OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization.

Most mitochondrial proteins originate from the cytosol and require transport into the organelle. Such precursor proteins must be unfolded to pass through translocation channels in mitochondrial membranes. Misfolding of transported proteins can result in their arrest and translocation failure. Arrested proteins block further import, disturbing mitochondrial functions and cellular proteostasis. Cellular responses to translocation failure have been defined in yeast. We developed the cell line-based translocase clogging model to discover molecular mechanisms that resolve failed import events in humans. The mechanism we uncover differs significantly from these described in fungi, where ATPase-driven extraction of blocked protein is directly coupled with proteasomal processing. We found human cells to rely primarily on mitochondrial factors to clear translocation channel blockage. The mitochondrial membrane depolarization triggered proteolytic cleavage of the stalled protein, which involved mitochondrial protease OMA1. The cleavage allowed releasing the protein fragment that blocked the translocase. The released fragment was further cleared in the cytosol by VCP/p97 and the proteasome.

In search for mitochondrial biomarkers of Parkinson's disease: Findings in parkin-mutant human fibroblasts.

Most cases of Parkinson's disease (PD) are idiopathic, with unknown aetiology and genetic background. However, approximately 10 % of cases are caused by defined genetic mutations, among which mutations in the parkin gene are the most common. There is increasing evidence of the involvement of mitochondrial dysfunction in the development of both idiopathic and genetic PD. However, the data on mitochondrial changes reported by different studies are inconsistent, which can reflect the variability in genetic background of the disease. Mitochondria, as a plastic and dynamic organelles, are the first place in the cell to respond to external and internal stress. In this work, we characterized mitochondrial function and dynamics (network morphology and turnover regulation) in primary fibroblasts from PD patients with parkin mutations. We performed clustering analysis of the obtained data to compare the profiles of mitochondrial parameters in PD patients and healthy donors. This allowed to extract the features characteristic for PD patients fibroblasts, which were a smaller and less complex mitochondrial network and decreased levels of mitochondrial biogenesis regulators and mitophagy mediators. The approach we used allowed a comprehensive characteristics of elements common for mitochondrial dynamics remodelling accompanying pathogenic mutation. This may be helpful in the deciphering key pathomechanisms of the PD disease.

Effect of Chronic Stress Present in Fibroblasts Derived from Patients with a Sporadic Form of AD on Mitochondrial Function and Mitochondrial Turnover.

Although the sporadic form of Alzheimer's disease (AD) is the prevalent form, the cellular events underlying the disease pathogenesis have not been fully characterized. Accumulating evidence points to mitochondrial dysfunction as one of the events responsible for AD progression. We investigated mitochondrial function in fibroblasts collected from patients diagnosed with the sporadic form of AD (sAD), placing a particular focus on mitochondrial turnover. We measured mitochondrial biogenesis and autophagic clearance, and evaluated the presence of bioenergetic stress in sAD cells. The mitochondrial turnover was clearly lower in the fibroblasts from sAD patients than in the fibroblasts from the control subjects, and the levels of many proteins regulating mitochondrial biogenesis, autophagy and mitophagy were decreased in patient cells. Additionally, the sAD fibroblasts had slightly higher mitochondrial superoxide levels and impaired antioxidant defense. Mitochondrial turnover undergoes feedback regulation through mitochondrial retrograde signaling, which is responsible for the maintenance of optimal mitochondrial functioning, and mitochondria-derived ROS participate as signaling molecules in this process. Our results showed that in sAD patients cells, there is a shift in the balance of mitochondrial function, possibly in response to the presence of cellular stress related to disease development.

Effects of plant alkaloids on mitochondrial bioenergetic parameters.

Mitochondria are among the first responders to various stress factors that challenge cell and tissue homeostasis. Various plant alkaloids have been investigated for their capacity to modulate mitochondrial activities. In this study, we used isolated mitochondria from mouse brain and liver tissues to assess nicotine, anatabine and anabasine, three alkaloids found in tobacco plant, for potential modulatory activity on mitochondrial bioenergetics parameters. All alkaloids decreased basal oxygen consumption of mouse brain mitochondria in a dose-dependent manner without any effect on the ADP-stimulated respiration. None of the alkaloids, at 1 nM or 1.25 µM concentrations, influenced the maximal rate of swelling of brain mitochondria. In contrast to brain mitochondria, 1.25 µM anatabine, anabasine and nicotine increased maximal rate of swelling of liver mitochondria suggesting a toxic effect. Only at 1 mM concentration, anatabine slowed down the maximal rate of Ca2+-induced swelling and increased the time needed to reach the maximal rate of swelling. The observed mitochondrial bioenergetic effects are probably mediated through a pathway independent of nicotinic acetylcholine receptors, as quantitative proteomic analysis could not confirm their expression in pure mitochondrial fractions isolated from mouse brain tissue.

Adaptation of mitochondrial network dynamics and velocity of mitochondrial movement to chronic stress present in fibroblasts derived from patients with sporadic form of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Only 10% of all cases are familial form, the remaining 90% are sporadic form with unknown genetic background. The etiology of sporadic AD is still not fully understood. Pathogenesis and pathobiology of this disease are limited due to the limited number of experimental models. We used primary culture of fibroblasts derived from patients diagnosed with sporadic form of AD for investigation of dynamic properties of mitochondria, including fission-fusion process and localization of mitochondria within the cell. We observed differences in mitochondrial network organization with decreased mitochondrial transport velocity, and a drop in the frequency of fusion-fission events. These studies show how mitochondrial dynamics adapt to the conditions of long-term mitochondrial stress that prevails in cells of sporadic form of AD.

Alteration of mitochondrial function in the livers of mice with glycogen branching enzyme deficiency.

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1+/-) mice. Mitochondria isolated from the livers of Gbe1+/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates.

Hallmarks of oxidative stress in the livers of aged mice with mild glycogen branching enzyme deficiency.

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1+/-), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1+/- mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1+/- livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress.

Mitochondrial Network and Biogenesis in Response to Short and Long-Term Exposure of Human BEAS-2B Cells to Aerosol Extracts from the Tobacco Heating System 2.2.

BACKGROUND/AIMS: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2). METHODS: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca2+ levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot. RESULTS: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process. CONCLUSION: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.

Mitochondria as a possible target for nicotine action.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

Mitochondria-associated membranes in aging and senescence: structure, function, and dynamics.

Sites of close contact between mitochondria and the endoplasmic reticulum (ER) are known as mitochondria-associated membranes (MAM) or mitochondria-ER contacts (MERCs), and play an important role in both cell physiology and pathology. A growing body of evidence indicates that changes observed in the molecular composition of MAM and in the number of MERCs predisposes MAM to be considered a dynamic structure. Its involvement in processes such as lipid biosynthesis and trafficking, calcium homeostasis, reactive oxygen species production, and autophagy has been experimentally confirmed. Recently, MAM have also been studied in the context of different pathologies, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, type 2 diabetes mellitus and GM1-gangliosidosis. An underappreciated amount of data links MAM with aging or senescence processes. In the present review, we summarize the current knowledge of basic MAM biology, composition and action, and discuss the potential connections supporting the idea that MAM are significant players in longevity.

Assessment of mitochondrial function following short- and long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product and reference cigarettes.

Mitochondrial dysfunction caused by cigarette smoke is involved in the oxidative stress-induced pathology of airway diseases. Reducing the levels of harmful and potentially harmful constituents by heating rather than combusting tobacco may reduce mitochondrial changes that contribute to oxidative stress and cell damage. We evaluated mitochondrial function and oxidative stress in human bronchial epithelial cells (BEAS 2B) following 1- and 12-week exposures to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), in comparison with TPM from the 3R4F reference cigarette. After 1-week exposure, 3R4F TPM had a strong inhibitory effect on mitochondrial basal and maximal oxygen consumption rates compared to TPM from THS2.2. Alterations in oxidative phosphorylation were accompanied by increased mitochondrial superoxide levels and increased levels of oxidatively damaged proteins in cells exposed to 7.5 µg/mL of 3R4F TPM or 150 µg/mL of THS2.2 TPM, while cytosolic levels of reactive oxygen species were not affected. In contrast, the 12-week exposure indicated adaptation of BEAS-2B cells to long-term stress. Together, the findings indicate that 3R4F TPM had a stronger effect on oxidative phosphorylation, gene expression and proteins involved in oxidative stress than TPM from the candidate modified-risk tobacco product THS2.2.

Quantifying ROS levels using CM-H2DCFDA and HyPer.

At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H2DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H2O2) levels using protein-based sensors (HyPer and SypHer).

[Mechanisms of mitochondrial transport and distribution within the cell]. / Mechanizmy transportu i dystrybucji mitochondriów w komórce.

Mitochondria are multifunctional, dynamic organelles, which are continuously undergoing fusion and fission and are actively distributed within the cell. Mitochondria travel along microtubules together with a mitochondrial trafficking complex, formed by motor and adaptor proteins. Proper mitochondrial movements are crucial for neurons, in which mitochondria translocate in two directions. Anterograde transport is an outward movement of mitochondria from the cell body to the synapse, whereas retrograde is an inward movement away from the synapse or plasma membrane toward the cell body. This article presents a summary of current knowledge about the intracellular transport of mitochondria and its regulation in mammalian cells.

Methods to monitor ROS production by fluorescence microscopy and fluorometry.

Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also lead to the premature death of the entire organism. Less severe increases in ROS levels may lead to the nonlethal oxidation of fundamental cellular components, such as proteins, phospholipids, and DNA, hence exerting a mutagenic effect that promotes oncogenesis and tumor progression. Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism.

Effect of mtDNA point mutations on cellular bioenergetics.

This overview discusses the results of research on the effects of most frequent mtDNA point mutations on cellular bioenergetics. Thirteen proteins coded by mtDNA are crucial for oxidative phosphorylation, 11 of them constitute key components of the respiratory chain complexes I, III and IV and 2 of mitochondrial ATP synthase. Moreover, pathogenic point mutations in mitochondrial tRNAs and rRNAs generate abnormal synthesis of the mtDNA coded proteins. Thus, pathogenic point mutations in mtDNA usually disturb the level of key parameter of the oxidative phosphorylation, i.e. the electric potential on the inner mitochondrial membrane (Δψ), and in a consequence calcium signalling and mitochondrial dynamics in the cell. Mitochondrial generation of reactive oxygen species is also modified in the mutated cells. The results obtained with cultured cells and describing biochemical consequences of mtDNA point mutations are full of contradictions. Still they help elucidate the biochemical basis of pathologies and provide a valuable tool for finding remedies in the future. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy.

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 µg/mL, however, lower PEI levels still exert some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 µg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or formation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI-mediated initiation of intrinsic apoptotic pathway is discussed.

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells.

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.

[Glycogenoses--genetic disorders leading to disturbed glycogen metabolism]. / Glikogenozy--choroby genetyczne zwiazane z zaburzeniami metabolizmu glikogenu.

Glycogen storage diseases (GSD, glycogenoses) is a group of genetic disorders resulting from abnormal metabolism of glycogen--a polymeric molecule involved in intercellular glucose storage. Currently 13 different types of glycogenoses are known. They all result from mutations in genes for different enzymes, which directly or indirectly regulate glycogen synthesis and degradation. The clinical manifestation of GSDs encompasses primarily liver, striated muscle and brain tissue dysfunction. In those tissues glycogen plays a particularly important role. spectrum and severity of symptoms is very diverse, depending on both the type and subtype of the disease as well as on the individual features of the patient. The therapy is based mainly on application of an appropriate diet. Enzyme replacement therapy is currently available for GSD type II. For some of the other types the possibility for gene therapy is intensively investigated.

Cytoprotective action of the potassium channel opener NS1619 under conditions of disrupted calcium homeostasis.

Cytoprotective properties of potassium channel openers (KCOs) have been demonstrated in several models of cell injury, mainly in ischemia-reperfusion-induced damage of cardiac muscle. The mechanism responsible for the observed cytoprotection and the relative contribution of plasma membrane or inner mitochondrial membrane potassium channels regarding the beneficial effects exerted by KCOs remain unclear. Our work demonstrates the cytoprotective properties of NS1619, an opener of large-conductance calcium-activated potassium channels (BKCa channels), using C2C12 myoblasts injured by calcium ionophore A23187 treatment. Application of two BKCa channel inhibitors, paxilline and iberiotoxin, abolished this cytoprotective effect. At concentrations of 10-100 µM, NS1619 increased the respiration rate and decreased mitochondrial membrane potential (Δψ) in C2C12 cells in a dose-dependent manner. At a concentration of 0.2 µM, paxilline, which effectively abolished the protective effect of NS1619, failed to counteract the opener-induced mitochondrial depolarization and increase in cellular respiration. This result indicates that the NS1619-mediated increase in the survival rate of A23187-treated C2C12 cells occurs in a manner distinct from its effect on mitochondrial functioning and suggests that activation of BKCa channels in the plasma membrane is the mechanism responsible for cytoprotection by NS1619.

The cytoprotective action of the potassium channel opener BMS-191095 in C2C12 myoblasts is related to the modulation of calcium homeostasis.

UNLABELLED: BMS-191095 is an opener of the mitochondrial ATP-regulated potassium channel, which has been shown to provide cytoprotection in models of ischemia-reperfusion induced injury in various tissues. This study aimed at checking the protective action of BMS-191095 under the conditions of oxidative stress or disruption of intracellular calcium homeostasis. METHODS: The cytoprotective potential of BMS-191095 was tested in C2C12 myoblasts injured by treatment with H(2)O(2) or calcium ionophore A23187. The influence of the opener on intracellular calcium levels, calpain activity and respiration rates were determined. RESULTS: BMS-191095 protected myoblasts from calcium ionophore A23187-induced injury, but not from H H(2)O(2)-induced injury. A23187-mediated cell damage was also prevented by calpain inhibitor PD 150606. A23187 administration led to a transient increase in cytosolic calcium levels, concomitant activation of calpains and a decrease in state 3 respiration rates, indicating mitochondrial dysfunction. Co-administration of BMS-191095 diminished calpain activation in A23187-treated cells but did not prevent mitochondrial damage. In the presence of BMS-191095, restoration of cytosolic calcium concentrations to basal levels after A23187 treatment was considerably faster which may underly the reduced activation of calpains. CONCLUSION: The BMS-191095-mediated cytoprotection observed in C2C12 myoblasts results probably from modulation of intracellular calcium transients leading to prevention of calpain activation.