Inside an enigma: do mitochondria contribute to cell death in Drosophila?

Mitochondria have been shown to play an important role in cell death in mammalian cells. However, the importance of mitochondria in Drosophila apoptosis is still under investigation. Many proteins involved in the regulation of apoptosis in mammals act at mitochondria or are released from mitochondria, resulting in caspase activation. In addition, these organelles undergo significant ultrastructural changes during apoptosis. This review highlights similarities and differences in the roles of mitochondria and mitochondrial factors in apoptosis between Drosophila and mammals. In Drosophila, many key regulators of apoptosis also appear to localize to this organelle, which also undergoes ultrastructural changes during apoptosis. Although many of the proteins important for the control of apoptosis in mammalian cells are conserved in Drosophila, the role that mitochondria play in apoptosis in this model system remains an area of controversy and active research.     

Apoptotic-like regulation of programmed cell death in plants

In plants, apoptotic-like programmed cell death (PCD) can be distinguished from other forms of plant cell death by protoplast condensation that results in a morphologically distinct cell corpse. In addition, there is a central regulatory role for the mitochondria and the degradation of the cell and its contents by PCD associated proteases. These distinguishing features are shared with animal apoptosis as it is probable that plant and animal cell death programmes arose in a shared unicellular ancestor. However, animal and plant cell death pathways are not completely conserved. The cell death programmes may have been further modified after the divergence of plant and animal lineages leading to converged, or indeed unique, features of their respective cell death programmes. In this review we will examine the features of apoptotic-like PCD in plants and examine the probable conserved components such as mitochondrial regulation through the release of apoptogenic proteins from the mitochondrial intermembrane space, the possible conserved or converged features such as “caspase-like” molecules which drive cellular destruction and the emerging unique features of plant PCD such as chloroplast involvement in cell death regulation.     

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.     

Mitochondria regulation in ferroptosis

Ferroptosis is recognized as a new form of regulated cell death which is initiated by severe lipid peroxidation relying on reactive oxygen species (ROS) generation and iron overload. This iron-dependent cell death manifests evident morphological, biochemical and genetic differences from other forms of regulated cell death, such as apoptosis, autophagy, necrosis and pyroptosis. Ferroptosis was primarily characterized by condensed mitochondrial membrane densities and smaller volume than normal mitochondria, as well as the diminished or vanished of mitochondria crista and outer membrane ruptured. Mitochondria take the center role in iron metabolism, as well as substance and energy metabolism as it’s the major organelle in iron utilization, catabolic and anabolic pathways. Interference of key regulators of mitochondrial lipid metabolism (e.g., ASCF2 and CS), iron homeostasis (e.g., ferritin, mitoferrin1/2 and NEET proteins), glutamine metabolism and other signaling pathways make a difference to ferroptotic sensitivity. Targeted induction of ferroptosis was also considered as a potential therapeutic strategy to some oxidative stress diseases, including neurodegenerative disorders, ischemia-reperfusion injury, traumatic spinal cord injury. However, the pertinence between mitochondria and ferroptosis is still in dispute. Here we systematic elucidate the morphological characteristics and metabolic regulation of mitochondria in the regulation of ferroptosis.     

Mitochondrial membrane hyperpolarization hijacks activated T lymphocytes toward the apoptotic-prone phenotype: homeostatic mechanisms of HIV protease inhibitors

A decrease of mitochondrial membrane potential has been hypothesized to be a marker of apoptotic cells, including activated T lymphocytes. It was recently demonstrated that HIV protease inhibitors, independently from any viral infection, can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis. To analyze the mechanisms underlying these effects, a specific study was undertaken in both resting and activated human PBL exposed to either receptor (e.g., anti-Fas)- or nonreceptor (e.g., radiation)-mediated apoptotic stimuli. T cell activation was found to be accompanied by a significant increase in mitochondrial membrane potential, or hyperpolarization, which was undetectable in resting cells. We also detected apoptotic hindering by HIV protease inhibitors only in activated T lymphocytes. This was apparently due to the ability of these drugs to block activation-associated mitochondria hyperpolarization, which, in turn, was paralleled by an impairment of cell cycle progression. Remarkably, protease inhibitors also prevented zidovudine-mediated mitochondrial toxicity. Finally, HIV-infected cells from naive patients behaved identically to activated T cells, displaying hyperpolarized mitochondria, while lymphocytes from patients under highly active antiretroviral therapy (which included HIV protease inhibitors) seemed to react as resting cells. Altogether these results clearly indicate that the hyperpolarization state of mitochondria may represent a prerequisite for the sensitization of lymphocytes to the so-called activation-induced cell death. They also suggest that HIV protease inhibitors, by interfering with induction of the mitochondrial hyperpolarization state, can result in cell survival even independent of any viral infection.     

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.     

Apoptosis in <Emphasis Type="Italic">Drosophila</Emphasis>: which role for mitochondria?

It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.     

Changes in astrocyte mitochondrial function with stress: effects of Bcl-2 family proteins

Mitochondria are central to both apoptotic and necrotic cell death, as well as to normal physiological function. Astrocytes are crucial for neuronal metabolic, antioxidant, and trophic support, as well as normal synaptic function. In the setting of stress, such as during cerebral ischemia, astrocyte dysfunction may compromise the ability of neurons to survive. Despite their central importance, the response of astrocyte mitochondria to stress has not been extensively studied. Limited data already suggest clear differences in the response of neuronal and astrocytic mitochondria to oxygen-glucose deprivation (GD). Prominent mitochondrial alterations during stress that can contribute to cell death include changes in production of reactive oxygen species (ROS) and release of death regulatory and signaling molecules from the intermembrane space. In response to stress mitochondrial respiratory function and membrane potential also change, and these changes appear to depend on cell type. Bcl-2 family proteins are the best studied regulators of cell death, especially apoptosis, and mitochondria are a major site of action for these proteins. Although much data supports the role of Bcl-2 family proteins in the regulation of some of these mitochondrial alterations, this remains an area of active investigation. This mini-review summarizes current knowledge regarding mitochondrial control of cell survival and death in astrocytes and the effects of anti-apoptotic Bcl-2 proteins on astrocyte mitochondrial function.     

Viral proteins and the mitochondrial apoptotic checkpoint

Regulated cell death by apoptosis constitutes a primary host defense for counteracting invading viral pathogens. In recent years, advances in the field of apoptosis research have revealed that mitochondria and mitochondria-derived factors play a central role in regulating cellular commitment to apoptosis. Here we explore the role of viral proteins in modulating cell death pathways that are relayed via this mitochondrial checkpoint.     

线粒体膜间隙蛋白在细胞凋亡中的作用

线粒体除了作为细胞内的“能量工厂”外,在控制细胞凋亡中起主导作用。细胞凋亡时,线粒体膜通透性增加,释放可溶性线粒体膜间隙蛋白质,进一步破坏细胞结构。在这些致死性蛋白质中,有些(cytc、Smac／DIABLO、Omi／HtrA2等)能够激活caspases,另一些(endo G、AIF、Omi/HtrA2等)则以非caspase依赖的方式发挥作用。多种线粒体因子参与细胞凋亡,强化了细胞器在凋亡控制中的核心作用。     

Viral proteins targeting mitochondria: controlling cell death

Mitochondrial membrane permeabilization (MMP) is a critical step regulating apoptosis. Viruses have evolved multiple strategies to modulate apoptosis for their own benefit. Thus, many viruses code for proteins that act on mitochondria and control apoptosis of infected cells. Viral proapoptotic proteins translocate to mitochondrial membranes and induce MMP, which is often accompanied by mitochondrial swelling and fragmentation. From a structural point of view, all the viral proapoptotic proteins discovered so far contain amphipathic alpha-helices that are necessary for the proapoptotic effects and seem to have pore-forming properties, as it has been shown for Vpr from human immunodeficiency virus-1 (HIV-1) and HBx from hepatitis B virus (HBV). In contrast, antiapoptotic viral proteins (e.g., M11L from myxoma virus, F1L from vaccinia virus and BHRF1 from Epstein-Barr virus) contain mitochondrial targeting sequences (MTS) in their C-terminus that are homologous to tail-anchoring domains. These domains are similar to those present in many proteins of the Bcl-2 family and are responsible for inserting the protein in the outer mitochondrial membrane leaving the N-terminus of the protein facing the cytosol. The antiapoptotic proteins K7 and K15 from avian encephalomyelitis virus (AEV) and viral mitochondria inhibitor of apoptosis (vMIA) from cytomegalovirus are capable of binding host-specific apoptosis-modulatory proteins such as Bax, Bcl-2, activated caspase 3, CAML, CIDE-B and HAX. In conclusion, viruses modulate apoptosis at the mitochondrial level by multiple different strategies.     

Cell death regulation by the Bcl-2 protein family in the mitochondria

An increase in the permeability of the outer mitochondrial membrane is central to apoptotic cell death, since it leads to the release of several apoptogenic factors, such as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream death programs. During apoptosis, the mitochondria also release AIF and endonuclease G, both of which are translocated to the nucleus and are implicated in apoptotic nuclear changes that occur in a caspase-independent manner. Mitochondrial membrane permeability is directly controlled by the major apoptosis regulator, i.e., the Bcl-2 family of proteins, mainly through regulation of the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane, although the precise molecular mechanisms are still not completely understood. Here, I focus on the mechanisms by which Bcl-2 family members control the permeability of mitochondrial membrane during apoptosis.     

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.     

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.     

Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes

A central mechanism in apoptosis is the activation of proteases of the caspase (cysteine aspartases) family. Protease activation has also been implicated in necrosis, but its role in this cell death process and the identity of the proteases involved and their substrates, are unknown. Using human autoantibodies to well characterized cellular proteins as detecting probes in immunoblotting, we observed that a defined and somewhat similar set of nuclear proteins, including poly (ADP-ribose) polymerase (PARP) and DNA topoisomerase I (Topo I), were selectively cleaved during both apoptosis and necrosis of cultured cells induced by various stimuli. The resulting cleavage products were distinctively different in the two cell death pathways. In contrast to apoptosis, the cleavages of PARP and Topo I during necrosis were not blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). These findings suggest that different proteases act in apoptosis and necrosis, and that although both cell death processes result in selective cleavage of almost identical cellular proteins, they can be distinguished immunochemically on the basis of their cleavage products.     

Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions (Berridge et al., 2000) [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes such as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death (Pinton et al., 2008) [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associates changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis (Kruman et al., 1998; Tombal et al., 1999; Lynch et al., 2000; Orrenius et al., 2003) , ,  and . Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b; Sancak et al., 2013)[8–10]. The next questions are how they are regulated for exquisite tight control of ER–mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.