Mechanisms of cell death
For years cell death has been a well known aspect of the development of multicellular organisms that continues throughout adulthood (Alberts, Johnson, Lewis, Raff, & Walter, 2008). In early fetal life it is the main way of removing the unnecessary cells in order to provide the desired shape and form of the new organism (Kindt, Goldsby, & Osborne, 2007). Cells have a built-in death program, termed programmed cell death (PCD), which enables them to kill themselves in a controlled way. The programmed cell death is not only a major participant in the normal development, but also in maintaining tissue homeostasis and in the elimination of damaged cells.
Nowadays, it is subdivided into three distinct categories: apoptosis (or PCD type 1), autophagy (or PCD type 2), and necrosis-like (or PCD type 3) (Bras, Queenan, & Susin, 2005). Increasing interest in the morphology of the programmed cell death has led to revision of the cell death classification. Thus apoptosis, the most common and best understood form of programmed cell death, should not be claimed as the only form of programmed cell death. Similarly, the previously considered non-programmed necrosis is nowadays established to have a programmed form which is shown to be dependent on reactive oxygen species (ROS) (Gogvadze, Orrenius, & Zhivotovsky, 2009).
1.1 Mechanisms of cell death
The different types of programmed cell death vary according to the morphological alterations taking place during cell death. Apoptosis is characterized mainly by a specific pathway of cell degradation which is followed by the formation of membrane-enclosed fragments, known as apoptotic bodies, which are finally phagocytosed by neighboring immune cells (Alberts et al., 2008).
Necrosis is characterized by organelle and cell swelling, followed by plasma membrane disruption and cell lysis (Gogvadze et al., 2009). A specific feature of necrosis is its early drastic drop of ATP production due to mitochondrial collapse.
Autophagy is characterized by a regulated lysosomal pathway involving the degradation and recycling of long-lived organelles and proteins. It induces cell death via excessive self-digestion. Autophagy is then an important mechanism for the removal of damaged mitochondria or other cell organelles (Gogvadze et al., 2009).
Recently, it has been shown that the main effectors of PCDI (apoptosis) are a family of cysteine proteases, known as caspases. There are two main pathways leading to the activation of the effector caspases - the death receptor pathway and the mitochondrial pathway (Alberts et al., 2008).Mitochondria, small, membrane-bound structures, are best known as the source of the reactions producing most of the cell's energy. However, recent evidence about their part in cell death pathways reveals them as main regulators of the decision between cellular survival and death (Bras et al., 2005)
The death receptor pathway (the extrinsic pathway) is initiated by the binding of cell-surface death receptors from the tumor necrosis factor (TNF) family to their ligands. The main ligands for those receptors are TNF itself and Fas ligand (Alberts et al., 2008). This extrinsic pathway activates the recruitment of procaspase-8 and procaspase-10 and the formation of the death-inducing signaling complex which activate downstream executioner caspase.
The second, mitochondria mediated, intrinsic pathway will be discussed in this review paper in more details. It involves caspase-dependent as well as caspase-independent pathways of cell death. The latter have been discovered by inhibiting or inactivating caspases, which still results in cell death. The underlying mechanisms of this pathway are suggested to involve mitochondrial alterations, such as disruption of electron transport chain , ROS production and decrease of ATP synthesis (Bras et al., 2005; Green & Reed, 1998).
2. Mitochondria in cell death pathways
2.1 Mitochondrial ATP production and Cell Death Induction
Adenosine triphosphate is prominently found in the inner membrane of mitochondria. The chemical structure of ATP is seen in Figure 1. The three terminal phosphate groups have a great reactivity with the chemical activities they involve in themselves (Alberts et al., 2008). The resulting product of these chemical activities i.e. of synthesis and hydrolysis is the yield of substantial amount of free energy.
The manner in which ATP is produced to yield energy is due to its establishment via oxidative reactions. The cristae of the mitochondria are membrane-containing compartments which hold the respiratory chain (or electron transport chain, ETC) and ATPase (Bereiter-Hahn, Vöth, Mai, & Jendrach, 2008). NADH that is produced by oxidative reactions in the cell induces hydrogen protons to be translocated from the mitochondrial matrix to the intermembrane space. Because of the continuous entry of hydrogen molecules, a proton gradient occurs. ATP synthase allows the entry of protons to occur via its proton channel. In other words, the proton-translocating ATPase is the complex which synthesizes ATP: ADP + Pi à ATP ("Cellular respiration,").
The mitochondrial permeability transition (MPT) phenomenon is an inducer of cell death involving ATP (Bristol).Normally, the proton-translocating ATPase initiates synthesis of ATP in the mitochondria. However, the effect of what is termed a “non-specific increase” can alter the permeability of the mitochondrial IM. This resulting state will negatively influence the permeability of the mitochondria by causing ATPase to hydrolyze, rather than synthesize ATP. Predictably, the final outcome is of the disrupted permeability is cell death (Bristol).
Cytochrome c as well as varying proteins are then released from the IM as a result from the swelling of the mitochondrial matrix (Desagher & Martinou, 2000). As it can be seen in Figure 2, the image to the left of the arrow indicates the normal state of the mitochondrion.
Furthermore, depiction of the aggregation of protons via electron transport re-entry through ATPase channel can be seen above, shown as V aka complex V of the respiratory chain (Desagher & Martinou, 2000). Without both a proper functioning voltage-dependent anion channel (VDAC) and ATP-ADP exchange, the aggregations of protons cannot be transported to the mitochondrial matrix. A subsequent rise in the mitochondrial membrane potential is then suggested to encourage osmotic swelling and thus initiate cell death (Desagher & Martinou, 2000).
In a review article by Bras et al (2005), an experiment involving leukemic cells demonstrated a role of oligomycin as a mediator of the apoptosis-necrosis switch. Oligomycin inhibits ATP synthesis thereby exhausting ATP levels. The “switch” between the two varying forms of cell death are due to certain implications: marked incrementing ATP levels will demonstrate the morphology of apoptosis which can be seen by way of electron microscopy (Nicotera & Melino, 2004). In contrast, a lack of ATP has been shown to die by way of necrosis. Bereiter-Hahn et al, (2008) have noted these situations as “extreme energectic conditions” which causes the migration of the mitochondria to be diminished.
How exactly is the motility affected in each of these conditions? With high levels of ATP, the movement of the mitochondrion is lost because damage to the motor molecules. The increased ATP in the cytoplasm destroys the vital link between the motor molecules and the cytoskeletal fibrils. The resulting condition is thus an inhibition of the motility of the mitochondrion (Bereiter-Hahn et al., 2008).
In the other circumstance, a lack of ATP, inhibition of the motility of the mitochondrion also occurs. The difference lies in the relationship between the motor molecules and the cytoskeletal fibrils. Instead of being destroyed, there is simply no binding activity because the mitochondrion becomes “trapped” (Bereiter-Hahn et al., 2008). Since there is a lack of ATP, ADP takes over the movement of the mitochondrion but this overtaking does not work for long. Eventually, the high energy demands of various organelles trap the mitochondria (Bereiter-Hahn et al., 2008). Furthermore, both the characteristic ladder pattern from the fragmentation of DNA and the presence of phosphatidylserine becomes absent (Bras et al., 2005)as demonstrated by gel electrophoresis and labeling with marker, Annexin V, respectively (Alberts et al., 2008).
A role of ATP in the induction of autophagic (type II) cell death is unclear yet there is a possibility that there is a dependency of ATP at the lysosomal level (Bras et al., 2005). Conversely, as aforementioned, the inhibition of ATP production can be seen in both apoptotic (type I) cell death and necrotic-like (type III) cell death.
In type I, ATP inhibition is said to occur relatively late in the course of apoptosis whilst in type III, there seems to be an early onset of diminishing ATP synthesis (Bras et al., 2005).
Clearly, the intracellular ATP concentration has a vital role in instigating a specified cell death pathway via a variety of possible metabolic conditions.
2.2 Mitochondrial release of apoptogenic proteins and Cell Death Induction
As mentioned before mitochondrial role in eukaryotic cells apoptosis can be expressed in different ways. The first mechanism to be discussed is the up-regulation of caspases by the release of apoptogenic proteins from the mitochondrial intermembrane space. Caspases, members of the cysteine proteases family, are specific as they only cleave their targets at specific aspartic acids (Alberts, 2008).
The regulation of caspases occurs by an activation pathway as these proteins are secreted in an inactive form, known as procaspase. Regulation of caspases is of vital importance as they are responsible for the breakdown of the nuclear lamina, cytoskeleton, and cell-to-cell adhesion molecules. Moreover, the activation of caspases must be tightly regulated as once activated, they initiate an irreversible pathway. Caspases activation is induced by different stimuli such as DNA damage or lack of oxygen. Moreover, the activation of caspases also depends on the cell type involved. Thus, caspases exist in numerous forms, and are involved in different pathways. Currently 18 distinct families have been identified (Aslan, 2009).
The pathways, involved in mitochondrial apoptosis, are divided into extrinsic (type I) and intrinsic (type II) pathways, of which the latter is involving mitochondria. The intrinsic pathway can be triggered by the stimuli previously mentioned. The caspases, which starts a pathway leading to apoptosis, are called initiator caspases. These initiators activate procaspases by proteolytic cleavage, which results in a subsequent chain reaction of other activator proteins (Bras, 2004). Eventually, the last caspases to be activated are executioner caspases, which cause the breakdown of cell structures.
Stimuli which activate the intrinsic pathway lead to the release of a mitochondrial intermembrane protein known as cytochrome c. The release of cytochrome c occurs via the formation of BH 123 oligomers in the mitochondrial membrane. The formation of these oligomers results in construction of a mitochondrial pore, which causes the release of cytochrome c. The formation of these oligomers is thought to be mediated by an increased release of Ca2+ from the ER into the cystol (Alberts, 2008). This signal is then thought to bind to Bcl -2 proteins located on the outer mitochondrial membrane. This binding causes the release of an anti-apoptotic protein which inhibits the mitochondrial pore formation (Bras, 2004). However, the exact mechanism of the formation and inhibition of the mitochondrial pore remains, as of yet, unknown.
When cytochrome c is, eventually, released into the cystol it binds to an apoptotic protease activating factor (Apaf-1), and procaspase-9 resulting in the formation of an apoptosome (Bras, 2005). The apoptosome is an important structure, responsible for the activation of executioner caspases. Activated caspases can, be caspase-3 and caspases-7, for example, which are responsible for the degradation of amyloid-beta proteins and most importantly apoptosis (Aslan, 2009). The activation of procaspases is, thus, the key step of apoptotic regulation, as it causes the formation of an apoptotic signal cascade.
Without an apoptotic signal, caspase-3 and 7 are unable to bind to an apoptosome. This is due to an apoptosis inhibiting protein known as XIAP. Thus, an apoptotic signal does not only release cytochrome c, but it also releases proteins from the mitochondrial intermembrane space to block the action of XIAP. These inhibitory proteins thus antagonize binding of the XIAP protein to the apoptosome.
The mitochondria can also release proteins which induce apoptosis in a caspase- independent way. Proteins involved in this mechanism are endonuclease G and protein AIF. These proteins move to the nucleus after being released from the mitochondria. Within the nucleus they control DNA degradation and chromatin condensation (Bras, 2004). Thus, the combination of cytochrome c and caspase independent proteins controls various actions during apoptosis. Their combined action then causes cell death via DNA degradation, breakdown of the cytoskeleton and cell adhesion molecules.
2.3 ROS production and Cell Death Induction
The electron transport chain function depends on the inner mitochondrial (IM) transmembrane potential which is vital for the proton gradient across the IM membrane and for the energetically-unfavorable synthesis of ATP (Bras et al., 2005). Thus, disruption of the ETC can lead to mitochondrial respiration dysfunction and induction of cell death, as already mentioned. In addition to that, as protons are pumped across the IM membrane to produce the essential for ATP synthesis electron gradient, very often an electron can escape from the NADH dehydrogenase (complex I of ETC) or cytochrome bc1 complex (complex III from the ETC). Those lost electrons can react with molecular oxygen to form oxygen radicals, which are consequently converted by the cell into hydrogen peroxide or reactive oxygen species (ROS) (Bras et al., 2005). It is estimated that 1 to 5 % of the electrons escape from the ETC and form ROS. Thus, everything that disrupts the ETC or decreases the coupling potential of the electron chain transport can lead to an increase of ROS production (Green & Reed, 1998).
ROS are important as their excessive quantity may cause lethal damages, leading to the mitochondria turnover via autophagy. Other type of PCD, leading to complete cell destruction, may be induced by high levels of ROS as well. One example is ROS-mediated destruction of the mitochondrial membrane, enhancing the cytochrome c leakage to the cytoplasm, thus enhancing apoptosis induction (Bras et al., 2005). A second example is the CD47 ligation-induced PCDIII (necrosis-like) where no cytochrome c involvement is shown (Bras et al., 2005).
Cell death may be induced by ETC dysfunction, excessive ROS, and cytochrome c leakage at the same time. However, variations of the mitochondrial alteration are possible in the different programmed cell death types (Bras et al., 2005)..
Multicellular organisms use programmed cell death as a method to remove unneeded structures, in order to control homeostasis. Three different types of cell death have been identified namely apoptosis, autophagy, and necrosis, respectively known as PCD type 1, 2, and 3. The various types are characterized by different morphological features. However, the common factor of cell death is the regulatory role of mitochondria. In case of apoptosis, cell death is caused by the release of cytochrome c from mitochondria, which subsequently activates caspases. Executioner caspases are responsible for the morphological characteristics of apoptosis, namely the breakdown of the cytoskeleton, and DNA degradation.
Another role of mitochondria in cell death is via the change in ATP production. Non specific mechanisms change the inner membrane permeability, which lead to the hydrolysis of ATP. Membrane transporters are disabled due to this reason, and protons will not be able to leave the mitochondria. This mechanism has been linked to swelling of the cytosol, and therefore necrosis is thought to be initiated due to disrupted ATP synthesis.
Another mechanism relating mitochondria to cell death is electron transport chain disruption and the subsequent formation of reactive oxygen species. Electrons escaping their transporters can react with oxygen, which results in the formation of oxygen radicals. These compounds are lethal for a cell as they can cause cytochrome c leakage, and CD47 ligation-induced necrosis. Thus, mitochondria occupy a central role in regulating the cellular responses to death signals