Apoptosis and Beyond: The Many Ways Cells Die

By James A. Radosevich

These volumes teach readers to think beyond apoptosis and describes all of the known processes that cells can undergo which result in cell death

This two-volume source on how cells dies is the first, comprehensive collection to cover all of the known processes that cells undergo when they die. It is also the only one of its kind to compare these processes. It seeks to enlighten those in the field about these many processes and to stimulate their thinking at looking at these pathways when their research system does not show signs of activation of the classic apoptotic pathway. In addition, it links activities like the molecular biology of one process (eg. Necrosis) to another process (eg. apoptosis) and contrasts those that are close to each.

Volume 1 of Apoptosis and Beyond: The Many Ways Cells Die begins with a general view of the cytoplasmic and nuclear features of apoptosis. It then goes on to offer chapters on targeting the cell death mechanism; microbial programmed cell death; autophagy; cell injury, adaptation, and necrosis; necroptosis; ferroptosis; anoikis; pyronecrosis; and more. Volume 2 covers such subjects as phenoptosis; pyroptosis; hematopoiesis and eryptosis; cyclophilin d-dependent necrosis; and the role of phospholipase in cell death.

• Covers all known processes that dying cells undergo
• Provides extensive coverage of a topic not fully covered before
• Offers chapters written by top researchers in the field
• Provides activities that link and contrast processes to each other

Apoptosis and Beyond: The Many Ways Cells Die will appeal to students and researchers/clinicians in cell biology, molecular biology, oncology, and tumor biology.

• Language: English
• Publisher: Wiley
• Release date: Sep 25, 2018
• ISBN: 9781119432432

Book preview

1

General View of the Cytoplasmic and Nuclear Features of Apoptosis

Humberto De Vitto,¹ Juan P. Valencia,² and James A. Radosevich³

¹Center of Health and Science, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

²University of Rio de Janeiro, Rio de Janeiro, Brazil

³Department of Oral Medicine and Diagnostic Sciences, University of Illinois at Chicago, Chicago, IL, USA

Abbreviations

1.1 Introduction

The normal development of a cell and the life cycles of the multicellular organism rely on a finely tuned balance between cell survival and death. In a biological context, cells need to grow, divide, and die. In regard to the latter process, cells have developed a very precisely regulated means of programmed cell death (PCD), which contributes to the maintenance of normal cell turnover, leading to reduced impact on tissues, organs, and the organism itself. Some cells have evolved a PCD process called apoptosis. Apoptosis can be simply defined as a set of biochemical cytoplasmic and mitochondrial events that may lead to the execution phase of nuclear events.

A wide array of stress stimuli can trigger the apoptotic process, and the biochemical signal can then be amplified in the cytoplasm and mitochondria by both extrinsic and intrinsic pathways. The convergence of the apoptotic signal is considered the activation of a family of cysteine aspartyl-specific proteases (caspases), composed of 12 proteins strictly involved in the apoptotic cell death process. The dying cells activate the execution pathway that leads to the appearance of blebs and to the pinching off of many of them, forming apoptotic bodies, which may be rounded and retracted from their own tissue. Subsequently, the immune system cells are able to eliminate the apoptotic bodies through an engulfment cell process. The morphological and biochemical features during the apoptotic process are not fully understood.

At the nuclear level, it is well established that endonucleases and exonucleases may hydrolyze the DNA into small fragments (200 pb) [1]. The nuclear events depend on caspase activation. Caspase 3 is considered the most important protease of the executioner pathway, and is activated by different initiator caspases. For instance, caspase 8 is activated from the death receptor, caspase 9 is involved in the mitochondrial apoptotic process, and caspase 10 is involved in the Perforin/granzyme (PFN/Gzm) pathways. The cleaved caspase 3 cleaves the endonuclease caspase-activated DNase (CAD), degrading the DNA at nucleosomal linkers [2,3], which generates small DNA fragments (∼50–300 kb). The subsequent processing of the DNA by exonucleases and endonucleases leads to the formation of 200 bp fragments. Many organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), lysosomes, and mitochondria, can be recycled or eliminated, depending on the apoptotic stimuli. It is important to note that mitochondria play a pivotal role in apoptosis, since they can release cytochrome c (cyt c) and endonuclease D (endo D), leading to cell death [4,5].

One of the apoptotic pathways is the extrinsic or death-receptor pathway. It depends for its activation on a death domain and a death ligand, such as tumor necrosis factor alpha (TNFα) and tumor necrosis factor receptor 1 (TNFR1). The ligand represents the external death signal, leading to the intracellular signaling of the effector pathway. The main receptors recruit adaptor proteins like Fas-associated death-domain protein (FADD), TNF receptor-associated death domain (TRADD), and receptor-interacting protein (RIP) [6–8], which in turn recruit other molecules such as pro-caspase 8. The dimerization of the death effector domain (DED) leads to the formation of a death-inducing signal complex (DISC), triggering the subsequent process of autocatalysis of pro-caspase 8 to an activated protein (caspase 8) [9]. Caspase 8 activation is considered the main feature that starts the extrinsic pathway, leading to cell death. In many cases, depending on the apoptotic stimuli, the extrinsic pathway can crosstalk with the intrinsic pathway through proteolysis of the BH3-only protein, BH3-interacting domain death agonist (BID), which is what promotes the release of cyt c from the mitochondria into the cytoplasm. In the cytoplasm, cyt c may be assembled with the adaptor protein apoptotic protease activating factor-1 (Apaf-1) and ATP, generating in the cytosol the multimolecular holoenzyme complex called the apoptosome (Figure 1.1) [10].

Figure 1.1 Schematic representation of the cytoplasmic and nuclear events of apoptosis. The Perforin/Granzyme pathway, extrinsic pathway, and intrinsic pathway represent the three main pathways of apoptosis. Through a vast array of death signals, all three pathways can be triggered. (A) The Perforin/Granzyme pathway is a unique pathway that partially works in a caspase-independent fashion (granzyme A branch), leading directly to DNA cleavage and cell death. However, the activation of the granzyme B branch can trigger initiator caspase 10, which activates executioner caspase 3. (B) The extrinsic pathway, when activated, can cleave pro-caspase 8 to caspase 8 by FAAD, then activate executioner caspase 3. Caspase 8 plays an important role in the activation of a truncated BID (tBID) protein, leading to the release of mitochondria proteins like cyt c. (C) Upon receiving incoming signals, the intrinsic pathway induces MPTP opening, leading to the release from the mitochondria of proteins such as cyt c, endo G/AIF, and Htra2/Omi. On the cytosol, cyt c forms the apoptosome, which cleaves pro-caspase 3, triggering the execution pathway. (D) The execution pathway is characterized by cell shrinkage, chromatin condensation, and the formation of cytoplasmic blebs and apoptotic bodies. cyt c, cytocrome c; FAAD, Fas-associated death domain; MTPT, mitochondrial permeability transition pore.

The PFN/Gzm pathway is considered part of the extrinsic pathway. It is activated when cells are infected by viruses and/or bacteria. Mechanistically, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells produce granzymes. The granzymes, together with the PFN, may facilitate the pore formation and action of Gzms that lead to cell death. Two types of Gzm, type A (GmzA) and type B (GmzB), have been described. GmzA is considered the most important serine-protease present in CTL and NK granules. GmzB relies on the mechanism of oligomerization of PFN to enter into the cell, and depends for its action on the activation of caspases, principally caspase 3 [11,12].

It is important to note that different stimuli and players are enlisted in the various apoptotic processes. Moreover, it is well known that immune-system cells, mitochondria, and nuclear events are involved in apoptosis. The entire biochemical process is still elusive. Accordingly, the current chapter will focus on cytoplasmic and nuclear events. We describe and highlight an overview of cytoplasmic events, including the extrinsic pathway and perforin/granzyme pathways (GmzA and GmzB), as well as the main nuclear features of the current process, by correlating these events with the intrinsic apoptotic pathway. We then use such pathway characteristics to explore in more detail the mechanisms of the regulation of important human diseases, including cancer and neurodegenerative diseases.

1.2 Cytoplasmic Events

During apoptosis, the dying cells become rounded and retracted from the tissue. Defined blebs appear within the cells. The process culminates in the pinching off of many blebs, producing apoptotic bodies. These bodies recruit phagocytes, which engulf them to recycle some of the molecules. The immune system represents the major mechanism capable of eliminating the apoptotic bodies, apparently in the same way that phagocytes eliminate non-self particles. These morphological and biochemical features of apoptosis have been extensively studied, but the whole mechanism is not fully understood.

It is well established that during the apoptotic process, the nuclear DNA is condensed and fragmented. Several proteins, such as endonucleases and exonucleases, may degrade the DNA chain by hydrolyzing it in small fragments, with approximately 200 bp [1]. In the cytoplasm, the Golgi apparatus, ER, lysosomes, and mitochondria can be eliminated or recycled, depending on the apoptotic stimuli. For instance, during oxidative stress involving an increase of reactive oxygen species (ROS), several mitochondrial proteins, such as cyt c and endo D, may be released from the mitochondrial intermembrane space into the cytoplasm and nucleus, leading to cell death [4,5]. Interesting, it has been indentified that the mitochondria, ER, and nucleus are targets of the Gzm pathway, which may trigger apoptosis, facilitating the PCD process.

1.2.1 The Extrinsic Pathway

The extrinsic pathway, also called the death receptor pathway, is involved in transmembrane receptor-mediated interactions, including those of the TNF family [13], which shares the features of the cysteine-rich extracellular domains and a cytoplasmatic domain (death domain) [14]. The death ligand represents the external death signal from the cell surface to the intracellular signaling and effector pathways. The mechanism requires the binding of the extracellular death ligands to the transmembrane cell receptors. The best-characterized ligands and their corresponding death receptors have been identified: (i) TNFα and TNFR1; (ii) fatty acid synthetase ligand and fatty acid synthetase receptor (FasL and FasR); (iii) Apo2 ligand and death receptor 4 (Apo2L and DR4); (iv) Apo2 ligand and death receptor 5 (Apo2L and DR5); and (v) Apo3 ligand and death receptor 3 (Apo3L and DR3) [14–18]. The receptors form clusters and can bind with their cognate trimeric ligands, leading to the recruitment of adaptor proteins, including FADD, TRADD, and RIP [6–8]. In turn, FADD or TRADD can recruit several molecules, such as pro-caspase 8, binding to them via dimerization of the DED, leading to DISC formation and subsequent autocatalysis of pro-caspase 8 and its active form (caspase 8) [9]. Caspase 8 activation is considered the main feature that triggers the extrinsic pathway, leading to cell death. Activated caspase 8 is involved in many proteolytic processes, including the activation of caspases 3, caspase 6, and caspase 7. These enzymes help induce the execution phase of apoptosis (Figure 1.1).

Depending on the apoptotic stimuli, the extrinsic pathway can crosstalk with the intrinsic pathway through proteolysis of the BH3-only protein, BID. The truncated BID (tBID) protein promotes release of mitochondrial cyt c into the cytoplasm, where it can assemble with the apoptosome complex, leading to cell death [10]. However, death receptor-mediated apoptosis can be inhibited by cellular FLICE-inhibitory protein (c-FLIP), which binds to both FADD and caspase 8, inactivating the autocatalytic effect of the caspase 8 complex [19,20]. Different mechanisms of inhibition of the extrinsic apoptosis pathway have also been described, including via the protein Toso, which blocks Fas-induced apoptosis, inhibiting the processing of caspase 8 in immune cells [21].

1.2.1.1 The Perforin/Granzyme Pathway

To eliminate potential dangerous cells like tumor cells or cells infected by viruses or bacteria, the immune system relies on CTLs and NK cells, both of which are produced by the action of Gzms. PFN, a protein capable of binding the membrane of the target cell, facilitate the pore formation that permits the action of Gzms. Gzms are considered specific serine-proteases involved in cell death. They are produced as inactive precursor molecules, designed to avoid the self-destruction of CTLs and NK cells. In human cells, five different Gzms have been reported. GzmA and GzmK are located on chromosome 5 and act as tryptases that cleave proteins following arginine or lysine (basic) residues. GzmB and GzmH are located on chromosome 14. GzmM is located on chromosome 19 and cleaves following methionine or leucine basic residues [22]. There are two Gzm-dependent pathways involved in cell death.

1.2.1.1.1 The Granzyme A Pathway

The GzmA is considered the most important serine-protease mechanism described in CTL and NK granulles. Unlike the GzmB pathway, which relies on the oligomerization of the PFN to enter into the target cell, the GzmA can activate a parallel pathway in a caspase-independent manner, leading to DNA degradation, such as single-stranded DNA damage [23]. Intracellular GzmA substrates have been found in the cytoplasm (Pro-IL-1β) [24], mitochondria (NDUFS3) [25], ER, and nucleus (SET1, APE1, HMGB2) [26–29], and are associated with histone H1, core histones, lamin A, B, and C, Ku70, and PARP1 [30–33]. Various stimuli can trigger the GzmA pathway, such as ROS generation, the loss of membrane potential, and mitochondrial swelling. This can lead to the disruption of the nuclear envelope, inhibition of DNA repair, and activation of cytokines, as a consequence of the accumulation of GzmA in the nucleus [34]. Between mitochondrial changes (within minutes) and phospathidyl serine externalization (30 minutes to 1 hour), dying cells can recruit the macrophage scavenger system [23].

GzmA is less cytotoxic than the GzmB pathway, which is active at micromolar-range concentrations [35]. At 2 hours after the stimulation of apoptosis, several features are present. This cell-death pathway does not activate caspases, because cell-death GzmA activation is known as a non-apoptotic death [36]. Moreover, GzmA does not permebilize the outer mitochondrial membrane (OMM), avoiding the releasing of mitochondrial apoptotic mediators like cyt c. The entry of GzmA into the mitochondria can be partially inhibited by cyclosporine A (CsA) and bongkrekic acid (BA), suggesting a role of permeability for the transition pore (PT) in GzmA mitochondrial damage [23,35].

The oxidative damage drives the ER to make the ER-associated oxidative stress response complex (SET), which contains two endonucleases (Ape1and NM23-H1) and a 5′-3′ exonuclease (Trex1), chromatin modifying proteins (SET1 and pp32), and DNA-binding proteins that protect against DNA distortion (HMGB2) [23,27,29,37]. GzmA enters into the nucleus and cleaves SET1, which inhibits NM23-H1 endonuclease activity, causing this complex to nick the DNA, and allowing Trex1 to act as an endonuclease [38]. In the same way, GzmA cleaves and inactivates HGMB2 and Ape1 [26], cleaves the linker histone H1, and removes the tail of core histones, allowing the nucleases to attack [30]. GzmA then cleaves and inactivates Ku70 and PARP-1, both of which are involved in DNA repair through the recognition of single- or double-strand breaks [32,33].

1.2.1.1.2 The Granzyme B Pathway

GzmB is produced by CTL and NK cells, which release it via granules. It binds its receptor, the mannose-6-phosphate/insulin-like growth factor II receptor, and is endocytosed but remains arrested in endocytic vesicles until it is released by PFN. The GzmB pathway relies on caspase activation, unlike the GzmA pathway.

The proteolitic activity of GzmB is similar to caspase activity, cleaving substrates after the aspartate (basic) residues. Caspase 3, 6, 7, 8, 9, and 10 have been found to serve as GzmB substrates in vitro [39–46], but only caspase 3 is believed to be important in vivo [11,12]. As a further mechanism, GzmB can process BID, promoting cyt c release, SMAC/Diablo activation, formation of apoptosis inducing factor (AIF), and release of Omi from the mitochondria. It does this by recruiting the inhibitor of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member, especially the Bax protein, to the mitochondrial membrane, leading to apoptosome formation [45–47]. GzmB can also process caspase 3 and 7, initiating the apoptotic process [48]. It has been demonstrated that pro-apoptotic caspase activation happens within minutes of target-cell recognition by CTLs. Unexpectedly, there is a rapid rate of caspase 3/7 biosensor activation following GzmB cversus Fas-mediated signal induction in murine CTLs. This Fas-mediated induction is detected after 90–120 minutes in porcine, murine, and human CTLs, consistent with FasL/Fas-induced activity [49]. Recently, key roles for GzmB have been described, positioning it as an allergic inflammatory response of NK [50]. It has also been shown that the major NK cell-activating receptor NKG2D and the NK cell effector are both mediated by GzmB.

1.2.2 Nuclear Features of Apoptosis

The first description of the apoptotic process as a basic biological phenomenon different from necrosis (based not only on morphological criteria) was given by Kerr et al. [51]. The authors described two characteristics of apoptosis: (i) cytoplasmic and nuclear condensation and the disruption of the cell into a number of membrane-bound, well-fragmented pieces; and (ii) formation of apoptotic bodies that are taken up by other cells for degradation. This study shed new light on the apoptotic mechanism as an important process of PCD that regulates several biological processes, including embryogenic development and aging, cell turnover in different tissues, and the control of the immune system. Inappropriate control of apoptosis appears in many human disorders, leading biologists to seek a better understanding of the entire process. Intriguingly, a wide variety of stimuli – both physiological and pathological conditions – can trigger apoptosis. In this section, we address the main biochemical features of apoptosis that focus on nuclear events, including the activation of the execution caspases (i.e., caspase 3, caspase 8, caspase 9, and caspase 10), chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies.

Early evidence described DNA fragmentation as a key feature of apoptosis. Using low concentrations of an exogenous agent like γ-irradiation to induce cell death, it was shown that the DNA of lymphocytes was completely degraded into oligonucleosomal fragments. Further, cells induced with near-physiological concentrations of glucocorticoid hormones showed chromatin condensation as an early structural change. In fact, this particular nuclear morphological change was associated with excision of the nucleosome chains from nuclear chromatin through activation of an intracellular, but non-lysosomal, endonuclease [52]. At this time, it was already known that some members of the caspase family, comprising 12 proteins, are strictly involved in the apoptotic cell death process [53,54]. These are the signals after mitochondrial outer-membrane permeabilization (MOMP) that activate the caspase pathway. However, the interconnection between the nuclear and cytoplasmic events involved in apoptosis became better appreciated when a nuclease protein (Nuc-1), a homolog of mammalian DNAase II, which plays a role in DNA degradation in the nematode Caenorhabditis elegans, was identified as acting downstream of Ced-3 and Ced-4 [56]. In particular, attempts have long been made to understand the link between the executioner caspases and subsequent nuclear apoptotic events, since a variety of death stimuli can activate these proteases, which amplify the signal of cell death.

Caspase 3 is considered the most important protease of the executioner pathway, and can be activated by any of the initiator caspases. Caspase 3 can be activated by caspase 8, which is activated from the death receptor; by caspase 9, which is involved in the mitochondrial apoptotic process; or by caspase 10, which is involved in the PFN/GzmB pathway. Each of these pathways is responsive to a wide range of stimuli capable of amplifying the cellular death signal in an energy-dependent manner. The cleavage of caspase 3 results in the activation of the endonuclease CAD. In apoptotic cells, activated caspase 3 cleaves inhibitor of caspase-activated DNase (ICAD) to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA. The CAD:ICAD complex inhibits the CAD activity as DNase. When CAD is cleaved by caspase 3, it can degrade chromosomal DNA like a scissor-like homodimer, cleaving double-strand DNA at nucleosomal linkers [2,3].

CAD is able to condense chromatin and to fragment chromosomal DNA in an irreversible manner that compromises DNA replication and gene transcription, leading to cell death. Accompanied by chromatin condensation, chromosomal DNA is cleaved into high-molecular-weight fragments of 50–300 kb, which are subsequently processed into low-molecular-weight fragments of approximately 180 bp. Several models have been designed to study the role of CAD in PCD. These studies have shown that the inhibition of CAD activity – for instance, by inducing degradation by a chaperon – can abolish internucleosomal DNA fragmentation. However, the inefficient DNA degradation activity detected in CAD-deficient cells suggests the existence of additional nuclease(s) during apoptosis. An interesting example that links the extrinsic and intrinsic pathways is related to the mammalian endonuclease G (endo G). Endo G is a nuclease that was first identified in the mitochondrial intermembrane space; upon apoptotic stimuli, it may be released from the mitochondria and translocated to the nucleus. The endonuclease activity is responsible for cleaving nucleic acids, representing a caspase-independent apoptotic pathway initiated from mitochondria. In mouse embryonic fibroblast (MEF) cells, taken from a DFF45/ICAD-knockout (KO) mouse, there was no detectable caspase 3-dependent activity, and it was shown that there was minimal DNA fragmentation. Moreover, the induction of apoptosis by ultraviolet irradiation or treatment with cyclohexamide (Chx) led to the release of both endo G and cyt c from the mitochondria to the cytosol and nuclei. The identification of DNA fragmentation has been used as a fundamental biological marker of apoptosis. The main method for detecting apoptotic PCD is known as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) [57].

There have been several further reports providing evidence for caspase-independent death programming in vitro and in vivo by cathepsins B and D, calpains, and serine proteases. Some of these death routines become evident only when the caspase-dependent pathway is inhibited, particularly in the case of ATP depletion or when using caspase inhibitors.

The evolutionarily conserved execution phase of apoptosis is characterized by cell morphology changes, including cell shrinking, plasma-membrane blebbing, and separation of cell fragments into apoptotic bodies. It is known that the actin–myosin system plays a key role in bleb formation through the activity of the Rho effector protein (ROCK I), which leads to the phosphorylation of myosin light-chain ATPase activity and coupling of actin–myosin filaments to the plasma membranes. Apoptotic bodies consist of cytoplasm-packed organelles that contain nuclear fragment. The integrity of the apoptotic bodies is maintained in order to avoid the release of their cellular constituents into the surrounding interstitial tissue, which would block activation of the inflammatory reaction; this permits the apoptotic bodies to be degraded efficiently within phagolysosomes by macrophages and various surrounding cells.

Although the evolutionarily conserved execution phase of apoptosis has been the theme of many studies, a full understanding of apoptosis at the molecular level is needed if we are to gain deeper insights into its basic and applied biology, particularly regarding new therapeutic strategies.

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2

Mitochondria in Focus: Targeting the Cell-Death Mechanism

Humberto De Vitto¹, Roberta Palorini,²,³ Giuseppina Votta², and Ferdinando Chiaradonna⁴

¹Center of Health and Science, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

²SYSBIO Center for Systems Biology, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy

³Luxembourg Centre for Systems Biomedicine, Esch-sur-Alzette, Luxembourg

⁴Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy

Abbreviations

2.1 Introduction

As a natural control, multicellular animals must tune their cell fate between life and death. Apoptosis, one of the most important and regulated processes of destruction, is a complex, energy-dependent biochemical network [1–3]. In organisms, apostosis is used to tightly control cell numbers and tissue size, and to protect against potentially dangerous cells and environmental signals that threaten homeostasis [4]. The term apoptosis was definitively inserted in the scientific literature by J.F. Kerr, A.H. Wyllie, and A.R. Currie [5]. In their seminal paper, these authors described a type of programmed cell death (PCD) observed in various tissues and cell types that had distinct morphological features as compared to cells undergoing pathological necrotic cell death [5].

In the 1980s, new insights were gained into apoptosis by the identification of the Bcl-2 gene as an important determinant for the development and maintenance of B-cell leukemia [6,7]. In particular, some authors described the ability of the Bcl-2 protein to promote cell immortalization and the survival of B-lymphoid cells, otherwise destined to die, by cooperating with the oncogene c-myc [8]. Along with these first indications regarding the role of Bcl-2 in cell survival, immunolocalization experiments clearly demonstrated that Bcl-2 was an outer mitochondrial membrane protein, suggesting a relationship between the mitochondria and apoptotic cell death [9]. However, the first indication regarding the connection between apoptosis and the mitochondria must be assigned to D.D. Neymeyer and colleagues, who showed that the apoptotic process could be blocked by the Bcl-2 protein [10]. Intensive efforts were promptly made to understand the key regulatory mechanisms linking the mitochondria and apoptosis, which permitted the identification of other proteins, including Bcl-xL, E1B 19K, CED-9, and Mcl-1, which, like Bcl-2, could act as inhibitors of cell death [11–13]. However, the exact mechanism by which Bcl-2 inhibited dependent cell-death protease activation was not established. Two scientific teams, advised by D.D. Newmeyer and X. Wang [14,15], simultaneously published papers characterizing, for the first time, the role of mitochondrial cytochrome c (cyt c) protein as a key element for apoptosis in Xenopus egg extract and in a human cell line. This protein normally resides in the space between the inner and outer mitochondrial membranes (IMM and OMM), where it functions to transfer electrons as part of oxidative phosphorylation (OXPHOS). However, upon certain stimuli triggering cell death, cyt c is released from the mitochondrion into the surrounding cytoplasm. The authors suggested that such a cytoplasmic localization permits cyt c to activate a cascade of events, especially characterized by specific proteases activation, which together lead to apoptotic death. Most interestingly, cyt c translocation, and consequently protease activation, was prevented by Bcl-2 expression and mitochondrial localization, underlining its main role in the inhibition of mitochondria-dependent apoptosis [14,15]. As the role of mitochondria in the apoptotic program was further clarified, other details regarding the cytoplasmic mechanisms were identified. Indeed, since cyt c is released into the cytosolic compartment, a dATP-dependent complex, called the apoptosome, forms through the action of apoptotic protease activating factor-1 (Apaf-1) and cyt c, which proceeds to activate a latent protease, namely procaspase 9, into caspase 9 [16]. The latter is a member of a family of cysteine aspartyl-specific proteases (caspases), comprising 12 proteins strictly involved in the apoptotic cell death process.

In 1998, D.R. Green and J.C. Reed published a special issue addressing the relation between mitochondria and apoptosis [17]. The authors identified and discussed the main mechanisms altering mitochondrial physiology and leading to this suicide event. They suggested that some signals coming to the mitochondria, such as calcium (Ca++), oxidants, proapoptotic proteins (e.g., Bax/Bak), and even caspase proteins, can modulate mitochondrial physiology and produce outgoing signaling, leading to PCD. In their seminal work, the authors hypothesized an apoptotic mechanism called the mitochondria swelling route, based on an alteration of the mitochondrial membrane features. In particular, this speculative mechanism is characterized by a mitochondrial inner transmembrane potential (ΔΨm) collapse, leading to mitochondrial channels opening and ultimately to mitochondrial permeability transition pore (MPTP) opening. This results in OXPHOS uncoupling, due to H+ gradient dissipation, and mitochondrial volume dysregulation. As a consequence, the mitochondrial matrix volume expansion can eventually induce outer-membrane rupture, releasing caspase-activating proteins into the cytosol and causing PCD via apoptosis [17]. On the other hand, pharmacological inhibitors of the MPTP can act as potent inhibitors of cyt c release, and hence prevent apoptosis [18]. Taken together, it was reasonably attractive to postulate that the mitochondria are a critical control point not only in cell death, but also in cell survival.

Given the advance of apoptosis issues, different mechanisms have been hypothesized to explain the role of the mitochondria in PCD. In fact, it has been proposed that a mitochondrion can act either as a passive organelle that releases cytotoxic proteins into the cytoplasm by passive leakage following the action of the proapoptotic BH-3-only proteins (e.g., Bid or Bim) or as an active organelle that induces, for instance, Ca++ mobilization from the endoplasmic reticulum (ER) to the mitochondria, leading to mitochondrial outer-membrane permeabilization (MOMP). In addition, morphological changes in the mitochondria, such as fission, induced by proteins like Drp1 and Fis, promote the production of reactive oxygen species (ROS), and inner-membrane changes may induce cyt c release across the outer membrane. This implies that, somehow, cytosolic proteins (such as Bid and other members of the Bcl-2 family that target the outer membranes of mitochondria) transduce signals into the interior of the organelle [19–21]. Nevertheless, as D.D. Newmeyer and S. Ferguson-Miller have said, the mitochondria and apoptosis scenario was turning diverse and controversial. In this regard, the authors have suggested that the next challenges to significantly improving our understanding will be to determine if these different proposed mechanisms may be utilized simultaneously, sequentially, and/or in a specific cellular context [19].

During the 2000s, some points of critical importance to understanding the complex and uncertain apoptotic process were addressed. Studies explaining the diverse array of positive and negative signals acting on the mitochondria, the diverse functions of pro- and antiapoptotic members of the Bcl-2 family, and the biochemical aspects of caspase activation must be considered the milestones [3,22,23]. All these aspects will be more extensively illustrated in later sections; here, we want just to underline that the identification of critical control points, such as the role of incoming signals, Bcl-2-family proteins, and caspase, has provided important clues to the role of mitochondrial-controlled apoptosis in maintaining physiological homeostasis, either during organism development [24–26] or in many pathological processes, for instance cancer and neurodegenerative diseases [27]. Otherwise, such dual roles are not unexpected, since mitochondria acting as a central hub of different stimuli (e.g., Ca++ or oxygen radicals), coming from different intracellular organelles (e.g., nucleus or ER) and with a different intensity (e.g., cytoplasmic Ca++ concentration) and duration, may control an array of cell outcomes that span from full survival to excessive apoptosis. All of these stimuli contribute to various human diseases, such as cancer, autoimmune diseases, viral infections, acquired immunodeficiency syndrome (AIDS), ischemia injury, and neurodegenerative diseases [27]. Cancer is a convincing example of human disease associated with increased cell survival; one for which evidence unquestionably exists in support of the idea that mitochondrial bioenergetics and cell metabolism are strongly associated with cell death/survival as critical features for tumor progression [28–32]. Thus, such identification has permitted the development of new therapeutic strategies addressing the relation between mitochondria and apoptosis (see Section 2.3).

In line with this, the current chapter is focused on the role of the mitochondria in apoptosis. We will describe and highlight an overall view of mitochondrial morphology, mitochondrial metabolism, and mitochondrial bioenergetics by correlating these features with the intrinsic apoptotic pathway. Such mitochondrial characteristics will then be used to explore in more detail the fascinating issue of how regulation of mitochondrial function can result either in cell accumulation or cell loss, especially in human diseases, including cancer and neurodegenerative diseases.

2.2 Mitochondria: Overview

2.2.1 Mitochondrial Morphology

In a somatic mammalian cell, it is well established that each mitochondrion harbors thousand of copies of mitochondrial DNA (mtDNA) and houses the OXPHOS system (see Figure 2.1) [33]. Mitochondrial morphology in mammalian cells has received a great deal of interest, especially after early findings that the mitochondrial network dynamic may be critical for their function and for cell homeostasis [34]. Although the molecular mechanisms regulating mitochondrial motility, fusion, and fission are not yet totally understood, they have together been related to a protective role in mitochondrial integrity and function. A weakness of the mitochondrial fusion and fission process has been linked to human diseases, including cancer and neurodegenerative disorders [35,36]. Mitochondria change their morphology in response to various stimuli. For example, during the early stages of apoptosis, mitochondrial fragmentation and cristae remodeling are recognized as important morphological alterations leading to cell death. Most interestingly, some proteins that control mitochondrial network dynamics appear to participate in the apoptotic process [37]. The mitochondrial fusion and fission machinery includes a set of outer-membrane proteins (e.g., Drp1 and Fis1) and one of inner-membrane proteins (e.g. Mfn1, Mnf2, and Opa1), which play a key role in apoptosis [38]. In fact, several studies performed in mammalian cells have shown that either decreasing Drp1 or Fis1 activity or increasing Mfn1 or Mfn2 activity may interfere with cyt c release and hence block PCD. On the other hand, it has also been shown that a reduction of Mfn1 or Mfn2 activity increases cell responsiveness to apoptotic stimuli [38]. Moreover, increased expression of the proapoptotic proteins Bax and Bak induces mitochondrial fragmentation and fusion of mitochondrial cristae, resulting in cell death [39]. In contrast, overexpression of the antiapoptotic Bcl-2 protein increases mitochondrial size and structural complexity, preventing cell death [40].

Figure 2.1 Mitochondria operate as a central hub, regulating their morphology, bioenergetics, and biosynthetic activity in order to respond to the metabolic demands of the cell. Bioenergetics: OXPHOS machinery uses electron transport chains (ETCs; complexes I–IV) to transport electrons to oxygen (the final electron acceptor), which is reduced to H2O. The electron transport generates a ΔΨm, which is required to activate the ATP synthase used to power the synthesis of the energy carrier molecule ATP from ADP + Pi. Metabolism: Glucose (glycolysis) and glutamine (glutaminolysis) are important nutrients required to govern catabolic and anabolic mitochondrial pathways (the tricarboxylic acid (TCA) cycle). This metabolic network shows as rate-limiting steps, with enzymes regulated by the oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) ratio, the ATP/ADP ratio, and the acetyl-CoA/CoA ratio. Morphology: Mitochondria harbor multiple copies of mtDNA, divided between two different compartments: the intermembrane space and the mitochondrial matrix (inner and outer membranes). They also harbor a complex enzymatic machinery of fusion (Mnf1/2 and Opa1) and fission (Drp1 and Fis), required to control mitochondrial dynamics in a vast spectrum of stimuli (e.g., ROS and Ca²+).

It is tempting to speculate that under metabolic stress, mitochondrial fusion may be an efficient mechanism by which to eliminate and recycle damaged mitochondria, avoiding the loss of an essential compartment and preserving energy production through OXPHOS. Conversely, mitochondrial fission has been described as an mtDNA and/or protein quality-control system that leads, in stressful conditions, to the establishment of healthy mitochondria. However, during high levels of stress (e.g., nutrient deprivation; see Figure 2.2), the fission machinery can trigger apoptosis [41]. As suggested by Youle and van der Bliek [41], in normal cells, the fusion and fission machinery helps to mitigate stress and to create new mitochondria, but disruptions in these processes affect normal development. Accordingly, further understanding of mitochondrial dynamics could help better elucidate the process of mitochondria cell death.

Figure 2.2 Representation of incoming death signals. Increased matrix metalloproteinase (MMP) is the point of no return in the cascade of events leading to apoptosis, allowing the release of apoptogenic factors. Different events lead to different MMP changes during apoptosis induction, such as an increase of Ca++ uptake (A), ROS formation (B), a drop in ΔΨm (C), or increased expression of Bax due to p53 activated by DNA damage (D). These events are driven by a wide range of stress conditions, including glucose and glutamine withdrawal (which activates almost all of them), growth-factor withdrawal, and viral infection, which are able to act in a direct way on Bax or Bak activation and through ΔΨm and ROS changes, respectively.

2.2.2 Mitochondria and Cell Metabolism

In the last 20 years, several findings have shown that cell metabolism and cell death are strictly associated [42]. In general, the mitochondrion is believed to act as a sensor capable of determining cell fate under several types of stress: whether to adapt and survive or die. The ability of mitochondria to keep cells alive or to lead to an irreversible catastrophic event is programmed into an array of mitochondrial metabolic pathways that are used to control the major metabolic signals. In mammalian cells, the most important mechanism that supports the mitochondria and cell metabolism is the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, coordinated by OXPHOS machinery. This complex metabolic network relies on nutrient availability (e.g., glucose, glutamine, lipids) and oxygen supply. Furthermore, OXPHOS activity is considered the mayor source of production of ROS, suggesting that mitochondria may sense oxidative stress leading to cell metabolic control and apoptosis [42].

Another class of metabolic signal that emerges with a substantial amount of interest in studies of metabolic control and apoptosis is the balance between oxidized and reduced nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH). It is remarkable that catabolic, anabolic, and waste-disposal mitochondrial pathways act as rate-limiting steps, with enzymes regulated by the NAD+/NADH and NADP+/NADPH ratio. For instance, in order to sustain metabolic pathways, those that synthetize macromolecules (anabolism), those that degrade molecules to release energy (catabolism), and those that eliminate toxic product (waste disposal) all depend on redox homeostasis, which may impact on cell-death pathways. Interestingly, some mitochondrial byproducts are found in the cytosolic environment, such as NADP+/NADPH plus ATP. The enzyme glutathione synthetase generates reduced glutathione (GSH). The redox balance between reduced and oxidized glutathione (GSSG) represents one of the most important antioxidant systems, playing a pivotal role in mitigating ROS and reactive nitrogen species (RNS) production. This mechanism emerges as a potentially key pathway that could be exploited for the development of novel strategies in human diseases (see Section 2.3.2) [43].

The third class of metabolic signals, the acetyl coenzyme A (acetyl-CoA)/CoA ratio is regulated by two enzymes: acetyl-CoA synthetases (ACS) and ATP-citrate lyase (ACL). This set of reactions provides acetyl-CoA for the lipogenesis and synthesis of other macromolecules, and for histone acetylation reactions used to regulate gene expression and enzyme function. Interestingly, interference in the acetyl-coenzymeA (acetyl-CoA)/CoA ratio may be sufficient to trigger the apoptotic process [44].

2.2.3 Mitochondria and Bioenergetics Metabolism

In the 1940s, mitochondria were described as housing OXPHOS machinery in eukaryotes cells, creating interest in the study of mitochondria as a biological component that unites many diseases. Structurally, mitochondria are organelles with an outer membrane and an inner membrane. Thus, they can be divided into two different compartments: the intermembrane space and the mitochondrial matrix. These compartments are structurally and functionally distinct. The particular biochemical anatomy of mitochondria provided underlying insights that allowed Peter Mitchell to introduce his chemiosmotic theory. The author hypothesized that biological oxidation reactions generate a proton gradient across the IMM that is transduced into a more useful form of chemical energy: the molecule of ATP [45]. In line with this observation, energy is generated in mitochondria by oxidizing hydrogen derived from our dietary carbohydrates (glycolysis), amino acids such as glutamine (glutaminolysis), and fatty acid (β-oxidation) through the tricarboxylic acid (TCA) cycle. These catabolic pathways can fuel the mitochondrial redox center, represented by universal acceptors (NADH and FADH2) that donate protons through the electron transport chain (ETC), and involve the reduction of O2 to H2O, generating ATP as a final product (see Section 2.3.1 for a detailed description) [46]. Mitochondria also encompass various others metabolic pathways, participating in lipogenesis, the urea cycle, and the synthesis of pirimidines, heme, and some amino acids [33]. Therefore, mitochondria must be considered organelles strongly responsible for the bioenergetics mechanisms controlling cell life and death. The entire role of mitochondrial dynamics and mitochondrial bioenergetics and metabolism is summarized in Figure 2.1.

Notably, nutrient shortage, hypoxia, oncogenes expression, ROS production, Ca²+ signaling, and other incoming and outgoing signals can influence cell metabolism, interfering in mitochondrial energy production and hence directly impacting on the rewiring of metabolic pathways. One important method of controlling cell fate involves regulation by Bcl-2 family proteins, which undergo MOMP and release cyt c and other mitochondrial proteins into the cytosol, triggering apoptosis. We address some examples in the next section.

2.3 Mitochondrial Apoptotic Pathways

2.3.1 Incoming Signals: Cytoplasmic and Nuclear Events

2.3.1.1 Cytoplasmic Events

Mitochondria have been described as playing a central role in the apoptotic process [17] at several levels: maintenance of ATP production [47], ΔΨm and mitochondrial permeability transition (MPT) for release of certain apoptogenic factors from the intermembrane space into the cytosol [17], and ROS production [48]. A major point of discussion, however, is whether mitochondria are the central decision maker or simply amplify, as a fundamental hub, the signaling pathways that link the detection of intracellular danger to adaptive responses by housing crucial signal transducers. This section is dedicated to the second aspect of mitochondrial involvement in apoptosis.

Mitochondria contain two membranes: the OMM fully surrounds the IMM, with a small intermembrane space between them. The IMM has restricted permeability and is loaded with proteins involved in electron transport and ATP synthesis. It surrounds the mitochondrial matrix, where the TCA cycle produces the electrons that are pumped from one protein complex to the next within the IMM. At the end of this ETC, the final electron acceptor is oxygen, and this ultimately forms H2O. At the same time, the ETC produces ATP. During electron transport, the participating protein complexes drive protons from the matrix out to the intermembrane space. This creates a concentration gradient of protons that then flows back into the mitochondria through another protein complex (ATP synthase) to power synthesis of the energy carrier molecule ATP [49]. The total force driving protons into the mitochondria (i.e., Δp) is a combination of ΔΨm, a charge or electrical gradient, and mitochondrial pH gradient (ΔpHm, an H+ chemical or concentration gradient).

The OMM has protein-based pores that allow the passage of ions and proteins through the MPTP. The MPTP consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), and cyclophilin D (CypD) [50]. These proteins cooperate to form a large conductance channel, and they are responsible for the permeability transition (PT) of mitochondria. The increase in PT is one of the major causes of cell death in a variety of conditions, and it may also play a role in mitochondrial autophagy [51].

In physiological conditions, mitochondria maintain a high ΔΨm, which is used to generate ATP and for the import of proteins. In this setting, ROS are kept to a minimum [52]. When the IMM becomes permeable to positively charged ions (K+, Mg++, and Ca++), which flow en masse into the mitochondrial matrix, driven by its electronegative nature, there is a dissipation of ΔΨm [17], acidification of the matrix (if the pH drops below 7.0) [53], abolition of mitochondrial ATP synthesis, and massive entry of H2O into the mitochondrial matrix, causing an osmotic imbalance that results in MOMP and the release into the cytosol of several factors that are normally confined within the intermembrane space, leading to apoptosis (see later). Most interestingly, several studies have shown that CyPD or ANT inhibition by cyclosporine A (CsA) and bongkrekic acid (BA), respectively, may block apoptosis in some systems, suggesting the involvement of the MPTP in apoptosis. However, Bossy-Wetzel et al. [54] have described a noncanonical system whereby cyt c release and caspase activation can occur before any detectable loss of MOMP, with the opening of the MPTP occurring downstream of apoptosome formation. Nonetheless, whether caspases can induce MPTP opening requires future elucidation [54]. However, MOMP represents an event that marks the point of no return of multiple signal-transduction cascades leading to cell death. Indeed, defects that alter the capacity of mitochondria to undergo MOMP are associated with a large array of human pathologies, including infectious diseases, ischemic conditions, neurodegenerative disorders, and cancer [55,56], and the dissipation of the ΔΨm constitutes an early and irreversible step in the cascade of events that leads to apoptotic cell death [57].

It is of note that the MPTP can operate in three distinct states: (i) a closed state, characterized by a high ΔΨm; (ii) a low-conductance state, characterized by a partly open pore, with a permeability to molecules <300 Da and a reversible decrease of the ΔΨm; (iii) the classic high-conductance state, characterized by an irreversible pore flip from the low-conductance state to the high-conductance state, with a permeability to molecules <1.5 kDa and an irreversible decrease of the ΔΨm collapse [58]. Maintenance of the ΔΨm is essential for cell survival; indeed, the MPTP remains tightly closed during normal mitochondrial function, in order to drive ATP synthesis and maintain OXPHOS activity [58]. Nonetheless, disruption of the ΔΨm by direct methods, such as the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), does not induce PT per se [59], suggesting that PT requires more than just dissipation of the ΔΨm in order to occur.

Mitochondrial membrane integrity is tightly controlled through interactions between pro- and antiapoptotic members of the Bcl-2 protein family [56]. Antiapoptotic Bcl-2 family members interact with and close the VDAC, whereas some but not all, proapoptotic members interact with the VDAC to open a protein-conducting pore through which apoptogenic factors pass (see later). The VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin-regulatory protein, which is able to converge a variety of cell-survival and cell-death signals to MPTP status [60].

The Bcl-2 family resides immediately upstream of the mitochondria, and can be divided into two classes with antagonistic properties: antiapoptotic (Bcl-2, Bcl-xL, Bcl-W, A1, Bag-1, and Mcl-1) and proapoptotic (Bax, Bak, Bad, Bid, Bcl-s, and Bok) [2]. The ratio between the antiapoptotic and proapoptotic Bcl-2 [52] members determines the susceptibility of cells to a death signal. All these proteins share in common at least a BH-3 domain: an amphipathic α-helical that serves as a critical death domain.

Proapoptotic Bcl-2 members can be subgrouped into the Bax family, which has several domains homologous to domains of Bcl-2, while the BH-3-only domain subgroup has only the BH-3 domain in common with Bcl-2. Members of the Bax family normally reside in an inactive form in the OMM or in the cytosol, whereas inactive members of the BH-3-only-domain family are normally localized into the cytosol. Both subgroups, upon activation by proapoptotic signals, need to pass from inactive monomeric conformation to active oligomeric complexes. BH-3-only-domain proteins are further subdivided into two groups: molecules typified by Bid, which can bind and activate proapoptotic Bax and Bak, and molecules such as Bim, Bad, and Noxa, which preferentially bind and inhibit the antiapoptotic Bcl-2/Bcl-xL proteins. Following multiple death stimuli, for instance, Bax translocates as a large homo-oligomer at the outer surface of the mitochondria, where it participates, in an almost unclear mode, in causing channel opening and mitochondrial fragmentation. Bak, when it activates the cell-death process, is arranged in large homo-oligomer complexes, apparently representing the active conformation [61].

Several ER-membrane proteins have been reported to interact with Bcl-2 family members and influence the apoptotic process. For example, Bax inhibitor 1 protein, a mammalian apoptosis suppressor, is localized specifically to the ER membrane [62]. Moreover, a member of the BH3-only family, Spike, localized in the ER, inhibits the formation of a complex between BAP31 and Bcl-xL [63]; in this way, BAP31 can be cleaved, and its amino-terminal fragment integrated in the ER, causing an early release of Ca++ from the ER, with an associated uptake of Ca++ into mitochondria and mitochondrial recruitment of a dynamin-related protein that mediates rupture of the OMM [64]. Mitochondria take up Ca++ electrophoretically from the cytosol through a uniport transporter. The energy-dependent Ca++ uptake, coupled to the release mediated by the exchange systems, constitutes an energy-dissipating mitochondrial Ca++ cycle. The affinity for Ca++ of the uniporter is low, and the size of the mitochondrial Ca++ pool is small under physiological conditions. However, under pathological conditions, intracellular Ca++ concentrations rise, becoming capable of stimulating numerous pathways, including activation of calcium-dependent proteases. Calpain-family cysteine proteases, implicated in the activation of Bax and Bid and in the inhibition of Bcl-2 and Bcl-xL [65], stimulate ROS production [66]. In turn, Bcl-2 resides in the ER, where it can regulate Ca++ homeostasis [67], and thus the induction of MPT (see Figure 2.2). In fact, MPT is also a Ca++-linked process, and many chemicals and radicals promote MPT formation. Typically, the effect of such inducers is to decrease the threshold amount of mitochondrial Ca++ needed to cause PT opening. In pathological settings, where the MPT contributes to cell killing, Ca++ may have several roles. First, increased Ca++ alone, and its uptake into mitochondria, may cause MPT onset. Second, other stressors may decrease the threshold for Ca²+-induced MPT, such that Ca²+ need not change but is still permissive for MPT onset. Lastly, stressors and increased Ca²+ may act synergistically to induce MPT.

Furthermore, Ca++ activates the calcium-sensitive mitochondrial fission protein Drp1, which has been implicated in Bax-induced channel opening and the release of cyt c [68].

Another intrinsic source leading to cell death by apoptosis is ROS. The most abundant and common ROS is the