Tauroursodeoxycholic acid prevents Bax-induced membrane perturbation and cytochrome C release in isolated mitochondria

Bax is a potent pro-apoptotic member of the Bcl-2 protein family that localizes to the mitochondrial membrane during apoptosis. Tauroursodeoxycholic acid (TUDCA) modulates the apoptotic threshold, in part, by preventing Bax translocation both in vitro and in vivo. The mechanisms by which Bax induces and TUDCA inhibits release of cytochrome c are unclear. We show here that recombinant Bax protein induced cytochrome c release in isolated mitochondria without detectable swelling. Co-incubation with TUDCA prevented efflux of mitochondrial factors and proteolytic processing of caspases in cytosolic extracts. Spectroscopic analyses of mitochondria exposed to Bax revealed increased polarity and fluidity of the membrane lipid core as well as altered protein order, indicative of Bax binding, together with loss of spin-label paramagnetism, characteristic of oxidative damage. TUDCA markedly abrogated the Bax-induced membrane perturbation. In conclusion, our results indicate that Bax protein directly induces cytochrome c release from mitochondria through a mechanism that does not require the permeability transition. Rather, it is accompanied by changes in the organization of membrane lipids and proteins. TUDCA is a potent inhibitor of Bax association with mitochondria. Thus, TUDCA modulates apoptosis by suppressing mitochondrial membrane perturbation through pathways that are also independent of the mitochondrial permeability transition.     

Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization

BACKGROUND: The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. MATERIALS AND METHODS: Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot. RESULTS: Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05). CONCLUSION: UCB and Abeta peptide activate the apoptotic machinery in neural cells. Toxicity occurs through a mitochondrial-dependent pathway, which in part involves opening of the permeability transition pore. Furthermore, membrane permeabilization is required for cytochrome c release from mitochondria and can be prevented by UDC or TUDC. These data suggest that the mitochondria is a pharmacological target for cytoprotection during unconjugated hyperbilirubinemia and neurodegenerative disorders, and that UDC or TUDC may be potential therapeutic agents.     

Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.     

Mitochondrial permeability transition pore is a potential drug target for neurodegeneration

Mitochondrial permeability transition pore (mPTP) plays a central role in alterations of mitochondrial structure and function leading to neuronal injury relevant to aging and neurodegenerative diseases including Alzheimer's disease (AD). mPTP putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and cyclophilin D (CypD). Reactive oxygen species (ROS) increase intra-cellular calcium and enhance the formation of mPTP that leads to neuronal cell death in AD. CypD-dependent mPTP can play a crucial role in ischemia/reperfusion injury. The interaction of amyloid beta peptide (Aβ) with CypD potentiates mitochondrial and neuronal perturbation. This interaction triggers the formation of mPTP, resulting in decreased mitochondrial membrane potential, impaired mitochondrial respiration function, increased oxidative stress, release of cytochrome c, and impaired axonal mitochondrial transport. Thus, the CypD-dependent mPTP is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of AD. Designing small molecules to block this interaction would lessen the effects of Aβ neurotoxicity. This review summarizes the recent progress on mPTP and its potential therapeutic target for neurodegenerative diseases including AD. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Chenodeoxycholate induction of mitochondrial permeability transition pore is associated with increased membrane fluidity and cytochrome c release: protective role of carvedilol

Chenodeoxycholate (CDCA) is a primary bile acid mostly implicated in cholestatic liver injury. In this study, we have investigated the involvement of membrane fluidity and cytochrome c release in CDCA-induced mitochondrial permeability transition (MPT), and the preventive role of carvedilol. Treatment of calcium-loaded hepatic mitochondria with CDCA was found to cause osmotic swelling and release of cytochrome c, associated with an increase in membrane fluidity, in both protein and lipid regions. Carvedilol and cyclosporine A (CyA) reduced both cytochrome c release and alterations in membrane fluidity induced by CDCA. The hydroxylated metabolite of carvedilol, BM-910228, had no effect. Thus, modulation of membrane fluidity, plays an important role in MPT pore opening promoted by CDCA. As a result, we have delineated a pathway for the preventive role of carvedilol in mitochondrial dysfunction induced by CDCA.     

Endomorphins and morphine limit anoxia-reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia-reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia-reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia-reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia-reperfusion and neurodegenerative diseases.     

Characterization of 8-epiprostaglandin F2alpha as a marker of amyloid beta-peptide-induced oxidative damage

The amyloid beta-peptide (A beta) is a major component of the neuritic plaques that are a defining histological characteristic of Alzheimer's disease. A beta can be directly toxic and pro-inflammatory to cells in vitro. Numerous reports have shown that oxidative damage and reactive oxygen species play a role in A beta-mediated neurotoxicity. 8-Epiprostaglandin F2alpha (8-isoprostane) is a well characterized product of lipid peroxidation that is formed nonenzymatically in cell membranes following an oxidative insult. We report a time- and concentration-dependent increase in 8-isoprostane levels in rat hippocampal cultures treated with A beta(1-40) or hydrogen peroxide. As evidence that 8-isoprostane production is part of an A beta toxic pathway, alkaline-treated peptide, which shows minimal toxic activity, resulted in greatly attenuated 8-isoprostane production. Although the increase in 8-isoprostane levels preceded cell death, exogenously added 8-isoprostane had no cytotoxic effects. The antioxidants vitamin E and propyl gallate attenuated A beta-induced 8-isoprostane formation yet had no effect on A beta-induced lactate dehydrogenase release. Neither vitamin E nor propyl gallate had any effect on A beta's ability to adopt a beta-pleated sheet structure and deposit on cells as determined by thioflavine S fluorescence. We conclude that 8-isoprostane is an indicator of A beta-induced damage but not necessarily a mediator of A beta-induced neurotoxicity. Also, 8-isoprostane could be a useful marker for assessing oxidative damage in the CNS.     

Myoglobin causes oxidative stress,increase of NO production and dysfunction of kidney's mitochondria

Rhabdomyolysis or crush syndrome is a pathology caused by muscle injury resulting in acute renal failure. The latest data give strong evidence that this syndrome caused by accumulation of muscle breakdown products in the blood stream is associated with oxidative stress with primary role of mitochondria. In order to evaluate the significance of oxidative stress under rhabdomyolysis we explored the direct effect of myoglobin on renal tubules and isolated kidney mitochondria while measuring mitochondrial respiratory control, production of reactive oxygen and nitrogen species and lipid peroxidation. In parallel, we evaluated mitochondrial damage under myoglobinurea in vivo. An increase of lipid peroxidation products in kidney mitochondria and release of cytochrome c was detected on the first day of myoglobinuria. In mitochondria incubated with myoglobin we detected respiratory control drop, uncoupling of oxidative phosphorylation, an increase of lipid peroxidation products and stimulated NO synthesis. Mitochondrial pore inhibitor, cyclosporine A, mitochondria-targeted antioxidant (SkQ1) and deferoxamine (Fe-chelator and ferryl-myoglobin reducer) abrogated these events. Similar effects (oxidative stress and mitochondrial dysfunction) were revealed when myoglobin was added to isolated renal tubules. Thus, rhabdomyolysis can be considered as oxidative stress-mediated pathology with mitochondria to be the primary target and possibly the source of reactive oxygen and nitrogen species. We speculate that rhabdomyolysis-induced kidney damage involves direct interaction of myoglobin with mitochondria possibly resulting in iron ions release from myoglobin's heme, which promotes the peroxidation of mitochondrial membranes. Usage of mitochondrial permeability transition blockers, Fe-chelators or mitochondria-targeted antioxidants, may bring salvage from this pathology.     

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.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.     

BID-dependent and BID-independent pathways for BAX insertion into mitochondria

In the absence of an apoptotic signal, BAX adopts a conformation that constrains the protein from integrating into mitochondrial membranes. Here, we show that caspases, including caspase-8, can initiate BAX insertion into mitochondria in vivo and in vitro. The cleavage product of caspase-8, tBID, induced insertion of BAX into mitochondria in vivo, and reconstitution in vitro showed that tBID, either directly or indirectly, relieved inhibition of the BAX transmembrane signal-anchor by the NH2-terminal domain, resulting in integration of BAX into mitochondrial membrane. In contrast to these findings, however, Bid-null mouse embryo fibroblasts supported Bax insertion into mitochondria in response to death signaling by either TNFalpha or E1A, despite the fact that cytochrome c release from the organelle was inhibited. We conclude, therefore, that a parallel Bid-independent pathway exists in these cells for mitochondrial insertion of Bax and that, in the absence of Bid, cytochrome c release can be uncoupled from Bax membrane insertion.     

Sequence and functional similarities between pro-apoptotic Bid and plant lipid transfer proteins

Pro-apoptotic proteins of the Bcl-2 family are known to act on mitochondria and facilitate the release of cytochrome c, but the biochemical mechanism of this action is unknown. Association with mitochondrial membranes is likely to be important in determining the capacity of releasing cytochrome c. The present work provides new evidence suggesting that some pro-apoptotic proteins like Bid have an intrinsic capacity of binding and exchanging membrane lipids. Detailed analysis indicates a significant sequence similarity between a subset of Bcl-2 family proteins including Bid and Nix and plant lipid transfer proteins. The similar structural signatures could be related to common interactions with membrane lipids. Indeed, isolated Bid shows a lipid transfer activity that is even higher than that of plant lipid transfer proteins. To investigate the possible relevance of these structure-function correlations to the apoptotic action of Bid, cell free assays were established with isolated mitochondria, recombinant Bid and a variety of exogenous lipids. Micromolar concentrations of lysolipids such as lysophosphatidylcholine were found to change the association of Bid with mitochondria and also stimulate the release of cytochrome c promoted by Bid. The changes in mitochondrial association and cytochrome c release were enhanced by the presence of liposomes of lipid composition similar to that of mitochondrial membranes. Thus, a mixture of liposomes, mitochondria and key lysolipids could reproduce the conditions enabling Bid to transfer lipids between donor and acceptor membranes, and also change its reversible association with mitochondria. Bid was also found to enhance the incorporation of a fluorescent lysolipid, but not of a related fatty acid, into mitochondria. On the basis of the results presented here, it is hypothesised that Bid action may depend upon its capacity of exchanging lipids and lysolipids with mitochondrial membranes. The hypothesis is discussed in relation to current models for the integrated action of pro-apoptotic proteins of the Bcl-2 family.     

Mitochondrial cholesterol import

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

Proapoptotic Bid binds to monolysocardiolipin,a new molecular connection between mitochondrial membranes and cell death

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.     

Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK(-) cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK(-) osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK(-) cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.     

Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products

Recent findings implicate that fibrillation products, the protein aggregates formed during the various steps leading to formation of mature fibrils, induce neurotoxicity predominantly in their intermediate oligomeric state. This has been shown to occur by increasing membrane permeability, eventually leading to cell death. Despite accumulating reports describing mechanisms of membrane permeabilization by oligomers in model membranes, studies directly targeted at characterizing the events occurring in biological membranes are rare. In the present report, we describe interaction of the original native structure, prefibrils and fibrils of hen egg white lysozyme (HEWL) with mitochondrial membranes, as an in vitro biological model, with the aim of gaining insight into possible mechanism of cytotoxicity at the membrane level. These structures were first characterized using a range of techniques, including fluorescence, size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were found to be flexible/hydrophobic structures with the capacity to interact with mitochondrial membranes. Possible permeabilization of mitochondria was explored utilizing sensitive fluorometric and luminometric assays. Results presented demonstrate release of mitochondrial enzymes upon exposure to HEWL oligomers, but not native enzyme monomer or mature fibrils, in a concentration-dependent manner. Release of cytochrome c was also observed, as reported earlier, and membrane stabilization promoted by addition of calcium prevented release. Moreover, the oligomer-membrane interaction was influenced by high concentrations of NaCl and spermine. The observed release of proteins from mitochondria is suggested to occur by a nonspecific perturbation mechanism.