RESUMEN
Friedreich ataxia (FRDA) is a life-threatening hereditary ataxia; its incidence is 1:50,000 individuals in the Caucasian population. A unique therapeutic drug for FRDA, the antioxidant Omaveloxolone, has been recently approved by the US Food and Drug Administration (FDA). FRDA is a multi-systemic neurodegenerative disease; in addition to a progressive neurodegeneration, FRDA is characterized by hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. Cardiomyopathy is the predominant cause of premature death. The onset of FRDA typically occurs between the ages of 5 and 15. Given the complexity and heterogeneity of clinical features and the variability of their onset, the identification of biomarkers capable of assessing disease progression and monitoring the efficacy of treatments is essential to facilitate decision making in clinical practice. We conducted an RNA-seq analysis in peripheral blood mononuclear cells from FRDA patients and healthy donors, identifying a signature of small non-coding RNAs (sncRNAs) capable of distinguishing healthy individuals from the majority of FRDA patients. Among the differentially expressed sncRNAs, microRNAs are a class of small non-coding endogenous RNAs that regulate posttranscriptional silencing of target genes. In FRDA plasma samples, hsa-miR-148a-3p resulted significantly upregulated. The analysis of the Receiver Operating Characteristic (ROC) curve, combining the circulating expression levels of hsa-miR-148a-3p and hsa-miR-223-3p (previously identified by our group), revealed an Area Under the Curve (AUC) of 0.86 (95%, Confidence Interval 0.77-0.95; p-value < 0.0001). An in silico prediction analysis indicated that the IL6ST gene, an interesting marker of neuroinflammation in FRDA, is a common target gene of both miRNAs. Our findings support the evaluation of combined expression levels of different circulating miRNAs as potent epi-biomarkers in FRDA. Moreover, we found hsa-miR-148a-3p significantly over-expressed in Intermediate and Late-Onset Friedreich Ataxia patients' group (IOG and LOG, respectively) compared to healthy individuals, indicating it as a putative prognostic biomarker in this pathology.
Asunto(s)
Biomarcadores , Ataxia de Friedreich , MicroARNs , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Ataxia de Friedreich/sangre , MicroARNs/genética , MicroARNs/sangre , Masculino , Biomarcadores/sangre , Pronóstico , Femenino , Adulto , RNA-Seq , Adolescente , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Niño , Adulto Joven , Persona de Mediana Edad , Preescolar , Curva ROC , Estudios de Casos y ControlesRESUMEN
Frataxin (FXN) deficiency is responsible for Friedreich's ataxia (FRDA) in which, besides the characteristic features of spinocerebellar ataxia, two thirds of patients develop hypertrophic cardiomyopathy that often progresses to heart failure and premature death. Different mechanisms might underlie FRDA pathogenesis. Among them, the role of miRNAs deserves investigations. We carried out an miRNA PCR-array analysis of plasma samples of early-, intermediate- and late-onset FRDA groups, defining a set of 30 differentially expressed miRNAs. Hsa-miR223-3p is the only miRNA shared between the three patient groups and appears upregulated in all of them. The up-regulation of hsa-miR223-3p was further validated in all enrolled patients (n = 37, Fc = +2.3; P < 0.0001). Using a receiver operating characteristic curve analysis, we quantified the predictive value of circulating hsa-miR223-3p for FRDA, obtaining an area under the ROC curve value of 0.835 (P < 0.0001) for all patients. Interestingly, we found a significant positive correlation between hsa-miR223-3p expression and cardiac parameters in typical FRDA patients (onset < 25 years). Moreover, a significant negative correlation between hsa-miR223-3p expression and HAX-1 (HCLS1-associated protein X-1) at mRNA and protein level was observed in all FRDA patients. In silico analyses suggested HAX-1 as a target gene of hsa-miR223-3p. Accordingly, we report that HAX-1 is negatively regulated by hsa-miR223-3p in cardiomyocytes (AC16) and neurons (SH-SY5Y), which are critically affected cell types in FRDA. This study describes for the first time the association between hsa-miR223-3p and HAX-1 expression in FRDA, thus supporting a potential role of this microRNA as non-invasive epigenetic biomarker for FRDA.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Ataxia de Friedreich , MicroARNs , Neuroblastoma , Proteínas Adaptadoras Transductoras de Señales/genética , Ataxia de Friedreich/patología , Humanos , MicroARNs/sangre , Miocitos Cardíacos/metabolismo , Neuroblastoma/metabolismo , ARN Mensajero/genéticaRESUMEN
Friedreich's ataxia (FRDA) is a rare monogenic disease characterized by multisystem, slowly progressive degeneration. Because of the genetic defect in a non-coding region of FXN gene, FRDA cells exhibit severe deficit of frataxin protein levels. Hence, FRDA pathophysiology is characterized by a plethora of metabolic disruptions related to iron metabolism, mitochondrial homeostasis and oxidative stress. Importantly, an impairment of the antioxidant defences exacerbates the oxidative damage. This appears closely associated with the disablement of key antioxidant proteins, such as the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the mitochondrial superoxide dismutase (MnSOD). The cytokine interferon gamma (IFN-γ) has been shown to increase frataxin expression in FRDA cells and to improve functional deficits in FRDA mice. Currently, IFN-γ represents a potential therapy under clinical evaluation in FRDA patients. Here, we show that IFN-γ induces a rapid expression of Nrf2 and MnSOD in different cell types, including FRDA patient-derived fibroblasts. Our data indicate that IFN-γ signals two separate pathways to enhance Nrf2 and MnSOD levels in FRDA fibroblasts. MnSOD expression increased through an early transcriptional regulation, whereas the levels of Nrf2 are induced by a post-transcriptional mechanism. We demonstrate that the treatment of FRDA fibroblasts with IFN-γ stimulates a non-canonical Nrf2 activation pathway through p21 and potentiates antioxidant responses under exposure to hydrogen peroxide. Moreover, IFN-γ significantly reduced the sensitivity to hydrogen peroxide-induced cell death in FRDA fibroblasts. Collectively, these results indicate the presence of multiple pathways triggered by IFN-γ with therapeutic relevance to FRDA.
Asunto(s)
Ataxia de Friedreich , Interferón gamma , Animales , Ratones , Interferón gamma/farmacología , Factor 2 Relacionado con NF-E2/genética , Antioxidantes/farmacología , Ataxia de Friedreich/genética , Peróxido de Hidrógeno , Superóxido DismutasaRESUMEN
Frataxin deficiency, responsible for Friedreich's ataxia (FRDA), is crucial for cell survival since it critically affects viability of neurons, pancreatic beta cells and cardiomyocytes. In FRDA, the heart is frequently affected with typical manifestation of hypertrophic cardiomyopathy, which can progress to heart failure and cause premature death. A microarray analysis performed on FRDA patient's lymphoblastoid cells stably reconstituted with frataxin, indicated HS-1-associated protein X-1 (HAX-1) as the most significantly upregulated transcript (FC = +2, P < 0.0006). quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and western blot analysis performed on (I) HEK293 stably transfected with empty vector compared to wild-type frataxin and (II) lymphoblasts from FRDA patients show that low frataxin mRNA and protein expression correspond to reduced levels of HAX-1. Frataxin overexpression and silencing were also performed in the AC16 human cardiomyocyte cell line. HAX-1 protein levels are indeed regulated through frataxin modulation. Moreover, correlation between frataxin and HAX-1 was further evaluated in peripheral blood mononuclear cells (PBMCs) from FRDA patients and from non-related healthy controls. A regression model for frataxin which included HAX-1, group membership and group* HAX-1 interaction revealed that frataxin and HAX-1 are associated both at mRNA and protein levels. Additionally, a linked expression of FXN, HAX-1 and antioxidant defence proteins MnSOD and Nrf2 was observed both in PBMCs and AC16 cardiomyocytes. Our results suggest that HAX-1 could be considered as a potential biomarker of cardiac disease in FRDA and the evaluation of its expression might provide insights into its pathogenesis as well as improving risk stratification strategies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cardiomiopatía Hipertrófica/patología , Ataxia de Friedreich/complicaciones , Regulación de la Expresión Génica , Insuficiencia Cardíaca/patología , Proteínas de Unión a Hierro/metabolismo , Miocitos Cardíacos/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Cardiomiopatía Hipertrófica/etiología , Cardiomiopatía Hipertrófica/metabolismo , Femenino , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Adulto Joven , FrataxinaRESUMEN
BACKGROUND: Friedreich's ataxia is an autosomal-recessive cerebellar ataxia caused by mutation of the frataxin gene, resulting in decreased frataxin expression, mitochondrial dysfunction, and oxidative stress. Currently, no treatment is available for Friedreich's ataxia patients. Given that levels of residual frataxin critically affect disease severity, the main goal of a specific therapy for Friedreich's ataxia is to increase frataxin levels. OBJECTIVES: With the aim to accelerate the development of a new therapy for Friedreich's ataxia, we took a drug repositioning approach to identify market-available drugs able to increase frataxin levels. METHODS: Using a cell-based reporter assay to monitor variation in frataxin amount, we performed a high-throughput screening of a library containing 853 U.S. Food and Drug Administration-approved drugs. RESULTS: Among the potentially interesting candidates isolated from the screening, we focused our attention on etravirine, an antiviral drug currently in use as an anti-human immunodeficiency virus therapy. Here, we show that etravirine can promote a significant increase in frataxin levels in cells derived from Friedreich's ataxia patients, by enhancing frataxin messenger RNA translation. Importantly, frataxin accumulation in treated patient cell lines is comparable to frataxin levels in unaffected carrier cells, suggesting that etravirine could be therapeutically relevant. Indeed, etravirine treatment restores the activity of the iron-sulphur cluster containing enzyme aconitase and confers resistance to oxidative stress in cells derived from Friedreich's ataxia patients. CONCLUSIONS: Considering its excellent safety profile along with its ability to increase frataxin levels and correct some of the disease-related defects, etravirine represents a promising candidate as a therapeutic for Friedreich's ataxia. © 2019 International Parkinson and Movement Disorder Society.
Asunto(s)
Ataxia de Friedreich/tratamiento farmacológico , Proteínas de Unión a Hierro/metabolismo , Piridazinas/uso terapéutico , Línea Celular , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Nitrilos , Pirimidinas , FrataxinaRESUMEN
Defective expression of frataxin is responsible for the inherited, progressive degenerative disease Friedreich's Ataxia (FRDA). There is currently no effective approved treatment for FRDA and patients die prematurely. Defective frataxin expression causes critical metabolic changes, including redox imbalance and ATP deficiency. As these alterations are known to regulate the tyrosine kinase Src, we investigated whether Src might in turn affect frataxin expression. We found that frataxin can be phosphorylated by Src. Phosphorylation occurs primarily on Y118 and promotes frataxin ubiquitination, a signal for degradation. Accordingly, Src inhibitors induce accumulation of frataxin but are ineffective on a non-phosphorylatable frataxin-Y118F mutant. Importantly, all the Src inhibitors tested, some of them already in the clinic, increase frataxin expression and rescue the aconitase defect in frataxin-deficient cells derived from FRDA patients. Thus, Src inhibitors emerge as a new class of drugs able to promote frataxin accumulation, suggesting their possible use as therapeutics in FRDA.
Asunto(s)
Ataxia de Friedreich/genética , Proteínas de Unión a Hierro/biosíntesis , Familia-src Quinasas/genética , Adenosina Trifosfato/deficiencia , Adenosina Trifosfato/genética , Inhibidores Enzimáticos/farmacología , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Unión a Hierro/genética , Oxidación-Reducción , Ubiquitinación/genética , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo , FrataxinaRESUMEN
Friedreich ataxia is an inherited neurodegenerative disease that leads to progressive disability. There is currently no effective treatment and patients die prematurely. The underlying genetic defect leads to reduced expression of the mitochondrial protein frataxin. Frataxin insufficiency causes mitochondrial dysfunction and ultimately cell death, particularly in peripheral sensory ganglia. There is an inverse correlation between the amount of residual frataxin and the severity of disease progression; therefore, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients' conditions. We previously discovered that a significant amount of frataxin precursor is degraded by the ubiquitin/proteasome system before its functional mitochondrial maturation. We also provided evidence for the therapeutic potential of small molecules that increase frataxin levels by docking on the frataxin ubiquitination site, thus preventing frataxin ubiquitination and degradation. We called these compounds ubiquitin-competing molecules (UCM). By extending our search for effective UCM, we identified a set of new and more potent compounds that more efficiently promote frataxin accumulation. Here we show that these compounds directly interact with frataxin and prevent its ubiquitination. Interestingly, these UCM are not effective on the ubiquitin-resistant frataxin mutant, indicating their specific action on preventing frataxin ubiquitination. Most importantly, these compounds are able to promote frataxin accumulation and aconitase rescue in cells derived from patients, strongly supporting their therapeutic potential.
Asunto(s)
Aconitato Hidratasa/metabolismo , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Fármacos Neuroprotectores/farmacología , Sitios de Unión , Línea Celular , Diseño de Fármacos , Fluorescencia , Células HEK293 , Humanos , Immunoblotting , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/genética , Simulación del Acoplamiento Molecular , Mutación , Fármacos Neuroprotectores/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitinación/efectos de los fármacos , FrataxinaRESUMEN
Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting â¼3 in 100 000 individuals in Caucasian populations. It is caused by intronic GAA repeat expansions that hinder the expression of the FXN gene, resulting in defective levels of the mitochondrial protein frataxin. Sensory neurons in dorsal root ganglia (DRG) are particularly damaged by frataxin deficiency. There is no specific therapy for FRDA. Here, we show that frataxin levels can be upregulated by interferon gamma (IFNγ) in a variety of cell types, including primary cells derived from FRDA patients. IFNγ appears to act largely through a transcriptional mechanism on the FXN gene. Importantly, in vivo treatment with IFNγ increases frataxin expression in DRG neurons, prevents their pathological changes and ameliorates the sensorimotor performance in FRDA mice. These results disclose new roles for IFNγ in cellular metabolism and have direct implications for the treatment of FRDA.
Asunto(s)
Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Interferón gamma/farmacología , Interferón gamma/fisiología , Proteínas de Unión a Hierro/biosíntesis , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/patología , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Células HeLa , Humanos , Interferón gamma/uso terapéutico , Proteínas de Unión a Hierro/genética , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Transcripción Genética , Activación Transcripcional , FrataxinaRESUMEN
Friedreich's ataxia (FRDA) is a devastating orphan disease, with no specific treatment. The disease is caused by reduced expression of the protein frataxin, which results in mitochondrial defects and oxidative damage. Levels of residual frataxin critically affect onset and progression of the disease. Understanding the molecular mechanisms that regulate frataxin stability and degradation may, therefore, be exploited for the design of effective therapeutics. Here we show that frataxin is degraded by the ubiquitin-proteasome system and that K(147) is the critical residue responsible for frataxin ubiquitination and degradation. Accordingly, a K(147)R substitution generates a more stable frataxin. We then disclose a set of lead compounds, computationally selected to target the molecular cleft harboring K(147), that can prevent frataxin ubiquitination and degradation, and increase frataxin levels in cells derived from FRDA patients. Moreover, treatment with these compounds induces substantial recovery of aconitase activity and adenosine-5'-triphosphate levels in FRDA cells. Thus, we provide evidence for the therapeutic potential of directly interfering with the frataxin degradation pathway.
Asunto(s)
Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Ataxia de Friedreich/tratamiento farmacológico , Ataxia de Friedreich/genética , Células HEK293 , Humanos , Proteínas de Unión a Hierro/genética , Mutación Missense , Complejo de la Endopetidasa Proteasomal/genética , Ubiquitina/genética , FrataxinaRESUMEN
The inability to produce normal levels of the mitochondrial protein frataxin causes the hereditary degenerative disorder Friedreich's Ataxia (FRDA), a syndrome characterized by progressive gait instability, cardiomyopathy and high incidence of diabetes. Frataxin is an iron-binding protein involved in the biogenesis of iron-sulfur clusters (ISC), prosthetic groups allowing essential cellular functions such as oxidative phosphorylation, enzyme catalysis and gene regulation. Although several evidence suggest that frataxin acts as an iron-chaperone within the mitochondrial compartment, we have recently demonstrated the existence of a functional extramitochondrial pool of mature frataxin in various human cell types. Here, we show that a similar proteolytic process generates both mature mitochondrial and extramitochondrial frataxin. To address the physiological function of human extramitochondrial frataxin, we searched for ISC-dependent interaction partners. We demonstrate that the extramitochondrial form of frataxin directly interacts with cytosolic aconitase/iron regulatory protein-1 (IRP1), a bifunctional protein alternating between an enzymatic and a RNA-binding function through the 'iron-sulfur switch' mechanism. Importantly, we found that the cytosolic aconitase defect and consequent IRP1 activation occurring in FRDA cells are reversed by the action of extramitochondrial frataxin. These results provide new insight into the control of cytosolic aconitase/IRP1 switch and expand current knowledge about the molecular pathogenesis of FRDA.
Asunto(s)
Aconitato Hidratasa/metabolismo , Citosol/metabolismo , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas de Unión a Hierro/farmacología , Aconitato Hidratasa/genética , Células Cultivadas , Ataxia de Friedreich/genética , Regulación de la Expresión Génica , Humanos , Proteína 1 Reguladora de Hierro/genética , FrataxinaRESUMEN
Friedreich ataxia is a rare neurodegenerative disorder caused by insufficient levels of the essential mitochondrial protein frataxin. It is a severely debilitating disease that significantly impacts the quality of life of affected patients and reduces their life expectancy, however, an adequate cure is not yet available for patients. Frataxin function, although not thoroughly elucidated, is associated with assembly of iron-sulfur cluster and iron metabolism, therefore insufficient frataxin levels lead to reduced activity of many mitochondrial enzymes involved in the electron transport chain, impaired mitochondrial metabolism, reduced ATP production and inefficient anti-oxidant response. As a consequence, neurons progressively die and patients progressively lose their ability to coordinate movement and perform daily activities. Therapeutic strategies aim at restoring sufficient frataxin levels or at correcting some of the downstream consequences of frataxin deficiency. However, the classical pathways of drug discovery are challenging, require a significant amount of resources and time to reach the final approval, and present a high failure rate. Drug repositioning represents a viable alternative to boost the identification of a therapy, particularly for rare diseases where resources are often limited. In this review we will describe recent efforts aimed at the identification of a therapy for Friedreich ataxia through drug repositioning, and discuss the limitation of such strategies.
RESUMEN
GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3.
Asunto(s)
Apoptosis , Gangliósidos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Acetilación , Acetilesterasa/genética , Acetilesterasa/metabolismo , Caspasas/metabolismo , Línea Celular , Activación Enzimática , Citometría de Flujo , Gangliósidos/química , Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Mitocondrias/metabolismo , Ácido N-Acetilneuramínico/química , Receptores de Superficie Celular/metabolismoRESUMEN
Novel strategies have been proposed for articular cartilage damage occurring during osteoarthritis (OA) and -among these- Extracorporeal Shock Wave Therapy (ESWT), intra-articular injections of Platelet-Rich Plasma (PRP) or Hyaluronic Acid (HA) revealed encouraging results. To investigate the possible mechanisms responsible for those clinical benefits, we established primary cultures of human chondrocytes derived from cartilage explants and measured the in vitro effects of ESW, PRP and HA therapies. After molecular/morphological cell characterization, we assessed those effects on the functional activities of the chondrocyte cell cultures, at the protein and molecular levels. ESWT significantly prevented the progressive dedifferentiation that spontaneously occurs during prolonged chondrocyte culture. We then attested the efficiency of all such treatments to stimulate the expression of markers of chondrogenic potential such as SOX9 and COL2A, to increase the Ki67 proliferation index as well as to antagonize the traditional marker of chondrosenescence p16INK4a (known as Cdkn2a). Furthermore, all our samples showed an ESW- and HA-mediated enhancement of migratory and anti-inflammatory activity onto the cytokine-rich environment characterizing OA. Taken together, those results suggest a regenerative effect of such therapies on primary human chondrocytes in vitro. Moreover, we also show for the first time that ESW treatment induces the surface expression of major hyaluronan cell receptor CD44 allowing the increase of COL2A/COL1A ratio upon HA administration. Therefore, this work suggests that ESW-induced CD44 overexpression enhances the in vitro cell susceptibility of human chondrocytes to HA, presumably favouring the repair of degenerated cartilage.
Asunto(s)
Condrocitos/fisiología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/administración & dosificación , Osteoartritis/terapia , Plasma Rico en Plaquetas/química , Regeneración , Anciano , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/fisiología , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Técnicas de Cocultivo , Tratamiento con Ondas de Choque Extracorpóreas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ácido Hialurónico/farmacología , Inyecciones Intraarticulares , Persona de Mediana Edad , Osteoartritis/inmunologíaRESUMEN
The regulation of dendritic cell (DC) survival is crucial for the modulation of adaptive immunity. Ceramide is a lipid mediator of the stress response, which accumulates intracellularly during DC differentiation. We found that ceramide levels are tightly regulated in human DCs and that the pharmacological inhibition of enzymes responsible for ceramide catabolism, such as ceramidases and sphingosine kinases, sensitizes DCs to ceramide-induced cell death. It is important that inhibition of sphingosine kinases, during lipopolysaccharide stimulation, causes extensive ceramide accumulation and death of DCs. These data indicate that ceramide catabolism regulates survival of human DCs and reveal novel potential targets for the pharmacological manipulation of the immune response.
Asunto(s)
Ceramidas/metabolismo , Células Dendríticas/enzimología , Inhibidores Enzimáticos/farmacología , Galactosidasas/antagonistas & inhibidores , Factores Inmunológicos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Ceramidas/inmunología , Células Dendríticas/inmunología , Galactosidasas/inmunología , Humanos , Inmunidad Activa/efectos de los fármacos , Inmunidad Activa/inmunología , Lipopolisacáridos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunologíaRESUMEN
Friedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. To date, there is no therapy to treat this condition. The amount of residual frataxin critically affects the severity of the disease; thus, attempts to restore physiological frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the frataxin E3 ligase may represent a strategy to achieve an increase in frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates frataxin ubiquitination and targets it for degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.
Asunto(s)
Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Catálisis , Línea Celular , Células HEK293 , Humanos , Proteínas Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , FrataxinaRESUMEN
Extracorporeal shock wave therapy (ESWT) is a non-invasive and innovative technology for the management of specific tendinopathies. In order to elucidate the ESWT-mediated clinical benefits, human Tendon-derived Stem/Progenitor cells (hTSPCs) explanted from 5 healthy semitendinosus (ST) and 5 ruptured Achilles (AT) tendons were established. While hTSPCs from the two groups showed similar proliferation rates and stem cell surface marker profiles, we found that the clonogenic potential was maintained only in cells derived from healthy donors. Interestingly, ESWT significantly accelerated hTSPCs differentiation, suggesting that the clinical benefits of ESWT may be ascribed to increased efficiency of tendon repair after injury.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Ondas de Choque de Alta Energía , Células Madre/citología , Traumatismos de los Tendones/patología , Tendones/citología , Adipogénesis/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Osteogénesis/efectos de la radiación , Células Madre/fisiología , Células Madre/efectos de la radiación , Traumatismos de los Tendones/radioterapia , Tendones/fisiología , Tendones/efectos de la radiaciónRESUMEN
Lipid and glycolipid mediators are important messengers of the adaptive responses to stress, including apoptosis. In mammalian cells, the intracellular accumulation of ganglioside GD3, an acidic glycosphingolipid, contributes to mitochondrial damage, a crucial event during the apoptopic program. GD3 is a minor ganglioside in most normal tissues. Its expression increases during development and in pathological conditions such as cancer and neurodegenerative disorders. Intriguingly, GD3 can mediate additional biological events such as cell proliferation and differentiation. These diverse and opposing effects indicate that tightly regulated mechanisms, including 9-O-acetylation, control GD3 function, by affecting intracellular levels, localization and structure of GD3, and eventually dictate biological outcomes and cell fate decisions.
Asunto(s)
Apoptosis/fisiología , Gangliósidos/fisiología , Animales , Diferenciación Celular , División Celular , Ceramidas/metabolismo , Gangliósidos/biosíntesis , Gangliósidos/química , Gangliósidos/metabolismo , Glicosiltransferasas/metabolismo , Humanos , Inflamación/metabolismo , Canales Iónicos/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , FN-kappa B/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Células Tumorales CultivadasRESUMEN
An accelerated activity of the GD3 synthase (ST8), with consequent GD3 accumulation, is part of the response to environmental stressors in different cell types. Depending on specific, yet largely undefined, cellular settings, this can be followed by adaptation or apoptosis, the latter mostly due to GD3-induced mitochondrial damage. Here we show that subcellular localization of ST8 could significantly affect the biological outcome of GD3 accumulation. Binding to the molecular chaperone calnexin causes the retention of ST8 within the endoplasmic reticulum (ER) and prevents its relocalization to the Golgi. Calnexin-dependent ER retention does not affect the activity of ST8; yet the de novo synthesized GD3 largely fails to reach the mitochondria. Accordingly, overexpression of calnexin suppresses the pro-apoptotic activity of ST8, while the loss of calnexin sensitizes the cells to ST8-induced apoptosis. Reconstitution of calnexin confers protection to deficient cells. Thus, calnexin controls the biological outcome of GD3 accumulation and reveals a novel role in the stress response.
Asunto(s)
Apoptosis , Calnexina/metabolismo , Sialiltransferasas/antagonistas & inhibidores , Sialiltransferasas/metabolismo , Animales , Calnexina/genética , Línea Celular , Pollos , Cricetinae , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , Mitocondrias/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Mitochondria are key players in fundamental processes such as energy production and adaptive responses to cellular stress, including apoptosis. Mitochondrial membranes may undergo permeabilization when perturbed by a number of intracellular stress mediators, and consequently may allow the release of intramitochondrial proteins. This event triggers and amplifies the cellular apoptotic program, provided sufficient energy is available. Mitochondrial membranes are therefore both targets and regulators of intracellular pathways controlling cell fate. Evidence is emerging that the integration and biological outcome of these pathways might be critically dependent on the unique lipid composition of mitochondrial membranes.
Asunto(s)
Apoptosis/fisiología , Lípidos/fisiología , Mitocondrias/fisiología , Animales , Permeabilidad de la Membrana Celular/fisiología , Humanos , Membranas Intracelulares/metabolismo , Lípidos/química , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
Lipid and glycolipid mediators are important components of the adaptive responses to stress, including apoptosis. In mammalian cells, the intracellular accumulation of ganglioside GD3, an acidic glycosphingolipid, contributes to mitochondrial damage, a crucial event during the apoptotic program. GD3 is a minor ganglioside in most normal tissues. Its expression increases during development and in pathological conditions such as cancer and neurodegenerative disorders. Interestingly, GD3 expression also increases with the normal ageing process. Moreover, GD3 can also mediate biological events like proliferation and differentiation. Since organism integrity requires a tight balance between cell proliferation, apoptosis and senescence, controlling the intracellular level of GD3 appears of particular importance for cell fate determination.