Megamitochondria formation - physiology and pathology

Mitochondria undergo structural changes simultaneously with their functional changes in both physiological and pathological conditions. These structural changes of mitochondria are classified into two categories: simple swelling and the formation of megamitochondria (MG). Data have been accumulated to indicate that free radicals play a crucial role in the mechanism of the MG formation induced by various experimental conditions which are apparently various. These include ethanol-, chloramphenicol- and hydrazine-induced MG formation. Involvement of free radicals in the mechanism of MG formation is showed by the fact that MG formation is successfully suppressed by free radical scavengers such as α-tocopherol, coenzyme Q₁₀, and 4-OH-TEMPO. Detailed mechanisms and pathophysiological meanings of MG formation still remain to be investigated. However, a body of evidence strongly suggests that enormous changes in physicochemical and biochemical properties of the mitochondrial membranes during MG formation take place and these changes are favorable for membrane fusion. A recent report showed that continous exposure of cells with MG to free radicals induces apoptosis, finding which suggests that MG formation is an adaptative process to unfavorable environments at the level of intracellular organelles. Mitochondria try to decrease intracellular reactive oxygen species (ROS) levels by decreasing the consume of oxygen via MG formation. If mitochondria succeed to suppress intracellular ROS levels, MG return to normal both structurally and functionally, and they restore the ability to actively synthesize ATP. If cells are additionally exposed to excess amounts of free radicals, MG become swollen, membrane potential of mitochondria (ΔΨm) decreases, cytochrome c is released from mitochondria, leading to activation of caspases and apoptosis is induced.     

A role for mitochondrial aquaporins in cellular life-and-death decisions?

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.     

Free radical–induced megamitochondria formation and apoptosis

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H₂O₂, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H₂-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G₁ phase, and apoptotic sub-G₁ populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H₂O₂ and O₂⁻ from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.     

A molecular mechanism for mimosine-induced apoptosis involving oxidative stress and mitochondrial activation

Mimosine, a non-protein amino acid, is mainly known for its action as a reversible inhibitor of DNA replication and, therefore, has been widely used as a cell cycle synchronizing agent. Recently, it has been shown that mimosine also induces apoptosis, as mainly reflected in its ability to elicit characteristic nuclear changes. The present study elucidates the mechanism underlying mimosine’s apoptotic effects, using the U-937 leukemia cell line. We now demonstrate that in isolated rat liver mitochondria, mimosine induces mitochondrial swelling that can be inhibited by cyclosporine A, indicative of permeability transition (PT) mega-channel opening. Mimosine-induced apoptosis was accompanied by formation of hydrogen peroxide and a decrease in reduced glutathione levels. The apoptotic process was partially inhibited by cyclosporine A and substantially blocked by the antioxidant N-acetylcysteine, suggesting an essential role for reactive oxygen species formation during the apoptotic processes. The apoptosis induced by mimosine was also accompanied by a decrease in mitochondrial membrane potential, cytochrome c release and caspase 3 and 9 activation. Our results thus imply that mimosine activates apoptosis through mitochondrial activation and formation of H₂O₂, both of which play functional roles in the induction of cell death. Maher Hallak and Liat Vazana have contributed equally to the work.     

Mitoparan and target-selective chimeric analogues: membrane translocation and intracellular redistribution induces mitochondrial apoptosis

Mastoparan, and structurally-related amphipathic peptides, may induce cell death by augmentation of necrotic and/or apoptotic pathways. To more precisely delineate cytotoxic mechanisms, we determined that [Lys(5,8)Aib(10)]mastoparan (mitoparan) specifically induces apoptosis of U373MG and ECV304 cells, as demonstrated by endonuclease and caspase-3 activation and phosphatidylserine translocation. Live cell imaging confirmed that, following translocation of the plasma membrane, mitoparan specifically co-localizes with mitochondria. Complementary studies indicated that mitoparan induces swelling and permeabilization of isolated mitochondria, through cooperation with a protein of the permeability transition pore complex VDAC, leading to the release of the apoptogenic factor, cytochrome c. N-terminal acylation of mitoparan facilitated the synthesis of chimeric peptides that incorporated target-specific address motifs including an integrin-specific RGD sequence and a Fas ligand mimetic. Significantly, these sychnologically-organised peptides demonstrated further enhanced cytotoxic potencies. We conclude that the cell penetrant, mitochondriotoxic and apoptogenic properties of mitoparan, and its chimeric analogues, offer new insights to the study and therapeutic induction of apoptosis.     

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.     

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.     

Bax does not directly participate in the Ca(2+)-induced permeability transition of isolated mitochondria

The mitochondrial permeability transition pore and Bax have both been proposed to be involved in the release of pro-apoptotic factors from mitochondria in the "intrinsic" pathway of apoptosis. The permeability transition pore is widely thought to be a supramolecular complex including or interacting with Bax. Given the relevance of the permeability transition in vivo, we have verified whether Bax influences the formation and/or the properties of the Ca(2+)/P(i)-induced permeability transition by using mitochondriaisolated from isogenic human colon cancer bax(+/-) and bax(-/-) HCT116 cell lines. We used mitochondria isolated from both types of cells and from Bax(+) cells exposed to apoptotic stimuli, as well as Bax-less mitochondria into which exogenous Bax had been incorporated. All exhibited the same behavior and pharmacological profile in swelling and Ca(2+)-retention experiments. Mitochondria from a bax(-)/bak(-) cell line also underwent an analogous Ca(2+)/P(i)-inducible swelling. This similarity indicates that Bax hasno major role in regulating the Ca(2+)-induced mitochondrial permeability transition.     

Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS.     

Apoptosis effector mechanisms: A requiem performed in different keys

Apoptosis is the regulated form of cell death utilized by metazoans to remove unneeded, damaged, or potentially deleterious cells. Certain manifestations of apoptosis may be associated with the proteolytic activity of caspases. These changes are often held as hallmarks of apoptosis in dying cells. Consequently, many regard caspases as the central effectors or executioners of apoptosis. However, this “caspase-centric” paradigm of apoptotic cell death does not appear to be as universal as once believed. In fact, during apoptosis the efficacy of caspases may be highly dependent on the cytotoxic stimulus as well as genetic and epigenetic factors. An ever-increasing number of studies strongly suggest that there are effectors in addition to caspases, which are important in generating apoptotic signatures in dying cells. These seemingly caspase-independent effectors may represent evolutionarily redundant or failsafe mechanisms for apoptotic cell elimination. In this review, we will discuss the molecular regulation of caspases and various caspase-independent effectors of apoptosis, describe the potential context and/or limitations of these mechanisms, and explore why the understanding of these processes may have relevance in cancer where treatment is believed to engage apoptosis to destroy tumor cells.     

Protective effects of methyl gallate on H2O2-induced apoptosis in PC12 cells

Neurodegenerative disorders are a class of diseases that have been linked to apoptosis induced by elevated levels of reactive oxygen species (ROS). ROS activates the apoptotic cascade through mitochondrial dysfunction and damage to lipids, proteins and DNA. Recently, fruit and tea-derived polyphenols have been found to be beneficial in decreasing oxidative stress and increasing overall health. Further, polyphenols including epigallocatechin gallate (EGCG) have been reported to inhibit apoptotic signaling and increase neural cell survival. In an effort to better understand the beneficial properties associated with polyphenol consumption, the aim of this study was to explore the neuroprotective effects of EGCG, methyl gallate (MG), gallic acid (GA) and N-acetylcysteine (NAC) on H₂O₂-induced apoptosis in PC12 cells and elucidate potential protective mechanisms. Cell viability data demonstrates that MG and NAC pre-treatments significantly increase viability of H₂O₂-stressed cells, while pre-treatments with EGCG and GA exacerbates stress. Quantitation of apoptosis and mitochondrial membrane potential shows that MG pre-treatment prevents mitochondria depolarization, however does not inhibit apoptosis and is thus evidence that MG can inhibit mitochondria-mediated apoptosis. Subsequent analysis of DNA degradation and caspase activation reveals that MG inhibits activation of caspase 9 and has a partial inhibitory effect on DNA degradation. These findings confirm the involvement of both intrinsic and extrinsic apoptotic pathways in H₂O₂-induced apoptosis and suggest that MG may have potential therapeutic properties against mitochondria-mediated apoptosis.     

Mitochondria and caspases in induced apoptosis in human luteinized granulosa cells

Apoptosis occurs as a physiologic process in the ovarian life cycle. Staurosporine, a protein kinase inhibitor, is reported to induce apoptosis. Here, we hypothesize that staurosporine will induce apoptosis in human luteinized granulosa cells and that mitochondria and the caspase cascade participate in this process. Luteinized granulosa cells isolated from in vitro fertilization patients were treated with staurosporine. Microscopy revealed that staurosporine treatment resulted in cells exhibiting evidence of apoptosis, including cell detachment, loss of cell processes, membrane shrinkage, and formation of apoptotic bodies. In the staurosporine-treated cells, flow cytometry and confocal microscopy showed a decrease in the mitochondrial cardiolipin levels. Western analysis showed cleavage of caspase-9, an initiator caspase, of caspase-3, an executioner caspase, and of a caspase substrate, poly-(ADP-ribose)-polymerase (PARP) in staurosporine-treated cells. These data support our hypothesis and that this is the first demonstration of the involvement of mitochondria and of cleavage of caspases in human luteinized granulosa cell apoptosis. This may serve as a useful model to delineate the mechanism of apoptosis in the ovary, such as corpus luteum regression.     

Mitochondrial membrane dynamics, cristae remodelling and apoptosis

Mitochondria form a highly dynamic reticular network in living cells, and undergo continuous fusion/fission events and changes in ultrastructural architecture. Although significant progress has been made in elucidating the molecular events underlying these processes, their relevance to normal cell function remains largely unexplored. Emerging evidence, however, suggests an important role for mitochondrial dynamics in cellular apoptosis. The mitochondria is at the core of the intrinsic apoptosis pathway, and provides a reservoir for protein factors that induce caspase activation and chromosome fragmentation. Additionally, mitochondria modulate Ca2+ homeostasis and are a source of various metabolites, including reactive oxygen species, that have the potential to function as second messengers in response to apoptotic stimuli. One of the mitochondrial factors required for activation of caspases in most intrinsic apoptotic pathways, cytochrome c, is largely sequestered within the intracristae compartment, and must migrate into the boundary intermembrane space in order to allow passage across the outer membrane to the cytosol. Recent evidence argues that inner mitochondrial membrane dynamics regulate this process. Here, we review the contribution of mitochondrial dynamics to the intrinsic apoptosis pathway, with emphasis on the inner membrane.     

ClC-3 chloride channel prevents apoptosis induced by thapsigargin in PC12 cells

Cell volume can be altered by two different ways, swelling and shrinkage. Cell swelling is regulated by volume-regulated Cl⁻ channel (VRC). It is not well understood whether shrinkage is regulated by VRC. We previously found that antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) prevented cell proliferation, which was related to cell swell volume regulation. In the present study, we further studied the role of ClC-3 Cl⁻ channel in cell apoptosis which was related to cell shrinkage volume regulation by using antisense oligonucleotide specific to ClC-3 (ClC-3 antisense) and ClC-3 cDNA transfection techniques. We found that thapsigargin (TG), a specific inhibitor of the endoplasmic reticulum calcium ATPase, evoked apoptotic morphological changes (including cytoplasmic blebbing, condensation of nuclear chromatin, and the formation of apoptotic bodies), DNA laddering, and caspase-3 activation in PC12 cells (Pheochromocytoma-derived cell line). TG increased the cell apoptotic population with a decrease in cell viability. These effects were consistent with the decrease in endogenous ClC-3 protein expression, which was also induced by TG. Overexpression of ClC-3 significantly inhibited TG effect on PC12 cell apoptosis, whereas the ClC-3 antisense produced opposite effects and facilitated apoptosis induced by TG. Our data strongly suggest that ClC-3 channel in PC12 cells mediates TG-induced apoptotic process through inhibitory mechanism. Thus, it appears that ClC-3 Cl⁻ channel mediates both cell proliferation and apoptosis through accelerative and inhibitory fashions, respectively. These authors contributed equally to this work.     

Botulinum neurotoxin C initiates two different programs for neurite degeneration and neuronal apoptosis

Clostridial neurotoxins are bacterial endopeptidases that cleave the major SNARE proteins in peripheral motorneurons. Here, we show that disruption of synaptic architecture by botulinum neurotoxin C1 (BoNT/C) in central nervous system neurons activates distinct neurodegenerative programs in the axo-dendritic network and in the cell bodies. Neurites degenerate at an early stage by an active caspase-independent fragmentation characterized by segregation of energy competent mitochondria. Later, the cell body mitochondria release cytochrome c, which is followed by caspase activation, apoptotic nuclear condensation, loss of membrane potential, and, finally, cell swelling and lysis. Recognition and scavenging of dying processes by glia also precede the removal of apoptotic cell bodies, in line with a temporal and spatial segregation of different degenerative processes. Our results suggest that, in response to widespread synaptic damage, neurons first dismantle their connections and finally undergo apoptosis, when their spatial relationships are lost.     

Apoptotic signaling in methylglyoxal-treated human osteoblasts involves oxidative stress, c-Jun N-terminal kinase, caspase-3, and p21-activated kinase 2

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. MG is cytotoxic through induction of cell death, and elevated MG levels in diabetes patients are believed to contribute to diabetic complications. In this report, we show for the first time that MG treatment triggers apoptosis in human osteoblasts. We further show that MG-induced apoptosis of osteoblasts involves specific apoptotic biochemical changes, including oxidative stress, c-Jun N-terminal kinase (JNK) activation, mitochondrial membrane potential changes, cytochrome C release, increased Bax/Bcl-2 protein ratios, and activation of caspases (caspase-9, caspase-3) and p21-activated protein kinase 2 (PAK2). Treatment of osteoblasts with SP600125, a JNK-specific inhibitor, led to a reduction in MG-induced apoptosis and decreased activation of caspase-3 and PAK2, indicating that JNK activity is upstream of these events. Experiments using anti-sense oligonucleotides against PAK2 further showed that PAK2 activation is required for MG-induced apoptosis in osteoblasts. Interestingly, we also found that MG treatment triggered nuclear translocation of NF-kappaB, although the precise regulatory role of NF-kappaB activation in MG-induced apoptosis remains unclear. Lastly, we examined the effect of MG on osteoblasts in vivo, and found that exposure of rats to dietary water containing 100-200 microM MG caused bone mineral density (BMD) loss. Collectively, these results reveal for the first time that MG treatment triggers apoptosis in osteoblasts via specific apoptotic signaling, and causes BMD loss in vivo.     

Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?

Several mitochondrial proteins, such as cytochrome c, are directly involved in the pathway for caspase activation following induction of apoptosis. Release of mitochondrial cytochrome c early in apoptosis is rapid and almost complete. Microinjection of cytochrome c into resting cells induces apoptosis, but the amount needed approaches the total cellular content. These observations suggest that mitochondrial protein release is an all-or-nothing process inside the cell and not an amplifiable apoptotic signal. To test this hypothesis, laser micro-irradiation was used to rupture membranes of individual mitochondria within living rat neural cells. Laser micro-irradiation caused swelling, fragmentation, depolarization, and cytochrome c depletion in targeted mitochondria. These effects were explained by correlative electron microscopic analysis showing local rupture of outer and inner membranes at the site of irradiation. In all cases, there were no detectable changes in the structure, membrane potential, or cytochrome c content of neighboring, non-irradiated organelles. Furthermore, irradiation of up to 15% of the mitochondria in a cell did not induce apoptosis. The results from these laser micro-irradiation experiments prove that local release of mitochondrial proteins does not constitute an amplifiable apoptotic signal in resting neural cells.     

Induction of Apoptosis in Rat Myocardium under Anoxic Conditions

The effect of anoxic incubation of small slices of isolated rat hearts on respiration, internucleosomal DNA fragmentation, and mitochondrial ultrastructure was investigated. Anoxic incubation for 72 h induced apoptosis accompanied by internucleosomal DNA fragmentation and changes in respiration and mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In a significant part of the mitochondrial population there were signs of mitochondrial swelling and appearance of electron-dense mitochondria. Anoxia also induced the appearance of an atypical (and previously unknown) population of small electron-dense mitochondria. They were characterized by unusual localization inside electron-light mitochondria. Under anoxic conditions the inner mitochondrial membrane formed electron-dense ordered structures. All changes described here reflect two opposing processes occurring in mitochondria: apoptotic destruction and compensatory processes responsible for maintenance of mitochondria.     

Chelerythrine induces apoptosis through a Bax/Bak-independent mitochondrial mechanism

Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.     

Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release

Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.