Apoptosis: mitochondrial and death receptor pathways
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Neuronal degeneration through inappropriate activation of apoptotic cell-death pathways explained. Mechanisms and key protein players.
By C. Hooper and R. Killick., Department of Neuroscience - Institute of Psychiatry, Kings College London
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Contents
Apoptosis
Apoptosis or programmed cell death (PCD) plays a pivotal role in development, cancer, normal aging and in neurological disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s disease (Thompson 1995). A common feature of many neurological diseases is the degeneration of neuronal cells. It is widely accepted that neuronal loss in such diseases occurs by the inappropriate activation of apoptotic cell-death pathways. Apoptosis is induced via two main routes involving either the mitochondria (the intrinsic pathway) or the activation of death receptors (the extrinsic pathway). Both pathways converge to induce the activation of caspases the final executioners of cell death, although, it should be noted that caspase-independent forms of apoptosis have been reported (Leist and Jaattela 2001). Ultimately, apoptotic cells are ingested by neighboring cells and phagocytes, preventing inflammation and tissue damage that might ensue upon cell lysis. The presence of the phospholipid phosphatidylserine (PS) on the outer leaflet of the plasma membrane acts as a signal for removal (Schlegel and Williamson 2001). Normally, cells maintain asymmetry of the inner and outer leaflets of the plasma membrane by actively translocating PS to the inner leaflet.
To date, at least 14 different caspases have been identified, which play distinct roles in apoptosis and inflammation (Wolf and Green 1999; Philchenkov 2004). Caspases are aspartate-specific cysteine proteases that are expressed as pro-enzymes containing three domains, including an NH2 terminal, a large subunit (~20 kDa) and a small subunit (~10 kDa). Caspase activation involves the proteolytic processing between domains allowing the association of the large and the small subunit. Active caspases function as a tetramer consisting of two heterodimers made up of a large and small subunit. A substantial body of evidence supports a cascade model for caspase activation. Initiator caspases such as caspase 2, 8, 9 and 10 instigate the apoptotic cascade and lead to the activation of effector caspases, which include caspase 3, 6 and 7. Caspases cause cell death by cleaving a number of cellular proteins including nuclear lamins (Lazebnik et al.,1995), DNA repair enzymes such as poly-ADP-ribose-polymerase (PARP) (Lazebnik et al., 1994), and cytoskeletal proteins such as actin (Mashima 1995), fodrin (Cryns et al., 1996) and gelsolin (Kothakota et al.,1997). The fragmentation of DNA during apoptosis is caused in part by an enzyme known as caspase-activated DNase (CAD). Normally CAD exists as an inactive complex with the inhibitor of CAD (ICAD). During apoptosis, ICAD is cleaved by caspase 3 resulting in the release of CAD, which in turn triggers the rapid fragmentation of DNA (Sakahira et al., 1998). Caspase activity is tightly regulated by a number of endogenous caspase inhibitors such as members of the inhibitor of apoptosis protein (IAP) family, which are characterized by the presence of at least one Baculoviral IAP repeat (BIR) domain. IAPs include c-IAP1, c-IAP2, NAIP, Survivin, X-linked IAP (XIAP), Bruce, ILP-2, and Livin (Liston et al, 2003; Nachmias et al., 2004).
Mitochondrial mediated apoptosis
DNA damage, ischemia and oxidative stress are all examples of apoptotic signals that lead to cell death through the mitochondria. The mitochondrial pathway of apoptosis begins with the permeabilization of the mitochondrial outer membrane. The mechanisms through which this occurs remain controversial, however, it is thought that permeabilization can be either permeability transition (PT) pore dependent or independent (Green and Kroemer 2004). The PT pore is comprised of the matrix protein cyclophilin D, the inner mitochondrial membrane protein adenine nucleotide translocator (ANT), and the outer mitochondrial membrane protein voltage-dependent anion channel (VDAC) (Crompton et al., 1998). The opening of the PT pore triggers the dissipation of the proton gradient created by electron transport, causing the uncoupling of oxidative phosphorylation. The opening of the PT pore also causes water to enter the mitochondrial matrix, which results in swelling of the intermembranal space and rupturing of the outer membrane causing the release of apoptogenic proteins (Crompton 1999; Green and Kroemer 2004). Released proteins include cytochrome c (Yang and Cortopassi 1998), apoptosis-inducing factor (AIF) (Susin et al., 1999) and endonuclease G (Li et al., 2001; van Loo et al., 2001). Cytochrome c in conjunction with apoptosis protease activating factor (APAF-1) and pro-caspase 9 form an ‘apoptosome’ (Zou et al., 1999). This complex promotes the activation of caspase 9, which in turn activates effector caspases that collectively orchestrate the execution of apoptosis. AIF (Susin et al., 1999) and endonuclease G (van Loo et al., 2001) both contribute to DNA fragmentation and subsequent chromosomal condensation, which are hallmark features of apoptosis. Other proteins released upon mitochondrial outer membrane permeabilisation include Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-associated binding protein with low pI) and Omi/HtrA2 (high temperature requirement A2), which antagonize IAPs thereby promoting caspase activation (Du et al., 2000; Suzuki et al., 2001).
PT pore independent mitochondrial membrane permeabilization is regulated by Bcl-2 family members, which are characterized by Bcl-2 homology (BH) domains (Green and Kroemer 2004). To date, four BH domains have been identified (BH1-4). The Bcl-2 family can be subdivided into anti-apoptotic members such as Bcl-2 and Bcl-xL and pro-apoptotic species. Pro-apoptotic members are grouped into two categories based on the expression of BH domains. Multi-domain proteins comprise BH domains 1-3 and include Bax, Bak, and Bok. The other sub-group, the BH3 only proteins consist of Bad, Bik, Bid, Puma, Bim, Bmf and Noxa. The BH3 only proteins activate multi-domain pro-apoptotic species (Wei et al., 2000; Letai et al., 2002) and disrupt the function of anti-apoptotic Bcl-2 family members (Letai et al., 2002). It is thought that multi-domain Bcl-2 family members form channels in the outer mitochondrial membrane through which apoptogenic proteins of the intermembranal space are released (Korsmeyer et al., 2000; Nechushtan et al., 2001; Kuwana et al., 2002).
Induction of p53 by pro-apoptotic stimuli results in apoptosis mediated by the mitochondrial pathway. Normally, p53 is maintained at low levels by murine double minute-2 (MDM2) or the human homolog (HDM2), which inhibits the transcriptional activity of p53 and promotes degradation of p53 via the proteasome (Brooks and Gu 2003). Activation of p53 involves stabilization of the protein by post-translational modifications, which disrupts the interaction between p53 and MDM2. p53 drives the expression of APAF-1 and certain pro-apoptotic Bcl-2 family members (Hofseth et al., 2004) as well as eliciting transcriptional independent death pathways (Caelles et al., 1994). p73 and p63 are recently discovered p53 homologs, which also play a role in apoptosis through the transactivation of certain p53 target genes (Yang et al., 2002). Unlike p53, there are multiple C-terminal splice variants of p73 and p63, as well as N-terminally truncated, DN, isoforms. These alternatively transcribed DN forms lack the transactivation domain (TA) and function as dominant negatives.
Mitochondrial-mediated apoptosis antibodies
Death receptor pathways
Death receptors are cell surface receptors belonging to the tumor necrosis factor (TNF) superfamily, which trigger apoptosis upon ligand binding. The best characterised death receptors are Fas (CD95/Apo1) (Dhein et al., 1995), TNF receptor 1 (p55) (Tartaglia et al., 1993), TRAMP (WSL-1/Apo3/DR3/LARD) (Kitson et al., 1996; Bodmer et al., 1997), TRAIL-R1 (DR4) (Pan et al., 1997) and TRAIL-R2 (DR5/Apo2/KILLER) (MacFarlane et al.,1997). Fas Ligand (CD95 ligand) binds Fas, TNF and lymphotoxin a bind to TNFR1 (Ashkenazi and Dixit 1998), TWEAK (Apo3 ligand) binds to TRAMP (Marsters et al., 1998) and TRAIL (Apo2 ligand) is the ligand for both TRAIL-R1 (Pan et al., 1997) and TRAIL-R2 (Walczak et al., 1998).
Death receptors contain an intracellular death domain (DD), which upon ligand binding associates with an adaptor protein called Fas-associated death domain (FADD) directly or indirectly via TNFR-associated death domain (TRADD) (Ashkenazi and Dixit 1998; Thorburn 2004). FADD also interacts with procaspase-8 to form a complex at the receptor called the death-inducing signaling complex (DISC). Once assembled, DISC induces the activation of caspase-8, which in turn precipitates the activation of downstream effector caspases. The BH3 only protein Bid is cleaved by pro-caspase 8 and translocates to the mitochondria to activate the intrinsic pathway (Luo et al., 1998), thereby linking the two death pathways.
In addition to death receptors, the TNF superfamily comprises decoy receptors (DcR), which inhibit death signaling through the sequestration of ligand. Decoy receptors include DcR1, DcR2 and osteoprotegerin (OPG), which bind to TRAIL and DcR3, which binds Fas ligand (Ashkenazi and Dixit 1999). Death receptor signaling is also regulated by cellular FLICE-like inhibitory protein (c-FLIP) an endogenous inhibitor that interacts with FADD to antagonize apoptosis (Irmler et al., 1997).
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