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1.
Dev Dyn ; 2023 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-37909656

RESUMEN

BACKGROUND: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. RESULTS: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. CONCLUSIONS: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes.

2.
Int J Mol Sci ; 24(1)2022 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-36613611

RESUMEN

Haploinsufficiency of the SETD5 gene, encoding a SET domain-containing histone methyltransferase, has been identified as a cause of intellectual disability and Autism Spectrum Disorder (ASD). Recently, the zebrafish has emerged as a valuable model to study neurodevelopmental disorders because of its genetic tractability, robust behavioral traits and amenability to high-throughput drug screening. To model human SETD5 haploinsufficiency, we generated zebrafish setd5 mutants using the CRISPR/Cas9 technology and characterized their morphological, behavioral and molecular phenotypes. According to our observation that setd5 is expressed in adult zebrafish brain, including those areas controlling social behavior, we found that setd5 heterozygous mutants exhibit defective aggregation and coordination abilities required for shoaling interactions, as well as indifference to social stimuli. Interestingly, impairment in social interest is rescued by risperidone, an antipsychotic drug used to treat behavioral traits in ASD individuals. The molecular analysis underscored the downregulation of genes encoding proteins involved in the synaptic structure and function in the adult brain, thus suggesting that brain hypo-connectivity could be responsible for the social impairments of setd5 mutant fishes. The zebrafish setd5 mutants display ASD-like features and are a promising setd5 haploinsufficiency model for drug screening aimed at reversing the behavioral phenotypes.


Asunto(s)
Trastorno del Espectro Autista , Metiltransferasas , Animales , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Metiltransferasas/genética , Metiltransferasas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Conducta Social
3.
Stem Cells ; 31(12): 2842-7, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24038725

RESUMEN

The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adjacent ectodermal territories. Unexpectedly, we also observed that Rx1, mainly through the activation of the transcriptional repressors TLE2 and Hes4, is necessary and sufficient to inhibit endomesodermal gene expression in retinal precursors of the eye field. In particular, Rx1 knockdown leads retinogenic blastomeres to adopt an endomesodermal fate, indicating a previously undescribed function for Rx1 in preventing the expression of endomesoderm determinants known to inhibit retinal fate. Altogether these data suggest that an essential requirement to establish a retinal precursor identity is the active inhibition of pathways leading to alternative fates.


Asunto(s)
Proteínas del Ojo/metabolismo , Proteínas Represoras/metabolismo , Retina/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Retina/citología , Xenopus laevis
4.
Genes (Basel) ; 15(4)2024 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-38674426

RESUMEN

Haploinsufficiency of the PRR12 gene is implicated in a human neuro-ocular syndrome. Although identified as a nuclear protein highly expressed in the embryonic mouse brain, PRR12 molecular function remains elusive. This study explores the spatio-temporal expression of zebrafish PRR12 co-orthologs, prr12a and prr12b, as a first step to elucidate their function. In silico analysis reveals high evolutionary conservation in the DNA-interacting domains for both orthologs, with significant syntenic conservation observed for the prr12b locus. In situ hybridization and RT-qPCR analyses on zebrafish embryos and larvae reveal distinct expression patterns: prr12a is expressed early in zygotic development, mainly in the central nervous system, while prr12b expression initiates during gastrulation, localizing later to dopaminergic telencephalic and diencephalic cell clusters. Both transcripts are enriched in the ganglion cell and inner neural layers of the 72 hpf retina, with prr12b widely distributed in the ciliary marginal zone. In the adult brain, prr12a and prr12b are found in the cerebellum, amygdala and ventral telencephalon, which represent the main areas affected in autistic patients. Overall, this study suggests PRR12's potential involvement in eye and brain development, laying the groundwork for further investigations into PRR12-related neurobehavioral disorders.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Proteínas de Pez Cebra , Animales , Encéfalo/metabolismo , Encéfalo/crecimiento & desarrollo , Retina/metabolismo , Retina/crecimiento & desarrollo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas de la Membrana/genética
5.
Int J Dev Biol ; 68(2): 85-91, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39016375

RESUMEN

The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Xenopus , Xenopus laevis , Animales , Xenopus laevis/metabolismo , Xenopus laevis/embriología , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Embrión no Mamífero/metabolismo , Embrión no Mamífero/embriología , Desarrollo Embrionario/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo
6.
Sci Rep ; 13(1): 6025, 2023 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-37055439

RESUMEN

In proliferating multipotent retinal progenitors, transcription factors dynamics set the fate of postmitotic daughter cells, but postmitotic cell fate plasticity driven by extrinsic factors remains controversial. Transcriptome analysis reveals the concurrent expression by postmitotic rod precursors of genes critical for the Müller glia cell fate, which are rarely generated from terminally-dividing progenitors as a pair with rod precursors. By combining gene expression and functional characterisation in single cultured rod precursors, we identified a time-restricted window where increasing cell culture density switches off the expression of genes critical for Müller glial cells. Intriguingly, rod precursors in low cell culture density maintain the expression of genes of rod and glial cell fate and develop a mixed rod/Muller glial cells electrophysiological fingerprint, revealing rods derailment toward a hybrid rod-glial phenotype. The notion of cell culture density as an extrinsic factor critical for preventing rod-fated cells diversion toward a hybrid cell state may explain the occurrence of hybrid rod/MG cells in the adult retina and provide a strategy to improve engraftment yield in regenerative approaches to retinal degenerative disease by stabilising the fate of grafted rod precursors.


Asunto(s)
Neuroglía , Retina , Retina/metabolismo , Neuroglía/metabolismo , Diferenciación Celular/genética , Factores de Transcripción/metabolismo , Técnicas de Cultivo de Célula
7.
Int J Antimicrob Agents ; 59(2): 106516, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-34999239

RESUMEN

High concentrations of ivermectin demonstrated antiviral activity against SARS-CoV-2 in vitro. The aim of this study was to assess the safety and efficacy of high-dose ivermectin in reducing viral load in individuals with early SARS-CoV-2 infection. This was a randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial. Participants were adults recently diagnosed with asymptomatic/oligosymptomatic SARS-CoV-2 infection. Exclusion criteria were: pregnant or lactating women; CNS disease; dialysis; severe medical condition with prognosis <6 months; warfarin treatment; and antiviral/chloroquine phosphate/hydroxychloroquine treatment. Participants were assigned (ratio 1:1:1) according to a randomised permuted block procedure to one of the following arms: placebo (arm A); single-dose ivermectin 600 µg/kg plus placebo for 5 days (arm B); and single-dose ivermectin 1200 µg/kg for 5 days (arm C). Primary outcomes were serious adverse drug reactions (SADRs) and change in viral load at Day 7. From 31 July 2020 to 26 May 2021, 32 participants were randomised to arm A, 29 to arm B and 32 to arm C. Recruitment was stopped on 10 June because of a dramatic drop in cases. The safety analysis included 89 participants and the change in viral load was calculated in 87 participants. No SADRs were registered. Mean (S.D.) log10 viral load reduction was 2.9 (1.6) in arm C, 2.5 (2.2) in arm B and 2.0 (2.1) in arm A, with no significant differences (P = 0.099 and 0.122 for C vs. A and B vs. A, respectively). High-dose ivermectin was safe but did not show efficacy to reduce viral load.


Asunto(s)
Antivirales/farmacocinética , Tratamiento Farmacológico de COVID-19 , Ivermectina/farmacocinética , SARS-CoV-2/efectos de los fármacos , Adulto , Antiparasitarios/sangre , Antiparasitarios/farmacocinética , Antiparasitarios/farmacología , Antivirales/sangre , Antivirales/farmacología , COVID-19/sangre , COVID-19/virología , Método Doble Ciego , Reposicionamiento de Medicamentos , Femenino , Humanos , Ivermectina/sangre , Ivermectina/farmacología , Masculino , Persona de Mediana Edad , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/patogenicidad , Resultado del Tratamiento , Carga Viral/efectos de los fármacos
8.
J Neurochem ; 119(6): 1317-29, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21988318

RESUMEN

Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity. In mice with OIR, beta-adrenergic receptor (ß-AR) blockade with propranolol has been shown to ameliorate different aspects of retinal dysfunction in response to hypoxia. In the present study, we used the OIR model to investigate the role of distinct ß-ARs on retinal proangiogenic factors, pathogenic neovascularization and electroretinographic responses. Our results demonstrate that ß(2) -AR blockade with ICI 118,551 decreases retinal levels of proangiogenic factors and reduces pathogenic neovascularization, whereas ß(1) - and ß(3) -AR antagonists do not. Determination of retinal protein kinase A activity is indicative of the fact that ß-AR blockers are indeed effective at the receptor level. In addition, the specificity of ICI 118,551 on retinal angiogenesis has been demonstrated by the finding that in mouse retinal explants, ß(2) -AR silencing prevents ICI 118,551 effects on hypoxia-induced vascular endothelial growth factor accumulation. In OIR mice, ICI 118,551 is effective in increasing electroretinographic responses suggesting that activation of ß(2) -ARs constitutes an important part of the retinal response to hypoxia. Lastly, immunohistochemical studies demonstrate that ß(2) -ARs are localized to several retinal cells, particularly to Müller cells suggesting the possibility that ß(2) -ARs play a role in regulating vascular endothelial growth factor production by these cells. The present results suggest that pathogenic angiogenesis, a key change in many hypoxic/ischemic vision-threatening retinal diseases, depends at least in part on ß(2) -AR activity and indicate that ß(2) -AR blockade can be effective against retinal angiogenesis.


Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Inhibidores de la Angiogénesis/uso terapéutico , Modelos Animales de Enfermedad , Oxígeno/efectos adversos , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/tratamiento farmacológico , Antagonistas Adrenérgicos beta/uso terapéutico , Animales , Animales Recién Nacidos , Atenolol/uso terapéutico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Recién Nacido , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Propanolaminas/uso terapéutico , ARN Mensajero , ARN Interferente Pequeño/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Neovascularización Retiniana/tratamiento farmacológico , Retinopatía de la Prematuridad/fisiopatología , Estadísticas no Paramétricas , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo
9.
Eur J Neurosci ; 33(3): 482-98, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21198982

RESUMEN

The vascular endothelial growth factor (VEGF) signalling pathway may represent an endogenous anti-convulsant in the rodent hippocampus although its exact contribution requires some clarification. In mouse hippocampal slices, the potassium channel blocker 4-aminopyridine (4-AP) in the absence of external Mg(2+)(0 Mg(2+)) produces both ictal and interictal activity followed by a prolonged period of repetitive interictal activity. In this model, we demonstrated that exogenous VEGF has clear effects on ictal and interictal activity as it reduces the duration of ictal-like events, but decreases the frequency and intensity of interictal discharges. VEGF affects epileptiform activity through its receptor VEGFR-2. We also demonstrated for the first time that the synaptic action of VEGF in the hippocampus is through VEGFR-2-mediated effects on NMDA and GABA(B) receptors and that VEGF does not affect the NMDA excytatory postsynaptic potential paired-pulse facilitation ratio. Exogenous VEGF does not affect the AMPA-mediated responses and the dendritic or the somatic GABA(A) inhibitory postsynaptic potentials. In addition, VEGF drastically reduces 0 Mg(2+)/4-AP-induced glutamate release through VEGFR-2 activation. In vitro epileptiform activity is sufficient to increase hippocampal expression of VEGF and VEGFR-2, and this up-regulation may serve a neuroprotective and/or anti-convulsant role. VEGFR-2 up-regulation has been localized to the CA1 region, which suggests that VEGF signalling may protect CA1 pyramidal cells from hyperexcitability. These results indicate that VEGF controls epileptic activity by influencing both glutamatergic and GABAergic transmission and further advance our understanding of the conditions required for endogenous VEGF up-regulation, and the mechanisms by which VEGF achieves an anti-convulsant effect.


Asunto(s)
Hipocampo/metabolismo , Neuronas/metabolismo , Convulsiones/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Potenciales Postsinápticos Excitadores/fisiología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Técnicas de Cultivo de Órganos , Receptores de GABA-B/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transmisión Sináptica/fisiología , Regulación hacia Arriba , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
10.
Microorganisms ; 9(8)2021 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-34442817

RESUMEN

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

11.
Clin Microbiol Infect ; 27(12): 1845-1850, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34329793

RESUMEN

OBJECTIVES: To assess the antibody response to BNT162b2 mRNA COVID-19 vaccine in a cohort of health-care workers (HCW), comparing individuals with previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and SARS-CoV-2-naive individuals. METHODS: HCW were tested at T0 (day of first dose), T1 (day of second dose) and T2 (2-3 weeks after second dose) for IgG anti-nucleocapsid protein, IgM anti-spike protein and IgG anti-receptor binding domain (IgG-RBD-S). The antibody response was compared between four main groups: group A, individuals with previous infection and positive antibodies at baseline; group B, individuals with the same history but negative antibodies; group C, individuals with no infection history but positive antibodies; group D, naive individuals. Repeated measures analysis was used to compare results over time-points. RESULTS: A total of 1935 HCW were included. Median IgG-RBD-S titre was significantly higher for group A (232 individuals) than for group B (56 individuals) both at T1 (A: 22 763 AU/mL, interquartile range (IQR) 14 222-37 204 AU/mL; B: 1373 AU/mL, IQR 783-3078 AU/mL, p 0.0003) and T2 (A: 30 765 AU/mL, IQR 19 841-42 813 AU/mL; B: 13 171 AU/mL, IQR 2324-22 688 AU/mL, p 0.0038) and for group D (1563 individuals; 796 AU/mL, IQR 379-1510 AU/mL at T1; 15 494 AU/mL, IQR 9122-23 916 AU/mL at T2, p < 0.0001 for both time-points). T1 values of group A were also significantly higher than T2 values of group D (p < 0.0001). Presence of symptoms, younger age and being female were associated with stronger antibody response. HCW infected in March showed a significantly stronger response (T1: 35 324 AU/mL, IQR 22 003-44 531 AU/mL; T2: 37 648 AU/mL, IQR 27 088-50 451 AU/mL) than those infected in November (T1: 18 499 AU/mL, IQR 11 492-27 283 AU/mL; T2: 23 210 AU/mL, IQR 18 074-36 086 AU/mL, p < 0.0001 for both time-points. CONCLUSIONS: Individuals with past SARS-CoV-2 infection had a strong antibody response after one single vaccine shot. A single dose might be sufficient for this group, regardless of the time elapsed since infection; however, the clinical correlation with antibody response needs to be studied.


Asunto(s)
Anticuerpos Antivirales/sangre , Formación de Anticuerpos , Vacuna BNT162/inmunología , COVID-19 , COVID-19/prevención & control , Personal de Salud , Humanos , Inmunoglobulina G/sangre , Estudios Prospectivos , ARN Mensajero
12.
Sci Rep ; 10(1): 17358, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-33060638

RESUMEN

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.


Asunto(s)
Berberina/toxicidad , Sistema Cardiovascular/embriología , Morfogénesis/efectos de los fármacos , Pez Cebra/embriología , Animales , Teratógenos/toxicidad
13.
J Neurochem ; 111(6): 1466-77, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19811607

RESUMEN

Somatostatin-14 (SRIF) co-localizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of hippocampal activity has been proposed, although the exact contribution of each SRIF receptor (sst(1)-sst(5)) in mediating SRIF action requires some clarification. We used hippocampal slices of wild-type and sst(1) knockout (KO) mice and selective pharmacological tools to provide conclusive evidence for a role of sst(1) in mediating SRIF inhibition of synaptic transmission. With single- and double-label immunohistochemistry, we determined the distribution of sst(1) in hippocampal slices and we quantified sst(1) colocalization with SRIF. With electrophysiology, we found that sst(1) activation with CH-275 inhibited both the NMDA- and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated responses. Results from sst(1) KO slices confirmed the specificity of CH-275 effects; sst(1) activation did not affect the inhibitory transmission which was in contrast increased by sst(4) activation with L-803,087 in both wild-type and sst(1) KO slices. The AMPA-mediated responses were increased by L-803,087. Functional interaction between sst(1) and sst(4) is suggested by the finding that their combined activation prevented the CH-275-induced inhibition of AMPA transmission. The involvement of pre-synaptic mechanisms in mediating inhibitory effects of sst(1) on excitatory transmission was demonstrated by the finding that CH-275 (i) increased the paired-pulse facilitation ratio, (ii) did not influence the AMPA depolarization in the presence of tetrodotoxin, and (iii) inhibited glutamate release induced by epileptiform treatment. We conclude that SRIF control of excitatory transmission through an action at sst(1) may represent an important contribution to the regulation of hippocampal activity.


Asunto(s)
Hipocampo/citología , Neuronas/fisiología , Receptores de Somatostatina/fisiología , Transmisión Sináptica/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Amidas/farmacología , Análisis de Varianza , Animales , Biofisica , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Técnicas In Vitro , Indoles/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/antagonistas & inhibidores , Receptores de Somatostatina/deficiencia , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Somatostatina/farmacología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética
14.
J Eukaryot Microbiol ; 56(3): 263-9, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19527354

RESUMEN

The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus. We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.


Asunto(s)
Apoptosis , Euplotes/efectos de los fármacos , Sesquiterpenos/toxicidad , Animales , Membrana Celular/ultraestructura , Fragmentación del ADN , Euplotes/metabolismo , Euplotes/ultraestructura , Etiquetado Corte-Fin in Situ/métodos , Viabilidad Microbiana , Orgánulos/ultraestructura , Sales de Tetrazolio/metabolismo , Tiazoles/metabolismo
15.
Int J Dev Biol ; 63(6-7): 311-316, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31250915

RESUMEN

Collagen prolyl 4-hydroxylases (c-P4Hs) are evolutionary conserved enzymes whose activity is essential for the correct folding of stable triple helical molecules of collagen and collagen-like proteins. They play crucial roles in embryo development, connective tissue functional organization, tumor growth and metastasis. Despite the important function of these enzymes, little is known about their expression during vertebrate development. In this study, we determine and compare the previously undescribed spatio-temporal expression patterns of the p4ha1 and p4ha2 genes, which encode the main subunits containing the enzyme active site, during Xenopus development. The two genes are maternally inherited and share expression in dorsal mesoderm, branchial arches and their derivatives, as well as in the central nervous system, although with distinct spatio-temporal patterns. A major co-expression domain for p4ha1 and p4ha2 is represented by the developing notochord, where these genes are transcribed from early neurula stage to stage 42 tadpole, thus paralleling the profile of collagen II production and suggesting a coordination between collagen synthesis and its post-translational modifications.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Procolágeno-Prolina Dioxigenasa/clasificación , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Procolágeno-Prolina Dioxigenasa/genética , Análisis Espacio-Temporal , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo
16.
Neuron ; 104(2): 271-289.e13, 2019 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-31515109

RESUMEN

Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.


Asunto(s)
Encéfalo/embriología , Cromatina/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Código de Histonas/genética , Metiltransferasas/genética , Proteínas de Pez Cebra/fisiología , Animales , Conducta Animal , Encéfalo/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Cognición , Epigénesis Genética , Histona Metiltransferasas/genética , Metiltransferasas/fisiología , Ratones , Mutación , Células-Madre Neurales/metabolismo , Empalme del ARN/genética , RNA-Seq , Conducta Social , Elongación de la Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/genética
17.
J Neurochem ; 106(5): 2224-35, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18624922

RESUMEN

Somatostatin acts at five G protein-coupled receptors, sst(1)-sst(5). In mouse ischaemic retinas, the over-expression of sst(2) (as in sst(1) knock-out mice) results in the reduction of cell death and glutamate release. In this study, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst(2) agonist octreotide decreased ischaemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst(2) with cyanamide. In sst(2) over-expressing ischaemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischaemic effect of somatostatin agonists in the presence of sst(2) over-expression, we tested sst(2) desensitisation because of internalisation or altered receptor function. We observed that (i) sst(2) was not internalised, (ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischaemia, (iii) both GRK1 and RGS1 were down-regulated by octreotide in wild-type ischaemic retinas, (iv) octreotide down-regulated GRK1 but not RGS1 in sst(2) over-expressing ischaemic retinas. These results demonstrate that sst(2) activation protects against retinal ischaemia. However, in the presence of sst(2) over-expression sst(2) is functionally desensitised by agonists, possibly because of sustained RGS1 levels.


Asunto(s)
Isquemia Encefálica/metabolismo , Degeneración Nerviosa/metabolismo , Receptores de Somatostatina/agonistas , Retina/metabolismo , Enfermedades de la Retina/metabolismo , Somatostatina/análogos & derivados , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Cianamida/farmacología , Femenino , Quinasa 1 del Receptor Acoplado a Proteína-G/efectos de los fármacos , Quinasa 1 del Receptor Acoplado a Proteína-G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Octreótido/farmacología , Oligopéptidos/farmacología , Proteínas RGS/efectos de los fármacos , Proteínas RGS/metabolismo , Receptores de Somatostatina/genética , Retina/efectos de los fármacos , Retina/fisiopatología , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/genética , Somatostatina/metabolismo
18.
Neuropharmacology ; 54(5): 874-84, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18329054

RESUMEN

The neuromodulatory peptide somatostatin-14 (SRIF) plays an important inhibitory role in epilepsy, but little is known on the signalling mechanisms coupled to this effect of SRIF. We have previously demonstrated that SRIF induces reduction of epileptiform bursting in a model of interictal-like activity in mouse hippocampal slices. In this same model, we investigated whether the cyclooxygenase 2 (COX-2)/prostaglandin E(2) (PGE(2)) pathway is part of those signalling mechanisms mediating SRIF anti-epileptic actions. Both the expression of COX-2 (mRNA and protein) and the endogenous release of PGE(2) increased in concomitance with epileptiform bursting. In particular, COX-2 protein increased in CA1/CA3 pyramidal layer and in the granular layer of the dentate gyrus. In addition, the selective inhibition of COX-2 by NS-398 markedly decreased endogenous PGE(2) release induced by epileptiform bursting and the epileptiform bursting itself. Similar effects on epileptiform bursting were obtained with another COX-2 inhibitor, i.e., meloxicam. SRIF application counteracted the increase of both COX-2 expression and PGE(2) release which occurred in concomitance with epileptiform bursting. Interestingly, SRIF and NS-398 comparably reduced epileptiform bursting in a non-additive manner and PGE(2) abolished the inhibitory effect of SRIF on epileptiform bursting. These results demonstrate that: i) the COX-2/PGE(2) pathway facilitates epileptiform bursting; and ii) SRIF exerts an anti-epileptic role by coupling to the COX-2/PGE(2) pathway. In conclusion, we have identified a key set of signalling events that underlie anti-convulsant effects of SRIF in a mouse model of hippocampal bursting, thus providing useful data not only to identify alternative intervention points for the modulation of SRIF function, but also to exploit new chemical space for drug-like molecules.


Asunto(s)
Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Epilepsia/patología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Transducción de Señal/fisiología , 4-Aminopiridina/farmacología , Animales , Ciclooxigenasa 2/genética , Dinoprostona/genética , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Epilepsia/inducido químicamente , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Nitrobencenos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Somatostatina , Sulfonamidas/farmacología
19.
Naunyn Schmiedebergs Arch Pharmacol ; 378(6): 563-77, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18665350

RESUMEN

The cyclic AMP pathway is major signal transduction system involved in hippocampal neurotransmission. Recently, the peptide somatostatin-14 (SRIF) has emerged as a key signal that, by activating its receptors, inhibits epileptiform bursting in the mouse hippocampus. Little is known on transduction mechanisms, which may mediate SRIF function in native cell/tissues. Using a well-established model of epileptiform activity induced by Mg(2+)-free medium with 4-aminopyridine [0 Mg(2+)/4-aminopyridine (4-AP)] in mouse hippocampal slices, we demonstrated that protein kinase A (PKA)-related signaling is upregulated by hippocampal bursting and that treatment with SRIF normalizes this upregulation. We also demonstrated that the SRIF-induced inhibition of PKA impairs phosphorylation of the NMDA receptor subunit NR1. Extracellular recordings of the 0 Mg(2+)/4-AP-induced hippocampal discharge from the CA3 region demonstrated that treating slices with compounds, which interfere with PKA activity, prevent SRIF inhibition of epileptiform bursting. Our results suggest that SRIF modulation of hippocampal activity may involve PKA-related signaling.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Epilepsia/fisiopatología , Hipocampo/fisiopatología , Somatostatina/fisiología , 4-Aminopiridina , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Epilepsia/inducido químicamente , Epilepsia/metabolismo , Femenino , Regulación de la Expresión Génica , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Transducción de Señal , Somatostatina/farmacología
20.
Invest Ophthalmol Vis Sci ; 48(8): 3480-9, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17652715

RESUMEN

PURPOSE: To determine whether the somatostatin receptor 2 (sst(2)) influences angiogenesis and its associated factors in a model of hypoxia-induced retinal neovascularization. METHODS: sst(1)-knockout (KO) mice, in which sst(2) is overexpressed and overfunctional, and sst(2)-KO mice were used. Angiogenesis was evaluated in fluorescein-perfused retinas. Angiogenesis-associated factors were determined by RT-PCR and immunohistochemistry. RESULTS: Retinal neovascularization was increased in sst(2)-KO mice, but remained unchanged in sst(1)-KO compared with wild-type (WT) mice. Retinal levels of sst(2) mRNA were not affected by hypoxia. Normoxic levels of angiogenesis regulators were similar in WT and KO retinas except for mRNA levels of IGF-1, Ang-2, and its receptor Tie-2. In WT, hypoxia induced an increase in mRNA levels of (1) VEGF and its receptors, (2) IGF-1R, and (3) Ang-2 and Tie-2. The increase in VEGF and IGF-1R mRNAs was more pronounced after sst(2) loss, but was less pronounced when sst(2) was overexpressed. In addition, in hypoxic retinas, sst(2) loss increased IGF-1 mRNA, whereas it decreased Ang-1, Tie-1, and Tie-2 mRNA levels. Moreover, Tie-1 mRNA increased when sst(2) was overexpressed. Immunohistochemistry confirmed the results in hypoxic retinas on increased expression of VEGF, IGF-1, and their receptors after sst(2) loss. It also allowed the localization of these factors to specific retinal cells. In this respect, VEGFR-2, IGF-1, and IGF-1R were localized to Müller cells. CONCLUSIONS: These results suggest that sst(2) may be protective against angiogenesis. The immediate clinical importance lies in the establishment of a potential pharmacological target based on sst(2) pharmacology.


Asunto(s)
Neovascularización Patológica/metabolismo , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Enfermedades de la Retina/metabolismo , Angiotensina I/genética , Angiotensina II/genética , Animales , Capilares/metabolismo , Capilares/patología , Modelos Animales de Enfermedad , Hipoxia/complicaciones , Factor I del Crecimiento Similar a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/etiología , Neovascularización Patológica/fisiopatología , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Receptor TIE-1/genética , Receptor TIE-2/genética , Enfermedades de la Retina/etiología , Enfermedades de la Retina/fisiopatología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
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