线粒体形态学改变与细胞凋亡

近年来,对于线粒体形态学以及其在凋亡过程中的改变和作用的研究打破了传统的观点。正常情况下,线粒体在细胞内相互连接成管网状结构,并发生着频繁的融合与分裂。融合和分裂由一系列蛋白质介导,二者之间的动态平衡维持着线粒体的形态和功能。在细胞凋亡的早期,线粒体融合和分裂失平衡,导致线粒体管网状结构碎裂和嵴的重构,这些改变对线粒体随后的变化以及凋亡的发生具有重要的意义。融合和分裂的蛋白质不仅调控线粒体形态和细胞凋亡过程,也和某些凋亡相关疾病有关。此外,促凋亡的Bcl-2蛋白可能通过改变线粒体的构形来调控凋亡过程。     

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.     

Tying trafficking to fusion and fission at the mighty mitochondria

The mitochondrion is a unique organelle that serves as the main site of ATP generation needed for energy in the cell. However, mitochondria also play essential roles in cell death through apoptosis and necrosis, as well as a variety of crucial functions related to stress regulation, autophagy, lipid synthesis and calcium storage. There is a growing appreciation that mitochondrial function is regulated by the dynamics of its membrane fusion and fission; longer, fused mitochondria are optimal for ATP generation, whereas fission of mitochondria facilitates mitophagy and cell division. Despite the significance of mitochondrial homeostasis for such crucial cellular events, the intricate regulation of mitochondrial fusion and fission is only partially understood. Until very recently, only a single mitochondrial fission protein had been identified. Moreover, only now have researchers turned to address the upstream machinery that regulates mitochondrial fusion and fission proteins. Herein, we review the known GTPases involved in mitochondrial fusion and fission, but also highlight recent studies that address the mechanisms by which these GTPases are regulated. In particular, we draw attention to a substantial new body of literature linking endocytic regulatory proteins, such as the retromer VPS35 cargo selection complex subunit, to mitochondrial homeostasis. These recent studies suggest that relationships and cross‐regulation between endocytic and mitochondrial pathways may be more widespread than previously assumed.      

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

Bax is essential for Drp1‐mediated mitochondrial fission but not for mitochondrial outer membrane permeabilization caused by photodynamic therapy

Bcl‐2 family proteins are critical for the regulation of apoptosis, with the pro‐apoptotic members Bax essential for the release of cytochrome c from mitochondria in many instances. However, we found that Bax was activated after mitochondrial depolarization and the completion of cytochrome c release induced by photodynamic therapy (PDT) with the photosensitizer Photofrin in human lung adenocarcinoma cells (ASTC‐a‐1). Besides, knockdown of Bax expression by gene silencing had no effect on mitochondrial depolarization and cytochrome c release, indicating that Bax makes no contribution to mitochondrial outer membrane permeabilization (MOMP) following PDT. Further study revealed that Bax knockdown only slowed down the speed of cell death induced by PDT, indicating that Bax is not essential for PDT‐induced apoptosis. The fact that Bax knockdown totally inhibited the mitochondrial accumulation of dynamin‐related protein (Drp1) and Drp1 knockdown attenuated cell apoptosis suggest that Bax can promote PDT‐induced apoptosis through promoting Drp1 activation. Besides, Drp1 knockdown also failed to inhibit PDT‐induced cell death finally, indicating that Bax‐mediated Drp1's mitochondrial translocation is not essential for PDT‐induced cell apoptosis. On the other hand, we found that protein kinase Cδ (PKCδ), Bim L and glycogen synthase kinase 3β (GSK3β) were activated upon PDT treatment and might contribute to the activation of Bax under the condition. Taken together, Bax activation is not essential for MOMP but essential for Drp1‐mediated mitochondrial fission during the apoptosis caused by Photofrin‐PDT. J. Cell. Physiol. 226: 530–541, 2011. © 2010 Wiley‐Liss, Inc.     

Molecular mechanisms and physiologic functions of mitochondrial dynamics

Mitochondria are highly dynamic organelles that continuously change their shape through frequent fusion, fission and movement throughout the cell, and these dynamics are crucial for the life and death of the cells as they have been linked to apoptosis, maintenance of cellular homeostasis, and ultimately to neurologic disorders and metabolic diseases. Over the past decade, a growing number of novel proteins that regulate mitochondrial dynamics have been discovered. Large GTPase family proteins and their regulators control these aspects of mitochondrial dynamics. In this review, we briefly summarize the current knowledge about molecular machineries regulating mitochondrial fusion/fission and the role of mitochondrial dynamics in cell pathophysiology.     

Overexpression of Bcl‐2 prevents neomycin‐induced hair cell death and caspase‐9 activation in the adult mouse utricle in vitro

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside‐induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside‐induced hair cell death can be prevented by broad‐spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside‐induced hair cell death requires activation of caspase‐9. Caspase‐9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside‐induced hair cell death is mediated by the mitochondrial (or “intrinsic”) cell death pathway. The Bcl‐2 family of pro‐apoptotic and anti‐apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl‐2 is an anti‐apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl‐2 to examine the role of Bcl‐2 in neomycin‐induced hair cell death. Overexpression of Bcl‐2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl‐2 overexpression prevented neomycin‐induced activation of caspase‐9 in hair cells. These results suggest that the expression level of Bcl‐2 has important effects on the pathway(s) important for the regulation of aminoglycoside‐induced hair cell death. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 89–100, 2004     

Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

Mitochondrial outer-membrane permeabilization and remodelling in apoptosis

Many human pathologies are associated with defects in mitochondria such as diabetes, neurodegenerative diseases or cancer. This tiny organelle is involved in a plethora of processes in mammalian cells, including energy production, lipid metabolism and cell death. In the so-called intrinsic apoptotic pathway, the outer mitochondrial membrane (MOM) is premeabilized by the pro-apoptotic Bcl-2 members Bax and Bak, allowing the release of apoptogenic factors such as cytochrome c from the inter-membrane space into the cytosol. At the same time, mitochondria fragment in response to Drp-1 activation suggesting that mitochondrial fission could play a role in mitochondrial outer-membrane permeabilization (MOMP). In this review, we will discuss the link that could exist between mitochondrial fission and fusion machinery, Bcl-2 family members and MOMP.     

MicroRNA‐181c targets Bcl‐2 and regulates mitochondrial morphology in myocardial cells

Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl‐2 family which includes pro‐ and anti‐apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA‐181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR‐181c could target the 3′ untranslated region of Bcl‐2, one of the anti‐apoptotic members of the Bcl‐2 family. Thus, we have suggested that miR‐181c was involved in regulation of Bcl‐2. In this study, we investigated this hypothesis using the Dual‐Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR‐181c. We found that the level of miR‐181c was inversely correlated with the Bcl‐2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR‐181c resulted in significant changes in the levels of caspases, Bcl‐2 and cytochrome C in these cells. The increased level of Bcl‐2 caused by the decrease in miR‐181c protected mitochondrial morphology from the tumour necrosis factor alpha‐induced apoptosis.     

Bh3 induced conformational changes in Bcl‐Xl revealed by crystal structure and comparative analysis

Apoptosis or programmed cell death is a regulatory process in cells in response to stimuli perturbing physiological conditions. The Bcl‐2 family of proteins plays an important role in regulating homeostasis during apoptosis. In the process, the molecular interactions among the three members of this family, the pro‐apoptotic, anti‐apoptotic and BH3‐only proteins at the mitochondrial outer membrane define the fate of a cell. Here, we report the crystal structures of the human anti‐apoptotic protein Bcl‐X_L in complex with BH3‐only BID^BH3 and BIM^BH3 peptides determined at 2.0 Å and 1.5 Å resolution, respectively. The BH3 peptides bind to the canonical hydrophobic pocket in Bcl‐X_L and adopt an alpha helical conformation in the bound form. Despite a similar structural fold, a comparison with other BH3 complexes revealed structural differences due to their sequence variations. In the Bcl‐X_L‐BID^BH3 complex we observed a large pocket, in comparison with other BH3 complexes, lined by residues from helices α1, α2, α3, and α5 located adjacent to the canonical hydrophobic pocket. These results suggest that there are differences in the mode of interactions by the BH3 peptides that may translate into functional differences in apoptotic regulation. Proteins 2015; 83:1262–1272. © 2015 Wiley Periodicals, Inc.     

Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics

Bcl-2 family proteins play central roles in apoptosis by regulating the release of mitochondrial intermembrane space proteins such as cytochrome c. Death-promoting Bcl-2 family members, such as Bax, can promote cytochrome c release and fragmentation of the mitochondrial network, whereas apoptosis-inhibitory members, such as Bcl-2 and Bcl-xL, can antagonize these events. It remains unclear whether CED-9, the worm Bcl-2 relative, can regulate mitochondrial fission/fusion dynamics or the release of proteins from the mitochondrial intermembrane space. Here, we show that CED-9 interacts with Mitofusin-2/fuzzy onions and can promote mitochondrial clustering and dramatic reorganization of mitochondrial networks. Consistent with its ability to neutralize CED-9 function, EGL-1 antagonized CED-9-dependent remodeling of the mitochondrial network. However, CED-9 failed to inhibit mitochondrial cytochrome c release or apoptosis induced by diverse triggers in mammalian cells. These data suggest that the ability to regulate mitochondrial fission/fusion dynamics is an evolutionarily conserved property of the Bcl-2 family.     

Proteins That Fuse and Fragment Mitochondria in Apoptosis: Con-Fissing a Deadly Con-Fusion?

During apoptosis, mitochondria undergo multiple changes that culminate in the release of cytochrome c and other proapoptotic cofactors. Recently, a role for previously overlooked morphological changes, fission of the mitochondrial reticulum and remodeling of mitochondrial cristae, has been suggested in mammalian cells and in developmental apoptosis of C. elegans. Mitochondrial morphology is determined by fusion and fission processes, controlled by a growing set of “mitochondria-shaping” proteins, whose levels and function appear to regulate the mitochondrial pathways of cell death. Expression of pro-fusion proteins, as well as of inhibition of pro-fission molecules reduces apoptosis, suggesting a linear relationship between fragmentation and death. Mechanisms by which mitochondrial fragmentation promotes apoptosis and interactions between fragmentation and remodeling of the inner membrane are largely unclear. A tempting, unifying hypothesis suggests that fission is coupled to cristae remodeling to maximize cytochrome c release.     

前凋亡因子与脑血管疾病的关系研究

Bim（bcl—2 interacting mediator of cell death）,又称前凋亡因子（pro-apoptotic Bcl-2 proteins）,是Bcl-2家族中BH3-only亚家族成员,具有促凋亡活性,是重要的凋亡诱导基因,在细胞凋亡过程中有重要作用,而最近研究表明凋亡是脑缺血后神经元死亡的重要形式,故Bim与脑血管疾病关系密切。本文就Bim的分子结构、生物学功能及其调节机制作一简单阐述,并重点讨论了其与脑血管疾病关系方面的研究进展,以望为脑血管疾病的防治提供一新的途径。     

Genome‐wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31^comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.     

The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis

Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.     

Platycodin D induces anoikis and caspase‐mediated apoptosis via p38 MAPK in AGS human gastric cancer cells

Mitogen‐activated protein kinases (MAPKs) cascades play important roles in cell proliferation, death, and differentiation in response to external stimuli. However, the precise role of MAPKs in platycodin D (PD)‐induced cytotoxicity remains unclear. In this study, we investigated the anticancer effect of PD and its underlying mechanism on AGS human gastric cancer cells. PD significantly inhibited cell proliferation and induced anoikis, which is a form of apoptosis in which cells detach from the substrate. It showed phosphatidylserine externalization, DNA fragmentation, increase of sub‐G1 phase, and activation of caspases in a dose‐ and time‐dependent manner. This apoptosis has been associated with the extrinsic pathway via Fas‐L and the intrinsic pathway via mitochondrial Bcl‐2 family members. Moreover, PD led to the phosphorylation of stresses‐activated protein kinases such as JNK and p38, followed by the activation of AP‐1. However, pretreatment with SB203580 (a p38 specific inhibitor) suppressed PD‐induced p38 and AP‐1 activation, and subsequently attenuated the PD‐induced apoptosis in AGS cells. These results suggest that p38 activation is responsible for PD‐induced apoptosis in AGS cells and PD might be useful for the development as the anticancer agent of gastric cancer. J. Cell. Biochem. 114: 456–470, 2013. © 2012 Wiley Periodicals, Inc.     

Mitochondrial death pathways in yeast and mammalian cells

In mammals, mitochondria are important mediators of programmed cell death, and this process is often regulated by Bcl-2 family proteins. However, a role for mitochondria-mediated cell death in non-mammalian species is more controversial. New evidence from a variety of sources suggests that mammalian mitochondrial fission/division proteins also have the capacity to promote programmed cell death, which may involve interactions with Bcl-2 family proteins. Homologues of these fission factors and several additional mammalian cell death regulators are conserved in flies, worms and yeast, and have been suggested to regulate programmed cell death in these species as well. However, the molecular mechanisms by which these phylogenetically conserved proteins contribute to cell death are not known for any species. Some have taken the conserved pro-death activity of mitochondrial fission factors to mean that mitochondrial fission per se, or failed attempts to undergo fission, are directly involved in cell death. Other evidence suggests that the fission function and the cell death function of these factors are separable. Here we consider the evidence for these arguments and their implications regarding the origins of programmed cell death.