RESUMEN
Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of ß-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca2+ stores and store-operated Ca2+ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca2+ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca2+ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.
Asunto(s)
Tetraploidía , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Senescencia Celular , Células Clonales , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína X Asociada a bcl-2/deficienciaRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Caspasa 3/deficiencia , Transducción de Señal/fisiología , Vesículas Sinápticas/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína Letal Asociada a bcl/deficiencia , Animales , Caspasa 3/genética , Células Cultivadas , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Imagen Molecular/métodos , Técnicas de Cultivo de Órganos , Vesículas Sinápticas/genética , Vesículas Sinápticas/ultraestructura , Proteína X Asociada a bcl-2/genética , Proteína Letal Asociada a bcl/genéticaRESUMEN
DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
Asunto(s)
Mutación , Oocitos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reparación del ADN por Recombinación/genética , Aneuploidia , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Femenino , Fertilización , Genes bcl-2 , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/citología , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Removal of the Bax gene from mice completely protects the somas of retinal ganglion cells (RGCs) from apoptosis following optic nerve injury. This makes BAX a promising therapeutic target to prevent neurodegeneration. In this study, Bax+/- mice were used to test the hypothesis that lowering the quantity of BAX in RGCs would delay apoptosis following optic nerve injury. RGCs were damaged by performing optic nerve crush (ONC) and then immunostaining for phospho-cJUN, and quantitative PCR were used to monitor the status of the BAX activation mechanism in the months following injury. The apoptotic susceptibility of injured cells was directly tested by virally introducing GFP-BAX into Bax-/- RGCs after injury. The competency of quiescent RGCs to reactivate their BAX activation mechanism was tested by intravitreal injection of the JNK pathway agonist, anisomycin. Twenty-four weeks after ONC, Bax+/- mice had significantly less cell loss in their RGC layer than Bax+/+ mice 3 weeks after ONC. Bax+/- and Bax+/+ RGCs exhibited similar patterns of nuclear phospho-cJUN accumulation immediately after ONC, which persisted in Bax+/- RGCs for up to 7 weeks before abating. The transcriptional activation of BAX-activating genes was similar in Bax+/- and Bax+/+ RGCs following ONC. Intriguingly, cells deactivated their BAX activation mechanism between 7 and 12 weeks after crush. Introduction of GFP-BAX into Bax-/- cells at 4 weeks after ONC showed that these cells had a nearly normal capacity to activate this protein, but this capacity was lost 8 weeks after crush. Collectively, these data suggest that 8-12 weeks after crush, damaged cells no longer displayed increased susceptibility to BAX activation relative to their naïve counterparts. In this same timeframe, retinal glial activation and the signaling of the pro-apoptotic JNK pathway also abated. Quiescent RGCs did not show a timely reactivation of their JNK pathway following intravitreal injection with anisomycin. These findings demonstrate that lowering the quantity of BAX in RGCs is neuroprotective after acute injury. Damaged RGCs enter a quiescent state months after injury and are no longer responsive to an apoptotic stimulus. Quiescent RGCs will require rejuvenation to reacquire functionality.
Asunto(s)
Traumatismos del Nervio Óptico/metabolismo , Nervio Óptico/metabolismo , Células Ganglionares de la Retina/citología , Proteína X Asociada a bcl-2/deficiencia , Animales , Apoptosis/fisiología , Modelos Animales de Enfermedad , Ratones Transgénicos , Neuroprotección/fisiología , Traumatismos del Nervio Óptico/tratamiento farmacológico , Retina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
IMPACT STATEMENT: Bax induces mitochondria-dependent programed cell death. While cytotoxic drugs activating Bax have been developed for cancer treatment, clinically effective therapeutics suppressing Bax-induced cell death rescuing essential cells have not been developed. This mini-review will summarize previously reported Bax inhibitors including peptides, small compounds, and antibodies. We will discuss potential applications and the future direction of these Bax inhibitors.
Asunto(s)
Apoptosis/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/uso terapéutico , Péptidos de Penetración Celular/toxicidad , Células Cultivadas , Modelos Animales de Enfermedad , Portadores de Fármacos , Diseño de Fármacos , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Autoantígeno Ku/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Poro de Transición de la Permeabilidad Mitocondrial , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/patología , Preservación de Órganos/métodos , Pinocitosis , Multimerización de Proteína/efectos de los fármacos , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/patología , Ratas , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/patología , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/inmunología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Withdrawal of the growth factor interleukin-3 (IL-3) from IL-3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprived Bax-/-Bak1-/- cells remain viable, but arrest and shrink. It was reported that withdrawal of IL-3 from Bax-/-Bak1-/- cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein (Txnip), which is induced in IL-3-dependent myeloid cells when growth factor is removed. We mutated Atg5 and Txnip by CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrested Bax-/-Bak1-/- cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL-3 withdrawal. Surprisingly, Atg5-deficient Bax/Bak1 double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL-3. Thus, Bax-/-Bak1-/- myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.
Asunto(s)
Glucosa/metabolismo , Glucosa/farmacología , Interleucina-3/deficiencia , Células Mieloides/citología , Células Mieloides/metabolismo , Animales , Apoptosis/fisiología , Proteína 5 Relacionada con la Autofagia/deficiencia , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Supervivencia Celular/fisiología , Técnicas de Inactivación de Genes , Interleucina-3/metabolismo , Ratones , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Immunosuppressive myeloid-derived suppressor cells (MDSC) subvert antitumor immunity and limit the efficacy of chimeric antigen receptor T cells (CAR-T). Previously, we reported that the GM-CSF/JAK2/STAT3 axis drives liver-associated MDSC (L-MDSC) proliferation and blockade of this axis rescued antitumor immunity. We extended these findings in our murine liver metastasis (LM) model, by treating tumor-bearing mice with STAT3 inhibitors (STATTIC or BBI608) to further our understanding of how STAT3 drives L-MDSC suppressive function. STAT3 inhibition caused significant reduction of tumor burden as well as L-MDSC frequencies due to decrease in pSTAT3 levels. L-MDSC isolated from STATTIC or BBI608-treated mice had significantly reduced suppressive function. STAT3 inhibition of L-MDSC was associated with enhanced antitumor activity of CAR-T. Further investigation demonstrated activation of apoptotic signaling pathways in L-MDSC following STAT3 inhibition as evidenced by an upregulation of the pro-apoptotic proteins Bax, cleaved caspase-3, and downregulation of the anti-apoptotic protein Bcl-2. Accordingly, there was also a decrease of pro-survival markers, pErk and pAkt, and an increase in pro-death marker, Fas, with activation of downstream JNK and p38 MAPK. These findings represent a previously unrecognized link between STAT3 inhibition and Fas-induced apoptosis of MDSCs. Our findings suggest that inhibiting STAT3 has potential clinical application for enhancing the efficacy of CAR-T cells in LM through modulation of L-MDSC.
Asunto(s)
Adenocarcinoma/secundario , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Benzofuranos/uso terapéutico , Óxidos S-Cíclicos/uso terapéutico , Neoplasias Hepáticas Experimentales/secundario , Terapia Molecular Dirigida , Células Supresoras de Origen Mieloide/patología , Naftoquinonas/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Proteína X Asociada a bcl-2/fisiología , Receptor fas/fisiología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Benzofuranos/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Óxidos S-Cíclicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica , Inmunoterapia , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Naftoquinonas/farmacología , Proteínas de Neoplasias/fisiología , Factor de Transcripción STAT3/fisiología , Transducción de Señal , Organismos Libres de Patógenos Específicos , Carga Tumoral , Escape del Tumor/fisiología , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Apoptotic cells expose Phosphatidylserine (PS), that serves as an "eat me" signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Fosfatidilserinas/farmacología , Células Receptoras Sensoriales/efectos de los fármacos , Degeneración Walleriana/metabolismo , Adenosina Trifosfato/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas del Dominio Armadillo/deficiencia , Proteínas del Dominio Armadillo/genética , Axotomía , Biomarcadores/metabolismo , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Embrión de Mamíferos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Expresión Génica , Ratones , Ratones Noqueados , Técnicas Analíticas Microfluídicas , Factor de Crecimiento Nervioso/farmacología , Plasticidad Neuronal/genética , Fosfatidilserinas/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología , Técnicas de Cultivo de Tejidos , Vincristina/farmacología , Degeneración Walleriana/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
The t-SNARE protein SNAP23 conventionally functions as a component of the cellular machinery required for intracellular transport vesicle fusion with target membranes and has been implicated in the regulation of fasting glucose levels, BMI, and type 2 diabetes. Surprisingly, we observed that adipocyte-specific KO of SNAP23 in mice resulted in a temporal development of severe generalized lipodystrophy associated with adipose tissue inflammation, insulin resistance, hyperglycemia, liver steatosis, and early death. This resulted from adipocyte cell death associated with an inhibition of macroautophagy and lysosomal degradation of the proapoptotic regulator BAX, with increased BAX activation. BAX colocalized with LC3-positive autophagic vacuoles and was increased upon treatment with lysosome inhibitors. Moreover, BAX deficiency suppressed the lipodystrophic phenotype in the adipocyte-specific SNAP23-KO mice and prevented cell death. In addition, ATG9 deficiency phenocopied SNAP23 deficiency, whereas ATG7 deficiency had no effect on BAX protein levels, BAX activation, or apoptotic cell death. These data demonstrate a role for SNAP23 in the control of macroautophagy and programmed cell death through an ATG9-dependent, but ATG7-independent, pathway regulating BAX protein levels and BAX activation.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Células 3T3-L1 , Animales , Apoptosis/fisiología , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia/deficiencia , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Lipodistrofia/metabolismo , Lipodistrofia/patología , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteolisis , Proteínas Qb-SNARE/deficiencia , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/deficiencia , Proteínas Qc-SNARE/genética , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Bax confirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuralgia/etiología , Traumatismos de los Nervios Periféricos/complicaciones , Células del Asta Posterior/metabolismo , Células del Asta Posterior/patología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Apoptosis , Supervivencia Celular , Dolor Crónico/etiología , Dolor Crónico/patología , Dolor Crónico/fisiopatología , Regulación hacia Abajo , Eliminación de Gen , Glutamatos/metabolismo , Masculino , Ratones Endogámicos C57BL , Inhibición Neural , Neuralgia/patología , Neuralgia/fisiopatología , Neuroprotección , Traumatismos de los Nervios Periféricos/fisiopatología , Transporte de Proteínas , Transducción de Señal , Transmisión Sináptica , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/metabolismo , Ácido gamma-Aminobutírico/biosíntesisRESUMEN
Fetal alcohol spectrum disorder is associated with defects in neuronal generation, migration, and differentiation. The present study tested the hypothesis that ethanol exposure during the period of naturally occurring neuronal death causes a time- and Bax-dependent neuronal loss. Wild-type and Bax knockout mice were given a pair of injections (two hours apart) of ethanol (2.5â¯g/kg) or saline on postnatal day (P) 4, P7, P10, or P13. Mean blood ethanol concentration was 435â¯mg/dl one hour after the second injection. The total numbers of neurons in individual layers of somatosensory cortex were stereologically determined in 30-day-old mice and the expressions of active caspase 3 immunopositivity and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) were determined 2-36 h after the first injection. On P30, ethanol caused up to 36% neuronal loss in cortical laminae of wild-type mice. In contrast, no ethanol-induced loss was detected in Bax knockout mice. Ethanol also caused an acute bilaminar (layers II/III and V) increase in caspase 3 immunoexpression and TUNEL in wild-type mice. The changes in the expression of these markers were age- and lamina-dependent. No ethanol-induced expression of caspase 3 or TUNEL was detected in Bax knockout animals. Thus, ethanol-induced death of cortical neurons is Bax-dependent, occurs concurrently in all layers, but does not correspond to lamina- and age-dependent expression of DNA fragmentation. Both ethanol-induced and naturally occurring death of cortical neurons rely on caspase 3-dependent and independent mechanisms.
Asunto(s)
Depresores del Sistema Nervioso Central/administración & dosificación , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/crecimiento & desarrollo , Etanol/efectos adversos , Neuronas/efectos de los fármacos , Proteína X Asociada a bcl-2/deficiencia , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Fragmentación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Trastornos del Espectro Alcohólico Fetal/metabolismo , Trastornos del Espectro Alcohólico Fetal/patología , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
Cells deficient in the pro-death Bcl-2 family members Bax and Bak are known to be resistant to apoptotic cell death, and previous we have shown that these two effectors are also needed for mitochondrial-dependent cellular necrosis (Karch et al., 2013). Here we show that mouse embryonic fibroblasts deficient in Bax/Bak1 are resistant to the third major form of cell death associated with autophagy through a mechanism involving lysosome permeability. Indeed, specifically targeting Bax only to the lysosome restores autophagic cell death in Bax/Bak1 null cells. Moreover, a monomeric-only mutant form of Bax is sufficient to increase lysosomal membrane permeability and restore autophagic cell death in Bax/Bak1 double-deleted mouse embryonic fibroblasts. Finally, increasing lysosomal permeability through a lysomotropic detergent in cells devoid of Bax/Bak1 restores autophagic cell death, collectively indicting that Bax/Bak integrate all major forms of cell death through direct effects on membrane permeability of multiple intracellular organelles.
Asunto(s)
Autofagia , Membrana Celular/metabolismo , Fibroblastos/fisiología , Lisosomas/metabolismo , Permeabilidad , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Células Cultivadas , Eliminación de Gen , Ratones , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína X Asociada a bcl-2/deficienciaRESUMEN
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) â wild-type (WT) chimera in which bone marrow derived cells are partially resistant to apoptosis, compared to WT(BM) â WT mice. These mice also showed delayed functional, histological recovery, increased tissue cytokines, and accelerated fibrosis. The circulating intercellular adhesion molecule-1 (ICAM-1)⺠Gr-1⺠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⺠neutrophils, faster resolution of inflammation and recovery. The presence of Tamm-Horsfall protein (THP) in circulating ICAM-1⺠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.
Asunto(s)
Lesión Renal Aguda/patología , Neutrófilos/metabolismo , Daño por Reperfusión/patología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/inmunología , Animales , Apoptosis/efectos de los fármacos , Quimiocinas/análisis , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Peróxido de Hidrógeno/toxicidad , Inmunoensayo , Molécula 1 de Adhesión Intercelular/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/citología , Neutrófilos/inmunología , Daño por Reperfusión/complicaciones , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Uromodulina/análisis , Uromodulina/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Interstitial fibrosis, a common pathological feature of chronic kidney diseases, is often associated with apoptosis in renal tissues. To determine the associated apoptotic pathway and its role in renal interstitial fibrosis, we established a mouse model in which Bax and Bak, two critical genes in the intrinsic pathway of apoptosis, were deleted specifically from kidney proximal tubules and used this model to examine renal apoptosis and interstitial fibrosis following unilateral urethral obstruction (UUO). It was shown that double knockout of Bax and Bak from proximal tubules attenuated renal tubular cell apoptosis and suppressed renal interstitial fibrosis in UUO. The results indicate that the intrinsic pathway of apoptosis contributes significantly to the tubular apoptosis and renal interstitial fibrosis in kidney diseases.
Asunto(s)
Apoptosis/genética , Túbulos Renales Proximales/metabolismo , Obstrucción Uretral/genética , Obstrucción Uretral/patología , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína X Asociada a bcl-2/deficiencia , Animales , Colágeno/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Fibrosis , Ratones , Ratones NoqueadosRESUMEN
The dentate gyrus (DG) principal cells are glutamatergic granule cells (GCs), and they are located in a compact cell layer. However, GCs are also present in the adjacent hilar region, but have been described in only a few studies. Therefore, we used the transcription factor prospero homeobox 1 (Prox1) to quantify GCs at postnatal day (PND) 16, 30, and 60 in a common mouse strain, C57BL/6J mice. At PND16, there was a large population of Prox1-immunoreactive (ir) hilar cells, with more in the septal than temporal hippocampus. At PND30 and 60, the size of the hilar Prox1-ir cell population was reduced. Similar numbers of hilar Prox1-expressing cells were observed in PND30 and 60 Swiss Webster mice. Prox1 is usually considered to be a marker of postmitotic GCs. However, many Prox1-ir hilar cells, especially at PND16, were not double-labeled with NeuN, a marker typically found in mature neurons. Most hilar Prox1-positive cells at PND16 co-expressed doublecortin (DCX) and calretinin, markers of immature GCs. Double-labeling with a marker of actively dividing cells, Ki67, was not detected. These results suggest that, surprisingly, a large population of cells in the hilus at PND16 are immature GCs (Type 2b and Type 3 cells). We also asked whether hilar Prox1-ir cell numbers are modifiable. To examine this issue, we conditionally deleted the proapoptotic gene BAX in Nestin-expressing cells at a time when there are numerous immature GCs in the hilus, PND2-8. When these mice were examined at PND60, the numbers of Prox1-ir hilar cells were significantly increased compared to control mice. However, deletion of BAX did not appear to change the proportion that co-expressed NeuN, suggesting that the size of the hilar Prox1-expressing population is modifiable. However, deleting BAX, a major developmental disruption, does not appear to change the proportion that ultimately becomes neurons.
Asunto(s)
Envejecimiento/fisiología , Giro Dentado/citología , Regulación del Desarrollo de la Expresión Génica/genética , Nestina/metabolismo , Neuronas/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Animales , Animales Recién Nacidos , Calbindina 2/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Nestina/genética , Neurogénesis/genética , Neuropéptidos/metabolismo , Especificidad de la Especie , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The proapoptotic protein Bak is implicated in the execution phase of apoptosis, a cell death program. Bak is essentially mitochondrial and during early steps of apoptosis undergoes conformational changes that lead to its full membrane integration in mitochondria and the subsequent liberation of pro-apoptotic mitochondrial proteins. Little is known about the partners and mechanisms implicated in the activation of Bak. We have recently shown that Bak is incorporated into a Voltage dependent anionic channel of type 2 (VDAC2)/Metaxin 1(Mtx1)/Metaxin 2 (Mtx2) multi-protein complex in both resting and dying cells. Here, we show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to a closer association with Mtx1. This change of partners is under the control of a tyrosine phosphorylation of Mtx1 by c-Abl.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/metabolismo , Glioma/patología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Eliminación de Gen , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Proteínas Proto-Oncogénicas/deficiencia , Trombocitopenia/genética , Proteína X Asociada a bcl-2/deficiencia , Animales , Proteínas Reguladoras de la Apoptosis/genética , Plaquetas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genotipo , Masculino , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Trombocitopenia/sangre , Proteína X Asociada a bcl-2/genéticaRESUMEN
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.
Asunto(s)
Apoptosis/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Dinaminas/genética , Fibroblastos/metabolismo , Dinámicas Mitocondriales/genética , Animales , Apoptosis/efectos de los fármacos , Factor Apoptótico 1 Activador de Proteasas/deficiencia , Factor Apoptótico 1 Activador de Proteasas/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Línea Celular , Citocromos c/metabolismo , Dinaminas/deficiencia , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Noqueados , Dinámicas Mitocondriales/efectos de los fármacos , Estrés Oxidativo , Transducción de Señal , Estaurosporina/farmacología , Tapsigargina/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genéticaRESUMEN
Angiogenesis is essential to match the size of blood vessel networks to the metabolic demands of growing tissues. While many genes and pathways necessary for regulating angiogenesis have been identified, those responsible for endothelial cell (EC) survival during angiogenesis remain largely unknown. We have investigated the in vivo role of myeloid cell leukemia 1 (MCL1), a pro-survival member of the BCL2 family, in EC survival during angiogenesis. EC-specific deletion of Mcl1 resulted in a dose-dependent increase in EC apoptosis in the angiogenic vasculature and a corresponding decline in vessel density. Our results suggest this apoptosis was independent of the BH3-only protein BIM. Despite the known link between apoptosis and blood vessel regression, this was not the cause of reduced vessel density observed in the absence of endothelial MCL1. Rather, the reduction in vessel density was linked to ectopic apoptosis in regions of the angiogenic vasculature where EC proliferation and new vessel growth occurs. We have therefore identified MCL1 as an essential survival factor for ECs that is required for blood vessel production during angiogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular , Células Cultivadas , Colágeno Tipo IV/metabolismo , Embrión de Mamíferos/citología , Células Endoteliales/citología , Riñón/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/deficiencia , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/deficiencia , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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