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BACKGROUND: Angelman syndrome (AS) is a rare neurodevelopmental genetic disorder caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, affecting approximately 1:15,000 live births. We have recently shown that mitochondrial function in AS is altered during mid to late embryonic brain development leading to increased oxidative stress and enhanced apoptosis of neural precursor cells. However, the overall alterations of metabolic processes are still unknown. Hence, as a follow-up, we aim to investigate the metabolic profiles of wild-type (WT) and AS littermates and to identify which metabolic processes are aberrant in the brain of AS model mice during embryonic development. METHODS: We collected brain tissue samples from mice embryos at E16.5 and performed metabolomic analyses using proton nuclear magnetic resonance (1H-NMR) spectroscopy. Multivariate and Univariate analyses were performed to determine the significantly altered metabolites in AS mice. Pathways associated with the altered metabolites were identified using metabolite set enrichment analysis. RESULTS: Our analysis showed that overall, the metabolomic fingerprint of AS embryonic brains differed from those of their WT littermates. Moreover, we revealed a significant elevation of distinct metabolites, such as acetate, lactate, and succinate in the AS samples compared to the WT samples. The elevated metabolites were significantly associated with the pyruvate metabolism and glycolytic pathways. LIMITATIONS: Only 14 metabolites were successfully identified and investigated in the present study. The effect of unidentified metabolites and their unresolved peaks was not determined. Additionally, we conducted the metabolomic study on whole brain tissue samples. Employing high-resolution NMR studies on different brain regions could further expand our knowledge regarding metabolic alterations in the AS brain. Furthermore, increasing the sample size could reveal the involvement of more significantly altered metabolites in the pathophysiology of the AS brain. CONCLUSIONS: Ube3a loss of function alters bioenergy-related metabolism in the AS brain during embryonic development. Furthermore, these neurochemical changes could be linked to the mitochondrial reactive oxygen species and oxidative stress that occurs during the AS embryonic development.
Assuntos
Síndrome de Angelman , Encéfalo , Modelos Animais de Doenças , Metabolômica , Espectroscopia de Prótons por Ressonância Magnética , Animais , Síndrome de Angelman/metabolismo , Síndrome de Angelman/genética , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Camundongos , Metaboloma , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , FemininoRESUMO
INTRODUCTION: Transcranial direct current stimulation (tDCS) is an evolving non-invasive neurostimulation technique. Despite multiple studies, its underlying molecular mechanisms are still unclear. Several previous human studies of the effect of tDCS suggest that it generates metabolic effects. The induction of metabolic effects by tDCS could provide an explanation for how it generates its long-term beneficial clinical outcome. AIM: Given these hints of tDCS metabolic effects, we aimed to delineate the metabolic pathways involved in its mode of action. METHODS: To accomplish this, we utilized a broad analytical approach of co-analyzing metabolomics and transcriptomic data generated from anodal tDCS in rat models. Since no metabolomic dataset was available, we performed a tDCS experiment of bilateral anodal stimulation of 200 µA for 20 min and for 5 consecutive days, followed by harvesting the brain tissue below the stimulating electrode and generating a metabolomics dataset using LC-MS/MS. The analysis of the transcriptomic dataset was based on a publicly available dataset. RESULTS: Our analyses revealed that tDCS alters the metabolic profile of brain tissue, affecting bioenergetic-related pathways, such as glycolysis and mitochondrial functioning. In addition, we found changes in calcium-related signaling. CONCLUSIONS: We conclude that tDCS affects metabolism by modulating energy production-related processes. Given our findings concerning calcium-related signaling, we suggest that the immediate effects of tDCS on calcium dynamics drive modifications in distinct metabolic pathways. A thorough understanding of the underlying molecular mechanisms of tDCS has the potential to revolutionize its applicability, enabling the generation of personalized medicine in the field of neurostimulation and thus contributing to its optimization.
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Estimulação Transcraniana por Corrente Contínua , Humanos , Ratos , Animais , Estimulação Transcraniana por Corrente Contínua/métodos , Cálcio , Cromatografia Líquida , Espectrometria de Massas em Tandem , Perfilação da Expressão GênicaRESUMO
Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder caused by the maternally inherited loss of function of the UBE3A gene. AS is characterized by a developmental delay, lack of speech, motor dysfunction, epilepsy, autistic features, happy demeanor, and intellectual disability. While the cellular roles of UBE3A are not fully understood, studies suggest that the lack of UBE3A function is associated with elevated levels of reactive oxygen species (ROS). Despite the accumulating evidence emphasizing the importance of ROS during early brain development and its involvement in different neurodevelopmental disorders, up to date, the levels of ROS in AS neural precursor cells (NPCs) and the consequences on AS embryonic neural development have not been elucidated. In this study we show multifaceted mitochondrial aberration in AS brain-derived embryonic NPCs, which exhibit elevated mitochondrial membrane potential (ΔΨm), lower levels of endogenous reduced glutathione, excessive mitochondrial ROS (mROS) levels, and increased apoptosis compared to wild-type (WT) littermates. In addition, we report that glutathione replenishment by glutathione-reduced ethyl ester (GSH-EE) corrects the excessive mROS levels and attenuates the enhanced apoptosis in AS NPCs. Studying the glutathione redox imbalance and mitochondrial abnormalities in embryonic AS NPCs provides an essential insight into the involvement of UBE3A in early neural development, information that can serve as a powerful avenue towards a broader view of AS pathogenesis. Moreover, since mitochondrial dysfunction and elevated ROS levels were associated with other neurodevelopmental disorders, the findings herein suggest some potential shared underlying mechanisms for these disorders as well.
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Síndrome de Angelman , Células-Tronco Neurais , Animais , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Espécies Reativas de Oxigênio , Células-Tronco Neurais/patologia , Neurônios/patologia , Glutationa , Ubiquitina-Proteína Ligases/genética , Modelos Animais de DoençasRESUMO
The UBE3A gene encodes the ubiquitin E3-ligase protein, UBE3A, which is implicated in severe neurodevelopmental disorders. Lack of UBE3A expression results in Angelman syndrome, while UBE3A overexpression, due to genomic 15q duplication, results in autism. The cellular roles of UBE3A are not fully understood, yet a growing body of evidence indicates that these disorders involve mitochondrial dysfunction and increased oxidative stress. We utilized bioinformatics approaches to delineate the effects of murine Ube3a deletion on the expression of mitochondrial-related genes and pathways. For this, we generated an mRNA sequencing dataset from mouse embryonic fibroblasts (MEFs) in which both alleles of Ube3a gene were deleted and their wild-type controls. Since oxidative stress and mitochondrial dysregulation might not be exhibited in the resting baseline state, we also activated mitochondrial functioning in the cells of these two genotypes using TNFα application. Transcriptomes of the four groups of MEFs, Ube3a+/+ and Ube3a-/-, with or without the application of TNFα, were analyzed using various bioinformatics tools and machine learning approaches. Our results indicate that Ube3a deletion affects the gene expression profiles of mitochondrial-associated pathways. We further confirmed these results by analyzing other publicly available human transcriptome datasets of Angelman syndrome and 15q duplication syndrome.
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Síndrome de Angelman/genética , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/patologia , Animais , Células Cultivadas , Biologia Computacional , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The UBE3A gene codes for a protein with two known functions, a ubiquitin E3-ligase which catalyzes ubiquitin binding to substrate proteins and a steroid hormone receptor coactivator. UBE3A is most famous for its critical role in neuronal functioning. Lack of UBE3A protein expression leads to Angelman syndrome (AS), while its overexpression is associated with autism. In spite of extensive research, our understanding of UBE3A roles is still limited. We investigated the cellular and molecular effects of Ube3a deletion in mouse embryonic fibroblasts (MEFs) and Angelman syndrome (AS) mouse model hippocampi. Cell cultures of MEFs exhibited enhanced proliferation together with reduced apoptosis when Ube3a was deleted. These findings were supported by transcriptome and proteome analyses. Furthermore, transcriptome analyses revealed alterations in mitochondria-related genes. Moreover, an analysis of adult AS model mice hippocampi also found alterations in the expression of apoptosis- and proliferation-associated genes. Our findings emphasize the role UBE3A plays in regulating proliferation and apoptosis and sheds light into the possible effects UBE3A has on mitochondrial involvement in governing this balance.
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The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-ß-related gene signature in mice. We also report elevated levels of IFN-ß in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-ß impairs, whereas treatment with exogenous IFN-ß enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-ß promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-ß produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.
Assuntos
Interferon beta/metabolismo , Macrófagos/imunologia , Peritonite/imunologia , Pneumonia Bacteriana/imunologia , Adulto , Idoso , Animais , Apoptose/imunologia , Modelos Animais de Doenças , Escherichia coli/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Interferon beta/genética , Interferon beta/imunologia , Células Jurkat , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos , Pneumonia Bacteriana/microbiologia , Cultura Primária de Células , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismoRESUMO
Angelman syndrome (AS) is a genetic disorder which entails autism, intellectual disability, lack of speech, motor deficits, and seizure susceptibility. It is caused by the lack of UBE3A protein expression, which is an E3-ubiquitin ligase. Despite AS equal prevalence in males and females, not much data on how sex affects the syndrome was reported. In the herein study, we thoroughly characterized many behavioral phenotypes of AS mice. The behavioral data acquired was analyzed with respect to sex. In addition, we generated a new mRNA sequencing dataset. We analyzed the coding transcriptome expression profiles with respect to the effects of genotype and sex observed in the behavioral phenotypes. We identified several neurobehavioral aspects, especially sensory perception, where AS mice either lack the male-to-female differences observed in wild-type littermates or even show opposed differences. However, motor phenotypes did not show male-to-female variation between wild-type (WT) and AS mice. In addition, by utilizing the mRNA sequencing, we identified genes and isoforms with expression profiles that mirror the sensory perception results. These genes are differentially regulated in the two sexes with inverse expression profiles in AS mice compared to WT littermates. Some of these are known pain-related and estrogen-dependent genes. The observed differences in sex-dependent neurobehavioral phenotypes and the differential transcriptome expression profiles in AS mice strengthen the evidence for molecular cross talk between Ube3a protein and sex hormone receptors or their elicited pathways. These interactions are essential for understanding Ube3a deletion effects, beyond its E3-ligase activity.
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Síndrome de Angelman/genética , Caracteres Sexuais , Transcriptoma/genética , Síndrome de Angelman/complicações , Síndrome de Angelman/fisiopatologia , Animais , Ansiedade/complicações , Ansiedade/fisiopatologia , Comportamento Animal , Comportamento Exploratório , Medo , Feminino , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Memória , Camundongos Endogâmicos C57BL , Atividade Motora , Odorantes , Dor/complicações , Dor/genética , Dor/fisiopatologia , Percepção da Dor , Fenótipo , Aprendizagem Espacial , TemperaturaRESUMO
Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b(-/-) mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.
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Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de Trombopoetina/metabolismo , Fator de Transcrição STAT5/metabolismo , Trombopoetina/metabolismo , Animais , Proliferação de Células/genética , Citometria de Fluxo , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Receptores de Citocinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de TempoRESUMO
The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.
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Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Loci Gênicos/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , MicroRNAs/metabolismo , Fator de Transcrição STAT5/metabolismo , Células-Tronco/fisiologia , Animais , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , MicroRNAs/genética , Modelos Biológicos , Gravidez , Análise de Sequência de RNARESUMO
Development of mammary alveolar epithelium during pregnancy is controlled by prolactin, through the transcription factors STAT5A/B that activate specific sets of target genes. Here we asked whether some of STAT5's functions are mediated by microRNAs. The miR-21 promoter sequence contains a bona-fide STAT5 binding site and miR-21 levels increased in HC11 mammary cells upon prolactin treatment. In vivo miR-21 was abundantly expressed in mammary epithelium at day 6 of pregnancy. Analysis of mice lacking miR-21 revealed that their mammary tissue developed normally during pregnancy and dams were able to nurse their pups. Our study demonstrated that although expression of miR-21 is under prolactin control through the transcription factors STAT5A/B its presence is dispensable for mammary development and lactation.
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Lactação/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , MicroRNAs/genética , Fator de Transcrição STAT5/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Epiteliais/fisiologia , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Camundongos Knockout , Camundongos Nus , MicroRNAs/metabolismo , Gravidez , Regiões Promotoras Genéticas , Ligação Proteica , Ativação Transcricional , TranscriptomaRESUMO
Generating heat and maintaining body temperature is the primary function of brown adipose tissue (BAT). Previous studies have implicated microRNAs, including miR-193b and miR-365-1, in BAT differentiation. We used mouse genetics to further understand the specific contributions of these two miRs. BAT function in mice with an inactivated miR-193b-365-1 locus, as determined by their response to the selective ß3 adrenergic receptor agonist CL316.243 and their tolerance to cold exposure, was normal and expression of genes associated with functional BAT, including Prdm16 and Ucp1, was unaffected. In addition, genome-wide expression profiles of miRNAs and mRNAs in BAT in the presence and absence of miR-193b-365-1 were determined. In summary, these data demonstrate, in contrast to earlier work, that the development, differentiation, and function of BAT do not require the presence of miR-193b and miR-365-1.
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Tecido Adiposo Marrom/crescimento & desenvolvimento , Tecido Adiposo Marrom/fisiologia , MicroRNAs/fisiologia , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxóis/farmacologia , Regulação da Expressão Gênica , Canais Iônicos/genética , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Desacopladora 1RESUMO
Genome wide analysis revealed the miR-17/92 cluster as a STAT5 target. This cluster encodes six microRNAs, which predictably target genes that play a role in mammary gland development. In this study, we have deleted the miR-17/92 cluster in mammary stem cells and evaluated in the mouse its function during mammary gland development. Loss of the miR-17/92 cluster did not affect mammary development from prepuberty to lactation. Our studies demonstrated that, while expression of the miR-17/92 cluster is under control of the key mammary transcription factor STAT5, its presence is not required for normal mammary development or lactation.
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Regulação da Expressão Gênica no Desenvolvimento/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , MicroRNAs/genética , Família Multigênica/fisiologia , Fator de Transcrição STAT5/genética , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Células Epiteliais/metabolismo , Feminino , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Lactação/genética , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Nus , Gravidez , RNA Mensageiro/genética , Fator de Transcrição STAT5/metabolismo , Análise de Sequência de DNA , Células-TroncoRESUMO
Serum concentrations of leptin and insulin were compared between gender-matched hypothyroid (n=25) and healthy (n=25) client-owned dogs within comparable age and body condition score (BCS) ranges. Fasted blood samples were collected from each dog and analysed for glucose, cholesterol, triglyceride, leptin and insulin concentrations. Leptin and insulin concentrations were significantly higher in the hypothyroid compared to normal dogs (P=0.006 and P=0.001, respectively) following adjustment for potential confounders. A nearly significant (P=0.051) interaction with BCS was found in the association between hypothyroidism and leptin. Leptin concentrations were significantly higher in hypothyroid dogs compared to normal dogs, in separate analyses for BCS 6 (P=0.036) and 7 (P=0.049). There was no significant difference in glucose concentration between the hypothyroid and normal groups (P=0.84) following adjustment for BCS. This study showed that canine hypothyroidism is associated with increased serum leptin and insulin concentrations, neither of which may be attributed to obesity alone.
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Doenças do Cão/sangue , Hipotireoidismo/veterinária , Insulina/sangue , Leptina/sangue , Obesidade/veterinária , Animais , Glicemia/análise , Constituição Corporal , Estudos de Casos e Controles , Colesterol/sangue , Cães , Feminino , Hipotireoidismo/sangue , Hipotireoidismo/complicações , Masculino , Obesidade/sangue , Obesidade/etiologia , Triglicerídeos/sangueRESUMO
Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.
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Glândulas Mamárias Animais/citologia , Fator de Transcrição STAT5/metabolismo , Células-Tronco/citologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Fator de Transcrição STAT5/genéticaRESUMO
Leptin, like estrogen, is one of the endo/paracrine factors, which are synthesized in and secreted from mature adipocytes. The roles of the mammary fat pad and mammary adipocytes in the initiation of lactation are not clear. In this study, we showed that combination of prolactin, leptin and estrogen elevated the expression of the milk protein beta-lactoglobulin. We also showed that after prolactin stimulate the secretion of leptin from the mammary fat, leptin upregulated the expression of estrogen receptor alpha in the mammary epithelial cells. Also, prolactin affected aromatase mRNA expression in the bovine mammary fat and we demonstrated that leptin and prolactin can affect cholesterol secretion from explants in culture to the medium. Therefore, we suggest that prolactin initiates estrogen expression (as represented by aromatase mRNA) in the mammary fat pad, whereas leptin stimulates estrogen receptor alpha expression in the mammary epithelial cells. We hypothesize that leptin and estrogen, secreted from the mammary fat regulate lactation after stimulation of prolactin.
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OBJECTIVES: To compare the expression of canine relaxin, relaxin-like factor (RLF), and relaxin receptors within the muscles of the pelvic diaphragm of dogs with perineal hernia (PH) and clinically normal dogs. STUDY DESIGN: In vivo comparative study. ANIMALS: Fifteen client-owned intact male dogs with PH were studied. Four mature intact male dogs with no evidence of perineal pathology served as controls. METHODS: Biopsy samples from the levator ani, coccygeus, and internal obturator muscles were obtained. RNA samples were reverse transcribed and analyzed by real-time PCR for the expression of canine relaxin receptor LRG7, relaxin, and RLF. RESULTS: Significantly higher expression levels of canine relaxin receptors occurred in the musculature of the pelvic diaphragm and internal obturator muscle in dogs with PH compared with normal dogs. Expression of canine RLF revealed no significant difference between dogs with PH and controls. The difference in the expression of canine relaxin between groups was not statistically significant. CONCLUSIONS: Relaxin receptor up-regulation occurs in the coccygeus, levator ani, and internal obturator muscles of dogs with PH. CLINICAL RELEVANCE: The higher expression of relaxin receptors within the muscles of the pelvic diaphragm in dogs with PH suggests that relaxin might play a role in the pathogenesis of PH. Atrophy of these muscles, which predisposes to PH, may be attributable to increased relaxin activity.