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1.
J Neurosci ; 42(47): 8897-8911, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36202617

RESUMO

Metabotropic glutamate receptor Type 3 (mGlu3) controls the sleep/wake architecture, which plays a role in the glutamatergic pathophysiology of schizophrenia. Interestingly, mGlu3 receptor expression is decreased in the brain of schizophrenic patients. However, little is known about the molecular mechanisms regulating mGlu3 receptors at the cell membrane. Subcellular receptor localization is strongly dependent on protein-protein interactions. Here we show that mGlu3 interacts with PICK1 and that this scaffolding protein is important for mGlu3 surface expression and function in hippocampal primary cultures. Disruption of their interaction via an mGlu3 C-terminal mimicking peptide or an inhibitor of the PDZ domain of PICK1 altered the functional expression of mGlu3 receptors in neurons. We next investigated the impact of disrupting the mGlu3-PICK1 interaction on hippocampal theta oscillations in vitro and in vivo in WT male mice. We found a decreased frequency of theta oscillations in organotypic hippocampal slices, similar to what was previously observed in mGlu3 KO mice. In addition, hippocampal theta power was reduced during rapid eye movement sleep, non-rapid eye movement (NREM) sleep, and wake states after intraventricular administration of the mGlu3 C-terminal mimicking peptide. Targeting the mGlu3-PICK1 complex could thus be relevant to the pathophysiology of schizophrenia.SIGNIFICANCE STATEMENT Dysregulation of the glutamatergic system might play a role in the pathophysiology of schizophrenia. Metabotropic glutamate receptors Type 3 (mGlu3) have been proposed as potential targets for schizophrenia. Understanding the molecular mechanisms regulating mGlu3 receptor at the cell membrane is critical toward comprehending how their dysfunction contributes to the pathogenesis of schizophrenia. Here we describe that the binding of the signaling and scaffolding protein PICK1 to mGlu3 receptors is important for their localization and physiological functions. The identification of new proteins that associate specifically to mGlu3 receptors will advance our understanding of the regulatory mechanisms associated with their targeting and function and ultimately might provide new therapeutic strategies to counter these psychiatric conditions.


Assuntos
Proteínas de Transporte , Hipocampo , Receptores de Glutamato Metabotrópico , Animais , Masculino , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Hipocampo/metabolismo , Domínios PDZ , Receptores de Glutamato Metabotrópico/metabolismo
2.
J Neurosci ; 41(26): 5567-5578, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193623

RESUMO

G-protein-coupled receptors can be constitutively activated following physical interaction with intracellular proteins. The first example described was the constitutive activation of Group I metabotropic glutamate receptors (mGluR: mGluR1,5) following their interaction with Homer1a, an activity-inducible early-termination variant of the scaffolding protein Homer that lacks dimerization capacity (Ango et al., 2001). Homer1a disrupts the links, maintained by the long form of Homer (cross-linking Homers), between mGluR1,5 and the Shank-GKAP-PSD-95-ionotropic glutamate receptor network. Two characteristics of the constitutive activation of the Group I mGluR-Homer1a complex are particularly interesting: (1) it affects a large number of synapses in which Homer1a is upregulated following enhanced, long-lasting neuronal activity; and (2) it mainly depends on Homer1a protein turnover. The constitutively active Group I mGluR-Homer1a complex is involved in the two main forms of non-Hebbian neuronal plasticity: "metaplasticity" and "homeostatic synaptic scaling," which are implicated in a large series of physiological and pathologic processes. Those include non-Hebbian plasticity observed in visual system, synapses modulated by addictive drugs (rewarded synapses), chronically overactivated synaptic networks, normal sleep, and sleep deprivation.


Assuntos
Encéfalo/fisiologia , Homeostase/fisiologia , Proteínas de Arcabouço Homer/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Humanos
3.
Mol Psychiatry ; 26(12): 7596-7609, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34331007

RESUMO

Shank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.


Assuntos
Transtorno Autístico , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Modelos Animais de Doenças , Endossomos/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo
4.
BMC Biol ; 18(1): 81, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620110

RESUMO

BACKGROUND: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells. RESULTS: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder. CONCLUSIONS: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.


Assuntos
Técnicas Biossensoriais/métodos , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , Animais , Diagnóstico por Imagem/métodos , Células HEK293 , Humanos , Camundongos , Músculo Quadríceps/fisiologia
5.
Neurobiol Dis ; 129: 13-28, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31051234

RESUMO

Finding new targets to control or reduce seizure activity is essential to improve the management of epileptic patients. We hypothesized that activation of the pre-synaptic and inhibitory metabotropic glutamate receptor type 7 (mGlu7) reduces spontaneous seizures. We tested LSP2-9166, a recently developed mGlu7/4 agonist with unprecedented potency on mGlu7 receptors, in two paradigms of epileptogenesis. In a model of chemically induced epileptogenesis (pentylenetetrazole systemic injection), LSP2-9166 induces an anti-epileptogenic effect rarely observed in preclinical studies. In particular, we found a bidirectional modulation of seizure progression by mGlu4 and mGlu7 receptors, the latter preventing kindling. In the intra-hippocampal injection of kainic acid mouse model that mimics the human mesial temporal lobe epilepsy, we found that LSP2-9166 reduces seizure frequency and hippocampal sclerosis. LSP2-9166 also acts as an anti-seizure drug on established seizures in both models tested. Specific modulation of the mGlu7 receptor could represent a novel approach to reduce pathological network remodeling.


Assuntos
Aminobutiratos/farmacologia , Anticonvulsivantes/farmacologia , Hipocampo/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/agonistas , Convulsões/metabolismo , Animais , Epilepsia/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/metabolismo , Excitação Neurológica/efeitos dos fármacos , Camundongos , Camundongos Mutantes
6.
Neurobiol Dis ; 113: 70-81, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29432809

RESUMO

BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.


Assuntos
Circulação Cerebrovascular/fisiologia , Progressão da Doença , Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Pericitos/metabolismo , Convulsões/metabolismo , Adolescente , Adulto , Animais , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Células Cultivadas , Criança , Pré-Escolar , Feminino , Hipocampo/irrigação sanguínea , Hipocampo/química , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/química , Pericitos/química , Convulsões/fisiopatologia
7.
Addict Biol ; 23(2): 735-749, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28872733

RESUMO

High-palatable and caloric foods are widely overconsumed due to hedonic mechanisms that prevail over caloric necessities leading to overeating and overweight. The nucleus accumbens (NAc) is a key brain area modulating the reinforcing effects of palatable foods and is crucially involved in the development of eating disorders. We describe that prolonged exposure to high-caloric chocolate cafeteria diet leads to overeating and overweight in mice. NAc functionality was altered in these mice, presenting structural plasticity modifications in medium spiny neurons, increased expression of neuroinflammatory factors and activated microglia, and abnormal responses after amphetamine-induced hyperlocomotion. Chronic inactivation of microglia normalized these neurobiological and behavioural alterations exclusively in mice exposed to cafeteria diet. Our data suggest that prolonged exposure to cafeteria diet produces neuroplastic and functional changes in the NAc that can modify feeding behaviour. Microglia activation and neuroinflammation play an important role in the development of these neurobiological alterations.


Assuntos
Dieta , Comportamento Alimentar/fisiologia , Hiperfagia/imunologia , Microglia/imunologia , Núcleo Accumbens/imunologia , Sobrepeso/imunologia , Anfetamina/farmacologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Chocolate , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/imunologia , Corpo Estriado/patologia , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Espinhas Dendríticas/patologia , Comportamento Alimentar/efeitos dos fármacos , Inflamação , Locomoção/efeitos dos fármacos , Camundongos , Microscopia Confocal , Minociclina/farmacologia , Plasticidade Neuronal , Neurônios/patologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/patologia , Células Piramidais/patologia
8.
J Neurosci ; 36(12): 3541-51, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27013682

RESUMO

Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor-Ca(2+)channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT: Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization.


Assuntos
Arrestinas/metabolismo , Benzamidas/administração & dosagem , Hiperalgesia/fisiopatologia , Percepção da Dor , Piperazinas/administração & dosagem , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Animais , Tolerância a Medicamentos , Feminino , Masculino , Camundongos , Camundongos Knockout , Isoformas de Proteínas
9.
J Cell Sci ; 127(Pt 16): 3451-62, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24938595

RESUMO

Quantitative spatio-temporal characterization of protein interactions in living cells remains a major challenge facing modern biology. We have investigated in living neurons the spatial dependence of the stoichiometry of interactions between two core proteins of the N-methyl-D-aspartate (NMDA)-receptor-associated scaffolding complex, GKAP (also known as DLGAP1) and DLC2 (also known as DYNLL2), using a novel variation of fluorescence fluctuation microscopy called two-photon scanning number and brightness (sN&B). We found that dimerization of DLC2 was required for its interaction with GKAP, which, in turn, potentiated GKAP self-association. In the dendritic shaft, the DLC2-GKAP hetero-oligomeric complexes were composed mainly of two DLC2 and two GKAP monomers, whereas, in spines, the hetero-complexes were much larger, with an average of ∼16 DLC2 and ∼13 GKAP monomers. Disruption of the GKAP-DLC2 interaction strongly destabilized the oligomers, decreasing the spine-preferential localization of GKAP and inhibiting NMDA receptor activity. Hence, DLC2 serves a hub function in the control of glutamatergic transmission by ordering GKAP-containing complexes in dendritic spines. Beyond illuminating the role of DLC2-GKAP interactions in glutamatergic signaling, these data underscore the power of the sN&B approach for quantitative spatio-temporal imaging of other important protein complexes.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Espinhas Dendríticas/metabolismo , Dimerização , Proteínas Ativadoras de GTPase , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/química , Ligação Proteica , Proteínas Associadas SAP90-PSD95 , Alinhamento de Sequência , Sinapses/química , Sinapses/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
10.
EMBO Rep ; 15(1): 103-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24357660

RESUMO

The orphan GluD2 receptor belongs to the ionotropic glutamate receptor family but does not bind glutamate. Ligand-gated GluD2 currents have never been evidenced, and whether GluD2 operates as an ion channel has been a long-standing question. Here, we show that GluD2 gating is triggered by type 1 metabotropic glutamate receptors, both in a heterologous expression system and in Purkinje cells. Thus, GluD2 is not only an adhesion molecule at synapses but also works as a channel. This gating mechanism reveals new properties of glutamate receptors that emerge from their interaction and opens unexpected perspectives regarding synaptic transmission and plasticity.


Assuntos
Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Sinalização do Cálcio , Cerebelo/citologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores , Glicina/análogos & derivados , Glicina/farmacologia , Células HEK293 , Humanos , Ativação do Canal Iônico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ramos Subendocárdicos/efeitos dos fármacos , Ramos Subendocárdicos/fisiologia , Resorcinóis/farmacologia
11.
Eur J Neurosci ; 41(2): 157-67, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25377770

RESUMO

Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling.


Assuntos
Córtex Cerebelar/metabolismo , Ácido Glutâmico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Western Blotting , Proteínas de Transporte/metabolismo , Córtex Cerebelar/ultraestrutura , Proteínas de Arcabouço Homer , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Células de Purkinje/metabolismo , Células de Purkinje/ultraestrutura , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Sinapses/ultraestrutura
12.
J Neurosci ; 33(23): 9699-715, 2013 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-23739967

RESUMO

Synaptic long-term potentiation (LTP) is a key mechanism involved in learning and memory, and its alteration is associated with mental disorders. Shank3 is a major postsynaptic scaffolding protein that orchestrates dendritic spine morphogenesis, and mutations of this protein lead to mental retardation and autism spectrum disorders. In the present study we investigated the role of a new Shank3-associated protein in LTP. We identified the Rho-GAP interacting CIP4 homolog 2 (Rich2) as a new Shank3 partner by proteomic screen. Using single-cell bioluminescence resonance energy transfer microscopy, we found that Rich2-Shank3 interaction is increased in dendritic spines of mouse cultured hippocampal neurons during LTP. We further characterized Rich2 as an endosomal recycling protein that controls AMPA receptor GluA1 subunit exocytosis and spine morphology. Knock-down of Rich2 with siRNA, or disruption of the Rich2-Shank3 complex using an interfering mimetic peptide, inhibited the dendritic spine enlargement and the increase in GluA1 subunit exocytosis typical of LTP. These results identify Rich2-Shank3 as a new postsynaptic protein complex involved in synaptic plasticity.


Assuntos
Exocitose/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Potenciação de Longa Duração/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sequência de Aminoácidos , Animais , Espinhas Dendríticas/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Ligação Proteica/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
13.
Hippocampus ; 24(12): 1466-81, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25044816

RESUMO

The acute activation of the dopamine D1-like receptors (D1R) is involved in a plethora of functions ranging from increased locomotor activity to the facilitation of consolidation, storage, and retrieval of memories. Although much less characterized, epileptiform activities, usually triggered by disruption of the glutamate and GABA balance, have also been reported to involve the dopaminergic transmission. Using a combination of biochemical, immunohistochemical, electrophysiological, and behavioral approaches we have investigated the consequences of repeated stimulation of D1R using the selective D1R-like agonist SKF81297. Here, we report that repeated systemic administration of SKF81297 induces kindled seizures in mice. These seizure episodes parallel the hyperactivation of the mTOR signaling in the hippocampus, leading to disrupted long-term potentiation (LTP) in the dentate gyrus (DG) and altered recognition memories. The mTOR inhibitor rapamycin delays the development of SKF81297-induced kindled seizures, and rescues LTP in the DG and object recognition. Our results show that repeated stimulation of D1R is sufficient to induce generalized seizures leading to the overactivation of mTOR signaling, disrupted hippocampal plasticity, and impaired long-term recognition memories. This work highlights the interest of mTOR inhibitors as therapeutic strategies to reverse plasticity and cognitive deficits.


Assuntos
Giro Denteado/fisiopatologia , Transtornos da Memória/fisiopatologia , Receptores de Dopamina D1/metabolismo , Convulsões/fisiopatologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Benzazepinas/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Giro Denteado/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Fármacos Neuroprotetores/farmacologia , Receptores de Dopamina D1/agonistas , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Técnicas de Cultura de Tecidos
14.
J Cell Sci ; 125(Pt 8): 2030-40, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22328512

RESUMO

At glutamatergic brain synapses, scaffolding proteins regulate receptor location and function. The targeting and organization of scaffolding proteins in the postsynaptic density (PSD) is poorly understood, but it is known that a core protein of the glutamatergic receptor postsynaptic scaffold complex, guanylate-kinase-associated protein (GKAP) interacts with dynein light chain 2 (DLC2, also known as DYNLL2), a protein associated with molecular motors. In the present study, we combined BRET imaging, immunostaining and electrophysiological recording to assess the role of the GKAP-DLC2 interaction in the functional organization of the glutamatergic synapse. We found that GKAP-DLC2 interaction in dendritic spine stabilizes scaffolding protein expression at the PSD and enhances synaptic NMDA receptor activity. Moreover, the GKAP-DLC2 functional interaction is favored by sustained synaptic activity. These data identify a regulatory pathway of synaptic transmission that depends on activity-induced remodelling of the postsynaptic scaffold protein complex.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Matriz Nuclear/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Matriz Nuclear/genética , Ligação Proteica , Receptores de N-Metil-D-Aspartato/genética , Proteínas Associadas SAP90-PSD95 , Sinapses/genética , Proteínas Supressoras de Tumor/genética
15.
J Immunol ; 189(5): 2131-7, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22815289

RESUMO

Inflammation is fundamental for protecting the organism against infection and injury. However, a failure to control immune response results in chronic inflammation and several associated disorders such as pain and loss of function. Initiation of inflammation is orchestrated by cytokines, among which IL-1ß is particularly important. IL-1ß is synthesized as an inactive protein that has to be processed by the inflammasome to generate the mature bioactive form. Conventional techniques cannot monitor IL-1ß activation with high spatial and temporal resolution. In this study, we present a ratiometric biosensor that allows monitoring IL-1ß processing in real time, with a temporal resolution of seconds and with a single-cell spatial resolution. Using this sensor, to our knowledge, we describe for the first time the kinetic of the inflammasome activity in living macrophages. With this new probe, we also demonstrated that the pro-IL-1ß processing occurs all over the cytoplasm.


Assuntos
Transferência de Energia/imunologia , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Medições Luminescentes/métodos , Macrófagos/imunologia , Animais , Proteínas de Bactérias/genética , Técnicas de Cultura de Células , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células HEK293 , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Proteínas Luminescentes/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia
16.
Br J Pharmacol ; 181(12): 1793-1811, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38369690

RESUMO

BACKGROUND AND PURPOSE: Voltage sensitivity is a common feature of many membrane proteins, including some G-protein coupled receptors (GPCRs). However, the functional consequences of voltage sensitivity in GPCRs are not well understood. EXPERIMENTAL APPROACH: In this study, we investigated the voltage sensitivity of the post-synaptic metabotropic glutamate receptor mGlu5 and its impact on synaptic transmission. Using biosensors and electrophysiological recordings in non-excitable HEK293T cells or neurons. KEY RESULTS: We found that mGlu5 receptor function is optimal at resting membrane potentials. We observed that membrane depolarization significantly reduced mGlu5 receptor activation, Gq-PLC/PKC stimulation, Ca2+ release and mGlu5 receptor-gated currents through transient receptor potential canonical, TRPC6, channels or glutamate ionotropic NMDA receptors. Notably, we report a previously unknown activity of the NMDA receptor at the resting potential of neurons, enabled by mGlu5. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that mGlu5 receptor activity is directly regulated by membrane voltage which may have a significant impact on synaptic processes and pathophysiological functions.


Assuntos
Receptor de Glutamato Metabotrópico 5 , Transmissão Sináptica , Animais , Humanos , Células HEK293 , Potenciais da Membrana , Neurônios/metabolismo , Neurônios/fisiologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Camundongos
17.
Environ Pollut ; 345: 123477, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38307239

RESUMO

Prolonged exposure to low levels of dietary contaminants is a context in modern life that could alter organ physiology gradually. Here, we aimed to investigate the impact of continuous exposure to acceptable daily intake (ADI) and non-observable adverse effect level (NOAEL) of glyphosate from gestation to adulthood using C57BL/6J mice and incorporating these levels into their food pellets. From adulthood, we analyzed neurophysiological and neuro-glia cellular adaptations in male and female animals. Using ex-vivo hippocampal slice electrophysiology, we found a reduced efficacy of Schaffer collateral-to-CA1 excitatory synapses in glyphosate-exposed dietary conditions, with ADI and NOAEL dose-dependent effects. Short-term facilitation of excitatory synaptic transmission was specifically increased in NOAEL conditions, with a predominant influence in males, suggesting a reduced probability of neurotransmitter release. Long-term synaptic potentiation (LTP) was decreased in NOAEL-exposed mice. Next, we explore whether these neurophysiological modifications are associated with neuro-glia changes in the somatosensory cortex and hippocampus. High-resolution confocal microscopy analyses unveil a dose-dependent increased density of excitatory Vglut1+ Homer1+ synapses. Microglial Iba1+ cells displayed a shortening of their ramifications, a sign of cellular reactivity that was more pronounced in males at NOAEL levels. The morphology of GFAP+ astrocytes was generally not modified. Finally, we asked whether mouse-specific cross-correlations exist among all data sets generated. This examination included the novel object recognition (NOR) test performed before ex vivo functional and immunohistochemical examinations. We report a negative linear regression between the number of synapses and NOR or LTP maintenance when plotting ADI and NOAEL datasets. These results outline synaptic and microglial cell adaptations resulting from prenatal and continuous dietary low levels of glyphosate, discernible in, but not limited to, adult males exposed to the NOAEL. We discuss the potential significance of these findings to real-world consumer situations and long-term brain resilience.


Assuntos
Glifosato , Microglia , Camundongos , Masculino , Feminino , Animais , Roedores , Exposição Dietética , Camundongos Endogâmicos C57BL , Encéfalo
18.
Annu Rev Pharmacol Toxicol ; 50: 89-109, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20055699

RESUMO

G protein-coupled receptors (GPCRs) are key transmembrane recognition molecules for regulatory signals such as light, odors, taste hormones, and neurotransmitters. In addition to activating guanine nucleotide binding proteins (G proteins), GPCRs associate with a variety of GPCR-interacting proteins (GIPs). GIPs contain structural interacting domains that allow the formation of large functional complexes involved in G protein-dependent and -independent signaling. At the cellular level, other functions of GIPs include targeting of GPCRs to subcellular compartments and their trafficking to and from the plasma membrane. Recently, roles of GPCR-GIP interactions in central nervous system physiology and pathologies have been revealed. Here, we highlight the role of GIPs in some important neurological and psychiatric disorders, as well as their potential for the future development of therapeutic drugs.


Assuntos
Encéfalo/fisiologia , Proteínas de Transporte/fisiologia , Animais , Antidepressivos/farmacologia , Ansiedade/etiologia , Epilepsia/etiologia , Síndrome do Cromossomo X Frágil/etiologia , Proteínas de Arcabouço Homer , Humanos , Transtornos do Humor/tratamento farmacológico , Plasticidade Neuronal , Proteínas Nucleares/fisiologia , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/fisiologia , Receptores de Serotonina/fisiologia , Esquizofrenia/etiologia , Transmissão Sináptica
19.
Sci Rep ; 13(1): 14763, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37679476

RESUMO

In the last two decades, safety concerns about general anesthesia (GA) arose from studies documenting brain cell death in various pharmacological conditions and animal models. Nowadays, a thorough characterization of sevoflurane-induced apoptosis in the entire neonatal mouse brain would help identify and further focus on underlying mechanisms. We performed whole-brain mapping of sevoflurane-induced apoptosis in post-natal day (P) 7 mice using tissue clearing and immunohistochemistry. We found an anatomically heterogenous increase in cleaved-caspase-3 staining. The use of a novel P7 brain atlas showed that the neocortex was the most affected area, followed by the striatum and the metencephalon. Histological characterization in cortical slices determined that post-mitotic neurons were the most affected cell type and followed inter- and intracortical gradients with maximal apoptosis in the superficial layers of the posterodorsal cortex. The unbiased anatomical mapping used here allowed us to confirm sevoflurane-induced apoptosis in the perinatal period, neocortical involvement, and indicated striatal and metencephalic damage while suggesting moderate hippocampal one. The identification of neocortical gradients is consistent with a maturity-dependent mechanism. Further research could then focus on the interference of sevoflurane with neuronal migration and survival during development.


Assuntos
Neocórtex , Feminino , Gravidez , Animais , Camundongos , Sevoflurano/farmacologia , Apoptose , Morte Celular , Anestesia Geral , Morte Encefálica
20.
Chemosphere ; 324: 138251, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36878369

RESUMO

Pesticides are omnipresent, and they pose significant environmental and health risks. Translational studies indicate that acute exposure to high pesticide levels is detrimental, and prolonged contact with low concentrations of pesticides, as single and cocktail, could represent a risk factor for multi-organ pathophysiology, including the brain. Within this research template, we focus on pesticides' impact on the blood-brain barrier (BBB) and neuroinflammation, physical and immunological borders for the homeostatic control of the central nervous system (CNS) neuronal networks. We examine the evidence supporting a link between pre- and postnatal pesticide exposure, neuroinflammatory responses, and time-depend vulnerability footprints in the brain. Because of the pathological influence of BBB damage and inflammation on neuronal transmission from early development, varying exposures to pesticides could represent a danger, perhaps accelerating adverse neurological trajectories during aging. Refining our understanding of how pesticides influence brain barriers and borders could enable the implementation of pesticide-specific regulatory measures directly relevant to environmental neuroethics, the exposome, and one-health frameworks.


Assuntos
Barreira Hematoencefálica , Praguicidas , Humanos , Praguicidas/toxicidade , Doenças Neuroinflamatórias , Encéfalo/patologia , Sistema Nervoso Central , Inflamação/induzido quimicamente
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