RESUMO
The parabrachial nucleus (PBN) is a major hub that receives sensory information from both internal and external environments. Specific populations of PBN neurons are involved in behaviors including food and water intake, nociceptive responses, breathing regulation, as well as learning and responding appropriately to threatening stimuli. However, it is unclear how many PBN neuron populations exist and how different behaviors may be encoded by unique signaling molecules or receptors. Here we provide a repository of data on the molecular identity, spatial location, and projection patterns of dozens of PBN neuron subclusters. Using single-cell RNA sequencing, we identified 21 subclusters of neurons in the PBN and neighboring regions. Multiplexed in situ hybridization showed many of these subclusters are enriched within specific PBN subregions with scattered cells in several other regions. We also provide detailed visualization of the axonal projections from 21 Cre-driver lines of mice. These results are all publicly available for download and provide a foundation for further interrogation of PBN functions and connections.
Assuntos
Núcleos Parabraquiais , Animais , Camundongos , Neurônios , AxôniosRESUMO
Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals.
Assuntos
Síndrome de Cushing , Animais , Domínio Catalítico/genética , Síndrome de Cushing/genética , Síndrome de Cushing/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hidrocortisona/metabolismo , Camundongos , ProteômicaRESUMO
BACKGROUND: Upregulation of cAMP-dependent and cAMP-independent PKA signaling is thought to promote cystogenesis in polycystic kidney disease (PKD). PKA-I regulatory subunit RIα is increased in kidneys of orthologous mouse models. Kidney-specific knockout of RIα upregulates PKA activity, induces cystic disease in wild-type mice, and aggravates it in Pkd1RC/RC mice. METHODS: PKA-I activation or inhibition was compared with EPAC activation or PKA-II inhibition using Pkd1RC/RC metanephric organ cultures. The effect of constitutive PKA (preferentially PKA-I) downregulation in vivo was ascertained by kidney-specific expression of a dominant negative RIαB allele in Pkd1RC/RC mice obtained by crossing Prkar1αR1αB/WT, Pkd1RC/RC , and Pkhd1-Cre mice (C57BL/6 background). The effect of pharmacologic PKA inhibition using a novel, selective PRKACA inhibitor (BLU2864) was tested in mIMCD3 3D cultures, metanephric organ cultures, and Pkd1RC/RC mice on a C57BL/6 × 129S6/Sv F1 background. Mice were sacrificed at 16 weeks of age. RESULTS: PKA-I activation promoted and inhibition prevented ex vivo P-Ser133 CREB expression and cystogenesis. EPAC activation or PKA-II inhibition had no or only minor effects. BLU2864 inhibited in vitro mIMCD3 cystogenesis and ex vivo P-Ser133 CREB expression and cystogenesis. Genetic downregulation of PKA activity and BLU2864 directly and/or indirectly inhibited many pro-proliferative pathways and were both protective in vivo. BLU2864 had no detectable on- or off-target adverse effects. CONCLUSIONS: PKA-I is the main PKA isozyme promoting cystogenesis. Direct PKA inhibition may be an effective strategy to treat PKD and other conditions where PKA signaling is upregulated. By acting directly on PKA, the inhibition may be more effective than or substantially increase the efficacy of treatments that only affect PKA activity by lowering cAMP.
Assuntos
Rim Policístico Autossômico Dominante , Rim Policístico Autossômico Recessivo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/farmacologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante/metabolismo , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods. The approach is based on selectively expressing a hemagglutinin A (HA)-tagged ribosomal protein in a target cell type and then using antibodies against HA to purify the polysomes and associated mRNAs from the target cell. The original approach makes use of a mouse line (RiboTag) harboring a modified allele of Rpl22 (Rpl22-HA) that is induced by the action of Cre recombinase. The Rpl22-HA gene can also be introduced into the animal by stereotaxic injection of an AAV-DIO-Rpl22-HA that is then activated in Cre-expressing cells. Both methods for tagging ribosomes facilitate the immunoprecipitation of ribosome-bound mRNAs and their analysis by qRT-PCR or RNA-Seq. This protocol will discuss the technical procedures and describe important considerations relevant to the analysis of the data. © 2019 by John Wiley & Sons, Inc.
Assuntos
RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ribossomos/genética , Análise de Sequência de RNA/métodos , Animais , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ribossomos/metabolismoRESUMO
Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1-/- mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca2+ homeostasis in neurons subjected to excitotoxic glutamate. Ca2+ deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca2+ deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.SIGNIFICANCE STATEMENT Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca2+ overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Isquemia Encefálica/metabolismo , Dinaminas/metabolismo , Dinâmica Mitocondrial/fisiologia , Acidente Vascular Cerebral/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Isquemia Encefálica/genética , Cálcio/metabolismo , Dinaminas/genética , Complexo II de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Fosforilação , Acidente Vascular Cerebral/genética , Superóxidos/metabolismoRESUMO
AIMS: Impaired Ca2 + cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca2+ cycling and excitation-contraction in cardiomyocytes. However, the role of the AKAP150 signalling complexes in the pathogenesis of heart failure has not been investigated. METHODS AND RESULTS: Here we examined how AKAP150 signalling complexes impact Ca2+ cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodelling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not affect calcineurin-nuclear factor of activated T cells signalling in cardiomyocytes or pressure overload- or agonist-induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca2+ regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca2+ cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. CONCLUSIONS: These findings define a critical role for AKAP150 in regulating Ca2+ cycling and myocardial ionotropy following pathological stress, suggesting the AKAP150 signalling pathway may serve as a novel therapeutic target for heart failure.
Assuntos
Proteínas de Ancoragem à Quinase A/deficiência , Sinalização do Cálcio , Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/metabolismo , Miócitos Cardíacos/metabolismo , Função Ventricular , Remodelação Ventricular , Proteínas de Ancoragem à Quinase A/genética , Animais , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Predisposição Genética para Doença , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Isoproterenol , Camundongos Knockout , Isquemia Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/complicações , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Fenótipo , Interferência de RNA , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Protein Kinase A (PKA) mediates synaptic plasticity and is widely implicated in learning and memory. The hippocampal dentate gyrus (DG) is thought to be responsible for processing and encoding distinct contextual associations in response to highly similar inputs. The mossy fiber (MF) axons of the dentate granule cells convey strong excitatory drive to CA3 pyramidal neurons and express presynaptic, PKA-dependent forms of plasticity. Here, we demonstrate an essential role for the PKA anchoring protein, AKAP7, in mouse MF axons and terminals. Genetic ablation of AKAP7 specifically from dentate granule cells results in disruption of MF-CA3 LTP directly initiated by cAMP, and the AKAP7 mutant mice are selectively deficient in pattern separation behaviors. Our results suggest that the AKAP7/PKA complex in the MF projections plays an essential role in synaptic plasticity and contextual memory formation.
Assuntos
Proteínas de Ancoragem à Quinase A/deficiência , Região CA3 Hipocampal/fisiologia , Núcleos Cerebelares/fisiologia , Técnicas de Inativação de Genes , Fibras Musgosas Hipocampais/fisiologia , Comportamento Espacial , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , CamundongosRESUMO
BACKGROUND: Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC. METHODS: The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors. RESULTS: The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases. CONCLUSION: FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Carcinoma Hepatocelular/enzimologia , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Neoplasias Hepáticas/enzimologia , Mutação , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Carcinoma Hepatocelular/genética , Domínio Catalítico , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Cirrose Hepática/complicações , Neoplasias Hepáticas/genética , Metástase Linfática , Recidiva Local de NeoplasiaRESUMO
Mice lacking the RIIß regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIß knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIß KO mice. During fasting, RIIß-PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIß KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIß KO mice. Our findings suggest that RIIß-PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin.
Assuntos
Adiposidade/genética , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Hipotálamo/metabolismo , Leptina/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores para Leptina/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dieta Hiperlipídica , Retroalimentação Fisiológica , Janus Quinases/metabolismo , Leptina/farmacologia , Camundongos , Camundongos Knockout , Neuropeptídeo Y/metabolismo , Obesidade/genética , Fosforilação , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismoRESUMO
Hypothalamic neuronal populations are central regulators of energy homeostasis and reproductive function. However, the ontogeny of these critical hypothalamic neuronal populations is largely unknown. We developed a novel approach to examine the developmental pathways that link specific subtypes of neurons by combining embryonic and adult ribosome-tagging strategies in mice. This new method shows that Pomc-expressing precursors not only differentiate into discrete neuronal populations that mediate energy balance (POMC and AgRP neurons), but also into neurons critical for puberty onset and the regulation of reproductive function (Kiss1 neurons). These results demonstrate a developmental link between nutrient-sensing and reproductive neuropeptide synthesizing neuronal populations and suggest a potential pathway that could link maternal nutrition to reproductive development in the offspring.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Hipotálamo/citologia , Kisspeptinas/metabolismo , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Células-Tronco/fisiologia , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , Dependovirus/genética , Embrião de Mamíferos , Vetores Genéticos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Kisspeptinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Pró-Opiomelanocortina/genética , RNA Mensageiro/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.
Assuntos
Aquaporina 2/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Diabetes Insípido/etiologia , Animais , Arginina Vasopressina/metabolismo , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Hipotálamo/metabolismo , Rim/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Receptores de Vasopressinas/metabolismo , Equilíbrio HidroeletrolíticoRESUMO
Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIß, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIß) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs.
Assuntos
Corpo Estriado/citologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Comportamento Alimentar/fisiologia , Regulação da Expressão Gênica/fisiologia , Atividade Motora/fisiologia , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Peso Corporal/genética , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Dopaminérgicos/farmacologia , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/genética , Transtornos dos Movimentos/genética , Mutação/genética , Obesidade/dietoterapia , Obesidade/genéticaRESUMO
Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.
Assuntos
Deficiência Intelectual/genética , Transtornos Mentais/genética , Proteínas Nucleares/genética , Distúrbios da Fala/genética , Sequência de Aminoácidos , Animais , Criança , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína/genética , Fatores de TranscriçãoRESUMO
Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.
Assuntos
Apetite/fisiologia , Neurônios/fisiologia , Ponte/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Adjuvantes Imunológicos/farmacologia , Proteína Relacionada com Agouti/deficiência , Proteína Relacionada com Agouti/genética , Animais , Anorexia/genética , Anorexia/fisiopatologia , Anorexia/prevenção & controle , Apetite/efeitos dos fármacos , Western Blotting , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Transtornos de Deglutição/genética , Transtornos de Deglutição/fisiopatologia , Transtornos de Deglutição/prevenção & controle , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Cloreto de Lítio/farmacologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenóis , Piperidinas/farmacologia , Ponte/citologia , Ponte/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Rombencéfalo/fisiologia , Fatores de Tempo , VagotomiaRESUMO
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
Targeted disruption of RIIß-protein kinase A (PKA) in mice leads to a lean phenotype, increased nocturnal locomotor activity, and activation of brown adipose tissue. Because RIIß is abundantly expressed in both white and brown adipose tissue as well as the brain, the contribution of neuronal vs. peripheral PKA to these phenotypes was investigated. We used a Cre-Lox strategy to reexpress RIIß in a tissue-specific manner in either adipocytes or neurons. Mice with adipocyte-specific RIIß reexpression remained hyperactive and lean, but pan-neuronal RIIß reexpression reversed both phenotypes. Selective RIIß reexpression in all striatal medium spiny neurons with Darpp32-Cre corrected the hyperlocomotor phenotype, but the mice remained lean. Further analysis revealed that RIIß reexpression in D2 dopamine receptor-expressing medium spiny neurons corrected the hyperlocomotor phenotype, which demonstrated that the lean phenotype in RIIß-PKA-deficient mice does not develop because of increased locomotor activity. To identify the neurons responsible for the lean phenotype, we used specific Cre-driver mice to reexpress RIIß in agouti-related peptide (AgRP)-, proopiomelanocortin (POMC)-, single-minded 1 (Sim1)-, or steroidogenic factor 1 (SF1)-expressing neurons in the hypothalamus, but observed no rescue of the lean phenotype. However, when RIIß was reexpressed in multiple regions of the hypothalamus and striatum driven by Rip2-Cre, or specifically in GABAergic neurons driven by Vgat-ires-Cre, both the hyperactive and lean phenotypes were completely corrected. Bilateral injection of adeno-associated virus1 (AAV1)-Cre directly into the hypothalamus caused reexpression of RIIß and partially reversed the lean phenotype. These data demonstrate that RIIß-PKA deficiency in a subset of hypothalamic GABAergic neurons leads to the lean phenotype.
Assuntos
Adiposidade/genética , Encéfalo/metabolismo , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/fisiologia , Homeostase/fisiologia , Locomoção/fisiologia , Neurônios/metabolismo , Análise de Variância , Animais , Western Blotting , Peso Corporal/genética , Calorimetria Indireta , Subunidade RIIbeta da Proteína Quinase Dependente de AMP Cíclico/genética , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Genótipo , Imuno-Histoquímica , Integrases/metabolismo , Leptina/sangue , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Reação em Cadeia da Polimerase , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Protein kinase A (PKA) is activated during sympathetic stimulation of the heart and phosphorylates key proteins involved in cardiac Ca(2+) handling, including the L-type Ca(2+) channel (Ca(V)1.2) and phospholamban (PLN). This results in acceleration and amplification of the beat-to-beat changes in cytosolic Ca(2+) in cardiomyocytes and, in turn, an increased rate and force of contraction. PKA is held in proximity to its substrates by protein scaffolds called A kinase anchoring proteins (AKAPs). It has been suggested that the short and long isoforms of AKAP7 (also called AKAP15/18) localize PKA in complexes with Ca(V)1.2 and PLN, respectively. We generated an AKAP7 KO mouse in which all isoforms were deleted and tested whether Ca(2+) current, intracellular Ca(2+) concentration, or Ca(2+) reuptake were impaired in isolated adult ventricular cardiomyocytes following stimulation with the ß-adrenergic agonist isoproterenol. KO cardiomyocytes responded normally to adrenergic stimulation, as measured by whole-cell patch clamp or a fluorescent intracellular Ca(2+) indicator. Phosphorylation of Ca(V)1.2 and PLN were also unaffected by genetic deletion of AKAP7. Immunoblot and RT-PCR revealed that only the long isoforms of AKAP7 were detectable in ventricular cardiomyocytes. The results indicate that AKAP7 is not required for regulation of Ca(2+) handling in mouse cardiomyocytes.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Animais , Southern Blotting , Primers do DNA/genética , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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
Neurons that produce kisspeptin play a critical role in reproduction. However, understanding the molecular physiology of kisspeptin neurons has been limited by the lack of an in vivo marker for those cells. Here, we report the development of a Kiss1-CreGFP knockin mouse, wherein the endogenous Kiss1 promoter directs the expression of a Cre recombinase-enhanced green fluorescent protein (GFP) fusion protein. The pattern of GFP expression in the brain of the knockin recapitulates what has been described earlier for Kiss1 in the male and female mouse, with prominent expression in the arcuate nucleus (ARC) (in both sexes) and the anteroventral periventricular nucleus (in females). Single-cell RT-PCR showed that the Kiss1 transcript is expressed in 100% of GFP-labeled cells, and the CreGFP transcript was regulated by estradiol in the same manner as the Kiss1 gene (i.e. inhibited in the ARC and induced in the anteroventral periventricular nucleus). We used this mouse to evaluate the biophysical properties of kisspeptin (Kiss1) neurons in the ARC of the female mouse. GFP-expressing Kiss1 neurons were identified in hypothalamic slice preparations of the ARC and patch clamped. Whole-cell (and loose attached) recordings revealed that Kiss1 neurons exhibit spontaneous activity and expressed both h- (pacemaker) and T-type calcium currents, and hyperpolarization-activated cyclic nucleotide-regulated 1-4 and CaV3.1 channel subtypes (measured by single cell RT-PCR), respectively. N-methyl-D-aspartate induced bursting activity, characterized by depolarizing/hyperpolarizing oscillations. Therefore, Kiss1 neurons in the ARC share molecular and electrophysiological properties of other CNS pacemaker neurons.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Kisspeptinas/metabolismo , Neurônios/metabolismo , Reprodução/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Orquiectomia , Ovariectomia , Reprodução/efeitos dos fármacosRESUMO
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.