4 Autophagic Cell Death in CIRI
4.1 Possible Autophagy Signaling Pathways in Cerebral Ischemia
The existence of autophagy in ischemic stroke has been found for many years; how- ever, it is not sure whether autophagy plays a protective role in ischemic cerebral injury or not yet [78, 79]. Generally, in the neuronal system, moderate autophagy is thought to be neuroprotective because autophagy helps to clear aggregated-protein associated with neurodegeneration. Inadequate or defective autophagy may lead to neuronal cell death, while excess autophagy, often triggered by intensive stress, can also promote neuronal cell death.
Almost any signal can be a trigger for autophagy, some activating the pathway and some suppressing the pathway. By far, energy depletion and oxygen deficient environment are the most powerful triggers for stimulating autophagy, while the reverse environment factors, hormones, receptors with cytokine activities, receptors with tyrosine kinase activities and receptors that recognize pathogen ligands can
Fig. 5.2 RIP1 and RIP3 are activated (phosphorylated) and combine with each other after CIRI. AIF is released from mitochondria and combines with RIP3 (perhaps phosphorylated RIP3) to form RIP3-AIF complexes. The RIP3-AIF complexes translocate into the nucleus resulting in chromatin condensation and DNA degradation, and then the neurons are triggered to undergo programmed necrosis. All of these changes after I/R injury are inhibited by pre-treatment with Nec-1 and 3-MA, except for the release of AIF from mitochondria in the 3-MA pre-treatment group. In neurons, the findings that caspase-8 expression was undetectable and caspase-3 was not activated indicate that caspase-dependent apoptosis is not involved in this process. Another necrop- tosis pathway in the cytoplasm induced by RIP1-RIP3-MLKL complexes, described by others, may also participate in this process
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also activate autophagy. Cerebral ischemia can activate multiple signaling pathways that subsequently feed into the autophagy pathway (Fig. 5.3).
The figure shows the many different signaling pathways involved in the activation of autophagy during cerebral ischemia. When activated, Akt and NF-κB activate mTOR to inhibit autophagy in cerebral ischemia. However, the activation of AMPK could inhibit the activity of mTOR and induce autophagy. Hypoxia caused by cerebral ischemia activates HIF-1α and induces autophagy through BNIP3 and p53.
Excitotoxicity could induce autophagy by ER stress and block autophagic flux by glutamate in cerebral ischemia. Autophagy could also be induced through ROS and inhibited through PPAR-γ. PPAR-γ: Peroxisome proliferator-activated receptor-γ; AMP: Adenosine 5′-monophosphate; PI3K: phosphatidylinositol 3-kinase; ROS:
reactive oxygen species; HIF-1α: hypoxia inducible factor 1α; Bcl- 2: B cell lymphoma/
leukmia-2; Bcl-xL: B-cell lymphoma-extra large; AMPK: AMP-activated protein kinase; AMPK: AMP-activated protein kinase; Akt/PKB: protein kinase B; NF-κB:
nuclear factor kappa B; ER: endoplasmic reticulum; BNIP3: Bcl-2 and adenovirus E1B 19 kDa interacting proteins 3; mTOR: mammalian target of rapamycin.
1. PI3K-Akt-mTORC1 mTOR is a 289 kDa serine/threonine protein kinase that regulates transcription, cytoskeleton organization, cell growth and cell survival.
The mTOR is a high energy sensor, which on the other hand is a negative
Fig. 5.3 Possible autophagy signaling pathways in CIRI 5 Programmed Cell Death in CIRI
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regulator of autophagy. By binding to different co-factors, mTOR can form two distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) [80]. mTORC1 is responsible for the inhibitory effect of rapamycin, more so than mTORC2. Recent studies suggest that the PI3K/Akt/mTOR pathway could regulate acute nervous system injury in cerebral hypoxia-isch- emia [81]. PI3K consists of class I, class II and class III. Class I PI3K plays an important role in the PI3K-Akt-mTOR pathway. PI3K phosphorylates and activates Akt which in turn phosphorylates and inactivates tuberous sclerosis complex (TSC) 1/2. Inactivated TSC1/2 increases the activation of Rheb which is part of the Ras family GTP-binding protein, and mTOR is subsequently activated. Autophagy is inhibited by activating mTOR [82]. Beclin-1, a component of the class III PI3K, is essential for the initial steps of autophagy and could also induce autophagy via the interaction with other components of the class III PI3K pathway in cerebral ischemia [83]. Peroxisome prolif-erator-acti- vated receptor-γ (PPAR-γ), a member of nuclear hormone receptor superfamily, is a ligand- activated transcription factor. PPAR-γ activation antagonizes beclin- 1-mediated autophagy via upregulation of Bcl-2/Bcl-xl which interact with beclin-1 in cerebral ischemia/reperfusion [84].
2. AMPK-mTORC1 AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase and consists of three subunits: a catalytic α-subunit and regulatory β and γ-subunits. Each subunit appears to have distinct functions. The most studied is the catalytic α-subunit which contains a threonine phosphorylation site that when phosphorylated, activates AMPK. The status of nutrient and energy depletion is sensed and modulated by kinase B1 (LKB1), Ca2+/calmodulin- dependent kinase kinase beta (CaMKKβ) and transforming growth factor β acti- vated kinase-1 (TAK1), resulting in phosphorylation of threonine residue at 172 position and activation of AMPK [85] and AMPK activation could subsequently inhibit the activity of mTOR to induce autophagy [86, 87].
3. Beclin 1-Bcl-2 complex.
Beclin 1 was identified as a Bcl-2-interacting protein through its BH3 domain [88]. The binding of Bcl-2 to Beclin 1 disrupts the association of Beclin 1 with PI3K, hVps34 and p150, therefore inhibiting autophagy [89]. Intriguingly, only ER-localized, but not mitochondria-localized, Bcl-2 inhibits autophagy [89].
Under stress conditions, Beclin 1 is released and induces autophagy [90, 91]. As previously demonstrated, the expression of Beclin 1 in neurons is dramatically increased in neonatal HI or focal cerebral ischemia [92, 93]. Ischemia stimulates autophagy through the AMPK–mTOR pathway, whereas ischemia/reperfusion stimulates autophagy through a Beclin 1-dependent but AMPK-independent pathway [94]. Although there are several different mechanisms to regulate the dissociation of Beclin 1 from Bcl-2 during autophagy in mammalian cells [95], the specific mechanism in cerebral ischemia is not yet established.
Hypoxia-inducible factor 1 (HIF-1) is a key transcriptional factor that is acti- vated in response to hypoxia during cerebral ischemia [96]. HIF-1 is composed of a constitutively expressed HIF-1β subunit and an inducibly expressed HIF-1α subunit. Since ubiquitination is inhibited under hypoxic conditions, HIF-1α can
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accumulate and dimerize with HIF-1β. This dimer activates transcription of a number of downstream hypoxia-responsive genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), glucose transporter 1, and glycolytic enzymes [97]. Bcl-2 and adenovirus E1B 19 kDa interacting proteins 3 (BNIP3) with a single Bcl-2 homology 3 (BH3) domain is a subfamily of Bcl-2 family proteins and also serves as an important target gene of HIF-1α [98].
BNIP3 can compete with beclin-1 for binding to Bcl-2 and beclin-1 is released to trigger autophagy [99]. BNIP3 also binds and inhibits Rheb, an upstream activator of mTOR, so it could activate autophagy by inhibiting mTOR activity.
The induced p53 stabilization by up-regulation of HIF-1α also plays an important role in post-ischemic autophagy activation [97].
4. p53 The tumor suppressor and transcription factor p53 has been reported to be pivotal in neuronal apoptosis [100]. Crighton et al. demonstrated that p53 induced autophagy through the upregulation of damage-regulated autophagy modulator (DRAM), the p53 target gene encoding a lysosomal protein [101].
Other study also demonstrated that the NF-κB-regulated p53 pathway contrib- utes to excitotoxic neuronal death by activating the autophagic process [102].
Overstimulation of N-methyl-d-aspartate receptors (NMDARs) induces the upregulation of p53, its target gene DRAM, and other autophagic proteins including LC3 and Beclin 1. Moreover, the NF-κB inhibitor SN50 inhibits the excitotoxin-induced upregulation of p53, its target gene DRAM, and other autophagic proteins.
Nuclear factor kappa B (NF-κB) is a transcription factor that regulates expression of multiple genes [103]. Recent experiments have demonstrated that the knockout of p50 (NF-κB1) enhanced autophagy by repression of mTOR in cerebral ischemic mice [104]. NF-κB-dependent p53 signal transduction path- way is also associated with autophagy and apoptosis in the rat hippocampus after cerebral ischemia/reperfusion insult [105]. Mitogen-activated protein kinases (MAPKs) include extracellular signal-related kinase (ERK), Jun NH2 terminal kinase (JNK) and p38 [106]. MAPK is one upstream regulator of mTORC1 and autophagy could also be induced via MAPK-mTOR signaling pathway in cerebral ischemia/reperfusion [107].
5. Others.
Autophagic cell death is activated in the nervous system in response to oxida- tive stress [108]. Oxidative stress can occur in cerebral ischemia and could increase reactive oxygen species such as superoxide, hydroxyl radical and hydro- gen peroxide. Recent studies have reported that selenium provides neuroprotec- tion through preserving mitochondrial function, decreasing reactive oxygen species production and reducing autophagy [109]. Autophagy can also be induced under conditions of excitotoxicity which can also occur in cerebral isch- emia [110]. Although excitotoxic glutamate blocks autophagic flux, it could also induce autophagy in hippocampal neurons [111]. Sustained elevations of Ca2+ in the mitochondrial matrix are a major feature of the intracellular cascade of lethal events during cerebral ischemia. Recently, it was reported that endoplasmic reticulum stress is one of the effects of excitotoxicity [1]. When endoplasmic
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reticula were exposed to toxic levels of excitatory neurotransmitters, Ca2+ was released via the activation of both ryanodine receptors and IP3R, leading to mitochondrial Ca2+ overload and activation of apoptosis. During endoplasmic reticulum stress, Ca2+ increase seems to be required for activating autophagy.