Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

Mitochondrial function is required for secretion of DAF-28/insulin in C. elegans

While insulin signaling has been extensively studied in Caenorhabditis elegans in the context of ageing and stress response, less is known about the factors underlying the secretion of insulin ligands upstream of the insulin receptor. Activation of the receptor governs the decision whether to progress through the reproductive lifecycle or to arrest growth and enter hibernation. We find that animals with reduced levels of the mitochondrial outer membrane translocase homologue TOMM-40 arrest growth as larvae and have decreased insulin signaling strength. TOMM-40 acts as a mitochondrial translocase in C. elegans and in its absence animals fail to import a mitochondrial protein reporter across the mitochondrial membrane(s). Inactivation of TOMM-40 evokes the mitochondrial unfolded protein response and causes a collapse of the proton gradient across the inner mitochondrial membrane. Consequently these broadly dysfunctional mitochondria render an inability to couple food abundance to secretion of DAF-28/insulin. The secretion defect is not general in nature since two other neuropeptides, ANF::GFP and INS-22::VENUS, are secreted normally. RNAi against two other putative members of the TOMM complex give similar phenotypes, implying that DAF-28 secretion is sensitive to mitochondrial dysfunction in general. We conclude that mitochondrial function is required for C. elegans to secrete DAF-28/insulin when food is abundant. This modulation of secretion likely represents an additional level of control over DAF-28/insulin function.     

Deleted in cancer 1 (DICE1) is an essential protein controlling the topology of the inner mitochondrial membrane in C. elegans

DICE1 (deleted in cancer 1), first identified in human lung carcinoma cell lines, is a candidate tumor suppressor, but the details of its activity remain largely unknown. We have found that RNA interference of its C. elegans homolog (DIC-1) produced inviable embryos with increased apoptosis, cavities in cells and abnormal morphogenesis. In the dic-1(RNAi) germ line, ced-3-dependent apoptosis increased, and cell cavities appeared at the late-pachytene/oogenic stage, leading to defective oogenesis. Immunofluorescence microscopy of DIC-1 revealed its ubiquitous expression in the form of cytoplasmic foci, and cryoelectron microscopy narrowed down the location of the foci to the inner membrane of mitochondria. After dic-1 RNAi, mitochondria had an irregular morphology and contained numerous internal vesicles. Homozygous embryos from a heterozygous dic-1 mother arrested at the L3 larval stage, in agreement with the essential role of DIC-1 in mitochondria. In summary, C. elegans DIC-1 plays a crucial role in the formation of normal morphology of the mitochondrial cristae/inner membrane. Our results suggest that human DICE1 may have several functions in multiple intracellular locations.     

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.     

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.     

Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione

Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS) and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 resides in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH), which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic HA14-1 induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was coimmunoprecipitated with GSH after chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by preincubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. After cotransfection of CHO cells, Bcl-2 was coimmunoprecipitated with OGC and this novel interaction was significantly enhanced by glutathione monoethyl ester. Similarly, recombinant Bcl-2 interacted with recombinant OGC in the presence of GSH. Bcl-2 and OGC cotransfection in CHO cells significantly increased the mitochondrial glutathione pool. Finally, the ability of Bcl-2 to protect CHO cells from apoptosis induced by hydrogen peroxide was significantly attenuated by the OGC inhibitor phenylsuccinate. These data suggest that GSH binding by Bcl-2 enhances its affinity for the OGC. Bcl-2 and OGC appear to act in a coordinated manner to increase the mitochondrial glutathione pool and enhance resistance of cells to oxidative stress. We conclude that regulation of mitochondrial glutathione transport is a principal mechanism by which Bcl-2 suppresses MOS.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals

The mitochondrial pathway of cell death, in which apoptosis proceeds following mitochondrial outer membrane permeabilization, release of cytochrome c, and APAF-1 apoptosome-mediated caspase activation, represents the major pathway of physiological apoptosis in vertebrates. However, the well-characterized apoptotic pathways of the invertebrates C. elegans and D. melanogaster indicate that this apoptotic pathway is not universally conserved among animals. This review will compare the role of the mitochondria in the apoptotic programs of mammals, nematodes, and flies, and will survey our knowledge of the apoptotic pathways of other, less familiar model organisms in an effort to explore the evolutionary origins of the mitochondrial pathway of apoptosis.     

M19 modulates skeletal muscle differentiation and insulin secretion in pancreatic β-cells through modulation of respiratory chain activity

Mitochondrial dysfunction due to nuclear or mitochondrial DNA alterations contributes to multiple diseases such as metabolic myopathies, neurodegenerative disorders, diabetes and cancer. Nevertheless, to date, only half of the estimated 1,500 mitochondrial proteins has been identified, and the function of most of these proteins remains to be determined. Here, we characterize the function of M19, a novel mitochondrial nucleoid protein, in muscle and pancreatic β-cells. We have identified a 13-long amino acid sequence located at the N-terminus of M19 that targets the protein to mitochondria. Furthermore, using RNA interference and over-expression strategies, we demonstrate that M19 modulates mitochondrial oxygen consumption and ATP production, and could therefore regulate the respiratory chain activity. In an effort to determine whether M19 could play a role in the regulation of various cell activities, we show that this nucleoid protein, probably through its modulation of mitochondrial ATP production, acts on late muscle differentiation in myogenic C2C12 cells, and plays a permissive role on insulin secretion under basal glucose conditions in INS-1 pancreatic β-cells. Our results are therefore establishing a functional link between a mitochondrial nucleoid protein and the modulation of respiratory chain activities leading to the regulation of major cellular processes such as myogenesis and insulin secretion.     

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.     

The mitochondrial apoptosis-induced channel (MAC) corresponds to a late apoptotic event

We have investigated the mechanism responsible for mitochondria permeabilization occurring during cell apoptosis. We have developed an in vivo model of apoptotic rat liver. Mitochondria appeared as an homogenous population in control liver. On the contrary, mitochondria varied in size, morphology, and the matrical density in apoptotic liver. Mitochondria were purified from control and apoptotic livers. In control conditions, a single mitochondrial population was identified; whereas three populations of mitochondria were purified from apoptotic liver. Our data show that these apoptotic populations correspond to early, intermediate, and late apoptotic mitochondria, which are characterized by an increasing extent of permeabilization of their outer membrane and a gradual enrichment in oligomerized Bax protein. Remarkably, a new ionic channel was observed in apoptotic but not in control mitochondria. The biophysical and pharmacological properties of this channel are in good agreement with those reported for a previously described mitochondrial apoptosis-induced channel (MAC) (Pavlov, E. V., Priault, M., Pietkiewicz, D., Cheng, E. H., Antonsson, B., Manon, S., Korsmeyer, S. J., Mannella, C. A., and Kinnally, K. W. (2001) J. Cell Biol. 155, 725-731). However, MAC activity was only observed in the late apoptotic mitochondrial population. Thus, our study establishes that MAC activity is related to the overall apoptotic process but corresponds to a late event.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Differential insulin‐like growth factor (IGF)‐independent interactions of IGF binding protein‐3 and IGF binding protein‐5 on apoptosis in human breast cancer cells. Involvement of the mitochondria.

We have demonstrated previously in Hs578T cells that insulin‐like growth factor binding protein (IGFBP)‐3 can significantly accentuate ceramide (C2)‐induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine‐glycine‐aspartic acid (RGD)‐containing peptide]. In contrast we found that IGFBP‐5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF‐independent actions of IGFBP‐3 and IGFBP‐5 on C2 and RGD‐induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 μmol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 μmol/L) significantly inhibited C2‐induced apoptosis, whereas it significantly accentuated RGD‐induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP‐3 but is not affected by IGFBP‐5. These data indicate that IGFBP‐3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP‐5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria. J. Cell. Biochem. 80:248–258, 2000. © 2000 Wiley‐Liss, Inc.     

Identification of a novel protein MICS1 that is involved in maintenance of mitochondrial morphology and apoptotic release of cytochrome c

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.     

Deficiency of cardiolipin synthase causes abnormal mitochondrial function and morphology in germ cells of Caenorhabditis elegans

Cardiolipin (CL) is a major membrane phospholipid specifically localized in mitochondria. At the cellular level, CL has been shown to have a role in mitochondrial energy production, mitochondrial membrane dynamics, and the triggering of apoptosis. However, the in vivo role of CL in multicellular organisms is largely unknown. In this study, by analyzing deletion mutants of a CL synthase gene (crls-1) in Caenorhabditis elegans, we demonstrated that CL depletion selectively caused abnormal mitochondrial function and morphology in germ cells but not in somatic cell types such as muscle cells. crls-1 mutants reached adulthood but were sterile with reduced germ cell proliferation and impaired oogenesis. In the gonad of crls-1 mutants, mitochondrial membrane potential was significantly decreased, and the structure of the mitochondrial cristae was disrupted. Contrary to the abnormalities in the gonad, somatic tissues in crls-1 mutants appeared normal with respect to cell proliferation, mitochondrial function, and mitochondrial morphology. Increased susceptibility to CL depletion in germ cells was also observed in mutants of phosphatidylglycerophosphate synthase, an enzyme responsible for producing phosphatidylglycerol, a precursor phospholipid of CL. We propose that the contribution of CL to mitochondrial function and morphology is different among the cell types in C. elegans.     

Differential insulin-like growth factor (IGF)-independent interactions of IGF binding protein-3 and IGF binding protein-5 on apoptosis in human breast cancer cells. Involvement of the mitochondria

We have demonstrated previously in Hs578T cells that insulin-like growth factor binding protein (IGFBP)-3 can significantly accentuate ceramide (C2)-induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine-glycine-aspartic acid (RGD)-containing peptide]. In contrast we found that IGFBP-5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF-independent actions of IGFBP-3 and IGFBP-5 on C2 and RGD-induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 micromol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 micromol/L) significantly inhibited C2-induced apoptosis, whereas it significantly accentuated RGD-induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP-3 but is not affected by IGFBP-5. These data indicate that IGFBP-3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP-5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria.     

B cell receptor cross-linking triggers a caspase-8-dependent apoptotic pathway that is independent of the death effector domain of Fas-associated death domain protein.

We have previously reported that B cell receptors, depending on the degree to which they are cross-linked, can promote apoptosis in various human B cell types. In this study, we show that B cell receptors can trigger two apoptotic pathways according to cross-linking and that these pathways control mitochondrial activation in human Burkitt's lymphoma cells. Whereas soluble anti-mu Ab triggers caspase-independent mitochondrial activation, cross-linked anti-mu Ab induces an apoptotic response associated with a caspase-dependent loss of mitochondrial transmembrane potential. This B cell receptor-mediated caspase-dependent mitochondrial activation is associated with caspase-8 activation. We show here that caspase-8 inhibitors strongly decrease cross-linking-dependent B cell receptor-mediated apoptosis in Burkitt's lymphoma BL41 cells. These inhibitors act upstream from the mitochondria as they prevented the loss of mitochondrial membrane potential observed in B cell receptor-treated BL41 cells. Caspase-8 activation in these cells was also evident from the detection of cleaved fragments of caspase-8 and the cleavage of specific substrates, including Bid. Our data show that cross-linked B cell receptors induced an apoptotic pathway involving sequential caspase-8 activation, loss of mitochondrial membrane potential, and the activation of caspase-9 and caspase-3. Cells expressing a dominant negative mutant of Fas-associated death domain protein were sensitive to cross-linked B cell receptor-induced caspase-8 activation and apoptosis; therefore, this caspase-8 activation was independent of the death effector domain of Fas-associated death domain protein.     

Different pathways lead to mitochondrial fragmentation during apoptotic and excitotoxic cell death in primary neurons

Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP‐1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 μM glutamate (l ‐Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP‐1 activity using overexpression of the dominant negative DRP‐1^K38A slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild‐type DRP‐1. In contrast, responses to l ‐Glu appeared DRP‐1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca²⁺ uptake or the ability of l ‐Glu to stimulate excitotoxic Ca²⁺ overload. Finally, apoptosis‐induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:335–341, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336