Rachael M. Lane

Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

Deletion of Bak and Bax, the effectors of mitochondrial apoptosis, does not affect platelet production, however, loss of prosurvival Bcl-xL results in megakaryocyte apoptosis and failure of platelet shedding.

Mcl-1 and Bcl-xL coordinately regulate megakaryocyte survival

Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.

Mcl-1 and Bcl-xL co-ordinately regulate megakaryocyte survival

Mature megakaryocytes depend on the function of Bcl-x(L), a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-x(L) does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1(Pf4Δ/Pf4Δ) animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-x(L), and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-x(L) in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-x(Pf4Δ/Pf4Δ) Mcl-1(Pf4Δ/Pf4Δ) embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-x(L) and Mcl-1 is essential for the viability of the megakaryocyte lineage.

IL-18 Production from the NLRP1 Inflammasome Prevents Obesity and Metabolic Syndrome

Interleukin-18 (IL-18) is activated by Caspase-1 in inflammasome complexes and has anti-obesity effects; however, it is not known which inflammasome regulates this process. We found that mice lacking the NLRP1 inflammasome phenocopy mice lacking IL-18, with spontaneous obesity due to intrinsic lipid accumulation. This is exacerbated when the mice are fed a high-fat diet (HFD) or a high-protein diet, but not when mice are fed a HFD with low energy density (high fiber). Furthermore, mice with an activating mutation in NLRP1, and hence increased IL-18, have decreased adiposity and are resistant to diet-induced metabolic dysfunction. Feeding these mice a HFD further increased plasma IL-18 concentrations and strikingly resulted in loss of adipose tissue mass and fatal cachexia, which could be prevented by genetic deletion of IL-18. Thus, NLRP1 is an innate immune sensor that functions in the context of metabolic stress to produce IL-18, preventing obesity and metabolic syndrome.

Platelet production proceeds independently of the intrinsic and extrinsic apoptosis pathways

BH3 mimetic drugs that target BCL-2 family pro-survival proteins to induce tumour cell apoptosis represent a new era in cancer therapy. Clinical trials of navitoclax (ABT-263, which targets BCL-2, BCL-XL and BCL-W) have shown great promise, but encountered dose-limiting thrombocytopenia. Recent work has demonstrated that this is due to the inhibition of BCL-XL, which is essential for platelet survival. These findings raise new questions about the established model of platelet shedding by megakaryocytes, which is thought to be an apoptotic process. Here we generate mice with megakaryocyte-specific deletions of the essential mediators of extrinsic (Caspase-8) and intrinsic (BAK/BAX) apoptosis. We show that megakaryocytes possess a Fas ligand-inducible extrinsic apoptosis pathway. However, Fas activation does not stimulate platelet production, rather, it triggers Caspase-8-mediated killing. Combined loss of Caspase-8/BAK/BAX does not impair thrombopoiesis, but can protect megakaryocytes from death in mice infected with lymphocytic choriomeningitis virus. Thus, apoptosis is dispensable for platelet biogenesis.

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-XL and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-XL for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-XL. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.

Physiological restraint of Bak by Bcl-xL is essential for cell survival

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.

BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis

The great escape Mitochondrial DNA (mtDNA) is a potent damage-associated molecular pattern that, if it reaches the cytoplasm or extracellular milieu, triggers innate immune pathways. mtDNA signaling has been implicated in a wide range of diseases; however, the mechanisms of mtDNA release are unclear, and the process has not been observed in real time thus far. McArthur et al. used live-cell lattice light-sheet microscopy to look at mtDNA release during intrinsic apoptosis. Activation of the pro-death proteins BAK and BAX resulted in the formation of large macro-pores in the mitochondrial outer membrane. These massive holes caused the inner mitochondrial membrane to balloon out into the cytoplasm, resulting in mitochondrial herniation. This process allowed the contents of the mitochondrial matrix, including mtDNA, to escape into the cytoplasm. Science, this issue p. eaao6047 Mitochondrial DNA is released from mitochondria in apoptotic cells as a result of BAK/BAX-induced mitochondrial herniation. INTRODUCTION There has been an explosion of interest in the role of cell death pathways and damage-associated molecular pattern (DAMP) signaling in shaping inflammatory and immune responses. Mitochondria are central to the intrinsic apoptosis pathway, the classical form of programmed cell death. Several mitochondrial constituents have been implicated as DAMPs, including mitochondrial DNA (mtDNA). Recent work has shown that activation of intrinsic BAK and BAX–mediated apoptosis results in mtDNA-dependent triggering of the innate immune cGAS/STING pathway, resulting in type I interferon production by dying cells. The apoptotic caspase cascade normally functions to suppress this mtDNA-induced cGAS/STING signaling, rendering apoptosis “immunologically silent.” RATIONALE It is thought that during apoptosis, mtDNA is released into the cytoplasm. In addition to apoptosis, loss of mtDNA from the matrix has been associated with conditions including HIV and dengue infection, calcium overload, irradiation, or inflammatory diseases such as systemic lupus erythematosus or rheumatoid arthritis. However, mtDNA escape from the mitochondria has not been documented in real time. RESULTS Using a combination of live-cell lattice light-sheet microscopy, 3D structured illumination microscopy, correlative light electron microscopy, and electron cryotomography, we found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner membrane an outlet through which it herniated, carrying with it mitochondrial matrix components, including the mitochondrial genome. A subset of the herniated inner membranes lost their integrity, allowing mtDNA to be exposed to the cytoplasm. CONCLUSION An extensive literature suggests that mtDNA is found outside the mitochondria—and, indeed, outside the cell—in a wide range of circumstances. Our study provides a mechanistic description of its release from the mitochondria. mtDNA release from mitochondria during apoptosis occurs irrespective of caspase activity, but in normal cells, caspases attenuate the subsequent cGAS/STING-mediated interferon response by driving rapid cellular collapse and clearance. Mitochondrial herniation might represent a general mechanism of mtDNA escape. In addition to BAK and BAX oligomerization, there may be alternative triggers—for example, other pore-forming proteins (host- or pathogen-derived) or mitochondrial stresses—that lead to the occurrence of this phenomenon. Schematic of apoptotic mitochondrial herniation. Inset images show the key stages as captured by lattice light-sheet microscopy, structured illumination microscopy, and correlative light and electron microscopy. Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion

The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.