Methylmalonyl acidemia: from mitochondrial metabolism to defective mitophagy and disease

ABSTRACT

Methylmalonic acidemia (MMA) is an autosomal recessive inborn error of metabolism due to the deficiency of mitochondrial MMUT (methylmalonyl-CoA mutase) – an enzyme that mediates the cellular breakdown of certain amino acids and lipids. The loss of MMUT leads to the accumulation of toxic organic acids causing severe organ dysfunctions and life-threatening complications. The mechanisms linking MMUT deficiency, mitochondrial alterations and cell toxicity remain uncharacterized. Using cell and animal-based models, we recently unveiled that MMUT deficiency impedes the PINK1-induced translocation of PRKN/Parkin to MMA-damaged mitochondria, thereby halting their delivery and subsequent degradation by macroautophagy/autophagy-lysosome systems. In turn, this defective mitophagy process instigates the accumulation of dysfunctional mitochondria that spark epithelial distress and tissue damage. Correction of PINK1-directed mitophagy defects or mitochondrial dysfunctions rescues epithelial distress in MMA cells and alleviates disease-relevant phenotypes in mmut?deficient zebrafish. Our findings suggest a link between primary MMUT deficiency and diseased mitochondria, mitophagy dysfunction and cell distress, offering potential therapeutic perspectives for MMA and other metabolic diseases.     

Identification and functional characterization of thromboxane A2 receptors in Schwann cells

Previous reports have demonstrated the presence of functional thromboxane A2 (TP) receptors in astrocytes and oligodendrocytes. In these experiments, the presence and function of TP receptors in primary rat Schwann cells (rSC) and a neurofibrosarcoma-derived human Schwann cell line (T265) was investigated. Immunocytochemical and immunoblot analyses using polyclonal anti-TP receptor antibodies demonstrate that both cell types express TP receptors. Treatment with the stable thromboxane A2 mimetic U46619 (10 microM) did not stimulate intracellular calcium mobilization in rSC, whereas T265 cells demonstrated a calcium response that was inhibited by prior treatment with TP receptor antagonists. U46619 also stimulated CREB phosphorylation on Ser133 in T265 cells and, to a lesser extent, in rSC. To identify potential mechanisms of CREB phosphorylation in rSC, we monitored intracellular cAMP levels following U46619 stimulation. Elevated levels of cAMP were detected in both rSC (20-fold) and T265 (15-fold) cells. These results demonstrate that TP receptor activation specifically stimulates CREB phosphorylation in T265 cells, possibly by a calcium- and/or cAMP-dependent mechanism. In contrast, TP receptor activation in rSC stimulates increases in cAMP and CREB phosphorylation but does not elicit changes in intracellular calcium.     

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.     

Neurotrophin-3-induced production of nerve growth factor is suppressed in Schwann cells exposed to high glucose: involvement of the polyol pathway

Development of hypesthesia, a loss of sensitivity to stimulation, is associated with impaired regeneration of peripheral sensory fibers, in which Schwann cells play a key role by secreting nerve growth factor (NGF). Recent clinical trials indicated that an inhibitor of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, significantly improved hypesthesia in diabetic patients. The fact that AR is localized in Schwann cells led us to investigate the role of the polyol pathway in NGF production of isolated Schwann cells. Among various endogenous factors examined, increased production of NGF was demonstrated in the cells treated with neurotrophin-3 (NT-3) for 24 h. NT-3-induced NGF production was significantly suppressed when cells were cultured in the medium containing high glucose. In these cells, the levels of glutathione (GSH) and cAMP-response element binding protein (CREB) were reduced, whereas the level of activated nuclear factor-kappaB (NF-kappaB) was elevated. These changes were abolished when an AR inhibitor fidarestat was included in the medium. NT-3-induced NGF production was further attenuated in the cells treated with an inhibitor of GSH synthesis. Together, the enhanced polyol pathway activity under high-glucose conditions seems to elicit reduced NT-3-induced NGF production in Schwann cells. Enhanced oxidative stress linked to the polyol pathway activity may mediate this process.     

Flux balance analysis of CHO cells before and after a metabolic switch from lactate production to consumption

Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C‐mol substrate consumed) six times greater than lactate producing cells. Biotechnol. Bioeng. 2013; 110: 660–666. © 2012 Wiley Periodicals, Inc.     

Using the “reverse Warburg effect” to identify high-risk breast cancer patients

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H2O2), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “Reverse Warburg Effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immuno-staining of human breast cancer tissue microarrays (TMAs; > 180 triple-negative patients) to directly assess the prognostic value of the “Reverse Warburg Effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis, and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (< 18% survival at 10 y post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-y survival rates of ~97% (p-value < 10^?32). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10^?14), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “Reverse Warburg Effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable-target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977, and AZD-3965).     

Proliferation‐ and migration‐enhancing effects of ginseng and ginsenoside Rg1 through IGF‐I‐ and FGF‐2‐signaling pathways on RSC96 Schwann cells

The aim of the present study is to evaluate the proliferation‐ and migration‐enhancing effects of ginseng and its component, ginsenoside (Rg1) on RSC96 Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs‐IGFIR‐Akt‐Bcl2 and proliferative signaling, cell cycle factors and mitogen‐activated protein kinase (MAPK) pathways, (2) migrating and anti‐scar signaling, FGF‐2‐uPA‐MMPs.We treated RSC96 cells with different concentrations (100, 200, 300, 400, 500 µg ml^?1) of ginseng and its constituent, Rg1 (5, 10, 15, 20, 25 µg ml^?1). We observed a proliferative effect in a dose‐dependent manner by PCNA western blotting assay, MTT assay, and wound healing test. Furthermore, we also found in the results of western blotting assay, ginseng and Rg1 enhance protein expression of IGF‐I pathway regulators, cell cycle controlling proteins, and MAPK signaling pathways to promote the cell proliferation. In addition, ginseng and Rg1 also stimulated the FGF‐2‐uPA‐MMP 9 migrating pathway to enhance the migration of RSC96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of ginseng and Rg1 on RSC96 cells were identified to be MAPK signaling‐dependent. On the basis of the results, applying appropriate doses of ginseng and Rg1 with biomedical materials would be a potential approach for enhancing neuron regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

The influence of oxygen supply on metabolism of neural cells cultured on a gas‐permeable PTFE foil

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen‐permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma‐derived cells (GOS‐3) and rat pheochromacytoma cells (PC12) were cultured on the gas‐permeable PTFE foil as well as a conventional non oxygen‐permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Extracellular methemoglobin promotes cyto-adherence of uninfected RBC to endothelial cells: Insight into cerebral malaria pathology

The endothelial cell barrier is tightly regulated, and disruption or the leaky behavior of the barrier leads to pathology. Disturbance of blood-brain barrier is observed during viral infection, cerebral malaria, and acute hemorrhagic encephalitis. Red blood cells (RBCs) bind to the endothelial cells (ECs) and their affinity towards ECs enhances in the presence of Plasmodium falciparum infection. ECs stimulated with methemoglobin (MetHb; 20 µM) for 1 hour exhibit high levels of cyto-adherence receptors CD36 and ICAM-1 on their cell surface compared with unstimulated cells. These ECs have acquired affinity towards uninfected RBCs in flow at arterial shear stress. SEM analysis indicates that EC–RBC cyto-adherence involved multiple attachment points. Initially, ECs bind single layer of RBCs and the number of RBCs increases over time to give high-order cyto-adherence with more than 30 RBCs adhered to each endothelial cell. The cyto-adherence complexes are stable to high shear stress and can withstand shear stress up to 450 dyne/cm ². MetHb-treated ECs exhibited high reactive oxygen species level, and preincubation of ECs with antioxidant (NAC or mannitol) abolished the formation of EC–RBC cyto-adherence complexes. In addition, gallic acid (present in red wine) and green tea extract has inhibited the formation of EC–RBC cyto-adherence complex. A better understanding of gallic acid and tea polyphenol targeting pathological cyto-adherence may allow us to develop a better adjuvant therapy for cerebral malaria and other noninfectious diseases.     

The metabolic flux phenotype of heterotrophic Arabidopsis cells reveals a flexible balance between the cytosolic and plastidic contributions to carbohydrate oxidation in response to phosphate limitation

Understanding the mechanisms that allow plants to respond to variable and reduced availability of inorganic phosphate is of increasing agricultural importance because of the continuing depletion of the rock phosphate reserves that are used to combat inadequate phosphate levels in the soil. Changes in gene expression, protein levels, enzyme activities and metabolite levels all point to a reconfiguration of the central metabolic network in response to reduced availability of inorganic phosphate, but the metabolic significance of these changes can only be assessed in terms of the fluxes supported by the network. Steady‐state metabolic flux analysis was used to define the metabolic phenotype of a heterotrophic Arabidopsis thaliana cell culture grown on a Murashige and Skoog medium containing 0, 1.25 or 5 mm inorganic phosphate. Fluxes through the central metabolic network were deduced from the redistribution of ¹³C into metabolic intermediates and end products when cells were labelled with [1‐¹³C], [2‐¹³C], or [¹³C₆]glucose, in combination with ¹⁴C measurements of the rates of biomass accumulation. Analysis of the flux maps showed that reduced levels of phosphate in the growth medium stimulated flux through phosphoenolpyruvate carboxylase and malic enzyme, altered the balance between cytosolic and plastidic carbohydrate oxidation in favour of the plastid, and increased cell maintenance costs. We argue that plant cells respond to phosphate deprivation by reconfiguring the flux distribution through the pathways of carbohydrate oxidation to take advantage of better phosphate homeostasis in the plastid.     

FoxM1 coordinates cell division,protein synthesis,and mitochondrial activity in a subset of β cells during acute metabolic stress

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The protective effects of resveratrol on Schwann cells with toxicity induced by ethanol in vitro

Schwann cells (SCs) are the myelin forming cells in the peripheral nervous system, they play a key role in the pathology of various polyneuropathies and provide trophic support to axons via expression of various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Ethanol (EtOH) adversely affected both SCs proliferation and myelin formation in culture. Resveratrol (Res) has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Whether Res has protective effects on SCs with EtOH-induced toxicity is still unclear. The protective efficacy of Res on EtOH-treated SCs in vitro was investigated in the present study. Res improved cell viability of the EtOH-treated SCs. Hoechst 33342 staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5′-adenosine monophosphate-activated protein kinase inhibitor Compound C and the silencing information regulator T1 inhibitor nicotinamide. Res could increase the mRNA and protein levels of BDNF and GDNF in the EtOH-treated SCs. However, the EtOH-induced increase of NGF in the SCs is inhibited by Res. The data from the present study indicate that Res protects SCs from EtOH-induced cell death and regulates the expression of neurotrophic factors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.     

Melanoma metabolism contributes to the cellular responses to MAPK/ERK pathway inhibitors

Background

Besides its influence on survival, growth, proliferation, invasion and metastasis, cancer cell metabolism also greatly influences the cellular responses to molecular-targeted therapies.

Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress

Background

Mitochondrial DNA (mtDNA) mutations are an important cause of mitochondrial diseases, for which there is no effective treatment due to complex pathophysiology. It has been suggested that mitochondrial dysfunction-elicited reactive oxygen species (ROS) plays a vital role in the pathogenesis of mitochondrial diseases, and the expression levels of several clusters of genes are altered in response to the elevated oxidative stress. Recently, we reported that glycolysis in affected cells with mitochondrial dysfunction is upregulated by AMP-activated protein kinase (AMPK), and such an adaptive response of metabolic reprogramming plays an important role in the pathophysiology of mitochondrial diseases.

Scope of review

We summarize recent findings regarding the role of AMPK-mediated signaling pathways that are involved in: (1) metabolic reprogramming, (2) alteration of cellular redox status and antioxidant enzyme expression, (3) mitochondrial biogenesis, and (4) autophagy, a master regulator of mitochondrial quality control in skin fibroblasts from patients with mitochondrial diseases.

Major conclusion

Induction of adaptive responses via AMPK–PFK2, AMPK–FOXO3a, AMPK–PGC-1α, and AMPK–mTOR signaling pathways, respectively is modulated for the survival of human cells under oxidative stress induced by mitochondrial dysfunction. We suggest that AMPK may be a potential target for the development of therapeutic agents for the treatment of mitochondrial diseases.

General significance

Elucidation of the adaptive mechanism involved in AMPK activation cascades would lead us to gain a deeper insight into the crosstalk between mitochondria and the nucleus in affected tissue cells from patients with mitochondrial diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.