RESUMEN
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.
Asunto(s)
Ataxia/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Naftiridinas/farmacología , Temblor/genética , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ataxia/tratamiento farmacológico , Ataxia/patología , Proliferación Celular/efectos de los fármacos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/antagonistas & inhibidores , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/patología , Células HeLa , Humanos , Ligandos , Neuronas/efectos de los fármacos , Neuronas/patología , Péptidos/genética , Biosíntesis de Proteínas/efectos de los fármacos , Resonancia por Plasmón de Superficie , Temblor/tratamiento farmacológico , Temblor/patología , Expansión de Repetición de Trinucleótido/genética , Repeticiones de Trinucleótidos/efectos de los fármacos , Repeticiones de Trinucleótidos/genéticaRESUMEN
By convention, carbon isotope ratios are expressed relative to VPDB defined by the calcite standard NBS19 in the 1980s. [See T. Coplen, Pure Appl. Chem. 1994, 66, 273-276.] To improve the realization of the VPDB scale, a second fixed point (lithium carbonate, LSVEC) was introduced in 2006 [T. Coplen et al. Anal. Chem. 2006, 78, 2439-2441], which is now known to be isotopically unstable. [Assonov, S. Rapid Commun. Mass Spectrom., 2018, 32, 827-830.] With the high-quality reference materials made available in 2020, it is now possible to realize the VPDB scale with high confidence. [Assonov, S. et al. Rapid Commun. Mass Spectrom., 2020, 34, e8867; Assonov, S. Rapid Commun. Mass Spectrom. 2021, 35, e9014; Qi, H. et al. Rapid Commun. Mass Spectrosc. 2021, 35, e9006.] Here, we report the analysis of 25 reference materials using isotope ratio combustion mass spectrometry, show the discontinuity between the values measured against the new IAEA reference materials and the values currently assigned to these reference materials on the VPDB2006, and provide a link bringing these materials onto the new VPDB2020.
Asunto(s)
Carbonato de Calcio , Isótopos de Carbono , Espectrometría de Masas , Estándares de ReferenciaRESUMEN
Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Mutación , Proteína FUS de Unión a ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Regiones no Traducidas 3' , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Neurociencias , Plásmidos/metabolismo , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting-an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.
Asunto(s)
Axones/patología , Factor Neurotrófico Derivado del Encéfalo/genética , Epilepsia del Lóbulo Temporal/genética , Convulsiones/genética , Transcripción Genética/genética , Animales , Epigénesis Genética/genética , Epilepsia del Lóbulo Temporal/patología , Masculino , Fibras Musgosas del Hipocampo/patología , Neurogénesis/genética , Plasticidad Neuronal/genética , Ratas , Convulsiones/patologíaRESUMEN
The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states.
Asunto(s)
Cromatina/metabolismo , Cromatina/ultraestructura , Algoritmos , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , ADN/ultraestructura , Humanos , Hibridación in Situ , Microscopía Confocal , Microscopía Electrónica , Microscopía Electrónica de RastreoRESUMEN
The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.
RESUMEN
Deleted in Liver Cancer (DLC) proteins belong to the family of RhoGAPs and are believed to operate as negative regulators of the Rho family of small GTPases. So far, the role of the first identified member from the DLC family, DLC1, was established as a tumor suppressor in hepatocellular carcinoma. The function of its close family relative, DLC2 is unequivocal. In the present study we attempted to determine whether the loss of DLC2 is a common feature of hepatocellular carcinoma tissue. We examined two types of hepatocellular carcinoma- typical and fibrolamellar one. Our analysis revealed that DLC2 protein is not diminished in cancer tissue when compared to non-cancerous liver specimens. What is more, we observed DLC2 to be more abundantly expressed in cancer tissue, particularly in tumors with the inflammation background. In addition, we found that DLC2 gene status was diploid in virtually all tumor samples examined. Our results indicate that DLC2 is not diminished in hepatocellular carcinoma cells. It appears that members of the DLC family, although structurally highly related, may function differently in cancer cells.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Diploidia , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/inmunología , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by expanded CGG (CGGexp) trinucleotides in the 5'UTR of the FMR1 gene encoding fragile X mental retardation protein (FMRP). The patients, with the number of the repeats ranging from 55 to 200, show specific manifestation of clinical symptoms that include intention tremor, gait ataxia, cognitive deficits, and brain atrophy. Accumulation of toxic polyglycine (FMRpolyG), a by-product of the CGGexp repeat-associated non-ATG (RAN) translation, is considered to be one of the main factors triggering neurodegenerative processes in FXTAS patients. Nevertheless, the nature of the FMRpolyG-induced cell damage, especially in the context of its soluble and inclusion-associated forms, is still elusive. Targeting either biosynthesis, cellular stability or aggregation capacity of toxic FMRpolyG could be considered as a potential therapeutic strategy for FXTAS. Therefore, we tested a variety of quantitative methods based on forced expression of genetic constructs carrying CGGexp repeats in the context of the FMR1 5'UTR fused to GFP, mCherry or Firefly luciferase gene in or out of frame to the polyglycine encoding sequence. We show that FMRpolyG translation either from native or an AUG-induced start codon as well as the translation yield of the FMRP open reading frame equivalent located downstream of the CGGexp element can be effectively estimated using fluorescence microscopy, flow cytometry or luciferase assay. We also quantitatively estimated soluble fraction and insoluble form of FMRpolyG aggregated in foci using an electrophoretic separation of cell lysates and fluorescence microscopy, respectively. Importantly, we show that dependent on a fusion tag, FMRpolyG has a different potential for aggregate formation. Our established protocols enable sensitive tracking of FMRP and FMRpolyG quantitative and qualitative changes after treatment with potential therapeutic agents for FXTAS. Furthermore, they can be modified for application to other RAN translation- and aggregation-related diseases.
RESUMEN
Differentiation of progenitor cells into adipocytes is accompanied by remarkable changes in cell morphology, cytoskeletal organization, and gene expression profile. Mature adipocytes are filled with a large lipid droplet and the nucleus tends to move to the cell periphery. It was hypothesized that the differentiation process is also associated with changes of nuclear organization. The aim of this study was to determine the number and distribution of selected components of nuclear architecture during porcine in vitro adipogenesis. The pig is an important animal model sharing many similarities to humans at the anatomical, physiological, and genetic levels and has been recognized as a good model for human obesity. Thus, understanding how cellular structures important for fundamental nuclear processes may be altered during adipocyte differentiation is of great importance. Mesenchymal stem cells (MSCs) were derived from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) and were cultured for 7 days in the adipogenic medium. A variable differentiation potential of these cell populations towards adipogenic lineage was observed, and for further study, a comparative characteristic of the nuclear organization in BM-MSCs and AD-MSCs was performed. Nuclear substructures were visualized by indirect immunofluorescence (nucleoli, nuclear speckles, PML bodies, lamins, and HP1α) or fluorescence in situ hybridization (telomeres) on fixed cells at 0, 3, 5, and 7 days of differentiation. Comprehensive characterization of these structures, in terms of their number, size, dynamics, and arrangement in three-dimensional space of the nucleus, was performed. It was found that during differentiation of porcine MSCs into adipocytes, changes of nuclear organization occurred and concerned: (1) the nuclear size and shape; (2) reduced lamin A/C expression; and (3) reorganization of chromocenters. Other elements of nuclear architecture such as nucleoli, SC-35 nuclear speckles, and telomeres showed no significant changes when compared to undifferentiated and mature fat cells. In addition, the presence of a low number of PML bodies was characteristic of the studied porcine mesenchymal stem cell adipogenesis system. It has been shown that the arrangement of selected components of nuclear architecture was very similar in MSCs derived from different sources, whereas adipocyte differentiation involves nuclear reorganization. This study adds new data on nuclear organization during adipogenesis using the pig as a model organism.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Núcleo Celular/metabolismo , Células Madre Mesenquimatosas/citología , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación Fluorescente in Situ , PorcinosRESUMEN
The deleted in liver cancer (DLC) protein family is composed of proteins that are hypothesized to function predominantly by regulating the activity of the small GTPases. The aim of the present study was to determine the expression and exact localization of DLC1 in hepatocellular carcinoma (HCC) tissue sections. In two types of HCC tissues, typical and fibrolamellar, immunohistochemical and immunofluorescent analysis were performed to assess DLC1 immunoreactivity. Additionally, the DLC1 gene status was determined by the fluorescence in situ hybridization. According to the observations, DLC1 is often lost in cancer cells; however, it can remain within the stromal component of tumor sections. The DLC1 immunoreactivity was particularly noticeable within the capsules surrounding the tumor masses. It was found that the DLC1 gene was deleted in 52% of HCC cases. In addition, the hemizygous deletion was observed to be independent of the HCC subtype. The results indicate that although the loss of DLC1 is a common step during hepatocarcinogenesis, this protein may be present in the tumor microenvironment.
RESUMEN
Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Epigénesis Genética/fisiología , Receptor trkB/fisiología , Convulsiones/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Masculino , Ratas , Ratas Wistar , Convulsiones/genética , Translocación Genética/fisiologíaRESUMEN
Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.
Asunto(s)
Epilepsia/enzimología , Epilepsia/genética , Hipocampo/anomalías , Metaloproteinasa 9 de la Matriz/genética , Sinapsis/metabolismo , Animales , Animales Modificados Genéticamente , Convulsivantes , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Modelos Animales de Enfermedad , Epilepsia/fisiopatología , Hipocampo/patología , Hipocampo/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Inmunoelectrónica , Fibras Musgosas del Hipocampo/anomalías , Fibras Musgosas del Hipocampo/patología , Fibras Musgosas del Hipocampo/fisiopatología , Vías Nerviosas/anomalías , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Plasticidad Neuronal/genética , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Sinapsis/patologíaRESUMEN
OBJECTIVES AND METHODS: To evaluate the results of thymectomy in myasthenia gravis we performed retrospective analysis of 82 consecutive patients in the mean age of 39 +/- 15 treated between 1991 and 2001. All patients underwent extended thymectomy by median sternotomy. Follow-up was assessed in 74 of 81(91.4%) patients, in the mean age of 39 +/- 15, discharged from the Department. RESULTS: Fifty three (71.6%) patients had symptoms of myasthenia gravis for less than 2 years. According to Osserman's classification 8 (10.8%) patients were assessed as class I, 32 (43.2%) as IIA 26 (35.2%) as IIB and 8 (10.8%) as IIC. In the postoperative period 8 (10.8%) patients had respiratory insufficiency, 5 (6.8%) were reoperated for bleeding. One patient died (1.4%) due to bilateral pneumonia and pulmonary insufficiency. After thymectomy the improvement of patient's clinical status was observed in 46 patients (86.4%) and complete remission was in 13 patients (17.6%). Prompt improvement after thymectomy (p = 0.008) and short duration of symptoms (p = 0.036) are positive predictive factors. Patients in class I had significantly better prognosis concerning complete remission (p = 0.036). Age, gender, histology of the thymus, and type of the thymoma had no influence on long time follow up. CONCLUSIONS: Extended thymectomy is a safe procedure leading to the improvement in majority of patients treated for myasthenia gravis.