RESUMEN

BACKGROUND: Newly generated cardiomyocytes (NGCs) concur with the recovery of human myocarditis occurring spontaneously in around 50% of cases. However, NGCs decline with age, and their modality of myocardial homing and integration are still unclear. METHODS: We retrospectively assessed NGCs in 213 consecutive patients with endomyocardial biopsy denoting acute myocarditis, with normal coronaries and valves. Tissue samples were processed for histology (H&E), immunohistochemistry for the evaluation of inflammatory infiltrates, immunostaining for alpha-sarcomeric-actin, junctional connexin-43, Ki-67, and phosphorylated STAT3 (p-STAT3), and Western blot (WB) for HMGB1. Frozen samples were analyzed using polymerase chain reaction (PCR) for cardiotropic viruses. Controls included 20 normal surgical biopsies. RESULTS: NGCs were defined as small myocytes (diameter < 10 µm) with nuclear positivity to Ki-67 and p-STAT3 and positive immunostaining for cytoplasmic α-sarcomeric actin and connexin-43. Their number/mm2 in relation to age and pathway of integration was evaluated. NGCs crossed the membrane and grew integrated within the empty necrotic myocytes. NGC mean diameter was 6.6 ± 3.34 vs. 22.5 ± 3.11 µm adult cells; their number, in comparison to LVEF, was 86.3 ± 10.3/mm2 in patients between 18 and 40 years, 50.4 ± 13.8/mm2 in those between 41 and 60, and 15.1 ± 5.7/mm2 in those between 61 and 80. Control NGCs' mean diameter was 0.2 ± 0.2 mm2. PCR was positive for viral genomes in 16% of cases; NGCs were not statistically different in viral and non-viral myocarditis. WB analysis revealed a higher expression of HMGB1 in myocarditis compared to myocardial controls. CONCLUSIONS: NGCs are constantly recognizable in acute human myocarditis. Their number declines with age. Their integration within necrotic myocytes allows for the preservation of the cardiac structure and function.

RESUMEN

The key DNA repair enzyme DNA-PKcs has several and important cellular functions. Loss of DNA-PKcs activity in mice has revealed essential roles in immune and nervous systems. In humans, DNA-PKcs is a critical factor for brain development and function since mutation of the prkdc gene causes severe neurological deficits such as microcephaly and seizures, predicting yet unknown roles of DNA-PKcs in neurons. Here we show that DNA-PKcs modulates synaptic plasticity. We demonstrate that DNA-PKcs localizes at synapses and phosphorylates PSD-95 at newly identified residues controlling PSD-95 protein stability. DNA-PKcs -/- mice are characterized by impaired Long-Term Potentiation (LTP), changes in neuronal morphology, and reduced levels of postsynaptic proteins. A PSD-95 mutant that is constitutively phosphorylated rescues LTP impairment when over-expressed in DNA-PKcs -/- mice. Our study identifies an emergent physiological function of DNA-PKcs in regulating neuronal plasticity, beyond genome stability.

Asunto(s)

Proteína Quinasa Activada por ADN , Homólogo 4 de la Proteína Discs Large , Potenciación a Largo Plazo , Plasticidad Neuronal , Estabilidad Proteica , Animales , Fosforilación , Proteína Quinasa Activada por ADN/metabolismo , Proteína Quinasa Activada por ADN/genética , Ratones , Homólogo 4 de la Proteína Discs Large/metabolismo , Homólogo 4 de la Proteína Discs Large/genética , Neuronas/metabolismo , Ratones Noqueados , Humanos , Sinapsis/metabolismo , Reparación del ADN , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al ADN

RESUMEN

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are clinically diagnosed using neuropsychological and cognitive tests, expensive neuroimaging-based approaches (MRI and PET) and invasive and time-consuming lumbar puncture for cerebrospinal fluid (CSF) sample collection to detect biomarkers. Thus, a rapid, simple and cost-effective approach to more easily access fluids and tissues is in great need. Here, we exploit the chemical direct reprogramming of patient skin fibroblasts into neurons (chemically induced neurons, ciNs) as a novel strategy for the rapid detection of different pathological markers of neurodegenerative diseases. We found that FAD fibroblasts have a reduced efficiency of reprogramming, and converted ciNs show a less complex neuronal network. In addition, ciNs from patients show misfolded protein accumulation and mitochondria ultrastructural abnormalities, biomarkers commonly associated with neurodegeneration. Moreover, for the first time, we show that microfluidic technology, in combination with chemical reprogramming, enables on-chip examination of disease pathological processes and may have important applications in diagnosis. In conclusion, ciNs on microfluidic devices represent a small-scale, non-invasive and cost-effective high-throughput tool for protein misfolding disease diagnosis and may be useful for new biomarker discovery, disease mechanism studies and design of personalised therapies.

Asunto(s)

Biomarcadores/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/metabolismo , Neuronas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Femenino , Humanos , Dispositivos Laboratorio en un Chip , Masculino , Microfluídica/métodos , Persona de Mediana Edad , Neuroimagen/métodos , Pruebas Neuropsicológicas , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología

RESUMEN

BACKGROUND: Emerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair. METHODS AND RESULTS: We used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage. CONCLUSIONS: Taken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.

Asunto(s)

Proteína Quinasa Activada por ADN/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo/fisiología , Animales , Apoptosis/fisiología , Cromonas , ADN/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/genética , Histonas/metabolismo , Peróxido de Hidrógeno/metabolismo , Morfolinas , Proteínas Nucleares/genética , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas

RESUMEN

(1) Background: Nicotine is implicated in the SARS-COV-2 infection through activation of the α7-nAChR and over-expression of ACE2. Our objective was to clarify the role of nicotine in SARS-CoV-2 infection exploring its molecular and cellular activity. (2) Methods: HBEpC or si-mRNA-α7-HBEpC were treated for 1 h, 48 h or continuously with 10-7 M nicotine, a concentration mimicking human exposure to a cigarette. Cell viability and proliferation were evaluated by trypan blue dye exclusion and cell counting, migration by cell migration assay, senescence by SA-ß-Gal activity, and anchorage-independent growth by cloning in soft agar. Expression of Ki67, p53/phospho-p53, VEGF, EGFR/pEGFR, phospho-p38, intracellular Ca2+, ATP and EMT were evaluated by ELISA and/or Western blotting. (3) Results: nicotine induced through α7-nAChR (i) increase in cell viability, (ii) cell proliferation, (iii) Ki67 over-expression, (iv) phospho-p38 up-regulation, (v) EGFR/pEGFR over-expression, (vi) increase in basal Ca2+ concentration, (vii) reduction of ATP production, (viii) decreased level of p53/phospho-p53, (ix) delayed senescence, (x) VEGF increase, (xi) EMT and consequent (xii) enhanced migration, and (xiii) ability to grow independently of the substrate. (4) Conclusions: Based on our results and on evidence showing that nicotine potentiates viral infection, it is likely that nicotine is involved in SARS-CoV-2 infection and severity.

Asunto(s)

COVID-19/patología , Células Epiteliales/efectos de los fármacos , Nicotina/efectos adversos , Sistema Respiratorio/efectos de los fármacos , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/virología , Humanos , Receptores Nicotínicos/metabolismo , Sistema Respiratorio/virología , SARS-CoV-2/patogenicidad , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Fumar/efectos adversos , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo

RESUMEN

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity.

Asunto(s)

Diferenciación Celular , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Hígado/citología , Benzazepinas/farmacología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Epigénesis Genética/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Histonas/metabolismo , Humanos , Indoles/farmacología , Lisina/metabolismo , Metilación , Análisis de Componente Principal , Piridonas/farmacología , Pirimidinas/farmacología

RESUMEN

Neurodegenerative diseases are characterized by a gradual loss of cognitive and physical functions. Medications for these disorders are limited and treat the symptoms only. There are no disease-modifying therapies available, which have been shown to slow or stop the continuing loss of neurons. Transdifferentiation, whereby somatic cells are reprogrammed into another lineage without going through an intermediate proliferative pluripotent stem cell stage, provides an alternative strategy for regenerative medicine and disease modeling. In particular, the transdifferentiation of somatic cells into specific subset of patient-specific neuronal cells offers alternative autologous cell therapeutic strategies for neurodegenerative disorders and presents a rich source of using diverse somatic cell types for relevant applications in translational, personalized medicine, as well as human mechanistic study, new drug-target identification, and novel drug screening systems. Here, we provide a comprehensive overview of the recent development of transdifferentiation research, with particular attention to chemical-induced transdifferentiation and perspectives for modeling and treatment of neurodegenerative diseases.

Asunto(s)

Transdiferenciación Celular , Reprogramación Celular , Enfermedades Neurodegenerativas/patología , Animales , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Medicina Regenerativa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTß) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes.

Asunto(s)

Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Interleucina-6/genética , Transcripción Genética , Replicación Viral/genética , Metilación de ADN/genética , ADN Circular/genética , ADN Viral/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Regulación Viral de la Expresión Génica , Células Hep G2 , Virus de la Hepatitis B/patogenicidad , Hepatitis B Crónica/virología , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/genética , Histonas/genética , Humanos , Interleucina-6/metabolismo , ARN/genética , Factor de Transcripción STAT3/genética

RESUMEN

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas F-Box/metabolismo , Hipocótilo/crecimiento & desarrollo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Complejo del Señalosoma COP9 , Tamaño de la Célula/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Proteínas F-Box/química , Proteínas F-Box/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Hipocótilo/genética , Hipocótilo/efectos de la radiación , Luz , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Mutación/genética , Péptido Hidrolasas/metabolismo , Fenotipo , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica/efectos de la radiación , Proteolisis/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ligasas SKP Cullina F-box/química , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitinación/efectos de la radiación