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1.
Hum Mol Genet ; 27(15): 2628-2643, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29750247

RESUMEN

Ribosome biogenesis is a global process required for growth and proliferation in all cells, but disruptions in this process surprisingly lead to tissue-specific phenotypic disorders termed ribosomopathies. Pathogenic variants in the RNA Polymerase (Pol) I subunit POLR1A cause Acrofacial Dysostosis-Cincinnati type, which is characterized by craniofacial and limb anomalies. In a zebrafish model of Acrofacial Dysostosis-Cincinnati type, we demonstrate that polr1a-/- mutants exhibit deficient 47S rRNA transcription, reduced monosomes and polysomes and, consequently, defects in protein translation. This results in Tp53-dependent neuroepithelial apoptosis, diminished neural crest cell proliferation and cranioskeletal anomalies. This indicates that POLR1A is critical for rRNA transcription, which is considered a rate limiting step in ribosome biogenesis, underpinning its requirement for neuroepithelial cell and neural crest cell proliferation and survival. To understand the contribution of the Tp53 pathway to the pathogenesis of Acrofacial Dysostosis-Cincinnati type, we genetically inhibited tp53 in polr1a-/- mutant embryos. Tp53 inhibition suppresses neuroepithelial apoptosis and partially ameliorates the polr1a mutant phenotype. However, complete rescue of cartilage development is not observed due to the failure to improve rDNA transcription and neural crest cell proliferation. Altogether, these data reveal specific functions for both Tp53-dependent and independent signaling downstream of polr1a in ribosome biogenesis during neural crest cell and craniofacial development, in the pathogenesis of Acrofacial Dysostosis-Cincinnati type. Furthermore, our work sets the stage for identifying Tp53-independent therapies to potentially prevent Acrofacial dysostosis-Cincinnati type and other similar ribosomopathies.


Asunto(s)
Deformidades Congénitas de las Extremidades/metabolismo , Disostosis Mandibulofacial/metabolismo , Cresta Neural/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Diferenciación Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Humanos , Deformidades Congénitas de las Extremidades/patología , Disostosis Mandibulofacial/patología , Mutación , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética
2.
J Biol Chem ; 292(16): 6431-6437, 2017 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-28292928

RESUMEN

Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.


Asunto(s)
Daño del ADN , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alfa-Amanitina/metabolismo , Línea Celular , Proteínas Cullin/metabolismo , Reparación del ADN , Elonguina , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Mutación , Plásmidos/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Factores de Transcripción/genética
3.
J Biol Chem ; 290(24): 15030-41, 2015 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-25878247

RESUMEN

Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription.


Asunto(s)
Mutágenos/farmacología , Estrés Oxidativo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Elonguina , Transferencia Resonante de Energía de Fluorescencia , Técnica del Anticuerpo Fluorescente Indirecta , Células HEK293 , Humanos , ARN Mensajero/genética , Tretinoina/farmacología , Rayos Ultravioleta
4.
Int J Neuropsychopharmacol ; 16(6): 1309-18, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23228615

RESUMEN

The biological mechanisms of autism spectrum disorders (ASDs) are largely unknown in spite of extensive research. ASD is characterized by altered function of multiple brain areas including the temporal cortex and by an increased synaptic excitation:inhibition ratio. While numerous studies searched for evidence of increased excitation in ASD, fewer have investigated the possibility of reduced inhibition. We characterized the cortical γ-amino butyric acid (GABA)ergic system in the rat temporal cortex of an ASD model [offspring of mothers prenatally injected with valproic acid (VPA)], by monitoring inhibitory post-synaptic currents (IPSCs) with patch-clamp. We found that numerous features of inhibition were severely altered in VPA animals compared to controls. Among them were the frequency of miniature IPSCs, the rise time and decay time of electrically-evoked IPSCs, the slope and saturation of their input/output curves, as well as their modulation by adrenergic and muscarinic agonists and by the synaptic GABAA receptor allosteric modulator zolpidem (but not by the extra-synaptic modulator gaboxadol). Our data suggest that both pre- and post-synaptic, but not extra-synaptic, inhibitory transmission is impaired in the offspring of VPA-injected mothers. We speculate that impairment in the GABAergic system critically contributes to an increase in the ratio between synaptic excitation and inhibition, which in genetically predisposed individuals may alter cortical circuits responsible for emotional, communication and social impairments at the core of ASD.


Asunto(s)
Trastorno Autístico/patología , Ambiente , Neuronas GABAérgicas/fisiología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Transmisión Sináptica/fisiología , Lóbulo Temporal/patología , Animales , Antimaníacos/toxicidad , Trastorno Autístico/etiología , Biofisica , Modelos Animales de Enfermedad , Estimulación Eléctrica , Femenino , GABAérgicos/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Técnicas de Placa-Clamp , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Lóbulo Temporal/efectos de los fármacos , Ácido Valproico/toxicidad
5.
Synapse ; 66(1): 20-8, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21905124

RESUMEN

Noradrenergic terminals from the locus coeruleus release norepinephrine (NE) throughout most brain areas, including the auditory cortex, where they affect neural processing by modulating numerous cellular properties including the inhibitory γ-aminobutyric acid (GABA)ergic transmission. We recently demonstrated that NE affects GABAergic signaling onto cortical pyramidal cells in a complex manner. In this study, we used a combination of patch-clamp recording and immunohistochemical techniques to identify the synaptic site and the location of the adrenergic receptors involved in the modulation of GABAergic signaling in cortical layer 2/3 of the rat. Our results showed that NE increases the frequency of spike-independent miniature inhibitory postsynaptic currents (mIPSCs), as well as the probability of release of unitary inhibitory postsynaptic currents (IPSCs) obtained with patch-clamp pair-recordings. The pharmacology of mIPSCs and the identification of adrenergic receptors in neurons containing the GABAergic marker parvalbumin (PV) suggest that NE increases the presynaptic probability of GABA release by activating α(2) - and ß-receptors on PV-positive neurons. On the contrary, bath-applied NE or phenylephrine, decreased the current mediated by pressure application of the GABA(A) -receptor agonist muscimol, as well as the amplitude-but not the frequency-of mIPSCs, indicating that activation of postsynaptic α(1) adrenoceptors reversibly depressed GABAergic currents. We speculate that while a generalized postsynaptic decrease of GABAergic inhibition might decrease the synaptic activation threshold for pyramidal neurons corresponding to an alert state, NE might promote perception and sensory binding by facilitating lateral inhibition as well as the production of γ-oscillations by a selective enhancement of perisomatic inhibition.


Asunto(s)
Corteza Auditiva/metabolismo , Norepinefrina/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Corteza Auditiva/efectos de los fármacos , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Norepinefrina/farmacología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos
6.
Biol Psychiatry ; 71(7): 574-82, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22196984

RESUMEN

BACKGROUND: Although it is known that stress elevates the levels of pro-inflammatory cytokines and promotes hyper-excitable central conditions, a causal relationship between these two factors has not yet been identified. Recent studies suggest that increases in interleukin 6 (IL-6) levels are specifically associated with stress. We hypothesized that IL-6 acutely and directly induces cortical hyper-excitability by altering the balance between synaptic excitation and inhibition. METHODS: We used patch-clamp to determine the effects of exogenous or endogenous IL-6 on electrically evoked postsynaptic currents on a cortical rat slice preparation. We used control subjects or animals systemically injected with lipopolysaccharide or subjected to electrical foot-shock as rat models of stress. RESULTS: In control animals, IL-6 did not affect excitatory postsynaptic currents but selectively and reversibly reduced the amplitude of inhibitory postsynaptic currents with a postsynaptic effect. The IL-6-induced inhibitory postsynaptic currents decrease was inhibited by drugs interfering with receptor trafficking and/or internalization, including wortmannin, Brefeldin A, 2-Br-hexadecanoic acid, or dynamin peptide inhibitor. In both animal models, stress-induced decrease in synaptic inhibition/excitation ratio was prevented by prior intra-ventricular injection of an analog of the endogenous IL-6 trans-signaling blocker gp130. CONCLUSIONS: Our results suggest that stress-induced IL-6 shifts the balance between synaptic inhibition and excitation in favor of the latter, possibly by decreasing the density of functional γ-aminobutyric acid A receptors, accelerating their removal and/or decreasing their insertion rate from/to the plasma membrane. We speculate that this mechanism could contribute to stress-induced detrimental long-term increases in central excitability present in a variety of neurological and psychiatric conditions.


Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Interleucina-6/fisiología , Estrés Psicológico/fisiopatología , Lóbulo Temporal/fisiopatología , Androstadienos/farmacología , Animales , Brefeldino A/farmacología , Receptor gp130 de Citocinas/antagonistas & inhibidores , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/farmacología , Lipopolisacáridos , Muscimol/farmacología , Oligopéptidos/farmacología , Palmitatos/farmacología , Ratas , Estrés Psicológico/inducido químicamente , Lóbulo Temporal/efectos de los fármacos , Wortmanina
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