Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice

“As previously reported, Bax/Bak1 double-knockout (DKO) MEFs were highly resistant to cell death by the apoptotic inducer staurosporine (Wei et al., 2001), while Wt MEFs showed high levels of killing (Figure 1A).”

“Necroptosis is induced in cells when stimulated with tumor necrosis factor α (TNFα) in the presence of caspase inhibitors, such as zVAD-fmk (Degterev et al., 2005).”

“Recently, mixed lineage kinase domain–like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5) were shown to be downstream of the RIP proteins (Sun et al., 2012; Wang et al., 2012).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“RIP1 is able to interact with RIP3 in controlling necroptosis (Cho et al., 2009; He et al., 2009; Zhang et al., 2009).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“Bax/Bak regulate cell death independent of ER Ca2+ Bax/Bak1 DKO cells were previously shown to have reduced Ca2+ levels in the endoplasmic reticulum (ER) as a protective mechanism against cell death (Scorrano et al., 2003).”

“To create heart- and liverspecific Bax and Bak1 knockout mice, Baxfl/fl Bak1−/− mice were crossed with α-myosin heavy chain (αMHC)-Cre and albumin-Cre transgenic lines (Postic et al., 1999; Oka et al., 2006).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“…that Bax/Bak function merely to regulate the MPTP by binding VDAC or ANT is not consistent with the data from Slc25a4/Slc25a5 (ANT1/2 protein) and Vdac1/2/3 gene–deleted mice, which showed that neither protein is directly required for MPTP formation (Kokoszka et al., 2004; Baines et al., 2007).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“More importantly, purified mitochondria from these DKO MEFs reconstituted with Wt or the nonoligomerizing Bax mutants (Hoppins et al., 2011) each showed partial restoration of MPTP function in the Ca2+ uptake assay (Figure 3E).”

“…that Bax/Bak function merely to regulate the MPTP by binding VDAC or ANT is not consistent with the data from Slc25a4/Slc25a5 (ANT1/2 protein) and Vdac1/2/3 gene–deleted mice, which showed that neither protein is directly required for MPTP formation (Kokoszka et al., 2004; Baines et al., 2007).”

“Recently, mixed lineage kinase domain–like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5) were shown to be downstream of the RIP proteins (Sun et al., 2012; Wang et al., 2012).”

“Over a decade ago, several reports suggested that Bax and/or Bak directly interacted with presumed components of the MPTP, the adenine nucleotide translocator (ANT) in the inner membrane and the VDAC in the outer membrane (Marzo et al., 1998; Narita et al., 1998; Shimizu et al., 1999).”

“Indeed, deletion of the gene encoding CypD renders mitochondria resistant to Ca2+ overload– induced swelling and the heart and brain partially resistant to cell death due to ischemic injury (Baines et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“DKO MEFs with reconstituted Bax and Bak were also previously described (Kim et al., 2009).”

“Indeed, Bcl-2 family members are known to alter membrane characteristics and have channel-like activities of multiple conductance states (Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997).”

“To create heart- and liverspecific Bax and Bak1 knockout mice, Baxfl/fl Bak1−/− mice were crossed with α-myosin heavy chain (αMHC)-Cre and albumin-Cre transgenic lines (Postic et al., 1999; Oka et al., 2006).”

“Since MPTP formation has been proposed to function as a Ca2+ release mechanism (Elrod et al., 2010; Bernardi and von Stockum, 2012), the observed augmentation in baseline Ca2+ in Bax/Bak-deficient mitochondria, and their greater uptake capacity further suggest that Bax/Bak are required for…”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“Ion selectivity was determined through reversal potentials after a 150:30 mM KCl gradient was established across the patch as previously reported (Pavlov et al., 2001).”

“More importantly, purified mitochondria from these DKO MEFs reconstituted with Wt or the nonoligomerizing Bax mutants (Hoppins et al., 2011) each showed partial restoration of MPTP function in the Ca2+ uptake assay (Figure 3E).”

“Histological analysis of the injury induced by myocardial ischemia/reperfusion was described previously (Kaiser et al., 2004).”

“The MPTP has been well characterized by direct patch-clamping of mitochondria and shown to have a specific conductance profile that is distinct from any other channels or permeating activities (Kinnally and Antonsson, 2007).”

“Bax-loxP (fl) Bak1−/− mice were described previously (Takeuchi et al., 2005).”

“Flow cytometry analysis of active Bax has been previously described (Panaretakis et al., 2002).”

“Mitochondrial Ca2+ uptake was measured with Calcium Green-5N (Invitrogen, Grand Island, NY) as previously described (Kwong et al., 2007).”

“Indeed, Nix/BNip3L, which is induced by disease/developmental signaling pathways that enhance cell death, can alter the activity and pro-death characteristics of Bax/ Bak as well as alter MPTP-dependent cell death (Chen et al., 2010).”

“However, restoration with either of the two mutants of Bax that cannot oligomerize, although both still localize to the mitochondria (George et al., 2007; Hoppins et al., 2011), restored ionomycin-induced killing but not apoptotic killing with staurosporine (Figure 3D).”

“Moreover, treatment with gossypol, a BH3-mimetic compound that can convert Bcl-2 and Bcl-xl into pore-forming pro-cell death agents (Lei et al., 2006) restored mitochondrial swelling in the absence of Bax/Bak protein, which was also inhibited with CsA (Figure 4C,D).”

“Seals were formed with these micropipettes on giant liposomes or mitochondria prepared as described previously (Martinez-Caballero et al., 2009).”

“Indeed, we and others have observed that Bax/ Bak1 null MEFs were resistant to this form of cell death (data not shown and Irrinki et al., 2011).”

“…is not a sieve but rather a membrane whose permeability is tightly regulated, despite the presence of other channels such as voltage-dependent anion channel (VDAC, comprising as many as three gene products, Vdac1, Vdac2, and Vdac3) and TOM (Jonas et al., 2004; Martinez-Caballero et al., 2005).”

“As another consideration, the α5/α6 domain of Bax is known to have membrane permeation activity and to permit pore formation on its own, the permeation size of which appears to reach an equilibrium of less than 10 kDa (Fuertes et al., 2010).”

“DOI: 10.7554/eLife.00772 16 of 21 Research article DKO MEFs were infected with a binary adenovirus expressing Bax (Ad-Bax) was previously described and obtained from Dr Bingliang Fang (Kagawa et al., 2000).”

“…recent studies have disputed such a direct role, suggesting that Bax/Bak only have a secondary effect, such as by regulating mitochondrial fission/fusion or no effect whatsoever, or just a secondary effect due to organelle rupture (De Marchi et al., 2004; Vaseva et al., 2012; Whelan et al., 2012).”

“Indeed, increasing concentrations of poloxamer-188 (P-188), which inherently reduces membrane permeability characteristics (Wu et al., 2004), prevented mitochondrial swelling induced by Ca2+ as analyzed in purified mitochondria in solution or by transmission electron microscopy (Figure 4E,F).”

“…is not a sieve but rather a membrane whose permeability is tightly regulated, despite the presence of other channels such as voltage-dependent anion channel (VDAC, comprising as many as three gene products, Vdac1, Vdac2, and Vdac3) and TOM (Jonas et al., 2004; Martinez-Caballero et al., 2005).”

“Cells at 80% confluence were harvested and mitochondria were isolated as previously described (Murphy et al., 2001).”

“However, gossypol, which can convert endogenous Bcl-2 proteins into a pro-death configuration and increase membrane permeability (de Peyster et al., 1986), enhanced or induced ionomycin-dependent cell death in Wt and DKO MEFs, respectively (Figure 7A).”

“DOI: 10.7554/eLife.00772 14 of 21 Research article one of which has a small pore size (0.9 nm) and is homogenous in the outer mitochondrial membrane, which could fulfill the permeability function we observed here to permit nonapoptotic cell death (Lin et al., 2011).”

“Bcl-2 family members appear to generally alter the permeability and rigidity of lipid membranes, and even the Bcl-2 homologue from Caenorhabditis elegans, CED-9 can induce leakiness of reconstituted lipid membranes (Tan et al., 2011).”

“In mitochondria from MEFs in which genes encoding Bax and Bak have been genetically inactivated, OMM permeability decreased without significantly altering PTP function in the IMM (306).”

“Recent genetic studies revealed that deletion of Bax/ Bak inhibits mPTP opening and necrotic cell death induced by Ca overload or ROS, suggesting the ability of Bax/Bak to facilitate mPTP-induced cell death.(113,114) Thus the aforementioned mitoHK-II-mediated inhibition of Bax binding to mitochondria could contribute…”

“ence of Bax and Bak, whose genetic ablation confers OMM resistance to swelling, thus preventing organelle rupture and cell death [123].”

“It has been proposed that such a “supercomplex” could be present preferentially at contact sites between the outer and inner membrane [23, 120, 121].”

“…inhibition of MPTP opening by deletion of MPTP components, notably the genes encoding CypD (Baines et al., 2005; Nakayama et al., 2007), PiC (Kwong et al., 2014), and Bax/ Bak (Karch et al., 2013; Whelan et al., 2012), reduced cardiac damage sustained in response to ischemia-reperfusion injury.”

“Recently, the proapoptotic proteins BAX and BAK were found to be critical mediators of primary necrosis.(163,186) Mice lacking BAX and BAK or BAX alone exhibit markedly decreased cardiac necrosis, apoptosis, and infarct size after ischemia-reperfusion in vivo.”

“Surprisingly, loss of BAX and BAK inhibits mitochondrial swelling and rupture following mPTP opening, indicating that some of the proteins involved in mitochondrial-mediated apoptosis may nonetheless affect the mPTP (Karch et al., 2013; Whelan et al., 2012).”

“Most recently, Molkentin's laboratory [52] have suggested that Bax/Bak proteins by regulating OMMpermeability are required forMPTP-dependent necrotic cell death.”

“This is illustrated by the apoptotic OMM proteins BAX and BAK, which appear to be necessary for some forms of cardiac necrosis (38,49).”

“These data were confirmed by Molkentin's group, who used genetic background knockout models for Bax and Bak [51].”

“However, the IMM mPTP machinery was still functional and the protection was attributed to preventing OMM rupture and release of IMS components (Whelan et al. 2012; Karch et al. 2013).”

“Moreover, mice lacking specifically Bax and Bak1 in cardiomyocytes have been attributed an increased resistance to cardiac ischemia/reperfusion, as compared to their wild-type littermates, and isolated Bax / Bak1 / mitochondria were unable to normally undergo MPT in vitro [40].”

“Moreover, this does not appear to be dependent on their ability to oligomerize and undergo MOMP as an oligomerization-deficient BAXmutant still permitsMPTPdependent mitochondrial swelling and necrotic cell death.(22,23) The mechanism by which BAX and BAK promote MPTP is still under investigation, with some data suppor…”

“In particular, the proapoptotic members Bax and Bcl-2 homologous antagonist killer (Bak) have a dramatic positive effect on mPTP opening, as confirmed in bax and bak knockout models (34).”

“Some data suggest that Bax on the outer mitochondrial membrane is in complex with MPTP.99 Another mechanism that the Kitsis group has explored is that Bax facilitates necrosis through its previously recognized ability to promote mitochondrial fusion.100,104 Interestingly, oligomerization-deficient Bax mutants, the sam…”

“Analysis of molecular mediators involved in mitochondrial MPT pore opening indicated that diclofenac induced early activation of Bax and Bak in immortalized human hepatocytes (HC-04), and caused mitochondrial translocation of Bax [106].”

“Outer membrane components, the voltage-dependent anion channel (VDAC) [15] and BCL-2 family members BAK/BAX [20] have further been suggested to play a role as modulators of pore opening.”

“Further, BAX and BAK are involved in the process of mitochondrial permeability transition pore (MPTP) formation in necrosis [145,146].”

“Structurally, the MPTP complex has been proposed to be comprised of the ATP synthase(17,18) and to be regulated by cyclophilin D (CypD),(19,20) ANT,(21) and the pro-apoptotic proteins Bax and Bak in the outer mitochondrial membrane.(22,23) PiC has also been suggested to be an inner-membrane component of the MPTP becau…”

“Beclin 1 can also influence apoptosis and necrosis of cells AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00162.2017 • www.ajplung.org through its interactions with the B-cell lymphoma-2 (BCL2) family of proteins, BCL2-associated X protein (BAX) and BCL2-antagonistic/killer (BAK; 53, 54, 138).”

“Bak form the components of the mitochondrial permeability transition pore (MPTP) in the outer membrane [90,91], the”

“(2013) provided evidence for B-cell lymphoma-2 (Bcl-2)-associated X (Bax) and Bcl-2-antagonist/killer 1 (Bak) members of the Bcl-2 family as mPTP components; using transgenic animal models, they demonstrated that the absence of both proteins promoted resistance to mitochondrial swelling and resulted in the lack of cha…”

“Bax and Bak are also required for PTPC-dependent necrotic cell death; in fact, the loss of Bax/Bak resulted in resistance to mitochondrial calcium overload and swelling [49].”

“…including Bcl-2 and Bcl-xL, have been suggested to modulate the mPTP by maintaining it in a close conformation, while pro-apoptotic proteins, such as Bax and Bak, engage ANTand/ or VDAC to induce mPTP opening (Marzo et al., 1998; Shimizu et al., 1999; Whelan et al., 2012; Karch et al., 2013).”

“18 19–21 This lack of translatable protection may be explained by potential compensatory forms of cell death during I/R, as many forms of cell death have been hypothesized to occur during this injury.”

“To explore the role of mPTP in the activation of the cGASSTING pathway, cyclosporin A (CsA), a mPTP inhibitor, was used to block mPTP opening.(46,47) HS-induced mPTP opening was reversed (Fig.”

“…outer membrane and allowing the exit of molecules less than 1,500 Da, including cytochrome c, apoptosis inducing factor (AIF) and endonuclease G. 46 Although release of cytochrome c and AIF is a hallmark of apoptosis, as they activate caspase 3/7 and lead to nuclear DNA cleavage, respectively,…”

“In addition, in some cases of apoptosis, the opening of the mPTP also contributes to mitochondrial swelling and rupture, as well as to necrotic cell death, which is regulated by Bax/Bak and cyclophilin D (CypD) [192, 193].”

“These data suggest that the MPTP is an IMM-regulated process, although in the absence of Bax/ Bak, the OMM resists swelling and prevents organelle rupture, which prevents necrosis [17].”

“The onset of MPT is characterized by the permeabilization of the MOM secondary to a sudden increase in permeability of the MIM, which allows the exit of molecules less than 1500 Da (Karch et al., 2013).”

“Ppif MEFs are partially resistant to necrosis induced by ionomycin or H2O2, but not against apoptosis induced by staurosporine [3, 4, 6].”

“Karch et al.(39) recently demonstrated that inhibition of MOMP by Bax and Bak”

“In light on these results, the authors concluded that Bax/Bak oligomers within the outer membrane are required for mitochondrial permeability pore-dependent cell death by serving as a necessary functional component of the MPTP [106].”

“The mPTP is a key effector in the pathway to cell death (Baines et al., 2003; Karch et al., 2013; Karch and Molkentin, 2014; Xu et al., 2019) in that opening of the mPTP further exacerbates mitochondrial dysfunction (Baines et al.”

“components of the OMM are still known to contribute to its induction [42,69].”

“mPTP is located at the inner mitochondrial membrane, while VDAC1 and Bak/Bax are located at the outer mitochondrial membrane [41,42].”

“For example, activation of the inner membrane permeability transition and loss of mitochondrial membrane potential can occur in absence of Bax and Bak (Karch et al., 2013), and Bax has been shown to form two distinct types of channels which can fine-tune outer membrane permeabilization (Lin et al.”

“…Karch et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. needed for mitochondrial-dependent cellular necrosis (Karch et al., 2013).”

“…the synthesis of mitochondrial proteins and increase mitochondrial permeability transition, which in turn allows the efflux of small molecules (Karch et al., 2013), causing mitochondria to become further depolarized, thus reducing the proton gradient leading to the collapse of the…”

“The essential apoptotic MOMP proteins, BAK and BAX, have been implicated in MPT by Karch et al. 94 to effect necrotic mitochondrial pore-dependent cell death through a mechanism that differs from apoptotic MOMP.94 The effect of mitochondrial disruption is thought to cause ATP-depletion and excessive ROS production to…”

“While subsequent studies have ruled out the requirement of VDAC as a component of the MPTP [125], other work has shown that the ANT c-state can specifically associate with BAX [126], which is consistent with our current understanding of BAX and BAK and their putative role in MPTP-dependent cell death [127].”

“Furthermore, the expression levels of Bax and Bak, which were recently suggested to govern the porosity of the mitochondrial outer membrane (22), were not altered by HAX-1 overexpression either.”

“ROS-induced oxidative stress can induce the occurrence of mitochondrial permeability transition (MPT) in hepatocytes, which also leads to changes in mitochondrial membrane potential [122,123].”

“To determine mitochondrial permeability transition pore (MPTP) activation, cardiac mitochondria were isolated by differential centrifugation [33,34].”

“Bcl-2 associated proteins contain various antiapoptotic proteins (bcl-2, bcl-xl) and proapoptotic proteins (bax, bak) which are prime regulators of apoptosis and necrotic death [50–52].”

“Of note, loss of the proapoptotic proteins BAK and BAX has been shown to reduce mitochondrial membrane permeability, resulting in resistance to mitochondrial calcium overload and necrotic cell death [74].”

“Also, in tissues with a low proliferative index, like the brain (Ren et al. 2010), heart, and liver (Whelan et al. 2012; Karch et al. 2013), BAX and BAK seem dispensable for homeostasis, as their codeletion does not seem to trigger a hyperplastic phenotype in these organs (Table 2).”

“Strikingly, triple knockout mice lacking Bax/Bak and cyclophilin D, the key regulator of mPTP opening and necrotic cell death, failed to display any further reduction in infarct size over those deficient in Bax/Bak suggesting that Bax and Bak regulate necrosis in vivo and confirming a connection between mitochondrial…”

“The role of Bax as an MPT pore component has been considered in some studies proposing that Bax and Bak may behave as the mitochondrial outer membrane component of the MPT pore [44].”

“Accordingly, BAX and BAK1 have been involved in RCD modalities other than apoptosis [74,75].”

“While the components of the MPTP on the inner membrane other than cyclophilin D are still being characterized, it is well recognized that the outer membrane components are composed of Bax, along with the protein Bak on the mitochondrial outer membrane (Karch et al., 2013; Karch et al., 2014).”

“reported that BCL-2 family members are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP [70].”

“Recently, double-knockout genetic experiments confirmed and extended the involvement of Bax and Bak, which are known as pro-apoptotic proteins, in mPTP action and in necrotic cell death (Karch et al., 2013).”

“During necrotic cell death, Bax and Bak as outer membrane components have been shown to promote mitochondrial swelling and rupture upon opening of the mitochondrial permeability transition pore (MPTP) by facilitating outer membrane permeability.(31) Opening of the MPTP, a channel in the inner mitochondrial membrane th…”