Metabolic control of adult neural stem cell self-renewal by the mitochondrial protease YME1L.

The transition between quiescence and activation in neural stem and progenitor cells (NSPCs) is coupled with reversible changes in energy metabolism with key implications for lifelong NSPC self-renewal and neurogenesis. How this metabolic plasticity is ensured between NSPC activity states is unclear. We find that a state-specific rewiring of the mitochondrial proteome by the i-AAA peptidase YME1L is required to preserve NSPC self-renewal. YME1L controls the abundance of numerous mitochondrial substrates in quiescent NSPCs, and its deletion activates a differentiation program characterized by broad metabolic changes causing the irreversible shift away from a fatty-acid-oxidation-dependent state. Conditional Yme1l deletion in adult NSPCs in vivo results in defective self-renewal and premature differentiation, ultimately leading to NSPC pool depletion. Our results disclose an important role for YME1L in coordinating the switch between metabolic states of NSPCs and suggest that NSPC fate is regulated by compartmentalized changes in protein network dynamics.

Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide.

The cytosolic appearance and propagation of bacteria cause overwhelming cellular stress responses that induce apoptosis under normal conditions. Therefore, successful bacterial colonization depends on the ability of intracellular pathogens to block apoptosis and to safeguard bacterial replicative niches. Here, we show that the cytosolic Gram-negative bacterium Shigella flexneri stalls apoptosis by inhibiting effector caspase activity. Our data identified lipopolysaccharide (LPS) as a bona fide effector caspase inhibitor that directly binds caspases by involving its O-antigen (O Ag) moiety. Bacterial strains that lacked the O Ag or failed to replicate within the cytosol were incapable of blocking apoptosis and exhibited reduced virulence in a murine model of bacterial infection. Our findings demonstrate how Shigella inhibits pro-apoptotic caspase activity, effectively delays coordinated host-cell demise and supports its intracellular propagation. Next to the recently discovered pro-inflammatory role of cytosolic LPS, our data reveal a distinct mode of LPS action that, through the disruption of the early coordinated non-lytic cell death response, ultimately supports the inflammatory breakdown of infected cells at later time points.

Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis.

Caspase-8 is the initiator caspase of extrinsic apoptosis1,2 and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality3, which can be rescued by deletion of either Ripk3 or Mlkl4-6. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8-/- mice3,7, Casp8C362S/C362S mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8C362S/C362S mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice8. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1ß. Both embryonic lethality and premature death were completely rescued in Casp8C362S/C362SMlkl-/-Asc-/- or Casp8C362S/C362SMlkl-/-Casp1-/- mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.

BAX/BAK-Induced Apoptosis Results in Caspase-8-Dependent IL-1ß Maturation in Macrophages.

IL-1ß is a cytokine of pivotal importance to the orchestration of inflammatory responses. Synthesized as an inactive pro-cytokine, IL-1ß requires proteolytic maturation to gain biological activity. Here, we identify intrinsic apoptosis as a non-canonical trigger of IL-1ß maturation. Guided by the discovery of the immunomodulatory activity of vioprolides, cyclic peptides isolated from myxobacteria, we observe IL-1ß maturation independent of canonical inflammasome pathways, yet dependent on intrinsic apoptosis. Mechanistically, vioprolides inhibit MCL-1 and BCL2, which in turn triggers BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Induction of MOMP results in the release of pro-apoptotic factors initiating intrinsic apoptosis, as well as the depletion of IAPs (inhibitors of apoptosis proteins). IAP depletion, in turn, operates upstream of ripoptosome complex formation, subsequently resulting in caspase-8-dependent IL-1ß maturation. These results establish the ripoptosome/caspase-8 complex as a pro-inflammatory checkpoint that senses the perturbation of mitochondrial integrity.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

IAP antagonization promotes inflammatory destruction of vascular endothelium.

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Embelin inhibits endothelial mitochondrial respiration and impairs neoangiogenesis during tumor growth and wound healing.

In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.

Dimethylfumarate induces apoptosis in human mast cells.

Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC-1 and in primary cord blood-derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca-MF). Our data show that only DMF potently induces apoptotic cell death in HMC-1 cells and CBMCs. DMF-mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase-9 and caspase-6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL- and dexamethasone-induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells.

Obesity promotes liver carcinogenesis via Mcl-1 stabilization independent of IL-6Rα signaling.

Obesity increases the incidence of hepatocellular carcinoma (HCC) development in part through the activation of obesity-associated proinflammatory signaling. Here, we show that in lean mice, abrogation of IL-6Rα signaling protects against diethylnitrosamine (DEN)-induced HCC development. HCC protection occurs via Mcl-1 destabilization, thus promoting hepatocyte apoptosis. IL-6 regulates Mcl-1 stability via the inhibition of PP-1α expression, promoting GSK-3ß inactivation. In addition, IL-6 suppresses expression of the Mcl-1 E3 ligase (Mule). Consequently, IL-6Rα deficiency activates PP-1α and Mule expression, resulting in increased Mcl-1 turnover and protection against HCC development. In contrast, in obesity, inhibition of PP-1α and Mule expression, leading to Mcl-1 stabilization, occurs independently of IL-6 signaling. Collectively, this study provides evidence that obesity inhibits hepatocyte apoptosis through Mcl-1 stabilization independent of IL-6 signaling, thus promoting liver carcinogenesis.

Anti-Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) differentially regulate apoptosis in normal and neoplastic human basophils.

Basophilia is associated with allergic and parasitic diseases and advanced chronic myeloid leukemia. In the present study, we characterized the expression and function of the death receptors Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in basophils from healthy donors compared to neoplastic basophils. Peripheral blood basophils obtained from healthy donors (HD-PBB) and from patients with chronic myeloid leukemia (CML-PBB) were found to express high levels of Fas/CD95 and low levels of TRAIL-R2, whereas the basophil-like chronic myeloid leukemia cell line KU-812 expressed significant levels of TRAIL-R1 and TRAIL-R2. HD-PBB underwent apoptosis in response to anti-Fas/CD95, but showed resistance to TRAIL, unless they were co-treated with actinomycin D. Interestingly, CML-PBB and KU-812 cells exhibited the opposite response pattern with resistance to anti-Fas/CD95, but significant susceptibility to TRAIL-induced apoptosis. Our data show that anti-Fas/CD95 and TRAIL differentially regulate apoptosis of normal and neoplastic human basophils, which may direct the development of novel therapeutic strategies.

Interleukin-6 is essential for zwitterionic polysaccharide-mediated abscess formation.

Abscess formation associated with secondary peritonitis causes severe morbidity and can be fatal. Formation of abscesses requires the presence of CD4+ T-cells. Zwitterionic polysaccharides (ZPSs) represent a novel class of immunomodulatory bacterial antigens that stimulate CD4+ T-cells in a major histocompatibility complex (MHC) class II-dependent manner. The capsular polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 possesses a zwitterionic charge with free amino groups and promotes T-cell-dependent abscess formation in an experimental mouse model. So far, nothing is known about the function of Interleukin (IL)-6 in intraperitoneal abscess formation. Here, we demonstrate that macrophages and dendritic cells (DCs), the most prevalent professional antigen-presenting cells involved in the formation of abscesses, secrete Interleukin (IL)-6 and are incorporated in the abscess capsule. Sp1 inhibits apoptosis of CD4+ T-cells and causes IL-17 expression by CD4+ T-cells in an IL-6-dependent manner. Abrogation of the Sp1-induced pleiotropic effects of IL-6 in IL-6-deficient mice and mice treated with an IL-6-specific neutralizing antibody results in significant inhibition of abscess formation. The data delineate the essential role of IL-6 in the linkage of innate and adaptive immunity in polysaccharide-mediated abscess formation.

PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR.

Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non-small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss.