The BAD-BAX-Caspase-3 Cascade Modulates Synaptic Vesicle Pools via Autophagy.

The BAD-BAX-caspase-3 cascade is a canonical apoptosis pathway. Macroautophagy ("autophagy" hereinafter) is a process by which organelles and aggregated proteins are delivered to lysosomes for degradation. Here, we report a new function of the BAD-BAX-caspase-3 cascade and autophagy in the control of synaptic vesicle pools. We found that, in hippocampal neurons of male mice, the BAD-BAX-caspase-3 pathway regulates autophagy, which in turn limits the size of synaptic vesicle pools and influences the kinetics of activity-induced depletion and recovery of synaptic vesicle pools. Moreover, the caspase-autophagy pathway is engaged by fear conditioning to facilitate associative fear learning and memory. This work identifies a new mechanism for controlling synaptic vesicle pools, and a novel, nonapoptotic, presynaptic function of the BAD-BAX-caspase-3 cascade.SIGNIFICANCE STATEMENT Despite the importance of synaptic vesicles for neurons, little is known about how the size of synaptic vesicle pools is maintained under basal conditions and regulated by neural activity. This study identifies a new mechanism for the control of synaptic vesicle pools, and a new, nonapoptotic function of the BAD-BAX-caspase-3 pathway in presynaptic terminals. Additionally, it indicates that autophagy is not only a homeostatic mechanism to maintain the integrity of cells and tissues, but also a process engaged by neural activity to regulate synaptic vesicle pools for optimal synaptic responses, learning, and memory.

Intrinsically determined cell death of developing cortical interneurons.

Cortical inhibitory circuits are formed by γ-aminobutyric acid (GABA)-secreting interneurons, a cell population that originates far from the cerebral cortex in the embryonic ventral forebrain. Given their distant developmental origins, it is intriguing how the number of cortical interneurons is ultimately determined. One possibility, suggested by the neurotrophic hypothesis, is that cortical interneurons are overproduced, and then after their migration into cortex the excess interneurons are eliminated through a competition for extrinsically derived trophic signals. Here we characterize the developmental cell death of mouse cortical interneurons in vivo, in vitro and after transplantation. We found that 40% of developing cortical interneurons were eliminated through Bax (Bcl-2-associated X)-dependent apoptosis during postnatal life. When cultured in vitro or transplanted into the cortex, interneuron precursors died at a cellular age similar to that at which endogenous interneurons died during normal development. Over transplant sizes that varied 200-fold, a constant fraction of the transplanted population underwent cell death. The death of transplanted neurons was not affected by the cell-autonomous disruption of TrkB (tropomyosin kinase receptor B), the main neurotrophin receptor expressed by neurons of the central nervous system. Transplantation expanded the cortical interneuron population by up to 35%, but the frequency of inhibitory synaptic events did not scale with the number of transplanted interneurons. Taken together, our findings indicate that interneuron cell death is determined intrinsically, either cell-autonomously or through a population-autonomous competition for survival signals derived from other interneurons.

DRP1-dependent apoptotic mitochondrial fission occurs independently of BAX, BAK and APAF1 to amplify cell death by BID and oxidative stress.

During apoptosis mitochondria undergo cristae remodeling and fragmentation, but how the latter relates to outer membrane permeabilization and downstream caspase activation is unclear. Here we show that the mitochondrial fission protein Dynamin Related Protein (Drp) 1 participates in cytochrome c release by selected intrinsic death stimuli. While Bax, Bak double deficient (DKO) and Apaf1(-/-) mouse embryonic fibroblasts (MEFs) were less susceptible to apoptosis by Bcl-2 family member BID, H(2)O(2), staurosporine and thapsigargin, Drp1(-/-) MEFs were protected only from BID and H(2)O(2). Resistance to cell death of Drp1(-/-) and DKO MEFs correlated with blunted cytochrome c release, whereas mitochondrial fragmentation occurred in all cell lines in response to all tested stimuli, indicating that other mechanisms accounted for the reduced cytochrome c release. Indeed, cristae remodeling was reduced in Drp1(-/-) cells, potentially explaining their resistance to apoptosis. Our results indicate that caspase-independent mitochondrial fission and Drp1-dependent cristae remodeling amplify apoptosis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation.

Adult hippocampal neurogenesis is a unique form of neural circuit plasticity that results in the generation of new neurons in the dentate gyrus throughout life. Neurons that arise in adults (adult-born neurons) show heightened synaptic plasticity during their maturation and can account for up to ten per cent of the entire granule cell population. Moreover, levels of adult hippocampal neurogenesis are increased by interventions that are associated with beneficial effects on cognition and mood, such as learning, environmental enrichment, exercise and chronic treatment with antidepressants. Together, these properties of adult neurogenesis indicate that this process could be harnessed to improve hippocampal functions. However, despite a substantial number of studies demonstrating that adult-born neurons are necessary for mediating specific cognitive functions, as well as some of the behavioural effects of antidepressants, it is unknown whether an increase in adult hippocampal neurogenesis is sufficient to improve cognition and mood. Here we show that inducible genetic expansion of the population of adult-born neurons through enhancing their survival improves performance in a specific cognitive task in which two similar contexts need to be distinguished. Mice with increased adult hippocampal neurogenesis show normal object recognition, spatial learning, contextual fear conditioning and extinction learning but are more efficient in differentiating between overlapping contextual representations, which is indicative of enhanced pattern separation. Furthermore, stimulation of adult hippocampal neurogenesis, when combined with an intervention such as voluntary exercise, produces a robust increase in exploratory behaviour. However, increasing adult hippocampal neurogenesis alone does not produce a behavioural response like that induced by anxiolytic agents or antidepressants. Together, our findings suggest that strategies that are designed to increase adult hippocampal neurogenesis specifically, by targeting the cell death of adult-born neurons or by other mechanisms, may have therapeutic potential for reversing impairments in pattern separation and dentate gyrus dysfunction such as those seen during normal ageing.

Baxbeta: a constitutively active human Bax isoform that is under tight regulatory control by the proteasomal degradation mechanism.

Although mRNAs of multiple isoforms of Bax, which encodes a central regulator of apoptosis signaling, have been reported, only Baxalpha protein has been well documented and studied. Baxalpha exists in latent form and is activated upon apoptosis induction through conformational changes. Here we demonstrate that Baxbeta protein is ubiquitously present among human cells, but its activity is restricted through stringent regulation by proteasomal degradation. In contrast to Baxalpha, native Baxbeta spontaneously integrates into mitochondrial membrane and is highly potent in inducing cytochrome c release from mitochondria. Remarkably, Baxbeta protein is upregulated by apoptotic stimuli via inhibition of its ubiquitination process, and stable expression of Baxbeta in HCT116-Bax(-/-) cells restores their sensitivity to multiple stimuli. Baxbeta associates with and promotes Baxalpha activation. Moreover, selective knockdown of Baxbeta desensitizes HCT116-Bax(+/-) cells to Bax-dependent apoptosis signaling. These observations underscore the plasticity of human Bax in serving its role as a "gatekeeper" for apoptosis.

Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury.

Effective clearance of inflammatory cells is required for resolution of inflammation. Here, we show in vivo evidence that apoptosis and reverse transendothelial migration (rTEM) are important mechanisms in eliminating neutrophils and facilitating recovery following ischemia/reperfusion injury (IRI) of the kidney. The clearance of neutrophils was delayed in the Bax knockout (KO)(BM) → wild-type (WT) chimera in which bone marrow derived cells are partially resistant to apoptosis, compared to WT(BM) → WT mice. These mice also showed delayed functional, histological recovery, increased tissue cytokines, and accelerated fibrosis. The circulating intercellular adhesion molecule-1 (ICAM-1)⁺ Gr-1⁺ neutrophils displaying rTEM phenotype increased during the recovery phase and blockade of junctional adhesion molecule-C (JAM-C), a negative regulator of rTEM, resulted in an increase in circulating ICAM-1⁺ neutrophils, faster resolution of inflammation and recovery. The presence of Tamm-Horsfall protein (THP) in circulating ICAM-1⁺ neutrophils could suggest that they are derived from injured kidneys. In conclusion, we suggest that apoptosis and rTEM are critically involved in the clearance mechanisms of neutrophils during the recovery phase of IRI.

Loss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1).

Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX.

Proapoptotic Bak and Bax guard against fatal systemic and organ-specific autoimmune disease.

Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak(-/-)bax(-/-) mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.

BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore.

BAX, the BCL-2-associated X protein, is a cardinal proapoptotic member of the BCL-2 family, which regulates the critical balance between cellular life and death. Because so many medical conditions can be categorized as diseases of either too many or too few cells, dissecting the biochemistry of BCL-2 family proteins and developing pharmacological strategies to target them have become high priority scientific objectives. Here, we focus on BAX, a latent, cytosolic and monomeric protein that transforms into a lethal mitochondrial oligomer in response to cellular stress. New insights into the structural location of BAX's 'on switch', and the multi-step conformational changes that ensue upon BAX activation, are providing fresh opportunities to modulate BAX for potential benefit in human diseases characterized by pathologic cell survival or unwanted cellular demise.

Regulation of mitochondrial ceramide distribution by members of the BCL-2 family.

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profile in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fibroblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosine-containing species.

Programmed cell death of retinal cone bipolar cells is independent of afferent or target control.

Programmed cell death contributes to the histogenesis of the nervous system, and is believed to be modulated through the sustaining effects of afferents and targets during the period of synaptogenesis. Cone bipolar cells undergo programmed cell death during development, and we confirm that the numbers of three different types are increased when the pro-apoptotic Bax gene is knocked out. When their cone afferents are selectively eliminated, or when the population of retinal ganglion cells is increased, however, cone bipolar cell number remains unchanged. Programmed cell death of the cone bipolar cell populations, therefore, may be modulated cell-intrinsically rather than via interactions with these synaptic partners.

Airway mesenchymal cell death by mevalonate cascade inhibition: integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins.

HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1α) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX(-/-)/BAK(-/-) murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease.

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

DNA damage and ER stress contribute to oblongifolin C-induced cell killing in Bax/Bak-deficient cells.

A key clinical problem in oncology is the treatment of apoptosis-resistant tumors. Tumor cells deficient in both of the proapoptotic proteins Bax and Bak are protected against most chemotherapeutic drug-induced apoptosis. We report here that a natural compound, oblongifolin C (OC), effectively eliminates Bax/Bak-deficient murine embryonic fibroblasts and colon carcinoma HCT116 cells. OC not only triggers DNA double-strand breaks and DNA damage response, but also inhibits repair of DNA damage. In addition, OC induces ER stress through upregulation of the transcription factor CHOP and activation of JNK kinases. Upon treatment with OC, cells undergo Bax/Bak-independent, caspase-mediated apoptosis. Taken together, our data establish a rationale for the broad use of OC to treat apoptosis deficient tumors.

Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function.

Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

Tie2(+) bone marrow endothelial cells regulate hematopoietic stem cell regeneration following radiation injury.

Hematopoietic stem cells (HSCs) reside in proximity to bone marrow endothelial cells (BM ECs) and maintenance of the HSC pool is dependent upon EC-mediated c-kit signaling. Here, we used genetic models to determine whether radioprotection of BM ECs could facilitate hematopoietic regeneration following radiation-induced myelosuppression. We developed mice bearing deletion of the proapoptotic proteins, BAK and BAX, in Tie2(+) ECs and HSCs (Tie2Bak/Bax(Fl/-) mice) and compared their hematopoietic recovery following total body irradiation (TBI) with mice which retained Bax in Tie2(+) cells. Mice bearing deletion of Bak and Bax in Tie2(+) cells demonstrated protection of BM HSCs, preserved BM vasculature, and 100% survival following lethal dose TBI. In contrast, mice that retained Bax expression in Tie2(+) cells demonstrated depletion of BM HSCs, disrupted BM vasculature, and 10% survival post-TBI. In a complementary study, VEcadherinBak/Bax(Fl/-) mice, which lack Bak and Bax in VEcadherin(+) ECs, also demonstrated increased recovery of BM stem/progenitor cells following TBI compared to mice which retained Bax in VEcadherin(+) ECs. Importantly, chimeric mice that lacked Bak and Bax in HSCs but retained Bak and Bax in BM ECs displayed significantly decreased HSC content and survival following TBI compared to mice lacking Bak and Bax in both HSCs and BM ECs. These data suggest that the hematopoietic response to ionizing radiation is dependent upon HSC-autonomous responses but is regulated by BM EC-mediated mechanisms. Therefore, BM ECs may be therapeutically targeted as a means to augment hematopoietic reconstitution following myelosuppression.

Disruption of dentate gyrus blocks effect of visual input on spatial firing of CA1 neurons.

The role of dentate gyrus in hippocampal mnemonic processing is uncertain. One proposed role of dentate gyrus is binding internally generated spatial representation with sensory information on external landmarks. To test this hypothesis, we compared effects of visual input on spatial firing of CA1 neurons in Bax knock-out mice in which dentate gyrus neural circuitry is selectively disrupted. Whereas spatial selectivity of CA1 neuronal firing was significantly higher under normal illumination than complete darkness in wild-type mice, it was similarly low in both illumination conditions in Bax knock-out mice. Also, whereas the spatial location of neuronal firing was more stably maintained in the light than in the dark condition in wild-type mice, it was similarly unstable in both illumination conditions in Bax knock-out mice. These results show that visual input allows selective and stable spatial firing of CA1 neurons in normal animals, but this effect is lost if dentate gyrus neural circuitry is disrupted. Our results provide empirical support for the proposed role of dentate gyrus in aligning internally generated spatial representation to external landmarks in building a unified representation of external space.

MFG-E8 mediates primary phagocytosis of viable neurons during neuroinflammation.

Milk-fat globule EGF factor-8 (MFG-E8, SED1, lactadherin) is known to mediate the phagocytic removal of apoptotic cells by bridging phosphatidylserine (PS)-exposing cells and the vitronectin receptor (VR) on phagocytes. However, we show here that MFG-E8 can mediate phagocytosis of viable neurons during neuroinflammation induced by lipopolysaccharide (LPS), thereby causing neuronal death. In vitro, inflammatory neuronal loss is independent of apoptotic pathways, and is inhibited by blocking the PS/MFG-E8/VR pathway (by adding PS blocking antibodies, annexin V, mutant MFG-E8 unable to bind VR, or VR antagonist). Neuronal loss is absent in Mfge8 knock-out cultures, but restored by adding recombinant MFG-E8, without affecting inflammation. In vivo, LPS-induced neuronal loss is reduced in the striatum of Mfge8 knock-out mice or by coinjection of an MFG-E8 receptor (VR) inhibitor into the rat striatum. Our data show that blocking MFG-E8-dependent phagocytosis preserves live neurons, implying that phagocytosis actively contributes to neuronal death during brain inflammation.

Bax and Bak have critical roles in ischemic acute kidney injury in global and proximal tubule-specific knockout mouse models.

Bax and Bak, two pro-apoptotic Bcl-2 family proteins, have been implicated in acute kidney injury following renal ischemia/reperfusion; however, definitive evidence for a role of these genes in the disease process is lacking. Here we first examined two Bax-deficient mouse models and found that only conditional Bax deletion specifically from proximal tubules could ameliorate ischemic acute kidney injury. Global (whole mouse) knockout of Bax enhanced neutrophil infiltration without significant effect on kidney injury. In contrast, global knockout of Bak protected mice from ischemic acute kidney injury with improved renal function. Interestingly, in these models, Bax or Bak knockout attenuated renal tubular cell apoptosis without significantly affecting necrotic tubular damage. Cytochrome c release in ischemic acute kidney injury was also suppressed in conditional Bax- or global Bak-knockout mice. In addition, Bak deficiency prevented mitochondrial fragmentation in ischemic acute kidney injury. Thus, our gene-knockout studies support a critical role of Bax and Bak in tubular cell apoptosis in ischemic acute kidney. Furthermore, necrosis and apoptosis have distinguishable regulatory functions.

Bcl-2 allows effector and memory CD8+ T cells to tolerate higher expression of Bim.

As acute infections resolve, most effector CD8(+) T cells die, whereas some persist and become memory T cells. Recent work showed that subsets of effector CD8(+) T cells, identified by reciprocal expression of killer cell lectin-like receptor G1 (KLRG1) and CD127, have different lifespans. Similar to previous reports, we found that effector CD8(+) T cells reported to have a longer lifespan (i.e., KLRG1(low)CD127(high)) have increased levels of Bcl-2 compared with their shorter-lived KLRG1(high)CD127(low) counterparts. Surprisingly, we found that these effector KLRG1(low)CD127(high) CD8(+) T cells also had increased levels of Bim compared with KLRG1(high)CD127(low) cells. Similar effects were observed in memory cells, in which CD8(+) central memory T cells expressed higher levels of Bim and Bcl-2 than did CD8(+) effector memory T cells. Using both pharmacologic and genetic approaches, we found that survival of both subsets of effector and memory CD8(+) T cells required Bcl-2 to combat the proapoptotic activity of Bim. Interestingly, inhibition or absence of Bcl-2 led to significantly decreased expression of Bim in surviving effector and memory T cells. In addition, manipulation of Bcl-2 levels by IL-7 or IL-15 also affected expression of Bim in effector CD8(+) T cells. Finally, we found that Bim levels were significantly increased in effector CD8(+) T cells lacking Bax and Bak. Together, these data indicate that cells having the highest levels of Bim are selected against during contraction of the response and that Bcl-2 determines the level of Bim that effector and memory T cells can tolerate.