RESUMEN
Dystrophin Dp71 is the most abundant product of the Duchenne muscular dystrophy gene in the nervous system, and mutations impairing its function have been associated with the neurodevelopmental symptoms present in a third of DMD patients. Dp71 is required for the clustering of neurotransmitter receptors and the neuronal differentiation of cultured cells; nonetheless, its precise role in neuronal cells remains to be poorly understood. In this study, we analyzed the effect of two pathogenic DMD gene point mutations on the Dp71 function in neurons. We engineered C272Y and E299del mutations to express GFP-tagged Dp71 protein variants in N1E-115 and SH-SY5Y neuronal cells. Unexpectedly, the ectopic expression of Dp71 mutants resulted in protein aggregation, which may be mechanistically caused by the effect of the mutations on Dp71 structure, as predicted by protein modeling and molecular dynamics simulations. Interestingly, Dp71 mutant variants acquired a dominant negative function that, in turn, dramatically impaired the distribution of different Dp71 protein partners, including ß-dystroglycan, nuclear lamins A/C and B1, the high-mobility group (HMG)-containing protein (BRAF35) and the BRAF35-family-member inhibitor of BRAF35 (iBRAF). Further analysis of Dp71 mutants provided evidence showing a role for Dp71 in modulating both heterochromatin marker H3K9me2 organization and the neuronal genes' expression, via its interaction with iBRAF and BRAF5.
Asunto(s)
Distrofina , Neuroblastoma , Distroglicanos/genética , Distrofina/genética , Heterocromatina , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Laminas/genética , Neuronas/metabolismo , Lámina Nuclear/metabolismo , Mutación Puntual , Agregado de Proteínas , Receptores de Neurotransmisores/genéticaRESUMEN
AIMS: Obesity and type 2 diabetes mellitus are two pathologies that share metabolic abnormalities in most of the cases; however, there are differences as well. Some studies have reported that approximately 30% of obese patients have normal glucose and lipid levels in blood despite an accumulation of abdominal adipose tissue. Here, we compare the gene expression in adipose tissue of several genes associated with obesity and/or diabetes between obese patients without T2D and obese patients with T2D. METHODS: Omental adipose tissue was collected during the patients elective bariatric surgery. Gene expression was determined by real-time PCR. Phenotypic variables were correlated with gene expression and 2^-ΔΔCt relative expression analysis between groups was performed. RESULTS: The stronger correlations in the obese without T2D or reference group was between ICAM1 and HbA1c; HP and TC and LDL while in the obese with diabetes or case group the correlation occurred between CSF1 and BMI. A correlation between HP and TC was found in the case group as well. The expression of VEGFA, CCND2, IL1R1 and PTEN was downregulated in the obese with T2D group. CONCLUSIONS: This study identified genes whose expression is different between obese subjects with and without diabetes. Those genes are related to inflammation, cholesterol transport, adipocyte differentiation/expansion and browning.
Asunto(s)
Tejido Adiposo/fisiología , Diabetes Mellitus Tipo 2/genética , Obesidad/genética , Adulto , Cirugía Bariátrica , Ciclina D2/genética , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Obesidad/cirugía , Fosfohidrolasa PTEN/genética , Fenotipo , Receptores Tipo I de Interleucina-1/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder characterized by cerebellar ataxia and retinopathy. SCA7 is caused by a CAG expansion in the ATXN7 gene, which results in an extended polyglutamine (polyQ) tract in the encoded protein, the ataxin-7. PolyQ expanded ataxin-7 elicits neurodegeneration in cerebellar Purkinje cells, however, its impact on the SCA7-associated retinopathy remains to be addressed. Since Müller glial cells play an essential role in retinal homeostasis, we generate an inducible model for SCA7, based on the glial Müller MIO-M1 cell line. The SCA7 pathogenesis has been explained by a protein gain-of-function mechanism, however, the contribution of the mutant RNA to the disease cannot be excluded. In this direction, we found nuclear and cytoplasmic foci containing mutant RNA accompanied by subtle alternative splicing defects in MIO-M1 cells. RNA foci were also observed in cells from different lineages, including peripheral mononuclear leukocytes derived from SCA7 patient, suggesting that this molecular mark could be used as a blood biomarker for SCA7. Collectively, our data showed that our glial cell model exhibits the molecular features of SCA7, which makes it a suitable model to study the RNA toxicity mechanisms, as well as to explore therapeutic strategies aiming to alleviate glial dysfunction.
RESUMEN
Radiotherapy, in addition to surgery and systemic chemotherapy, remains the core of the current clinical management of cancer. Radioresistance is one of the major causes of disease progression and mortality in cancer; therefore, it is a significant challenge in the treatment of locally advanced, recurrent and metastatic cancer. Epigenetic mechanisms that control hallmarks of cancer have a key role in the development of radiation resistance of cancer cells. Recent advances in DNA methylation, histone modification, chromatin remodeling and non-coding RNAs identified in the control of signal transduction pathways in cancer and cancer stem cells have provided even greater promise in the improvement of understanding cancer radioresistance. Many epigenetic drugs that target epigenetic enzymes revert the radioresistant phenotypes decreasing the possibility that resistant cancer cells will develop refractory tumors to radiotherapy. Epigenetic profiles identified as regulators of DNA damage repair, hypoxia, cell survival, apoptosis and invasion are determinants in the development of tumor radioresistance; hence, they also are promising in personalized medicine to develop novel targeted therapies or biomarkers to follow-up the effectiveness of radiotherapy. Now, it is clear that radiotherapy can influence a complex epigenetic network for transcriptional reprogramming, enabling the cells to adapt and avoid the effect of radiotherapy. This review aims to highlight the epigenetic modifications identified in cancer radioresistance and to discuss approaches to disable epigenetic networks to increase the sensitivity and specificity of radiotherapy.
Asunto(s)
Neoplasias , Apoptosis , Metilación de ADN , Epigénesis Genética , Humanos , Neoplasias/genética , Neoplasias/radioterapia , Transducción de SeñalRESUMEN
Myotonic dystrophy type 1 (DM1), the most frequent inherited muscular dystrophy in adults, is caused by the CTG repeat expansion in the 3'UTR of the DMPK gene. Mutant DMPK RNA accumulates in nuclear foci altering diverse cellular functions including alternative splicing regulation. DM1 is a multisystemic condition, with debilitating central nervous system alterations. Although a defective neuroglia communication has been described as a contributor of the brain pathology in DM1, the specific cellular and molecular events potentially affected in glia cells have not been totally recognized. Thus, to study the effects of DM1 mutation on glial physiology, in this work, we have established an inducible DM1 model derived from the MIO-M1 cell line expressing 648 CUG repeats. This new model recreated the molecular hallmarks of DM1 elicited by a toxic RNA gain-of-function mechanism: accumulation of RNA foci colocalized with MBNL proteins and dysregulation of alternative splicing. By applying a microarray whole-transcriptome approach, we identified several gene changes associated with DM1 mutation in MIO-M1 cells, including the immune mediators CXCL10, CCL5, CXCL8, TNFAIP3, and TNFRSF9, as well as the microRNAs miR-222, miR-448, among others, as potential regulators. A gene ontology enrichment analyses revealed that inflammation and immune response emerged as major cellular deregulated processes in the MIO-M1 DM1 cells. Our findings indicate the involvement of an altered immune response in glia cells, opening new windows for the study of glia as potential contributor of the CNS symptoms in DM1.
Asunto(s)
Mutación , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica/genética , Neuroglía/metabolismo , Transcriptoma , Regiones no Traducidas 3' , Empalme Alternativo , Línea Celular , Núcleo Celular/metabolismo , Sistema Nervioso Central/metabolismo , Exones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Humanos , Sistema Inmunológico , Inflamación , Distrofia Miotónica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
Nuclear ß-dystroglycan (ß-DG) is involved in the maintenance of nuclear architecture and function. Nonetheless, its relevance in defined nuclear processes remains to be determined. In this study we generated a C2C12 cell-based DG-null model using CRISPR-Cas9 technology to provide insights into the role of ß-DG on nuclear processes. Since DG-null cells exhibited decreased levels of lamin B1, we aimed to elucidate the contribution of DG to senescence, owing to the central role of lamin B1 in this pathway. Remarkably, the lack of DG enables C2C12 cells to acquire senescent features, including cell-cycle arrest, increased senescence-associated-ß-galactosidase activity, heterochromatin loss, aberrant nuclear morphology and nucleolar disruption. We demonstrated that genomic instability is one driving cause of the senescent phenotype in DG-null cells via the activation of a DNA-damage response associated with mitotic failure, as shown by the presence of multipolar mitotic spindles, which in turn induced the formation of micronuclei and γH2AX foci (DNA-damage marker), telomere shortening and p53/p21 upregulation. Altogether, these events might ultimately lead to premature senescence, impeding the replication of the damaged genome. In summary, we present evidence supporting a role for DG in protecting against senescence, through the maintenance of proper lamin B1 expression/localization and proper mitotic spindle organization.
Asunto(s)
Senescencia Celular/genética , Distroglicanos/genética , Inestabilidad Genómica/genética , Mitosis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Puntos de Control del Ciclo Celular/genética , Línea Celular , Núcleo Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN/genética , Histonas/genética , Lamina Tipo B/genética , Ratones , Ratones Noqueados , Huso Acromático/genética , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genética , beta-Galactosidasa/genéticaRESUMEN
ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.
Asunto(s)
Nucléolo Celular/metabolismo , Distroglicanos/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , ARN Ribosómico/biosíntesis , Animales , Proliferación Celular/genética , Citoplasma/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Distroglicanos/antagonistas & inhibidores , Distroglicanos/genética , Ratones , Mioblastos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Estrés Oxidativo , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Dominios Proteicos/genética , ARN Ribosómico/genética , Ribosomas/metabolismo , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
Sepsis is a life-threatening organ-dysfunction condition caused by a dysregulated response to an infectious condition that can cause complications in patients with major trauma. Burns are one of the most destructive forms of trauma; despite the improvements in medical care, infections remain an important cause of burn injury-related mortality and morbidity, and complicated sepsis predisposes patients to diverse complications such as organ failure, lengthening of hospital stays, and increased costs. Accurate diagnosis and early treatment of sepsis may have a beneficial impact on clinical outcome of burn-injured patients. In this review, we offer a comprehensive description of the current and traditional markers used as indicative of sepsis in burned patients. However, although these are markers of the inflammatory post-burn response, they usually fail to predict sepsis in severely burned patients due to that they do not reflect the severity of the infection. Identification and measurement of biomarkers in early stages of infection is important in order to provide timely response and effective treatment of burned patients. Therefore, we compiled important experimental evidence, demonstrating novel biomarkers, including molecular markers such as genomic DNA variations, alterations of transcriptome profiling (mRNA, miRNAs, lncRNAs and circRNAs), epigenetic markers, and advances in proteomics and metabolomics. Finally, this review summarizes next-generation technologies for the identification of markers for detection of sepsis after burn injuries.
Asunto(s)
Biomarcadores/metabolismo , Quemaduras/complicaciones , Sepsis/diagnóstico , Diagnóstico Precoz , Perfilación de la Expresión Génica , Genómica , Humanos , Pruebas en el Punto de Atención , Sepsis/complicaciones , Sepsis/genética , Sepsis/metabolismoRESUMEN
We have adopted the PC12 cell line as in vitro cell model for studying Dp71 function in neuronal cells. These cells express a cytoplasmic (Dp71f) and a nuclear (Dp71d) isoform of Dp71 as well as various dystrophin-associated proteins (DAPs). In this study, we revealed by confocal microscopy analysis and Western blotting evaluation of cell fractions the presence of different DAPs (beta-dystroglycan, beta-dystrobrevin, epsilon-sarcoglycan and gamma1-syntrophin) in the nucleus of PC12 cells. Furthermore, we established by immunoprecipitation assays that Dp71d and the DAPs form a dystrophin-associated protein complex (DAPC) in the nucleus. Interestingly, depletion of Dp71 by antisense treatment (antisense-Dp71 cells) provoked a drastic reduction of nuclear DAPs, which indicates that Dp71d is critical for DAPs stability within the nucleus. Although Up71, the utrophin gene product homologous to Dp71, exhibited increased expression in the antisense-Dp71 cells, its scarce nuclear levels makes unlikely that could compensate for Dp71 nuclear deficiency.