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1.
Cereb Cortex ; 32(3): 479-489, 2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-34247243

RESUMO

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.


Assuntos
Proteínas Nucleares , Receptores Acoplados a Proteínas G , Animais , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
2.
Brain ; 143(10): 2911-2928, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33103737

RESUMO

Human post-natal neurodevelopmental delay is often associated with cerebral alterations that can lead, by themselves or associated with peripheral deficits, to premature death. Here, we report the clinical features of 10 patients from six independent families with mutations in the autosomal YIF1B gene encoding a ubiquitous protein involved in anterograde traffic from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus morphology. The patients displayed global developmental delay, motor delay, visual deficits with brain MRI evidence of ventricle enlargement, myelination alterations and cerebellar atrophy. A similar profile was observed in the Yif1b knockout (KO) mouse model developed to identify the cellular alterations involved in the clinical defects. In the CNS, mice lacking Yif1b displayed neuronal reduction, altered myelination of the motor cortex, cerebellar atrophy, enlargement of the ventricles, and subcellular alterations of endoplasmic reticulum and Golgi apparatus compartments. Remarkably, although YIF1B was not detected in primary cilia, biallelic YIF1B mutations caused primary cilia abnormalities in skin fibroblasts from both patients and Yif1b-KO mice, and in ciliary architectural components in the Yif1b-KO brain. Consequently, our findings identify YIF1B as an essential gene in early post-natal development in human, and provide a new genetic target that should be tested in patients developing a neurodevelopmental delay during the first year of life. Thus, our work is the first description of a functional deficit linking Golgipathies and ciliopathies, diseases so far associated exclusively to mutations in genes coding for proteins expressed within the primary cilium or related ultrastructures. We therefore propose that these pathologies should be considered as belonging to a larger class of neurodevelopmental diseases depending on proteins involved in the trafficking of proteins towards specific cell membrane compartments.


Assuntos
Cílios/genética , Complexo de Golgi/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas de Transporte Vesicular/genética , Animais , Células Cultivadas , Cílios/patologia , Feminino , Complexo de Golgi/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/diagnóstico por imagem
3.
J Psychiatry Neurosci ; 45(5): 344-355, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459080

RESUMO

Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.


Assuntos
Transtorno Depressivo Maior/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Serotonina/metabolismo , Comportamento Social , Proteínas de Transporte Vesicular/efeitos dos fármacos , Proteínas de Transporte Vesicular/metabolismo , Animais , Autopsia , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Feminino , Fluoxetina/farmacologia , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleos da Rafe/efeitos dos fármacos , Núcleos da Rafe/fisiologia , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/fisiologia , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Caracteres Sexuais
4.
Cell Mol Life Sci ; 76(24): 4995-5009, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31139847

RESUMO

Protein interacting with Amyloid Precursor Protein (APP) tail 1 (PAT1) also called APPBP2 or Ara 67 has different targets such as APP or androgen receptor and is expressed in several tissues. PAT1 is known to be involved in the subcellular trafficking of its targets. We previously observed in primary neurons that PAT1 is poorly associated with APP at the cell surface. Here we show that PAT1 colocalizes with vesicles close to the cell surface labeled with Rab5, Rab4, EEA1 and Rabaptin-5 but not with Rab11 and Rab7. Moreover, PAT1 expression regulates the number of EEA1 and Rab5 vesicles, and endocytosis/recycling of the transferrin receptor. In addition, low levels of PAT1 decrease the size of transferrin-colocalized EEA1 vesicles with time following transferrin uptake. Finally, overexpression of the APP binding domain to PAT1 is sufficient to compromise endocytosis. Altogether, these data suggest that PAT1 is a new actor in transferrin early endocytosis. Whether this new function of PAT1 may have consequences in pathology remains to be determined.


Assuntos
Sistemas de Transporte de Aminoácidos/genética , Simportadores/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Endocitose/genética , Endossomos/genética , Endossomos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Neurônios/metabolismo , Transporte Proteico , Receptores Androgênicos/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7
5.
Traffic ; 16(9): 978-93, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26077767

RESUMO

Yif1B is an intracellular membrane-bound protein belonging to the Yip family, shown previously to control serotonin 5-HT1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.


Assuntos
Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/ultraestrutura , Transporte Proteico , Ratos , Proteínas de Transporte Vesicular/genética
6.
J Neurosci ; 36(5): 1456-70, 2016 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-26843630

RESUMO

The 5-HT3 receptors are serotonin-gated ion channels that physically couple with purinergic P2X2 receptors to trigger a functional cross-inhibition leading to reciprocal channel occlusion. Although this functional receptor-receptor coupling seems to serve a modulatory role on both channels, this might not be its main physiological purpose. Using primary cultures of rat hippocampal neurons as a quantitative model of polarized targeting, we show here a novel function for this interaction. In this model, 5-HT3A receptors did not exhibit by themselves the capability of distal targeting in dendrites and axons but required the presence of P2X2R for their proper subcellular localization. 5-HT3AR distal targeting occurred with a delayed time course and exhibited a neuron phenotype dependency. In the subpopulation of neurons expressing endogenous P2X2R, 5-HT3AR distal neuritic localization correlated with P2X2R expression and could be selectively inhibited by P2X2R RNA interference. Cotransfection of both receptors revealed a specific colocalization, cotrafficking in common surface clusters, and the axonal rerouting of 5-HT3AR. The physical association between the two receptors was dependent on the second intracellular loop of the 5-HT3A subunit, but not on the P2X2R C-terminal tail that triggers the functional cross-inhibition with the 5-HT3AR. Together, these data establish that 5-HT3AR distal targeting in axons and dendrites primarily depends on P2X2R expression. Because several P2XR have now been shown to functionally interact with several other members of the 4-TMD family of receptor channels, we propose to reconsider the real functional role for this receptor family, as trafficking partner proteins dynamically involved in other receptors targeting. SIGNIFICANCE STATEMENT: So far, receptor targeting mechanisms were found to involve intracellular partner proteins or supramolecular complexes that couple receptors to cytoskeletal elements and recruit them into cargo vesicles. In this paper, we describe a new trafficking mechanism for the neuronal serotonin 5-HT3A ionotropic channel receptor, in which the role of routing partner is endowed by a functionally interacting purinergic receptor: the P2X2 receptor. This work not only unveils the mechanism by which 5-HT3 receptors can reach their axonal localization required for the control of neurotransmitter release, but also suggests that, in addition to their modulatory role, the family of P2X receptors could have a previously undescribed functional role of trafficking partner proteins dynamically involved in the targeting of other receptors.


Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Canais Iônicos de Abertura Ativada por Ligante/química , Camundongos , Neurônios/metabolismo , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X2/química , Receptores 5-HT3 de Serotonina/química , Xenopus laevis
7.
J Pineal Res ; 60(1): 95-108, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26514267

RESUMO

In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT1 , but not with MT2 , we identified several presynaptic proteins, suggesting a potential role of MT1 in neurotransmission. Presynaptic localization of MT1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT1 physically interacts with the voltage-gated calcium channel Cav 2.2 and inhibits Cav 2.2-promoted Ca(2+) entry in an agonist-independent manner. In conclusion, we show that MT1 is part of the presynaptic protein network and negatively regulates Cav 2.2 activity, providing a first hint for potential synaptic functions of MT1.


Assuntos
Encéfalo/metabolismo , Canais de Cálcio Tipo N/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptor MT1 de Melatonina/metabolismo , Canais de Cálcio Tipo N/genética , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/genética , Receptor MT1 de Melatonina/genética
8.
J Neurosci ; 34(1): 282-94, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24381289

RESUMO

Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, this expected effect has a slow onset after starting SSRI treatment because of initial activation of 5-HT(1A) autoreceptor-mediated negative feedback of 5-HT release. After chronic SSRI treatment, 5-HT(1A) autoreceptors desensitize, which allows 5-HT tone elevation. Because 5-HT(1A) receptor (5-HT(1A)R) internalization has been proposed as a possible mechanism underlying 5-HT(1A) autoreceptor desensitization, we examined whether this receptor could internalize under well controlled in vitro conditions in the LLC-CPK1 cell line and in raphe or hippocampal neurons from rat embryos. To this goal, cells were transfected with recombinant lentiviral vectors encoding N-terminal tagged 5-HT(1A)R, and exposed to various pharmacological conditions. Constitutive endocytosis and plasma membrane recycling of tagged-5-HT(1A)R was observed in LLC-PK1 cells as well as in neurons. Acute exposure (for 1 h) to the full 5-HT(1A)R agonists, 5-HT and 5-carboxamido-tryptamine, but not the partial agonist 8-OH-DPAT, triggered internalization of tagged 5-HT(1A)R in serotonergic neurons only. In contrast, sustained exposure (for 24 h) to all agonists induced tagged-5-HT(1A)R endocytosis in raphe serotonergic neurons and a portion of hippocampal neurons, but not LLC-PK1 cells and partial agonist displayed an effect only in serotonergic neurons. In all cases, agonist-induced tagged 5-HT(1A)R endocytosis was prevented by the 5-HT(1A)R antagonist, WAY-100635, which was inactive on its own. These data showed that agonist-induced 5-HT(1A)R internalization does exist in neurons and depends on agonist efficacy and neuronal phenotype. Its differential occurrence in serotonergic neurons supports the idea that 5-HT(1A)R internalization might underlie 5-HT(1A) autoreceptor desensitization under SSRI antidepressant therapy.


Assuntos
Autorreceptores/agonistas , Autorreceptores/metabolismo , Neurônios/metabolismo , Fenótipo , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Feminino , Células LLC-PK1 , Neurônios/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Suínos
9.
Traffic ; 12(11): 1501-20, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21801291

RESUMO

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.


Assuntos
Axônios/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Endocitose/fisiologia , Células HeLa , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Células LLC-PK1 , Dados de Sequência Molecular , Neurônios/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Receptor 5-HT1A de Serotonina/metabolismo , Relação Estrutura-Atividade , Suínos , Células Tumorais Cultivadas
10.
J Neurosci ; 32(41): 14227-41, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-23055492

RESUMO

Although essential for their neuronal function, the molecular mechanisms underlying the dendritic targeting of serotonin G-protein-coupled receptors are poorly understood. Here, we characterized a Yif1B-dependent vesicular scaffolding complex mediating the intracellular traffic of the rat 5-HT(1A) receptor (5-HT(1A)R) toward dendrites. By combining directed mutagenesis, GST-pull down, and surface plasmon resonance, we identified a tribasic motif in the C-tail of the 5-HT(1A)R on which Yif1B binds directly with high affinity (K(D) ≈ 37 nM). Moreover, we identified Yip1A, Rab6, and Kif5B as new partners of the 5-HT(1A)R/Yif1B complex, and showed that their expression in neurons is also crucial for the dendritic targeting of the 5-HT(1A)R. Live videomicroscopy revealed that 5-HT(1A)R, Yif1B, Yip1A, and Rab6 traffic in vesicles exiting the soma toward the dendritic tree, and also exhibit bidirectional motions, sustaining their role in 5-HT(1A)R dendritic targeting. Hence, we propose a new trafficking pathway model in which Yif1B is the scaffold protein recruiting the 5-HT(1A)R in a complex including Yip1A and Rab6, with Kif5B and dynein as two opposite molecular motors coordinating the traffic of vesicles along dendritic microtubules. This targeting pathway opens new insights for G-protein-coupled receptors trafficking in neurons.


Assuntos
Dendritos/fisiologia , Regiões de Interação com a Matriz/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Receptor 5-HT1A de Serotonina/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Células Cultivadas , Dendritos/genética , Marcação de Genes/métodos , Humanos , Regiões de Interação com a Matriz/genética , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular/genética
11.
Nat Commun ; 14(1): 8003, 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38049397

RESUMO

Directed cell migration requires sustained cell polarisation. In migrating cortical interneurons, nuclear movements are directed towards the centrosome that organises the primary cilium signalling hub. Primary cilium-elicited signalling, and how it affects migration, remain however ill characterised. Here, we show that altering cAMP/cGMP levels in the primary cilium by buffering cAMP, cGMP or by locally increasing cAMP, influences the polarity and directionality of migrating interneurons, whereas buffering cAMP or cGMP in the apposed centrosome compartment alters their motility. Remarkably, we identify CXCL12 as a trigger that targets the ciliary cAMP/cGMP ratio to promote sustained polarity and directed migration. We thereby uncover cAMP/cGMP levels in the primary cilium as a major target of extrinsic cues and as the steering wheel of neuronal migration.


Assuntos
Polaridade Celular , Cílios , Cílios/fisiologia , GMP Cíclico , Interneurônios/fisiologia , Movimento Celular/fisiologia
12.
J Neurosci ; 28(32): 8063-73, 2008 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-18685031

RESUMO

The 5-HT(1A) receptor (5-HT(1A)R) is the most extensively characterized serotonin (5-HT) receptor mainly because of its involvement in the mode of action of antidepressants. The 5-HT(1A)R is confined to the somatodendritic domain of central neurons, where it mediates serotonin-evoked hyperpolarization. Our previous studies underlined the role of the short 5-HT(1A)R C-terminal domain in receptor targeting to dendrites. We used this 17 aa region as bait in a yeast two-hybrid screen, and identified, for the first time, an intracellular protein interacting with the 5-HT(1A)R. This protein is homologous to the yeast Yif1p, previously implicated in vesicular trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, but not yet characterized in mammals. We confirmed 5-HT(1A)R-Yif1B interaction by glutathione S-transferase pull-down experiments using rat brain extracts and transfected cell lines. Yif1B is highly expressed in the brain, and specifically in raphe 5-HT(1A)R-expressing neurons. Colocalization of Yif1B and 5-HT(1A)R was observed in small vesicles involved in transient intracellular trafficking. Last, inhibition of endogenous expression of Yif1B in primary neuron cultures by small interfering RNA specifically prevented the addressing of 5-HT(1A)R to distal portions of the dendrites, without affecting other receptors, such as sst2A, P2X(2), and 5-HT(3A) receptors. Together, our results provide strong evidence that Yif1B is a member of the ER/Golgi trafficking machinery, which plays a key role in specific targeting of 5-HT(1A)R to the neuronal dendrites. This finding opens up new pathways for the study of 5-HT(1A)R regulation by partner proteins and for the development of novel antidepressant drugs.


Assuntos
Dendritos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Glutationa Transferase/metabolismo , Células LLC-PK1 , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Suínos , Distribuição Tecidual , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
13.
FASEB J ; 22(7): 2340-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18267982

RESUMO

Using the Tph1-invalidated mouse line, in which blood is depleted in serotonin (5-hydroxytryptamine, 5-HT), we have demonstrated previously that maternal 5-HT is required for normal embryonic development. Here, we address the issue of the influence of the maternal 5-HT concentration on the cardiac function of the offspring as adults. We investigated the cardiac phenotype of Tph1-invalidated mice born to Tph1 heterozygous and null mothers. Functionally, all mutants display a significant decrease of cardiac contractility, indicative of impaired left ventricular function. They exhibit progressive dilated cardiomyopathy and are unable to adapt appropriately to a pharmacological stress. Moreover, we show that the cardiopathy is more severe in adult Tph1(-/-) mice born to homozygous mothers than to heterozygous mothers. Importantly, the severity of the cardiac phenotype is inversely correlated with the plasma 5-HT concentration but not the whole-blood 5-HT concentration. Thus, plasma 5-HT concentration may be a useful index of heart failure. These findings show that cardiac function, through the plasma 5-HT concentration, is influenced by the maternal serotonergic status.


Assuntos
Desenvolvimento Embrionário/fisiologia , Coração/fisiologia , Serotonina/fisiologia , Animais , Eletrocardiografia , Feminino , Triagem de Portadores Genéticos , Testes de Função Cardíaca , Homozigoto , Camundongos , Camundongos Knockout , Transdução de Sinais , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/fisiologia
14.
Neuroscience ; 396: 175-186, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472430

RESUMO

Significant alterations in glutamatergic neurotransmission have been reported in major depressive disorder (MDD) that could underlie psychiatric traits. Studies were mainly interested in synaptic dysfunction in the prefrontal cortex, a key structure involved in depressive-like behavior, however hippocampus has been shown to be important in MDD. As cognitive deficits such as hippocampus-memory process were observed in MDD, we investigated in a mild hypoglutamatergic model behaviors related to depression and memory, synaptic transmission parameters and glutamatergic state specifically in the hippocampus. We thus characterized these phenotypes in adult male mice partially depleted in glutaminase type 1 or GLS1 (GLS1 HET), the enzyme responsible for glutamate synthesis in neurons, that we previously characterized as displaying moderate lower levels of glutamate in brain. We showed that GLS1 mutant mice display AMPA-R-mediated response deficits after prolonged repetitive stimulation with electrophysiological recording and inability to sustain glutamate release by microdialysis experiments with no consequences on behavioral spatial learning performances. However, their ability to escape from unpleasant but repeated escapable condition was attenuated whereas they were more immobile in the unescapable situation in the FST during re-test. These results show that GLS1 mutant mice display moderate impairments of hippocampal glutamatergic neurotransmission and moderate changes in adaptive behaviors that have been shown to participate to the development of depressive-like state.


Assuntos
Aprendizagem da Esquiva/fisiologia , Ácido Glutâmico/fisiologia , Glutaminase/fisiologia , Hipocampo/fisiologia , Resposta de Imobilidade Tônica/fisiologia , Aprendizagem Espacial/fisiologia , Transmissão Sináptica/fisiologia , Animais , Corticosterona/sangue , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ácido Glutâmico/metabolismo , Glutaminase/genética , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Microdiálise , Mutação , Restrição Física/fisiologia
15.
J Neurosci ; 26(12): 3141-53, 2006 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-16554465

RESUMO

The type 1 cannabinoid receptor (CB1R) is one of the most abundant G-protein-coupled receptors (GPCRs) in the brain, predominantly localized to axons of GABAergic neurons. Like several other neuronal GPCRs, CB1R displays significant in vitro constitutive activity (i.e., spontaneous activation in the absence of ligand). However, a clear biological role for constitutive GPCR activity is still lacking. This question was addressed by studying the consequences of constitutive activation on the intracellular trafficking of endogenous or transfected CB1Rs in cultured hippocampal neurons using optical and electron microscopy. We found that constitutive activity results in a permanent cycle of endocytosis and recycling, which is restricted to the somatodendritic compartment. Thus, CB1Rs are continuously removed by endocytosis from the plasma membrane in the somatodendritic compartment but not in axons, where CB1Rs accumulate on surface. Blocking constitutive activity by short-term incubation with inverse agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) results in sequestration of recycled CB1Rs on the somatodendritic plasma membrane. Long-term inhibition of endocytosis by cotransfection of dominant-negative proteins results in impaired axonal polarization of surface-bound CB1Rs. Kinetic analysis shows that the majority of newly synthesized CB1Rs arrive first to the somatodendritic plasma membrane, from where they are rapidly removed by AM281-sensitive constitutive endocytosis before being delivered to axons. Thus, constitutive-activity driven somatodendritic endocytosis is required for the proper axonal targeting of CB1R, representing a novel, conformation-dependent targeting mechanism for axonal GPCRs.


Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Endocitose/fisiologia , Hipocampo/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Moduladores de Receptores de Canabinoides/metabolismo , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Endocitose/efeitos dos fármacos , Hipocampo/ultraestrutura , Ligantes , Microscopia Eletrônica de Transmissão , Morfolinas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Pirazóis/farmacologia , Ratos , Receptor CB1 de Canabinoide/efeitos dos fármacos
16.
J Neurosci ; 26(17): 4660-71, 2006 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-16641247

RESUMO

Neurotransmitter glutamate has been thought to derive mainly from glutamine via the action of glutaminase type 1 (GLS1). To address the importance of this pathway in glutamatergic transmission, we knocked out GLS1 in mice. The insertion of a STOP cassette by homologous recombination produced a null allele that blocked transcription, encoded no immunoreactive protein, and abolished GLS1 enzymatic activity. Null mutants were slightly smaller, were deficient in goal-directed behavior, hypoventilated, and died in the first postnatal day. No gross or microscopic defects were detected in peripheral organs or in the CNS. In cultured neurons from the null mutants, miniature EPSC amplitude and duration were normal; however, the amplitude of evoked EPSCs decayed more rapidly with sustained 10 Hz stimulation, consistent with an observed reduction in depolarization-evoked glutamate release. Because of this activity-dependent impairment in glutamatergic transmission, we surmised that respiratory networks, which require temporal summation of synaptic input, would be particularly affected. We found that the amplitude of inspirations was decreased in vivo, chemosensitivity to CO2 was severely altered, and the frequency of pacemaker activity recorded in the respiratory generator in the pre-Bötzinger complex, a glutamatergic brainstem network that can be isolated in vitro, was increased. Our results show that although alternate pathways to GLS1 glutamate synthesis support baseline glutamatergic transmission, the GLS1 pathway is essential for maintaining the function of active synapses, and thus the mutation is associated with impaired respiratory function, abnormal goal-directed behavior, and neonatal demise.


Assuntos
Encéfalo/enzimologia , Ácido Glutâmico/metabolismo , Glutaminase/deficiência , Hipoventilação/fisiopatologia , Rim/enzimologia , Transtornos Mentais/fisiopatologia , Transmissão Sináptica , Animais , Animais Recém-Nascidos , Objetivos , Camundongos , Camundongos Knockout , Vias Neurais/metabolismo , Transtornos Respiratórios , Mecânica Respiratória , Taxa de Sobrevida
17.
J Comp Neurol ; 524(14): 2776-802, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-26918661

RESUMO

GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Núcleo Celular/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Acoplados a Proteínas G/biossíntese , Fatores Etários , Animais , Animais Recém-Nascidos , Córtex Cerebral/química , Citoplasma/química , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/análise , Adulto Jovem
18.
Prog Mol Biol Transl Sci ; 132: 97-126, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26055056

RESUMO

Serotonin receptors (5-HTRs) mediate both central and peripheral control on numerous physiological functions such as sleep/wake cycle, thermoregulation, food intake, nociception, locomotion, sexual behavior, gastrointestinal motility, blood coagulation, and cardiovascular homeostasis. Six families of the G-protein-coupled receptors comprise most of serotonin receptors besides the conserved 5-HT3R Cys-loop type which belongs to the family of Cys-loop ligand-gated cation channel receptors. Many of these receptors are targets of pharmaceutical drugs, justifying the importance for elucidating their coupling, signaling and functioning. Recently, special interest has been focused on their trafficking inside cell lines or neurons in conjunction with their interaction with partner proteins. In this review, we describe the trafficking of 5-HTRs including their internalization, desensitization, or addressing to the plasma membrane depending on specific mechanisms which are peculiar for each class of serotonin receptor.


Assuntos
Membrana Celular/metabolismo , Receptores de Serotonina/metabolismo , Animais , Células CHO , Caveolinas/metabolismo , Sistema Nervoso Central/embriologia , Cricetulus , Cisteína/química , Endocitose , Células HEK293 , Hipocampo/metabolismo , Humanos , Ligantes , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transfecção
19.
Neuroreport ; 13(17): 2365-9, 2002 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-12488828

RESUMO

Within the nucleus tractus solitarii (NTS), serotonin (5-HT) exerts modulatory effects on central mechanisms controlling autonomic functions, notably through the stimulation of 5-HT2 receptors. Using double immunocytochemical labeling with specific anti-5-HT2A receptor antibodies and antibodies directed against the catecholamine (CA) synthesizing enzymes, i.e. tyrosine hydroxylase, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase, we investigated whether 5-HT effects could be mediated by 5-HT2A receptors located on CA perikarya and/or processes within the NTS. A relatively high density of 5-HT2A immunoreactive processes was observed throughout the NTS. 5-HT2A neuronal perikarya were also found within the NTS except in its rostrolateral part. Double immunolabeling experiments revealed many 5-HT2A receptors on CA processes but only few 5-HT2A/TH and 5-HT2A/DBH perikarya. These data support the idea that, within the NTS, 5-HT2A-mediated control of autonomic functions involves CA neurons.


Assuntos
Catecolaminas/metabolismo , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Núcleo Solitário/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Dopamina beta-Hidroxilase/metabolismo , Imunofluorescência , Masculino , Neurônios/citologia , Feniletanolamina N-Metiltransferase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina , Serotonina/metabolismo , Núcleo Solitário/citologia , Transmissão Sináptica/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo
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