RESUMO
Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo.
Assuntos
Axônios/metabolismo , Biossíntese de Proteínas , Proteoma/metabolismo , Células Ganglionares da Retina/metabolismo , Processamento Alternativo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteoma/análise , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Colículos Superiores/metabolismo , Transmissão SinápticaRESUMO
A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Integrina alfa4/metabolismo , Animais , Western Blotting , Células CHO , Movimento Celular , Células Cultivadas , Cromatografia de Afinidade , Cricetinae , Cricetulus , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cães , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Integrina alfa4/genética , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.
Assuntos
Técnicas Genéticas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Epitopos , Éxons , Hemaglutininas/química , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Células de Sertoli/metabolismoRESUMO
Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE(2)). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.
Assuntos
Quimiocinas/metabolismo , AMP Cíclico/metabolismo , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Inibidores da Fosfodiesterase 4 , Inibidores de Fosfodiesterase/farmacologia , Fator 3 Ativador da Transcrição/fisiologia , Quimiocinas/genética , Humanos , Macrófagos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica/fisiologiaRESUMO
We report the effects of mixed omega-7 fatty acid supplementation on changes in serum hsCRP, TNFα, and IL-6 levels and self-reported outcomes in people with non-specific chronic musculoskeletal discomfort. DESIGN: A double-blind, placebo-controlled, 1:1 randomized single crossover trial composed of 688 mg/day palmiteolate for the verum and an equivalent amount of medium-chain triglycerides for the placebo. METHOD: Data were analyzed in two independent groups and as a crossover group. RESULTS: From 211 screened participants in 2017-2019, 56 were randomized. Six participants dropped out and fifty completers contributed to the statistical analyses. At baseline, none of the investigated biomarkers were significantly correlated to subjectively assessed musculoskeletal discomfort levels. For the two-group analysis (n = 26 and n = 24), none of the serum biomarkers reached statistical significance; however, a statistically significant placebo effect was found in the subjective outcomes. CONCLUSION: For the crossover analysis (n = 50), three weeks of supplementation with n7FA containing 688 mg per day of palmiteolate did not reduce serum inflammatory biomarkers nor did it improve subjectively measured quality of life (QoL) compared to placebo. Future studies should explore appropriate biomarkers, sufficient power, length of dosing, inclusion criteria for volunteers with higher BMI, and the verification of cis-palmiteolate versus trans-palmiteolate.
Assuntos
Suplementos Nutricionais , Ácidos Graxos Insaturados/farmacologia , Inflamação , Adulto , Idoso , Biomarcadores/sangue , Proteína C-Reativa/análise , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Inflamação/sangue , Inflamação/metabolismo , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Placebos , Fator de Necrose Tumoral alfa/sangueRESUMO
Muscle differentiation is controlled by positive and negative signals. While much attention has been placed on proteins that promote muscle formation, the importance of negative regulators has been underemphasized. MBNL3/CHCR belongs to the muscleblind family of Cys3His zinc finger proteins implicated in myotonic dystrophy. MBNL3 is expressed in myoblasts, muscle precursor cells, and during the early stages of myogenesis, but is detected at very low levels in terminally differentiated myotubes. Constitutive expression of MBNL3 inhibits myotube formation and antagonizes myogenin and myosin heavy chain expression. To identify MBNL3 target genes, we compared the expression profile of C2C12 mouse myoblasts that constitutively express MBNL3 with control cells. From the 15,247 genes represented on the DNA microarray, classification by biological function indicated that genes involved in muscle development/contraction and cell adhesion were down-regulated by MBNL3 expression. mRNA and protein levels for the muscle transcription factor MyoD and E-box regulated transcription were reduced in C2C12-MBNL3 expressing cells. We hypothesize that MBNL3 serves to antagonize muscle differentiation by suppressing MyoD expression levels to prevent unwanted myogenic gene transcription. These findings are the first indication that a mammalian muscleblind-like (MBNL) protein plays a regulatory role in muscle differentiation under nonpathogenic conditions.
Assuntos
Proteínas de Transporte/fisiologia , Diferenciação Celular , Proteína MyoD/fisiologia , Transcrição Gênica , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Regulação para Baixo , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas de Ligação a RNARESUMO
BACKGROUND: US healthcare consumers increasingly demand more integrative medical care. Collaboration among clinicians trained in different professional disciplines and specialties may require particular character traits and/or training that focus on factors that facilitate effective collaborative work. Dispositional humility may be a factor that balances self-focused desire for recognition with other-focused professional collaboration to serve patients. The objective of this paper is to create a concept map of dispositional humility in healthcare professionals as a factor to enhance collaboration. METHODS: Articles published between 1997 and 2017 were searched using the term "dispositional humility" or titles containing "humility" AND either "leadership," "cultural," "religious," "relational," or "personality." The abstracts were screened for relevance and full articles were located. To strengthen the scientific rigor of qualitative work by systematizing a method of concept analysis, the Walker and Avant's eight-step concept analysis was used. RESULTS: Ninety-five articles were reviewed in the qualitative synthesis, including 82 full-text articles from the original search and 13 full-text articles containing the concepts "empathy," "professionalism" or "openness" identified from references found in the 82 articles. A concept map was created after interpreting the contents of these articles. CONCLUSIONS: Collaboration requires not only professional competency but also positive dispositional factors. Dispositional humility allows clinicians to have an accurate self-assessment, to be open to new ideas, to appreciate the contribution of others, and to develop generosity. Dispositional humility in leaders can facilitate character development of team members and create an environment characterized by fairness and equality, transparency, non-punitive consequences for reporting errors and near-misses, and a safe and encouraging environment. Nonetheless, dispositional humility must be nurtured and developed through professional training because high educational attainment, career and financial success, and busy schedules may lead to a sense of self-importance and entitlement that promotes separation of team members into hierarchies based on professional disciplines and specialties.
RESUMO
OBJECTIVES: Dispositional humility in professionals is a character trait that allows one to monitor self-centered occupational drive and to pay attention to the needs of other professionals. The aim of this study is to test whether or not clinicians working in interprofessional team care environments identify the character trait of humility as an important factor for successful collaborative relationships. This study aimed to revise a concept map of dispositional humility created through literature review. DESIGN: An explanatory sequential mixed-methods study was composed of the HEXACO personality test and the Integrative Medicine Attitude Questionnaire, followed by summative and directed content analyses of one-on-one interview data in order to identify the element of dispositional humility. SETTING: In the State of Washington, USA, where physicians (MD/DO), nurse practitioners (NP) and naturopathic clinicians (ND) serve Medicaid patients in community clinics. PARTICIPANTS: 6 MDs, 4 NPs, and 11 NDs. RESULTS: Twenty-one primary care clinicians were enrolled. Fifteen clinicians completed the interview. It was observed that the: 1) honesty-humility trait (p<0.01), conscientiousness (&p<0.01), and openness to experience (p<0.05) domains of primary care clinicians were statistically significantly higher than the reference standards; 2) attitudes toward integrative medicine did not differentiate the different clinician types; and 3) qualitative data supported the component of dispositional humility as a desirable trait in professionals with whom they would like to work. CONCLUSION: To maintain high-quality patient care while working as a team, limiting self-interest while focusing on the needs of others may be necessary and in the best interest of patients. An attitude of accepting the principles of integrative medicine has permeated this sample of primary healthcare workers. Both quantitative and qualitative analyses revealed that humility was viewed as an important character trait for successful interprofessional collaboration. A revised concept map of dispositional humility to enhance collaborative relationships was created.
RESUMO
Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5'UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension.
Assuntos
Axônios/metabolismo , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/genética , Regeneração Nervosa/genética , Crescimento Neuronal/genética , Traumatismos dos Nervos Periféricos/genética , Biossíntese de Proteínas/genética , Células Receptoras Sensoriais/metabolismo , Animais , Técnicas de Cultura de Células , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica , Ratos , Ratos WistarRESUMO
Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing "extraribosomal," regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-ß signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.
Assuntos
Morfogênese , Precursores de RNA/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Embrião não Mamífero/metabolismo , Éxons/genética , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteína Smad2/metabolismo , Frações Subcelulares/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Two main genes encoding the catalytic subunits Calpha and Cbeta of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cbeta genes encode several splice variants, including the splice variant Cbeta2. In mouse Cbeta2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cbeta2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. RESULTS: We identified a novel 47 kDa splice variant encoded by the mouse Cbeta gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cbeta2. Based on the present results and the existence of a human brain-specifically expressed Cbeta splice variant designated Cbeta4 that is identical to the former mouse Cbeta2 splice variant, the mouse splice variant has now been renamed mouse Cbeta4. CONCLUSION: Murine lymphoid tissues express a protein that is a homologue of human and bovine Cbeta2. The murine Cbeta gene encodes the splice variants Cbeta1, Cbeta2, Cbeta3 and Cbeta4, as is the case with the human Cbeta gene.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Isoenzimas/genética , Tecido Linfoide/enzimologia , Baço/enzimologia , Processamento Alternativo , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Variação Genética , Humanos , Isoenzimas/metabolismo , Camundongos , Peso Molecular , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The 75 kDa neurotrophin receptor (p75NTR) and two neurotrophin receptor homologs (NRH1, NRH2) constitute a subfamily of the nerve growth factor/tumor necrosis factor receptor superfamily. NRH1 coexists with p75NTR in fish, amphibians, and birds but is absent in mammals, whereas NRH2 exists only in mammals. Unlike p75NTR and NRH1, NRH2 lacks a canonical extracellular ligand binding domain. The similarity of NRH2 to the product of metalloproteinase cleavage of p75NTR prompted us to examine the cleavage of p75NTR in greater detail. p75NTR, NRH1, and NRH2 undergo multiple proteolytic cleavages that ultimately release cytoplasmic fragments. For p75NTR, cleavage in the extracellular domain by a PMA-inducible membrane metalloproteinase is followed by cleavage within or near the transmembrane domain, releasing the intracellular domain into the cytoplasm. This processing resembles the alpha- and gamma-secretase-mediated processing of beta-amyloid precursor protein and the similar processing of Notch. Although neurotrophins did not regulate p75NTR processing, the alpha- and gamma-secretase-mediated cleavage of p75 is modulated by receptor tyrosine kinases (Trks) TrkA and TrkB but not TrkC. Surprisingly, although NRH1 and NRH2 also undergo proteolytic cytoplasmic release of intracellular domains, a different protease mediates the cleavage. Furthermore, whereas the p75NTR soluble intracellular domain accumulates only in the presence of proteasome inhibitors, the equivalent fragment of NRH2 is stable and localizes in the nucleus. Because soluble intracellular domains of p75NTR and NRH2 were found to activate NF-kappaB in concert with TNF receptor associated factor 6 (TRAF6), we propose that cleavage of these proteins may serve conserved cytoplasmic and nuclear signaling functions through distinct proteases.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/fisiologia , Secretases da Proteína Precursora do Amiloide , Animais , Proteínas Reguladoras de Apoptose , Ácido Aspártico Endopeptidases , Proteínas de Transporte/genética , Linhagem Celular , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Endopeptidases/metabolismo , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , NF-kappa B/metabolismo , Fragmentos de Peptídeos/biossíntese , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Proteínas/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/genética , Homologia de Sequência de Aminoácidos , Fator 6 Associado a Receptor de TNF , XenopusRESUMO
Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RI alpha regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RI alpha as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RI alpha has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RI alpha regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RI alpha in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RI alpha knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RI alpha knockout embryos can be rescued by crossing RI alpha knockout mice to C alpha knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RI alpha knockout embryos reveals profound alterations in the actin-based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Embrião de Mamíferos/fisiologia , Animais , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog , Humanos , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Neoplasia Endócrina Múltipla/enzimologia , Neoplasia Endócrina Múltipla/genética , Mixoma/enzimologia , Mixoma/genética , Oócitos/fisiologia , Transtornos da Pigmentação/enzimologia , Transtornos da Pigmentação/genética , Transdução de Sinais , Síndrome , Transativadores/metabolismoRESUMO
An understanding of the molecular mechanisms by which androgens drive spermatogenesis has been thwarted by the fact that few consistent androgen receptor (AR) target genes have been identified. Here, we addressed this issue using next-generation sequencing coupled with the RiboTag approach, which purifies translated mRNAs expressed in cells that express cyclic recombinase (CRE). Using RiboTag mice expressing CRE in Sertoli cells (SCs), we identified genes expressed specifically in SCs in both prepubertal and adult mice. Unexpectedly, this analysis revealed that the SC-specific gene program is already largely defined at the initiation of spermatogenesis despite the subsequent dramatic maturational changes known to occur in SCs. To identify AR-regulated genes, we generated triple-mutant mice in which the SCs express the RiboTag but lack ARs. RNA sequencing analysis revealed hundreds of SC-expressed AR-regulated genes that had previously gone unnoticed, including suppressed genes involved in ovarian development. Comparison of the SC-enriched dataset with that from the whole testes allowed us to classify genes in terms of their degree of expression in SCs. This revealed that a greater fraction of AR-up-regulated genes than AR-down-regulated genes were expressed predominantly in SCs. Our results also revealed that AR signaling in SCs causes a large number of genes not detectably expressed in SCs to undergo altered expression, thereby providing genome-wide evidence for wide-scale communication between SCs and other cells. Taken together, our results identified novel classes of genes expressed in a hormone-dependent manner in different testicular cell subsets and highlight a new approach to analyze cell type-specific gene regulation.
Assuntos
Regulação da Expressão Gênica , Técnicas Genéticas , Genoma/genética , Biossíntese de Proteínas/genética , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Androgênios/farmacologia , Animais , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Células de Sertoli/efeitos dos fármacos , Maturidade Sexual/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The primary transcript of the mammalian Fragile X Mental Retardation-1 gene (Fmr1), like many transcripts in the central nervous system, is alternatively spliced to yield mRNAs encoding multiple proteins, which can possess quite different biochemical properties. Despite the fact that the relative levels of the 12 Fmr1 transcript isoforms examined here vary by as much as two orders of magnitude amongst themselves in both adult and embryonic mouse brain, all are associated with polyribosomes, consistent with translation into the corresponding isoforms of the protein product, FMRP (Fragile X Mental Retardation Protein). Employing the RiboTag methodology developed in our laboratory, the relative proportions of the 7 most abundant transcript isoforms were measured specifically in neurons and found to be similar to those identified in whole brain. Measurements of isoform profiles across 11 regions of adult brain yielded similar distributions, with the exceptions of the hippocampus and the olfactory bulb. These two regions differ from most of the brain in relative amounts of transcripts encoding an alternate form of one of the KH RNA binding domains. A possible relationship between patterns of expression in the hippocampus and olfactory bulb and the presence of neuroblasts in these two regions is suggested by the isoform patterns in early embryonic brain and in cultured neural progenitor cells. These results demonstrate that the relative levels of the Fmr1 isoforms are modulated according to developmental stage, highlighting the complex ramifications of losing all the protein isoforms in individuals with Fragile X Syndrome. It should also be noted that, of the eight most prominent FMRP isoforms (1-3, 6-9 and 12) in mouse, only two have the major site of phosphorylation at Ser-499, which is thought to be involved in some of the regulatory interactions of this protein.
Assuntos
Encéfalo/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Regulação da Expressão Gênica , Polirribossomos/metabolismo , Isoformas de RNA , Processamento Alternativo , Animais , Encéfalo/embriologia , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Masculino , Camundongos , Células-Tronco Neurais/metabolismoRESUMO
Male spermatogenesis is a complex biological process that is regulated by hormonal signals from the hypothalamus (GnRH), the pituitary gonadotropins (LH and FSH) and the testis (androgens, inhibin). The two key somatic cell types of the testis, Leydig and Sertoli cells, respond to gonadotropins and androgens and regulate the development and maturation of fertilization competent spermatozoa. Although progress has been made in the identification of specific transcripts that are translated in Sertoli and Leydig cells and their response to hormones, efforts to expand these studies have been restricted by technical hurdles. In order to address this problem we have applied an in vivo ribosome tagging strategy (RiboTag) that allows a detailed and physiologically relevant characterization of the "translatome" (polysome-associated mRNAs) of Leydig or Sertoli cells in vivo. Our analysis identified all previously characterized Leydig and Sertoli cell-specific markers and identified in a comprehensive manner novel markers of Leydig and Sertoli cells; the translational response of these two cell types to gonadotropins or testosterone was also investigated. Modulation of a small subset of Sertoli cell genes occurred after FSH and testosterone stimulation. However, Leydig cells responded robustly to gonadotropin deprivation and LH restoration with acute changes in polysome-associated mRNAs. These studies identified the transcription factors that are induced by LH stimulation, uncovered novel potential regulators of LH signaling and steroidogenesis, and demonstrate the effects of LH on the translational machinery in vivo in the Leydig cell.
Assuntos
Células Intersticiais do Testículo/metabolismo , RNA Mensageiro/genética , Células de Sertoli/metabolismo , Animais , Linhagem Celular , Hormônio Foliculoestimulante/metabolismo , Imunoprecipitação , Hormônio Luteinizante/metabolismo , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1(Cre/+) and Kiss1(Cre/Cre)) have â¼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1(Cre/Cre) mice sire normal-sized litters, female Kiss1(Cre/Cre) mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success.
Assuntos
Kisspeptinas/metabolismo , Neurônios/metabolismo , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Animais , Dinorfinas/genética , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Expressão Gênica , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Kisspeptinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Precursores de Proteínas/genética , Receptores da Neurocinina-3/genética , Reprodução/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Caracteres Sexuais , Fatores Sexuais , Maturidade Sexual/genética , Maturidade Sexual/fisiologia , Transdução de Sinais/genética , Taquicininas/genéticaRESUMO
The striatum regulates motor control, reward and learning. Abnormal function of striatal GABAergic medium spiny neurons (MSNs) is believed to contribute to the deficits in these processes that are observed in many neuropsychiatric diseases. The orphan G protein-coupled receptor GPR88 is robustly expressed in MSNs and is regulated by neuropharmacological drugs, but its contribution to MSN physiology and behavior is unclear. We found that, in the absence of GPR88, MSNs showed increased glutamatergic excitation and reduced GABAergic inhibition, which promoted enhanced firing rates in vivo, resulting in hyperactivity, poor motor coordination and impaired cue-based learning in mice. Targeted viral expression of GPR88 in MSNs rescued the molecular and electrophysiological properties and normalized behavior, suggesting that aberrant MSN activation in the absence of GPR88 underlies behavioral deficits and its dysfunction may contribute to behaviors observed in neuropsychiatric disease.
Assuntos
Sinais (Psicologia) , Atividade Motora/genética , Neurônios/fisiologia , Receptores Acoplados a Proteínas G/deficiência , Análise de Variância , Animais , Aprendizagem da Esquiva/fisiologia , Benzilaminas/farmacologia , Biofísica , Células Cultivadas , Cromonas/farmacologia , Corpo Estriado/citologia , Estimulação Elétrica , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Antagonistas GABAérgicos/farmacologia , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neurônios/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ácidos Fosfínicos/farmacologia , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Teste de Desempenho do Rota-Rod , Ácido gama-Aminobutírico/farmacologiaRESUMO
We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Genes Dominantes , Mutação , Alelos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células-Tronco Embrionárias/metabolismo , Glucose/metabolismo , Integrases/genética , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Brown adipose tissue (BAT) is rich in mitochondria and can uncouple oxidative phosphorylation to produce heat as a by-product of fatty acid metabolism. This thermogenic effect helps to maintain body temperature and also plays a critical role in energy homeostasis and the regulation of body weight. Both cyclic adenosine monophosphate and cyclic guanosine monophosphate (cGMP) contribute to the intracellular regulation of mitochondrial biogenesis and the differentiation of BAT. New evidence has defined the essential role of the cGMP-dependent protein kinase I in a pathway that modulates the RhoA-ROCK pathway and insulin receptor signaling to elicit BAT differentiation and stimulate thermogenesis.