Mechanisms of non-apoptotic programmed cell death in diabetes and heart failure

Programmed cell elimination is an important pathological mediator of disease. Multiple pathways to programmed cell death have been delineated, including apoptosis, autophagy and programmed necrosis. Cross-talk between the signaling pathways mediating each process has made it difficult to define specific mechanisms of in vivo programmed cell death. For this reason, many “apoptotic” diseases may involve other death signaling pathways. Recent advances in genetic complementation using mouse knockout models are helping to dissect apoptotic and necrotic cell death in different pathological states. The current state of research in this area is reviewed, focusing upon new findings describing the role of programmed necrosis induced by the mitochondrial permeability transition in mouse models of heart failure and diabetes.Key words: apoptosis, necrosis, mitochondrial permeability transition pore     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Role of apoptotic and necrotic cell death under physiologic conditions

Apoptosis is considered to be a programmed and controlled mode of cell death, whereas necrosis has long been described as uncontrolled and accidental cell death resulting from extremely harsh conditions. In the following review, we will discuss the features and physiological meanings as well as recent advances in the elucidation of the signaling pathways of both apoptotic cell death and programmed necrotic cell death.     

Necrosis: a specific form of programmed cell death?

For a long time necrosis was considered as an alternative to programmed cell death, apoptosis. Indeed, necrosis has distinct morphological features and it is accompanied by rapid permeabilization of plasma membrane. However, recent data indicate that, in contrast to necrosis caused by very extreme conditions, there are many examples when this form of cell death may be a normal physiological and regulated (programmed) event. Various stimuli (e.g., cytokines, ischemia, heat, irradiation, pathogens) can cause both apoptosis and necrosis in the same cell population. Furthermore, signaling pathways, such as death receptors, kinase cascades, and mitochondria, participate in both processes, and by modulating these pathways, it is possible to switch between apoptosis and necrosis. Moreover, antiapoptotic mechanisms (e.g., Bcl-2/Bcl-x proteins, heat shock proteins) are equally effective in protection against apoptosis and necrosis. Therefore, necrosis, along with apoptosis, appears to be a specific form of execution phase of programmed cell death, and there are several examples of necrosis during embryogenesis, a normal tissue renewal, and immune response. However, the consequences of necrotic and apoptotic cell death for a whole organism are quite different. In the case of necrosis, cytosolic constituents that spill into extracellular space through damaged plasma membrane may provoke inflammatory response; during apoptosis these products are safely isolated by membranes and then are consumed by macrophages. The inflammatory response caused by necrosis, however, may have obvious adaptive significance (i.e., emergence of a strong immune response) under some pathological conditions (such as cancer and infection). On the other hand, disturbance of a fine balance between necrosis and apoptosis may be a key element in development of some diseases.     

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.     

AIF-Mediated Programmed Necrosis: A Highly Orchestrated Way to Die

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly regulated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.     

Programmed cell death pathways in cancer: a review of apoptosis,autophagy and programmed necrosis

Programmed cell death (PCD), referring to apoptosis, autophagy and programmed necrosis, is proposed to be death of a cell in any pathological format, when mediated by an intracellular program. These three forms of PCD may jointly decide the fate of cells of malignant neoplasms; apoptosis and programmed necrosis invariably contribute to cell death, whereas autophagy can play either pro‐survival or pro‐death roles. Recent bulk of accumulating evidence has contributed to a wealth of knowledge facilitating better understanding of cancer initiation and progression with the three distinctive types of cell death. To be able to decipher PCD signalling pathways may aid development of new targeted anti‐cancer therapeutic strategies. Thus in this review, we present a brief outline of apoptosis, autophagy and programmed necrosis pathways and apoptosis‐related microRNA regulation, in cancer. Taken together, understanding PCD and the complex interplay between apoptosis, autophagy and programmed necrosis may ultimately allow scientists and clinicians to harness the three types of PCD for discovery of further novel drug targets, in the future cancer treatment.     

Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation

An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. Biochemical and genetic analyses of apoptosis provide an increasingly detailed picture of the intracellular signaling pathways involved. Nevertheless, it remains to be determined whether apoptosis can become a clinically important approach to modulate plaque progression. In this review, we have outlined some of the most recent results concerning apoptosis in atherosclerosis with a special focus on oxidized lipids, inflammation and therapeutic regulation of the apoptotic cell death process.     

Chronicles of a death foretold: Dual sequential cell death checkpoints in TNF signaling

The kinase RIP1 wears a coat of many colors during TNF receptor signaling and can regulate both activation of pro-survival NFkB and programmed cell death pathways. In this review, we outline how coating RIP1 with K63-linked ubiquitin chains forms a protective layer that prevents RIP1 from binding apoptotic regulators and serves as an early guard against cell death. Further on, binding of NFkB signaling components to the ubiquitin coat of RIP1 activates long-term pro-survival signaling and forms a more impenetrable suit of armor against cell death. If RIP1 is not decorated with ubiquitin chains it becomes an unstoppable harbinger of bad news: programmed cell death.     

Programmed cell death in fission yeast Schizosaccharomyces pombe

Yeasts have proven to be invaluable, genetically tractable systems to study various fundamental biological processes including programmed cell death. Recent advances in the elucidation of the molecular pathways underlying apoptotic cell death in yeasts have revealed remarkable similarities to mammalian apoptosis at cellular, organelle and macromolecular levels, thus making a strong case for the relevance of yeast models of regulated cell death. Programmed cell death has been reported in fission yeast Schizosaccharomyces pombe, primarily in the contexts of perturbed intracellular lipid metabolism, defective DNA replication, improper mitotic entry, chronological and replicative aging. Here we review the current understanding of the programmed cell death in fission yeast, paying particular attention to lipid-induced cell death. We discuss our recent findings that fission yeast exhibits plasticity of apoptotic and non-apoptotic modes of cell death in response to different lipid stimuli and growth conditions, and that mitochondria, reactive oxygen species and novel cell death mediators including metacaspase Pca1, SpRad9 and Pck1 are involved in the lipotoxic cell death. We also present perspectives on how various aspects of the cell and molecular biology of this organism can be explored to shed light on the governing principles underlying lipid-mediated signaling and cell demise.     

Oxidative stress and neuronal death/survival signaling in cerebral ischemia

It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways could provide novel therapeutic strategies in clinical stroke.     

Caspase-independent programmed cell death with necrotic morphology.

Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.     

Toll-like Receptor 3-mediated Necrosis via TRIF,RIP3, and MLKL

Toll-like receptor (TLR) signaling is triggered by pathogen-associated molecular patterns that mediate well established cytokine-driven pathways, activating NF-κB together with IRF3/IRF7. In addition, TLR3 drives caspase 8-regulated programmed cell death pathways reminiscent of TNF family death receptor signaling. We find that inhibition or elimination of caspase 8 during stimulation of TLR2, TLR3, TLR4, TLR5, or TLR9 results in receptor interacting protein (RIP) 3 kinase-dependent programmed necrosis that occurs through either TIR domain-containing adapter-inducing interferon-β (TRIF) or MyD88 signal transduction. TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3). In fibroblasts, this pathway proceeds independent of RIP1 or its kinase activity, but it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase. Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes. Cell fate decisions following TLR signaling parallel death receptor signaling and rely on caspase 8 to suppress RIP3-dependent programmed necrosis whether initiated directly by a TRIF-RIP3-MLKL pathway or indirectly via TNF activation and the RIP1-RIP3-MLKL necroptosis pathway.     

Executionary pathway for apoptosis：lessons from mutant mice

Apoptosis or programmed cell death(PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms.Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer.The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals.In this review,we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.     

Plant lectins: targeting programmed cell death pathways as antitumor agents

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.     

Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

Microglia are the resident immune cells in the central nervous system and key players against pathogens and injury. However, persistent microglial activation often exacerbates pathological damage and has been implicated in many neurological diseases. Despite their pivotal physiological and pathophysiological roles, how the survival and death of activated microglia is regulated remains poorly understood. We report here that microglia activated through Toll-like receptors (TLRs) undergo RIP1/RIP3-dependent programmed necrosis (necroptosis) when exposed to the pan caspase inhibitor zVAD-fmk. Although zVAD-fmk and the caspase-8 inhibitor IETD-fmk had no effect on unstimulated primary microglia, they markedly sensitized microglia to TLR1/2,3,4,7/8 ligands or TNF treatment, triggering programmed necrosis that was completely blocked by R1P1 kinase inhibitor necrostatin-1. Interestingly, necroptosis induced by TLR ligands and zVAD was restricted to microglial cells and was not observed in astrocytes, neurons or oligodendrocytes even though they are known to express certain TLRs. Deletion of genes encoding TNF or TNFR1 failed to prevent lipopolysaccharide- and poly(I:C)-induced microglial necroptosis, unveiling a TNF-independent programmed necrosis pathway in TLR3- and TLR4-activated microglia. Microglia from mice lacking functional TRIF were fully protected against TLR3/4 activation and zVAD-fmk-induced necrosis, and genetic deletion of rip3 also prevented microglia necroptosis. Activation of c-jun N-terminal kinase and generation of specific reactive oxygen species were downstream signaling events required for microglial cell death execution. Taken together, this study reveals a robust RIP3-dependent necroptosis signaling pathway in TLR-activated microglia upon caspase blockade and suggests that TLR signaling and programmed cell death pathways are closely linked in microglia, which could contribute to neuropathology and neuroinflammation when dysregulated.     

The prevalence of TNFα-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay

Death receptor-induced programmed necrosis is regarded as a secondary death mechanism dominating only in cells that cannot properly induce caspase-dependent apoptosis. Here, we show that in cells lacking TGFβ-activated Kinase-1 (TAK1) expression, catalytically active Receptor Interacting Protein 1 (RIP1)-dependent programmed necrosis overrides apoptotic processes following Tumor Necrosis Factor-α (TNFα) stimulation and results in rapid cell death. Importantly, the activation of the caspase cascade and caspase-8-mediated RIP1 cleavage in TNFα-stimulated TAK1 deficient cells is not sufficient to prevent RIP1-dependent necrosome formation and subsequent programmed necrosis. Our results demonstrate that TAK1 acts independently of its kinase activity to prevent the premature dissociation of ubiquitinated-RIP1 from TNFα-stimulated TNF-receptor I and also to inhibit the formation of TNFα-induced necrosome complex consisting of RIP1, RIP3, FADD, caspase-8 and cFLIP(L). The surprising prevalence of catalytically active RIP1-dependent programmed necrosis over apoptosis despite ongoing caspase activity implicates a complex regulatory mechanism governing the decision between both cell death pathways following death receptor stimulation.     

Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.     

Antiapoptotic signaling via MCL1 confers resistance to caspase-3-mediated apoptotic cell death in the pregnant human uterine myocyte

Our group has previously identified elevated levels of nonapoptotic active caspase 3 (CASP3) accompanied by increased prosurvival, antiapoptotic signaling in the pregnant mouse uterus during late gestation. We speculated that increased antiapoptotic signaling desensitized the pregnant uterine myocyte to the apoptotic action of uterine CASP3. This current study examines the mechanism by which the pregnant myocyte gains resistance to the apoptotic effects of increased uterine CASP3. Using both primary human pregnant fundal myometrial cultures and the telomerase-immortalized human uterine myocyte cell line (hTERT) as our model systems, uterine myocytes were exposed to UV irradiation and Fas ligand to stimulate both the intrinsic and extrinsic apoptotic pathways. Stimulation of either the intrinsic or extrinsic apoptotic pathways resulted in elevated levels of uterine myocyte CASP3. However, apoptotic cell death was restricted to CASP3 activated by intrinsic stimulation via UV light. In contrast Fas ligand-mediated CASP3 activation was accompanied by increased antiapoptotic signaling mimicking our in vivo observations in the pregnant mouse uterus. Using small interfering RNA to inhibit antiapoptotic signaling, we determined the ability of the human uterine myocyte to resist apoptotic cell death in the absence of the prosurvival, antiapoptotic signaling. Accordingly, suppression of antiapoptotic signaling specifically mediated by myeloid cell leukemia sequence 1 was sufficient to sensitize the uterine myocyte to undergo apoptotic cell death. These data demonstrate that elevated myeloid cell leukemia sequence 1 levels are sufficient to confer apoptotic resistance on the human uterine myocyte despite highly elevated levels of active CASP3.