Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia‐reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia‐reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n‐acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system. Copyright © 2009 John Wiley & Sons, Ltd.     

Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.     

Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death

Ultraviolet A (UVA), the long wavelength part of the sun's ultraviolet radiation, elicits its harmful effects through production of reactive oxygen species. In this study, we have tested the hypothesis that the mitochondrial electron transport chain, the main source of reactive oxygen species in cells, importantly contributes to UVA-induced cell damage. Model cell lines completely lacking a mitochondrial electron transport chain (rho(0)-cells) were not protected against UVA-induced cell death. Also, primary human fibroblasts and keratinocytes with induced depletion of electron transport chain activity were not better protected against UVA-induced cell death. On the other hand, diphenyleneiodonium and resiniferatoxin, inhibitors of plasma membrane oxidases, protected primary human fibroblasts against UVA, as potently as the lipid peroxidation chain breaker Trolox. These data indicate that plasma membrane electron transport systems, but not the mitochondrial electron transport chain, play a major role in UVA-induced cell death.     

The role of lipid radicals in electron transport and energy transformation

Data given propose two regimes of lipid radicals and oxygen utilization realized in microsomal and mitochondrial membranes. The first one, lipid peroxidation, i.e. interaction of lipid radicals and oxygen is an empty step. In converting this regime to the functional one NADPH-dependent lipid peroxidation is inhibited. A change of this regime to the functional one in microsome demand the presence of hydroxylation substrates. Setting lipid radical-dependent coupling apparatus on phosphorylation in mitochondria occur in the presence of ADP and Pi-phosphorylation substrates.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Mitochondrial control of cell death

In many instances, permeabilization of mitochondrial membranes is a rate-limiting step of apoptotic or necrotic cell demise. This has important consequences for the pathophysiology of cell death, as well as for its pharmacological control.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Cell death caused by selenium deficiency and protective effect of antioxidants

Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although alpha-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and alpha-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides.     

Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage.     

Mitochondrial regulation of apoptotic cell death

Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

The death domain kinase RIP has an important role in DNA damage-induced, p53-independent cell death

Tumor suppressor p53 plays a critical role in cellular responses, such as cell cycle arrest and apoptosis following DNA damage. DNA damage-induced cell death can be mediated by a p53-dependent or p53-independent pathway. Although p53-mediated apoptosis has been well documented, little is known about the signaling components of p53-independent cell death. Here we report that the death domain kinase, RIP (receptor-interacting protein), is important for DNA damage-induced, p53-independent cell death. DNA damage induces cell death in both wild-type and p53-/- mouse embryonic fibroblast cells. We found that RIP-/- mouse embryonic fibroblast cells, which have a mutant form of the p53 protein, are resistant to DNA damage-induced cell death. The reconstitution of RIP protein expression in RIP-/- cells restored the sensitivity of cells to DNA damage-induced cell death. We also found that RIP mediates this process through activating mitogen-activated protein kinase, JNK1. Furthermore, knocking down the expression of RIP blocked DNA damage-induced cell death in the human colon cancer cell line, p53 null HCT 116. Taken together, our study demonstrates that RIP is one of the critical components involved in mediating DNA damage-induced, p53-independent cell death.     

Mitochondrial membrane permeability transition and cell death

Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received much attention. In apoptotic and necrotic death, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase in the permeability of the mitochondrial membrane that leads to loss of Deltapsi, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (PTP) and putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Our studies of mice lacking Cyp D have revealed that it is essential for occurrence of the MPT and that the Cyp D-dependent MPT regulates some forms of necrotic cell death, but not apoptotic death. We have also shown that two anti-apoptotic proteins, Bcl-2 and Bcl-x(L), block the MPT by directly inhibition of VDAC activity. Here we summarize a role of the MPT in cell death.     

Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.     

Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_m exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca²⁺ release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

Mitochondrial dynamics in yeast cell death and aging

Mitochondria play crucial roles in programmed cell death and aging. Different stimuli activate distinct mitochondrion-dependent cell death pathways, and aging is associated with a progressive increase in mitochondrial damage, culminating in oxidative stress and cellular dysfunction. Mitochondria are highly dynamic organelles that constantly fuse and divide, forming either interconnected mitochondrial networks or separated fragmented mitochondria. These processes are believed to provide a mitochondrial quality control system and enable an effective adaptation of the mitochondrial compartment to the metabolic needs of the cell. The baker's yeast, Saccharomyces cerevisiae, is an established model for programmed cell death and aging research. The present review summarizes how mitochondrial morphology is altered on induction of cell death or on aging and how this correlates with the induction of different cell death pathways in yeast. We highlight the roles of the components of the mitochondrial fusion and fission machinery that affect and regulate cell death and aging.