Bcl-X(L) and calyculin A prevent translocation of Bax to mitochondria during apoptosis

During many forms of apoptosis, Bax, a pro-apoptotic protein of the Bcl-2 family, translocates from the cytosol to the mitochondria and induces cytochrome c release, followed by caspase activation and DNA degradation. Both Bcl-X(L) and the protein phosphatase inhibitor calyculin A have been shown to prevent apoptosis, and here we investigated their impact on Bax translocation. ML-1 cells incubated with either anisomycin or staurosporine exhibited Bax translocation, cytochrome c release, caspase 8 activation, and Bid cleavage; only the latter two events were caspase-dependent, confirming that they are consequences in this apoptotic pathway. Both Bcl-X(L) and calyculin A prevented Bax translocation and cytochrome c release. Bcl-X(L) is generally thought to heterodimerize with Bax to prevent cytochrome c release and yet they remain in different cellular compartments, suggesting that their heterodimerization at the mitochondria is not the primary mechanism of Bcl-X(L)-mediated protection. Using chemical cross-linking agents, Bax appeared to exist as a monomer in undamaged cells. Upon induction of apoptosis, Bax formed homo-oligomers in the mitochondrial fraction with no evidence for cross-linking to Bcl-2 or Bcl-X(L). Considering that both Bcl-X(L) and calyculin A inhibit Bax translocation, we propose that Bcl-X(L) may regulate Bax translocation through modulation of protein phosphatase or kinase signaling.     

The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a non-classical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.     

Deadly Conversations: Nuclear-Mitochondrial Cross-Talk

Neuronal damage following stroke or neurodegenerative diseases is thought to stem in part from overexcitation of N -methyl-D-aspartate (NMDA) receptors by glutamate. NMDA receptors triggered neurotoxicity is mediated in large part by activation of neuronal nitric oxide synthase (nNOS) and production of nitric oxide (NO). Simultaneous production of superoxide anion in mitochondria provides a permissive environment for the formation of peroxynitrite (ONOO-). Peroxynitrite damages DNA leading to strand breaks and activation of poly(ADP-ribose) polymerase-1 (PARP-1). This signal cascade plays a key role in NMDA excitotoxicity, and experimental models of stroke and Parkinson's disease. The mechanisms of PARP-1-mediated neuronal death are just being revealed. While decrements in ATP and NAD are readily observed following PARP activation, it is not yet clear whether loss of ATP and NAD contribute to the neuronal death cascade or are simply a biochemical marker for PARP-1 activation. Apoptosis-inducing factor (AIF) is normally localized to mitochondria but following PARP-1 activation, AIF translocates to the nucleus triggering chromatin condensation, DNA fragmentation and nuclear shrinkage. Additionally, phosphatidylserine is exposed and at a later time point cytochrome c is released and caspase-3 is activated. In the setting of excitotoxic neuronal death, AIF toxicity is caspase independent. These observations are consistent with reports of biochemical features of apoptosis in neuronal injury models but modest to no protection by caspase inhibitors. It is likely that AIF is the effector of the morphologic and biochemical events and is the commitment point to neuronal cell death, events that occur prior to caspase activation, thus accounting for the limited effects of caspase inhibitors. There exists significant cross talk between the nucleus and mitochondria, ultimately resulting in neuronal cell death. In exploiting this pathway for the development of new therapeutics, it will be important to block AIF translocation from the mitochondria to the nucleus without impairing important physiological functions of AIF in the mitochondria.     

Bid-induced release of AIF from mitochondria causes immediate neuronal cell death

Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.     

Spatial and temporal changes in Bax subcellular localization during anoikis

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.     

The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria in response to apoptotic stimuli, where it induces cytochrome c release. In this study, we show that the phosphatidylinositol 3-OH kinase (PI3K)-Akt pathway plays an important role in the regulation of Bax subcellular localization. We found that LY294002, a PI3K inhibitor, blocked the effects of serum to prevent Bax translocation to mitochondria and that expression of an active form of PI3K suppressed staurosporine-induced Bax translocation, suggesting that PI3K activity is essential for retaining Bax in the cytoplasm. In contrast, both U0126, a MEK inhibitor, and active MEK had little effect on Bax localization. In respect to downstream effectors of PI3K, we found that expression of active Akt, but not serum and glucocorticoid-induced protein kinase (SGK), suppressed staurosporine-induced translocation of Bax, whereas dominant negative Akt moderately promoted Bax translocation. Expression of Akt did not alter the levels of Bax, Bcl-2, Bcl-X(L), or phosphorylated JNK under the conditions used, suggesting that there were alternative mechanisms for Akt in the suppression of Bax translocation. Collectively, these results suggest that the PI3K-Akt pathway inhibits Bax translocation from cytoplasm to mitochondria and have revealed a novel mechanism by which the PI3K-Akt pathway promotes survival.     

A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120

Bax, a pro-apoptotic Bcl-2 family protein, translocates to mitochondria during apoptosis, where it causes MOMP (mitochondrial outer membrane permeabilization). MOMP releases pro-apoptotic factors, such as cytochrome c and SMAC (second mitochondrial activator of caspases)/Diablo, into the cytosol where they activate caspases. It is often inferred that Bax activation occurs in a single step, a conformational change in the protein causing its translocation and oligomerization into high-molecular-mass membrane pores. However, a number of studies have shown that Bax translocation to mitochondria does not necessarily induce MOMP. Indeed, Bax translocation can occur several hours prior to release of cytochrome c, indicating that its regulation may be a complex series of events, some of which occur following its association with mitochondria. In the present study, we have examined endogenous Bax in epithelial cells undergoing anoikis, a physiologically relevant form of apoptosis that occurs when normal cells lose contact with the ECM (extracellular matrix). Using BN-PAGE (blue native PAGE), we show that Bax forms a 200 kDa complex before caspase activation. Furthermore, Bax in this 200 kDa complex is not in the active conformation, as determined by exposure of N-terminal epitopes. These results indicate that Bax oligomerization is an event that must be interpreted differently from the currently held view that it represents the apoptotic pore.     

Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis

Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-X(L), correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.     

Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

Background  

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).     

p38 MAP kinase mediates bax translocation in nitric oxide-induced apoptosis in neurons

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, we determined if NO-induced cell death in neurons was dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), an NO donor, elevated caspase activity and induced death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminished caspase induction and protected SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk did not provide significant protection. A role for p38 MAP kinase was further substantiated by the observation that SB203580 blocked translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promoted Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons were resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway.     

Rho GTPases regulate axon growth through convergent and divergent signaling pathways

Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. Here we show that the actin depolymerization factor cofilin is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rok or Pak, respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection.     

The N-terminal conformation of Bax regulates cell commitment to apoptosis

The Bcl-2 protein Bax normally resides in the cytosol, but during apoptosis it translocates to mitochondria where it is responsible for releasing apoptogenic factors. Using anoikis as a model, we have shown that Bax translocation does not commit cells to apoptosis, and they can be rescued by reattachment to extracellular matrix within a specific time. Bax undergoes an N-terminal conformational change during apoptosis that has been suggested to regulate conversion from its benign, cytosolic form to the active, membrane bound pore. We now show that the Bax N-terminus regulates commitment and mitochondrial permeabilisation, but not the translocation to mitochondria. We identify Proline 13 within the N-terminus of Bax as critical for this regulation. The subcellular distribution of Proline 13 mutant Bax was identical to wild-type Bax in both healthy and apoptotic cells. However, Proline 13 mutant Bax induced rapid progression to commitment, mitochondrial permeabilisation and death. Our data identify changes in Bax controlling commitment to apoptosis that are mechanistically distinct from those controlling its subcellular localisation. Together, they indicate that multiple regulatory steps are required to activate the proapoptotic function of Bax.     

Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.Subject terms: Apoptosis, Cell death in the nervous system, Neurodegeneration     

Ceramide generated by sphingomyelin hydrolysis and the salvage pathway is involved in hypoxia/reoxygenation-induced Bax redistribution to mitochondria in NT-2 cells

Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.     

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.     

Tauroursodeoxycholic acid prevents amyloid-beta peptide-induced neuronal death via a phosphatidylinositol 3-kinase-dependent signaling pathway

Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates cell death by interrupting classic pathways of apoptosis. Amyloid-beta (Abeta) peptide has been implicated in the pathogenesis of Alzheimer's disease, where a significant loss of neuronal cells is thought to occur by apoptosis. In this study, we explored the cell death pathway and signaling mechanisms involved in Abeta-induced toxicity and further investigated the anti-apoptotic effect(s) of TUDCA. Our data show significant induction of apoptosis in isolated cortical neurons incubated with Abeta peptide. Apoptosis was associated with translocation of pro-apoptotic Bax to the mitochondria, followed by cytochrome c release, caspase activation, and DNA and nuclear fragmentation. In addition, there was almost immediate but weak activation of the serine/threonine protein kinase Akt. Inhibition of the phosphatidylinositide 3 prime-OH kinase (PI3K) pathway with wortmannin did not markedly affect Abeta-induced cell death, suggesting that this signaling pathway is not crucial for Abeta-mediated toxicity. Notably, co-incubation with TUDCA significantly modulated each of the Abeta-induced apoptotic events. Moreover, wortmannin decreased TUDCA protection against Abeta-induced apoptosis, reduced Akt phosphorylation, and increased Bax translocation to mitochondria. Together, these findings indicate that Abeta-induced apoptosis of cortical neurons proceeds through a Bax mitochondrial pathway. Further, the PI3K signaling cascade plays a role in regulating the anti-apoptotic effects of TUDCA.     

BimL involvement in Bax activation during UV irradiation-induced apoptosis

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but translocates to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors in response to apoptotic stimuli. However, the molecular mechanism of Bax activation is not fully understood. We show here the role of BimL in Bax activation during UV irradiation-induced apoptosis. In this study, GFP-BimL plasmid was constructed. The dynamic interaction between BimL and Bax during UV irradiation-induced apoptosis was observed using fluorescence resonance energy transfer (FRET) technique. Our experimental results showed that BimL translocation to mitochondria occurred before Bax translocation, and that BimL activated Bax indirectly. Moreover, inhibition of c-Jun N-terminal protein kinase (JNK) activation blocked BimL translocation, delayed and attenuated Bax translocation and subsequent apoptosis. These results demonstrate that BimL is involved in UV irradiation-induced apoptosis by indirectly activating Bax.     

Neuronal Apoptosis: BH3-Only Proteins the Real Killers?

At present there is a poor understanding of the events that lead up to neuronal apoptosis that occurs in neurodegenerative diseases and following acute ischemic episodes. Apoptosis is critical for the elimination of unwanted neurons within the developing nervous system. The Bcl-2 family of proteins contains pro- and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. There is increasing interest in a subfamily of the Bcl-2 family, the BH3-only proteins, and their pro-apoptotic effects within neurons. Recently ischemic and seizure-induced neuronal injury has been shown to result in the activation of the BH3-only protein, Bid. This protein is cleaved and the truncated protein (tBid) translocates to the mitochondria. The translocation of tBid to the mitochondria is associated with the activation of outer mitochondrial membrane proteins Bax/Bak and the release of cytochrome C from the mitochondria. ER stress also has been implicated as a factor for the induction of apoptosis in ischemic neuronal injury. The induction of ER stress in hippocampal neurons has been shown to activate expression of bb3/PUMA, a member of the BH3-only gene family. Activation of PUMA is associated with the activation and clustering of the pro-apoptotic Bcl-2 family member Bax and the loss of cytochrome C from the mitochondria.