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The biological functions of macroautophagy have been examined by generating mice lacking several Atg genes, including AtgS (ref. 4), beclin 1 (Becnl) (7,8) and Atg7 (ref. 5), considered essential for [...], Macroautophagy is a process that leads to the bulk degradation of subcellular constituents by producing autophagosomes/autolysosomes (1-3). It is believed that AtgS (ref. 4) and Atg7 (ref. 5) are essential genes for mammalian macroautophagy. Here we show, however, that mouse cells lacking AtgS or Atg7 can still form autophagosomes/ autolysosomes and perform autophagy-mediated protein degradation when subjected to certain stressors. Although lipidation of the microtubule-associated protein light chain 3 (LC3, also known as Mapllc3a) to form LC3-II is generally considered to be a good indicator of macroautophagy (6), it did not occur during the AtgS/Atg7independent alternative process of macroautophagy. We also found that this alternative process of macroautophagy was regulated by several autophagic proteins, including Unc-5l-like kinase 1 (Ulkl) and beclin 1. Unlike conventional macroautophagy, autophagosomes seemed to be generated in a Rab9-dependent manner by the fusion of isolation membranes with vesicles derived from the traps-Golgi and late endosomes. In vivo, AtgS-independent alternative macroautophagy was detected in several embryonic tissues. It also had a function in clearing mitochondria during erythroid maturation. These results indicate that mammalian macroautophagy can occur through at least two different pathways: an AtgS/Atg7-dependent conventional pathway and an AtgS/Atg7-independent alternative pathway.

Author(s): Takashi Nakagawa [1, 2, 4]; Shigeomi Shimizu [1, 4]; Tetsuya Watanabe [3]; Osamu Yamaguchi [3]; Kinya Otsu [3]; Hirotaka Yamagata [1]; Hidenori Inohara [2]; Takeshi Kubo [2]; Yoshihide Tsujimoto [...]

Apoptosis is distinguished by changes including nuclear chromatin condensation and nuclear fragmentation, but the molecular basis of these events is still unclear. A new nuclear factor, named Acinus, has been identified which is necessary for apoptotic chromatin condensation, but not for DNA fragmentation. The new factor is present in the nucleus and activated through cleavage during apoptosis.

Bcl-2 family proteins may target voltage-dependent anion channel (VDAC) directly in order to modulate channel activity. It is probable that Bax/Bak prompts a conformational change in VDAC, thus allowing the cytochrome c to pass through the channel. However, it is also possible that Bax/Bak and VDAC could together create a larger channel. It appears that the Bcl-2 family of proteins target VDAC to regulate apoptosis-associated mitochondrial changes that play a key role in determining cell survival or death.

Author(s): Yuya Nishida; Satoko Arakawa; Kenji Fujitani; Hirofumi Yamaguchi; Takeshi Mizuta; Toku Kanaseki; Masaaki Komatsu; Kinya Otsu; Yoshihide Tsujimoto; Shigeomi Shimizu Nature 461, 654658 (2009); doi:10.1038/nature08455 In Supplementary Fig. 19a [...]

Research demonstrates that the SMN gene expressed in the motor neurone disease spinal muscular atrophy is co-expressed with the anti-apoptotic protein Bcl-2. The co-expression suppresses Bax-induced or Fas-mediated apoptosis. Evidence from genetic mutation experiments suggests that reduced SMN activity results in the onset of SMA. Co-immunoprecipitation results featuring human Bcl-2 and SMN are shown.

Bcl-2 and Bcl-xL controls the proteases present in the cell which might be how they prevent death due to oxygen withdrawal. In this type of cell death the amount of free oxygen radicals formed becomes less which affects the oxidation of lipids, proteins and DNA in the cell. The prevention of hypoxia-induced death by Bcl-2 and Bcl-xL is not because of their action on reactive oxygen species (ROS) since ROS is not involved even in very small amounts.

Small intestinal enterocytes are continuously renewed. Shedding/death of enterocytes involves receptor-interacting protein kinase 1 (RIPK1)-dependent (but RIPK3-independent) necrotic death, but the regulatory mechanism of the processes is not fully understood. Here, we show that mouse housing conditions, such as the type of bedding material and the presence or absence of a Shepherd Shack, affect enterocyte turnover rate and determine whether enterocyte shedding/death is RIPK1-independent or -dependent. Mice housed with ALPHA-dri (αDri, hard paper chip) bedding material without a Shepherd Shack had a higher, largely RIPK1-dependent enterocyte turnover rate and higher blood corticosterone levels, suggesting the involvement of minor stress, whereas mice housed with αDri plus a Shepherd Shack or with Soft Chip had a lower, RIPK1-independent turnover rate and lower blood corticosterone levels. Corticosterone administration to a small intestine culture derived from mice housed with αDri plus a Shepherd Shack or with Soft Chip increased enterocyte shedding/death and turnover. By using kinase inhibitors and knockout mice, we showed that the switch from RIPK1-independent to RIPK1-dependent enterocyte shedding/death and turnover involves suppression of TANK-binding kinase 1. Our results demonstrate that housing conditions may cause minor stress, which alters the mode of enterocyte shedding/death and enterocyte turnover rate in mice., (© 2023. The Author(s).)

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Programmed cell death, which is required for the development and homeostasis of metazoans, includes mechanisms such as apoptosis, autophagic cell death, and necrotic (or type III) death. Members of the Bcl2 family regulate apoptosis, among which Bax and Bak act as a mitochondrial gateway. Although embryonic fibroblasts from Bax/Bak double-knockout (DKO) mice are resistant to apoptosis, we previously demonstrated that these cells die through an autophagy-dependent mechanism in response to various types of cellular stressors. To determine the physiological role of autophagy-dependent cell death, we generated Atg5/Bax/Bak triple-knockout (TKO) mice, in which autophagy is greatly suppressed compared with DKO mice. Embryonic fibroblasts and thymocytes from TKO mice underwent autophagy much less frequently, and their viability was much higher than DKO cells in the presence of certain cellular stressors, providing genetic evidence that DKO cells undergo Atg5-dependent death. Compared with wild-type embryos, the loss of interdigital webs was significantly delayed in DKO embryos and was even further delayed in TKO embryos. Brain malformation is a distinct feature observed in DKO embryos on the 129 genetic background, but not in those on a B6 background, whereas such malformations appeared in TKO embryos even on a B6 background. Taken together, our data suggest that Atg5-dependent cell death contributes to the embryonic development of DKO mice, implying that autophagy compensates for the deficiency in apoptosis.

Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease., Competing Interests: The authors declare no conflict of interest.

Autophagy is a cellular process that degrades subcellular constituents, and is conserved from yeast to mammals. Although autophagy is believed to be essential for living cells, cells lacking Atg5 or Atg7 are healthy, suggesting that a non-canonical degradation pathway exists to compensate for the lack of autophagy. In this study, we show that the budding yeast Saccharomyces cerevisiae, which lacks Atg5, undergoes bulk protein degradation using Golgi-mediated structures to compensate for autophagy when treated with amphotericin B1, a polyene antifungal drug. We named this mechanism Golgi membrane-associated degradation (GOMED) pathway. This process is driven by the disruption of PI(4)P-dependent anterograde trafficking from the Golgi, and it also exists in Atg5-deficient mammalian cells. Biologically, when an Atg5-deficient β-cell line and Atg7-deficient β-cells were cultured in glucose-deprived medium, a disruption in the secretion of insulin granules from the Golgi occurred, and GOMED was induced to digest these (pro)insulin granules. In conclusion, GOMED is activated by the disruption of PI(4)P-dependent anterograde trafficking in autophagy-deficient yeast and mammalian cells., (© 2016 The Authors.)

Extensive studies have unveiled the intracellular molecular signaling pathways of cell death. To better understand cell death in tissues, it is important to investigate the influence of neighboring cells on the response to death stimuli. By time-lapse microscopy, we found that cells in couplets (two hepatocytes attached to each other) died independently when stimulated with anti-Fas antibody and staurosporine, whereas acetaminophen (APAP) and aryl alcohol caused synchronized cell death although its timing varied among different couplets. Synchronized death of couplets was not caused by APAP when hepatocytes were deficient in both Connexin26 and Connexin32, indicating a crucial role of gap junctions in the synchronized death process. We also demonstrated that APAP-sensitive male hepatocytes were protected by attachment to APAP-insensitive female hepatocytes, with this protection being dependent on gap junctions. These findings indicate that APAP-induced and aryl alcohol-induced necrotic death of hepatocytes is modulated by attached neighboring cells via gap junctions.

During the process of death receptor-mediated apoptosis, Bid is cleaved by activated caspase-8, and then cleaved Bid conveys apoptotic signals to the mitochondria by activating Bax/Bak. In the present study, we found that D609 (an antitumor drug with multiple activities) blocks Fas-induced apoptosis. D609 did not interfere with activation of caspase-8 and cleavage of Bid, whereas it blocked cytochrome c release from the mitochondria by inhibiting the activation of Bax and Bak. D609 had no protective effect against apoptosis of SKW6.4 cells, which are typical type I cells. Studies using permeabilized cells revealed that in addition to activation of caspase-8, Fas activated a distinct and D609-sensitive signaling pathway that transmitted signal(s) sensitizing the mitochondria to apoptotic stimuli, and that D609 itself promoted mitochondrial resistance to apoptotic stimuli.

Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis.

The Bcl-2 family of proteins regulates apoptosis chiefly by controlling mitochondrial membrane permeability. It has previously been shown that the BH4 domain of Bcl-2/Bcl-xL is essential for the prevention of apoptotic mitochondrial changes, including the release of cytochrome c and apoptotic cell death. We have previously reported that BH4 peptide fused to the protein transduction domain of HIV-1 TAT protein (TAT-BH4) significantly inhibits etoposide-induced apoptosis in a cell line. This time, we investigated whether TAT-BH4 peptide was cytoprotective in ex vivo and in vivo rodent models. Intraperitoneal injection of TAT-BH4 peptide greatly inhibited X-ray-induced apoptosis in the small intestine of mice and partially suppressed Fas-induced fulminant hepatitis. In addition, this peptide markedly suppressed heart failure after ischemia-reperfusion injury in isolated rat heart, probably by preventing mitochondrial dysfunction. These findings demonstrate that TAT-BH4 peptide exerts anti-apoptotic activity both in vivo and ex vivo, and imply that it may be a useful therapeutic agent for diseases involving mitochondrial dysfunction and apoptosis.

We have previously shown that the toxic pro-oxidant methylselenol is released from selenomethionine (SeMET) by cancer cells transformed with the adenoviral methionine alpha,gamma-lyase (methioninase, MET) gene cloned from Pseudomonas putida. Methylselenol damaged the mitochondria via oxidative stress, and caused cytochrome c release into the cytosol thereby activating caspase enzymes and thereby apoptosis. However, gene therapy strategies are less effective if tumor cells overexpress the antiapoptotic mitochondrial protein bcl-2. In this study, we investigated whether rAdMET/SeMET was effective against bcl-2-overproducing A549 lung cancer cells. We established two clones of the human lung cancer A549 cell line that show moderate and high expression levels of bcl-2, respectively, compared to the parent cell line, which has very low bcl-2 expression. Staurosporine-induced apoptosis was inhibited in the bcl-2-overproducing clones as well as in the parental cell line. In contrast to staurosporine, apoptosis was induced in the bcl-2-overproducing clones as well as the parental cell line by AdMET/SeMET. Apoptosis in the rAdMET-SeMET-treated cells was determined by fragmentation of nuclei, and release of cytochrome c from mitochondria to the cytosol. A strong bystander effect of AdMET/SeMET was observed on A549 cells as well as the bcl-2-overproducing clones. rAdMET/SeMET prodrug gene therapy is therefore a promising novel strategy effective against bcl-2 overexpression, which has blocked other gene therapy strategies.

Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial membrane permeability. We have previously shown that the voltage-dependent anion channel (VDAC) plays a crucial role in apoptotic changes of the mitochondria and its activity is directly regulated by some Bcl-2 family members, including Bcl-2/Bcl-x(L) and Bax/Bak but not Bid. Here, we showed that in isolated mitochondria, Bim induced loss of membrane potential and cytochrome c release like Bax/Bak, with these changes being inhibited by an anti-VDAC antibody. In addition, microinjection of the anti-VDAC antibody significantly reduced Bim-induced apoptosis. Study using purified proteins indicated that Bim directly interacts with the VDAC. Immunoprecipitation analysis revealed that Bim interacts with the VDAC and the interaction is remarkably enhanced during apoptosis. An experiment using liposomes indicated that Bim enhanced VDAC activity, as did Bax/Bak. Furthermore, Bim (but not tBid) was able to induce apoptotic changes of yeast mitochondria in a VDAC-dependent manner, and also induced the lysis of red blood cells, with this effect being inhibited by the anti-VDAC antibody. These results indicate that Bim has an ability to activate directly the VDAC, which plays an important role in apoptosis of mammalian cells.