RESUMEN
The study aims to elucidate the therapeutic mechanism of Baicalin (BAI) in alleviating cartilage injury in osteoarthritic (OA) rat models, concentrating on its regulation of the miR-766-3p/AIFM1 axis. An OA rat model was developed with unilateral anterior cruciate ligament transection (ACLT). Interventions comprised of BAI treatment and intra-articular administration of miR-766-3p inhibitor. For evaluation, histopathological staining was conducted to investigate the pathological severity of knee cartilage injury. The levels of oxidative stress (OS) indicators including MDA, SOD, and GSH-Px, were quantified using colorimetric assays. Inflammatory factors (IFs; TNF-?, IL-1?, and IL-6) in knee joint lavage fluids were assessed using ELISA, while RT-PCR was employed to quantify miR-766-3p expression. TUNEL apoptosis staining was utilized to detect chondrocyte apoptosis, and western blotting examined autophagy-related markers (LC3, Beclin, p62), extracellular matrix (ECM) synthesis-associated indices (COL2A, ACAN, MMP13), and apoptosis-inducing factor mitochondrion-associated 1 (AIFM1). Histological examination revealed a marked amelioration of cartilage injury in the BAI-treated OA rat models compared to controls. BAI treatment significantly reduced inflammation and OS of knee joint fluid, activated autophagy, and decreased chondrocyte apoptosis and ECM degradation. Interestingly, the inhibitory effects of BAI on these pathological markers were significantly decreased by the miR-766-3p inhibitor. Further assessment revealed that BAI efficiently promoted miR-766-3p expression while inhibiting AIFM1 protein expression. BAI potentially mitigates articular cartilage injury in OA rats, likely through modulation of miR-766-3p/AIFM1 axis. Keywords: Baicalin, microRNA, AIFM1, Osteoarthritisv, Rat.
Asunto(s)
Flavonoides , MicroARNs , Ratas Sprague-Dawley , Animales , Flavonoides/farmacología , Flavonoides/uso terapéutico , MicroARNs/metabolismo , MicroARNs/genética , MicroARNs/biosíntesis , Ratas , Masculino , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Factor Inductor de la Apoptosis/metabolismo , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
Urate nephropathy, a common complication of hyperuricemia, has garnered increasing attention worldwide. However, the exact pathogenesis of this condition remains unclear. Currently, inflammation is widely accepted as the key factor in urate nephropathy. Therefore, the aim of this study was to elucidate the interaction of lincRNA-p21/AIF-1/CMPK2/NLRP3 via exosomes in urate nephropathy. This study evaluated the effect of lincRNA-p21/AIF-1/CMPK2/NLRP3 using clinical data collected from patients with urate nephropathy and human renal tubular epithelial cells (HK2) cultured with different concentrations of urate. In clinical research section, the level of lincRNA-p21/AIF-1 in exosomes of urine in patients with hyperuricemia or urate nephropathy was found to be increased, particularly in patients with urate nephropathy. In vitro study section, the level of exosomes, inflammation, autophagy, and apoptosis was increased in HK2 cells induced by urate. Additionally, the expression of lincRNA-p21, AIF-1, CMPK2, and NLRP3 was upregulated in exosomes and HK2 cells. Furthermore, manipulating the activity of lincRNA-p21, AIF-1, CMPK2, and NLRP3 through overexpression or interference vectors regulated the level of inflammation, autophagy, and apoptosis in HK2 cells. In conclusion, the pathway of lincRNA-p21/AIF-1/CMPK2/NLRP3 contributed to inflammation, autophagy, and apoptosis of human renal tubular epithelial cell induced by urate via exosomes. Additionally, the specific exosomes in urine might serve as novel biomarkers for urate nephropathy.
Asunto(s)
Apoptosis , Autofagia , Células Epiteliales , Exosomas , Proteína con Dominio Pirina 3 de la Familia NLR , ARN Largo no Codificante , Ácido Úrico , Humanos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ácido Úrico/metabolismo , Exosomas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Transducción de Señal , Inflamación/metabolismo , Inflamación/patología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Línea Celular , Masculino , Factor Inductor de la Apoptosis/metabolismo , Femenino , Persona de Mediana Edad , Hiperuricemia/metabolismo , Hiperuricemia/orina , Proteínas de Unión al Calcio , Proteínas de MicrofilamentosRESUMEN
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
BACKGROUND/AIMS: Traumatic brain injury is a significant public problem with an incidence of 10 million people per year, causing the largest deaths and disabilities worldwide. Head injuries can be classified into primary and secondary head injuries. Secondary head injuries can be caused by several factors such as ischemia, cerebral edema, and neuroinflammation. AIF and MMP-9 are two parameters that can be indicators in measuring the effect of Oleuropein on traumatic brain injury in rats. Oleuropein itself has many activities such as antioxidant, anti-apoptotic, antimicrobial, anti-inflammatory, and neuroprotective. METHODS: Adult male Sprague-Dawley rats (250-350 grams) were exposed to head injury, with or without intraperitoneal administration of Oleuropein. Within 24-72 hours brain tissue was isolated for immunohistochemical analysis, ELISA, and TUNEL. AIF, GFAP, MMP-9, and HMGB-1 levels were determined using immunohistochemistry in both the control and treatment groups. Statistical analysis was made using the One-Way Analysis of Variance (ANOVA) and paired t-test. RESULTS: The results showed that Oleuropein was able to reduce AIF and MMP-9 levels in rats with traumatic brain injury. This indicates that Oleuropein has a neuroprotective effect by reducing inflammation and apoptosis. CONCLUSION: Oleuropein has a potential neuroprotective effect in traumatic brain injury by reducing inflammation and apoptosis. Therefore, Oleuropein can be considered as a potential therapeutic agent for traumatic brain injury in the future.
Asunto(s)
Factor Inductor de la Apoptosis , Lesiones Traumáticas del Encéfalo , Modelos Animales de Enfermedad , Glucósidos Iridoides , Iridoides , Metaloproteinasa 9 de la Matriz , Ratas Sprague-Dawley , Animales , Glucósidos Iridoides/farmacología , Glucósidos Iridoides/uso terapéutico , Metaloproteinasa 9 de la Matriz/metabolismo , Masculino , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/patología , Iridoides/farmacología , Iridoides/uso terapéutico , Ratas , Factor Inductor de la Apoptosis/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Proteína HMGB1/metabolismo , Apoptosis/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/efectos de los fármacosRESUMEN
OBJECTIVES: Diabetes is closely linked to hearing loss, yet the exact mechanisms remain unclear. Cochlear stria vascularis and pericytes (PCs) are crucial for hearing. This study investigates whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes via elevated ROS levels due to oxidative stress, impacting hearing loss. METHODS: We established a type II diabetes model in C57BL/6J mice and used auditory brainstem response (ABR), Evans blue staining, HE staining, immunohistochemistry, and immunofluorescence to observe changes in hearing, blood-labyrinth barrier (BLB) permeability, stria vascularis morphology, and apoptosis protein expression. Primary cultured stria vascularis pericytes were subjected to high glucose, and apoptosis levels were assessed using flow cytometry, Annexin V-FITC, Hoechst 33342 staining, Western blot, Mitosox, and JC-1 probes. RESULTS: Diabetic mice showed decreased hearing thresholds, reduced stria vascularis density, increased oxidative stress, cell apoptosis, and decreased antioxidant levels. High glucose exposure increased apoptosis and ROS content in pericytes, while mitochondrial membrane potential decreased, with AIF and cytochrome C (CytC) released from mitochondria to the cytoplasm. Adding oxidative scavengers reduced AIF and CytC release, decreasing pericyte apoptosis. DISCUSSION: Hyperglycemia may induce mitochondrial apoptosis of cochlear stria vascularis pericytes through oxidative stress.
Asunto(s)
Factor Inductor de la Apoptosis , Apoptosis , Citocromos c , Hiperglucemia , Ratones Endogámicos C57BL , Mitocondrias , Estrés Oxidativo , Pericitos , Proteínas Proto-Oncogénicas c-bcl-2 , Especies Reactivas de Oxígeno , Estría Vascular , Animales , Pericitos/metabolismo , Pericitos/efectos de los fármacos , Pericitos/patología , Estría Vascular/metabolismo , Estría Vascular/patología , Ratones , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Citocromos c/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Hiperglucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Masculino , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Cóclea/metabolismo , Cóclea/patologíaRESUMEN
This study aimed to evaluate the influence of combined intermittent fasting (IF) and high-intensity interval training (HIIT) on morphology, caspase-independent apoptosis signaling pathway, and myostatin expression in soleus and gastrocnemius (white portion) muscles from healthy rats. Sixty-day-old male Wistar rats (n = 60) were divided into four groups: control (C), IF, high-intensity-interval training (T), and high-intensity-interval training and intermittent fasting (T-IF). The C and T groups received ad libitum chow daily; IF and T-IF received the same standard chow every other day. Animals from T and T-IF underwent a HIIT protocol five times a week for 12 weeks. IF reduced gastrocnemius mass and increased pro-apoptotic proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in soleus and cleaved-to-non-cleaved PARP-1 ratio and myostatin expression in gastrocnemius white portion. HIIT increased AIF and apoptosis repressor with caspase recruitment domain expression in soleus and cleaved-to-total PARP-1 ratio in gastrocnemius muscle white portion. The combination of IF and HIIT reduced fiber cross-sectional area in both muscles, increased EndoG and AIF expression, and decreased cleaved-to-non-cleaved PARP-1 ratio in gastrocnemius muscle white portion. Muscle responses to IF and HIIT are directly impacted by the muscle fiber type composition and are modulated, at least in part, by myostatin and caspase-independent apoptosis signaling.
Asunto(s)
Factor Inductor de la Apoptosis , Apoptosis , Ayuno , Entrenamiento de Intervalos de Alta Intensidad , Fibras Musculares de Contracción Lenta , Atrofia Muscular , Miostatina , Ratas Wistar , Transducción de Señal , Animales , Masculino , Apoptosis/fisiología , Ayuno/metabolismo , Ayuno/fisiología , Miostatina/metabolismo , Entrenamiento de Intervalos de Alta Intensidad/métodos , Ratas , Transducción de Señal/fisiología , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Factor Inductor de la Apoptosis/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Rápida/patología , Endodesoxirribonucleasas/metabolismo , Condicionamiento Físico Animal/métodos , Condicionamiento Físico Animal/fisiología , Músculo Esquelético/metabolismo , Ayuno Intermitente , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
BACKGROUND: Heart failure (HF) is characterized by oxidative stress and mitochondrial dysfunction. This study investigates the therapeutic potential of Necrostatin-1 (Nec-1) delivered through exosomes derived from induced pluripotent stem cells (iPSCs) to address these pathologies in HF. METHODS: An HF rat model was established, and comprehensive assessments were performed using echocardiography, hemodynamics, and ventricular mass index measurements. iPSCs were used to isolate exosomes, loaded with Nec-1, and characterized for efficient delivery into cardiomyocytes. The interaction between Nec-1-loaded exosomes (Nec-1-Exos), poly (ADP-ribose) polymerase 1 (PARP1), and apoptosis-inducing factor mitochondria-associated 1 (AIFM1) was explored. Gain-of-function experiments assessed changes in cardiomyocyte parameters, and histological analyses were conducted on myocardial tissues. RESULTS: Cardiomyocytes successfully internalized Nec-1-loaded exosomes, leading to downregulation of PARP1, inhibition of AIFM1 nuclear translocation, increased ATP and superoxide dismutase levels, reduced reactive oxygen species and malonaldehyde levels, and restored mitochondrial membrane potential. Histological examinations confirmed the modulation of the PARP1/AIFM1 axis by Nec-1, mitigating HF. CONCLUSIONS: iPSC-derived exosomes carrying Nec-1 attenuate oxidative stress and mitochondrial dysfunction in HF by targeting the PARP1/AIFM1 axis. This study proposes a promising therapeutic strategy for HF management and highlights the potential of exosome-mediated drug delivery.
Asunto(s)
Exosomas , Insuficiencia Cardíaca , Imidazoles , Indoles , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1 , Exosomas/metabolismo , Animales , Estrés Oxidativo/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Insuficiencia Cardíaca/metabolismo , Indoles/farmacología , Masculino , Imidazoles/farmacología , Cardiotónicos/farmacología , Ratas Sprague-Dawley , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Factor Inductor de la Apoptosis/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , RatasRESUMEN
Subarachnoid hemorrhage (SAH) is a neurological condition with high mortality and poor prognosis, and there are currently no effective therapeutic drugs available. Poly (ADP-ribose) polymerase 1 (PARP-1) dependent cell death pathway-parthanatos is closely associated with stroke. We investigated improvements in neurological function, oxidative stress, blood-brain barrier and parthanatos-related protein expression in rats with SAH after intraperitoneal administration of PARP-1 inhibitor (AG14361). Our study found that the expression of parthanatos-related proteins was significantly increased after SAH. Immunofluorescence staining showed increased expression of apoptosis-inducing factor (AIF) in the nucleus after SAH. Administration of PARP-1 inhibitor significantly reduced malondialdehyde (MDA) level and the expression of parthanatos-related proteins. Immunofluorescence staining showed that PARP-1 inhibitor reduced the expression of 8-hydroxy-2' -deoxyguanosine (8-OHdG) and thus reduced oxidative stress. Moreover, PARP-1 inhibitor could inhibit inflammation-associated proteins level and neuronal apoptosis, protect the blood-brain barrier and significantly improve neurological function after SAH. These results suggest that PARP-1 inhibitor can significantly improve SAH, and the underlying mechanism may be through inhibiting parthanatos pathway.
Asunto(s)
Barrera Hematoencefálica , Lesiones Encefálicas , Muerte Celular , Parthanatos , Poli(ADP-Ribosa) Polimerasa-1 , Hemorragia Subaracnoidea , Animales , Masculino , Ratas , Factor Inductor de la Apoptosis/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/etiología , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/patología , Muerte Celular/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Parthanatos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Sprague-Dawley , Hemorragia Subaracnoidea/metabolismo , Hemorragia Subaracnoidea/complicaciones , Hemorragia Subaracnoidea/tratamiento farmacológico , Hemorragia Subaracnoidea/patologíaRESUMEN
The functional integrity of the central nervous system relies on complex mechanisms in which the mitochondria are crucial actors because of their involvement in a multitude of bioenergetics and biosynthetic pathways. Mitochondrial diseases are among the most prevalent groups of inherited neurological disorders, affecting up to 1 in 5000 adults and despite considerable efforts around the world there is still limited curative treatments. Harlequin mice correspond to a relevant model of recessive X-linked mitochondrial disease due to a proviral insertion in the first intron of the Apoptosis-inducing factor gene, resulting in an almost complete depletion of the corresponding protein. These mice exhibit progressive degeneration of the retina, optic nerve, cerebellum, and cortical regions leading to irremediable blindness and ataxia, reminiscent of what is observed in patients suffering from mitochondrial diseases. We evaluated the progression of cerebellar degeneration in Harlequin mice, especially for Purkinje cells and its relationship with bioenergetics failure and behavioral damage. For the first time to our knowledge, we demonstrated that Harlequin mice display cognitive and emotional impairments at early stage of the disease with further deteriorations as ataxia aggravates. These functions, corresponding to higher-order cognitive processing, have been assigned to a complex network of reciprocal connections between the cerebellum and many cortical areas which could be dysfunctional in these mice. Consequently, Harlequin mice become a suitable experimental model to test innovative therapeutics, via the targeting of mitochondria which can become available to a large spectrum of neurological diseases.
Asunto(s)
Factor Inductor de la Apoptosis , Disfunción Cognitiva , Modelos Animales de Enfermedad , Metabolismo Energético , Células de Purkinje , Animales , Células de Purkinje/metabolismo , Células de Purkinje/patología , Ratones , Factor Inductor de la Apoptosis/metabolismo , Factor Inductor de la Apoptosis/genética , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Disfunción Cognitiva/genética , Masculino , Mitocondrias/metabolismo , Mitocondrias/patología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Enfermedades Mitocondriales/genética , Ratones Endogámicos C57BLRESUMEN
AIMS: Synaptic vesicle protein 2A (SV2A) is a unique therapeutic target for pharmacoresistant epilepsy (PRE). As seizure-induced neuronal programmed death, parthanatos was rarely reported in PRE. Apoptosis-inducing factor (AIF), which has been implicated in parthanatos, shares a common cytoprotective function with SV2A. We aimed to investigate whether parthanatos participates in PRE and is mitigated by SV2A via AIF. METHODS: An intraperitoneal injection of lithium chloride-pilocarpine was used to establish an epileptic rat model, and phenytoin and phenobarbital sodium were utilized to select PRE and pharmacosensitive rats. The expression of SV2A was manipulated via lentivirus delivery into the hippocampus. Video surveillance was used to assess epileptic ethology. Biochemical tests were employed to test hippocampal tissues following a successful SV2A infection. Molecular dynamic calculations were used to simulate the interaction between SV2A and AIF. RESULTS: Parthanatos core index, PARP1, PAR, nuclear AIF and MIF, γ-H2AX, and TUNEL staining were all increased in PRE. SV2A is bound to AIF to form a stable complex, successfully inhibiting AIF and MIF nuclear translocation and parthanatos and consequently mitigating spontaneous recurrent seizures in PRE. Moreover, parthanatos deteriorated after the SV2A reduction. SIGNIFICANCE: SV2A protected hippocampal neurons and mitigated epileptic seizures by inhibiting parthanatos via binding to AIF in PRE.
Asunto(s)
Factor Inductor de la Apoptosis , Epilepsia Refractaria , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso , Parthanatos , Ratas , Ratas Sprague-Dawley , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Epilepsia Refractaria/metabolismo , Regulación de la Expresión Génica , Regulación hacia Arriba , Proteínas de Neoplasias/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Transporte Activo de Núcleo Celular , Fosforilación , Fragmentación del ADN , Hipocampo/citología , Hipocampo/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
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
Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states.
Asunto(s)
Factor Inductor de la Apoptosis , Dispersión del Ángulo Pequeño , Regulación Alostérica , Relación Estructura-Actividad , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/metabolismo , Difracción de Rayos X/métodos , Humanos , Descubrimiento de Drogas/métodos , Modelos Moleculares , Cinética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.
Asunto(s)
Apoptosis , Quemaduras , Fibroblastos , Calor , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , Fibroblastos/patología , Fibroblastos/metabolismo , Quemaduras/patología , Calor/efectos adversos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Parthanatos , Necrosis , Células Cultivadas , Muerte Celular , Piel/patología , Piel/citología , Piel/lesiones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Caspasas/metabolismo , Roturas del ADN de Doble Cadena , Adenosina Trifosfato/metabolismoRESUMEN
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
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 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
Accumulating evidence indicates that microbial communities in the human body crucially affect health through the production of chemical messengers. However, the relationship between human microbiota and cancer has been underexplored. As a result of a biochemical investigation of the commensal oral microbe, Corynebacterium durum, we identified the non-enzymatic transformation of tryptamine into an anticancer compound, durumamide A (1). The structure of 1 was determined using LC-MS and NMR data analysis as bis(indolyl)glyoxylamide, which was confirmed using one-pot synthesis and X-ray crystallographic analysis, suggesting that 1 is an oxidative dimer of tryptamine. Compound 1 displayed cytotoxic activity against various cancer cell lines with IC50 values ranging from 25 to 35⯵M. A drug affinity responsive target stability assay revealed that survivin is the direct target protein responsible for the anticancer effect of 1, which subsequently induces apoptosis-inducing factor (AIF)-mediated apoptosis. Inspired by the chemical structure and bioactivity of 1, a new derivative, durumamide B (2), was synthesized using another indole-based neurotransmitter, serotonin. The anticancer properties of 2 were similar to those of 1; however, it was less active. These findings reinforce the notion of human microbiota-host interplay by showing that 1 is naturally produced from the human microbial metabolite, tryptamine, which protects the host against cancer.