RESUMEN
Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.
RESUMEN
Mutations in the TM4SF2 gene, which encodes TSPAN7, cause a severe form of intellectual disability (ID) often comorbid with autism spectrum disorder (ASD). Recently, we found that TM4SF2 loss in mice affects cognition. Here, we report that Tm4sf2-/y mice, beyond an ID-like phenotype, display altered sociability, increased repetitive behaviors, anhedonic- and depressive-like states. Cognition relies on the integration of information from several brain areas. In this context, the lateral habenula (LHb) is strategically positioned to coordinate the brain regions involved in higher cognitive functions. Furthermore, in Tm4sf2-/y mice we found that LHb neurons present hypoexcitability, aberrant neuronal firing pattern and altered sodium and potassium voltage-gated ion channels function. Interestingly, we also found a reduced expression of voltage-gated sodium channel and a hyperactivity of the PKC-ERK pathway, a well-known modulator of ion channels activity, which might explain the functional phenotype showed by Tm4sf2-/y mice LHb neurons. These findings support Tm4sf2-/y mice as useful in modeling some ASD-like symptoms. Additionally, we can speculate that LHb functional alteration in Tm4sf2-/y mice might play a role in the disease pathophysiology.
Asunto(s)
Habénula/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/genética , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Anhedonia , Animales , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/fisiopatología , Depresión , Modelos Animales de Enfermedad , Habénula/fisiopatología , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/fisiopatología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/fisiopatología , Proteína Quinasa C/metabolismo , Conducta Social , Conducta EstereotipadaRESUMEN
Tetraspanins are a family of proteins largely expressed in mammals. These proteins share very similar structures and are involved in several biological processes spanning from the immune system to cancer growth regulation. Moreover, tetraspanins are scaffold proteins that are able to interact with each other and with a subset of proteins involved in the regulation of the central nervous system, including synapse formation, function and plasticity. In this review, we will focus on the analysis of the literature on tetraspanins, highlighting their involvement in synapse formation and function through direct or indirect modulation of synaptic proteins.
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Sinapsis/metabolismo , Tetraspaninas/metabolismo , Animales , Humanos , Plasticidad Neuronal , Transporte de Proteínas , Receptores de Neurotransmisores/metabolismo , Sinapsis/fisiología , Tetraspaninas/genéticaRESUMEN
Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.
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Proteínas del Citoesqueleto/genética , Proteínas Activadoras de GTPasa/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Discapacidad Intelectual/genética , Proteínas Nucleares/genética , Animales , Dendritas/efectos de los fármacos , Dendritas/genética , Dendritas/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Enfermedades Genéticas Ligadas al Cromosoma X/tratamiento farmacológico , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Humanos , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/patología , Interneuronas/efectos de los fármacos , Interneuronas/patología , Ratones Noqueados , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/patología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.
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Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Psicotrópicos/farmacología , Receptores AMPA/metabolismo , Regulación Alostérica , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/ultraestructura , Discapacidad Intelectual/patología , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
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Quinasa del Factor 2 de Elongación/metabolismo , Epilepsia/enzimología , Neuronas/enzimología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Epilepsia/patología , Miedo/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The capacity to guarantee the proper excitatory/inhibitory balance is one of the most critical steps during early development responsible for the correct brain organization, function, and plasticity. GABAergic neurons guide this process leading to the right structural organization, brain circuitry, and neuronal firing. Here, we identified the ataxia telangiectasia mutated (ATM), a serine/threonine protein kinase linked to DNA damage response, as crucial in regulating neurotransmission. We found that reduced levels of ATM in the hippocampal neuronal cultures produce an excitatory/inhibitory unbalance toward inhibition as indicated by the higher frequency of miniature inhibitory postsynaptic current events and an increased number of GABAergic synapses. In vivo, the increased inhibition still persists and, even if a higher excitation is also present, a reduced neuronal excitability is found as indicated by the lower action potential frequency generated in response to high-current intensity stimuli. Finally, we found an elevated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in heterozygous hippocampi associated with lower expression levels of the ERK1/2 phosphatase PP1. Given that the neurodegenerative condition associated with genetic mutations in the Atm gene, ataxia telangiectasia, presents a variable phenotype with impairment in cognition, our molecular findings provide a logical frame for a more clear comprehension of cognitive defects in the pathology, opening to novel therapeutic strategies.
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Hipocampo/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipocampo/citología , Hipocampo/embriología , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibición Neural/fisiología , Neuronas/citología , Fosforilación , Simportadores/metabolismo , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/administración & dosificación , Cotransportadores de K ClRESUMEN
BACKGROUND: Loss of function mutations in PCDH19 gene causes an X-linked, infant-onset clustering epilepsy, associated with intellectual disability and autistic features. The unique pattern of inheritance includes random X-chromosome inactivation, which leads to pathological tissue mosaicism. Females carrying PCDH19 mutations are affected, while males have a normal phenotype. No cure is presently available for this disease. METHODS: Fibroblasts from a female patient carrying frameshift mutation were reprogrammed into human induced pluripotent stem cells (hiPSCs). To create a cell model of PCDH19-clustering epilepsy (PCDH19-CE) where both cell populations co-exist, we created mosaic neurons by mixing wild-type (WT) and mutated (mut) hiPSC clones, and differentiated them into mature neurons with overexpression of the transcriptional factor Neurogenin 2. RESULTS: We generated functional neurons from patient-derived iPSC using a rapid and efficient method of differentiation through overexpression of Neurogenin 2. Was revealed an accelerated maturation and higher arborisation in the mutated neurons, while the mosaic neurons showed the highest frequency of action potential firing and hyperexcitability features, compared to mutated and WT neurons. CONCLUSIONS: Our findings provide evidence that PCDH19 c.2133delG mutation affects proper metaphases with increased numbers of centrosomes in stem cells and accelerates neuronal maturation in premature cells. PCDH19 mosaic neurons showed elevated excitability, representing the situation in PCDH19-CE brain. We suggest Ngn2 hiPSC-derived PCDH19 neurons as an informative experimental tool for understanding the pathogenesis of PCDH19-CE and a suitable approach for use in targeted drug screening strategies.
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Epilepsia , Células Madre Pluripotentes Inducidas , Masculino , Humanos , Femenino , Cadherinas/genética , Protocadherinas , Epilepsia/genética , Mutación , Análisis por ConglomeradosRESUMEN
Interest in the function of ataxia-telangiectasia-mutated protein (ATM) is extensively growing as evidenced by preclinical studies that continuously link ATM with new intracellular pathways. Here, we exploited Atm+/- and Atm-/- mice and demonstrate that cognitive defects are rescued by the delivery of the antidepressant Fluoxetine (Fluox). Fluox increases levels of the chloride intruder NKCC1 exclusively at hippocampal level suggesting an ATM context-specificity. A deeper investigation of synaptic composition unveils increased Gluk-1 and Gluk-5 subunit-containing kainate receptors (KARs) levels in the hippocampus, but not in the cortex, of Atm+/- and Atm-/- mice. Analysis of postsynaptic fractions and confocal studies indicates that KARs are presynaptic while in vitro and ex vivo electrophysiology that are fully active. These changes are (i) linked to KCC2 activity, as the KCC2 blockade in Atm+/- developing neurons results in reduced KARs levels and (ii) developmental regulated. Indeed, the pharmacological inhibition of ATM kinase in adults produces different changes as identified by RNA-seq investigation. Our data display how ATM affects both inhibitory and excitatory neurotransmission, extending its role to a variety of neurological and psychiatric disorders.
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Proteínas de la Ataxia Telangiectasia Mutada , Hipocampo , Simportadores , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Receptores de Ácido Kaínico , Simportadores/genética , Simportadores/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.
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Cadherinas , Epilepsia , Genes Inmediatos-Precoces , Protocadherinas , Cadherinas/genética , Cadherinas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Regulación de la Expresión Génica , Humanos , Protocadherinas/genética , Protocadherinas/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de SeñalRESUMEN
Rho GTPases are a class of G-proteins involved in several aspects of cellular biology, including the regulation of actin cytoskeleton. The most studied members of this family are RHOA and RAC1 that act in concert to regulate actin dynamics. Recently, Rho GTPases gained much attention as synaptic regulators in the mammalian central nervous system (CNS). In this context, ARHGAP22 protein has been previously shown to specifically inhibit RAC1 activity thus standing as critical cytoskeleton regulator in cancer cell models; however, whether this function is maintained in neurons in the CNS is unknown. Here, we generated a knockout animal model for arhgap22 and provided evidence of its role in the hippocampus. Specifically, we found that ARHGAP22 absence leads to RAC1 hyperactivity and to an increase in dendritic spine density with defects in synaptic structure, molecular composition, and plasticity. Furthermore, arhgap22 silencing causes impairment in cognition and a reduction in anxiety-like behavior in mice. We also found that inhibiting RAC1 restored synaptic plasticity in ARHGAP22 KO mice. All together, these results shed light on the specific role of ARHGAP22 in hippocampal excitatory synapse formation and function as well as in learning and memory behaviors.
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Cognición/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Sinapsis/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Ansiedad/genética , Ansiedad/metabolismo , Conducta Animal/fisiología , Espinas Dendríticas/metabolismo , Proteínas Activadoras de GTPasa/genética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Plasticidad Neuronal/genética , Neuropéptidos/genética , Sinapsis/genética , Sinaptosomas/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
Impairment of the GABAergic system has been reported in epilepsy, autism, attention deficit hyperactivity disorder, and schizophrenia. We recently demonstrated that ataxia telangiectasia mutated (ATM) directly shapes the development of the GABAergic system. Here, we show for the first time to our knowledge how the abnormal expression of ATM affects the pathological condition of autism. We exploited 2 different animal models of autism, the methyl CpG binding protein 2-null (Mecp2y/-) mouse model of Rett syndrome and mice prenatally exposed to valproic acid, and found increased ATM levels. Accordingly, treatment with the specific ATM kinase inhibitor KU55933 (KU) normalized molecular, functional, and behavioral defects in these mouse models, such as (a) delayed GABAergic development, (b) hippocampal hyperexcitability, (c) low cognitive performances, and (d) social impairments. Mechanistically, we demonstrate that KU administration to WT hippocampal neurons leads to (a) higher early growth response 4 activity on Kcc2b promoter, (b) increased expression of Mecp2, and (c) potentiated GABA transmission. These results provide evidence and molecular substrates for the pharmacological development of ATM inhibition in autism spectrum disorders.
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Trastorno del Espectro Autista/tratamiento farmacológico , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Trastorno del Espectro Autista/fisiopatología , Trastorno del Espectro Autista/psicología , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Reparación del ADN , Modelos Animales de Enfermedad , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG/deficiencia , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Embarazo , Efectos Tardíos de la Exposición Prenatal , Inhibidores de Proteínas Quinasas/farmacología , Pironas/farmacología , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/fisiopatología , Síndrome de Rett/psicología , Simportadores/genética , Simportadores/metabolismo , Ácido Valproico/toxicidad , Cotransportadores de K ClRESUMEN
Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABA(A)-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABA(A)-Rs, and a tonic component mediated by extrasynaptic GABA(A)-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3alpha,5alpha-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the delta subunit of the GABA(A)-R and a concomitant decrease in that of the gamma(2) subunit in the hippocampus at P19. Expression of the alpha(4) subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5alpha-reductase inhibitor finasteride prevented the changes in tonic current and in delta and gamma(2) subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABA(A)-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/fisiología , Periodo Posparto/fisiología , Preñez/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Animales , Femenino , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Embarazo , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/biosíntesisRESUMEN
PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons from embryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.
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Potenciales de Acción , Cadherinas/metabolismo , Epilepsia/metabolismo , Epilepsia/fisiopatología , Hipocampo/patología , Inhibición Neural/fisiología , Neuronas/patología , Receptores de GABA-A/metabolismo , Animales , Regulación hacia Abajo , Cinética , ARN Interferente Pequeño/metabolismo , Ratas Sprague-DawleyRESUMEN
Homeostatic and hedonic pathways distinctly interact to control food intake. Dysregulations of circuitries controlling hedonic feeding may disrupt homeostatic mechanisms and lead to eating disorders. The anorexigenic peptides nucleobindin-2 (NUCB2)/nesfatin-1 may be involved in the interaction of these pathways. The endogenous levels of this peptide are regulated by the feeding state, with reduced levels following fasting and normalized by refeeding. The fasting state is associated with biochemical and behavioral adaptations ultimately leading to enhanced sensitization of reward circuitries towards food reward. Although NUCB2/nesfatin-1 is expressed in reward-related brain areas, its role in regulating motivation and preference for nutrients has not yet been investigated. We here report that both dopamine and GABA neurons express NUCB2/nesfatin-1 in the VTA. Ex vivo electrophysiological recordings show that nesfatin-1 hyperpolarizes dopamine, but not GABA, neurons of the VTA by inducing an outward potassium current. In vivo, central administration of nesfatin-1 reduces motivation for food reward in a high-effort condition, sucrose intake and preference. We next adopted a 2-bottle choice procedure, whereby the reward value of sucrose was compared with that of a reference stimulus (sucralose + optogenetic stimulation of VTA dopamine neurons) and found that nesfatin-1 fully abolishes the fasting-induced increase in the reward value of sucrose. These findings indicate that nesfatin-1 reduces energy intake by negatively modulating dopaminergic neuron activity and, in turn, hedonic aspects of food intake. Since nesfatin-1´s actions are preserved in conditions of leptin resistance, the present findings render the NUCB2/nesfatin-1 system an appealing target for the development of novel therapeutical treatments towards obesity.
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Proteínas de Unión al Calcio , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Motivación , Proteínas del Tejido Nervioso/metabolismo , Nucleobindinas , RecompensaRESUMEN
Tetraspanins are a class of evolutionarily conserved transmembrane proteins with 33 members identified in mammals that have the ability to organize specific membrane domains, named tetraspanin-enriched microdomains (TEMs). Despite the relative abundance of different tetraspanins in the CNS, few studies have explored their role at synapses. Here, we investigate the function of TSPAN5, a member of the tetraspanin superfamily for which mRNA transcripts are found at high levels in the mouse brain. We demonstrate that TSPAN5 is localized in dendritic spines of pyramidal excitatory neurons and that TSPAN5 knockdown induces a dramatic decrease in spine number because of defects in the spine maturation process. Moreover, we show that TSPAN5 interacts with the postsynaptic adhesion molecule neuroligin-1, promoting its correct surface clustering. We propose that membrane compartmentalization by tetraspanins represents an additional mechanism for regulating excitatory synapses.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Espinas Dendríticas/metabolismo , Microdominios de Membrana/metabolismo , Tetraspaninas/química , Tetraspaninas/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Hipocampo/metabolismo , Humanos , Ratones Endogámicos C57BL , Unión Proteica , Células Piramidales/metabolismo , Ratas Wistar , Sinapsis/metabolismoRESUMEN
Neurodevelopmental disorders (NDDs) are a group of diseases whose symptoms arise during childhood or adolescence and that impact several higher cognitive functions such as learning, sociability and mood. Accruing evidence suggests that a shared pathogenic mechanism underlying these diseases is the dysfunction of glutamatergic synapses. We summarize present knowledge on autism spectrum disorders (ASD), intellectual disability (ID), Down syndrome (DS), Rett syndrome (RS) and attention-deficit hyperactivity disorder (ADHD), highlighting the involvement of glutamatergic synapses and receptors in these disorders. The most commonly shared defects involve α-amino-3-hydroxy-5-methyl- 4-isoxazole propionic acid receptors (AMPARs), N-methyl-d-aspartate receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), whose functions are strongly linked to synaptic plasticity, affecting both cell-autonomous features as well as circuit formation. Moreover, the major scaffolding proteins and, thus, the general structure of the synapse are often deregulated in neurodevelopmental disorders, which is not surprising considering their crucial role in the regulation of glutamate receptor positioning and functioning. This convergence of defects supports the definition of neurodevelopmental disorders as a continuum of pathological manifestations, suggesting that glutamatergic synapses could be a therapeutic target to ameliorate patient symptomatology.
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Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Receptores de Glutamato/metabolismo , Sinapsis/patología , Animales , HumanosRESUMEN
The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15-/- mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.
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Dendritas/genética , Neuritas/fisiología , Neuropéptidos/genética , Células Piramidales/fisiología , Proteína de Unión al GTP rac1/genética , Actinas/genética , Actinas/metabolismo , Animales , Axones/metabolismo , Proteínas Activadoras de GTPasa/genética , Conos de Crecimiento/metabolismo , Mutación con Pérdida de Función/genética , Ratones , Neuritas/metabolismo , Fosforilación , Células Piramidales/metabolismo , Transducción de Señal/genéticaRESUMEN
Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.
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Hipocampo/metabolismo , Proteínas de Microfilamentos/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Transmisión Sináptica/genética , Animales , Giro Dentado/metabolismo , Giro Dentado/patología , Giro Dentado/ultraestructura , Embrión de Mamíferos , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patología , Regulación de la Expresión Génica , Células HEK293 , Hipocampo/patología , Hipocampo/ultraestructura , Humanos , Inyecciones Intraventriculares , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural , Neurogénesis/genética , Neuronas/patología , Neuronas/ultraestructura , Cultivo Primario de Células , Ratas , Ratas Wistar , Receptor Cross-Talk , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinapsis/metabolismo , Sinapsis/patología , Sinapsis/ultraestructuraRESUMEN
Thiocolchicoside (TCC) is used clinically for its muscle relaxant, anti-inflammatory, and analgesic properties, and it has been shown to interact with gamma-aminobutyric acid (GABA) type A receptors (GABAARs) and strychnine-sensitive glycine receptors in the rat central nervous system. In contrast to a proposed agonistic action at these two types of inhibitory receptors, pharmacological evidence has shown that, under certain conditions, TCC manifests convulsant activity in animals and humans. We now show that the phasic and tonic GABAAR-mediated currents recorded from Purkinje cells and granule neurons, respectively, in parasagittal cerebellar slices from adult male rats were inhibited by TCC in a concentration-dependent manner. The median inhibitory concentrations of TCC for these effects were approximately 0.15 and approximately 0.9 microM, respectively. TCC did not potentiate GABABR-mediated currents in hippocampal slices, suggesting that its muscle relaxant action is not mediated by GABABRs. Intraperitoneal injection of TCC in rats either alone or in combination with negative modulators of GABAergic transmission revealed convulsant and proconvulsant actions of this drug. Our data, consistent with clinical observations of the epileptogenic effect of this compound, suggest that TCC is a potent competitive antagonist of GABAAR function.