RESUMEN
The transition from the swimming larval stage to the settlement stage represents a significant node in the marine sponge developmental process. Previous research has shown that the outer membrane vesicles (OMVs) from the bacterial species Tenacibaculum mesophilum associated with the sponge Tedania sp. influence larval settlement: low concentrations of OMVs increase the attachment rate, whereas high concentrations decrease the attachment rate. Here, by comparing the transcriptomes of sponge larvae in filtered seawater (FSW group) and in FSW supplemented with OMVs (FSW-OMV group), the results indicated that bacterial OMVs affected larval settlement by modulating the expression levels of apoptosis-inducing factor (AIF) in the host. Subsequently, quantitative real-time PCR revealed a decrease in aif expression near the time of settlement (SE) compared to that in the control group. RNA interference (RNAi) was used to target the aif gene, and the rate of larval settlement was significantly reduced, confirming the inhibitory effect of high concentrations of OMVs. Moreover, small RNA (sRNA) sequencing of OMVs revealed the existence of abundant AIF-sRNAs of 30 nt, further suggesting that one pathway for the involvement of sponge-associated bacteria in host development is the transport of OMVs and the direct function of cargo loading.
Asunto(s)
Factor Inductor de la Apoptosis , Larva , Poríferos , Animales , Poríferos/microbiología , Poríferos/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Factor Inductor de la Apoptosis/genética , Larva/microbiología , Larva/metabolismo , Larva/crecimiento & desarrollo , SimbiosisRESUMEN
Neuroglobin, a member of the globin superfamily, is abundant in the brain, retina, and cerebellum of mammals and localizes to mitochondria. The protein exhibits neuroprotective capacities by participating in electron transfer, oxygen supply, and protecting against oxidative stress. Our objective was to determine whether neuroglobin overexpression can be used to treat neurological disorders. We chose Harlequin mice, which harbor a retroviral insertion in the first intron of the apoptosis-inducing factor gene resulting in the depletion of the corresponding protein essential for mitochondrial biogenesis. Consequently, Harlequin mice display degeneration of the cerebellum and suffer from progressive blindness and ataxia. Cerebellar ataxia begins in Harlequin mice at the age of 4 months and is characterized by neuronal cell disappearance, bioenergetics failure, and motor and cognitive impairments, which aggravated with aging. Mice aged 2 months received adeno-associated viral vectors harboring the coding sequence of neuroglobin or apoptosis-inducing factor in both cerebellar hemispheres. Six months later, Harlequin mice exhibited substantial improvements in motor and cognitive skills; probably linked to the preservation of respiratory chain function, Purkinje cell numbers and connectivity. Thus, without sharing functional properties with apoptosis-inducing factor, neuroglobin was efficient in reducing ataxia in Harlequin mice.
Asunto(s)
Ataxia Cerebelosa , Cerebelo , Globinas , Mitocondrias , Proteínas del Tejido Nervioso , Neuroglobina , Animales , Ratones , Factor Inductor de la Apoptosis/metabolismo , Factor Inductor de la Apoptosis/genética , Ataxia Cerebelosa/metabolismo , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/terapia , Cerebelo/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Globinas/metabolismo , Globinas/genética , Homeostasis , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Neuroglobina/metabolismo , Neuronas/metabolismoRESUMEN
Studies have highlighted oxidative damage in the inner ear as a critical pathological basis for sensorineural hearing loss, especially the presbycusis. Poly(ADP-ribose) polymerase-1 (PARP1) activation responds to oxidative stress-induced DNA damage with pro-repair and pro-death effects resembling two sides of the same coin. PARP1-related cell death, known as parthanatos, whose underlying mechanisms are attractive research hotspots but remain to be clarified. In this study, we observed that aged rats showed stria vascularis degeneration and oxidative damage, and PARP1-dependent cell death was prominent in age-related cochlear disorganization and dysfunction. Based on oxidative stress model of primary cultured stria marginal cells (MCs), we revealed that upregulated PARP1 and PAR (Poly(ADP-ribose)) polymers are responsible for MCs oxidative death with high mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse, while inhibition of PARP1 ameliorated the adverse outcomes. Importantly, the PARylation of apoptosis-inducing factor (AIF) is essential for its conformational change and translocation, which subsequently causes DNA break and cell death. Concretely, the interaction of PAR and truncated AIF (tAIF) is the mainstream in the parthanatos pathway. We also found that the effects of AIF cleavage and release were achieved through calpain activity and mPTP opening, both of which could be regulated by PARP1 via mediation of mitochondria Ca2+ concentration. In conclusion, the PAR-Ca2+-tAIF signaling pathway in parthanatos contributes to the oxidative stress damage observed in MCs. Targeting PAR-Ca2+-tAIF might be a potential therapeutic strategy for the early intervention of presbycusis and other oxidative stress-associated sensorineural deafness.
Asunto(s)
Factor Inductor de la Apoptosis , Calcio , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1 , Presbiacusia , Animales , Factor Inductor de la Apoptosis/metabolismo , Factor Inductor de la Apoptosis/genética , Ratas , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Calcio/metabolismo , Presbiacusia/metabolismo , Presbiacusia/patología , Presbiacusia/genética , Parthanatos/genética , Potencial de la Membrana Mitocondrial , Estría Vascular/metabolismo , Estría Vascular/patología , Apoptosis , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Ratas Sprague-Dawley , Daño del ADN , Envejecimiento/metabolismo , Envejecimiento/patología , Cóclea/metabolismo , Cóclea/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Masculino , Humanos , Células CultivadasRESUMEN
Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway.
Asunto(s)
Factor Inductor de la Apoptosis , Modelos Animales de Enfermedad , Pérdida Auditiva , Animales , Humanos , Masculino , Ratones , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Técnicas de Sustitución del Gen , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patología , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Pérdida Auditiva/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/patología , Atrofia Muscular/metabolismo , Mutación , Transporte de Proteínas , Ganglio Espiral de la Cóclea/metabolismo , Ganglio Espiral de la Cóclea/patologíaRESUMEN
Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.
Asunto(s)
Factor Inductor de la Apoptosis , Dominio Catalítico , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales , Modelos Moleculares , Unión Proteica , Factor Inductor de la Apoptosis/metabolismo , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/genética , Humanos , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Regulación Alostérica , Cristalografía por Rayos X , NAD/metabolismo , NAD/química , Sitios de Unión , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.
Asunto(s)
Stichopus , Vibrio , Animales , Stichopus/genética , beta Carioferinas , Inmunidad Innata/genética , Factor Inductor de la Apoptosis/genética , Vibrio/fisiología , ApoptosisRESUMEN
Apoptosis-inducing factor 1 (AIF1) overexpression is intimately linked to the sensitivity of yeast cells towards hydrogen peroxide or acetic acid. Therefore, studying the mechanism of AIF1 regulation in the cell would provide a significant understanding of the factors guiding yeast apoptosis. In this report, we show the time-dependent induction of AIF1 under hydrogen peroxide stress. Additionally, we find that AIF1 expression in response to hydrogen peroxide is mediated by two transcription factors, Yap5 (DNA binding) and Cdc73 (non-DNA binding). Furthermore, substituting the H3K36 residue with another amino acid significantly abrogates AIF1 expression. However, substituting the lysine (K) in H3K4 or H3K79 with alanine (A) does not affect AIF1 expression level under hydrogen peroxide stress. Altogether, reduced AIF1 expression in cdc73Δ is plausibly due to reduced H3K36me3 levels in the cells.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Metilación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Our previous study found that receptor interacting protein 3 (RIP3) and apoptosis-inducing factor (AIF) were involved in neuronal programmed necrosis during global cerebral ischemia-reperfusion (I/R) injury. Here, we further studied its downstream mechanisms and the role of the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BAF). A 20-min global cerebral I/R injury model was constructed using the 4-vessel occlusion (4-VO) method in male rats. 3-MA and BAF were injected into the lateral ventricle 1 h before ischemia. Spatial and activation changes of proteins were detected by immunofluorescence (IF), and protein interaction was determined by immunoprecipitation (IP). The phosphorylation of H2AX (γ-H2AX) and activation of mixed lineage kinase domain-like protein (p-MLKL) occurred as early as 6 h after reperfusion. RIP3, AIF, and cyclophilin A (CypA) in the neurons after I/R injury were spatially overlapped around and within the nucleus and combined with each other after reperfusion. The survival rate of CA1 neurons in the 3-MA and BAF groups was significantly higher than that in the I/R group. Autophagy was activated significantly after I/R injury, which was partially inhibited by 3-MA and BAF. Pretreatment with both 3-MA and BAF almost completely inhibited nuclear translocation, spatial overlap, and combination of RIP3, AIF, and CypA proteins. These findings suggest that after global cerebral I/R injury, RIP3, AIF, and CypA translocated into the nuclei and formed the DNA degradation complex RIP3/AIF/CypA in hippocampal CA1 neurons. Pretreatment with autophagy inhibitors could reduce neuronal necroptosis by preventing the formation of the RIP3/AIF/CypA complex and its nuclear translocation.
Asunto(s)
Isquemia Encefálica , Macrólidos , Daño por Reperfusión , Ratas , Masculino , Animales , Ciclofilina A/genética , Ciclofilina A/metabolismo , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Necroptosis , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Apoptosis , Neuronas/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , AutofagiaRESUMEN
Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment involving disruptions to inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself. The outcomes of cochlear implants (CI) for ANSD are variable and dependent on the location of lesion sites. Discovering a potential therapeutic agent for ANSD remains an urgent requirement. Here, 293T stable transfection cell lines and patient induced pluripotent stem cells (iPSCs)-derived auditory neurons carrying the apoptosis inducing factor (AIF) p.R422Q variant were used to pursue a therapeutic regent for ANSD. Nicotinamide adenine dinucleotide (NADH) is a main electron donor in the electron transport chain (ETC). In 293T stable transfection cells with the p.R422Q variant, NADH treatment improved AIF dimerization, rescued mitochondrial dysfunctions, and decreased cell apoptosis. The effects of NADH were further confirmed in patient iPSCs-derived neurons. The relative level of AIF dimers was increased to 150.7 % (P = 0.026) from 59.2 % in patient-neurons upon NADH treatment. Such increased AIF dimerization promoted the mitochondrial import of coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4), which further restored mitochondrial functions. Similarly, the content of mitochondrial calcium (mCa2+) was downregulated from 136.7 % to 102.3 % (P = 0.0024) in patient-neurons upon NADH treatment. Such decreased mCa2+ levels inhibited calpain activity, ultimately reducing the percentage of apoptotic cells from 30.5 % to 21.1 % (P = 0.021). We also compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD. NADH treatment had comparable restorative effects on functions of ANSD patient-specific cells to that of gene correction. Our findings offer evidence of the molecular mechanisms of ANSD and introduce NADH as a potential therapeutic agent for ANSD therapy.
Asunto(s)
Factor Inductor de la Apoptosis , Apoptosis , Pérdida Auditiva Central , NAD , Células Receptoras Sensoriales , Pérdida Auditiva Central/genética , Pérdida Auditiva Central/metabolismo , Pérdida Auditiva Central/fisiopatología , Apoptosis/efectos de los fármacos , NAD/farmacología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Dimerización , Mitocondrias/efectos de los fármacos , Células HEK293 , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Calpaína/metabolismo , Activación Enzimática/efectos de los fármacos , Genotipo , Humanos , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismoRESUMEN
AIFM1 is a mitochondrial flavoprotein involved in caspase-independent cell death and regulation of respiratory chain complex biogenesis. Mutations in the AIFM1 gene have been associated with multiple clinical phenotypes, but the effectiveness of riboflavin treatment remains controversial. Furthermore, few studies explored the reasons underlying this controversy. We reported a 7-year-old boy with ataxia, sensorimotor neuropathy and muscle weakness. Genetic and histopathological analyses were conducted, along with assessments of mitochondrial function and apoptosis level induced by staurosporine. Riboflavin deficiency and supplementation experiments were performed using fibroblasts. A missense c.1019T > C (p. Met340Thr) variant of AIFM1 was detected in the proband, which caused reduced expression of AIFM1 protein and mitochondrial dysfunction as evidenced by downregulation of mitochondrial complex subunits, respiratory deficiency and collapse of ΔΨm. The proportion of apoptotic cells in mutant fibroblasts was lower than controls after induction of apoptosis. Riboflavin deficiency resulted in decreased AIFM1 protein levels, while supplementation with high concentrations of riboflavin partially increased AIFM1 protein levels in variant fibroblasts. In addition, mitochondrial respiratory function of mutant fibroblasts was partly improved after riboflavin supplementation. Our study elucidated the pathogenicity of the AIFM1 c.1019T > C variant and revealed mutant fibroblasts was intolerant to riboflavin deficiency. Riboflavin supplementation is helpful in maintaining the level of AIFM1 protein and mitochondrial respiratory function. Early riboflavin treatment may serve as a valuable attempt for patients with AIFM1 variant.
Asunto(s)
Enfermedades Mitocondriales , Deficiencia de Riboflavina , Masculino , Humanos , Niño , Deficiencia de Riboflavina/genética , Deficiencia de Riboflavina/metabolismo , Riboflavina/uso terapéutico , Riboflavina/genética , Riboflavina/metabolismo , Mutación Missense , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismoRESUMEN
Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.
Asunto(s)
Factor Inductor de la Apoptosis , Calcio , Células Madre Pluripotentes Inducidas , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Niño , Humanos , Recién Nacido , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Calcio/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales/metabolismoRESUMEN
Charcot-Marie-Tooth disease(CMT) is a hereditary peripheral neuropathy, characterized by progressive distal hypoesthesia and amyotrophia. CMT is characterized by an X- linked recessive inheritance pattern. The apoptosis-inducing factor mitochondria associated-1 (AIFM1) is the main pathogenic gene of the X-linked recessive Charcot-Marie-Tooth disease-4 with or without cerebellar ataxia (CMTX4), also known as Cowchock syndrome. In this study, we enrolled a family with CMTX from the southeast region of China and identified a novel AIFM1 variant (NM_004208.3: c.931C>G; p.L311V) using whole exon sequencing technology. The results of our study may also be useful for genetic counseling, embryo screening of in vitro fertilization embryos, and prenatal genetic diagnosis.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Humanos , Enfermedad de Charcot-Marie-Tooth/genética , Factor Inductor de la Apoptosis/genética , Secuenciación del Exoma , Pueblos del Este de Asia , Linaje , MutaciónRESUMEN
Parthanatos is a type of programmed cell death initiated by over-activated poly (ADP-ribose) polymerase 1 (PARP1). Nuclear translocation of apoptosis inducing factor (AIF) is a prominent feature of parthanatos. But it remains unclear how activated nuclear PARP1 induces mitochondrial AIF translocation into nuclei. Evidence has shown that deoxypodophyllotoxin (DPT) induces parthanatos in glioma cells via induction of excessive ROS. In this study we explored the downstream signal of activated PARP1 to induce nuclear translocation of AIF in DPT-triggered glioma cell parthanatos. We showed that treatment with DPT (450 nM) induced PARP1 over-activation and Tax1 binding protein 1 (TAX1BP1) distribution to mitochondria in human U87, U251 and U118 glioma cells. PARP1 activation promoted TAX1BP1 distribution to mitochondria by depleting nicotinamide adenine dinucleotide (NAD+). Knockdown of TAX1BP1 with siRNA not only inhibited TAX1BP1 accumulation in mitochondria, but also alleviated nuclear translocation of AIF and glioma cell death. We demonstrated that TAX1BP1 enhanced the activity of respiratory chain complex I not only by upregulating the expression of ND1, ND2, NDUFS2 and NDUFS4, but also promoting their assemblies into complex I. The activated respiratory complex I generated more superoxide to cause mitochondrial depolarization and nuclear translocation of AIF, while the increased mitochondrial superoxide reversely reinforced PARP1 activation by inducing ROS-dependent DNA double strand breaks. In mice bearing human U87 tumor xenograft, administration of DPT (10 mg· kg-1 ·d-1, i.p., for 8 days) markedly inhibited the tumor growth accompanied by NAD+ depletion, TAX1BP1 distribution to mitochondria, AIF distribution to nuclei as well as DNA DSBs and PARP1 activation in tumor tissues. Taken together, these data suggest that TAX1BP1 acts as a downstream signal of activated PARP1 to trigger nuclear translocation of AIF by activation of mitochondrial respiratory chain complex I.
Asunto(s)
Glioma , Parthanatos , Humanos , Ratones , Animales , Factor Inductor de la Apoptosis/genética , Superóxidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , NAD/metabolismo , Transporte de Electrón , Complejo I de Transporte de Electrón , Glioma/metabolismo , Proteínas de Neoplasias/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
The AIFM1 gene encodes a mitochondrial protein that acts as a flavin adenine dinucleotide-dependent nicotinamide adenine dinucleotide oxidase and apoptosis regulator. Monoallelic pathogenic AIFM1 variants result in a spectrum of X-linked neurological disorders, including Cowchock syndrome. Common features in Cowchock syndrome include a slowly progressive movement disorder, cerebellar ataxia, progressive sensorineural hearing loss, and sensory neuropathy. We identified a novel maternally inherited hemizygous missense AIFM1 variant, c.1369C>T p.(His457Tyr), in two brothers with clinical features consistent with Cowchock syndrome using next-generation sequencing. Both individuals had a progressive complex movement disorder phenotype, including disabling tremor poorly responsive to medications. Deep brain stimulation (DBS) of the ventral intermediate thalamic nucleus ameliorated contralateral tremor and improved their quality of life; this suggests the beneficial role for DBS in treatment-resistant tremor within AIFM1-related disorders.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Estimulación Encefálica Profunda , Humanos , Masculino , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Calidad de Vida , Temblor/genética , Temblor/terapiaRESUMEN
Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.|T260A, p.|R422W, and p.|R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%|â|49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%|â|17.9%, which was significantly higher than that (6.9%|â|7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
Asunto(s)
Factor Inductor de la Apoptosis , NAD , Humanos , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , NAD/metabolismo , Dimerización , ApoptosisRESUMEN
The natural product dehydrocurvularin (DSE2) is a fungal-derived macrolide with potent anticancer activity, but the mechanism is still unclear. We found that DSE2 effectively inhibited the growth of gastric cancer cells and induced the apoptosis by activating Poly(ADP-ribose) polymerase 1 (PARP-1) and caspase-3. Pharmacological inhibition and genetic knockdown with PARP-1 or caspase-3 suppressed DSE2-induced apoptosis. PARP-1 was previously reported to be cleaved into fragments during apoptosis. However, PARP-1 was barely cleaved in DSE2-induced apoptosis. DSE2 induced PARP-1 activation as indicated by rapid depletion of NAD+ and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). Interestingly, the PARP-1 inhibitor (Olaparib) attenuated the cytotoxicity of DSE2. Moreover, the combination of Olaparib and Z-DEVD-FMK (caspase-3 inhibitor) further reduced the cytotoxicity. It has been shown that PARP-1 activation triggers cytoplasm-nucleus translocation of apoptosis-inducing factor (AIF). Caspase-3 inhibitors inhibited PARP-1 activation and suppressed PARP-1-induced AIF nuclear translocation. These results indicated that DSE2-induced caspase-3 activation may occur before PARP-1 activation. The ROS inhibitor, N-acetyl-cysteine, significantly inhibited the activation of caspase-3 and PARP-1, indicating that ROS overproduction contributed to DSE2-induced apoptosis. Using an in vivo approach, we further found that DSE2 significantly inhibited gastric tumor growth and promoted translocation of AIF to the nucleus. In conclusion, DSE2 induces gastric cell apoptosis by activating caspase-3 and PARP-1, and shows potent antitumor activity against human gastric carcinoma in vitro and in vivo.
Asunto(s)
Antineoplásicos , Neoplasias Gástricas , Humanos , Apoptosis , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Antineoplásicos/farmacologíaRESUMEN
Chagas disease is endemic in 22 Latin American countries, with approximately 8 million individuals infected worldwide and 10,000 deaths yearly. Trypanosoma cruzi presents an intracellular life cycle in mammalian hosts to sustain infection. Parasite infection activates host cell responses, promoting an unbalance in reactive oxygen species (ROS) in the intracellular environment inducing genomic DNA lesions in the host cell during infection. To further understand changes in host cell chromatin induced by parasite infection, we investigated alterations in chromatin caused by infection by performing quantitative proteomic analysis. DNA Damage Repair proteins, such as Poly-ADP-ribose Polymerase 1 (PARP-1) and X-Ray Repair Cross Complementing 6 (XRRC6), were recruited to the chromatin during infection. Also, changes in chromatin remodeling enzymes suggest that parasite infection may shape the epigenome of the host cells. Interestingly, the abundance of oxidative phosphorylation mitochondrial and vesicle-mediated transport proteins increased in the host chromatin at the final stages of infection. In addition, Apoptosis-inducing Factor (AIF) is translocated to the host cell nucleus upon infection, suggesting that cells enter parthanatos type of death. Altogether, this study reveals how parasites interfere with the host cells' responses at the chromatin level leading to significant crosstalk that support and disseminate infection. SIGNIFICANCE: The present study provides novel insights into the effects of Trypanosoma cruzi on the chromatin from the host cell. This manuscript investigated proteomic alterations in chromatin caused by parasite infection at early and late infection phases by performing a quantitative proteomic analysis. In this study, we revealed that parasites interfere with DNA metabolism in the early and late stages of infection. We identified that proteins related to DNA damage repair, oxidative phosphorylation, and vesicle-mediated transport have increased abundance at the host chromatin. Additionally, we have observed that Apoptosis-inducing Factor is translocated to the host cell nucleus upon infection, suggesting that the parasites could lead the cells to enter Parthanatos as a form of programmed cell death. The findings improve our understanding on how the parasites modulate the host cell chromatin to disseminate infection. In this study, we suggest a mechanistic parasite action towards host nucleus that could be used to indicate targets for future treatments.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Animales , Humanos , Proteoma/metabolismo , Cromatina/metabolismo , Proteómica , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMEN
Excitotoxicity is the underlying mechanism for all acute neuronal injury, from cerebral ischemia, status epilepticus, traumatic CNS injury, and hypoglycemia. It causes morphological neuronal necrosis, and it triggers a programmed cell death program. Excessive calcium entry through the NMDA-receptor-operated cation channel activates two key enzymes-calpain I and neuronal nitric oxide synthase (nNOS). Calpain I, a cytosolic enzyme, translocates to mitochondrial and lysosomal membranes, causing release of cytochrome c, endonuclease G, and apoptosis-inducing factor (AIF) from mitochondria and DNase II and cathepsins B and D from lysosomes. These all translocate to neuronal nuclei, creating DNA damage, which activates poly(ADP) ribose polymerase-1 (PARP-1) to form excessive amounts of poly(ADP) ribose (PAR) polymers, which translocate to mitochondrial membranes, causing release of truncated AIF (tAIF). The free radicals that are released from mitochondria and peroxynitrite, formed from nitric oxide (NO) from nNOS catalysis of L-arginine to L-citrulline, damage mitochondrial and lysosomal membranes and DNA. The end result is the necrotic death of neurons. Another programmed necrotic pathway, necroptosis, occurs through a parallel pathway. As investigators of necroptosis do not recognize the excitotoxic pathway, it is unclear to what extent each contributes to programmed neuronal necrosis. We are studying the extent to which each contributes to acute neuronal necrosis and the extent of cross-talk between these pathways.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas , Estado Epiléptico , Humanos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Calpaína/metabolismo , Ribosa/metabolismo , Necrosis/metabolismo , Neuronas/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Membranas Mitocondriales/metabolismo , Factor Inductor de la Apoptosis/genética , Factor Inductor de la Apoptosis/metabolismoRESUMEN
Ischemic stroke (IS) poses a heavy burden on the healthcare system, and revascularization is the most effective treatment. However, ischemia/reperfusion (I/R) injury, one main cause of revascularization complications, significantly hinders IS recovery. Unfortunately, none of the neuroprotectants tested to date has been successfully translated clinically for post-revascularization I/R injury therapy. In multiple pathophysiological processes, apoptosis antagonizing transcription factor (AATF) serves as a cell protector, but its role in neuronal I/R injury is unknown. Therefore, we firstly demonstrated the expression profiles of AATF in a distal middle cerebral artery occlusion/reperfusion (dMCAO/R) model and found that AATF expression was increased in cortical neuron after dMCAO/R. Over-expressing AATF reduced infarct volume, alleviated neuronal death, and promoted neurological functions. Next, we used an oxygen-glucose deprivation/reoxygenation (OGD/R) model to investigate the mechanism of AATF. Results indicated that AATF alleviated OGD/R-induced large-scale DNA fragmentation, which suggested that the protective effect of AATF may be attributed to parthanatos inhibition. After that, we examined the regulatory mechanism of AATF. We found that AATF did not affect poly (ADP-ribose) accumulation and apoptosis-inducing factor (AIF) nucleus translocation. AATF competitively interacted with nuclear AIF, which inhibited AIF from binding DNA. At last, we verified the effect and mechanism of AATF in dMCAO/R model. The present study, for the first time, demonstrates the expression, function, and mechanism of AATF in the context of neuronal I/R injury via dMCAO/R and OGD/R model, which provides new evidence in this area and may facilitate exploring new therapeutic targets.