Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation.

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS.

Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy.

Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria.

Impaired placental autophagy in placental malaria.

BACKGROUND: Placental malaria is a major cause of low birthweight, principally due to impaired fetal growth. Intervillositis, a local inflammatory response to placental malaria, is central to the pathogenesis of poor fetal growth as it impairs transplacental amino acid transport. Given the link between inflammation and autophagy, we investigated whether placental malaria-associated intervillositis increased placental autophagy as a potential mechanism in impaired fetal growth. METHODS: We examined placental biopsies collected after delivery from uninfected women (n = 17) and from women with Plasmodium falciparum infection with (n = 14) and without (n = 7) intervillositis. Western blotting and immunofluorescence staining coupled with advanced image analysis were used to quantify the expression of autophagic markers (LC3-II, LC3-I, Rab7, ATG4B and p62) and the density of autophagosomes (LC3-positive puncta) and lysosomes (LAMP1-positive puncta). RESULTS: Placental malaria with intervillositis was associated with higher LC3-II:LC3-I ratio, suggesting increased autophagosome formation. We found higher density of autophagosomes and lysosomes in the syncytiotrophoblast of malaria-infected placentas with intervillositis. However, there appear to be no biologically relevant increase in LC3B/LAMP1 colocalization and expression of Rab7, a molecule involved in autophagosome/lysosome fusion, was lower in placental malaria with intervillositis, indicating a block in the later stage of autophagy. ATG4B and p62 expression showed no significant difference across histological groups suggesting normal autophagosome maturation and loading of cargo proteins into autophagosomes. The density of autophagosomes and lysosomes in the syncytiotrophoblast was negatively correlated with placental amino acid uptake. CONCLUSIONS: Placental malaria-associated intervillositis is associated with dysregulated autophagy that may impair transplacental amino acid transport, possibly contributing to poor fetal growth.

Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. RESULTS: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. CONCLUSIONS: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

Evidence for the recruitment of autophagic vesicles in human brain after stroke.

Autophagy is a homeostatic process for recycling proteins and organelles that is increasingly being proposed as a therapeutic target for acute and chronic neurodegenerative diseases, including stroke. Confirmation that autophagy is present in the human brain after stroke is imperative before prospective therapies can begin the translational process into clinical trials. Our current study using human post-mortem tissue observed an increase in staining in microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1; also known as p62) and the increased appearance of autophagic vesicles after stroke. These data confirm that alterations in autophagy take place in the human brain after stroke and suggest that targeting autophagic processes after stroke may have clinical significance.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.

Analysis of the Relative Contribution of Phagocytosis, LC3-Associated Phagocytosis, and Canonical Autophagy During Helicobacter pylori Infection of Macrophages.

BACKGROUND: Previous findings have suggested that Helicobacter pylori induces autophagic processes and subsequently takes refuge in autophagosomes, thereby contributing to persistent infection. Recently, a noncanonical form of autophagy, LC3 (microtubule-associated protein 1 light chain 3)-associated phagocytosis (LAP), has been shown to be required for efficient clearance of some intracellular bacteria. Whether H. pylori infection induces LAP had not been examined previously. In this study, we determined the extent to which H. pylori infection induces canonical autophagy or LAP in macrophages, and the involvement of the H. pylori cag pathogenicity island (cagPAI) with these processes. METHODS: Immunofluorescence confocal microscopy was used to analyze the formation of GFP-LC3 puncta and their colocalization with H. pylori. Transmission electron microscopy was used to detect the ultrastructure of H. pylori-containing compartments. RESULTS: The majority of intracellular bacteria (85-95%) were found in phagosomes that were LC3-negative, with a small proportion (4-14%) appearing "free" in the cytosol. Only a very small percentage (0.5-6%) of intracellular H. pylori was sequestered in autophagosomes. Furthermore, no statistically significant difference in the relative distribution of H. pylori in the various compartments was observed between wild-type and cagPAI-mutant bacteria. CONCLUSIONS: In macrophages, H. pylori infection does not induce LAP, but can induce canonical autophagy, which entraps a very small fraction of intracellular bacteria. We propose that this subpopulation of intracellular H. pylori might have escaped from phagosomes into the cytosol before being sequestered by autophagosomes. The cagPAI of H. pylori has only minor influence, if any, on the extent of these processes.

X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein.

Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low ΦF, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123T69V (2.0 Å) and eCGP123H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.

Autophagy: starvation relieves transcriptional repression of ATG genes.

Autophagy is a highly regulated process about which relatively little is known, particularly concerning the transcriptional control of autophagy regulation. A new study identifies a key regulator of the expression of autophagy-related genes, thereby providing insights into the signalling pathways modulating autophagy.

Burkholderia pseudomallei type III secretion system cluster 3 ATPase BsaS, a chemotherapeutic target for small-molecule ATPase inhibitors.

Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacterium Burkholderia pseudomallei, to enter and survive in host cells. B. pseudomallei can escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded by bsaS, as a target for chemotherapeutic treatment of infection. A B. pseudomallei bsaS deletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with the bsaS deletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis.

Autophagy and burkholderia.

Autophagy has become increasingly viewed as an important component of the eukaryotic innate immune system. The elimination of intracellular pathogens by autophagy in mammalian cells (xenophagy) results not only in the degradation of invading bacteria, viruses, fungi and parasites, but also liberation of metabolites that may have been utilized during pathogen infection, thus promoting cell survival. After gaining entry into the cell, intracellular bacterial pathogens attempt to escape from phagosomes (or endosomes) into the cytosol where they endeavour to continue the infection cycle unhindered by host cell protective mechanisms. Bacterial recognition resulting from either their cytosolic location, the secretion of bacterial products, or phagosomal membrane damage, can induce autophagy. In this context, induction of autophagy results in the clearance of some bacterial pathogens, whereas other bacteria are able to manipulate autophagy for their own benefit and appear to effectively replicate within autophagosome-like vesicles. Some bacteria are seemingly able to evade autophagy and Burkholderia pseudomallei is one of them. This review will discuss the autophagic processes that may be activated by host cells to provide protection against infection by this bacterial pathogen.

Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy.

Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ∼2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.

Phanta: a non-fluorescent photochromic acceptor for pcFRET.

We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.

Nucleophagy at a glance.

Under certain circumstances, the removal of damaged or non-essential parts of the nucleus, or even an entire nucleus, is crucial in order to promote cell longevity and enable proper function. A selective form of autophagy, known as nucleophagy, can be used to accomplish the degradation of nucleus-derived material. In this Cell Science at a Glance article and the accompanying poster, we summarize the similarities and differences between the divergent modes of nucleophagy that have been described to date, emphasizing, where possible, the molecular mechanism, the membrane interactions and rearrangements, and the nature of the nucleus-derived material that is degraded. In turn, we will consider nucleophagy processes in the lower eukaryotes, the budding yeast Saccharomyces cerevisiae, filamentous fungi Aspergillus and Magnaporthe oryzae and the ciliated protozoan Tetrahymena thermophila, and finally in mammalian cells. We will also briefly discuss the emerging links between nucleophagy and human disease.

The impact of autophagic processes on the intracellular fate of Helicobacter pylori: more tricks from an enigmatic pathogen?

Helicobacter pylori is a Gram-negative pathogen that colonizes the gastric epithelium of 50-60% of the world's population. Approximately one-fifth of the infected individuals manifest severe diseases such as peptic ulcers or gastric cancer. H. pylori infection has proven difficult to cure despite intensive antibiotic treatment. One possible reason for the relatively high resistance to antimicrobial therapy is the ability of H. pylori to reside inside host cells. Although considered by most as an extracellular pathogen, H. pylori can invade both gastric epithelial cells and immunocytes to some extent. The intracellular survival of H. pylori has been implicated in its ability to persist in the stomach, evade host immune responses and resist eradication by membrane-impermeable antibiotics. Interestingly, recent evidence suggests that macroautophagy, a cellular self-degradation process characterized by the formation of double-membraned autophagosomes, plays an important role in determining the intracellular fate of H. pylori. Detailed understanding of the interaction between H. pylori and host cell autophagic processes is anticipated to provide novel insights into the molecular mechanisms of macroautophagy and H. pylori pathogenesis, opening new avenues for the therapeutic intervention of autophagy-related and H. pylori-related disorders.

Beclin 1 is required for starvation-enhanced, but not rapamycin-enhanced, LC3-associated phagocytosis of Burkholderia pseudomallei in RAW 264.7 cells.

LC3-associated phagocytosis (LAP) of Burkholderia pseudomallei by murine macrophage (RAW 264.7) cells is an intracellular innate defense mechanism. Beclin 1, a protein with several roles in autophagic processes, is known to be recruited to phagosomal membranes as a very early event in LAP. We sought to determine whether knockdown of Beclin 1 by small interfering RNA (siRNA) would affect recruitment of LC3 and subsequent LAP of infecting B. pseudomallei. Both starvation and rapamycin treatment can induce Beclin 1-dependent autophagy. Therefore, we analyzed the consequences of Beclin 1 knockdown for LAP in infected cells that had been either starved or treated with rapamycin by determining the levels of bacterial colocalization with LC3 and intracellular survival. Concurrently, we confirmed the location of bacteria as either contained in phagosomes or free in the cytoplasm. We found that both rapamycin and starvation treatment enhanced LAP of B. pseudomallei but that the rapamycin response is Beclin 1 independent whereas the starvation response is Beclin 1 dependent.

A green fluorescent protein containing a QFG tri-peptide chromophore: optical properties and X-ray crystal structure.

Rtms5 is an deep blue weakly fluorescent GFP-like protein ([Formula: see text], 592 nm; [Formula: see text], 630nm; Φ(F), 0.004) that contains a (66)Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5(Y67F) and Rtms5(Y67F/H146S) in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5(Y67F) spectra were characterised by two absorbing species ([Formula: see text], 440 nm and 513 nm) and green fluorescence emission ([Formula: see text], 440 nm; [Formula: see text], 508 nm; Φ(F), 0.11), whilst Rtms5(Y67F/H146S) spectra were characterised by a single absorbing species ([Formula: see text], 440 nm) and a relatively high fluorescence quantum yield (Φ(F,) 0.75; [Formula: see text], 440 nm; [Formula: see text], 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3-11). These are the first GFP-like proteins with green emissions (500-520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5(Y67F) results from an unusual form of the chromophore protonated at the acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.

Autophagy vitalizes the pathogenicity of pathogenic fungi.

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.

Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death.

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both autophagic cell death and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (-3, -7, and -9). Substantial depolarization of mitochondria occurred after 1h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.