, lymphocytes and tumor associated-macrophages orchestrate most of the biological pathways. In epithelial ovarian cancer, high rates of activated pericytes determine a poorer prognosis, defining a common signature promoting ovarian cancer proliferation, local invasion and distant spread. Mesenchymal cells also release chemokines and cytokines under hormonal influence, such as estrogens that drive most of the endometrial cancers. Interestingly, the architecture of the cervical cancer milieu is shaped by the synergy of high-risk Human Papilloma Virus oncoproteins and the activity of stromal estrogen receptor α. Lymphocytes represent a shield against cancer cells but some cell subpopulation could lead to immunosuppression, tumor growth and dissemination. Cytotoxic tumor infiltrating lymphocytes can be eluded by over-adapted cancer cells in a scenario of immune-tolerance driven by T-regulatory cells. Therefore, the tumor microenvironment has a high translational potential offering many targets for biological and immunological therapies.

Monoamine oxidase-dependent histamine catabolism accounts for post-ischemic cardiac redox imbalance and injury.

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Down-regulation of the mitochondrial aspartate-glutamate carrier isoform 1 AGC1 inhibits proliferation and N-acetylaspartate synthesis in Neuro2A cells.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines.

Significant metabolic changes occur in the shift from resting to activated cellular status in inflammation. Thus, changes in expression of a large number of genes and extensive metabolic reprogramming gives rise to acquisition of new functions (e.g. production of cytokines, intermediates for biosynthesis, lipid mediators, PGE, ROS and NO). In this context, mitochondrial carriers, which catalyse the transport of solute across mitochondrial membrane, change their expression to transport mitochondrially produced molecules, among which citrate and succinate, to be used as intracellular signalling molecules in inflammation. This review summarises the mitochondrial carriers studied so far that are, directly or indirectly, involved in inflammation.

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

Clinical implications from proteomic studies in neurodegenerative diseases: lessons from mitochondrial proteins.

Mitochondria play a key role in eukaryotic cells, being mediators of energy, biosynthetic and regulatory requirements of these cells. Emerging proteomics techniques have allowed scientists to obtain the differentially expressed proteome or the proteomic redox status in mitochondria. This has unmasked the diversity of proteins with respect to subcellular location, expression and interactions. Mitochondria have become a research 'hot spot' in subcellular proteomics, leading to identification of candidate clinical targets in neurodegenerative diseases in which mitochondria are known to play pathological roles. The extensive efforts to rapidly obtain differentially expressed proteomes and unravel the redox proteomic status in mitochondria have yielded clinical insights into the neuropathological mechanisms of disease, identification of disease early stage and evaluation of disease progression. Although current technical limitations hamper full exploitation of the mitochondrial proteome in neurosciences, future advances are predicted to provide identification of specific therapeutic targets for neurodegenerative disorders.

The mitochondrial side of epigenetics.

The bidirectional cross talk between nuclear and mitochondrial DNA is essential for cellular homeostasis and proper functioning. Mitochondria depend on nuclear contribution for much of their functionality, but their activities have been recently recognized to control nuclear gene expression as well as cell function in many different ways. Epigenetic mechanisms, which tune gene expression in response to environmental stimuli, are key regulatory events at the interplay between mitochondrial and nuclear interactions. Emerging findings indicate that epigenetic factors can be targets or instruments of mitochondrial-nuclear cross talk. Additionally, the growing interest into mtDNA epigenetic modifications opens new avenues into the interaction mechanisms between mitochondria and nucleus. In this review we summarize the points of mitochondrial and nuclear reciprocal control involving epigenetic factors, focusing on the role of mitochondrial genome and metabolism in shaping epigenetic modulation of gene expression. The relevance of the new findings on the methylation of mtDNA is also highlighted as a new frontier in the complex scenario of mitochondrial-nuclear communication.

The Saccharomyces cerevisiae gene YPR011c encodes a mitochondrial transporter of adenosine 5'-phosphosulfate and 3'-phospho-adenosine 5'-phosphosulfate.

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.

Hyperhomocysteinemia: related genetic diseases and congenital defects, abnormal DNA methylation and newborn screening issues.

Homocysteine, a sulfur-containing amino acid derived from the methionine metabolism, is located at the branch point of two pathways of the methionine cycle, i.e. remethylation and transsulfuration. Gene abnormalities in the enzymes catalyzing reactions in both pathways lead to hyperhomocysteinemia. Hyperhomocysteinemia is associated with increased risk for congenital disorders, including neural tube closure defects, heart defects, cleft lip/palate, Down syndrome, and multi-system abnormalities in adults. Since hyperhomocysteinemia is known to affect the extent of DNA methylation, it is likely that abnormal DNA methylation during embryogenesis, may be a pathogenic factor for these congenital disorders. In this review we highlight the importance of homocysteinemia by describing the genes encoding for enzymes of homocysteine metabolism relevant to the clinical practice, especially cystathionine-ß-synthase and methylenetetrahydrofolate reductase mutations, and the impairment of related metabolites levels. Moreover, a possible correlation between hyperhomocysteine and congenital disorders through the involvement of abnormal DNA methylation during embryogenesis is discussed. Finally, the relevance of present and future diagnostic tools such as tandem mass spectrometry and next generation sequencing in newborn screening is highlighted.

Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.

Mitochondrial DNA methylation as a next-generation biomarker and diagnostic tool.

Recent expansion of our knowledge on epigenetic changes strongly suggests that not only nuclear DNA (nDNA), but also mitochondrial DNA (mtDNA) may be subjected to epigenetic modifications related to disease development, environmental exposure, drug treatment and aging. Thus, mtDNA methylation is attracting increasing attention as a potential biomarker for the detection and diagnosis of diseases and the understanding of cellular behavior in particular conditions. In this paper we review the current advances in mtDNA methylation studies with particular attention to the evidences of mtDNA methylation changes in diseases and physiological conditions so far investigated. Technological advances for the analysis of epigenetic variations are promising tools to provide insights into methylation of mtDNA with similar resolution levels as those reached for nDNA. However, many aspects related to mtDNA methylation are still unclear. More studies are needed to understand whether and how changes in mtDNA methylation patterns, global and gene specific, are associated to diseases or risk factors.

OPA1 drives macrophage metabolism and functional commitment via p65 signaling.

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

Identification of mitochondrial thiamin diphosphate carriers from Arabidopsis and maize.

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.

Deletion of Lactate Dehydrogenase-A Impairs Oncogene-Induced Mouse Hepatocellular Carcinoma Development.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a multistep process whereby abnormally proliferating cancer cells undergo extensive metabolic reprogramming. Metabolic alterations in hepatocarcinogenesis depend on the activation of specific oncogenes, thus partially explaining HCC heterogeneity. c-Myc oncogene overexpression, frequently observed in human HCCs, leads to a metabolic rewiring toward a Warburg phenotype and production of lactate, resulting in the acidification of the extracellular space, favoring the emergence of an immune-permissive tumor microenvironment. Here, we investigated whether Ldha genetic ablation interferes with metabolic reprogramming and HCC development in the mouse. METHODS: We characterized the metabolic reprogramming in tumors induced in C57BL/6J mice hydrodynamically cotransfected with c-Myc and h-Ras. Using the same experimental model, we investigated the effect of Ldha inhibition-gained through the inducible and hepatocyte-specific Ldha knockout-on cancer cell metabolic reprogramming, number and size of HCC lesions, and tumor microenvironment alterations. RESULTS: c-Myc/h-Ras-driven tumors display a striking glycolytic metabolism, suggesting a switch to a Warburg phenotype. The tumors also exhibited enhanced pentose phosphate pathway activity, the switch of glutamine to sustain glutathione synthesis instead of the tricarboxylic acid cycle, and the impairment of oxidative phosphorylation. In addition, Ldha abrogation significantly hampered tumor number and size together with an evident inhibition of the Warburg-like metabolic feature and a remarkable increase of CD4+ lymphocytes compared with Ldha wild-type livers. CONCLUSIONS: These results demonstrate that Ldha deletion significantly impairs mouse HCC development and suggest lactate dehydrogenase as a potential target to enhance the efficacy of the current therapeutic options.

Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3.

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.