RESUMO
Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.
Assuntos
Blefarofimose/genética , Blefaroptose/genética , Deficiência Intelectual/genética , Ubiquitina-Proteína Ligases/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Blefarofimose/diagnóstico , Blefaroptose/diagnóstico , Encéfalo/patologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sistema Nervoso Central , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Genótipo , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Mutação , Estresse Oxidativo , Síndrome , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
We conducted a genome-wide association study (GWAS) and a follow-up study of bipolar disorder (BD), a common neuropsychiatric disorder. In the GWAS, we investigated 499,494 autosomal and 12,484 X-chromosomal SNPs in 682 patients with BD and in 1300 controls. In the first follow-up step, we tested the most significant 48 SNPs in 1729 patients with BD and in 2313 controls. Eight SNPs showed nominally significant association with BD and were introduced to a meta-analysis of the GWAS and the first follow-up samples. Genetic variation in the neurocan gene (NCAN) showed genome-wide significant association with BD in 2411 patients and 3613 controls (rs1064395, p = 3.02 × 10(-8); odds ratio = 1.31). In a second follow-up step, we replicated this finding in independent samples of BD, totaling 6030 patients and 31,749 controls (p = 2.74 × 10(-4); odds ratio = 1.12). The combined analysis of all study samples yielded a p value of 2.14 × 10(-9) (odds ratio = 1.17). Our results provide evidence that rs1064395 is a common risk factor for BD. NCAN encodes neurocan, an extracellular matrix glycoprotein, which is thought to be involved in cell adhesion and migration. We found that expression in mice is localized within cortical and hippocampal areas. These areas are involved in cognition and emotion regulation and have previously been implicated in BD by neuropsychological, neuroimaging, and postmortem studies.
Assuntos
Transtorno Bipolar/genética , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Neurocam/genética , Animais , Encéfalo/patologia , Estudos de Casos e Controles , Seguimentos , Humanos , Camundongos , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Schizophrenia is a complex neuropsychiatric disorder of unclear etiology. The strongest known genetic risk factor is the 22q11.2 microdeletion. Research has yet to confirm which genes within the deletion region are implicated in schizophrenia. The minimal 1.5 megabase deletion contains MIR185, which encodes microRNA 185. METHODS: We determined miR-185 expression in embryonic and adult mouse brains. Common and rare variants at this locus were then investigated using a human genetics approach. First, we performed gene-based analyses for MIR185 common variants and target genes using Psychiatric Genomics Consortium genome-wide association data. Second, MIR185 was resequenced in German patients (n = 1000) and controls (n = 500). We followed up promising variants by genotyping an additional European sample (patients, n = 3598; controls, n = 4082). RESULTS: In situ hybridization in mice revealed miR-185 expression in brain regions implicated in schizophrenia. Gene-based tests revealed association between common variants in 3 MIR185 target genes (ATAT1, SH3PXD2A, NTRK3) and schizophrenia. Further analyses in mice revealed overlapping expression patterns for these target genes and miR-185. Resequencing identified 2 rare patient-specific novel variants flanking MIR185. However, follow-up genotyping provided no further evidence of their involvement in schizophrenia. LIMITATIONS: Power to detect rare variant associations was limited. CONCLUSION: Human genetic analyses generated no evidence of the involvement of MIR185 in schizophrenia. However, the expression patterns of miR-185 and its target genes in mice, and the genetic association results for the 3 target genes, suggest that further research into the involvement of miR-185 and its downstream pathways in schizophrenia is warranted.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Acetiltransferases/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Variação Genética , Estudo de Associação Genômica Ampla , Técnicas de Genotipagem , Compostos Heterocíclicos , Humanos , Hibridização In Situ , Camundongos Endogâmicos C57BL , Proteínas dos Microtúbulos , Receptor trkC/genética , População Branca/genéticaRESUMO
At the imprinted Rasgrf1 locus in mouse, a cis-acting sequence controls DNA methylation at a differentially methylated domain (DMD). While characterizing epigenetic marks over the DMD, we observed that DNA and H3K27 trimethylation are mutually exclusive, with DNA and H3K27 methylation limited to the paternal and maternal sequences, respectively. The mutual exclusion arises because one mark prevents placement of the other. We demonstrated this in five ways: using 5-azacytidine treatments and mutations at the endogenous locus that disrupt DNA methylation; using a transgenic model in which the maternal DMD inappropriately acquired DNA methylation; and by analyzing materials from cells and embryos lacking SUZ12 and YY1. SUZ12 is part of the PRC2 complex, which is needed for placing H3K27me3, and YY1 recruits PRC2 to sites of action. Results from each experimental system consistently demonstrated antagonism between H3K27me3 and DNA methylation. When DNA methylation was lost, H3K27me3 encroached into sites where it had not been before; inappropriate acquisition of DNA methylation excluded normal placement of H3K27me3, and loss of factors needed for H3K27 methylation enabled DNA methylation to appear where it had been excluded. These data reveal the previously unknown antagonism between H3K27 and DNA methylation and identify a means by which epigenetic states may change during disease and development.
Assuntos
Metilação de DNA , Impressão Genômica , Histonas/metabolismo , Lisina/metabolismo , ras-GRF1/genética , Alelos , Animais , Células Cultivadas , Feminino , Masculino , Metilação , Camundongos , Camundongos Endogâmicos , Modelos Genéticos , Especificidade da Espécie , ras-GRF1/metabolismoRESUMO
OBJECTIVE: Long-term respiratory consequences of bronchopulmonary dysplasia (BPD) in preterm infants born in the post-surfactant era ("new" BPD) remain partially unknown. The present study aimed to evaluate the respiratory outcomes of "new" BPD in adolescents who were born preterm. METHODS: This multicenter, cross-sectional study included 286 adolescents born between 2003 and 2005 (mean age: 14.2 years); among them, 184 and 102 were born extremely preterm (EP; <28 weeks' gestation) and moderate-late preterm (32 to <37 weeks' gestation), respectively. Among EP adolescents, 92 had BPD, and 92 did not. All participants underwent lung function tests, skin prick testing, and questionnaires on asthma symptoms and quality of life. RESULTS: EP adolescents with BPD had significantly lower forced expiratory volume in 1 s (FEV1 ), forced vital capacity (FVC), FEV1 /FVC ratio, and forced expiratory flow between 25% and 75% of FVC than other included adolescents. FEV1 /FVC ratios were below the lower limit of normal (z-score <-1.645) in 30.4% of EP adolescents with BPD, 13.0% of EP adolescents without BPD, and 11.8% of adolescents who were born moderate-late preterm. Bronchodilator response and air-trapping were significantly higher in BPD adolescents than in other adolescents. Diffusion capacity was significantly lower in EP adolescents than in moderate-late preterm adolescents. Asthma symptoms and quality-of-life scores were similar among groups. CONCLUSION: EP adolescents with "new" BPD had poorer pulmonary function than EP adolescents without BPD or moderate-late preterm adolescents. Further studies are needed to determine whether "new" BPD is associated with early-onset chronic obstructive pulmonary disease in adulthood.
Assuntos
Displasia Broncopulmonar , Adolescente , Displasia Broncopulmonar/complicações , Estudos Transversais , Feminino , Volume Expiratório Forçado , Humanos , Recém-Nascido Prematuro , Gravidez , Qualidade de VidaRESUMO
Changes in brain cyclic AMP (cAMP) have been suggested to underlie the clinical action of antidepressant treatments. Also, a regionally-selective regulation of cAMP-specific phosphodiesterases (PDEs) has been demonstrated for some antidepressants. To further investigate the effects of antidepressant treatments on PDEs, we examined the expression of different cAMP-specific PDEs in the brain of rats treated (1 and 14 days) with fluoxetine 3 mg/kg day. The mRNAs coding for PDE4A, PDE4B, PDE4D, and the five known PDE4D splice variants were analyzed by in situ hybridization on 45 brain structures of acute and chronic fluoxetine-treated rats. We also examined the binding sites for the putative antidepressant drug [(3)H]rolipram, a PDE4-selective inhibitor. In some brain areas single fluoxetine administration increased the density of the mRNA of all PDE4 isozymes, except PDE4D and PDE4D5. Chronic fluoxetine treatment increased PDE4A mRNA levels and decreased those for PDE4B, PDE4D and PDE4D1 mRNAs in some brain regions. The study was complemented with the analysis of the expression of the transcripts of BDNF. Chronic fluoxetine treatment down-regulated the expression of BDNF. These results show that the expression of PDE4 isozymes is modulated by a clinically relevant fluoxetine dose. The significance of these changes in PDE4 expression to the antidepressant effect of fluoxetine is discussed.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/biossíntese , Encéfalo/efeitos dos fármacos , Fluoxetina/administração & dosagem , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ/métodos , Animais , Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Isoenzimas/biossíntese , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
OBJECTIVE: Genome-wide association has been reported between the NCAN gene and bipolar disorder. The aims of this study were to characterize the clinical symptomatology most strongly influenced by NCAN and to explore the behavioral phenotype of Ncan knockout (Ncan(-/-)) mice. METHOD: Genotype/phenotype correlations were investigated in patients with bipolar disorder (N=641) and the genetically related disorders major depression (N=597) and schizophrenia (N=480). Principal components and genotype association analyses were used to derive main clinical factors from 69 lifetime symptoms and to determine which of these factors were associated with the NCAN risk allele. These analyses were then repeated using the associated factor(s) only in order to identify the more specific clinical subdimensions that drive the association. Ncan(-/-) mice were tested using diverse paradigms, assessing a range of behavioral traits, including paradigms corresponding to bipolar symptoms in humans. RESULTS: In the combined patient sample, the NCAN risk allele was significantly associated with the "mania" factor, in particular the subdimension "overactivity." Ncan(-/-) mice were hyperactive and showed more frequent risk-taking and repetitive behaviors, less depression-like conduct, impaired prepulse inhibition, amphetamine hypersensitivity, and increased saccharin preference. These aberrant behavioral responses normalized after the administration of lithium. CONCLUSIONS: NCAN preferentially affected mania symptoms in humans. Ncan(-/-) mice showed behavioral abnormalities that were strikingly similar to those of the human mania phenotype and may thus serve as a valid mouse model.
Assuntos
Comportamento Animal/fisiologia , Transtorno Bipolar/genética , Estudos de Associação Genética/estatística & dados numéricos , Neurocam/genética , Neurocam/fisiologia , Alelos , Animais , Comportamento Animal/efeitos dos fármacos , Transtorno Depressivo Maior/genética , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença/genética , Genótipo , Humanos , Lítio/farmacologia , Masculino , Camundongos , Camundongos Knockout , Análise de Componente Principal/métodos , Esquizofrenia/genéticaRESUMO
The regeneration of diseased hyaline cartilage continues to be a great challenge, mainly because degeneration--caused either by major injury or by age-related processes--can overextend the tissue's self-renewal capacity. We show that repair tissue from human articular cartilage during the late stages of osteoarthritis harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPCs). These exhibit stem cell characteristics such as clonogenicity, multipotency, and migratory activity. The isolated CPCs, which exhibit a high chondrogenic potential, were shown to populate diseased tissue ex vivo. Moreover, downregulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9. This, in turn, increased the matrix synthesis potential of the CPCs without altering their migratory capacity. Our results offer new insights into the biology of progenitor cells in the context of diseased cartilage tissue. Our work may be relevant in the development of novel therapeutics for the later stages of osteoarthritis.
Assuntos
Cartilagem Articular/citologia , Movimento Celular , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoartrite/patologia , Células-Tronco/fisiologia , Proteína Morfogenética Óssea 6/farmacologia , Cartilagem Articular/patologia , Cartilagem Articular/fisiologia , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Colágeno Tipo II/agonistas , Colágeno Tipo II/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Técnicas de Silenciamento de Genes , Humanos , Osteoartrite/metabolismo , Osteoblastos/metabolismo , Osteogênese/fisiologia , RNA Interferente Pequeno/genética , Regeneração/fisiologia , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/ultraestrutura , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Polycomb proteins are epigenetic regulators of gene expression. Human central nervous system (CNS) malformations are congenital defects of the brain and spinal cord. One example of a human CNS malformation is Chiari malformation (CM), which presents as abnormal brainstem growth and cerebellar herniation, sometimes accompanied by spina bifida and cortical defects; it can occur in families. Clinically, CM ranges from an asymptomatic condition to one with incapacitating or lethal symptoms, including neural tube defects and hydrocephalus. However, no genes that are causally involved in any manifestation of CM or similar malformations have been identified. Here, we show that a pathway that involves Zac1 (also known as Plagl1 or Lot1) and controls neuronal proliferation is altered in mice that are heterozygous for the polycomb gene Suz12, resulting in a phenotype that overlaps with some clinical manifestations of the CM spectrum. Suz12 heterozygotes show cerebellar herniation and an enlarged brainstem, accompanied by occipital cortical alterations and spina bifida. Downward displacement of the cerebellum causes hydrocephalus in the most severely impaired cases. Although the involvement of polycomb genes in human disease is starting to be recognized, this is the first demonstration of their role in nervous system malformations. Our work strongly suggests that brain malformations such as CM can result from altered epigenetic regulation of genes involved in cell proliferation in the brain.
Assuntos
Encéfalo/anormalidades , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Defeitos do Tubo Neural/genética , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Animais , Proliferação de Células , Epigênese Genética , Heterozigoto , Hibridização In Situ , Camundongos , Modelos Genéticos , Fenótipo , Complexo Repressor Polycomb 2 , Fatores de TempoRESUMO
Phosphodiesterases (PDE) control intracellular cyclic adenosine monophosphate (cAMP) levels, which appear to play an important role in the regulation of inflammation. PDE4B is especially important in this process. Using in situ hybridization histochemistry we first mapped the expression sites of the four PDE4B splicing forms in rat brain. Using the systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) as an inflammation model in rats, we found an increase in PDEB2 mRNA expression in choroid plexus. The differential expression of PDE4B spliced forms and the differential regulation of PDE4B2 in an inflammatory model further supports an involvement of this splicing variant in the inflammatory response.
Assuntos
Encéfalo/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Processamento Alternativo , Animais , Encéfalo/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Expressão Gênica/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Mensageiro/análise , RatosRESUMO
cAMP plays an important role as a second-messenger molecule controlling multiple cellular processes. Its hydrolysis provides an important mechanism by which cAMP levels are regulated. This is performed by a large multigene family of cyclic nucleotide phosphodiesterases (PDEs). Members of the PDE4 enzyme family are selectively inhibited by rolipram. Five different mRNA splice forms for PDE4D have been isolated. Here, we analyzed the regional distribution of the mRNAs coding for the splice variants PDE4D1, PDE4D2, PDE4D3, PDE4D4, and PDE4D5 in the rat brain by in situ hybridization histochemistry using specific radiolabeled oligonucleotides. We found that all five splice variants showed a distinct distribution pattern and, in some cases, in association with specific brain pathways. The most relevant differences were in hippocampal formation, medial habenula, basal ganglia, and area postrema, at both the regional and cellular level. The dorsal and median raphe nuclei exclusively contained PDE4D2 mRNA transcripts, probably located on serotonergic cells. PDE4D1 mRNA was expressed in some white matter cells. PDE4D1 and PDE4D2 mRNA splice forms presented a similar distribution in the area postrema, whereas for PDE4D4 and PDE4D5 the cellular distribution presented a complementary pattern. The differential expression of PDE4D mRNA splice variants in the area postrema is consistent with their possible involvement in emesis control and suggests new molecular targets for a more selective drug design.