RESUMO
The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.
Assuntos
Cloretos , Água , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Cloretos/metabolismo , Mutagênese , Cátions/metabolismo , Água/metabolismoRESUMO
A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.
Assuntos
Síndrome de Down/terapia , Terapia Genética/métodos , Membro 2 da Família 12 de Carreador de Soluto/genética , Animais , Cloretos/metabolismo , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/psicologia , Técnicas de Silenciamento de Genes , Homeostase , Masculino , Camundongos , Neurônios/metabolismo , Interferência de RNARESUMO
The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.
Assuntos
Caderinas/metabolismo , Moduladores GABAérgicos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Animais , Células COS , Chlorocebus aethiops , Epilepsia/genética , Feminino , Células HEK293 , Hipocampo/citologia , Humanos , Potenciais Pós-Sinápticos Inibidores , Plasticidade Neuronal , Protocaderinas , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismoRESUMO
Intracellular chloride ([Cl-]i) and pH (pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-]i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl- and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-]i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-]i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-]i in response to hypercapnia and seizure activity.
Assuntos
Cloretos/metabolismo , Citoplasma/metabolismo , Hipocampo/metabolismo , Imagem Óptica/métodos , Fótons , Células Piramidais/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Animais Recém-Nascidos , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Camundongos , Células Piramidais/citologiaRESUMO
Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases and associated downstream signalling have been strongly implicated in both neurodevelopment and autism spectrum disorders. We found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) similarly affects neuronal migration and spine density during mouse cortical development in vivo and results in impaired core behaviours related to autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signalling by decreasing FGFR2 degradation from the plasma membrane. Accordingly, FGFR2 overexpression rescues all defects due to Negr1 knockdown in vivo. Negr1 knockout mice present phenotypes similar to Negr1-downregulated animals. These data indicate that NEGR1 and FGFR2 cooperatively regulate cortical development and suggest a role for defective NEGR1-FGFR2 complex and convergent downstream ERK and AKT signalling in autism spectrum disorders.
Assuntos
Transtorno do Espectro Autista/fisiopatologia , Moléculas de Adesão Celular Neuronais/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/fisiologia , Animais , Transtorno do Espectro Autista/metabolismo , Comportamento Animal/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/metabolismo , Movimento Celular , Córtex Cerebral/crescimento & desenvolvimento , Espinhas Dendríticas/fisiologia , Modelos Animais de Doenças , Regulação para Baixo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
KCC2 is the major chloride extruder in neurons. The spatiotemporal regulation of KCC2 expression orchestrates the developmental shift towards inhibitory GABAergic drive and the formation of glutamatergic synapses. Whether KCC2's role in synapse formation is similar in different brain regions is unknown. First, we found that KCC2 subcellular localization, but not overall KCC2 expression levels, differed between cortex and hippocampus during the first postnatal week. We performed site-specific in utero electroporation of KCC2 cDNA to target either hippocampal CA1 or somatosensory cortical pyramidal neurons. We found that a premature expression of KCC2 significantly decreased spine density in CA1 neurons, while it had the opposite effect in cortical neurons. These effects were cell autonomous, because single-cell biolistic overexpression of KCC2 in hippocampal and cortical organotypic cultures also induced a reduction and an increase of dendritic spine density, respectively. In addition, we found that the effects of its premature expression on spine density were dependent on BDNF levels. Finally, we showed that the effects of KCC2 on dendritic spine were dependent on its chloride transporter function in the hippocampus, contrary to what was observed in cortex. Altogether, these results demonstrate that KCC2 regulation of dendritic spine development, and its underlying mechanisms, are brain-region specific.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Região CA1 Hipocampal/crescimento & desenvolvimento , Espinhas Dendríticas/fisiologia , Córtex Somatossensorial/crescimento & desenvolvimento , Simportadores/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Região CA1 Hipocampal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células Piramidais/fisiologia , Ratos Sprague-Dawley , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation by PKA and Cdk5 in the modulation of this process is unknown. We expressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression of WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5-dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival. Significance statement: Synapsin III is an atypical member of the synapsin family of synaptic vesicle-associated phosphoproteins that is precociously expressed in neurons and is downregulated afterward. Although experimental evidence suggests a specific role for Synapsin III in neuronal development, the molecular mechanisms are still largely unknown. We found that Synapsin III plays a central role in early stages of neuronal development involving neuronal survival, polarization, and neuritic growth and that these effects are dependent on phosphorylation by cAMP-dependent protein kinase and cyclin-dependent protein kinase-5. These results explain the recently described neurodevelopmental defects in the migration and orientation of Synapsin III-depleted cortical neurons and support the potential association of Synapsin III with neurodevelopmental disorders such as schizophrenia.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Neurônios/fisiologia , Sinapsinas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Proteínas Quinases Dependentes de AMP Cíclico/genética , Quinase 5 Dependente de Ciclina/genética , Embrião de Mamíferos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinapsinas/genética , Tubulina (Proteína)/metabolismoRESUMO
Atypical febrile seizures are considered a risk factor for epilepsy onset and cognitive impairments later in life. Patients with temporal lobe epilepsy and a history of atypical febrile seizures often carry a cortical malformation. This association has led to the hypothesis that the presence of a cortical dysplasia exacerbates febrile seizures in infancy, in turn increasing the risk for neurological sequelae. The mechanisms linking these events are currently poorly understood. Potassium-chloride cotransporter KCC2 affects several aspects of neuronal circuit development and function, by modulating GABAergic transmission and excitatory synapse formation. Recent data suggest that KCC2 downregulation contributes to seizure generation in the epileptic adult brain, but its role in the developing brain is still controversial. In a rodent model of atypical febrile seizures, combining a cortical dysplasia and hyperthermia-induced seizures (LHS rats), we found a premature and sustained increase in KCC2 protein levels, accompanied by a negative shift of the reversal potential of GABA. In parallel, we observed a significant reduction in dendritic spine size and mEPSC amplitude in CA1 pyramidal neurons, accompanied by spatial memory deficits. To investigate whether KCC2 premature overexpression plays a role in seizure susceptibility and synaptic alterations, we reduced KCC2 expression selectively in hippocampal pyramidal neurons by in utero electroporation of shRNA. Remarkably, KCC2 shRNA-electroporated LHS rats show reduced hyperthermia-induced seizure susceptibility, while dendritic spine size deficits were rescued. Our findings demonstrate that KCC2 overexpression in a compromised developing brain increases febrile seizure susceptibility and contribute to dendritic spine alterations.
Assuntos
Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Convulsões Febris/patologia , Simportadores/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Suscetibilidade a Doenças/metabolismo , Epilepsia/fisiopatologia , Transtornos da Memória/metabolismo , Neurogênese/fisiologia , Células Piramidais/metabolismo , Ratos Sprague-Dawley , Convulsões Febris/metabolismo , Convulsões Febris/fisiopatologia , Cotransportadores de K e Cl-RESUMO
A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.
Assuntos
Bioensaio/métodos , Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Animais , Biotinilação , Diferenciação Celular , Forma Celular , Células Cultivadas , Espinhas Dendríticas/metabolismo , Inativação Gênica , Células HEK293 , Humanos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese , Sinapses/metabolismoRESUMO
Down syndrome (DS) is the most common genetic cause of cognitive disability. However, it is largely unclear how triplication of a small gene subset may impinge on diverse aspects of DS brain physiopathology. Here, we took a multi-omic approach and simultaneously analyzed by RNA-seq and proteomics the expression signatures of two diverse regions of human postmortem DS brains. We found that the overexpression of triplicated genes triggered global expression dysregulation, differentially affecting transcripts, miRNAs, and proteins involved in both known and novel biological candidate pathways. Among the latter, we observed an alteration in RNA splicing, specifically modulating the expression of genes involved in cytoskeleton and axonal dynamics in DS brains. Accordingly, we found an alteration in axonal polarization in neurons from DS human iPSCs and mice. Thus, our study provides an integrated multilayer expression database capable of identifying new potential targets to aid in designing future clinical interventions for DS.
Assuntos
Encéfalo , Síndrome de Down , Síndrome de Down/metabolismo , Síndrome de Down/genética , Síndrome de Down/patologia , Humanos , Encéfalo/metabolismo , Encéfalo/patologia , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica/métodos , Masculino , Neurônios/metabolismo , Axônios/metabolismo , Axônios/patologia , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Splicing de RNA , MultiômicaRESUMO
Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However, no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages, including adulthood. Here, we showed that such mosaicism is present in wild-type (WT) and down syndrome (DS) neuronal networks, as assessed at increasing scales of complexity (cultures, brain slices, behaving mice). Nevertheless, WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover, we found heterogeneous GABAergic responses in developed control and trisomic human induced-pluripotent-stem-cells-derived neurons. Thus, a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons, possibly contributing to disease-associated behaviors.
RESUMO
Besides recent advances in neonatal care, preterm newborns still develop sex-biased behavioral alterations. Preterms fail to receive placental insulin-like growth factor-1 (IGF-1), a major fetal growth hormone in utero, and low IGF-1 serum levels correlate with preterm poor neurodevelopmental outcomes. Here, we mimicked IGF-1 deficiency of preterm newborns in mice by perinatal administration of an IGF-1 receptor antagonist. This resulted in sex-biased brain microstructural, functional, and behavioral alterations, resembling those of ex-preterm children, which we characterized performing parallel mouse/human behavioral tests. Pharmacological enhancement of GABAergic tonic inhibition by the U.S. Food and Drug Administration-approved drug ganaxolone rescued functional/behavioral alterations in mice. Establishing an unprecedented mouse model of prematurity, our work dissects the mechanisms at the core of abnormal behaviors and identifies a readily translatable therapeutic strategy for preterm brain disorders.
Assuntos
Encefalopatias , Fator de Crescimento Insulin-Like I , Estados Unidos , Criança , Humanos , Recém-Nascido , Gravidez , Feminino , Animais , Camundongos , Receptor IGF Tipo 1 , Placenta , Recém-Nascido Prematuro , Encefalopatias/tratamento farmacológicoRESUMO
The question of whether material stiffness enhances cell adhesion and clustering is still open to debate. Results from the literature are seemingly contradictory, with some reports illustrating that adhesion increases with surface stiffness and others suggesting that the performance of a system of cells is curbed by high values of elasticity. To address the role of elasticity as a regulator in neuronal cell adhesion and clustering, we investigated the topological characteristics of networks of neurons on polydimethylsiloxane (PDMS) surfaces - with values of elasticity (E) varying in the 0.55-2.65 MPa range. Results illustrate that, as elasticity increases, the number of neurons adhering on the surface decreases. Notably, the small-world coefficient - a topological measure of networks - also decreases. Numerical simulations and functional multi-calcium imaging experiments further indicated that the activity of neuronal cells on soft surfaces improves for decreasing E. Experimental findings are supported by a mathematical model, that explains adhesion and clustering of cells on soft materials as a function of few parameters - including the Young's modulus and roughness of the material. Overall, results indicate that - in the considered elasticity interval - increasing the compliance of a material improves adhesion, improves clustering, and enhances communication of neurons.
Assuntos
Adesão Celular , Elasticidade , Neurônios , Neurônios/fisiologia , Animais , Dimetilpolisiloxanos/química , Propriedades de Superfície , Módulo de Elasticidade , Células Cultivadas , RatosRESUMO
Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 2 (KCC2) lead to impaired intracellular chloride concentration in neurons and imbalanced excitation/inhibition in the brain. These alterations have been observed in several neurological disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compound ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclinical characterization of ARN23746 toward its development as a clinical candidate. ARN23746 shows an overall excellent metabolism profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the effective dose. These results further support ARN23746 as a solid candidate for clinical trial-enabling studies.
RESUMO
Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.
Assuntos
Bumetanida , Simportadores , Bumetanida/farmacologia , Cátions/metabolismo , Cloretos/metabolismo , Diuréticos/farmacologia , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Protocadherin 19 gene-related epilepsy or protocadherin 19 clustering epilepsy is an infantile-onset epilepsy syndrome characterized by psychiatric (including autism-related), sensory, and cognitive impairment of varying degrees. Protocadherin 19 clustering epilepsy is caused by X-linked protocadherin 19 protein loss of function. Due to random X-chromosome inactivation, protocadherin 19 clustering epilepsy-affected females present a mosaic population of healthy and protocadherin 19-mutant cells. Unfortunately, to date, no current mouse model can fully recapitulate both the brain histological and behavioural deficits present in people with protocadherin 19 clustering epilepsy. Thus, the search for a proper understanding of the disease and possible future treatment is hampered. By inducing a focal mosaicism of protocadherin 19 expression using in utero electroporation in rats, we found here that protocadherin 19 signalling in specific brain areas is implicated in neuronal migration, heat-induced epileptic seizures, core/comorbid behaviours related to autism and cognitive function.
RESUMO
The action of visual experience on visual cortical circuits is maximal during a critical period of postnatal development. The long-term effects of this experience are likely mediated by signaling cascades regulating experience-dependent gene transcription. Developmental modifications of these pathways could explain the difference in plasticity between the young and adult cortex. We studied the pathways linking experience-dependent activation of ERK to CREB-mediated gene expression in vivo. In juvenile mice, visual stimulation that activates CREB-mediated gene transcription also induced ERK-dependent MSK and histone H3 phosphorylation and H3-H4 acetylation, an epigenetic mechanism of gene transcription activation. In adult animals, ERK and MSK were still inducible; however, visual stimulation induced weak CREB-mediated gene expression and H3-H4 posttranslational modifications. Stimulation of histone acetylation in adult animals by means of trichostatin promoted ocular dominance plasticity. Thus, differing, experience-dependent activations of signaling molecules might be at the basis of the differences in experience-dependent plasticity between juvenile and adult cortex.
Assuntos
Histonas/metabolismo , Plasticidade Neuronal/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Córtex Visual/fisiologia , Acetilação/efeitos dos fármacos , Fatores Etários , Animais , Período Crítico Psicológico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dominância Cerebral/fisiologia , Regulação para Baixo/genética , Epigênese Genética/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Histonas/genética , Ácidos Hidroxâmicos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Estimulação Luminosa , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Córtex Visual/citologia , Córtex Visual/crescimento & desenvolvimentoRESUMO
Experience-dependent plasticity in the cortex is often higher during short critical periods in postnatal development. The mechanisms limiting adult cortical plasticity are still unclear. Maturation of intracortical GABAergic inhibition is suggested to be crucial for the closure of the critical period for ocular dominance (OD) plasticity in the visual cortex. We find that reduction of GABAergic transmission in the adult rat visual cortex partially reactivates OD plasticity in response to monocular deprivation (MD). This is accompanied by an enhancement of activity-dependent potentiation of synaptic efficacy but not of activity-dependent depression. We also found a decrease in the expression of chondroitin sulfate proteoglycans in the visual cortex of MD animals with reduced inhibition, after the reactivation of OD plasticity. Thus, intracortical inhibition is a crucial limiting factor for the induction of experience-dependent plasticity in the adult visual cortex.
Assuntos
Dominância Ocular/fisiologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Córtex Visual/fisiologia , Fatores Etários , Animais , Potenciais Evocados Visuais/fisiologia , Ratos , Ratos Long-EvansRESUMO
Cation-coupled chloride cotransporters (CCCs) modulate the transport of sodium and/or potassium cations coupled with chloride anions across the cell membrane. CCCs thus help regulate intracellular ionic concentration and consequent cell volume homeostasis. This has been largely exploited in the past to develop diuretic drugs that act on CCCs expressed in the kidney. However, a growing wealth of evidence has demonstrated that CCCs are also critically involved in a great variety of other pathologies, motivating most recent drug discovery programs targeting CCCs. Here, we examine the structure-function relationship of CCCs. By linking recent high-resolution cryogenic electron microscopy (cryo-EM) data with older biochemical/functional studies on CCCs, we discuss the mechanistic insights and opportunities to design selective CCC modulators to treat diverse pathologies.
RESUMO
The chloride importer NKCC1 and the chloride exporter KCC2 are key regulators of neuronal chloride concentration. A defective NKCC1/KCC2 expression ratio is associated with several brain disorders. Preclinical/clinical studies have shown that NKCC1 inhibition by the United States FDA-approved diuretic bumetanide is a potential therapeutic strategy in preclinical/clinical studies of multiple neurological conditions. However, bumetanide has poor brain penetration and causes unwanted diuresis by inhibiting NKCC2 in the kidney. To overcome these issues, a growing number of studies have reported more brain-penetrating and/or selective bumetanide prodrugs, analogs, and new molecular entities. Here, we review the evidence for NKCC1 pharmacological inhibition as an effective strategy to manage neurological disorders. We also discuss the advantages and limitations of bumetanide repurposing and the benefits and risks of new NKCC1 inhibitors as therapeutic agents for brain disorders.