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Programmed Cell Death

Programmed cell death plays an essential role in tissue remodelling during embryonic development and in the removal of damaged cells to ensure homeostasis of whole organisms. There are two main types of cell death, with distinct morphological and molecular features.

Apoptosis (Programmed Cell Death)

Apoptosis, from the Greek meaning falling off, was introduced as a concept in its modern sense by Kerr in 1972 (Kerr et al., 1972), and it has since been recognised that deregulation of apoptosis is a major contributing factor to a range of pathological disorders, including autoimmune disorders such as AIDS and rheumatoid arthritis, neurodegenerative disorders such as Alzheimers disease, and cancer (Duke et al., 1996). Certain morphological characteristics distinguish the progression of apoptosis from necrosis. In contrast to necrotic swelling, the apoptotic cell reduces in size and detaches from adjacent cells. The plasma membrane becomes convoluted, known as blebbing, and the chromatin within the nucleus becomes highly condensed at the nuclear envelope (pyknosis).

Figure 1 (Video): Neutrophils undergoing programmed cell death (apoptosis) following the treatment with cyclohexide. Video on Youtube. Link: https://www.youtube.com/watch?v=ukxNXSPckjQ

Necrosis

Necrosis is a largely unregulated cell death pathway, and is typically induced by trauma, infectious agents or toxins. It is characterised by swelling of the cells prior to the degradation of DNA and eventual cell lysis leading to an inflammatory response in the surrounding tissue (Majno et al., 1995). Recently, reports have indicated that some necrosis may occur via a programmed pathway termed necroptosis, which is mediated by an array of signalling proteins including the tumour necrosis factor receptor (TNFR), and the receptor-interacting protein RIP1 and RIP3 (Vandenabeele et al., 2010). As this unwarranted inflammatory response is damaging to tissue, the preferred method for cell death is by a pathway of programmed cell death, known as apoptosis. The morphological hallmarks of apoptosis were identified microscopically as long ago as 1885 by Walther Flemming (Flemming, 1885), who termed the process chromatolysis.

DNA cleavage & blebbing in programmed cell death

Endogenous nucleases cleave DNA at internucleosomal regions, forming multiples of 180bp oligonucleosomal DNA fragments, and producing a characteristic DNA laddering pattern which is the biochemical hallmark of apoptosis. The nuclear envelope then fragments (karyorrhexis), and DNA is eventually completely degraded (karyolysis). Individual blebs may separate from the body of the cell as vesicles containing cellular material, known as apoptotic bodies. Finally, the phospholipid phosphatidylserine, normally localised to the cytoplasmic face of the plasma membrane, is flipped to the external face of the plasma membrane by the action of a group of enzymes known as scramblases (Sahu et al., 2007). Phosphatidylserine acts as a signal that promotes phagocytosis of the apoptotic bodies, thus ensuring the removal of apoptotic corpse, and preventing the inflammatory response induced by necrotic cell death. Apoptosis can progress via 2 distinct pathways, known as extrinsic and intrinsic apoptosis (Fulda et al., 2006).

Programmed Cell Death Pathways

Extrinsic Cell Death

The extrinsic pathway of apoptosis is initiated either by an extracellular signal which activates a death receptor (e.g. Fas, TNF), or by the release of Granzyme B and perforin from cytotoxic lymphocytes as part of the innate immune response to infection (Goping et al., 2003). Activation of the death receptors leads to their oligomerisation and recruitment of the cytosolic adaptor proteins (e.g. FADD, TRADD), facilitating the assembly of the death-inducing signalling complex (DISC), which is comprised of the death receptors and associated adaptor proteins in conjunction with procaspase-8 (Ashkenazi et al., 1998).

Cell Death Receptors

The extrinsic pathway of apoptosis is mediated by death receptors (DRs) belonging to the tumour necrosis factor (TNF) receptor superfamily. Members of this family include, TNFR1, CD95 /Fas, TRAIL-R1/DR4 and TRAIL-R2/DR5 (Itoh et al., 1991; Pan et al., 1997; Tartaglia et al., 1993). These receptors are commonly expressed on the surface of cells as homotrimeric type I transmembrane proteins and are characterised by the presence of cysteine rich extracellular domains (Ashkenazi and Dixit, 1998). Death receptors also contain a cytoplasmic domain of about 80 amino acids which is referred to as a death domain (DD), which plays a vital part in transmitting the death signal from the cells surface to intracellular signalling pathways.

CD95/Fas Receptor

One of the best studied and most prominent of the death receptors is the CD95/Fas death receptor, which participates in maintaining homeostasis within the immune system. CD95 is implicated in the deletion of mature T-cells, killing of inflammatory cells, the destruction of cancer cells or virus infected cells by cytotoxic lymphocytes (Ashkenazi and Dixit, 1998; Nagata, 1997). Ligation of the CD95 receptors results in trimerisation, clustering of the death domain receptors and recruitment of the adaptor molecule the Fas-associated death domain (FADD) to its cytosolic tail (Algeciras-Schimnich et al., 2002).

Death Inducing Signalling Complex (DISC)

FADD in turn recruits caspase-8 by homophillic interactions with its N-terminal death effector domains (DED). This then forms the CD95 death inducing signalling complex (DISC) (Kischkel et al., 1995), resulting in activation of the initiator caspase, caspase-8 (Boldin et al., 1995). Caspase-8 activation results in autoproteolyitc activation. The active subunits p10 and p18 are released into the cytoplasm, with part of the pro-domain remaining attached to the DISC.

Caspase-8 activation mechanism & DISC formation

Upon activation caspase-8 can propagate the apoptotic signal by cleavage of further downstream effector caspases such as caspase-3 (Medema et al., 1997; Murphy et al., 2004). However, it has also been reported that caspase-8 can be activated in similar manner to caspase-9 where processing is not required and caspase-8 is activated by dimerisation (Boatright et al., 2003; Donepudi and Grutter, 2002).

Under some conditions caspases-8 cannot directly activate downstream caspases and hence there have been two cell types described both of which utilise the CD95 receptor /ligand complex (Scaffidi et al., 1998). In what have been termed type I cells, death is instigated by large amounts of active caspase-8 produced at the DISC, resulting in the direct cleavage of caspase-3 prior to loss of mitochondrial transmembrane potential. In comparison in the type II cells the activation of the death receptor pathway alone is insufficient to induce apoptosis. In these cells little DISC is formed and hence smaller amount of active caspase-8 is available and thus the apoptotic signal requires amplification by the simultaneous engagement of the mitochondrial pathway.

This pathway is carried out by caspase-8 mediated cleavage of a BH3-domain-containing Bcl-2 family member Bid. Active caspase-8 cleaves Bid to produce a truncated form (tBid), which either alone or in combination with other molecules induces mitochondria to release pro-apoptotic factors such as cytochrome c resulting in the formation of the apoptosome complex (Li et al., 1998; Luo et al., 1998).

FLIP - Death Inducing Signalling Complex (DISC)

Another component of the DISC is the protein, FLIP (FLICE-like Inhibitory protein) which is a homolog of caspase-8. v-FLIP or viral FLIP has been recognized in several herpes viruses (Bertin et al., 1997). The human homolog of v-FLIP is c-FLIP or cellular-FLIP. c-FLIP is incapable of proteolytic activity as it is lacking the catalytic cysteine residue which is present in caspase-8. Two splice variants of c-FLIP exist, a long form c-FLIPL and a short form, c-FLIPS (Krueger et al., 2001). The exact role of c-FLIP remains controversial as may have reported it to have both pro-and anti-apoptotic functions. Its impact on apoptosis appears to vary between cell types as over-expression of FLIP in some cell lines did confer protection (Hu et al., 1997; Irmler et al., 1997; Kataoka et al., 1998; Srinivasula et al., 2003) whereas in contrast others studies reported that FLIP actually enhanced apoptosis (Han et al., 1997; Inohara et al., 1997). In an effort to shed some light on the role of c-FLIP, Chang and colleagues showed that at low levels of expression, c-FLIP enhances Fas-induced caspase-8 activation and only at higher levels, similar to those found in tumours can c-FLIP inhibit caspase-8 (Chang et al., 2002; Micheau et al., 2002).

DR4 (TRAIL-R1) and DR5 (TRAIL-R2)

Alternative death receptor pathways exist such as TNF-related apoptosis inducing ligand (TRAIL/Apo2L) and its receptors. Apo2L/TRAIL interacts with an unusually complex receptor system of two DRs and three decoys. The two agonistic receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) contain a conserved death domain motif which enables them to engage the cells apoptotic machinery upon ligand binding (MacFarlane et al., 1997; Pan et al., 1997; Schneider et al., 1997; Sheridan et al., 1997; Walczak et al., 1997). The remaining three receptors act as decoys, DcR1 (TRAIL-R3) and DcR2 (TRAIL-R4) have close homology to DR4 and DR5. However, DcR1 lacks both a transmembrane and a death domain (Pan et al., 1997) and DcR2 has a truncated non-functional death domain (Degli-Esposti et al., 1997), therefore rendering them incapable of transmitting an apoptotic signal. Signalling via TRAIL receptors is very similar to that of CD95, involving receptor trimerisation and recruitment of FADD.

TNF-alpha trimer activation

Tumour necrosis factor-alpha, has two surface receptors which are referred to as TNF-RI (p55/p60) and TNF-RII (p75/p80) (Vandenabeele et al., 1995). Both TNF receptors have significant homology in their extracellular domains; however their cytoplasmic domains are structurally different. The TNF-RI has a death domain, whereas TNF-RII does not. TNF-RI mediates signalling for both survival (via NF-kB) and cell death (Hsu et al., 1996a; Hsu et al., 1996b; Rothe et al., 1995). Apoptotic cell death is mediated by TNF-RI. Upon ligation, TNF-alpha trimerises TNF-RI and induces association of the receptors death domain (DD) and subsequent recruitment of an adapter protein containing a homologous DD, the TNF receptor-associated death-domain (TRADD).

TNF receptor-associated death-domain (TRADD) mediated Cell Death

TRADD is then responsible for the recruitment of several signalling molecules, including FADD, TNF-R-associated factor-2 (TRAF-2) and receptor interactive protein (RIP) (Ashkenazi and Dixit, 1998; Harper et al., 2003; Micheau and Tschopp, 2003). TRAF-2 and RIP stimulate pathways leading to activation of NF-kB, whereas FADD mediates activation of apoptosis. FADD is a common conduit in both CD95-and TNF-RI-mediated apoptosis. Recruitment of TRADD and FADD following ligation of TNFR-I with TNF-alph results in the recruitment and auto catalytic activation of pro-caspase 8 to mature active caspase 8. Although caspase-8 and FADD are critical for TNF alpha-induced apoptosis, whether they are actually recruited to the TNFR-1 signalling complex is debatable (Harper et al., 2003; Micheau and Tschopp, 2003).

Intrinsic Cell Death

Mitochondrial outer membrane permeabilisation (MOMP)

Pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins mediate apoptosis by regulating mitochondrial outer membrane permeabilisation (MOMP) (Green et al., 2004). MOMP is the point of no return where cells become irreversibly committed to cell death (Tait et al., 2010), and causes release of apoptogenic proteins such as cytochrome C, endoG, Smac/DIABLO, AIF-1, and Omi/HtrA2 from the intermembrane space into the cytoplasm. Cytochrome C release into the cytoplasmic compartment leads to its interaction with Apaf-1, allowing the cleavage of procaspase-9, the formation of the Apaf-1/caspase-9 apoptosome complex, and the subsequent activation of effector caspases (Li et al., 1997; Zou et al., 1999). The fate of the cell is ultimately thought to be determined by the relative abundance and activity of the opposing Bcl-2 proteins.

Figure 2 (Video): Animation of programmed cell death activation pathways through mitochondrial outer membrane permeabilization (MOMP) and extrinsic cell death pathways. Video on Youtube. Link: https://www.youtube.com/watch?v=SyvOPXeg4ig

Bcl-2 family proteins and cell death regulation

Bcl-2 family proteins are key regulators of apoptosis. All family members share homology with the archetypal member of the family, B-cell lymphoma-2 protein (Bcl-2), which contains four Bcl-2 homology domains classified BH 1-4. Bcl-2 family proteins can be functionally subdivided into pro-apoptotic and anti-apoptotic members. Pro-apoptotic Bcl-2 proteins may be further subdivided into 2 groups based on structure and function, as either BH3-only or multidomain members.

BH3-only proteins and structure

BH3-only members are simpler in structure, possessing only a catalytic domain, a BH3 domain, and in some cases a transmembrane region, but are otherwise unstructured. The primary function of the BH3-only proteins is the activation of the multidomain family members Bax and Bak. In addition multidomain members all possess BH domains 1-3 as well as a transmembrane region.

Bax & Bak - MOMP regulating proteins

Bax and Bak function by forming homo- or hetero-dimeric pores in the outer mitochondrial membrane (MOMP) and catalysing the release of apoptogenic factors into the cytosol. Recent reports indicate that Bid, Bim, and PUMA are the primary activators of Bax/Bak dimerisation, with the other BH3-only proteins acting in a facilitatory fashion (Kim et al., 2006). Anti-apoptotic Bcl-2 members all possess BH domains 1-4, and all except Bcl-2A1 have a transmembrane region. Anti-apoptotic members act to prevent the formation of Bax/Bak dimers. The primary function of pro-apoptotic Bcl-2 proteins, therefore, is to form stable heterodimers with, and thus sequester, the anti-apoptotic members of the family, allowing Bax/Bak dimerisation and subsequent MOMP formation.

Two contrasting mechanisms for MOMP formation have been proposed. In the indirect model, all pro-apoptotic BH3-only proteins act only by interaction with anti-apoptotic members (Willis et al., 2005; Willis et al., 2007). In the direct model, BH3-only proteins are further subdivided into sensitisers (Bad, Bmf, Noxa) which antagonise the anti-apoptotic members, and activators (Bim, Puma, tBid) which directly promote Bak/Bax dimerisation (Galonek et al., 2006; Hyungjin et al., 2006).

Assays to measure programmed cell death

19th Jan 2020 Seán Mac Fhearraigh PhD

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