线粒体稳定高钙信号诱发超氧炫

线粒体对于细胞钙信号和活性氧信号转导有重要的调控作用．超氧炫是新近发现的单个线粒体超氧阴离子短时程爆发现象,反映了活性氧生成动力学的一种新形式．线粒体钙信号作为重要的细胞功能调控信号,能否及如何调控超氧炫尚待深入研究．本研究对HeLa细胞进行高胞外钙和离子霉素刺激,或用皂苷穿孔细胞质膜后置于高钙细胞内液中,两种方法均显著增加了超氧炫发生的频率．其中,穿孔细胞胞浆高钙诱导的超氧炫依赖于线粒体钙单向转运体,表明超氧炫由线粒体基质内高钙信号所诱发．重要的是,离子霉素诱导的超氧炫发生频率与线粒体稳态钙水平线性相关,而与瞬态线粒体钙无相关性,提示钙离子对超氧炫的调控是一个多步骤、相对缓慢的过程．综上,线粒体基质的稳态高钙是超氧炫的重要调控因子．     

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.     

Ammonia Neurotoxicity and the Mitochondrial Permeability Transition

Ammonia is a neurotoxin that predominantly affects astrocytes. Disturbed mitochondrial function and oxidative stress, factors implicated in the induction of the mitochondrial permeability transition (MPT), appear to be involved in the mechanism of ammonia neurotoxicity. We have recently shown that ammonia induces the MPT in cultured astrocytes. To elucidate the mechanisms of the MPT, we examined the role of oxidative stress and glutamine, a byproduct of ammonia metabolism. The ammonia-induced MPT was blocked by antioxidants, suggesting a causal role of oxidative stress. Direct application of glutamine (4.5-7.0 mM) to cultured astrocytes increased free radical production and induced the MPT. Treatment of astrocytes with the mitochondrial glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, completely blocked free radical formation and the MPT, suggesting that high ammonia concentrations in mitochondria resulting from glutamine hydrolysis may be responsible for the effects of glutamine. These studies suggest that oxidative stress and glutamine play major roles in the induction of the MPT associated with ammonia neurotoxicity.     

微丝骨架通过调控线粒体膜透性影响拟南芥耐盐性

植物微丝参与了许多重要的细胞生理活动,与植物耐盐性有密切的联系。在微丝解聚剂Latrunculin B（LatB）存在的情况下,拟南芥会表现出盐胁迫敏感。本研究结果表明盐胁迫下LatB可增加拟南芥线粒体膜通透性转换孔（mitochondrial permeability transition pore,MPTP）开放度,导致线粒体膜电势下降和细胞色素C的释放。而加入MPTP抑制剂环孢素A（CsA）后,膜电势下降程度降低,细胞色素C释放减少,解聚微丝造成的盐敏感表型得到一定程度恢复。     

Redox Regulation of the Mitochondrial Permeability Transition Pore

The recent data on redox regulation of the mitochondrial cyclosporin-sensitive pore are reviewed here. They indicate that the pore is modulated by the redox state of pyridine nucleotides and glutathione at two independent sites. Special attention is paid to experimental approaches for studying this phenomenon in isolated mitochondria. The relation between oxidative stress and the opening of the mitochondrial pore in some cases of cell injury and in programmed cell death (apoptosis) is discussed.     

Proinflammatory Cytokines Stimulate Mitochondrial Superoxide Flashes in Articular Chondrocytes In Vitro and In Situ

Objective

Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.

Methods

Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by “superoxide flash” activity recorded with time lapse scanning.

Results

Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or “superoxide flashes” occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm²*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.

Conclusions

The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.     

The Mitochondrial Permeability Transition as a Target for Neuroprotection

Mitochondria serve as checkpoints and amplifiers on cell death pathways. In the central nervous system, mitochondrial involvement seems essential for normal expression of cell death phenotypes, and interference with these pathways thus seems a reasonable approach to neuroprotection. We have been involved in examining the potential involvement of the mitochondrial permeability transition (mPT) as one of several possible mechanisms by which mitochondria may be drawn into these death cascades. This possibility, though still controversial, is supported by evidence that factors that may stimulate mPT induction are associated with some forms of cell death (e.g., in stroke) and are modulated by diseases of the central nervous system (e.g., Huntington's). Evidence of neuroprotection seen with compounds such as N-Met-Val cyclosporine also support this possibility.     

Role of the Mitochondrial Permeability Transition Pore in Apoptosis

Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial megachannel (=mitochondrial PT pore). PT has important metabolic consequences, namely the collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins. Recent evidence suggests that PT is a critical, rate limiting event of apoptosis (programmed cell death): (i) induction of PT suffices to cause apoptosis; (ii) one of the immediate consequences of PT, disruption of the mitochondrial transmembrane potential (_m), is a constant feature of early apoptosis; (iii) prevention of PT impedes the _m collapse as well as all other features of apoptosis at the levels of the cytoplasma, the nucleus, and the plasma membrane; (iv) PT is modulated by members of the apoptosis-regulatory bcl-2 gene family. Recent data suggest that the acquisition of the apoptotic phenotype, including characteristic changes in nuclear morphology and biochemistry (chromatin condensation and DNA fragmentation), depends on the action of apoptogenic proteins released from the mitochondrial intermembrane space.     

活性氧、线粒体通透性转换与细胞凋亡

线粒体是真核细胞中非常重要的细胞器,细胞中的活性氧等自由基主要来源于此,线粒体膜的通透性转换(mitochondrial permeability transition,MPT)及其孔道(mitochondrialpermeability transition pore,MPTP)更是在内源性细胞凋亡中发挥了关键作用。持续性的线粒体膜通透性转换在凋亡的效应阶段起决定性作用,可介导细胞色素c等促凋亡因子从线粒体释放到胞浆中,进一步激活下游的信号通路,导致细胞不可逆地走向凋亡。瞬时性的线粒体膜通透性转换及其偶联的线粒体局部的活性氧爆发同样具有促凋亡的作用。线粒体通透性孔道的开放释放出大量活性氧,这些活性氧又能够进一步激活该孔道,以正反馈的形式进一步加剧孔道的打开,放大凋亡信号。活性氧、线粒体通透性转换与细胞凋亡之间具有密不可分的联系,本文根据已知的研究结果集中讨论了这三者的关系,并着重论述了该领域中的最新发现和成果。     

Mitochondrial Permeability Transition Induced by Different Concentrations of Zinc

Zinc is one of the required trace elements in animals, and it serves an important role in biological systems. However, high levels of zinc are poisonous to organisms. So far, there exist conflicting reports about zinc ions-induced mitochondrial permeability transition (MPT). We analyzed the effects of Zn²⁺ on MPT by monitoring mitochondrial swelling with the ultraviolet–visible light absorption spectrum, characterizing the fluidity of the membrane with fluorescence anisotropy, detecting the transmembrane potential (Δψ) with fluorescence intensity, and observing mitochondrial ultrastructure with transmission electron microscopy. Data reveal that low concentrations of zinc ions can trigger MPT while high levels of zinc ions cannot, which implies that zinc ions’ toxicity cannot be the result of a single simple mechanism.     

Mitochondrial Superoxide Radicals Differentially Affect Muscle Activity and Neural Function

Cellular superoxide radicals (O₂⁻) are mostly generated during mitochondrial oxygen metabolism. O₂⁻ serves as the raw material for many reactive oxygen species (ROS) members like H₂O₂ and OH^.− radicals following its catalysis by superoxide dismutase (SOD) enzymes and also by autocatalysis (autodismutation) reactions. Mitochondrial ROS generation could have serious implications on degenerative diseases. In model systems overproduction of mitochondrial O₂⁻ resulting from the loss of SOD2 function leads to movement disorders and drastic reduction in life span in vertebrates and invertebrates alike. With the help of a mitochondrial SOD2 loss-of-function mutant, Sod2ⁿ²⁸³, we measured the sensitivity of muscles and neurons to ROS attack. Neural outputs from flight motor neurons and sensory neurons were unchanged in Sod2ⁿ²⁸³ and the entire neural circuitry between the giant fiber (GF) and the dorsal longitudinal muscles (DLM) showed no overt defect due to elevated ROS. Such insensitivity of neurons to mitochondrial superoxides was further established through neuronal expression of SOD2, which failed to improve survival or locomotive ability of Sod2ⁿ²⁸³. On the other hand, ultrastructural analysis of Sod2ⁿ²⁸³ muscles revealed fewer mitochondria and reduced muscle ATP production. By targeting the SOD2 expression to the muscle we demonstrate that the early mortality phenotype of Sod2ⁿ²⁸³ can be ameliorated along with signs of improved mobility. In summary, muscles appear to be more sensitive to superoxide attack relative to the neurons and such overt phenotypes observed in SOD2-deficient animals can be directly attributed to the muscle.BETWEEN Drosophila, mouse, and human, the enzymatic antioxidant defense system shares similar organization both structurally (Landis and Tower 2005) and functionally. Besides having a good degree of homology (Duttaroy et al. 1994; Landis and Tower 2005), other significant similarities include the presence of a single copy of Sod1 and Sod2 genes in each with no degree of functional complementation between these enzymes (Copin et al. 2000). While vertebrates have developed additional antioxidant defense enzymes such as glutathione peroxidase (Gpx) and extracellular superoxide dismutase (EcSOD or Sod3), neither Gpx nor an active SOD3 has been demonstrated in Drosophila, although a Sod3-like sequence has been identified (Landis and Tower 2005). Complete loss of SOD2 function is fatally injurious for both mice and Drosophila (Li et al. 1995; Lebovitz et al. 1996; Kirby et al. 2002; Duttaroy et al. 2003). The severe phenotypic effects of SOD2 loss of function have been attributed to elevated DNA damage and protein carbonylation (Golden and Melov 2001). SOD2 loss of function has also been attributed to “free radical attack” or “oxidative insult” on mitochondria where obvious mitochondrial damage was apparent from the inactivation of mitochondrial Fe-S cluster enzymes aconitase and succinate dehydrogenase (Melov et al. 1999; Kirby et al. 2002; Paul et al. 2007). Furthermore, impairment of cellular signaling, specifically those induced by reactive oxygen species (ROS) (Klotz 2005), might also play a very significant role in the early mortality effects of SOD2-deficient flies as indicated recently (Wicks et al. 2009).Sod2 null mice with damaged mitochondria display a number of pathologies including cardiomyopathy (Li et al. 1995), neurodegeneration, and seizures (Melov et al. 1998). Drosophila mutants of mitochondrial dysfunction are also claimed to be associated with neurodegeneration (Kretzschmar et al. 1997; Min and Benzer 1997, 1999; Rogina et al. 1997; Palladino et al. 2002, 2003; Celotto et al. 2006). In addition to the neurons, muscles are important targets for oxidative modification (Choksi and Papaconstantinou 2008; Choksi et al. 2008). Aerobic muscles with high mitochondrial content and high myoglobin levels, for example, show a significant increase in oxidative modification of all electron transport chain proteins compared to muscles with fewer mitochondria and less myoglobin (anaerobic muscle) (Choksi and Papaconstantinou 2008; Choksi et al. 2008). Mice lacking the Cu-ZnSOD enzyme suffer from a rapid loss of skeletal muscle mass, resembling an accelerated sarcopenia (Jackson 2006; Muller et al. 2006). We therefore set out to measure the impact of heightened superoxide concentration on neurons and muscles of Sod2ⁿ²⁸³ flies that are devoid of SOD2, the principal scavenger of superoxide radicals in mitochondria (Duttaroy et al. 2003; Belton et al. 2006).     

Synergistic Triggering of Superoxide Flashes by Mitochondrial Ca2+ Uniport and Basal Reactive Oxygen Species Elevation

Mitochondrial superoxide flashes reflect a quantal, bursting mode of reactive oxygen species (ROS) production that arises from stochastic, transient opening of the mitochondrial permeability transition pore (mPTP) in many types of cells and in living animals. However, the regulatory mechanisms and the exact nature of the flash-coupled mPTP remain poorly understood. Here we demonstrate a profound synergistic effect between mitochondrial Ca²⁺ uniport and elevated basal ROS production in triggering superoxide flashes in intact cells. Hyperosmotic stress potently augmented the flash activity while simultaneously elevating mitochondrial Ca²⁺ and ROS. Blocking mitochondrial Ca²⁺ transport by knockdown of MICU1 or MCU, newly identified components of the mitochondrial Ca²⁺ uniporter, or scavenging mitochondrial basal ROS markedly diminished the flash response. More importantly, whereas elevating Ca²⁺ or ROS production alone was inefficacious in triggering the flashes, concurrent physiological Ca²⁺ and ROS elevation served as the most powerful flash activator, increasing the flash incidence by an order of magnitude. Functionally, superoxide flashes in response to hyperosmotic stress participated in the activation of JNK and p38. Thus, physiological levels of mitochondrial Ca²⁺ and ROS synergistically regulate stochastic mPTP opening and quantal ROS production in intact cells, marking the flash as a coincidence detector of mitochondrial Ca²⁺ and ROS signals.     

Inactivation of Mitochondrial Permeability Transition Pore by Octylguanidine and Octylamine

Mitochondrial permeability transition occurs through a Ca²⁺-dependent opening of atransmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase(ANT). In this work, we induced permeability transition by adding 0.5 M carboxyatractyloside.The process was evaluated analyzing Ca²⁺ efflux, a drop in transmembrane electric gradient,and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at theconcentration of 100 M, inhibited, almost completely, nonspecific membrane permeability.Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to doso. The inhibition was reversed after the addition of 40 mM Li⁺, Na⁺ K⁺,Rb⁺, or Cs⁺; K⁺ wasthe most effective. We propose that the positive charge of the amines interact with negativecharges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetratesinto the hydrophobic milieu of the inner membrane, fixing the reagent.     

Inhibitory Effects of Adenine Nucleotides on Brain Mitochondrial Permeability Transition

The adenine nucleotides ADP and ATP are probably the most important endogenous inhibitors of the mitochondrial permeability transition (MPT). We studied the inhibitory effects of adenine nucleotides on brain MPT by measuring mitochondrial swelling and Ca²⁺ and cytochrome c release. We observed that in the presence of either ADP or ATP, at 250 μM, brain mitochondria accumulated more than 1 μmol Ca²⁺ × mg protein⁻¹. ADP or ATP also prevented Ca²⁺-induced mitochondrial swelling and cytochrome c release. Interestingly, ATP lost most of its inhibitory effects on MPT when the experiments were carried out in the presence of ATP-regenerating systems. These results indicate that MPT inhibition observed in the presence of added ATP could be mainly due to hydrolysis of ATP to ADP. From mitochondrial swelling measurements, half-maximal inhibitory values (K _i) of 4.5 and 98 μM were obtained for ADP and ATP, respectively. In addition, a delayed mitochondrial swelling sensitive to higher ADP concentrations was observed. Mitochondrial anoxia/reoxygenation did not interfere with the inhibitory effect of ADP on Ca²⁺-induced MPT, but oxidative phosphorylation markedly decreased this effect. We conclude that ADP is a potent inhibitor of brain MPT whereas ATP is a weaker inhibitor of this phenomenon. Our results suggest that ADP can have an important protective role against MPT-mediated tissue damage under conditions of brain ischemia and hypoglycemia.     

Hypertriglyceridemia Increases Mitochondrial Resting Respiration and Susceptibility to Permeability Transition

High plasma level of triglycerides (TGs) is a common feature in atherosclerosis, obesity, diabetes, alcoholism, stress, and infection. Since mitochondria have been implicated in cell death under a variety of metabolic disorders, we examined liver mitochondrial functions in hypertriglyceridemic transgenic mice. Hypertriglyceridemia increased resting respiration and predisposed to mitochondrial permeability transition (MPT). Ciprofibrate therapy reduced plasma TG levels, normalized respiration, and prevented MPT. The higher resting respiration in transgenic mitochondria remained in the presence of the adenine nucleotide carrier inhibitor, carboxyatractyloside, bovine serum albumin, and the uncoupling proteins (UCPs) inhibitor, GDP. UCP2 content was similar in both control and transgenic mitochondria. We propose that faster resting respiration represents a regulated adaptation to oxidize excess free fatty acid in the transgenic mice.     

Exploiting the Role of Resveratrol in Rat Mitochondrial Permeability Transition

Resveratrol (RSV), a natural polyphenolic antioxidant, has been considered an anticarcinogenic agent as it triggers tumor cell apoptosis through activation of the mitochondrial pathway. In our study, the effects of RSV on mitochondria, especially on the mitochondrial permeability transition (MPT) process, were investigated by multiple methods. We found that RSV induced a collapse of membrane potential and matrix swelling related to MPT. We further demonstrated that Ca²⁺ was necessary for this RSV-induced MPT opening. In addition, RSV induced the inner membrane permeabilization to H⁺ and K⁺, the depression of respiration and changes in membrane fluidity. The results suggested that RSV-induced MPT was accompanied by mitochondrial dysfunction. But the prohibition on lipid peroxidation and different effects of low- and high-dose RSV on membrane fluidity and respiration showed that the interaction of RSV and the mitochondria could not be the result of a single simple mechanism.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Increased Neuronal Differentiation of Neural Progenitor Cells Derived from Phosphovimentin-Deficient Mice

Vimentin is an intermediate filament (also known as nanofilament) protein expressed in several cell types of the central nervous system, including astrocytes and neural stem/progenitor cells. Mutation of the vimentin serine sites that are phosphorylated during mitosis (VIM ^SA/SA ) leads to cytokinetic failures in fibroblasts and lens epithelial cells, resulting in chromosomal instability and increased expression of cell senescence markers. In this study, we investigated morphology, proliferative capacity, and motility of VIM ^SA/SA astrocytes, and their effect on the differentiation of neural stem/progenitor cells. VIM ^SA/SA astrocytes expressed less vimentin and more GFAP but showed a well-developed intermediate filament network, exhibited normal cell morphology, proliferation, and motility in an in vitro wound closing assay. Interestingly, we found a two- to fourfold increased neuronal differentiation of VIM ^SA/SA neurosphere cells, both in a standard 2D and in Bioactive3D cell culture systems, and determined that this effect was neurosphere cell autonomous and not dependent on cocultured astrocytes. Using BrdU in vivo labeling to assess neural stem/progenitor cell proliferation and differentiation in the hippocampus of adult mice, one of the two major adult neurogenic regions, we found a modest increase (by 8%) in the fraction of newly born and surviving neurons. Thus, mutation of the serine sites phosphorylated in vimentin during mitosis alters intermediate filament protein expression but has no effect on astrocyte morphology or proliferation, and leads to increased neuronal differentiation of neural progenitor cells.     

Integrin-Associated Protein Promotes Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural stem/progenitor cells (NSPCs) proliferate and differentiate depending on their intrinsic properties and local environment. During the development of the mammalian nervous system, NSPCs generate neurons and glia sequentially. However, little is known about the mechanism that determines the timing of switch from neurogenesis to gliogenesis. In this study, we established a culture system in which the neurogenic potential of NSPCs is decreased in a time-dependent manner, so that short-term-cultured NSPCs differentiate into more neurons compared with long-term-cultured NSPCs. We found that short-term-cultured NSPCs express high levels of integrin-associated protein form 2 (IAP2; so-called CD47) mRNA using differential display analysis. Moreover, IAP2 overexpression in NSPCs induced neuronal differentiation of NSPCs. These findings reveal a novel mechanism by which IAP2 induces neuronal differentiation of NSPCs.     

红莲型细胞质雄性不育性与线粒体渗透性转换

以水稻红莲型细胞质雄性不育(HL-CMS)的不育系粤泰A(YTA)、保持系粤泰B(YTB)以及杂种F1代红莲2号(HL2)的黄化苗为材料,研究了在不同pH及离子强度下线粒体渗透性转换(MPT)的发生及其差异。结果表明,YTA、YTB和HL2间MPT的发生均存在差别,不育系YTA线粒体渗透性转换孔(PTP)的开启与关闭对pH及离子强度的变化较保持系YTB和HL2敏感。HL2与YTA虽然具有相同的细胞质来源,但两者之间PTP及MPT的特性明显不同,前者MPT的变化与具有正常生理功能的YTB线粒体的PTP和MPT的发生特点与特性相似。这些说明红莲型水稻细胞质雄性不育的发生可能与其MPT的发生有关。