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Disruption of the inhibitory control provided by the glycinergic system is one of the major mechanisms underlying chronic pain. In line with this concept, recent studies have provided robust proof that pharmacological intervention of glycine receptors (GlyRs) restores the inhibitory function and exerts anti-nociceptive effects on preclinical models of chronic pain. A targeted regulation of the glycinergic system requires the identification of the GlyR subtypes involved in chronic pain states. Nevertheless, the roles of individual GlyR subunits in nociception and in chronic pain are yet not well defined. This review aims to provide a systematic outline on the contribution of GlyR subtypes in chronic pain mechanisms, with a particular focus on molecular pathways of spinal glycinergic dis-inhibition mediated by post-translational modifications at the receptor level. The current experimental evidence has shown that phosphorylation of synaptic α1ß and α3ß GlyRs are involved in processes of spinal glycinergic dis-inhibition triggered by chronic inflammatory pain. On the other hand, the participation of α2-containing GlyRs and of ß subunits in pain signaling have been less studied and remain undefined. Although many questions in the field are still unresolved, future progress in GlyR research may soon open new exciting avenues into understanding and controlling chronic pain.
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The original version of this Article contained an error in the spelling of the author Anna K Radke, which was incorrectly given as Anna R Radke. This has now been corrected in both the PDF and HTML versions of the Article.
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Obsessive-compulsive disorder (OCD) is a severe, chronic neuropsychiatric disorder with a strong genetic component. The SLC1A1 gene encoding the neuronal glutamate transporter EAAT3 has been proposed as a candidate gene for this disorder. Gene variants affecting SLC1A1 expression in human brain tissue have been associated with OCD. Several mouse models fully or partially lacking EAAT3 have shown no alterations in baseline anxiety-like or repetitive behaviors. We generated a transgenic mouse model (EAAT3glo) to achieve conditional, Cre-dependent EAAT3 overexpression and evaluated the overall impact of increased EAAT3 expression at behavioral and synaptic levels. Mice with EAAT3 overexpression driven by CaMKIIα-promoter (EAAT3glo/CMKII) displayed increased anxiety-like and repetitive behaviors that were both restored by chronic, but not acute, treatment with fluoxetine or clomipramine. EAAT3glo/CMKII mice also displayed greater spontaneous recovery of conditioned fear. Electrophysiological and biochemical analyses at corticostriatal synapses of EAAT3glo/CMKII mice revealed changes in NMDA receptor subunit composition and altered NMDA-dependent synaptic plasticity. By recapitulating relevant behavioral, neurophysiological, and psychopharmacological aspects, our results provide support for the glutamatergic hypothesis of OCD, particularly for the increased EAAT3 function, and provide a valuable animal model that may open novel therapeutic approaches to treat this devastating disorder.
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Ansiedade/metabolismo , Comportamento Animal/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Neostriado/metabolismo , Plasticidade Neuronal/fisiologia , Transtorno Obsessivo-Compulsivo/metabolismo , Animais , Linhagem Celular , Clomipramina/farmacologia , Modelos Animais de Doenças , Transportador 3 de Aminoácido Excitatório/genética , Fluoxetina/farmacologia , Expressão Gênica/genética , Camundongos , Camundongos Transgênicos , Neuroblastoma , Técnicas de Patch-Clamp , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
The participation of reactive oxygen species (ROS) generated by NOX1 and NOX2/NADPH oxidase has been documented during inflammatory pain. However, the molecular mechanism involved in their activation is not fully understood. We reported earlier a key role of Cyclin-dependent kinase 5 (Cdk5) during inflammatory pain. In particular, we demonstrated that TNF-α increased p35 expression, a Cdk5 activator, causing Cdk5-mediated TRPV1 phosphorylation followed by an increment in Ca2+ influx in nociceptive neurons and increased pain sensation. Here we evaluated if Cdk5 activation mediated by p35 transfection in HEK293 cells or by TNF-α treatment in primary culture of nociceptive neurons could increase ROS production. By immunofluorescence we detected the expression of catalytic subunit (Nox1 and Nox2) and their cytosolic regulators (NOXO1 and p47phox) of NOX1 and NOX2/NADPH oxidase complexes, and their co-localization with Cdk5/p35 in HEK293 cells and in nociceptive neurons. By using a hydrogen peroxide sensor, we detected a significant increase of ROS production in p35 transfected HEK293 cells as compared with control cells. This effect was significantly blocked by VAS2870 (NADPH oxidase inhibitor) or by roscovitine (Cdk5 activity inhibitor). Also by using another ROS probe named DCFH-DA, we found a significant increase of ROS production in nociceptive neurons treated with TNF-α and this effect was also blocked by VAS2870 or by roscovitine treatment. Interestingly, TNF-α increased immunodetection of p35 protein and NOX1 and NOX2/NADPH oxidase complexes in primary culture of trigeminal ganglia neurons. Finally, the cytosolic regulator NOXO1 was significantly translocated to plasma membrane after TNF-α treatment and roscovitine blocked this effect. Altogether these results suggest that Cdk5 activation is implicated in the ROS production by NOX1 and NOX2/NADPH oxidase complexes during inflammatory pain.
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The purinergic P2X2 receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.
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Ativação do Canal Iônico/fisiologia , Receptores Purinérgicos P2X2/metabolismo , Células Receptoras Sensoriais/fisiologia , Treonina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Camundongos , Mutação/genética , Oócitos , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Ratos , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X3/metabolismo , Roscovitina , Células Receptoras Sensoriais/efeitos dos fármacos , Treonina/genética , XenopusRESUMO
Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS) upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that can yield new insights on the molecular mechanisms behind the effects of prenatal stress.
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Moléculas de Adesão Celular Neuronais/genética , Metilação de DNA , Proteínas da Matriz Extracelular/genética , Exposição Materna , Transtornos Mentais/etiologia , Proteínas do Tecido Nervoso/genética , Efeitos Tardios da Exposição Pré-Natal , Regiões Promotoras Genéticas , Serina Endopeptidases/genética , Estresse Fisiológico , Estresse Psicológico , Animais , Animais Recém-Nascidos , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Comportamento Animal , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Feminino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Gravidez , Ratos , Proteína Reelina , Serina Endopeptidases/metabolismo , Transdução de SinaisRESUMO
Neuronal processes (neurites and axons) have an important role in brain cells communication and, generally, they are damaged in neurodegenerative diseases. Recent evidence has showed that the activation of PPARγ pathway promoted neuronal differentiation and axon polarity. In addition, activation of PPARγ using thiazolidinediones (TZDs) prevented neurodegeneration by reducing neuronal death, improving mitochondrial function, and decreasing neuroinflammation in neuropathic pain. In this review, we will discuss important evidence that supports a possible role of PPARγ in neuronal development, improvement of neuronal health, and pain signaling. Therefore, activation of PPARγ is a potential target with therapeutic applications against neurodegenerative disorders, brain injury, and pain regulation.
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Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.
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Encéfalo/metabolismo , Quinase 5 Dependente de Ciclina/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Linhagem Celular , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Deleção de Genes , Espectrometria de Massas , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada , Fosfoproteínas/química , Fosforilação , Proteômica , Purinas/farmacologia , RoscovitinaRESUMO
Rap1 signaling is important for migration, differentiation, axonal growth, and during neuronal polarity. Rap1 can be activated by external stimuli, which in turn regulates specific guanine nucleotide exchange factors such as C3G, among others. Cdk5 functions are also important to neuronal migration and differentiation. Since we found that pharmacological inhibition of Cdk5 by using roscovitine reduced Rap1 protein levels in COS-7 cells and also C3G contains three putative phosphorylation sites for Cdk5, we examined whether the Cdk5-dependent phosphorylation of C3G could affect Rap1 expression and activity. We co-transfected C3G and tet-OFF system for p35 over-expression, an activator of Cdk5 activity into COS-7 cells, and then we evaluated phosphorylation in serine residues in C3G by immunoprecipitation and Western blot. We found that p35 over-expression increased C3G-serine-phosphorylation while inhibition of p35 expression by tetracycline or inhibition of Cdk5 activity with roscovitine decreased it. Interestingly, we found that MG-132, a proteasome inhibitor, rescue Rap1 protein levels in the presence of roscovitine. Besides, C3G-serine-phosphorylation and Rap1 protein levels were reduced in brain from Cdk5(-/-) as compared with the Cdk5(+/+) brain. Finally, we found that p35 over-expression increased Rap1 activity while inhibition of p35 expression by tetracycline or roscovitine decreased Rap1 activity. These results suggest that Cdk5-mediated serine-phosphorylation of C3G may control Rap1 stability and activity, and this may potentially impact various neuronal functions such as migration, differentiation, and polarity.
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Quinase 5 Dependente de Ciclina/fisiologia , Proteínas rap1 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Inibidores de Cisteína Proteinase , Fator 2 de Liberação do Nucleotídeo Guanina/fisiologia , Imunoprecipitação , Subunidade p35 da Interleucina-12/biossíntese , Leupeptinas/farmacologia , Proteínas de Fusão de Membrana , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Tetraciclina/farmacologia , TransfecçãoRESUMO
Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that is mostly active in the nervous system, where it regulates several processes such as neuronal migration, actin and microtubule dynamics, axonal guidance, and synaptic plasticity, among other processes. In addition to these known functions, in the past few years, novel roles for Cdk5 outside of the nervous system have been proposed. These include roles in gene transcription, vesicular transport, apoptosis, cell adhesion, and migration in many cell types and tissues such as pancreatic cells, muscle cells, neutrophils, and others. In this review, we will summarize the recently studied non-neuronal functions of Cdk5, with a thorough analysis of the biological consequences of these novel roles.