Rapidly fatal poisoning with an insecticide containing rotenone.

Introduction: Rotenone is a botanical pesticide derived from extracts of Derris roots, which is traditionally used as piscicide, but also as an industrial insecticide for home gardens. Its mechanism of action is potent inhibition of mitochondrial respiratory chain by uncoupling oxidative phosphorylation by blocking electron transport at complex-I. Despite its classification as mild to moderately toxic to humans (estimated LD50, 300-500 mg/kg), there is a striking variety of acute toxicity of rotenone depending on the formulation (solvents). Human fatalities with rotenone-containing insecticides have been rarely reported, and a rapid deterioration within a few hours of the ingestion has been described previously in one case. Case report: A 49-year-old Tamil man with a history of asthma, ingested 250 mL of an insecticide containing 1.24% of rotenone (3.125 g, 52.1-62.5 mg/kg) in a suicide attempt at home. The product was not labeled as toxic. One hour later, he vomited repeatedly and emergency services were alerted. He was found unconscious with irregular respiration and was intubated. On arrival at the emergency department, he was comatose (GCS 3) with fixed and dilated pupils, and absent corneal reflexes. Physical examination revealed hemodynamic instability with hypotension (55/30 mmHg) and bradycardia (52 bpm). Significant laboratory findings were lactic acidosis (pH 6.97, lactate 17 mmol/L) and hypokalemia (2 mmol/L). Cranial computed tomography (CT) showed early cerebral edema. A single dose of activated charcoal was given. Intravenous hydration, ephedrine, repeated boli of dobutamine, and a perfusor with 90 micrograms/h norepinephine stabilized blood pressure temporarily. Atropine had a minimal effect on heart rate (58 bpm). Intravenous lipid emulsion was considered (log Pow 4.1), but there was a rapid deterioration with refractory hypotension and acute circulatory failure. The patient died 5h after ingestion of the insecticide. No autopsy was performed. Quantitative analysis of serum performed by high-resolution/accurate mass-mass spectrometry and liquid chromatography (LC-HR/AM-MS): 560 ng/mL rotenone. Other substances were excluded by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Conclusion: The clinical course was characterized by early severe symptoms and a rapidly fatal evolution, compatible with inhibition of mitochondrial energy supply. Although rotenone is classified as mild to moderately toxic, physicians must be aware that suicidal ingestion of emulsified concentrates may be rapidly fatal. (n=3): stridor, cyanosis, cough (one each). Local swelling after chewing or swallowing soap developed at the earliest after 20 minutes and persisted beyond 24 hours in some cases. Treatment with antihistamines and/or steroids relieved the symptoms in 9 cases. Conclusion: Bar soap ingestion by seniors carries a risk of severe local reactions. Half the patients developed symptoms, predominantly swellings of tongue and/or lips (38%). Cognitive impairment, particularly in the cases of dementia (37%), may increase the risk of unintentional ingestion. Chewing and intraoral retention of soap leads to prolonged contact with the mucosal membranes. Age-associated physiological changes of oral mucosa probably promote the irritant effects of the surfactants. Medical treatment with antihistamines and corticosteroids usually leads to rapid decline of symptoms. Without treatment, there may be a risk of airway obstruction.

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BACKGROUND: In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata). RESULTS: Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse. CONCLUSIONS: Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

Protective role of amantadine in mitochondrial dysfunction and oxidative stress mediated by hepatitis C virus protein expression.

Amantadine is an antiviral and antiparkinsonian drug that has been evaluated in combination therapies against hepatitis C virus (HCV) infection. Controversial results have been reported concerning its efficacy, and its mechanism of action remains unclear. Data obtained in vitro suggested a role of amantadine in inhibiting HCV p7-mediated cation conductance. In keeping with the fact that mitochondria are responsible to ionic fluxes and that HCV infection impairs mitochondrial function, we investigated a potential role of amantadine in modulating mitochondrial function. Using a well-characterized inducible cell line expressing the full-length HCV polyprotein, we found that amantadine not only prevented but also rescued HCV protein-mediated mitochondrial dysfunction. Specifically, amantadine corrected (i) overload of mitochondrial Ca(2+); (ii) inhibition of respiratory chain activity and oxidative phosphorylation; (iii) reduction of membrane potential; and (iv) overproduction of reactive oxygen species. The effects of amantadine were observed within 15 min following drug administration and confirmed in Huh-7.5 cells transfected with an infectious HCV genome. These effects were also observed in cells expressing subgenomic HCV constructs, indicating that they are not mediated or only in part mediated by p7. Single organelle analyzes carried out on isolated mouse liver mitochondria demonstrated that amantadine induces hyperpolarization of the membrane potential. Moreover, amantadine treatment increased the calcium threshold required to trigger mitochondrial permeability transition opening. In conclusion, these results support a role of amantadine in preserving cellular bioenergetics and redox homeostasis in HCV-infected cells and unveil an effect of the drug which might be exploited for a broader therapeutic utilization.

Expanding view of phenotype and oxidative stress in Friedreich's ataxia patients with and without idebone.

Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia, is characterised by progressive ataxia with dysarthria of speech, loss of deep-tendon reflexes, impaired vibratory and proprioceptive sensations and corticospinal weakness with a Babinski's sign. Patients eventually also develop kyphoscoliosis, cardiomyopathy and diabetes mellitus. The disease is a GAA repeat disorder resulting in severely reduced levels of frataxin, with secondary increased sensitivity to oxidative stress. The anti-oxidative drug, idebenone, is effective against FRDA-associated cardiomyopathy. We provide detailed clinical, electrophysiological and biochemical data from 20 genetically confirmed FRDA patients and have analysed the relationship between phenotype, genotype and malondialdehyde (MDA), which is a marker of superoxide formation. We assessed the effects of idebenone biochemically by measuring blood MDA and clinically by serial measurements of the International Cooperative Ataxia Rating Scale (ICARS). The GAA repeat length influenced the age at onset (p <0.001), the severity of ataxia (p = 0.02), the presence of cardiomyopathy (p = 0.04) and of low-frequency hearing loss (p = 0.009). Multilinear regression analysis showed (p = 0.006) that ICARS was dependent on the two variables of disease duration (p = 0.01) and size of the GAA expansion (p = 0.02). We found no correlation to bilateral palpebral ptosis, visual impairment, diabetes mellitus or skeletal deformities, all of which appear to be signs of disease progression rather than severity. We discuss more thoroughly two underrecognised clinical findings: palpebral ptosis and GAA length-dependent low-frequency hearing loss. The average ICARS remained unchanged in 10 patients for whom follow-up on treatment was available (mean 2.9 years), whereas most patients treated with idebenone reported an improvement in dysarthria (63%), hand dexterity (58%) and fatigue (47%) after taking the drug for several weeks or months. Oxidative stress analysis showed an unexpected increase in blood MDA levels in patients on idebenone (p = 0.04), and we discuss the putative underlying mechanism for this result, which could then explain the unique efficacy of idebenone in treating the FRDA-associated cardiomyopathy, as opposed to other antioxidative drugs. Indeed, idebenone is not only a powerful stimulator of complexes II and III of the respiratory chain, but also an inhibitor of complex I activity, then promoting superoxide formation. Our preliminary clinical observations are the first to date supporting an effect of idebenone in delaying neurological worsening. Our MDA results point to the dual effect of idebenone on oxidative stress and to the need for controlled studies to assess its potential toxicity at high doses on the one hand, and to revisit the exact mechanisms underlying the physiopathology of Friedreich's ataxia on the other hand, while recent reports suggest non-oxidative pathophysiology of the disease.

Hepatitis C virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor 1 alpha-mediated glycolytic adaptation.

Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

Bistability of mitochondrial respiration underlies paradoxical reactive oxygen species generation induced by anoxia

Increased production of reactive oxygen species (ROS) in mitochondria underlies major systemic diseases, and this clinical problem stimulates a great scientific interest in the mechanism of ROS generation. However, the mechanism of hypoxia-induced change in ROS production is not fully understood. To mathematically analyze this mechanism in details, taking into consideration all the possible redox states formed in the process of electron transport, even for respiratory complex III, a system of hundreds of differential equations must be constructed. Aimed to facilitate such tasks, we developed a new methodology of modeling, which resides in the automated construction of large sets of differential equations. The detailed modeling of electron transport in mitochondria allowed for the identification of two steady state modes of operation (bistability) of respiratory complex III at the same microenvironmental conditions. Various perturbations could induce the transition of respiratory chain from one steady state to another. While normally complex III is in a low ROS producing mode, temporal anoxia could switch it to a high ROS producing state, which persists after the return to normal oxygen supply. This prediction, which we qualitatively validated experimentally, explains the mechanism of anoxia-induced cell damage. Recognition of bistability of complex III operation may enable novel therapeutic strategies for oxidative stress and our method of modeling could be widely used in systems biology studies.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

BRAIN DAMAGE IN METHYLMALONIC ACIDURIA: 2-METHYLCITRATE LEADS TO AMMONIA INCREASE AND APOPTOSIS

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in methylmalonic aciduria. 1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate (PA) were repeatedly added to the culture media at two different time points of the cultures. In cultures treated with 2-MCA, we observed a significant increase of lactate in the medium, consistent with a possible inhibition of Krebs cycle and respiratory chain, as described earlier in the literature. Interestingly, we further observed that 2-MCA induced an important increase in ammonia production with concomitant decrease of glutamine concentrations, which suggests an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease with deleterious effects on early stages of brain development. By immunohistochemistry we could show that 2-MCA substantially increased the number of apoptotic cells. On the cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth stimulating effect on the cultures. We can conclude that 2-MCA was the most toxic metabolite in our model for methylmalonic aciduria inducing ammonia accumulation and massive apoptosis in brain cells.

Expanding view of phenotype and oxidative stress in Friedreich's ataxia patients with and without idebenone

Summary: Friedreich's ataxia (FRDA), the most common autosomal recessive ataxia, is characterised by progressive ataxia with dysarthria of speech, loss of deep-tendon reflexes, impaired vibratory and proprioceptive sensations and corticospinal weakness with a Babinski's sign. Patients eventually also develop kyphoscoliosis, cardiomyopathy and diabetes mellitus. The disease is a GAA repeat disorder resulting in severely reduced levels of frataxin, with secondary increased sensitivity to oxidative stress. The anti-oxidative drug, idebenone, is effective against FRDA-associated cardiomyopathy. We provide detailed clinical, electrophysiological and biochemical data from 20 genetically confirmed FRDA patients and have analysed the relation-ship between phenotype, genotype and malondialdehyde (MDA), which is a marker of superoxide formation. We assessed the effects of idebenone biochemically by measuring blood M DA and clinically by serial measurements of the International Cooperative Ataxia Rating Scale (ICARS). The GAA repeat length influenced the age at onset (p <0.001), the severity of ataxia (p= 0.02), the presence of cardiomyopathy (p =0.04) and of low-frequency hearing loss (p = 0.009). Multilinear regression analysis showed (p = 0.006) that ICARS was dependent on the two variables of disease duration (p = 0.01) and size of the GAA expansion (p = 0.02). We found no correlation to bilateral palpebral ptosis visual impairment, diabetes mellitus or skeletal deformities, all of which appear to be signs of disease progression rather than severity. We discuss more thoroughly two underrecognised clinical findings: palpebral ptosis and GAA length-dependent low-frequency hearing loss. The average ICARS remained unchanged in 10 patients for whom follow-up on treatment was available (mean 2.9 years), whereas most patients treated with idebenone reported an improvement in dysarthria (63%), hand dexterity (.58%) and fatigue (47%) after taking the drug for several weeks or months. Oxidative stress analysis showed an unexpected increase in blood MDA levels in patients on idebenone (p = 0.04), and we discuss the putative underlying mechanism for this result, which could then explain the unique efficacy of idebenone in treating the FRDA-associated cardiomyopathy, as opposed to other antioxidative drugs. Indeed, idebenone is not only a powerful stimulator of complexes II and III of the respiratory chain, but also an inhibitor of complex I activity, then promoting superoxide formation. Our preliminary clinical observations are the first to date supporting an effect of idebenone in delaying neurological worsening. Our MDA results point to the dual effect of idebenone on oxidative stress and to the need for controlled studies to assess its potential toxicity at high doses on the one hand, and to revisit the exact mechanisms underlying the .physiopathology of Friedreich's ataxia on the other hand, while recent reports suggest non-oxidative pathophysiology of the disease.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Drug-induced mitochondrial dysfunction and cardiotoxicity.

Mitochondria has an essential role in myocardial tissue homeostasis; thus deterioration in mitochondrial function eventually leads to cardiomyocyte and endothelial cell death and consequent cardiovascular dysfunction. Several chemical compounds and drugs have been known to directly or indirectly modulate cardiac mitochondrial function, which can account both for the toxicological and pharmacological properties of these substances. In many cases, toxicity problems appear only in the presence of additional cardiovascular disease conditions or develop months/years following the exposure, making the diagnosis difficult. Cardiotoxic agents affecting mitochondria include several widely used anticancer drugs [anthracyclines (Doxorubicin/Adriamycin), cisplatin, trastuzumab (Herceptin), arsenic trioxide (Trisenox), mitoxantrone (Novantrone), imatinib (Gleevec), bevacizumab (Avastin), sunitinib (Sutent), and sorafenib (Nevaxar)], antiviral compound azidothymidine (AZT, Zidovudine) and several oral antidiabetics [e.g., rosiglitazone (Avandia)]. Illicit drugs such as alcohol, cocaine, methamphetamine, ecstasy, and synthetic cannabinoids (spice, K2) may also induce mitochondria-related cardiotoxicity. Mitochondrial toxicity develops due to various mechanisms involving interference with the mitochondrial respiratory chain (e.g., uncoupling) or inhibition of the important mitochondrial enzymes (oxidative phosphorylation, Szent-Györgyi-Krebs cycle, mitochondrial DNA replication, ADP/ATP translocator). The final phase of mitochondrial dysfunction induces loss of mitochondrial membrane potential and an increase in mitochondrial oxidative/nitrative stress, eventually culminating into cell death. This review aims to discuss the mechanisms of mitochondrion-mediated cardiotoxicity of commonly used drugs and some potential cardioprotective strategies to prevent these toxicities.

Mitochondrial disease in Southwestern Finland. Population-based molecular genetic and clinical studies

Mitochondria are present in all eukaryotic cells. They enable these cells utilize oxygen in the production of adenosine triphosphate in the oxidative phosphorylation system, the mitochondrial respiratory chain. The concept ‘mitochondrial disease’ conventionally refers to disorders of the respiratory chain that lead to oxidative phosphorylation defect. Mitochondrial disease in humans can present at any age, and practically in any organ system. Mitochondrial disease can be inherited in maternal, autosomal dominant, autosomal recessive, or X-chromosomal fashion. One of the most common molecular etiologies of mitochondrial disease in population is the m.3243A>G mutation in the MT-TL1 gene, encoding mitochondrial tRNALeu(UUR). Clinical evaluation of patients with m.3243A>G has revealed various typical clinical features, such as stroke-like episodes, diabetes mellitus and sensorineural hearing loss. The prevalence and clinical characteristics of mitochondrial disease in population are not well known. This thesis consists of a series of studies, in which the prevalence and characteristics of mitochondrial disease in the adult population of Southwestern Finland were assessed. Mitochondrial haplogroup Uk was associated with increased risk of occipital ischemic stroke among young women. Large-scale mitochondrial DNA deletions and mutations of the POLG1 gene were the most common molecular etiologies of progressive external ophthalmoplegia. Around 1% of diabetes mellitus emerging between the ages 18 – 45 years was associated with the m.3243A>G mutation. Moreover, among these young diabetic patients, mitochondrial haplogroup U was associated with maternal family history of diabetes. These studies demonstrate the usefulness of carefully planned molecular epidemiological investigations in the study of mitochondrial disorders.