RESUMEN
In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP." In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.
Asunto(s)
Apoptosis , Caspasas , Supervivencia Celular , Mitocondrias , Apoptosis/fisiología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Caspasas/metabolismo , Supervivencia Celular/genética , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismoRESUMEN
CCR6+CXCR3+CCR4-CD4+ memory T cells, termed Th1*, are important for long-term immunity to Mycobacterium tuberculosis and the pathogenesis of autoimmune diseases. Th1* cells express a unique set of lineage-specific transcription factors characteristic of both Th1 and Th17 cells and display distinct gene expression profiles compared with other CD4+ T cell subsets. To examine molecules and signaling pathways important for the effector function of Th1* cells, we performed loss-of-function screening of genes selectively enriched in the Th1* subset. The genetic screen yielded candidates whose depletion significantly impaired TCR-induced IFN-γ production. These included genes previously linked to IFN-γ or M. tuberculosis susceptibility and novel candidates, such as ISOC1, encoding a metabolic enzyme of unknown function in mammalian cells. ISOC1-depleted T cells, which produced less IFN-γ and IL-17, displayed defects in oxidative phosphorylation and glycolysis and impairment of pyrimidine metabolic pathway. Supplementation with extracellular pyrimidines rescued both bioenergetics and IFN-γ production in ISOC1-deficient T cells, indicating that pyrimidine metabolism is a key driver of effector functions in CD4+ T cells and Th1* cells. Results provide new insights into the immune-stimulatory function of ISOC1 as well as the particular metabolic requirements of human memory T cells, providing a novel resource for understanding long-term T cell-driven responses.
Asunto(s)
Hidrolasas/metabolismo , Interferón gamma/genética , Interleucina-17/genética , Células TH1/inmunología , Regulación de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Células HEK293 , Voluntarios Sanos , Humanos , Hidrolasas/genética , Memoria Inmunológica/genética , Cultivo Primario de Células , Pirimidinas/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Células TH1/metabolismoRESUMEN
Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.
Asunto(s)
GTP Fosfohidrolasas/genética , Mitocondrias Hepáticas/genética , Membranas Mitocondriales/metabolismo , Forma de los Orgánulos/genética , Proteína X Asociada a bcl-2/genética , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Potencial de la Membrana Mitocondrial/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/ultraestructura , Dinámicas Mitocondriales/genética , Membranas Mitocondriales/ultraestructura , Permeabilidad , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2 , Células Cultivadas , Citocromos c/metabolismo , Humanos , Leupeptinas/farmacología , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Proteínas Mutantes/metabolismo , Péptidos/farmacología , Permeabilidad/efectos de los fármacos , Estructura Cuaternaria de Proteína , Proteína Destructora del Antagonista Homólogo bcl-2/químicaRESUMEN
Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for "intrinsic" apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the "hit-and-run" hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at â¼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
Asunto(s)
Biocatálisis , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Multimerización de Proteína , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Biocatálisis/efectos de los fármacos , Dinamina I/metabolismo , Humanos , Cinética , Liposomas/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Quinazolinonas/farmacología , Ratas , Ratas Sprague-Dawley , TermodinámicaRESUMEN
BACKGROUND: Oral antiviral drugs with improved antiviral potency and safety are needed to address current challenges in clinical practice for treatment of COVID-19, including the risks of rebound, drug-drug interactions, and emerging resistance. METHODS: Olgotrelvir (STI-1558) is designed as a next-generation antiviral targeting the SARS-CoV-2 main protease (Mpro), an essential enzyme for SARS-CoV-2 replication, and human cathepsin L (CTSL), a key enzyme for SARS-CoV-2 entry into host cells. FINDINGS: Olgotrelvir is a highly bioavailable oral prodrug that is converted in plasma to its active form, AC1115. The dual mechanism of action of olgotrelvir and AC1115 was confirmed by enzyme activity inhibition assays and co-crystal structures of AC1115 with SARS-CoV-2 Mpro and human CTSL. AC1115 displayed antiviral activity by inhibiting replication of all tested SARS-CoV-2 variants in cell culture systems. Olgotrelvir also inhibited viral entry into cells using SARS-CoV-2 Spike-mediated pseudotypes by inhibition of host CTSL. In the K18-hACE2 transgenic mouse model of SARS-CoV-2-mediated disease, olgotrelvir significantly reduced the virus load in the lungs, prevented body weight loss, and reduced cytokine release and lung pathologies. Olgotrelvir demonstrated potent activity against the nirmatrelvir-resistant Mpro E166 mutants. Olgotrelvir showed enhanced oral bioavailability in animal models and in humans with significant plasma exposure without ritonavir. In phase I studies (ClinicalTrials.gov: NCT05364840 and NCT05523739), olgotrelvir demonstrated a favorable safety profile and antiviral activity. CONCLUSIONS: Olgotrelvir is an oral inhibitor targeting Mpro and CTSL with high antiviral activity and plasma exposure and is a standalone treatment candidate for COVID-19. FUNDING: Funded by Sorrento Therapeutics.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19 , Inhibidores de Proteasa de Coronavirus , SARS-CoV-2 , Animales , Humanos , Ratones , Antivirales/farmacología , Antivirales/uso terapéutico , Catepsina L/antagonistas & inhibidores , COVID-19/prevención & control , Modelos Animales de Enfermedad , Ratones Transgénicos , Inhibidores de Proteasa de Coronavirus/química , Inhibidores de Proteasa de Coronavirus/farmacología , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Tratamiento Farmacológico de COVID-19/métodosRESUMEN
BACKGROUND: Olgotrelvir is an oral antiviral with dual mechanisms of action targeting severe acute respiratory syndrome coronavirus 2 main protease (i.e., Mpro) and human cathepsin L. It has potential to serve as a single-agent treatment of coronavirus disease 2019 (Covid-19). METHODS: We conducted a phase 3, double-blind, randomized, placebo-controlled trial to evaluate the efficacy and safety of olgotrelvir in 1212 nonhospitalized adult participants with mild to moderate Covid-19, irrespective of risk factors, who were randomly assigned to receive orally either 600 mg of olgotrelvir or placebo twice daily for 5 days. The primary and key secondary end points were time to sustained recovery of a panel of 11 Covid-19-related symptoms and the viral ribonucleic acid (RNA) load. The safety end point was incidence of treatment-emergent adverse events. RESULTS: The baseline characteristics of 1212 participants were similar in the two groups. In the modified intention-to-treat population (567 patients in the placebo group and 558 in the olgotrelvir group), the median time to symptom recovery was 205 hours in the olgotrelvir group versus 264 hours in the placebo group (hazard ratio, 1.29; 95% confidence interval [CI], 1.13 to 1.46; P<0.001). The least squares mean (95% CI) changes of viral RNA load from baseline were -2.20 (-2.59 to -1.81) log10 copies/ml in olgotrelvir-treated participants and -1.40 (-1.79 to -1.01) in participants receiving placebo at day 4. Skin rash (3.3%) and nausea (1.5%) were more frequent in the olgotrelvir group than in the placebo group; there were no treatment-related serious adverse events, and no deaths were reported. CONCLUSIONS: Olgotrelvir as a single-agent treatment significantly improved symptom recovery. Adverse effects were not dose limiting. (Funded by Sorrento Therapeutics, a parent company of ACEA Therapeutics; ClinicalTrials.gov number, NCT05716425.).
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , Humanos , Masculino , Método Doble Ciego , Femenino , Persona de Mediana Edad , Antivirales/uso terapéutico , Antivirales/efectos adversos , Antivirales/administración & dosificación , Adulto , COVID-19/virología , SARS-CoV-2 , Anciano , Resultado del Tratamiento , Compuestos OrgánicosRESUMEN
The HIV-1 entry pathway into permissive cells has been a subject of debate. Accumulating evidence, including our previous single virus tracking results, suggests that HIV-1 can enter different cell types via endocytosis and CD4/coreceptor-dependent fusion with endosomes. However, recent studies that employed indirect techniques to infer the sites of HIV-1 entry into CD4+ T cells have concluded that endocytosis does not contribute to infection. To assess whether HIV-1 enters these cells via endocytosis, we probed the role of intracellular trafficking in HIV-1 entry/fusion by a targeted shRNA screen in a CD4+ T cell line. We performed a screen utilizing a direct virus-cell fusion assay as readout and identified several host proteins involved in endosomal trafficking/maturation, including Rab5A and sorting nexins, as factors regulating HIV-1 fusion and infection. Knockdown of these proteins inhibited HIV-1 fusion irrespective of coreceptor tropism, without altering the CD4 or coreceptor expression, or compromising the virus' ability to mediate fusion of two adjacent cells initiated by virus-plasma membrane fusion. Ectopic expression of Rab5A in non-permissive cells harboring Rab5A shRNAs partially restored the HIV-cell fusion. Together, these results implicate endocytic machinery in productive HIV-1 entry into CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Endocitosis , VIH-1/fisiología , Nexinas de Clasificación/genética , Internalización del Virus , Proteínas de Unión al GTP rab5/genética , Línea Celular , Humanos , Membranas Intracelulares/virología , ARN Interferente Pequeño/genética , Replicación ViralRESUMEN
The mitochondrial Na(+)-Ca(2+) exchanger (mNCE) mediates efflux of Ca(2+) from mitochondria in exchange for influx of Na(+). We show that inhibition of the mNCE enhances mitochondrial oxidative metabolism and increases glucose-stimulated insulin secretion in rat islets and INS-1 cells. The benzothiazepine CGP37157 inhibited mNCE activity in INS-1 cells (50% inhibition at IC(50) = 1.5 micro mol/l) and increased the glucose-induced rise in mitochondrial Ca(2+) ([Ca(2+)](m)) 2.1 times. Cellular ATP content was increased by 13% in INS-1 cells and by 49% in rat islets by CGP37157 (1 micro mol/l). Krebs cycle flux was also stimulated by CGP37157 when glucose was present. Insulin secretion was increased in a glucose-dependent manner by CGP37157 in both INS-1 cells and islets. In islets, CGP37157 increased insulin secretion dose dependently (half-maximal efficacy at EC(50) = 0.06 micro mol/l) at 8 mmol/l glucose and shifted the glucose dose response curve to the left. In perifused islets, mNCE inhibition had no effect on insulin secretion at 2.8 mmol/l glucose but increased insulin secretion by 46% at 11 mmol/l glucose. The effects of CGP37157 could not be attributed to interactions with the plasma membrane sodium calcium exchanger, L-type calcium channels, ATP-sensitive K(+) channels, or [Ca(2+)](m) uniporter. In hyperglycemic clamp studies of Wistar rats, CGP37157 increased plasma insulin and C-peptide levels only during the hyperglycemic phase of the study. These results illustrate the potential utility of agents that affect mitochondrial metabolism as novel insulin secretagogues.
Asunto(s)
Clonazepam/análogos & derivados , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Mitocondrias/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Aequorina/genética , Animales , Calcio/análisis , Línea Celular , Membrana Celular/química , Clonazepam/farmacología , Expresión Génica , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/ultraestructura , Masculino , NAD/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Intercambiador de Sodio-Calcio/análisis , Tiazepinas/farmacología , TransfecciónRESUMEN
Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy. DOA presents in the first decade of life and manifests as progressive vision loss. In DOA retinal ganglion cells and the optic nerve degenerate by an unknown mechanism. The gene mutated in DOA, Optic Atrophy Type 1 (OPA1), encodes a dynamin-related GTPase implicated in mitochondrial fusion and maintenance of the mitochondrial network and genome. Here, we determine which cell types in the normal retina and the optic nerve express OPA1. In the normal rat retina, OPA1 is expressed in the ganglion cell layer as well as in the outer plexiform layer, the inner nuclear layer, and the inner plexiform layer. In the ganglion cell layer, OPA1 is expressed predominantly in retinal ganglion cells. By contrast, OPA1 protein is low or undetectable in astrocytes and oligodendrocytes of the optic nerve. Additionally, OPA1 protein is present in axonal mitochondria. Last, OPA1 expression is present in mitochondria of processes and cell bodies of purified retinal ganglion cells and of the RGC-5 cell line. Thus, OPA1 is predominantly expressed in retinal ganglion cells of the normal rat retina and axons of the optic nerve. These findings may explain the selective vulnerability of retinal ganglion cells to OPA1 loss of function.
Asunto(s)
Axones/metabolismo , GTP Fosfohidrolasas/metabolismo , Nervio Óptico/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Astrocitos/metabolismo , Masculino , Oligodendroglía/metabolismo , Nervio Óptico/citología , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Retina/citologíaRESUMEN
Mitochondrial bioenergetic function is a key to cell life and death. Cells need energy not only to support their vital functions but also to die gracefully. Execution of an apoptotic program includes energy-dependent steps, including kinase signaling, formation of the apoptosome, and effector caspase activation. Under conditions of bioenergetic collapse, cells are diverted toward necrotic demise. Mitochondrial outer membrane permeabilization (MOMP) is a decisive event in the execution of apoptosis. It is also causally linked to a decline in bioenergetic function via different mechanisms, not merely due to cytochrome c dispersion. MOMP-induced bioenergetic deficiency is usually irreversible and commits cells to die, even when caspases are inactive. Here, we discuss the mechanisms by which MOMP impacts bioenergetics in different cell death paradigms.
Asunto(s)
Apoptosis , Muerte Celular/fisiología , Metabolismo Energético , Mitocondrias/metabolismo , NAD/metabolismo , Oxígeno/química , Caspasas/metabolismo , Supervivencia Celular , Citocromos c/metabolismo , Activación Enzimática , Células HeLa , Humanos , Membranas Mitocondriales/metabolismo , Necrosis , Proteína X Asociada a bcl-2/metabolismoRESUMEN
In apoptosis, mitochondrial outer membrane permeabilization (MOMP) triggers caspase-dependent death. However, cells undergo clonogenic death even if caspases are blocked. One proposed mechanism involved the release of cytotoxic proteins (e.g., AIF and endoG) from mitochondria. To initiate MOMP directly without side effects, we created a tamoxifen-switchable BimS fusion protein. Surprisingly, even after MOMP, caspase-inhibited cells replicated DNA and divided for approximately 48 h before undergoing proliferation arrest. AIF and endoG remained in mitochondria. However, cells gradually lost mitochondrial membrane potential and ATP content, and DNA synthesis slowed to a halt by 72 h. These defects resulted from a partial loss of respiratory function, occurring 4-8 h after MOMP, that was not merely due to dispersion of cytochrome c. In particular, Complex I activity was completely lost, and Complex IV activity was reduced by approximately 70%, whereas Complex II was unaffected. Later, cells exhibited a more profound loss of mitochondrial protein constituents. Thus, under caspase inhibition, MOMP-induced clonogenic death results from a progressive loss of mitochondrial function, rather than the release of cytotoxic proteins from mitochondria.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Caspasas/metabolismo , Muerte Celular/fisiología , Respiración de la Célula/fisiología , Mitocondrias/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular , Proliferación Celular , Inhibidores de Cisteína Proteinasa/metabolismo , Citocromos c/metabolismo , Activación Enzimática , Glucosa/metabolismo , Glucólisis/fisiología , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Desacopladores/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.
Asunto(s)
Dinaminas/fisiología , GTP Fosfohidrolasas/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/ultraestructura , Proteínas Mitocondriales/fisiología , Neuronas/ultraestructura , Óxido Nítrico/fisiología , Adenosina Trifosfato/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Autofagia , Células Cultivadas , Corteza Cerebral/citología , Metabolismo Energético , Radicales Libres/metabolismo , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Ratas , Rotenona/farmacología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Adenosina Difosfato/química , Adenosina Trifosfato/química , Animales , Calcimicina/farmacología , Calcio/metabolismo , Muerte Celular , Supervivencia Celular , Células Cultivadas , Cerebelo/citología , Citoplasma/metabolismo , Electrones , Ácido Glutámico/química , Ácido Glutámico/farmacología , Ionóforos/farmacología , Iones , Potenciales de la Membrana , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Abstract We have investigated the role of Ca2+ accumulation and neuronal injury in cerebellar granule neurons after glutamate receptor overactivation. After the removal of the free cytosolic Ca2+ we identified an extensive second Ca2+ fraction (SCF) that is retained within the neurons after glutamate receptor overactivation. The SCF reaches a plateau within 10 min with the magnitude of this SCF accumulation reflecting the extent of the neuronal injury that occurs within the neurons. The existence of this SCF is sensitive to both NMDA receptor antagonists and mitochondrial inhibitors but is unaffected by agents that deplete endoplasmic reticulum Ca2+, indicating that this Ca2+ fraction may be located within the mitochondria. Through the isolation of mitochondria from cerebellar granule neurons treated with glutamate we have shown that the majority of the SCF is mitochondrial in location. On the removal of the glutamate stimulus the SCF recovers at a slower rate than the free Ca2+ concentration within the neuron. This is intriguing, as it implies a capacity to remember previous excitatory events. Most significantly we have shown that a short pre-application of subthreshold glutamate or kainate blocks both SCF Ca2+ accumulation and extensive neuronal injury in response to high concentrations of glutamate. These findings may be relevant to the observations of pre-conditioning in the brain and heart.
Asunto(s)
Calcio/metabolismo , Cerebelo/citología , Ácido Glutámico/toxicidad , Espacio Intracelular/efectos de los fármacos , Neuronas/efectos de los fármacos , Factores de Edad , Alameticina , Análisis de Varianza , Animales , Animales Recién Nacidos , Encefalopatías/inducido químicamente , Encefalopatías/metabolismo , Isótopos de Calcio/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Recuento de Células , Muerte Celular/efectos de los fármacos , Fraccionamiento Celular/métodos , Células Cultivadas , Diagnóstico por Imagen/métodos , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Fura-2/metabolismo , Glicina/farmacología , Hidroliasas/metabolismo , Indoles/farmacología , Ionóforos/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/metabolismo , Propidio , Quinoxalinas , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders.
Asunto(s)
Encéfalo/ultraestructura , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Mitocondrias/química , Neuronas/fisiología , Animales , Secuencia de Bases , Supervivencia Celular , Corteza Cerebral/citología , AMP Cíclico , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/fisiología , Ensayo de Cambio de Movilidad Electroforética , Humanos , Ratones , Ratones Transgénicos , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Datos de Secuencia Molecular , Enfermedades Neurodegenerativas , Neuronas/ultraestructura , Nitrocompuestos/farmacología , Consumo de Oxígeno/fisiología , Fosforilación , Propionatos/farmacología , Ratas , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD(+)-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)(+) pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/L-beta-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50 mV more electronegative than the NADH/NAD(+) couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD(+), accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a.