Update on the Histoenzymatic Methods for Visualization of the Activity of Individual Mitochondrial Respiratory Chain Complexes in the Human Frozen Sections.

Investigation of mitochondrial metabolism perturbations and successful diagnosis of patients with mitochondrial abnormalities often requires assessment of human samples like muscle or liver biopsy as well as autopsy material. Immunohistochemical and histochemical examination is an important technique to investigate mitochondrial dysfunction that combined with spectrophotometric and Blue Native electrophoresis techniques can be an important tool to provide diagnosis of mitochondrial disorders. In this chapter, we focus on technical description of the methods that are suitable to detect the activity of complex I, II, and IV of mitochondrial respiratory chain in frozen sections of brain, heart, muscle, and liver biopsies/autopsy. The protocols provided can be useful not only for general assessment of mitochondrial activity in studied material, but they are also successfully used in the diagnostic procedures in case of suspicion of mitochondrial disorders. In the age of high-performance NGS sequencing, these methods can be used to confirm whether mutations are pathogenic by proving their impact on the activity of individual respiratory chain complexes.

Modified Blue Native Gel Approach for Analysis of Respiratory Supercomplexes.

In mitochondrial oxidative phosphorylation (Ox-Phos), individual electron transport chain complexes are thought to assemble into supramolecular entities termed supercomplexes (SCs). The technique of blue native (BN) gel electrophoresis has emerged as the method of choice for analyzing SCs. However, the process of sample extraction for BN gel analysis is somewhat tedious and introduces the possibility for experimental artifacts. Here we outline a streamlined method that eliminates a centrifugation step and provides a more representative sampling of a population of mitochondria on the final gel. Using this method, we show that SC composition does not appear to change dynamically with altered mitochondrial function.

Incorporating Distance-Based Top-n-gram and Random Forest To Identify Electron Transport Proteins.

Cellular respiration provides direct energy substances for living organisms. Electron storage and transportation should be completed through electron transport chains during the cellular respiration process. Thus, identifying electron transport proteins is an important research task. In protein identification, selection of the feature extraction method and classification algorithm has a direct bearing on classification. The distance-based Top-n-gram method, which was proposed based on the frequency profile and considered evolutionary information, was used in this study for feature extraction. The Max-Relevance-Max-Distance algorithm was adopted for feature selection. The first 4D features that greatly influenced the classification result were selected to form the feature data set. Finally, the random forest algorithm was used to identify electron transport proteins. Under the 10-fold cross-validation of the model constructed in this study, sensitivity, specificity, and accuracy rates surpassed 85%, 80%, and 82%, respectively. In the testing set, F-measure, AUC value, and accuracy exceeded 74%, 95%, and 86%, respectively. These experimental results indicated that the classification model built in this study is an effective tool in identifying electron transport proteins.

Time Course of Decrease in Skeletal Muscle Mitochondrial Biogenesis after Discontinuation of High-Fat Diet.

It is known that a high-fat diet induces an increase in mitochondrial biogenesis in skeletal muscle. To examine the time course of decrease in mitochondrial biogenesis in skeletal muscle after discontinuing a high-fat diet feeding, C57BL/6 mice were fed a high-fat diet for 4 wk and then switched to the control diet for another 3 or 7 d. During the high-fat diet withdrawal period, the protein content of the mitochondrial respiratory chain decreased faster than the fatty acid oxidation enzymes. The mitochondrial DNA copy number remained high for at least 1 wk after withdrawing the high-fat diet. These results suggested that after switching to the control diet following a period of high-fat diet, the increased mitochondrial biogenesis levels are maintained for a few days, and the rate of decline is divergent between the different mitochondrial components.

Immunolocalisation pattern of complex I-V in ageing human retina: Correlation with mitochondrial ultrastructure.

Earlier studies reported accumulation of mitochondrial DNA mutations in ageing and age-related macular degeneration. To know about the mitochondrial status with age, we examined immunoreactivity (IR) to markers of mitochondria (anti-mitochondrial antibody and voltage-dependent anion channel-1) and complex I-V (that mediate oxidative phosphorylation, OXPHOS) in donor human retinas (age: 19-94years; N=26; right eyes). In all samples, at all ages, IR to anti-mitochondrial antibody and voltage-dependent anion channel-1 was prominent in photoreceptor cells. Between second and seventh decade of life, strong IR to complex I-V was present in photoreceptors over macular to peripheral retina. With progressive ageing, the photoreceptors showed a decrease in complex I-IR (subunit NDUFB4) at eighth decade, and a weak or absence of IR in 10 retinas between ninth and tenth decade. Patchy IR to complex III and complex IV was detected at different ages. IR to ND1 (complex I) and complex II and V remained unaltered with ageing. Nitrosative stress (evaluated by IR to a nitro-tyrosine antibody) was found in photoreceptors. Superoxide dismutase-2 was found upregulated in photoreceptors with ageing. Mitochondrial ultrastructure was examined in two young retinas with intact complex IR and six aged retinas whose counterparts showed weak to absence of IR. Observations revealed irregular, photoreceptor inner segment mitochondria in aged maculae and mid-peripheral retina between eighth and ninth decade; many cones possessed autophagosomes with damaged mitochondria, indicating age-related alterations. A trend in age-dependent reduction of complex I-IR was evident in aged photoreceptors, whereas patchy complex IV-IR (subunits I and II) was age-independent, suggesting that the former is prone to damage with ageing perhaps due to oxidative stress. These changes in OXPHOS system may influence the energy budget of human photoreceptors, affecting their viability.

Mitochondrial tRNA mutations in 2070 Chinese Han subjects with hypertension.

BACKGROUND: Mitochondria have the profound impact on vascular function in both health and disease. However, mitochondrial genetic determinants for the development of hypertension remain poorly explored. METHODS AND RESULTS: The Sanger sequence analysis of 22 mitochondrial tRNA genes were performed in a cohort of 2070 Han Chinese hypertensive and 512 control subjects. This analysis identified 165 variants among 22 tRNA genes. These variants were evaluated for the pathogenicity using the following criteria: (1) present in <1% of the controls; (2) evolutional conservation; (3) potential structural and functional alterations. We identified 47 (5 known and 42 novel/putative) hypertension-associated tRNA variants in 80 hypertensive subjects. These variants could have potential structural alterations and functional significance of tRNAs. By using lymphoblastoid cell lines derived from 6 probands carrying one of 6 represented variants (tRNA(Ala) 5655T>C, tRNA(Gly) 10003T>C, tRNA(Leu(UUR)) 3253T>C, tRNA(Asp) 7551A>G, tRNA(Glu) 14692A>G, tRNA(Thr) 15909A>G) and 6 control subjects lacking these variants, we showed marked reductions in the steady-state level of corresponding 5 tRNAs, but not tRNA(Thr), in mutant cell lines, compared with control cells lines. The various decreases in the activities of complexes I, III and IV were observed in mutant cells carrying one of five tRNA variants, except tRNA(Thr) 15909A>G variant. The deficient respirations were responsible for the decrease in the mitochondrial ATP production and increasing production of reactive oxygen species in mutant cell lines carrying one of five tRNA variants. CONCLUSION: Mitochondrial tRNA variants are the important causes of hypertension, accounting for 3.9% cases of 2070 Han Chinese hypertensive subjects. Our findings may provide new insights into the pathophysiology of hypertension that were manifested by mitochondrial dysfunction.

Evaluating clinical mitochondrial respiratory chain enzymes from biopsy specimens presenting skewed probability distribution of activity data.

Due to the relative rarity of mitochondrial diseases, generating reference ranges is problematic in evaluation of respiratory chain activities particularly in pediatric cases. We determined the sample distribution of respiratory chain enzyme activities in skeletal muscle biopsies collected from pediatric patients suspected of neuromuscular disorders. Activities of NADH-ubiquinone reductase, NADH-cytochrome c reductase, succinate-cytochrome c reductase; ubiquinol-cytochrome c reductase and cytochrome c oxidase activities have log-normal distributions even when confirmed mitochondrial diseases were ruled out. Impact of the log-normal distribution of the respiratory chain enzyme activities on clinical diagnostics is discussed.

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Role of altered mitochondria functions in the pathogenesis of systemic lupus erythematosus.

Mitochondria, main producers of reactive-oxygen species (ROS), were studied to examine their role in the pathogenesis of systemic lupus erythematosus (SLE). PBMCs and mitochondria were isolated from SLE patients and healthy volunteers for various parameters. Mitochondrial ROS, swelling, hyperpolarization and levels of cytochrome c, caspase3 in the cells were assessed by flow cytometry. ROS was significantly increased in SLE patients (SLE vs controls: 1.83 ± 1.03 vs 1.10 ± 0.35; p < 0.0001). Depolarized state of mitochondria was greater in patients (SLE vs controls: 7.10 ± 5.50% vs 2.5 ± 1.8%; p < 0.05). Mitochondria swelling was found to be significantly altered in patients (SLE vs controls: 112.65 ± 36.56 vs 60.49 ± 20.69; p < 0.001). Expression of cytochrome c and caspase 3 (SLE vs controls: 1.37 ± 0.37% vs 1.01 ± 0.03%; 1.57 ± 0.46% vs 1.06 ± 0.07%; p < 0.05) respectively was found to be significantly increased in SLE. Further, the enzymatic activity of mitochondrial complex was assessed in isolated mitochondria. A significant decrease in activity of Complex I (SLE vs controls: 11.79 ± 3.18 vs 15.10 ± 6.38 nmol NADH oxidized/min/mg protein, p < 0.05); Complex IV (SLE vs control: 9.41 ± 5.16 vs 13.56 ± 5.92 nmol cytochrome c oxidized/min/mg protein, p < 0.05) and Complex V (SLE vs controls: 4.85 ± 1.39 vs 6.17 ± 2.02 nmol ATP hydrolyzed/min/mg protein, p < 0.05) was found in SLE patients in comparison to healthy controls. However, Complex II did not show significant variation in either group (SLE vs controls: 42.2 ± 28.6 vs 61.71 ± 42.3 nmol succinate oxidized/min/mg protein; ns). The decrease in enzyme activities of mitochondrial Complexes I, IV and V on one hand and ROS, hyperpolarization and apoptosis on the other points toward a possible role of mitochondria in the pathogenesis of lupus.

In vitro studies in VCP-associated multisystem proteinopathy suggest altered mitochondrial bioenergetics.

Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).

The characterization of neuroenergetic effects of chronic L-tyrosine administration in young rats: evidence for striatal susceptibility.

Tyrosinemia type II is an inborn error of metabolism caused by a deficiency in hepatic cytosolic aminotransferase. Affected patients usually present a variable degree of mental retardation, which may be related to the level of plasma tyrosine. In the present study we evaluated effect of chronic administration of L-tyrosine on the activities of citrate synthase, malate dehydrogenase, succinate dehydrogenase and complexes I, II, II-III and IV in cerebral cortex, hippocampus and striatum of rats in development. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old); rats were killed 12 h after last injection. Our results demonstrated that L-tyrosine inhibited the activity of citrate synthase in the hippocampus and striatum, malate dehydrogenase activity was increased in striatum and succinate dehydrogenase, complexes I and II-III activities were inhibited in striatum. However, complex IV activity was increased in hippocampus and inhibited in striatum. By these findings, we suggest that repeated administrations of L-tyrosine cause alterations in energy metabolism, which may be similar to the acute administration in brain of infant rats. Taking together the present findings and evidence from the literature, we hypothesize that energy metabolism impairment could be considered an important pathophysiological mechanism underlying the brain damage observed in patients with tyrosinemia type II.

No excess of mitochondrial DNA deletions within muscle in progressive multiple sclerosis.

BACKGROUND: Mitochondrial dysfunction is an established feature of multiple sclerosis (MS). We recently described high levels of mitochondrial DNA (mtDNA) deletions within respiratory enzyme-deficient (lacking mitochondrial respiratory chain complex IV with intact complex II) neurons and choroid plexus epithelial cells in progressive MS. OBJECTIVES: The objective of this paper is to determine whether respiratory enzyme deficiency and mtDNA deletions in MS were in excess of age-related changes within muscle, which, like neurons, are post-mitotic cells that frequently harbour mtDNA deletions with ageing and in disease. METHODS: In progressive MS cases (n=17), known to harbour an excess of mtDNA deletions in the central nervous system (CNS), and controls (n=15), we studied muscle (paraspinal) and explored mitochondria in single fibres. Histochemistry, immunohistochemistry, laser microdissection, real-time polymerase chain reaction (PCR), long-range PCR and sequencing were used to resolve the single muscle fibres. RESULTS: The percentage of respiratory enzyme-deficient muscle fibres, mtDNA deletion level and percentage of muscle fibres harbouring high levels of mtDNA deletions were not significantly different in MS compared with controls. CONCLUSION: Our findings do not provide support to the existence of a diffuse mitochondrial abnormality involving multiple systems in MS. Understanding the cause(s) of the CNS mitochondrial dysfunction in progressive MS remains a research priority.

Mitochondrial abnormalities in temporal lobe of autistic brain.

Autism spectrum disorder (ASD) consists of a group of complex developmental disabilities characterized by impaired social interactions, deficits in communication and repetitive behavior. Multiple lines of evidence implicate mitochondrial dysfunction in ASD. In postmortem BA21 temporal cortex, a region that exhibits synaptic pathology in ASD, we found that compared to controls, ASD patients exhibited altered protein levels of mitochondria respiratory chain protein complexes, decreased Complex I and IV activities, decreased mitochondrial antioxidant enzyme SOD2, and greater oxidative DNA damage. Mitochondrial membrane mass was higher in ASD brain, as indicated by higher protein levels of mitochondrial membrane proteins Tom20, Tim23 and porin. No differences were observed in either mitochondrial DNA or levels of the mitochondrial gene transcription factor TFAM or cofactor PGC1α, indicating that a mechanism other than alterations in mitochondrial genome or mitochondrial biogenesis underlies these mitochondrial abnormalities. We further identified higher levels of the mitochondrial fission proteins (Fis1 and Drp1) and decreased levels of the fusion proteins (Mfn1, Mfn2 and Opa1) in ASD patients, indicating altered mitochondrial dynamics in ASD brain. Many of these changes were evident in cortical pyramidal neurons, and were observed in ASD children but were less pronounced or absent in adult patients. Together, these findings provide evidence that mitochondrial function and intracellular redox status are compromised in pyramidal neurons in ASD brain and that mitochondrial dysfunction occurs during early childhood when ASD symptoms appear.

Mitochondrial function in human brains is affected by pre- and post mortem factors.

AIM: Mitochondrial function and the ensuing ATP synthesis are central to the functioning of the brain and contribute to neuronal physiology. Most studies on neurodegenerative diseases have highlighted that mitochondrial dysfunction is an important event contributing to pathology. However, studies on the human brain mitochondria in various neurodegenerative disorders heavily rely on post mortem samples. As post mortem tissues are influenced by pre- and post mortem factors, we investigated the effect of these variables on mitochondrial function. METHODS: We examined whether the mitochondrial function (represented by mitochondrial enzymes and antioxidant activities) in post mortem human brains (n=45) was affected by increased storage time (11.8-104.1 months), age of the donor (2 days to 80 years), post mortem interval (2.5-26 h), gender difference and agonal state [based on Glasgow Coma Scale: range=3-15] in the frontal cortex, as a prototype. RESULTS: We observed that the activities of citrate synthase, succinate dehydrogenase and mitochondrial reductase (MTT) were significantly affected only by gender difference (citrate synthase: P=0.005; succinate dehydrogenase: P=0.01; mitochondrial reductase: P=0.006), being higher in females, but not by any other factor. Mitochondrial complex I activity was significantly inhibited by increasing age (r=-0.40; P=0.05). On the other hand, the mitochondrial antioxidant enzyme glutathione reductase decreased with severe agonal state (P=0.003), while the activity of glutathione-S-transferase declined with increased storage time (P=0.005) and severe agonal state (P=0.02). CONCLUSION: Our data highlight the influence of pre- and post mortem factors on preservation of mitochondrial function with implications for studies on brain pathology employing stored human samples.

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.

High-frequency minisatellite instability of the mitochondrial genome in colorectal cancer tissue associated with clinicopathological values.

Most studies of mitochondrial DNA (mtDNA) mutations in colorectal cancer have used case-control and case-database comparisons without searching their clinical relevance. This study was to investigate colorectal cancer tissue-specific mtDNA mutations from 54 matched colorectal cancer and adjacent normal tissues and then to evaluate their clinical values. This study focused on analyzing control region including mtDNA minisatellites and coding regions. Cancer tissue-specific mtDNA mutations were found in over half of the patients (59%). The patterns of mtDNA mutations were substitution only (13%), mtDNA minisatellite instability (mtMSI) (20%) and both mutations combined (26%). mtMSI in colorectal cancer was mainly occurred in the 303 polyC (35%) and 16184 poly C (19%) minisatellite. mtDNA copy number and hydrogen peroxide level were significantly increased in colorectal cancer tissue. The amount of mtDNA large deletions was significantly decreased in colorectal cancer tissue compared with those from matched normal mucosa (p = 0.03). The activity of the mitochondrial respiratory chain enzyme complexes I, II and III in colorectal cancer tissues was impaired. mtDNA haplogroup B4 might be closely associated with colorectal cancer risk. The patient group harboring cancer tissue-specific mtDNA mutations showed larger tumor sizes (p = 0.005) and more advanced TNM stages (p = 0.002). Thus, mtDNA mutations in colorectal cancer might be implicated in risk factors that induce poor outcomes and tumorigenesis.

Blue native-gel electrophoresis proteomics.

The importance of the mitochondrion in maintaining normal cellular physiology has long been appreciated. Recently there has been an upsurge in mitochondrial research due to increased recognition that a number of diseases are caused by defective functioning of this key intracellular organelle. Given this, along with advances made in proteomics technologies, the mitochondrion is clearly recognized as a top candidate for proteomics analysis. However, mitochondrial proteomics is not a trivial undertaking due to physicochemical properties that impair the resolution of inner mitochondrial membrane proteins when using conventional proteomic gel electrophoresis procedures. To circumvent such problems, many laboratories have adapted blue native-gel electrophoresis (BN-PAGE), a specialized type of native-gel electrophoresis, to generate high-resolution proteomic maps of the oxidative phosphorylation system. In this short overview the concepts and methods of BN-PAGE are presented, which demonstrate the power of using this complementary proteomics approach to identify alterations in the mitochondrial proteome that contribute to disease.

Quantitative shotgun proteomics of enriched heterocysts from Nostoc sp. PCC 7120 using 8-plex isobaric peptide tags.

The filamentous cyanobacterium Nostoc sp. strain PCC 7120 is capable of fixing atmospheric nitrogen. The labile nature of the core process requires the terminal differentiation of vegetative cells to form heterocysts, specialized cells with altered cellular and metabolic infrastructure to mediate the N2-fixing process. We present an investigation targeting the cellular proteomic expression of the heterocysts compared to vegetative cells of a population cultured under N2-fixing conditions. New 8-plex iTRAQ reagents were used on enriched replicate heterocyst and vegetative cells, and replicate N2-fixing and non-N2-fixing filaments to achieve accurate measurements. With this approach, we successfully identified 506 proteins, where 402 had confident quantifications. Observations provided by purified heterocyst analysis enabled the elucidation of the dominant metabolic processes between the respective cell types, while emphasis on the filaments enabled an overall comparison. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production.

Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes.

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.