Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P20 pot

176 S.E. Alway and P.M. Siu slightly with age (i.e., less nuclei/cytoplasm area). With age there is a loss of satel- lite cells or muscle precussor cells (MPCs), which reduces the muscle’s ability to replace nuclei (Brack et al. 2005, 2007; Bruusgaard et al. 2006; Brack and Rando 2007). This results in a somewhat transient increase in the nuclear domain with aging, but the excessive domain size triggers fibre atrophy (Brack et al. 2005) which in turn restores the original nuclear domain size, but also contributes to sar- copenia (Fig. 1). 3 Apoptosis Signaling Pathways in Muscle In single nucleated cell populations, apoptosis functions to destroy and eliminate the entire cell through a cascade of cellular suicide steps. One of the distinctive characteristics of apoptosis is that it allows the execution of cells in the absence of inflammation and therefore it does not disturb neighboring cells. This characteristic of apoptosis permits highly selective dismissal of targeted individual cells among the whole cell population. Apoptosis-induced myonuclear debris removal likely involves the ubiquitin-proteasome pathway, as well as autophagy in many cell Fig. 1 Muscle fibres are illustrated in cross section (a, c, f) or longitudinally (b, d, e, g). Myonuclei in muscle fibres control a fixed cytoplasmic domain (c)). Nuclear are targeted for elimination by apoptosis (red; c, d). Fewer nuclei are unable to maintain the cytoplasmic area (e) and these results in fibre atrophy and ultimately sarcopenia (f and g) 177Nuclear Apoptosis and Sarcopenia types (Yang et al. 2009; Korolchuk et al. 2009) including skeletal muscle (Attaix et al. 2005; Combaret et al. 2009). Literature relating to how the ubiquitin-protea- some and autophagy pathways are associated with apoptosis in muscle is currently scarce and further investigation in this area is warranted. Three primary apoptotic pathways have been identified in mediating cellular signalling transduction leading to the implementation of apoptosis in muscle cells (Fig. 2). These apoptotic pathways include mitochondria-dependent (intrinsic), death receptor-mediated (extrinsic), and endoplasmic reticulum-calcium stress- induced pathways (Li et al. 1998; Gorman et al. 2000; Nakagawa et al. 2000; Phaneuf and Leeuwenburgh 2002; Green and Kroemer 2004; Spierings et al. 2005). These apoptotic pathways are named based on the origin of stimulus and the subcellular site that carries out the signalling events. Various gene products play a role in regulating the process of apoptosis. These proteins include B-cell leukaemia/lymphoma-2 (BCL-2) family proteins, caspases, inhibitors of apoptosis proteins (IAPs), caspase-independent apoptotic factors including apoptosis inducing factor (AIF), endonuclease G (EndoG) and heat requirement A2 protein Fig. 2 Three apoptotic pathways have been identified in sarcopenia. These include the intrinsic (mitochondria pathway) which involves mitochondria dysfunction and increased mitochondria permeability. A series of downstream signalling events results in activation of initiator caspases (e.g., caspase 9) and effector caspases (e.g., caspase 3) and finally apoptosis. The endoplasmic reticulum (ER)-calcium stress pathway activates initiator caspases (e.g., caspase 12) then effector caspases (e.g., caspase 3 or 7). The extrinsic pathway is activated by a ligand (e.g., TNF-a) and activates initiator caspases (e.g., caspase 8) and the effector caspases (e.g., caspase 3) and through this to apoptosis 178 S.E. Alway and P.M. Siu (HtrA2/Omi), and other apoptosis-related proteins like cytochrome c, apoptosis protease activating factor-1 (Apaf-1), apoptosis repressor with caspase recruitment domain (ARC), Smac/DIABLO, p53, heat shock proteins (HSPs) and others. The participation of these apoptotic factors are selective in nature and are largely dependent on the apoptotic pathway being invoked. For example, initiator cas- pases-8, -9, and -12 are activated when cells are exposed to an appropriate stress stimulus. When apoptosis is stimulated by TNF-a and FasL which subsequently activate the death receptor apoptotic pathway, caspase 8 is the initiator caspase being triggered and responsible for the mediation of the corresponding subsequent signalling transduction (Li et al. 1998; Sun et al. 1999). Smac/Diablo is also thought to mediate the pro-apoptotic function of TNF-a- regulated PUMA (Yu et al. 2007). Apoptotic signalling initiated by intracellular calcium disturbance and endoplasmic reticulum stress is attributed to initial activation of caspase-12 (Nakagawa et al. 2000; Nakagawa and Yuan 2000) whereas caspase 9 mediates the mitochondria-dependent apoptosis through the interaction of procaspase 9 with Apaf-1, dATP/ATP, and cytochrome c. Although different initiator caspases (cas- pase 8, -12, and -9) are responsible for the initial signalling transduction in differ- ent apoptotic pathways, the signals eventually converge on the activation of common effector caspases-3, -6, or -7, which function to progress to the final dismissal of the target cell. 4 Intrinsic Apoptotic Pathway 4.1 Role of Mitochondria in the Intrinsic Apoptosis Pathway in Muscle An accumulating body of evidence suggests that disruptions in mitochondrial function precedes the initiation of the intrinsic apoptotic pathway in sarcopenia of aging (Siu et al. 2005b; Pistilli et al. 2006b; Chabi et al. 2008; Seo et al. 2008) as well as disuse-associated muscle atrophy (Siu and Alway 2005b; Adhihetty et al. 2007b). Mitochondria play a critical role in maintaining cellular integrity through the regulation of apoptosis (Fig. 3). When mitochondria localized proteins are released to the cytosol, they can initiate a cascade of proteolytic events that converge on the nucleus leading to the fragmentation of DNA and elimination of the nucleus. This compromises muscle cell viability and ultimately leads to cell death (Bernardi 1999) in non-muscle cells. The release of these apoptotic proteins, include cyto- chrome c, endonuclease G (EndoG), Smac/Diablo and apoptosis-inducing factor (AIF), through either the mitochondrial permeability transition pore (mtPTP) (Kroemer and Reed 2000; Precht et al. 2005; Forte and Bernardi 2006; Rasola and Bernardi 2007; Knudson and Brown 2008) or the homooligomeric Bax mitochondria apoptotic channels (MAC) in the outer mitochondria membrane, occurs in response 179Nuclear Apoptosis and Sarcopenia to cellular stressors including ROS (Dejean et al. 2006a, b; Martin et al. 2007). Putative components of the MAC channel are Bax and Bak, whereas Bcl2 acts as a negative regulator of this channel (Dejean et al. 2005, 2006a, b). Thus, this intimate connection between mitochondrial function and the viability of skeletal muscle suggests that this organelle likely plays a significant role in the progression of aging and sarcopenia. Indeed, it is evident that in skeletal muscle of aged individuals, the induction of apoptosis is greater when compared to younger subjects. Oxidative stress is greater in muscles of old vs. young adult animals (Ryan et al. 2008) and may have a role in regulating increased apoptotic signalling (Siu et al. 2008). Furthermore, increased oxidative stress may increase peroxida- tion of the mitochondrial lipid cardiolipin, Bax mobilization and release of cyto- chrome c (Huang et al. 2008). The increase in cytochrome c and EndoG release from the mitochondria of aged individuals (Tamilselvan et al. 2007) is paralleled by an increase in cleavage and activation of caspase 3 (Alway et al. 2003b; Siu et al. 2005b; Chabi et al. 2008), and p53 mediated apoptosis (Tamilselvan et al. 2007). A consequence of apoptosis is a loss in myonuclear number, resulting in a Oxidative stress BcI-2 Apaf1 AIF XIAP APOPTOSIS Endo G Smac Cytochrome c Caspase 3 Caspase 9 Procaspase 9 B A X B A X B A X B A X BcI-2 Mitochondria Intrinsic (Mitochondria) Pathway in Sarcopenia Fig. 3 The intrinsic (mitochondria) pathway is activated in sarcopenia. Pro-apoptotic factors (e.g., Bax) heterodimerise to form a mitochondria channel which releases caspase dependent (e.g., cytochrome c) or caspase independent (e.g., AIF, Endo G, Smac/Diablo) pro-apoptotic factors and result in DNA fragmentation and nuclear apoptosis in muscle 180 S.E. Alway and P.M. Siu reduction in myofiber diameter to maintain a constant myonuclear domain size (Dirks and Leeuwenburgh 2005; Pistilli et al. 2006b; Wang et al. 2008; Alway and Siu 2008; Pistilli and Alway 2008). This decrease in fibre area results in whole muscle atrophy, especially in fast muscles which have a high percentage of type II myosin heavy chain. This suggests that there is a significant mitochondrial involvement in the progression of sarcopenia. Greater mitochondrial dysfunction is also evident in muscles with higher type II muscle fibre composition, and this may be key to the preferential loss of type II fibres found in the elderly (Conley et al. 2007a). 4.2 Oxidative Stress and Mitochondria The free radical theory of aging first proposed by Harman more than five decades ago (Harman 1956), suggests that mitochondria dysfunction from oxidative dam- age to mitochondria DNA (mtDNA) caused by reactive oxygen species (ROS) is a central factor contributing to aging (Harman 1992, 2003, 2006, 2009; Malinska et al. 2009; Kadenbach et al. 2009). The mitochondrion is the main cellular site for ROS; however, it is not the only site for ROS production. Nevertheless, it is reasonable to expect that mitochon- drial components will be susceptible to oxidative damage. In particular, mtDNA in muscle is particularly susceptible to oxidative damage (Hagen et al. 2004; Murray et al. 2007; Ricci et al. 2007; Meissner 2007; Meissner et al. 2008) due to its proximity to the electron transport chain (ETC), the lack of protective histones and an inefficient repair system compared to nuclear DNA (Wei and Lee 2002; Lee and Wei 2007; Ma et al. 2009). Mutations in mtDNA can lead to the synthesis of defective respiratory chain elements, which may impair oxidative phosphory- lation, increase ROS production or decrease ATP availability (Harman 2006; Malinska et al. 2009; Kadenbach et al. 2009; Ma et al. 2009). Several lines of evidence support the idea that mtDNA damage and mutations contribute to aging in muscle (reviewed in (Dirks et al. 2006; Dirks Naylor and Leeuwenburgh 2008; Marzetti et al. 2009b). For example, mice expressing a mutated mtDNA poly- merase accumulate mtDNA mutations and display a premature aging phenotype, which includes extensive sarcopenia, compared to wild-type littermates (Kujoth et al. 2005, 2006). Oxidative stress is greater in muscles of old vs. young adult animals (Ryan et al. 2008) and may have a role in regulating increased apoptotic signalling (Siu et al. 2008). Furthermore, increased oxidative stress may function to elevate per- oxidation of the mitochondrial lipid cardiolipin, as well as Bax mobilization and release of cytochrome c (Huang et al. 2008). The increase in cytochrome c and EndoG release from the mitochondria of aged individuals (Tamilselvan et al. 2007) is paralleled by an increase in cleavage and activation of caspase 3 (Alway et al. 2003b; Siu et al. 2005b; Chabi et al. 2008), and p53 mediated apoptosis (Tamilselvan et al. 2007). 181Nuclear Apoptosis and Sarcopenia 4.3 BCL2 Protein Family The BCL-2 family serves as an important upstream intracellular checkpoint which plays a crucial role in the coordination of the apoptotic signalling (Danial and Korsmeyer 2004). BCL-2 family members share homology within four conserved sequence motifs which are: BH1, BH2, BH3, and BH4 family proteins. In general, the BCL-2 family consists of three subclasses: (a) anti-apoptotic proteins (e.g., Bcl-2, Bcl-XL, Bcl-W, A1, and Mcl-1), (b) multidomain pro-apoptotic proteins (Bax, Bak, and Bok), and (c) BH3-only pro-apoptotic proteins (Bid, Bad, Bim, Bik, Dp5/Hrk, Noxa, and Puma) (Chao and Korsmeyer 1998; Mikhailov et al. 2001, 2003; Danial and Korsmeyer 2004). All pro-apoptotic members and most anti-apoptotic members con- tain the BH3 domain and this domain is believed to be essential for the interactions among the family members (Korsmeyer 1995; Chao and Korsmeyer 1998; Korsmeyer 1999). The BH3 sequence motif has a hydrophobic a-helix which is favourable for protein interaction, and this is the putative region responsible for the association among the BCL-2 family members through homo- or hetero- oligomerisation (Chao and Korsmeyer 1998; Mikhailov et al. 2001, 2003; Er et al. 2007). The strict control which balances cell survival and apoptotic cell death is believed to be primarily regu- lated by the relative ratio of pro- and anti-apoptotic BCL-2 members (Chao et al. 1995; Korsmeyer et al. 1995; Chao and Korsmeyer 1998; Danial and Korsmeyer 2004). Among the BCL-2 family members, pro-apoptotic Bax and anti-apoptotic Bcl-2 have been well-studied. These proteins are thought to constitute the main components in the regulation of mitochondria apoptotic channels or pores. Essentially, Bcl-2 forms a homodimer with Bax and prevents its translocation to the mitochondria in non-apop- totic conditions. However, an apoptotic stimulus translocates Bax to mitochondria and phosphorylates it. Bax undergoes conformational change to expose its N-terminus (Hsu et al. 1997; Wolter et al. 1997; Basanez et al. 1999; Desagher and Martinou 2000; Cartron et al. 2002) to allow the Bax–Bax-oligomerisation and insertion of Bax into the outer mitochondrial membrane (Zha et al. 1996), which mediates the subsequent release of the apoptogenic factors (e.g., cytochrome, EndoG, AIF etc.) from the mito- chondrial intermembrane space (Narita et al. 1998; Reed et al. 1998; Shimizu et al. 1999; Tsujimoto and Shimizu 2000; Tsujimoto et al. 2006; Kroemer et al. 2007). Bcl-2 functions to prevent the Bax–Bax-oligomerisation and therefore opposes the pro- apoptotic activity of Bax (Yin et al. 1994; Korsmeyer 1995, 1999; Korsmeyer et al. 1995; Reed 1997, 2006; Reed et al. 1998; Antonsson et al. 2000; Kroemer et al. 2007; Lalier et al. 2007). 4.4 Caspase (Cysteine-dependent Aspartic Acid Specific Protease) Dependent Signalling The involvement of the pro-apoptotic role of cysteine-dependent aspartate pro- teases (caspases) has been extensively studied and several members appear to have 182 S.E. Alway and P.M. Siu a critical role in apoptotic signaling transduction (Earnshaw et al. 1999; Chang and Yang 2000; Grutter 2000; Degterev et al. 2003). Although caspase 9 is an exception (Stennicke and Salvesen 1999; Stennicke et al. 1999), other caspases are synthe- sized as inactive zymogens (i.e., procaspases). When procaspases undergo cleavage or oligomerisation-mediated self-/auto-activation by an apoptotic signal, they are converted from their inactive procaspases to the active protease (Earnshaw et al. 1999; Deveraux et al. 1999; Stennicke and Salvesen 1999; Stennicke et al. 1999; Deveraux and Reed 1999; Chang and Yang 2000; Grutter 2000). Caspase 9 is an initiator caspase which has been shown to mediate the signalling of mitochondria- mediated apoptosis. Caspase 9 participates in a protein complex, the apoptosome. The interaction of procaspase 9 with Apaf-1, cytochrome c (which is released from the mitochondria), and ATP/dATP in the cytosol activates caspase 9 which cleaves procaspase 3 and activates it (Chang and Yang 2000; Shiozaki et al. 2002; Acehan et al. 2002; Shi 2002a, b, 2004). Caspase 3 is a common downstream effector (executer) caspase for initiating DNA destruction. Cellular substrates for caspase 3 cleavage include proteins responsible for cell cycle regulation (e.g., p21 Cip1/Waf1 ), apoptotic cell death (e.g., Bcl-2 and IAP), DNA repair (e.g., poly(ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD), cell signal transduction (e.g., Akt/PKB), and cytoskeletal structural scaffold (e.g., gelsolin), etc. (Chang and Yang 2000). 4.5 Caspase-independent Apoptotic Signalling Mitochondria-housed proteins including apoptosis-inducing factor (AIF), endonu- clease G (EndoG) and high temperature requirement protein A2 (HtrA2/Omi) have been shown to be able to induce apoptosis without the involvement of caspases (Joza et al. 2001; Li et al. 2001; Blink et al. 2004). AIF is a mitochondrial flavo- protein that has both oxidoreductase and apoptosis-inducing activities (Joza et al. 2001, 2005; Cande et al. 2002a, b). Although the full physiologic importance of AIF is not yet completely known, it is clear that AIF has an important role in mitochondrial-mediated apoptosis. The apoptotic function of AIF may be the result of a putative DNA binding site which results in chromatin condensation and DNA fragmentation (Lipton and Bossy-Wetzel 2002; Ye et al. 2002). EndoG is an well- conserved nuclear-encoded endonuclease, which can induce chromosomal DNA cleavage in a caspase-independent manner (Li et al. 2001). In contrast, the apop- totic properties of a serine protease HtrA2/Omi are less well defined. It has been thought that HtrA2/Omi induces apoptosis via the mechanism similar to Smac/ DIABLO, in which the apoptosis-suppressing activities of IAPs are removed through a caspase-regulated process (Hegde et al. 2002; Shi 2004; Shiozaki and Shi 2004). However, it has also been shown that the apoptosis-inducing ability of HtrA2/Omi can function via its proteolytic activity in the absence of caspase activa- tion (Blink et al. 2004; Suzuki et al. 2004). These caspase-independent proteins are normally housed in the mitochondrial intermembrane space, but they are released 183Nuclear Apoptosis and Sarcopenia into cytosol once in response to an apoptotic stimulus (Joza et al. 2001; Li et al. 2001; Cande et al. 2002b; Blink et al. 2004). It is known that cytosolic and nuclear levels of AIF and EndoG are elevated in skeletal muscles of old and senescent animals (Leeuwenburgh et al. 2005; Siu and Alway 2006a; Marzetti et al. 2008c). This confirms a central role for apoptosis in sarcopenia, but the extent to which caspase-dependent vs. caspase-independent signalling dominates apoptotic elimination of nuclei has not yet been established. Although a role for HtrA2/Omi has been suggested in response to myocardial injury or heart failure (Siu et al. 2007; Bhuiyan and Fukunaga 2007), it has not been established that HtrA2/Omi is elevated in sarcopenia. 4.6 Mitochondria-associated Apoptotic Suppressors A group of mitochondrially stored endogenous proteins have been shown to func- tion in suppressing pro-apoptotic signaling. Members of this Inhibitor of Apoptosis (IAP) family include X-linked inhibitor of apoptosis (XIAP), apoptosis repressor with caspases recruitment domain protein (ARC), and Fas-associated death domain protein-like interleukin 1a-converting enzyme-like inhibitory protein (FLIP). XIAP is a fundamental conserved gene product among many species (Deveraux et al. 1998; Shi 2002b). The anti-apoptotic ability of XIAP is attributed to the con- served baculovirus inhibitor of apoptosis repeat (BIR) motif which is the essential part for the inhibition on initiator as well as effector caspases and all protein mem- bers in IAP family are found to carry at least one of this BIR motif (Deveraux et al. 1998, 1999; Salvesen and Duckett 2002; Sanna et al. 2002; Chowdhury et al. 2008). ARC and FLIP are two endogenous apoptosis-suppressing proteins with high expression levels in muscle tissue (Irmler et al. 1997; Koseki et al. 1998). It is pos- sible that the high resistance of mature muscle tissues to apoptosis is related to the abundant expressions of these two apoptotic suppressors, although this has not been definitively shown. The apoptotic suppressive effects of ARC and FLIP are thought to be due to their inhibiting interactions with selective caspases, in particular, cas- pase 8 which is the initiator caspase in the death receptor-mediated apoptosis (Irmler et al. 1997; Koseki et al. 1998; Abmayr et al. 2004; Heikaus et al. 2008; Yu et al. 2009b). Additional observations indicate that ARC is able to interact with pro-apoptotic Bax protein and so exhibits the apoptosis suppressive effect by influ- encing the mitochondria-mediated apoptotic signaling (Gustafsson et al. 2004). Regulation of the extrinsic pathway is very complex, with some proteins appear- ing to have dual roles. For example, c-FLIP (L) is widely regarded as an inhibitor of initiator caspase 8 activation and cell death in the extrinsic pathway; however, it is also capable of enhancing procaspase 8 activation through heterodimerisation of their respective protease domains. Cleavage of the inter-subunit linker of c-FLIP(L) by procaspase 8 potentiates the activation process by enhancing heterodimerisation between the two proteins and elevates the proteolytic activity of unprocessed cas- pase-(C)8 (Yu et al. 2009b). FLIP’s role in regulation of apoptosis may be in part 184 S.E. Alway and P.M. Siu related to the individual splice variants (i.e., protein isoforms). For example, FLIP S versus FLIP L or FLIP c . For example, disruption of NF-Kappa B regulation of FLIPc has been implicated in muscle wasting diseases such as Limb-girdle muscular dys- trophy type 2A (Benayoun et al. 2008) although it is not known if similar deregula- tions occur in aging muscles. 4.7 Sarcopenia-associated Mitochondria Mediated Signalling in Apoptosis Sarcopenia is a complex pathology which is not fully understood. Several factors are thought to contribute to sarcopenia including increases in inflammation and oxidative stress, loss of systemically or locally generated growth signals, neural factors and reduced muscle progenitor stem cell function. Not only do post-mitotic myocytes exhibit apoptosis during atrophy induced by denervation and unloading (Allen et al. 1997; Jin et al. 2001; Jejurikar et al. 2002; Alway et al. 2003a, b; Siu and Alway 2005a; Siu et al. 2005c), but apoptosis is thought to have an important role in the aging associated loss of muscle mass or sarcopenia (Dirks and Leeuwenburgh 2002; Pollack et al. 2002; Alway et al. 2002a, b; Leeuwenburgh 2003; Dirks and Leeuwenburgh 2004; Siu et al. 2005c). Evidence for myonuclei undergoing apoptosis via the intrinsic pathway in aging has been shown by increases in TUNEL positive nuclei, increases in the frequency of nuclei with DNA strand breaks and in the expression of pro-apoptotic genes and proteins including Bax, caspase 3, apoptosis-inducing factor (AIF) and apoptotic protease- activating factor (Apaf1) in aged and atrophied muscles in mammals and non-mammals including birds, worms and flies (Alway et al. 2002a, b; Senoo-Matsuda et al. 2003; Siu et al. 2004, 2005c; Zheng et al. 2005; Siu and Alway 2005a, 2006a, b; Dirks and Leeuwenburgh 2006; Pistilli et al. 2006b; li-Youcef et al. 2007; Dirks Naylor and Leeuwenburgh 2008). 5 Extrinsic Apoptotic Signalling in Skeletal Muscle One potential mechanism contributing to the onset of sarcopenia may be the increase in circulating cytokines which activates the extrinsic apoptotic pathway. The circulating concentrations of specific cytokines have been shown to be elevated in the serum as a result of aging. In humans, serum levels of catabolic cytokines, such as TNF-a (Sandmand et al. 2003; Schaap et al. 2009) and IL-6 (Bruunsgaard 2002; Forsey et al. 2003; Pedersen et al. 2003; Schaap et al. 2009), are increased in healthy elderly compared to young adults. Serum concentrations of TNF-a have been proposed as a prognostic marker of all cause-mortality in centenarians (Bruunsgaard et al. 2003b) and with age-associated pathology and mortality in 80-year old adults (Bruunsgaard et al. 2003a). 185Nuclear Apoptosis and Sarcopenia 5.1 Tumour Necrosis Factor-a (TNF-a) and Death Receptor Signalling Several studies have also drawn associations between the increases in circulating cytokines and the sarcopenic process (Visser et al. 2002; Pedersen et al. 2003; Schaap et al. 2006, 2009). Specifically, elevated circulating levels of TNF-a are associated with lower appendicular skeletal muscle mass (Pedersen et al. 2003) and reduced knee extensor and grip strength (Visser et al. 2002). Tumour necrosis factor-a (TNF-a) is a pleiotropic cytokine that has an impor- tant role in many different physiological and pathological processes including immune and inflammatory responses (Wajant et al. 2003; Wajant 2009). TNF-a- induced apoptosis is mediated by its interactions with cell-surface receptors such as extrinsic signalling through TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) (Wajant et al. 2003; Wajant 2009). The extrinsic death ligand associated apoptotic pathway in sarcopenia is thought to be activated by ligands such as TNF-a. Ligand binding induces trimerisation of death receptors, activation of caspase 8 and subse- quently executioner caspases, such as caspase 3 (Ricci et al. 2007). The contribu- tion of the extrinsic apoptotic pathway to skeletal muscle mass losses, especially during aging, has been less well studied than the intrinsic pathway (Phillips and Leeuwenburgh 2005). However, activation of this pathway does appear to play a role in aging associated muscle loss (Fig. 4). The increase in circulating concentrations TNF-a in aged animals may initiate pro-apoptotic signalling upon binding to the type I TNF receptor (TNFR). Upon binding, a death inducing signalling complex (DISC) is formed at the cytoplasmic portion of the TNFR, composed of adaptor proteins such as Fas associated death domain protein (FADD), TNFR associated death domain protein (TRADD) and procaspase 8 (reviewed in Sprick and Walczak (2004)). Formation of the DISC stimulates cleavage of procaspase 8 into the functional initiator caspase 8. Once cleaved, caspase 8 stimulates cleavage and activation of the executioner caspase 3, which is directly linked to pro-apoptotic changes. Thus, this pathway represents an extrinsic pathway of apoptosis activated by binding of a ligand (TNF-a) to a cell surface death receptor (type-I TNFR). Nuclear factor-kB (NF-kB) is the best-known mediator of TNF-a-associated cellular responses. NF-kB is a group of dimeric transcription factors which are members of the NF-kB/Rel family, including p50, p52, p65 (Rel-A), Rel-B, and c-Rel (Shih et al. 2009; Kearns and Hoffmann 2009). The activity of NF-kB is normally regulated by the IkB family of inhibitors, which bind to and sequester NF-kB in the cytoplasm (Shih et al. 2009). Activation of NF-kB is triggered by IkB phosphorylation by IKK kinases and subsequent proteasomal degradation, which allows NF-kB to translocate to the nucleus, where it binds to the kB consensus sequences and modulates specific target genes (Kearns and Hoffmann 2009; Vallabhapurapu and Karin 2009). NF-kB is thought to provide a protective role in TNF-a-induced apoptosis. This is because NF-kB is a transcriptional activator of anti-apoptotic proteins including c-FLIP, Bcl-2 and Bcl-XL (Vallabhapurapu and . area (e) and these results in fibre atrophy and ultimately sarcopenia (f and g) 177Nuclear Apoptosis and Sarcopenia types (Yang et al. 2009; Korolchuk et al. 2009) including skeletal muscle (Attaix. ligand (e.g., TNF-a) and activates initiator caspases (e.g., caspase 8) and the effector caspases (e.g., caspase 3) and through this to apoptosis 178 S.E. Alway and P.M. Siu (HtrA2/Omi), and. factors and result in DNA fragmentation and nuclear apoptosis in muscle 180 S.E. Alway and P.M. Siu reduction in myofiber diameter to maintain a constant myonuclear domain size (Dirks and Leeuwenburgh