Victor J. Yuste

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis.

ABSTRACT Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N′-nitro-N′-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase‐independent mode of cell death mediated by apoptosis‐inducing factor (AIF). After poly(ADP‐ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C‐terminal proline‐rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA‐degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C‐terminal PBD abolishes AIF/H2AX interaction and AIF‐mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA‐deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase‐independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Cysteine protease inhibition prevents mitochondrial apoptosis-inducing factor (AIF) release

During the last years, research on the molecular mechanisms governing caspase-dependent and -independent cell death has yielded a significant quantity of exciting works. As part of those efforts, apoptosis-inducing factor (AIF) was the first identified mitochondrial protein involved in caspase-independent cell death. Under physiological conditions, AIF is a mitochondrial FAD-dependent oxidoreductase that plays a role in oxidative phosphorylation. However, after a cellular insult, AIF is released from mitochondria and translocates to cytosol and nucleus where it achieves its proapoptotic function. Importantly, AIF seems a key factor in neuronal cell death and is involved in the early stages of development. Although alternative studies demonstrate that AIF could also be released in a caspase-dependent manner, AIF has been generally implicated in the caspase-independent mode of cell death. In this context, two recent papers from Otera et al. and Uren et al. demonstrated that AIF is a membrane-integrated protein that needs to be cleaved for becoming a soluble and apoptogenic protein. In fact, Otera et al. states that AIF is released from mitochondria by a twostep process. First, AIF is cleaved in the mitochondrial matrix by the mitochondrial processing peptidase to form an innermembrane-anchored form. In a second step, AIF is processed, by an unidentified protease, in the intermembrane space of mitochondria (IMS) to yield a soluble and proapoptotic protein released to cytosol. But how is AIF cleaved in the IMS? Is it by a caspase? Apparently not. A third study from Polster et al. reported that after incubation of isolated mitochondria with different amounts of Ca2þ , mitochondrial AIF release occurs through an N-terminal cleavage mediated by the Ca2þ -dependent protease calpain I (also called m-calpain). These data provide a critical clue to understanding the regulation of AIF action. However, we here demonstrate that the mechanism governing AIF cleavage in the mitochondrial IMS is more complex than initially expected. We first separate mitochondria from other organelles and debris on a discontinuous density gradient. The use of specific antibodies against AIF, LAMP1, and ERK, three markers of the mitochondrial, lysosomal, or cytoplasmic compartments, further confirmed the mitochondrial enrichment of our preparation. In fact, and as shown in Figure 1a, mitochondria are greatly enriched after Percoll gradient (AG) and the lysosomal and cytoplasmic cellular compartments are eliminated or reduced to a large extent. To address the underlying mechanisms by which AIF is cleaved into the IMS and released from mitochondria to induce death, we first determined by Edman microsequencing the N-terminal amino acids of the two forms of AIF: the inner-membrane-anchored and the soluble and apoptogenic form. Our working hypothesis is that the protease responsible for the AIF cleavage must specifically yield the form released from mitochondria. In this way, the mouse anchored AIF presents an N-terminal sequence starting at the amino acid A54 of the AIF precursor. On the other hand, when treating mitochondria in a calciumdepleted medium with atractyloside (Atr), an agent that induces mitochondrial AIF liberation, the protein became soluble and showed a lower apparent molecular weight (Figure 1b). This form, which we called tAIF, reveals an N-terminal sequence starting at the amino acid L103 of the AIF precursor (indicating a proteolytic processing at position G102/L103). Intriguingly, like Atr, Ca2þ treatment induced in a dose-dependent manner an AIF proteolytic processing that yields the same soluble form of AIF: tAIF (Figure 1b). These results indicate that it is possible to induce AIF cleavage in the IMS in a Ca2þ -dependent or in Ca2þ -independent manner. Our results also implicate that proteases that are different from the previously identified m-calpain (which works exclusively in a Ca2þ environment) are implicated in AIF processing. Interestingly enough, the N-terminal sequence of tAIF, obtained in our mitochondrial in vitro assays, is similar to the soluble form of AIF purified from cytosols of HeLa cells treated with etoposide, camptothecin, cisplatin, staurosporine, and MNNG (data not shown). This fact indicates that tAIF is the apoptogenic form of the protein released from mitochondria to cytosol after an apoptotic stimulus. Using a fluorometric assessment, we next looked for a proteolytic activity provoking the Ca2þ -independent AIF cleavage. As expected, in the absence of Ca2þ no relevant m-calpain activity was visualized (Figure 1c). Thus, we searched for other proteolytic activities that could regulate the Atr-dependent mitochondrial AIF cleavage. As shown in Figure 1c, supernatants from Atr-treated mitochondria contained a panel of proteases able to process z-Arg-Arg-amc (zRR), H-Arg-amc (R), and z-Phe-Arg-amc (zFR), three wellknown substrates used for the measurement of cathepsin B, cathepsin H, and cathepsin L/S activity, respectively. In contrast, no caspase-3 activity (measured with Ac-Asp-GluVal-Asp-afc) was found (Figure 1c). Similar protease activities were measured in the supernatant obtained after the addition of digitonin to mitochondria (data not shown). These results Cell Death and Differentiation (2005) 12, 1445–1448 & 2005 Nature Publishing Group All rights reserved 1350-9047/05

Characterization of the Cell Death Process Induced by Staurosporine in Human Neuroblastoma Cell Lines

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Drp1 mediates caspase-independent type III cell death in normal and leukemic cells

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.

The prevention of the staurosporine‐induced apoptosis by Bcl‐XL, but not by Bcl‐2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

ABSTRACT Ligation of CD47 triggers caspase-independent programmed cell death (PCD) in normal and leukemic cells. Here, we characterize the morphological and biochemical features of this type of death and show that it displays the hallmarks of type III PCD. A molecular and biochemical approach has led us to identify a key mediator of this type of death, dynamin-related protein 1 (Drp1). CD47 ligation induces Drp1 translocation from cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 provokes an impairment of the mitochondrial electron transport chain, which results in dissipation of mitochondrial transmembrane potential, reactive oxygen species generation, and a drop in ATP levels. Surprisingly, neither the activation of the most representative proapoptotic members of the Bcl-2 family, such as Bax or Bak, nor the release of apoptogenic proteins AIF (apoptosis-inducing factor), cytochrome c, endonuclease G (EndoG), Omi/HtrA2, or Smac/DIABLO from mitochondria to cytosol is observed. Responsiveness of cells to CD47 ligation increases following Drp1 overexpression, while Drp1 downregulation confers resistance to CD47-mediated death. Importantly, in B-cell chronic lymphocytic leukemia cells, mRNA levels of Drp1 strongly correlate with death sensitivity. Thus, this previously unknown mechanism controlling caspase-independent type III PCD may provide the basis for novel therapeutic approaches to overcome apoptotic avoidance in malignant cells.

AIFsh, a Novel Apoptosis-inducing Factor (AIF) Pro-apoptotic Isoform with Potential Pathological Relevance in Human Cancer

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti‐apoptotic strategies in staurosporine‐treated SH‐SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z‐VAD.fmk a broad‐spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH‐SY5Y cells. At higher concentrations, z‐VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over‐expression of Bcl‐2 in SH‐SY5Y protected cells from death at doses of staurosporine up to 1 µm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl‐2 increased the threshold for apoptotic cell death commitment. Over‐expression of Bcl‐XL was far more effective than Bcl‐2. Bcl‐XL transfected cells showed a remarkable resistance staurosporine‐induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 µm staurosporine. In these conditions, SH‐SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP‐2 neuronal specific proteins.

The long form of fas apoptotic inhibitory molecule is expressed specifically in neurons and protects them against death receptor-triggered apoptosis

AIF is a main mediator of caspase-independent cell death. It is encoded by a single gene located on chromosome X, region q25–26 and A6 in humans and mice, respectively. Previous studies established that AIF codes for two isoforms of the protein, AIF and AIF-exB. Here, we identify a third AIF isoform resulting from an alternate transcriptional start site located at intron 9 of AIF. The resulting mRNA encodes a cytosolic protein that corresponds to the C-terminal domain of AIF (amino acids 353–613). We named this new isoform AIFshort (AIFsh). AIFsh overexpression in HeLa cells results in nuclear translocation and caspase-independent cell death. Once in the nucleus, AIFsh provokes the same effects than AIF, namely chromatin condensation and large scale (50 kb) DNA fragmentation. In contrast, these apoptogenic effects are not precluded by the AIF-inhibiting protein Hsp70. These findings identify AIFsh as a new pro-apoptotic isoform of AIF, and also reveal that the first N-terminal 352 amino acids of AIF are not required for its apoptotic activity. In addition, we demonstrate that AIFsh is strongly down-regulated in tumor cells derived from kidney, vulva, skin, thyroid, and pancreas, whereas, γ-irradiation treatment provokes AIFsh up-regulation. Overall, our results identify a novel member of the AIF-dependent pathway and shed new light on the role of caspase-independent cell death in tumor formation/suppression.

Malonate induces cell death via mitochondrial potential collapse and delayed swelling through an ROS‐dependent pathway

Death receptors (DRs) and their ligands are expressed in developing nervous system. However, neurons are generally resistant to death induction through DRs and rather their activation promotes neuronal outgrowth and branching. These results suppose the existence of DRs antagonists expressed in the nervous system. Fas apoptosis inhibitory molecule (FAIMS) was first identified as a Fas antagonist in B-cells. Soon after, a longer alternative spliced isoform with unknown function was identified and named FAIML. FAIMS is widely expressed, including the nervous system, and we have shown previously that it promotes neuronal differentiation but it is not an anti-apoptotic molecule in this system. Here, we demonstrate that FAIML is expressed specifically in neurons, and its expression is regulated during the development. Expression could be induced by NGF through the extracellular regulated kinase pathway in PC12 (pheochromocytoma cell line) cells. Contrary to FAIMS, FAIML does not increase the neurite outgrowth induced by neurotrophins and does not interfere with nuclear factor κB pathway activation as FAIMS does. Cells overexpressing FAIML are resistant to apoptotic cell death induced by DRs such as Fas or tumor necrosis factor R1. Reduction of endogenous expression by small interfering RNA shows that endogenous FAIML protects primary neurons from DR-induced cell death. The detailed analysis of this antagonism shows that FAIML can bind to Fas receptor and prevent the activation of the initiator caspase-8 induced by Fas. In conclusion, our results indicate that FAIML could be responsible for maintaining initiator caspases inactive after receptor engagement protecting neurons from the cytotoxic action of death ligands.

Neuronal survival induced by neurotrophins requires calmodulin

1 Herein we study the effects of the mitochondrial complex II inhibitor malonate on its primary target, the mitochondrion. 2 Malonate induces mitochondrial potential collapse, mitochondrial swelling, cytochrome c (Cyt c) release and depletes glutathione (GSH) and nicotinamide adenine dinucleotide coenzyme (NAD(P)H) stores in brain‐isolated mitochondria. 3 Although, mitochondrial potential collapse was almost immediate after malonate addition, mitochondrial swelling was not evident before 15 min of drug presence. This latter effect was blocked by cyclosporin A (CSA), Ruthenium Red (RR), magnesium, catalase, GSH and vitamin E. 4 Malonate added to SH‐SY5Y cell cultures produced a marked loss of cell viability together with the release of Cyt c and depletion of GSH and NAD(P)H concentrations. All these effects were not apparent in SH‐SY5Y cells overexpressing Bcl‐xL. 5 When GSH concentrations were lowered with buthionine sulphoximine, cytoprotection afforded by Bcl‐xL overexpression was not evident anymore. 6 Taken together, all these data suggest that malonate causes a rapid mitochondrial potential collapse and reactive oxygen species production that overwhelms mitochondrial antioxidant capacity and leads to mitochondrial swelling. Further permeability transition pore opening and the subsequent release of proapoptotic factors such as Cyt c could therefore be, at least in part, responsible for malonate‐induced toxicity.