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1.
Biochem Biophys Res Commun ; 530(1): 209-214, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828287

RESUMO

More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.


Assuntos
Proliferação de Células , MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Ribonuclease III/genética , Transcriptoma
2.
Immunity ; 48(2): 286-298.e6, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29396162

RESUMO

Glucocorticoids are steroid hormones with strong anti-inflammatory and immunosuppressive effects that are produced in a diurnal fashion. Although glucocorticoids have the potential to induce interleukin-7 receptor (IL-7R) expression in T cells, whether they control T cell homeostasis and responses at physiological concentrations remains unclear. We found that glucocorticoid receptor signaling induces IL-7R expression in mouse T cells by binding to an enhancer of the IL-7Rα locus, with a peak at midnight and a trough at midday. This diurnal induction of IL-7R supported the survival of T cells and their redistribution between lymph nodes, spleen, and blood by controlling expression of the chemokine receptor CXCR4. In mice, T cell accumulation in the spleen at night enhanced immune responses against soluble antigens and systemic bacterial infection. Our results reveal the immunoenhancing role of glucocorticoids in adaptive immunity and provide insight into how immune function is regulated by the diurnal rhythm.


Assuntos
Ritmo Circadiano/fisiologia , Glucocorticoides/farmacologia , Receptores CXCR4/fisiologia , Receptores de Interleucina-7/fisiologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Quimiocina CXCL12/biossíntese , Feminino , Memória Imunológica , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Glucocorticoides/fisiologia
3.
Cell Death Dis ; 8(5): e2813, 2017 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-28542144

RESUMO

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (DicerHET) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (DicerCKO). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.


Assuntos
Envelhecimento/metabolismo , Neurônios Dopaminérgicos/metabolismo , MicroRNAs/metabolismo , Neuroproteção , Ribonuclease III/metabolismo , Alelos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Neurônios Dopaminérgicos/patologia , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Deleção de Genes , Mesencéfalo/metabolismo , Camundongos Knockout , MicroRNAs/genética , Atividade Motora/efeitos dos fármacos , Degeneração Neural/genética , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neuroproteção/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tapsigargina/farmacologia
4.
Am J Physiol Renal Physiol ; 311(5): F901-F906, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27582101

RESUMO

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl- cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.


Assuntos
Túbulos Renais Coletores/metabolismo , Túbulos Renais Distais/metabolismo , Néfrons/metabolismo , Animais , Aquaporina 2/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Simportadores de Cloreto de Sódio/metabolismo
5.
Sci Rep ; 6: 19206, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26755241

RESUMO

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54(-/-)Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54(-/-)Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells.


Assuntos
Fertilidade/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Retroalimentação Fisiológica , Feminino , Gonadotropinas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ovário/metabolismo , Ovário/ultraestrutura , Fenótipo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Reprodução , Testículo/metabolismo
6.
J Clin Invest ; 126(2): 695-705, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26690700

RESUMO

Systemic inflammation causes malaise and general feelings of discomfort. This fundamental aspect of the sickness response reduces the quality of life for people suffering from chronic inflammatory diseases and is a nuisance during mild infections like common colds or the flu. To investigate how inflammation is perceived as unpleasant and causes negative affect, we used a behavioral test in which mice avoid an environment that they have learned to associate with inflammation-induced discomfort. Using a combination of cell-type­specific gene deletions, pharmacology, and chemogenetics, we found that systemic inflammation triggered aversion through MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Further, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor­expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, we demonstrated that inflammation-induced aversion was not an indirect consequence of fever or anorexia but that it constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic motivational circuitry is a key mechanism underlying the negative affect induced by inflammation.


Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Dinoprostona/metabolismo , Neurônios Dopaminérgicos/metabolismo , Endotélio Vascular/metabolismo , Transmissão Sináptica , Animais , Encéfalo/patologia , Encefalopatias/genética , Encefalopatias/patologia , Linhagem Celular , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/genética , Neurônios Dopaminérgicos/patologia , Endotélio Vascular/patologia , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo
7.
Nat Commun ; 6: 6693, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25827749

RESUMO

Skeletal muscle has a pleiotropic role in organismal energy metabolism, for example, by storing protein as an energy source, or by excreting endocrine hormones. Muscle proteolysis is tightly controlled by the hypothalamus-pituitary-adrenal signalling axis via a glucocorticoid-driven transcriptional programme. Here we unravel the physiological significance of this catabolic process using skeletal muscle-specific glucocorticoid receptor (GR) knockout (GRmKO) mice. These mice have increased muscle mass but smaller adipose tissues. Metabolically, GRmKO mice show a drastic shift of energy utilization and storage in muscle, liver and adipose tissues. We demonstrate that the resulting depletion of plasma alanine serves as a cue to increase plasma levels of fibroblast growth factor 21 (FGF21) and activates liver-fat communication, leading to the activation of lipolytic genes in adipose tissues. We propose that this skeletal muscle-liver-fat signalling axis may serve as a target for the development of therapies against various metabolic diseases, including obesity.


Assuntos
Tecido Adiposo/metabolismo , Metabolismo Energético , Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Receptores de Glucocorticoides/genética , Adaptação Fisiológica , Alanina/sangue , Animais , Hipertrofia , Lipólise/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Proteólise , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais
8.
Hepatology ; 61(3): 979-89, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25266280

RESUMO

UNLABELLED: The ubiquitously expressed transcriptional regulator serum response factor (SRF) is controlled by both Ras/MAPK (mitogen-activated protein kinase) and Rho/actin signaling pathways, which are frequently activated in hepatocellular carcinoma (HCC). We generated SRF-VP16iHep mice, which conditionally express constitutively active SRF-VP16 in hepatocytes, thereby controlling subsets of both Ras/MAPK- and Rho/actin-stimulated target genes. All SRF-VP16iHep mice develop hyperproliferative liver nodules that progresses to lethal HCC. Some murine (m)HCCs acquire Ctnnb1 mutations equivalent to those in human (h)HCC. The resulting transcript signatures mirror those of a distinct subgroup of hHCCs, with shared activation of oncofetal genes including Igf2, correlating with CpG hypomethylation at the imprinted Igf2/H19 locus. CONCLUSION: SRF-VP16iHep mHCC reveal convergent Ras/MAPK and Rho/actin signaling as a highly oncogenic driver mechanism for hepatocarcinogenesis. This suggests simultaneous inhibition of Ras/MAPK and Rho/actin signaling as a treatment strategy in hHCC therapy.


Assuntos
Neoplasias Hepáticas Experimentais/etiologia , Fator de Resposta Sérica/fisiologia , Animais , Proliferação de Células , Ilhas de CpG , Metilação de DNA , Perfilação da Expressão Gênica , Hepatócitos/patologia , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Fator de Crescimento Insulin-Like II/genética , Linfócitos/patologia , Camundongos , Mutação , beta Catenina/genética
9.
J Neurosci ; 34(46): 15297-305, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25392497

RESUMO

Sex differences in brain function underlie robust differences between males and females in both normal and disease states. Although alternative mechanisms exist, sexual differentiation of the male mammalian brain is initiated predominantly by testosterone secreted by the testes during the perinatal period. Despite considerable advances in understanding how testosterone and its metabolite estradiol sexually differentiate the brain, little is known about the mechanism that generates the male-specific perinatal testosterone surge. In mice, we show that a male-specific activation of GnRH neurons occurs 0-2 h following birth and that this correlates with the male-specific surge of testosterone occurring up to 5 h after birth. The necessity of GnRH signaling for the sexually differentiating effects of the perinatal testosterone surge was demonstrated by the persistence of female-like brain characteristics in adult male, GnRH receptor knock-out mice. Kisspeptin neurons have recently been identified to be potent, direct activators of GnRH neurons. We demonstrate that a population of kisspeptin neurons appears in the preoptic area of only the male between E19 and P1. The importance of kisspeptin inputs to GnRH neurons for the process of sexual differentiation was demonstrated by the lack of a normal neonatal testosterone surge, and disordered brain sexual differentiation of male mice in which the kisspeptin receptor was deleted selectively from GnRH neurons. These observations demonstrate the necessity of perinatal GnRH signaling for driving brain sexual differentiation and indicate that kisspeptin inputs to GnRH neurons are essential for this process to occur.


Assuntos
Hormônio Liberador de Gonadotropina/fisiologia , Neurônios/fisiologia , Área Pré-Óptica/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Diferenciação Sexual/fisiologia , Transdução de Sinais , Animais , Animais Recém-Nascidos , Feminino , Hormônio Liberador de Gonadotropina/genética , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Gravidez , Receptores Acoplados a Proteínas G/genética , Receptores de Kisspeptina-1 , Receptores LHRH/genética , Receptores LHRH/fisiologia , Caracteres Sexuais , Testosterona/sangue , Tirosina 3-Mono-Oxigenase/metabolismo , Vasopressinas/metabolismo
10.
J Neurosci ; 34(32): 10659-74, 2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-25100599

RESUMO

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.


Assuntos
Hiperfagia/complicações , Hipotálamo/metabolismo , MicroRNAs/metabolismo , Obesidade/etiologia , Obesidade/patologia , Absorciometria de Fóton , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Serina-Treonina Quinases TOR/metabolismo , Transdução Genética
12.
Front Behav Neurosci ; 8: 212, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24966820

RESUMO

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB.

13.
Mol Endocrinol ; 28(6): 899-911, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24713037

RESUMO

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.


Assuntos
Receptor alfa de Estrogênio/genética , Ativação Transcricional , Animais , Sequência de Bases , Sequência Consenso , Estradiol/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Ligação Proteica , Elementos de Resposta , Útero/metabolismo
14.
Biochem Biophys Res Commun ; 447(3): 407-12, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24713303

RESUMO

Although the mineralocorticoid receptor (MR) is expressed in osteoblasts and osteocytes and frequently co-localizes with the glucocorticoid receptors (GR), its pathophysiological functions in bone remain elusive. We report here that pharmacologic inhibition of MR function with eplerenone resulted in increased bone mass, with stimulation of bone formation and suppression of resorption, while specific genetic deletion of MR in osteoblast lineage cells had no effect. Further, treatment with eplerenone as well as specific deletion of MR in osteocytes ameliorated the cortical bone thinning caused by slow-release prednisolone pellets. Thus, MR may be involved in the deleterious effects of glucocorticoid excess on cortical bone.


Assuntos
Doenças Ósseas Metabólicas/induzido quimicamente , Osso e Ossos/efeitos dos fármacos , Glucocorticoides/efeitos adversos , Osteogênese/efeitos dos fármacos , Receptores de Mineralocorticoides/fisiologia , Animais , Doenças Ósseas Metabólicas/metabolismo , Osso e Ossos/metabolismo , Eplerenona , Camundongos , Camundongos Endogâmicos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Prednisona/efeitos adversos , Espironolactona/análogos & derivados , Espironolactona/farmacologia
15.
Front Cell Neurosci ; 7: 207, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24273493

RESUMO

Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimer's disease (AD) and may play a role in dementia. Moreover, aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3, and dentate gyrus (DG). Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that 36 transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF) binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the DG a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving neuronal function.

16.
Nat Commun ; 4: 2492, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24051579

RESUMO

Signaling between kisspeptin and its receptor, G-protein-coupled receptor 54 (Gpr54), is now recognized as being essential for normal fertility. However, the key cellular location of kisspeptin-Gpr54 signaling is unknown. Here we create a mouse with a GnRH neuron-specific deletion of Gpr54 to assess the role of gonadotropin-releasing hormone (GnRH) neurons. Mutant mice are infertile, fail to go through puberty and exhibit markedly reduced gonadal size and follicle-stimulating hormone levels alongside GnRH neurons that are unresponsive to kisspeptin. In an attempt to rescue the infertile phenotype of global Gpr54⁻/⁻ mutants, we use BAC transgenesis to target Gpr54 to the GnRH neurons. This results in mice with normal puberty onset, estrous cyclicity, fecundity and a recovery of kisspeptin's stimulatory action upon GnRH neurons. Using complimentary cell-specific knockout and knockin approaches we demonstrate here that the GnRH neuron is the key site of kisspeptin-Gpr54 signaling for fertility.


Assuntos
Hormônio Liberador de Gonadotropina/genética , Hipotálamo/metabolismo , Infertilidade/genética , Kisspeptinas/genética , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Animais , Feminino , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/patologia , Infertilidade/metabolismo , Infertilidade/patologia , Kisspeptinas/metabolismo , Camundongos , Camundongos Knockout , Neurônios/patologia , Tamanho do Órgão , Ovário/metabolismo , Ovário/patologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Kisspeptina-1 , Maturidade Sexual
17.
J Neurosci ; 33(38): 15132-44, 2013 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-24048844

RESUMO

Dendrite development is controlled by the interplay of intrinsic and extrinsic signals affecting initiation, growth, and maintenance of complex dendrites. Bone morphogenetic proteins (BMPs) stimulate dendrite growth in cultures of sympathetic, cortical, and hippocampal neurons but it was unclear whether BMPs control dendrite morphology in vivo. Using a conditional knock-out strategy to eliminate Bmpr1a and Smad4 in immature noradrenergic sympathetic neurons we now show that dendrite length, complexity, and neuron cell body size are reduced in adult mice deficient of Bmpr1a. The combined deletion of Bmpr1a and Bmpr1b causes no further decrease in dendritic features. Sympathetic neurons devoid of Bmpr1a/1b display normal Smad1/5/8 phosphorylation, which suggests that Smad-independent signaling paths are involved in dendritic growth control downstream of BMPR1A/B. Indeed, in the Smad4 conditional knock-out dendrite and cell body size are not affected and dendrite complexity and number are increased. Together, these results demonstrate an in vivo function for BMPs in the generation of mature sympathetic neuron dendrites. BMPR1 signaling controls dendrite complexity postnatally during the major dendritic growth period of sympathetic neurons.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Dendritos/metabolismo , Gânglios Simpáticos/citologia , Células Receptoras Sensoriais/citologia , Transdução de Sinais/fisiologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas Arqueais/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proteínas Morfogenéticas Ósseas/genética , Células Cultivadas , DNA Polimerase Dirigida por DNA/metabolismo , Embrião de Mamíferos , Corantes Fluorescentes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína Smad4/deficiência , Proteína Smad4/genética , Estatísticas não Paramétricas , Fatores de Transcrição/metabolismo
18.
PLoS One ; 8(8): e72632, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977333

RESUMO

The aim of this study was to investigate whether conditional inactivation of the glucocorticoid receptors (GRs) in noradrenergic neurons affects animal behavior in mice. Selective ablation of GRs in the noradrenergic system was achieved using the Cre/loxP approach. We crossed transgenic mice expressing the Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter with animals harboring the floxed GR gene. The resulting GR(DBHCre) mutant mice exhibited no alterations in terms of normal cage behavior, weight gain, spatial memory or spontaneous locomotor activity, regardless of gender. To assess depressive- and anxiety-like behaviors we performed the Tail Suspension Test and the Light-Dark Box Test. While male mutant animals did not show any alternations in both tests, female GR(DBHCre) mutants displayed depressive- and anxiety-like behavior. Additionally, male GR(DBHCre) mice were exposed to chronic restraint stress but still exhibited immobility times and anxiety statuses similar to those of non-stressed animals while stressed control mice clearly revealed depressive- and anxiety-like phenotype. Thus, in males the effects of the mutation were precipitated only after chronic restraint stress procedure. Our data reveal a possible gender-dependent role of GRs in the noradrenergic system in anxiety- and depressive-like behavior in mice.


Assuntos
Neurônios Adrenérgicos/metabolismo , Transtornos de Ansiedade/metabolismo , Comportamento Animal , Depressão/metabolismo , Receptores de Glucocorticoides/metabolismo , Neurônios Adrenérgicos/patologia , Animais , Transtornos de Ansiedade/sangue , Transtornos de Ansiedade/psicologia , Doença Crônica , Corticosterona/sangue , Depressão/sangue , Depressão/psicologia , Feminino , Locus Cerúleo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Psicológico/metabolismo
19.
Mol Endocrinol ; 27(10): 1655-65, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23979842

RESUMO

The glucocorticoid receptor (GR) regulates hypothalamic-pituitary-adrenal (HPA) axis activity during the stress response. The paraventricular nucleus (PVN) is a major site of negative feedback to coordinate the degree of the HPA axis activity with the magnitude of the exposed stressor. To define the function of endogenous PVN GR, we used Cre-loxP technology to disrupt different GR exons in Sim1-expressing neurons of the hypothalamus. GR exon 2-deleted mice (Sim1Cre-GRe2Δ) demonstrated 43% loss of PVN GR compared with an 87% GR loss in exon 3-deleted mice (Sim1Cre-GRe3Δ). Sim1Cre-GRe3Δ mice display stunted growth at birth but develop obesity in adulthood and display impaired stress-induced glucose release. We observed elevated basal and stress-induced corticosterone levels in Sim1Cre-GRe3Δ mice, compared with control and Sim1Cre-GRe2Δ mice, and impaired dexamethasone suppression, indicating an inability to negatively regulate corticosterone secretion. Sim1Cre-GRe3Δ mice also showed increased CRH mRNA in the PVN, increased basal plasma ACTH levels, and reduced locomotor behavior. We observed no differences in Sim1Cre-GRe2Δ mice compared with control mice in any measure. Our behavioral data suggest that GR deletion in Sim1-expressing neurons has no effect on anxiety or despair-like behavior under basal conditions. We conclude that loss of PVN GR results in severe HPA axis hyperactivity and Cushing's syndrome-like phenotype but does not affect anxiety and despair-like behaviors.


Assuntos
Adiposidade , Sistema Hipotálamo-Hipofisário/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Corticosterona/sangue , Feminino , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Neurônios/metabolismo , Obesidade/metabolismo , Núcleo Hipotalâmico Paraventricular/patologia , Receptores de Glucocorticoides/genética
20.
Neuropsychopharmacology ; 38(8): 1585-97, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23446450

RESUMO

Brain kappa-opioid receptors (KORs) are implicated in states of motivation and emotion. Activation of KORs negatively regulates mesolimbic dopamine (DA) neurons, and KOR agonists produce depressive-like behavioral effects. To further evaluate how KOR function affects behavior, we developed mutant mice in which exon 3 of the KOR gene (Oprk1) was flanked with Cre-lox recombination (loxP) sites. By breeding these mice with lines that express Cre-recombinase (Cre) in early embryogenesis (EIIa-Cre) or only in DA neurons (dopamine transporter (DAT)-Cre), we developed constitutive KOR knockouts (KOR(-/-)) and conditional knockouts that lack KORs in DA-containing neurons (DAT-KOR(lox/lox)). Autoradiography demonstrated complete ablation of KOR binding in the KOR(-/-) mutants, and reduced binding in the DAT-KOR(lox/lox) mutants. Quantitative reverse transcription PCR (qPCR) studies confirmed that KOR mRNA is undetectable in the constitutive mutants and reduced in the midbrain DA systems of the conditional mutants. Behavioral characterization demonstrated that these mutant lines do not differ from controls in metrics, including hearing, vision, weight, and locomotor activity. Whereas KOR(-/-) mice appeared normal in the open field and light/dark box tests, DAT-KOR(lox/lox) mice showed reduced anxiety-like behavior, an effect that is broadly consistent with previously reported effects of KOR antagonists. Sensitization to the locomotor-stimulating effects of cocaine appeared normal in KOR(-/-) mutants, but was exaggerated in DAT-KOR(lox/lox) mutants. Increased sensitivity to cocaine in the DAT-KOR(lox/lox) mutants is consistent with a role for KORs in negative regulation of DA function, whereas the lack of differences in the KOR(-/-) mutants suggests compensatory adaptations after constitutive receptor ablation. These mouse lines may be useful in future studies of KOR function.


Assuntos
Ansiolíticos/metabolismo , Encéfalo/metabolismo , Cocaína/farmacologia , Neurônios Dopaminérgicos/metabolismo , Plasticidade Neuronal/fisiologia , Receptores Opioides kappa/deficiência , Animais , Benzenoacetamidas/metabolismo , Benzenoacetamidas/farmacologia , Encéfalo/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasticidade Neuronal/efeitos dos fármacos , Ligação Proteica/fisiologia , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Receptores Opioides kappa/agonistas
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