RESUMEN
BACKGROUND: Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS: Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS: Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS: Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.).
Asunto(s)
Corteza Cerebral/anomalías , Análisis Mutacional de ADN/métodos , Malformaciones del Desarrollo Cortical/genética , Mutación , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/genética , Humanos , Lisencefalia/genética , Imagen por Resonancia Magnética , Malformaciones del Desarrollo Cortical/patología , Heterotopia Nodular Periventricular/genéticaRESUMEN
The capacity of pancreatic ß cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote ß cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, ß cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and ß cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes.
RESUMEN
We present a new strategy for the differentiation of embryonic stem (ES) cells into insulin-producing cells via a multi-step process without selection and induction of nestin-positive cells. During ES cell differentiation, transcript levels of genes characteristic of early and mature beta cells including Pdx1, Pax4, insulin and islet amyloid pancreatic peptide are up regulated. Islet-like clusters are characterized by expression of C-peptide, insulin and partially cytokeratin 19 as well as by ion channel activity similar to that found in embryonic beta cells. Cells of islet-like clusters show glucose-dependent insulin release at terminal stage. At an intermediate stage, nestin is partially co-expressed with C-peptide and cytokeratin 19, whereas islet-like clusters at the terminal stage are nestin-negative. We conclude that expression of nestin and cytokeratin 19 is a normal property of ES cells preceding differentiation into C-peptide/insulin-producing cells without any selection for nestin-positive phenotypes.
Asunto(s)
Embrión de Mamíferos/citología , Insulina/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/embriología , Proteínas del Tejido Nervioso/metabolismo , Células Madre/citología , Animales , Péptido C/química , Anhidrasa Carbónica II/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Electrofisiología , Ensayo de Inmunoadsorción Enzimática , Proteínas de Homeodominio/metabolismo , Islotes Pancreáticos/metabolismo , Queratinas/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Modelos Biológicos , Nestina , Técnicas de Placa-Clamp , Péptidos/química , Fenotipo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estreptozocina/farmacología , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
Irs2-mediated insulin/IGF1 signaling in the CNS modulates energy balance and glucose homeostasis; however, the site for Irs2 function is unknown. The hormone leptin mediates energy balance by acting on leptin receptor (LepR-b)-expressing neurons. To determine whether LepR-b neurons mediate the metabolic actions of Irs2 in the brain, we utilized Lepr(cre) together with Irs2(L/L) to ablate Irs2 expression in LepR-b neurons (Lepr(ΔIrs2)). Lepr(ΔIrs2) mice developed obesity, glucose intolerance, and insulin resistance. Leptin action was not altered in young Lepr(ΔIrs2) mice, although insulin-stimulated FoxO1 nuclear exclusion was reduced in Lepr(ΔIrs2) mice. Indeed, deletion of Foxo1 from LepR-b neurons in Lepr(ΔIrs2) mice normalized energy balance, glucose homeostasis, and arcuate nucleus gene expression. Thus, Irs2 signaling in LepR-b neurons plays a crucial role in metabolic sensing and regulation. While not required for leptin action, Irs2 suppresses FoxO1 signaling in LepR-b neurons to promote energy balance and metabolism.
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Factores de Transcripción Forkhead/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Leptina/metabolismo , Neuronas/metabolismo , Receptores de Leptina/metabolismo , Animales , Encéfalo/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Metabolismo Energético , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Expresión Génica , Glucosa/genética , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Homeostasis , Insulina/genética , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Resistencia a la Insulina/genética , Leptina/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Obesidad/genética , Obesidad/metabolismo , Receptores de Leptina/genética , Transducción de SeñalRESUMEN
Aging is a major risk factor for the progression of neurodegenerative diseases, including Huntington disease (HD). Reduced neuronal IGF1 or Irs2 signaling have been shown to extend life span in mice. To determine whether Irs2 signaling modulates neurodegeneration in HD, we genetically modulated Irs2 concentrations in the R6/2 mouse model of HD. Increasing Irs2 levels in the brains of R6/2 mice significantly reduced life span and increased neuronal oxidative stress and mitochondrial dysfunction. In contrast, reducing Irs2 levels throughout the body (except in ß cells, where Irs2 expression is needed to prevent diabetes onset; R6/2â¢Irs2+/-â¢Irs2ßtg mice) improved motor performance and extended life span. The slower progression of HD-like symptoms was associated with increased nuclear localization of the transcription factor FoxO1 and increased expression of FoxO1-dependent genes that promote autophagy, mitochondrial function, and resistance to oxidative stress. Mitochondrial function improved and the number of autophagosomes increased in R6/2â¢Irs2+/-â¢Irs2ßtg mice, whereas aggregate formation and oxidative stress decreased. Thus, our study suggests that Irs2 signaling can modulate HD progression. Since we found the expression of Irs2 to be normal in grade II HD patients, our results suggest that decreasing IRS2 signaling could be part of a therapeutic approach to slow the progression of HD.
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Enfermedad de Huntington/fisiopatología , Proteínas Sustrato del Receptor de Insulina/fisiología , Mitocondrias/fisiología , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas Sustrato del Receptor de Insulina/deficiencia , Proteínas Sustrato del Receptor de Insulina/genética , Longevidad/genética , Longevidad/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Estrés Oxidativo , Transducción de SeñalRESUMEN
The aim of this study was to define when an insulin-positive cell becomes functional in vivo and starts to exocytose insulin in a regulated nutrient-dependent manner. Insulin-positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage-activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basal insulin release drops and, after postnatal day 2, the insulin-positive cells show both adult-like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin-positive cells during late-embryonic life, functional beta cells appear only 2 days after birth.
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Exocitosis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Canales de Calcio/metabolismo , Forma de la Célula , Electrofisiología , Glucosa/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/crecimiento & desarrollo , Ratones , Microscopía Electrónica de Transmisión , Canales de Potasio/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing beta-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single beta-cells. The insulin signal in the embryonic beta-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in beta-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of beta-cells.
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Insulina/metabolismo , Islotes Pancreáticos/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Biomarcadores , Compartimento Celular/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Citosol/metabolismo , Femenino , Ganglios Autónomos/citología , Ganglios Autónomos/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Inmunohistoquímica , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/embriología , Queratinas/metabolismo , Masculino , Potenciales de la Membrana/fisiología , Ratones , Técnicas de Cultivo de Órganos/métodos , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Proteínas tau/metabolismoRESUMEN
This study addressed some electrophysiological mechanisms enabling neonatal rat spinal networks in vitro to generate spontaneous rhythmicity. Networks, made up by excitatory connections only after block of GABAergic and glycinergic transmission, develop regular bursting (disinhibited bursts) suppressed by the Na(+) pump blocker strophanthidin. Thus the Na(+) pump is considered important to control bursts. This study, however, shows that, after about 1 h in strophanthidin solution, networks of the rat isolated spinal cord surprisingly resumed spontaneous bursting ("strophanthidin bursting"), which consisted of slow depolarizations with repeated oscillations. This pattern, recorded from lumbar ventral roots, was synchronous on both sides, of irregular periodicity, and lasted for > or =12 h. Assays of (86)Rb(+) uptake by spinal tissue confirmed Na(+) pump block by strophanthidin. The strophanthidin rhythm was abolished by glutamate receptor antagonists or tetrodotoxin, indicating its network origin. N-methyl-D-aspartate (NMDA), serotonin, or high K(+) could not accelerate it. The size of each burst was linearly related to the length of the preceding pause. Bursts could also be generated by dorsal root electrical stimulation and possessed similar dependence on the preceding pause. Conversely, disinhibited bursts could be evoked at short intervals from the preceding one unless repeated pulses were applied in close sequence. These data suggest that rhythmicity expressed by excitatory spinal networks could be controlled by Na(+) pump activity or slow synaptic depression. A model based on the differential time course of pump operation and synaptic depression could simulate disinhibited and strophanthidin bursting, indicating two fundamental, activity-dependent processes for regulating network discharge.
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Potenciales de Acción , Inhibición Neural , Redes Neurales de la Computación , Periodicidad , ATPasa Intercambiadora de Sodio-Potasio , Médula Espinal/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , N-Metilaspartato/farmacología , Inhibición Neural/efectos de los fármacos , Neuronas/fisiología , Potasio/farmacología , Ratas , Ratas Wistar , Serotonina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Estrofantidina/farmacología , Estricnina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
The cellular distribution of GABAC receptor rho1 and rho2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum rho1 and rho2 mRNAs were expressed in Purkinje cells and basket-like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the rho2 signal was consistently stronger than rho1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both rho1 and rho2 mRNAs showed somatodendritic localization, extending out for >100 microm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of rho1 and rho2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.