PMC:3023298 / 34805-57440 JSONTXT

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and Molecular Regulation of Apoptosis\nApoptosis is a structurally and biochemically organized form of cell death (Figure 2). Apoptotic molecular networks are conserved in yeast, hydra, nematode, fruit fly, zebra fish, mouse, and human [120]. The current understanding of the molecular mechanisms of apoptosis in cells is built on studies by Robert Horvitz and colleagues on PCD in a nematode Caenorhabditis elegans [121]. They pioneered the understanding of the genetic control of developmental cell death by showing that it is regulated predominantly by three genes (ced-3, ced-4, and ced-9) [121]. This seminal work led to the identification of several families of apoptosis-regulation genes (Table 2) in mammals, including the Bcl-2 family [37,38,122] and the caspase family of cysteine-containing, aspartate-specific proteases [123]. Other regulators of apoptotic cell death, most of which are mitochondrial proteins or influence mitochondria, are the p53 gene family, cell surface death receptors, cytochrome c, apoptosis inducing factor (AIF), second mitochondrial activator of caspases (Smac), the inhibitor of apoptosis protein (IAP) family, and HtrA2/Omi [100,124,125,126,127,128,129].\nSpecific organelles, including mitochondria and the ER, have been identified as critical for the apoptotic process. In seminal work by Li, Wang, and colleagues, it was discovered that the mitochondrion integrates death signals engaged by proteins in the Bcl-2 family and releases molecules residing in the mitochondrial intermembrane space, such as cytochrome c, that complexes with cytoplasmic proteins (e.g., apoptotic protease activating factor 1, Apaf1) to activate caspase proteases leading to internucleosomal cleavage of DNA (Figure 1 and Figure 2) [125,126]. The ER, which regulates intracellular Ca2+ levels, participates in a loop with mitochondria to modulate mPT and cytochrome c release through the actions of Bcl-2 protein family members (Figure 1) [130].\nTable 2 Some Molecular Regulators of Apoptosis Relevant to Neurodegeneration and Potential Drug Targeting for Neuroprotection.\nBcl-2 Family Caspase Family IAP Family Tumor Suppressor\nAnti-apoptotic proteins Pro-apoptotic proteins\nBcl-2 Bax Apoptosis “initiators”: caspase-2, 8, 9, 10 NAIP p53\nBcl-xL Bak1 Apollon p63\nMcl-1 Bcl-xS Survivin p73\nBoo/Diva Bad Apoptosis “executioners”: caspase-2, 3, 6, 7 IAP1 　\n　 Bid IAP2 　\n　 Bik XIAP 　\n　 Bim Cytokine processors: caspase-1, 4, 5, 11, 12, 14 　 　\n　 Noxa 　 　\n　 Puma 　 　\n\nBcl-2 family of Cell Survival and Cell Death Proteins\nMitochondria can control cell death (Figure 1) using Bcl-2 family members to regulate apoptosis by modulating the release of cytochrome c from mitochondria into the cytosol. Two models can account for this process: the Bcl-2-associated X protein (Bax)/Bak1 channel model and the mitochondrial apoptosis-induced channel or MAC) (Figure 1). The bcl-2 proto-oncogene family is a large group of apoptosis regulatory genes encoding about 20 different proteins. These proteins are defined by at least one of four conserved B-cell lymphoma (Bcl) homology domains (BH1-BH4) in their amino acid sequence that function in protein-protein interactions [37,38,122]. Some of the proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1) have all four BH1-BH4 domains and are anti-apoptotic (Table 2). Other proteins that are pro-apoptotic have BH1-BH3 sequences (e.g., Bax and Bak1) or only the BH3 domain (e.g., Bad, Bid, Bim, Bik, Noxa, and Puma) that contains the critical death domain (Table 2). Bcl-xL and Bax have α-helices resembling the pore-forming subunit of diphtheria toxin [131]; thus, Bcl-2 family members appear to function by conformation-induced insertion into the outer mitochondrial membrane to form channels or pores that can regulate release apoptogenic factors (Figure 1). Bcl-2 family members can form homodimers or heterodimers and higher-order multimers with other family members. Bax/Bak1 heterodimerization with either Bcl-2 or Bcl-xL neutralizes their pro-apoptotic activity. When Bax and Bak1 are present in excess, the anti-apoptotic activity of Bcl-2 and Bcl-xL is antagonized, and apoptosis is promoted.\nThe expression of many of these proteins is regulated developmentally, and they have differential tissue distributions and subcellular localizations. Most of these proteins are found in CNS. The subcellular distributions of Bax, Bak1, and Bad in healthy adult rodent CNS tissue [132] are consistent with what is known about these proteins in cultured mammalian non-neuronal cells [133,134]. Bax, Bad, and Bcl-2 reside primarily in the cytosol, whereas Bak1 resides primarily in mitochondria.\nRelease of cytochrome c from mitochondria (Figure 1) can occur through mechanisms that involve the formation of membrane channels comprised of Bax or Bak1 [135] and Bax and the VDAC [136]. In the Bax/Bak1 channel model (Figure 1, left), after specific cell death inducing stimuli Bax undergoes a conformation shift and translocates to the OMM where it inserts. Bak1 is a similar pro-apoptotic protein localized mostly to the OMM. Bax/Bak1 monomers physically interact to form oligomeric or heteromeric channels that are permeable to cytochrome c. The formation of these channels is blocked by Bcl-2 and Bcl-xL at multiple sites. BH3-only members (Bad, Bid, Noxa, Puma) are pro-apoptotic and can modulate the conformation of Bax/Bak1 to sensitize this channel, possibly by exposing its membrane insertion domain. The MAC could be a channel similar to the Bax/Bak1 channel, but it might also have additional components such as VDAC.\nReleased cytochrome c then triggers the assembly of the cytoplasmic apoptosome. The apoptosime is a protein complex of apoptotic protease activating factor 1 (Apaf1), cytochrome c, and procaspase-9; this is the engine that drives caspase-3 activation in mammalian cells (Figure 1) [125]. Bcl-2 and Bcl-xL block the release of cytochrome c [137,138] from mitochondria and thus the activation of caspase-3 (Figure 1) [125,126]. This Bcl-2 and Bcl-xL mediated retention of mitochondrial cytochrome c [126,139] is caused by inhibition of Bax channel-forming activity in the outer mitochondrial membrane [135] or by modulation mitochondrial membrane potential and volume homeostasis [139]. BH3-only proteins such as Bim, Bid, Puma, and Noxa appear to induce a conformational change in Bax or they serve as decoys for Bcl-xL that allow Bax to form pores in the outer mitochondrial membrane [140]. Cells without bax and bak genes are resistant to mitochondrial cytochrome c release during apoptosis [141].\nSome anti-apoptotic proteins also have functions downstream of mitochondria. For, example Bcl-xL has anti-apoptotic activity by interacting with Apaf1 and caspase-9 and inhibiting Apaf1-mediated autocatalytic maturation of caspase-9 [142]. Boo can inhibit Bak- and Bik-induced apoptosis (but not Bax-induced cell death) possibly through heterodimerization and by interactions with Apaf1 and caspase-9 [143].\nProtein phosphorylation regulates the functions of some Bcl-2 family members having downstream mitochondrial consequences. Bcl-2 loses its anti-apoptotic activity following serine phosphorylation, possibly because its antioxidant function is inactivated [144]. Bcl-2 can also associate with non-homologous proteins, including the protein kinase Raf-1 [145]. This association can target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate Bad at serine residues [145]. The phosphatidylinositol 3-kinase (PI3-K) –Akt pathway also regulates the function of Bad [146,147] and caspase-9 [148] through phosphorylation. In the presence of sufficient trophic factors, Bad is phosphorylated. Phosphorylated Bad is sequestered in the cytosol by interacting with soluble protein 14-3-3 and, when bound to protein 14-3-3, Bad is unable to interact with Bcl-2 and Bcl-xL, thereby promoting cell survival [149]. Conversely, when Bad is dephosphorylated by calcineurin [150], it dissociates from protein 14-3-3 in the cytosol and translocates to the mitochondria where it exerts pro-apoptotic activity. Non-phosphorylated Bad heterodimerizes with membrane-associated Bcl-2 or Bcl-xL to liberate Bax from Bax-Bcl-2 and Bax-Bcl-XL dimers, thus promoting cell death [109]. In liver mitochondria, Bad and glucokinase exist in a complex that functions in mitochondrial-based glucokinase activity and mitochondrial respiration in response to glucose [152]. Glucose deprivation results Bad dephosphorylation and Bad-dependent cell death, thereby linking glucose metabolism to apoptosis [152].\n\nCaspase Family of Cell Demolition Proteases\nCaspases (cysteinyl aspartate-specific proteinases) are cysteine proteases that have a near absolute substrate requirement for aspartate in the P1 position of the peptide bond. Fourteen caspase genes have been identified in mammals [153]. Some caspases (e.g., caspase-12) in human and mouse function differently and have different contributions to cell death mechanisms. Caspases exist as constitutively expressed inactive pro-enzymes (30–50 kDa) in healthy cells. Caspase zymogens are found in different proportions at different subcellular locations. In HeLa cells, most caspase-3 pro-enzyme is found in the cytoplasm, while only 10% is found in mitochondria [154]. In rat heart and brain, 90% of caspase-9 pro-enzyme is mitochondrial [155]. The zymogens contain 3 domains: an amino-terminal pro-domain; a large subunit (~20 kDa); and a small subunit (~10 kDa). Caspases are activated through regulated proteolysis of pro-enzyme with “initiator” caspases activating “executioner” caspases (Table 1; Figure 1). Other caspase family members function in inflammation by processing cytokines (Table 1) [153].\nThe pro-domain of initiator caspases contains amino acid sequences that are caspase recruitment domains (CARD) or death effector domains (DED) that enable the caspases to interact with other molecules that regulate their activation. Activation of caspases involves proteolytic processing between domains, and then association of large and small subunits to form a heterodimer with both subunits contributing to the catalytic site. Two heterodimers associate to form a tetramer that has 2 catalytic sites that function independently. Some isoforms of caspases (e.g., caspase-9, isoform 2) are inactive proteolytically and function as dominant negative inhibitors of active forms.\nActive caspases have many target proteins [112] that are cleaved during regulated and organized cell death. Caspases cleave nuclear proteins (e.g., DNases, poly(ADP) ribose polymerase, DNA-dependent protein kinase, heteronuclear ribonucleoproteins, transcription factors or lamins), cytoskeletal proteins (e.g., actin and fodrin), and cytosolic proteins (e.g., other caspases, protein kinases, Bid).\nIn human cell line models of apoptosis (Figure 1), activation of caspase-3 occurs when caspase-9 pro-enzyme (also known as Apaf3) is bound by Apaf1 that then oligomerizes in a process initiated by cytochrome c (identified as Apaf2) and either ATP or dATP [125]. Cytosolic ATP or dATP are required cofactors for cytochrome c-induced caspase activation [125]. Apaf1, a 130 kDa cytoplasmic protein, serves as a docking protein for procaspase-9 and cytochrome c [125]. Apaf1 becomes activated when ATP is bound and hydrolyzed, with the hydrolysis of ATP and the binding of cytochrome c promoting Apaf1 oligomerization [113]. This oligomeric complex recruits procaspase-9 (forming the apoptosome) and mediates the autocatalytic activation of caspase-9 that disassociates from the complex and becomes available to activate caspase-3 (Figure 1). Once activated, caspase-3 cleaves a protein with DNase activity (i.e., DFF-45), and this cleavage activates a process leading to the internucleosomal fragmentation of genomic DNA (Figure 2, top left) [103].\nSo far three caspase-related signaling pathways have been identified that can lead to apoptosis [103,125,126,157], but crosstalk among these pathways is possible. The intrinsic mitochondria-mediated pathway is controlled by Bcl-2 family proteins. It is regulated by cytochrome c release from mitochondria, promoting the activation of caspase-9 through Apaf1 and then caspase-3 activation. The extrinsic death receptor pathway involves the activation of cell-surface death receptors (see below), including Fas and tumor necrosis factor receptor, leading to the formation of the death-inducing signaling complex (DISC) and caspase-8 activation that in turn cleaves and activates downstream caspases such as caspase-3, -6, and -7. Caspase-8 can also cleave Bid leading to the translocation, oligomerization, and insertion of Bax or Bak1 into the mitochondrial membrane. Another pathway involves the activation of caspase-2 by DNA damage or ER stress as a pre-mitochondrial signal [158].\n\nInhibitor of Apoptosis Protein (IAP) Family\nThe activity of pro-apoptotic proteins is blocked to prevent untimely apoptosis in normal cells. Apoptosis can be antagonized by the IAP family in mammalian cells [159,160,161]. This family includes X chromosome-linked IAP (XIAP), IAP1, IAP2, neuronal apoptosis inhibitory protein (NAIP), Survivin, Livin, and Apollon. These proteins are characterized by 1 to 3 baculoviral IAP repeat domains consisting of a zinc finger domain of ~70–80 amino acids [160]. Apollon is a huge (530 kDa) protein that also has a ubiquitin-conjugating enzyme domain. The main identified anti-apoptotic function of IAPs is the suppression of caspase activity [161]. Procaspase-9 and procaspase-3 are major targets of several IAPs. IAPs reversibly interact directly with caspases to block substrate cleavage. Apollon also ubiquitinates and facilitates proteasomal degradation of active caspase-9 and second mitochondria-derived activator of caspases (Smac) [162]. However, IAPs do not prevent caspase-8-induced proteolytic activation of procaspase-3. IAPs can also block apoptosis by reciprocal interactions with the nuclear transcription factor NFκB [160].\nScant information is available on IAPs in the nervous system. Survivin is essential for nervous system development in mouse because conditional deletion of survivin gene in neuronal precursor cells causes reduced brain size and severe multifocal degeneration and death shortly after birth [163]. NAIP is expressed throughout the CNS in neurons [164]. XIAP is enriched highly in mouse spinal motor neurons [165]. The importance of the IAP gene family in pediatric neurodegeneration is underscored by the finding that NAIP is deleted partially in a significant proportion of children with spinal muscular atrophy [166].\nMitochondrial proteins exist that inhibit mammalian IAPs. A murine mitochondrial protein called Smac and its human ortholog DIABLO (for direct IAP-binding protein with low pI) inactivate the anti-apoptotic actions of IAPs and thus exert pro-apoptotic actions [167,168]. Smac/DIABLO are released into the cytosol to inactivate the anti-apoptotic actions of inhibitor of apoptosis proteins that inhibit caspases (Figure 1). These IAP inhibitors are 23 kDa mitochondrial proteins (derived from 29 kDa precursor proteins processed in the mitochondria) that are released into the cytosol from the intermembrane space to sequester IAPs. High temperature requirement protein A2 (HtrA2), also called Omi, is another mitochondrial serine protease that exerts pro-apoptotic activity by inhibiting IAPs [169]. HtrA2/Omi functions as a homotrimeric protein that cleaves IAPs irreversibly, thus facilitating caspase activity. The intrinsic mitochondrial-mediated cell death pathway is regulated by Smac and HtrA2/Omi [169]. Mutations in the htra2 gene, identified as PARK13 (Table 3), have been linked to the development of Parkinson’s disease [170], but this linkage is controversial [171].\n\nApoptosis Inducing Factor (AIF)\nAIF is a mammalian cell mitochondrial protein identified as a flavoprotein oxidoreductase [172]. AIF has an N-terminal mitochondrial localization signal that is cleaved off to generate a mature protein of 57 kDa after import into the inter-mitochondrial membrane space. Under normal physiological conditions AIF might function as a ROS scavenger targeting H2O2 [127] or in redox cycling with nicotinamide adenine dinucleotide phosphate [173]. After some apoptotic stimuli, AIF is released from mitochondria (Figure 1) and translocates to the nucleus [172]. Over-expression of AIF in cultured cells induces cardinal features of apoptosis, including chromatin condensation, high molecular weight DNA fragmentation, and loss of mitochondrial transmembrane potential [172].\n\np53/p63/p73 Family of Tumor Suppressors\nCell death by apoptosis can be triggered by DNA damage. p53 and related DNA binding proteins identified as p73 and p63 are involved in this process [124]. p53, p73 and p63 function in apoptosis as well as growth arrest and repair. They can commit to death cells that have sustained DNA damage from ROS, irradiation, and other genotoxic stresses [124]. p53 and p73 have similar oligomerization and DNA sequence transactivation properties. p73 exists as a group of full-length isoforms (including p73α and p73β) and as truncated isoforms that lack the transactivation domain (∆N-p73). p53 is the most well studied of this family of proteins.\np53 is a short-lived protein with a half-life of ~5-20 min in most types of cells studied but can rapidly accumulate several-fold in response to DNA damage. This rapid regulation is mediated by posttranslational modification such as phosphorylation and acetylation as well as intracellular redox state [174]. The elevation in p53 protein levels occurs through stabilization and prevention of degradation. p53 is degraded rapidly in a ubiquitination-dependent proteasomal pathway [175]. Murine double minute 2 (Mdm2, the human homolog is Hdm2) has a crucial role in this degradation pathway [176]. Mdm2 functions in a feedback loop to limit the duration or magnitude of the p53 response to DNA damage. Expression of the mdm2 gene is controlled by p53 [176]. Mdm2 binds to the N-terminal transcriptional activation domain of p53 and regulates its DNA binding activity and stability by direct association. Mdm2 has ubiquitin ligase activity for p53 through the ubiquitin-conjugating enzyme E2. Stabilization of p53 is achieved through phosphorylation of serine15 resulting in inhibition of formation of Mdm2-p53 complexes. Activated p53 binds the promoters of several genes encoding proteins associated with growth control and cell cycle checkpoints (e.g., p21, growth-arrest and DNA damage-45, Mdm2) and apoptosis (e.g., Bax, Bcl-2, Bcl-xL, and Fas). The BH3-only proteins Puma and Noxa are critical mediators of p53-mediated apoptosis [137].\np53 can mediate cell death through extra-nuclear transcriptional-independent mechanisms. p53 can translocate rapidly to mitochondria in response to genotoxic, hypoxic, and oxidative stresses in non-neuronal cells [178] and in neurons [90]. This localization can mediate mitochondrial membrane permeabilization through direct physical interaction with Bax [179] and activation of Bak through disruption of the Bak-Mcl1 complex [180].\np53 can drive apoptosis in cultured sympathetic ganglion neurons in response to neurotrophin withdrawal [181] and in cultured mouse cortical neurons in response to DNA damage [90]. A small-molecule inhibitor of p53 binding to mitochondria protects against neuronal apoptosis in cultured mouse cortical neurons [90]. p53-mediated neuronal apoptosis in vitro can be blocked by the ∆N-p73 isoform by direct binding and inactivation of p53 [141]. In vivo experiments show that p53 gene ablation protects against neuronal apoptosis induced by axotomy and target deprivation in rodent brain and spinal cord [182,183].\n\nCell Surface Death Receptors\nCell death can also be initiated at the cell membrane by surface death receptors of the tumor necrosis factor (TNF) receptor family. Fas (CD95/Apo-1) and the 75-kDa neurotrophin receptor (p75NTR) are members of the large TNF receptor family [100]. Signals for apoptosis are initiated at the cell surface by aggregation (trimerization) of the death domain containing members of this receptor family by their specific ligand. Fas death receptor-mediated apoptosis is a well described pathway for death receptor signaling and is independent of new RNA or protein synthesis. Activation of Fas is induced by binding of the multivalent Fas ligand (FasL), a member of the TNF-cytokine family. FasL is expressed on activated T cells and natural killer cells. Clustering of Fas on the target cell by FasL recruits Fas-associated death domain (FADD), a cytoplasmic adapter molecule that functions in the activation of the caspase 8-Bid pathway, thus forming the DISC [185]. Signaling for apoptosis then proceeds via the extrinsic or intrinsic pathway. In the extrinsic pathway, active caspase-8 then directly cleaves caspase-3 [100]. Activation of the mitochondrial or intrinsic pathway proceeds via caspase 8 mediated cleavage of cytosolic Bid [185]. The truncated form of Bid then translocates to mitochondria, thereby functioning as a BH3-only transducer of Fas activation signal at the cell plasma membrane to mitochondria [185]. Bid translocation from the cytosol to mitochondrial membranes is associated with a conformational change in Bax (that is prevented by Bcl-2 and Bcl-xL) and is accompanied by release of cytochrome c from mitochondria [186].\nApoptosis can also be mediated by p75NTR [187]. Activation of p75NTR occurs by binding of nerve growth factor. When p75NTR is activated without tropomyosin receptor kinases, neurotrophin binding induces homodimer formation and generation of ceramide through sphingomyelin hydrolysis. Ceramide production is associated with the activation of Jun N-terminal kinase (JNK) that phosphorylates and activates c-Jun and other transcription factors. p75NTR mediates hippocampal neuron death in response to neurotrophin withdrawal, involving cytochrome c, Apaf1, and caspases-9, -6, and -3 (but not caspase-8), and thus is different from Fas-mediated cell death [187].\nEvidence for the importance of these signaling pathways in experimental brain injury is growing. Activation of multiple components of the Fas death receptor signaling pathway have been found in rat and mouse models of motor neuron degeneration [188] and blocking Fas death receptor signaling by genetic means affords protection in these models [188]. Neuron degeneration caused by target deprivation in vivo appears to be driven in part by a death receptor-dependent pathway [18]."}