RESUMEN
Since 1967, it is known that the loss of GLI3 causes very severe defects in murine eye development. GLI3 is able to act as a transcriptional activator (GLI3-A) or as a transcriptional repressor (GLI3-R). Soon after the discovery of these GLI3 isoforms, the question arose which of the different isoforms is involved in eye formation - GLI3-A, GLI3-R or even both. For several years, this question remained elusive. By analysing the eye morphogenesis of Gli3XtJ/XtJ mouse embryos that lack GLI3-A and GLI3-R and of Gli3Δ699/Δ699 mouse embryos in which only GLI3-A is missing, we revealed that GLI3-A is dispensable in vertebrate eye formation. Remarkably, our study shows that GLI3-R is sufficient for the creation of morphologically normal eyes although the molecular setup deviates substantially from normality. In depth-investigations elucidated that GLI3-R controls numerous key players in eye development and governs lens and retina development at least partially via regulating WNT/ß-CATENIN signalling.
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Embrión de Mamíferos/embriología , Proteínas del Tejido Nervioso/metabolismo , Organogénesis , Retina/embriología , Vía de Señalización Wnt , Proteína Gli3 con Dedos de Zinc/metabolismo , Animales , Embrión de Mamíferos/citología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Retina/citología , Proteína Gli3 con Dedos de Zinc/genéticaRESUMEN
Protein degradation is a pivotal process for eukaryotic development and homeostasis. The majority of proteins are degraded by the ubiquitinâ»proteasome system and by autophagy. Recent studies describe a crosstalk between these two main eukaryotic degradation systems which allows for establishing a kind of safety mechanism. If one of these degradation systems is hampered, the other compensates for this defect. The mechanism behind this crosstalk is poorly understood. Novel studies suggest that primary cilia, little cellular protrusions, are involved in the regulation of the crosstalk between the two degradation systems. In this review article, we summarise the current knowledge about the association between cilia, the ubiquitinâ»proteasome system and autophagy.
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Autofagia , Cilios/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Ciliopatías/patología , Humanos , Transducción de SeñalRESUMEN
Initially, the function of the fat mass and obesity associated (Fto) gene seemed to be primarily the regulation of the body weight. Here we show that loss of Fto results in a hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis. In consequence, Fto-/- mice display an anxiety-like behavior and impairments in working memory. Furthermore, differentiation of neurons is affected in the hippocampus. As a cause of these impairments we identified a processing defect of the neurotrophin BDNF which is most likely the result of a reduced expression of MMP-9. Therefore, we propose FTO as a possible new target to develop novel approaches for the treatment of diseases associated with hippocampal disorders. In parallel, we also would like to make the point that any anti-obesity therapy via blocking FTO function can have negative effects on the proper function of the hippocampus.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Procesamiento Proteico-Postraduccional , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Ansiedad/genética , Ansiedad/metabolismo , Ansiedad/patología , Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/genética , Hipocampo/patología , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipotálamo-Hipofisario/patología , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Memoria a Corto Plazo , Ratones , Ratones Noqueados , Sistema Hipófiso-Suprarrenal/metabolismo , Sistema Hipófiso-Suprarrenal/patologíaRESUMEN
RPGRIP1L is an evolutionary highly conserved gene encoding a protein that localises at the transition zone of primary cilia. Mutations in RPGRIP1L result in ciliopathies, severe human diseases caused by dysfunctional cilia. Patients with mutations in this gene often suffer from an impaired development of not only one but various organs. To elucidate the function of Rpgrip1l in human development and the mechanisms underlying ciliopathies, different model organisms are used. In this review article, we summarise the findings of these investigations comprising novel functions of Rpgrip1l and the most promising therapeutic approaches.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cilios/genética , Cilios/metabolismo , Modelos Animales de Enfermedad , Humanos , Mutación , Proteostasis , Transducción de SeñalRESUMEN
Ciliopathies are life-threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans By performing quantitative immunofluorescence studies in RPGRIP1L-/- mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type-specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate-specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Cilios/fisiología , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos de Neoplasias , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular , Estructuras de la Membrana Celular , Células Cultivadas , Proteínas del Citoesqueleto , Embrión de Mamíferos/citología , Fibroblastos/citología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
Previously, macroautophagy/autophagy was demonstrated to be regulated inter alia by the primary cilium. Mutations in RPGRIP1L cause ciliary dysfunctions resulting in severe human diseases summarized as ciliopathies. Recently, we showed that RPGRIP1L deficiency leads to a decreased proteasomal activity at the ciliary base in mice. Importantly, the drug-induced restoration of proteasomal activity does not rescue ciliary length alterations in the absence of RPGRIP1L indicating that RPGRIP1L affects ciliary function also via other mechanisms. Based on this knowledge, we analyzed autophagy in Rpgrip1l-negative mouse embryos. In these embryos, autophagic activity was decreased due to an increased activation of the MTOR complex 1 (MTORC1). Application of the MTORC1 inhibitor rapamycin rescued dysregulated MTORC1, autophagic activity and cilia length but not proteasomal activity in Rpgrip1l-deficient mouse embryonic fibroblasts demonstrating that RPGRIP1L seems to regulate autophagic and proteasomal activity independently from each other.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Fibroblastos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Células Cultivadas , Cilios/metabolismo , Citoplasma/metabolismo , Ratones , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: Myelosuppression is a life-threatening complication of thiopurine therapy, and the incidence of thiopurine-induced myelosuppression is higher in East Asians than in Europeans. We investigated genetic factors associated with thiopurine-induced leukopenia in patients with IBD. DESIGN: A genome-wide association study (GWAS) was conducted in thiopurine-treated patients with IBD, followed by high-throughput sequencing of genes identified as significant in the GWAS or those involved in thiopurine metabolism (n=331). Significant loci associated with thiopurine-induced leukopenia were validated in two additional replication cohorts (n=437 and n=330). Functional consequences of FTO (fat mass and obesity-associated) variant were examined both in vitro and in vivo. RESULTS: The GWAS identified two loci associated with thiopurine-induced leukopenia (rs16957920, FTO intron; rs2834826, RUNX1 intergenic). High-throughput targeted sequencing indicated that an FTO coding variant (rs79206939, p.A134T) linked to rs16957920 is associated with thiopurine-induced leukopenia. This result was further validated in two replication cohorts (combined p=1.3×10-8, OR=4.3). The frequency of FTO p.A134T is 5.1% in Koreans but less than 0.1% in Western populations. The p.A134T variation reduced FTO activity by 65% in the nucleotide demethylase assay. In vivo experiments revealed that Fto-/- and Fto+/- mice were more susceptible to thiopurine-induced myelosuppression than wild-type mice. CONCLUSIONS: The results suggest that the hypomorphic FTO p.A134T variant is associated with thiopurine-induced leukopenia. These results shed light on the novel physiological role of FTO and provide a potential pharmacogenetic biomarker for thiopurine therapy.
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Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Azatioprina/efectos adversos , Inmunosupresores/efectos adversos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Leucopenia/inducido químicamente , Mercaptopurina/efectos adversos , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Anciano , Animales , Azatioprina/uso terapéutico , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/genética , Leucopenia/genética , Masculino , Mercaptopurina/uso terapéutico , Ratones , Ratones Noqueados , Persona de Mediana Edad , República de Corea , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
An incomplete septation of the ventricles in the vertebrate heart that disturbes the strict separation between the contents of the two ventricles is termed a ventricular septal defect (VSD). Together with bicuspid aortic valves, it is the most frequent congenital heart disease in humans. Until now, life-threatening VSDs are usually treated surgically. To avoid surgery and to develop an alternative therapy (e.g., a small molecule therapy), it is necessary to understand the molecular mechanisms underlying ventricular septum (VS) development. Consequently, various studies focus on the investigation of signalling pathways, which play essential roles in the formation of the VS. In the past decade, several reports found evidence for an involvement of Hedgehog (HH) signalling in VS development. In this review article, we will summarise the current knowledge about the association between HH signalling and VS formation and discuss the use of such knowledge to design treatment strategies against the development of VSDs.
RESUMEN
The primary cilium is an essential structure for the mediation of numerous signaling pathways involved in the coordination and regulation of cellular processes essential for the development and maintenance of health. Consequently, ciliary dysfunction results in severe human diseases called ciliopathies. Since many of the cilia-mediated signaling pathways are oncogenic pathways, cilia are linked to cancer. Recent studies demonstrate the existence of a cilia-regulated proteasome and that this proteasome is involved in cancer development via the progression of oncogenic, cilia-mediated signaling. This review article investigates the association between primary cilia and cancer with particular emphasis on the role of the cilia-regulated proteasome.
RESUMEN
The Hedgehog signalling pathway is evolutionarily highly conserved and essential for embryonic development of invertebrates and vertebrates. Consequently, impaired Hedgehog signalling results in very severe human diseases, ranging from holoprosencephaly to Pallister-Hall syndrome. Due to this great importance for human health, the focus of numerous research groups is placed on the investigation of the detailed mechanisms underlying Hedgehog signalling. Today, it is known that tiny cell protrusions, known as primary cilia, are necessary to mediate Hedgehog signalling in vertebrates. Although the Hedgehog pathway is one of the best studied signalling pathways, many questions remain. One of these questions is: How do primary cilia control Hedgehog signalling in vertebrates? Recently, it was shown that primary cilia regulate a special kind of proteasome which is essential for proper Hedgehog signalling. This review article will cover this novel cilia-proteasome association in embryonic Hedgehog signalling and discuss the possibilities provided by future investigations on this topic.
RESUMEN
BACKGROUND: Single nucleotide polymorphisms (SNPs) within the Fat mass and obesity associated (FTO) gene have been linked with increased body weight. However, the data on an association of FTO with cardiovascular diseases remains conflicting. Therefore, we ascertained whether FTO is associated with aortic valve stenosis (AVS), one of the most frequent cardiovascular diseases in the Western world. METHODS AND FINDINGS: In this population-based case-control study the FTO SNP rs9939609 was analyzed in 300 German patients with AVS and 429 German controls of the KORA survey S4, representing a random population. Blood samples were collected prior to aortic valve replacement in AVS cases and FTO rs9939609 was genotyped via ARMS-PCR. Genotype frequencies differed significantly between AVS cases and KORA controls (p = 0.004). Separate gender-analyses uncovered an association of FTO with AVS exclusively in males; homozygote carriers for the risk-allele (A) had a higher risk to develop AVS (p = 0.017, odds ratio (OR) 1.727; 95% confidence interval (CI) 1.087-2.747, recessive model), whereas heterozygote carriers for the risk-allele showed a lower risk (p = 0.002, OR 0.565, 95% CI 0.384-0.828, overdominant model). After adjustment for multiple co-variables, the odds ratios of heterozygotes remained significant for an association with AVS (p = 0.008, OR 0.565, 95% CI 0.369-0.861). CONCLUSIONS: This study revealed an association of FTO rs9939609 with AVS. Furthermore, this association was restricted to men, with heterozygotes having a significantly lower chance to develop AVS. Lastly, the association between FTO and AVS was independent of BMI and other variables such as diabetes mellitus.
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Estenosis de la Válvula Aórtica/genética , Sobrepeso/genética , Polimorfismo de Nucleótido Simple , Proteínas/genética , Caracteres Sexuales , Anciano , Alelos , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/epidemiología , Índice de Masa Corporal , Estudios de Casos y Controles , Comorbilidad , Diabetes Mellitus/epidemiología , Diabetes Mellitus/genética , Femenino , Frecuencia de los Genes , Alemania/epidemiología , Heterocigoto , Humanos , Hipertensión/epidemiología , Hipertensión/genética , Masculino , Persona de Mediana Edad , Sobrepeso/epidemiología , Estudios Prospectivos , Riesgo , UltrasonografíaRESUMEN
Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cilios/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Centrosoma , Femenino , Masculino , Ratones Endogámicos C3H , Ratones Noqueados , Mitosis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de ProteínasRESUMEN
AIMS: Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia. RESULTS: A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only. INNOVATION: Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease. CONCLUSION: eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation.
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Endotelio Vascular/metabolismo , Neointima/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo , Alanina/metabolismo , Animales , Aorta/metabolismo , Bovinos , Citrulina/metabolismo , Cisteína/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Superóxidos/metabolismo , VasodilataciónRESUMEN
Besides the active Wnt signalling itself, the extracellular inhibition by Dkk1 is important for various embryonic developmental processes, such as optic vesicle differentiation and facial outgrowth. Although a feedback crosstalk of the active Wnt/ß-catenin signaling and Dkk1 regulation has been suggested, the control of Dkk1 transcription by the Tcf/Lef1 mediated Wnt signalling and its connection to additional signalling factors has not been elucidated in vivo. Here, we used a combination of transgenic mouse approaches and biochemical analyses to unravel the direct Dkk1 transcriptional regulation via Tcf/Lefs. By using site directed mutagenesis, we tested several conserved Tcf/Lef1 binding sites within Dkk1 conserved non-coding elements (CNEs) and found that these are required for tissue specific reporter expression. In addition a conserved Msx1/2 binding site is required for retinal reporter expression and Msx2 but not Msx1 binds its conserved binding site within CNE195 in the optic cups. Within craniofacial expression domains, Lef1 interferes with Dkk1 directly via two conserved Tcf/Lef1 binding sites in the craniofacial enhancer CNE114, both of which are required for the general craniofacial Dkk1 reporter activation. Furthermore, these Tcf/Lef1 sites are commonly bound in the whisker hair bud mesenchyme but specifically Tcf/Lef1 (no. 2) is required for mandibular activation and repression of maxillar Dkk1 activation. Lastly, we tested the Tcf/Lef1 binding capacities of the Dkk1 promoter and found that although Lef1 binds the Dkk1 promoter, these sites are not sufficient for tissue specific Dkk1 activation. Together, we here present the importance of conserved Tcf/Lef1 and Msx1/2 sites that are required for differential Dkk1 transcriptional reporter activation in vivo. This requirement directly correlates with Lef1 and Msx1/2 interaction with these genomic loci.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Factor de Transcripción MSX1/metabolismo , Animales , Sitios de Unión , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/metabolismo , Ratones , Especificidad de Órganos , Unión Proteica , Activación TranscripcionalRESUMEN
The metabolic syndrome is a worldwide problem mainly caused by obesity. FTO was found to be a obesity-risk gene in humans and FTO deficiency in mice led to reduction in adipose tissue. Thus, FTO is an important factor for the development of obesity. Leptin-deficient mice are a well characterized model for analysing the metabolic syndrome. To determine the relevance of FTO for the development of the metabolic syndrome we analysed different parameters in combined homozygous deficient mice (Lep(ob/ob);Fto(-/-)). Lep(ob/ob);Fto(-/-) mice showed an improvement in analysed hallmarks of the metabolic syndrome in comparison to leptin-deficient mice wild type or heterozygous for Fto. Lep(ob/ob);Fto(-/-) mice did not develop hyperglycaemia and showed an improved glucose tolerance. Furthermore, extension of beta-cell mass was prevented in Lep(ob/ob);Fto(-/-)mice and accumulation of ectopic fat in the liver was reduced. In conclusion this study demonstrates that FTO deficiency has a protective effect not only on the development of obesity but also on the metabolic syndrome. Thus, FTO plays an important role in the development of metabolic disorders and is an interesting target for therapeutic agents.
Asunto(s)
Síndrome Metabólico/genética , Oxigenasas de Función Mixta/genética , Oxo-Ácido-Liasas/genética , Tejido Adiposo/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Peso Corporal/genética , Modelos Animales de Enfermedad , Hígado Graso/metabolismo , Hígado Graso/patología , Femenino , Predisposición Genética a la Enfermedad , Hepatocitos/metabolismo , Hiperglucemia/genética , Hiperglucemia/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Leptina/deficiencia , Leptina/genética , Metabolismo de los Lípidos , Masculino , Síndrome Metabólico/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Astrocytes express two sodium-coupled transporters, glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT-1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high-resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100ß. In CA1, the density of GFAP-positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT-1 was completed only at postnatal days (P) P20-25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3-5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT-1 exhibited a laminar expression pattern from P10-15 on, with the highest immunoreactivity in the stratum lacunosum-moleculare. At the cellular level, GLT-1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT-1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT-1 expression in the developing hippocampus. The clustering of GLT-1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood-brain barrier during formation of the hippocampal network.
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Astrocitos/metabolismo , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Animales , Western Blotting , Femenino , Proteína Ácida Fibrilar de la Glía , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Fluorescente , Proteínas del Tejido Nervioso/metabolismo , Fracciones Subcelulares/metabolismo , Regulación hacia ArribaRESUMEN
Polymorphisms in the fat mass and obesity-associated gene (FTO) are associated with human obesity and obesity-prone behaviors, including increased food intake and a preference for energy-dense foods. FTO demethylates N6-methyladenosine, a potential regulatory RNA modification, but the mechanisms by which FTO predisposes humans to obesity remain unclear. In adiposity-matched, normal-weight humans, we showed that subjects homozygous for the FTO "obesity-risk" rs9939609 A allele have dysregulated circulating levels of the orexigenic hormone acyl-ghrelin and attenuated postprandial appetite reduction. Using functional MRI (fMRI) in normal-weight AA and TT humans, we found that the FTO genotype modulates the neural responses to food images in homeostatic and brain reward regions. Furthermore, AA and TT subjects exhibited divergent neural responsiveness to circulating acyl-ghrelin within brain regions that regulate appetite, reward processing, and incentive motivation. In cell models, FTO overexpression reduced ghrelin mRNA N6-methyladenosine methylation, concomitantly increasing ghrelin mRNA and peptide levels. Furthermore, peripheral blood cells from AA human subjects exhibited increased FTO mRNA, reduced ghrelin mRNA N6-methyladenosine methylation, and increased ghrelin mRNA abundance compared with TT subjects. Our findings show that FTO regulates ghrelin, a key mediator of ingestive behavior, and offer insight into how FTO obesity-risk alleles predispose to increased energy intake and obesity in humans.
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Apetito , Ghrelina/sangre , Proteínas/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adolescente , Adulto , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Encéfalo/fisiología , Ingestión de Alimentos/psicología , Alimentos , Neuroimagen Funcional , Expresión Génica , Regulación de la Expresión Génica , Estudios de Asociación Genética , Células HEK293 , Humanos , Imagen por Resonancia Magnética , Masculino , Metilación , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recompensa , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Adulto JovenRESUMEN
Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.
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Dopamina/fisiología , Neuronas Dopaminérgicas/enzimología , Mesencéfalo/fisiología , Oxigenasas de Función Mixta/fisiología , Oxo-Ácido-Liasas/fisiología , Adenina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Animales , Cocaína/farmacología , Cuerpo Estriado/fisiología , Neuronas Dopaminérgicas/fisiología , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Metilación , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxigenasas de Función Mixta/deficiencia , Oxigenasas de Función Mixta/genética , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Fenotipo , Quinpirol/farmacología , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Receptores de Dopamina D2/deficiencia , Receptores de Dopamina D2/fisiología , Receptores de Dopamina D3/fisiología , Recompensa , Transducción de Señal/efectos de los fármacosRESUMEN
Ventricular septal defects (VSDs) are the most common congenital heart defects in humans. Despite several studies of the molecular mechanisms involved in ventricular septum (VS) development, very little is known about VS-forming signaling. We observed perimembranous and muscular VSDs in Fantom (Ftm)-negative mice. Since Ftm is a ciliary protein, we investigated presence and function of cilia in murine hearts. Primary cilia could be detected at distinct positions in atria and ventricles at embryonic days (E) 10.5-12.5. The loss of Ftm leads to shortened cilia and a reduced proliferation in distinct atrial and ventricular ciliary regions at E11.5. Consequently, wall thickness is diminished in these areas. We suggest that ventricular proliferation is regulated by cilia-mediated Sonic hedgehog (Shh) and platelet-derived growth factor receptor α (Pdgfrα) signaling. Accordingly, we propose that primary cilia govern the cardiac proliferation which is essential for proper atrial and ventricular wall development and hence for the fully outgrowth of the VS. Thus, our study suggests ciliopathy as a cause of VSDs.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Cilios/metabolismo , Tabiques Cardíacos/embriología , Ventrículos Cardíacos/embriología , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C3H , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A block of single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity associated) gene is associated with variation in body weight. Previous works suggest that increased expression of FTO, which encodes a 2-oxoglutarate-dependent nucleic acid demethylase, leads to increased body weight, although the underlying mechanism has remained unclear. To elucidate the function of FTO, we examined the consequences of altered FTO levels in cultured cells and murine brain. Here we show that a knockdown of FTO in HEK293 cells affects the transcripts levels of genes involved in the response to starvation, whereas overexpression of FTO affects the transcript levels of genes related to RNA processing and metabolism. Subcellular localization of FTO further strengthens the latter notion. Using immunocytochemistry and confocal laser scanning microscopy, we detected FTO in nuclear speckles and--to a lesser and varying extent--in the nucleoplasm and nucleoli of HEK293, HeLa and MCF-7 cells. Moreover, RNA modification analyses revealed that loss of Fto affects the 3-methyluridine/uridine and pseudouridine/uridine ratios in total brain RNA. We conclude that altered levels of FTO have multiple and diverse consequences on RNA modifications and the transcriptome.