Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I–IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.     

Fenproporex Increases Locomotor Activity and Alters Energy Metabolism,and Mood Stabilizers Reverse These Changes: a Proposal for a New Animal Model of Mania

Fenproporex (Fen) is converted in vivo into amphetamine, which is used to induce mania-like behaviors in animals. In the present study, we intend to present a new animal model of mania. In order to prove through face, construct, and predictive validities, we evaluated behavioral parameters (locomotor activity, stereotypy activity, and fecal boli amount) and brain energy metabolism (enzymes citrate synthase; malate dehydrogenase; succinate dehydrogenase; complexes I, II, II–III, and IV of the mitochondrial respiratory chain; and creatine kinase) in rats submitted to acute and chronic administration of fenproporex, treated with lithium (Li) and valproate (VPA). The administration of Fen increased locomotor activity and decreased the activity of Krebs cycle enzymes, mitochondrial respiratory chain complexes, and creatine kinase, in most brain structures evaluated. In addition, treatment with mood stabilizers prevented and reversed this effect. Our results are consistent with the literature that demonstrates behavioral changes and mitochondrial dysfunction caused by psychostimulants. These findings suggest that chronic administration of Fen may be a potential animal model of mania.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

Non-Nucleoside Reverse Transcriptase Inhibitors Efavirenz and Nevirapine Inhibit Cytochrome C Oxidase in Mouse Brain Regions

The highly active antiretroviral therapy completely changed the clinical evolution of HIV infection, reducing the morbidity and mortality among human immunodeficiency virus (HIV)-1 infected patients. Therefore, in the present study we evaluated the effect of chronic efavirenz (EFV) and nevirapine (NVP) administration on mitochondrial respiratory chain complexes activities (I, II, II-III, IV) in different brain regions of mice. Mice were orally administered via gavage with EFV 10 mg/kg, NVP 3.3 mg/kg or vehicle (controls) once a day for 36 days. We observed that the complex IV activity was inhibited by both EFV and NVP in cerebral cortex, striatum and hippocampus of mice, but not in cerebellum, as compared to control group. In contrast, chronic EFV and NVP administration did not alter complexes I, II and II-III. We speculated that brain energy metabolism dysfunction could be involved in the CNS-related adverse effects.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

In vitro effects of silver nanoparticles on the mitochondrial respiratory chain

Silver has been used for years in medicine; it has known antimicrobial properties. Additionally, silver has been used in water and air filtration to eliminate microorganisms, and, more recently, as a biocide to prevent infections in burns. In contact with the human body, nanoparticles can elicit a spectrum of tissue responses such as the generation of reactive oxygen species, decreased function of mitochondria and even cell death. Mitochondries are intracellular organelles that play a crucial role in ATP production. In the present work, we evaluate the in vitro effect of silver nanoparticles (AgN) on the activities of mitochondrial respiratory chain complexes from the brain, skeletal muscle, heart, and liver of rats. Our results demonstrated that AgN (10, 25, and 50 mg l^?1) decreases the activity of mitochondrial respiratory chain complexes I, II, III, and IV from all tissues.     

Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

Thioacetamide-Induced Fulminant Hepatic Failure Induces Cerebral Mitochondrial Dysfunction by Altering the Electron Transport Chain Complexes

Fulminant hepatic failure (FHF) is an acute form of hepatic encephalopathy resulting from severe inflammatory or necrotic liver damage without any previously established liver damage. This develops as a complication due to viral infections, and drug abuse. FHF also occurs in acute disorders like Reye’s syndrome. Although the exact mechanisms in the etiology of FHF are not understood, elevated levels of brain ammonia have been consistently reported. Such increased ammonia levels are suggested to alter neurotransmission signals and impair cerebral energy metabolism due to mitochondrial dysfunctions. In the present study we have examined the role of cerebral electron transport chain complexes, including complex I, II, III IV, and pyruvate dehydrogenase in the non-synaptic mitochondria isolated from the cortex of the thioacetamide-induced FHF rats. Further, we have examined if the structure of mitochondria is altered. The results of the current study demonstrated a decrease in the activity of the complex I by 31 and 48% at 18 and 24 h respectively after the thioacetamide injection. Similarly, the activity of electron transport chain complex III was inhibited by 35 and 52% respectively, at 18 and 24 h, respectively. The complex II and complex IV, on the other hand, revealed unaltered activity. Further the activity of pyruvate dehydrogenase at 18 and 24 h after the induction of FHF was inhibited by 29 and 43%, respectively. Our results also suggest mitochondrial swelling in FHF induced rats. The inhibition of the respiratory complexes III and I and pyruvate dehydrogenase might lead to the increased production of free radical resulting in oxidative stress and cerebral energy disturbances thereby leading to mitochondrial swelling and further contributing to the pathogenesis of FHF.     

Inhibition of complex I by neuroleptics in normal human brain cortex parallels the extrapyramidal toxicity of neuroleptics

There is increasing evidence that a defect of the mitochondrial respiratory chain is implicated in the development of Parkinson disease. Decreased complex I activity of the mitochondrial respiratory chain has been reported in platelets, muscle, and brain of patients with Parkinson disease. Extrapyramidal symptoms (e.g. parkinsonism and dystonic reactions) are major limiting side effects of neuroleptics. Experimental evidence suggests that neuroleptics inhibit complex I in rat brain. There has not been a study of the effects of neuroleptics in human tissue, however. We therefore analyzed the activities of complexes I + III, complexes II + III, succinate dehydrogenase, complex IV (cytochrome c oxidase), and of citrate synthase in normal human brain cortex after the addition of haloperidol and chlorpromazine and the atypical neuroleptics risperidone, zotepine, and clozapine. Activity of complex I was progressively inhibited by all neuroleptics. Half maximal inhibition (IC₅₀) was 0.1 mM fo r haloperidol, 0.4 mM for chlorpromazine, and 0.5 mM for risperidone and zotepine. Clozapine had no effect on enzyme activity at concentrations up to 0.5 mM, followed by a slow decline with a maximum inhibition of 70% at 10 mM. IC50 was at about 2.5 mM. Thus, the concentration of clozapine needed to cause 50% inhibition of the activity of complexes I and III was about 5 times that of zotepine and risperidone, about 6 times that of chlorpromazine, and 25 times that of haloperidol. The inhibition thus paralleled the incidence of extrapyramidal effects caused by the different neuroleptics as they are known from numerous clinical studies. Our data support the hypothesis that neuroleptic-induced extrapyramidal side effects may be due to inhibition of the mitochondrial respiratory chain. (Mol Cell Biochem 174: 255–259, 1997)     

Inhibition of mitochondrial respiratory chain in brain of rats subjected to an experimental model of depression

Depressive disorders, including major depression, are serious and disabling. However, the exact pathophysiology of depression is not clearly understood. Life stressors contribute in some fashion to depression and are an extension of what occurs normally. In this context, chronic stress has been used as an animal model of depression. Based on the hypothesis that metabolism impairment might be involved in the pathophysiology of depression, in the present work we evaluated the activities of mitochondrial respiratory chain complexes and creatine kinase in brain of rats subjected to chronic stress. After 40 days of mild stress, a reduction in sweet food ingestion was observed, as well as increased adrenal gland weight, when compared to control group. We also verified that control group gained weight after 40 days, but stressed group did not. Moreover, our findings showed that complex I, III and IV were inhibited in stress group only in cerebral cortex and cerebellum. On the other hand, complex II and creatine kinase were not affected in stressed group. Although it is difficult to extrapolate our findings to the human condition, the inhibition of mitochondrial respiratory chain by chronic stress may be one mechanism in the pathophysiology of depressive disorders.     

Inhibition of mitochondrial respiratory chain in brain of rats subjected to an experimental model of depression

Depressive disorders, including major depression, are serious and disabling. However, the exact pathophysiology of depression is not clearly understood. Life stressors contribute in some fashion to depression and are an extension of what occurs normally. In this context, chronic stress has been used as an animal model of depression. Based on the hypothesis that metabolism impairment might be involved in the pathophysiology of depression, in the present work we evaluated the activities of mitochondrial respiratory chain complexes and creatine kinase in brain of rats subjected to chronic stress. After 40 days of mild stress, a reduction in sweet food ingestion was observed, as well as increased adrenal gland weight, when compared to control group. We also verified that control group gained weight after 40 days, but stressed group did not. Moreover, our findings showed that complex I, III and IV were inhibited in stress group only in cerebral cortex and cerebellum. On the other hand, complex II and creatine kinase were not affected in stressed group. Although it is difficult to extrapolate our findings to the human condition, the inhibition of mitochondrial respiratory chain by chronic stress may be one mechanism in the pathophysiology of depressive disorders.     

Respiratory chain supercomplexes in plant mitochondria

Supercomplexes are defined associations of protein complexes, which are important for several cellular functions. This "quintenary" organization level of protein structure recently was also described for the respiratory chain of plant mitochondria. Except succinate dehydrogenase (complex II), all complexes of the oxidative phosphorylation (OXPOS) system (complexes I, III, IV and V) were found to form part of supercomplexes. Compositions of these supramolecular structures were systematically investigated using digitonin solubilizations of mitochondrial fractions and two-dimensional Blue-native (BN) polyacrylamide gel electrophoresis. The most abundant supercomplex of plant mitochondria includes complexes I and III at a 1:2 ratio (I1 + III2 supercomplex). Furthermore, some supercomplexes of lower abundance could be described, which have I2 + III4, V2, III2 + IV(1-2), and I1 + III2 + IV(1-4) compositions. Supercomplexes consisting of complexes I plus III plus IV were proposed to be called "respirasome", because they autonomously can carry out respiration in the presence of ubiquinone and cytochrome c. Plant specific alternative oxidoreductases of the respiratory chain were not associated with supercomplexes under all experimental conditions tested. However, formation of supercomplexes possibly indirectly regulates alternative respiratory pathways in plant mitochondria on the basis of electron channeling. In this review, procedures to characterize the supermolecular organization of the plant respiratory chain and results concerning supercomplex structure and function are summarized and discussed.     

Significance of respirasomes for the assembly/stability of human respiratory chain complex I

We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.     

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.     

Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells

Glutamate is an excitotoxin responsible for causing neuronal damage associated with mitochondria dysfunction. We have analyzed the relationship between the mitochondrial respiratory rate, the membrane potential (delta psi) and the activity of mitochondrial complexes in retinal cells in culture, used as neuronal models. Glutamate (10 microM-10 mM) dose-dependently decreased the O2 consumption and the membrane potential. A linear correlation was found between these parameters, suggesting that the mitochondrial respiratory function was affected. Exposure to glutamate (100 microM) for 10 min, in the absence of Mg2+, inhibited the activity of complex I (26.3%), complexes II/III (22.2%) and complex IV (26.7%). MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), a non-competitive antagonist of the NMDA (N-methyl-D-aspartate) receptors, completely reversed the effect exerted by 100 microM glutamate at the level of complexes I, II/III and IV. These results suggest that NMDA receptor-mediated inhibition of mitochondrial respiratory chain complexes may be responsible for the alteration in the respiratory rate of chick retinal cells submitted to glutamate.     

Inhibition of the Mitochondrial Respiratory Chain by Phenylalanine in Rat Cerebral Cortex

Phenylketonuria (PKU) is biochemically characterized by the accumulation of phenylalanine (Phe) and its metabolites in tissues of affected children. Neurological damage is the clinical hallmark of PKU, and Phe is considered the main neurotoxic metabolite in this disorder. However, the mechanisms of neurotoxicity are poorly known. The main objective of the present work was to measure the activities of the mitochondrial respiratory chain complexes (RCC) and succinate dehydrogenase (SDH) in brain cortex of Wistar rats subjected to chemically induced hyperphenylalaninemia (HPA). We also investigated the in vitro effect of Phe on SDH and RCC activities in the cerebral cortex of 22-day-old rats. HPA was induced by subcutaneous administration of 2.4 mol/g body weight -methylphenylalanine, a phenylalanine hydroxylase inhibitor, once a day, plus 5.2 M/g body weight phenylalanine, twice a day, from the 6th-21st postnatal day. The results showed a reduction of SDH and complex I + III activity in brain cortex of rats subjected to HPA. We also verified that Phe inhibited the in vitro activity of complexes I + III, possibly by competition with NADH. Considering the importance of SDH and RCC for the maintenance of energy supply to brain, our results suggest that energy deficit may contribute to the Phe neurotoxicity in PKU.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Mitochondrial Respiratory Dysfunction and Oxidative Stress after Chronic Malathion Exposure

Malathion is a pesticide used on a large scale and with high potential risk for human exposure. However, it is reasonable to hypothesize that while the malathion is metabolizing reactive oxygen species (ROS) can be generated and subsequently there is onset of an oxidative stress in central nervous system (CNS) structures: hippocampus, cortex, striatum and cerebellum of intoxicated rats due to mitochondrial respiratory chain disfunctions. The present study was therefore undertaken to evaluate malathion-induced lipid peroxidation (LPO), superoxide production from sub-mitochondrial particles and the activity of complexes II and IV of the mitochondrial respiratory chain. Malathion was administered in doses of 25, 50, 100 and 150 mg malathion/kg. After malathion administration LPO increased in hippocampus and striatum. This was accompanied by an increase in the formation of superoxide in submitochondrial particles in the hippocampus. Complex IV suffered significant inhibition of its activity. We could demonstrate in this study that malathion induces oxidative stress and it could be due to inactivation of mitochondrial respiratory complexes.     

Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat

We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration ofEscherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0–0.8 Hz) in systemic arterial pressure signals. LPS also elicited progressive hypotension, and death ensued within 4 h. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochromec reductase (Complexes I + III) and cytochromec oxidase (Complex IV) in the RVLM during all three phases of endotoxemia. On the other hand, the activity of succinate cytochromec reductase (Complexes II + III) remained unaltered. We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat.     

线粒体电子传递系统的组装及其生物学意义

电子传递链亦称呼吸链,由位于线粒体内膜的I、II、III、IV 4种复合物组成,负责电子传递和产生质子梯度。电子主要从复合物I进入电子传递链,经复合物III传递至复合物IV。电子传递系统的组装是一个十分复杂的过程,目前已知主要有约69个结构亚基以及至少16个组装因子参与了人类复合物I、III、IV的组装,这些蛋白质由核基因组与线粒体基因组共同编码。对线粒体电子传递系统的蛋白质组成及其结构已研究得较为清楚,但对它们的组装了解得还比较初步。许多人类线粒体疾病是由于电子传递系统的功能障碍引起的,其中又有许多是由于该系统中一个或多个部件的错误组装引起的。研究这些缺陷不仅能够加深对线粒体疾病发病机理的了解,也有助于揭示线粒体功能的调控机制。将着重对电子传递系统复合物的组装及其与人类疾病关系的研究进展进行综述。