RESUMEN
Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.
Asunto(s)
Mutación Missense , Receptores de Ácido Kaínico , Masculino , Femenino , Humanos , Ratones , Animales , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/metabolismo , Neuronas/fisiología , Hipocampo/fisiología , Células Piramidales/fisiologíaRESUMEN
Seizures are a frequent complication of adult-type diffuse gliomas, and are often difficult to control with medications. Gliomas with mutations in isocitrate dehydrogenase 1 or 2 (IDHmut) are more likely than IDH-wild type (IDHwt) gliomas to cause seizures as part of their initial clinical presentation. However, whether IDHmut is also associated with seizures during the remaining disease course, and whether IDHmut inhibitors can reduce seizure risk, are unclear. Clinical multivariable analyses showed that preoperative seizures, glioma location, extent of resection, and glioma molecular subtype (including IDHmut status) all contributed to postoperative seizure risk in adult-type diffuse glioma patients, and that postoperative seizures were often associated with tumor recurrence. Experimentally, the metabolic product of IDHmut, d-2-hydroxyglutarate, rapidly synchronized neuronal spike firing in a seizure-like manner, but only when non-neoplastic glial cells were present. In vitro and in vivo models recapitulated IDHmut glioma-associated seizures, and IDHmut inhibitors currently being evaluated in glioma clinical trials inhibited seizures in those models, independent of their effects on glioma growth. These data show that postoperative seizure risk in adult-type diffuse gliomas varies in large part by molecular subtype, and that IDHmut inhibitors could play a key role in mitigating such risk in IDHmut glioma patients.
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Neoplasias Encefálicas , Glioma , Adulto , Humanos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Recurrencia Local de Neoplasia , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Progresión de la Enfermedad , Isocitrato Deshidrogenasa/genética , MutaciónRESUMEN
Perampanel (PMP) is a third generation antiseizure drug reported to be a potent and selective noncompetitive negative allosteric modulator of one sub-family of ionotropic glutamate receptor (iGluR), the α-amino-3-hydroxy-S-methylisoxazole-4-propionic acid receptors (AMPARs). However, the recent structural resolution of AMPARs in complex with PMP revealed that its binding pocket is formed from residues that are largely conserved in two members of another family of iGluRs, the GluK4 and GluK5 kainate receptor (KAR) subunits. We show here that PMP inhibits both recombinant and neuronal KARs, contrary to the previous reports, and that the NAM activity requires GluK5 subunits to be channel constituents. PMP inhibited heteromeric GluK1/GluK5 and GluK2/GluK5 KARs at IC50 values comparable to that for AMPA receptors but was much less potent on homomeric GluK1 or GluK2 KARs. The auxiliary subunits Neto1 or Neto2 also made GluK2-containing KARs more sensitive to inhibition. Finally, PMP inhibited mouse neuronal KARs containing GluK5 subunits and Neto proteins in nociceptive dorsal root ganglia neurons and hippocampal mossy fiber - CA3 pyramidal neuron synapses. These data suggest that clinical actions of PMP could arise from differential inhibition of AMPAR or KAR signaling and that more selective drugs might maintain antiseizure efficacy while reducing adverse effects.Significance Statement:Perampanel (PMP) is a regulatory approved antiseizure drug used for refractory partial-onset and generalized tonic-clonic seizures that acts as a selective negative allosteric modulator of α-amino-3-hydroxy-S-methylisoxazole-4-propionic acid receptors (AMPARs). Here we demonstrate that PMP inhibits kainate receptors (KARs), a second family of ionotropic glutamate receptors, in addition to AMPARs. NAM activity on KARs required GluK5 subunits or Neto auxiliary subunits as channel constituents. KAR inhibition therefore could contribute to PMP antiseizure action or the adverse effects that are significant with this drug. Drug discovery aimed at more selective allosteric modulators that discriminate between AMPARs and KARs could yield next-generation drugs with improved therapeutic profiles for treatment of epilepsy.
RESUMEN
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Asunto(s)
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Animales , Sistema Nervioso Central , Ácido Glutámico , Humanos , Neurotransmisores , Receptores Ionotrópicos de Glutamato/genéticaRESUMEN
Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite's satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.
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Dendritas/metabolismo , Aparato de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Autoantígenos/metabolismo , Proliferación Celular , Retículo Endoplásmico/metabolismo , Glicosilación , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Polisacáridos/metabolismo , Proteoma/metabolismo , Ratas , Receptores Nicotínicos/metabolismoRESUMEN
Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.
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Encéfalo/metabolismo , Discapacidades del Desarrollo/genética , Epilepsia/genética , Discapacidad Intelectual/genética , Mutación , Receptores de Ácido Kaínico/genética , Adolescente , Adulto , Alelos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , Preescolar , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Epilepsia/diagnóstico por imagen , Epilepsia/metabolismo , Epilepsia/patología , Potenciales Evocados/fisiología , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Heterocigoto , Homocigoto , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Activación del Canal Iónico , Masculino , Modelos Moleculares , Neuronas/metabolismo , Neuronas/patología , Conformación Proteica , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
Dravet syndrome (DS) is a developmental and epileptic encephalopathy with an increased incidence of sudden death. Evidence of interictal breathing deficits in DS suggests that alterations in subcortical projections to brainstem nuclei may exist, which might be driving comorbidities in DS. The aim of this study was to determine whether a subcortical structure, the bed nucleus of the stria terminalis (BNST) in the extended amygdala, is activated by seizures, exhibits changes in excitability, and expresses any alterations in neurons projecting to a brainstem nucleus associated with respiration, stress response, and homeostasis. Experiments were conducted using F1 mice generated by breeding 129.Scn1a+/- mice with wild-type C57BL/6J mice. Immunohistochemistry was performed to quantify neuronal c-fos activation in DS mice after observed spontaneous seizures. Whole-cell patch-clamp and current-clamp electrophysiology recordings were conducted to evaluate changes in intrinsic and synaptic excitability in the BNST. Spontaneous seizures in DS mice significantly enhanced neuronal c-fos expression in the BNST. Further, the BNST had altered AMPA/NMDA postsynaptic receptor composition and showed changes in spontaneous neurotransmission, with greater excitation and decreased inhibition. BNST to parabrachial nucleus (PBN) projection neurons exhibited intrinsic excitability in wild-type mice, while these projection neurons were hypoexcitable in DS mice. The findings suggest that there is altered excitability in neurons of the BNST, including BNST-to-PBN projection neurons, in DS mice. These alterations could potentially be driving comorbid aspects of DS outside of seizures, including respiratory dysfunction and sudden death.
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Epilepsias Mioclónicas , Núcleos Parabraquiales , Amígdala del Cerebelo , Animales , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.1 , Transmisión SinápticaRESUMEN
Store-operated Orai1 calcium channels function as highly Ca2+-selective ion channels and are broadly expressed in many tissues including the central nervous system, but their contributions to cognitive processing are largely unknown. Here, we report that many measures of synaptic, cellular, and behavioral models of learning are markedly attenuated in mice lacking Orai1 in forebrain excitatory neurons. Results with focal glutamate uncaging in hippocampal neurons support an essential role of Orai1 channels in amplifying NMDA-receptor-induced dendritic Ca2+ transients that drive activity-dependent spine morphogenesis and long-term potentiation at Schaffer collateral-CA1 synapses. Consistent with these signaling roles, mice lacking Orai1 in pyramidal neurons (but not interneurons) exhibit striking deficits in working and associative memory tasks. These findings identify Orai1 channels as essential regulators of dendritic spine Ca2+ signaling, synaptic plasticity, and cognition.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Espinas Dendríticas/metabolismo , Ácido Glutámico/metabolismo , Animales , Hipocampo/metabolismo , Memoria , Ratones , Proteína ORAI1 , Células Piramidales/metabolismo , Transducción de SeñalRESUMEN
Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in preclinical and clinical studies. Their therapeutic window is narrow, however, and clinical application will likely only occur if greater discrimination in activity is achieved. Toward that end, we compared the modulatory activity of two PAMs recently considered as clinical candidates, LY451395 (mibampator) and PF-04958242/BIIB104, on recombinant and native AMPA receptors (AMPARs). We found that the principle molecular determinant that shaped modulatory activity of both PAMs on deactivation (recombinant) and decay (synaptic) of AMPARs was the auxiliary protein incorporated into the receptor complexes. AMPARs containing the stargazin/γ2 transmembrane AMPAR regulatory protein (TARP) were slowed to a >10-fold degree by both PAMs as compared with those incorporating γ8 TARP. Neither subunit composition nor flip/flop splice variation had substantive effect. Similarly, stargazin/γ2-containing mossy fiber EPSCs in cerebellar granule neurons were slowed to a â¼5-fold greater degree than EPSCs in hippocampal CA1 pyramidal cell neurons, which express the γ8 TARP. LY451395 exhibited greater efficacy than BIIB104 at both synapses. These studies provide insight into the receptor constituents that determine efficacy of sulfonamide PAMs. We conclude that compounds that discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/γ2 efficacy such as BIIB104, could act as viable procognitive therapeutics. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of AMPA receptors enhance cognitive function in a variety of preclinical models. A clearer understanding of the critical determinants of PAM activity could yield critical insight into pathways to maximize their therapeutic index. Here we show that auxiliary proteins for AMPARs play a major, but thus far underappreciated, role in shaping recombinant and neuronal AMPAR modulation by two clinical candidate PAMs. These data will inform both clinical outcomes as well as future rational development of new modulators.
Asunto(s)
Proteínas de la Membrana/metabolismo , Receptores AMPA/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Compuestos de Bifenilo/farmacología , Cerebelo/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Multimerización de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Resultado del Tratamiento , Triazoles/farmacologíaRESUMEN
Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser237 of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser237 is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b-HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.
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Epilepsia del Lóbulo Temporal/metabolismo , Proteínas de la Membrana/metabolismo , Peroxinas/metabolismo , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Dendritas/metabolismo , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Activación del Canal Iónico , Ácido Kaínico , Proteínas de la Membrana/química , Ratones Endogámicos C57BL , Peroxinas/química , Fosforilación , Fosfoserina/metabolismo , Subunidades de Proteína/química , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4+ and CD8+ T cells. Significantly, proinflammatory (IL-17- and GM-CSF-producing) γδ T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both γδ T cell- and IL-17RA-deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.
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Epilepsia Refractaria/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T Reguladores/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/metabolismo , Encéfalo/patología , Supervivencia Celular/efectos de los fármacos , Niño , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Epilepsia Refractaria/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Inmunidad Innata/efectos de los fármacos , Inflamación/patología , Interleucina-17/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Hilar mossy cells (HMCs) in the hippocampus receive glutamatergic input from dentate granule cells (DGCs) via mossy fibers (MFs) and back-projections from CA3 pyramidal neuron collateral axons. Many fundamental features of these excitatory synapses have not been characterized in detail despite their potential relevance to hippocampal cognitive processing and epilepsy-induced adaptations in circuit excitability. In this study, we compared pre- and postsynaptic parameters between MF and CA3 inputs to HMCs in young and adult mice of either sex and determined the relative contributions of the respective excitatory inputs during in vitro and in vivo models of hippocampal hyperexcitability. The two types of excitatory synapses both exhibited a modest degree of short-term plasticity, with MF inputs to HMCs exhibiting lower paired-pulse (PP) and frequency facilitation than was described previously for MF-CA3 pyramidal cell synapses. MF-HMC synapses exhibited unitary excitatory synaptic currents (EPSCs) of larger amplitude, contained postsynaptic kainate receptors, and had a lower NMDA/AMPA receptor ratio compared to CA3-HMC synapses. Pharmacological induction of hippocampal hyperexcitability in vitro transformed the abundant but relatively weak CA3-HMC connections to very large amplitude spontaneous bursts of compound EPSCs (cEPSCs) in young mice (â¼P20) and, to a lesser degree, in adult mice (â¼P70). CA3-HMC cEPSCs were also observed in slices prepared from mice with spontaneous seizures several weeks after intrahippocampal kainate injection. Strong excitation of HMCs during synchronous CA3 activity represents an avenue of significant excitatory network generation back to DGCs and might be important in generating epileptic networks.
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Fibras Musgosas del Hipocampo/fisiología , Vías Nerviosas/fisiología , Plasticidad Neuronal/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Animales , Región CA3 Hipocampal/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Transmisión Sináptica/fisiologíaRESUMEN
Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by CDH10 within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions.SIGNIFICANCE STATEMENT The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.
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Cadherinas/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Sinapsis/metabolismo , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Unión Proteica/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4ß2-type nicotinic receptors (α4ß2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4ß2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4ß2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4ß2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4ß2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.
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Agonistas Nicotínicos/metabolismo , Receptores Nicotínicos/metabolismo , Cese del Hábito de Fumar , Vareniclina/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Nicotina/metabolismo , Regulación hacia ArribaRESUMEN
KEY POINTS: Ionotropic glutamate receptor (iGluR) subunits are N-glycosylated at 4-12 sites, and Golgi processing produces mature receptors that contain high-mannose, hybrid and complex oligosaccharides. N-glycosylation is crucial for receptor biogenesis, influences receptor trafficking and provides a binding site for carbohydrate binding proteins. Glycan moieties are large, polar and occasionally charged, and they are attached at sites along iGluRs that position them for involvement in the structural changes underlying gating. Altering glycan content on kainate receptors (KARs), a subfamily of iGluRs, changes functional properties of the receptor, such as desensitization, recovery from desensitization and deactivation. We report the first observation that the charged trisaccharide HNK-1 is conjugated to native KARs, and we find that it substantially alters recombinant KAR functional properties. Our results show that the molecular composition of N-glycans can influence KAR biophysical properties, revealing a potential mechanism for fine-tuning the function of these receptors. ABSTRACT: Ionotropic glutamate receptors (iGluRs) are tetrameric proteins with between four and 12 consensus sites for N-glycosylation on each subunit, which potentially allows for a high degree of structural diversity conferred by this post-translational modification. N-glycosylation is required for proper folding of iGluRs in mammalian cells, although the impact of oligosaccharides on the function of successfully folded receptors is less clear. Glycan moieties are large, polar, occasionally charged and mediate many protein-protein interactions throughout the nervous system. Additionally, they are attached at sites along iGluR subunits that position them for involvement in the structural changes underlying gating. In the present study, we show that altering glycan content on kainate receptors (KARs) changes the functional properties of the receptors in a manner dependent on the identity of both the modified sugars and the subunit composition of the receptor to which they are attached. We also report that native KARs carry the complex capping oligosaccharide human natural killer-1. Glycosylation patterns probably differ between cell types, across development or with pathologies, and thus our findings reveal a potential mechanism for context-specific fine-tuning of KAR function through diversity in glycan structure.
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Polisacáridos/química , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/fisiología , Alcaloides/farmacología , Animales , Femenino , Glicosilación , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Ácido Kaínico/genética , Swainsonina/farmacología , alfa-Manosidasa/antagonistas & inhibidoresRESUMEN
OBJECTIVE: To identify inherited or de novo mutations associated with a suite of neurodevelopmental abnormalities in a 10-year-old patient displaying ataxia, motor and speech delay, and intellectual disability. METHODS: We performed whole-exome sequencing of the proband and her parents. A pathogenic gene variant was identified as damaging based on sequence conservation, gene function, and association with disorders having similar phenotypic profiles. Functional characterization of the mutated protein was performed in vitro using a heterologous expression system. RESULTS: A single de novo point mutation in the GRIK2 gene was identified as causative for the neurologic symptoms of the proband. The mutation is predicted to change a codon for alanine to that of a threonine at position 657 (A657T) in the GluK2 kainate receptor (KAR) subunit, a member of the ionotropic glutamate receptor gene family. Whole-cell voltage-clamp recordings revealed that KARs incorporating the GluK2(A657T) subunits show profoundly altered channel gating and are constitutively active in nominally glutamate-free extracellular media. CONCLUSIONS: In this study, we associate a de novo gain-of-function mutation in the GRIK2 gene with deficits in motor and higher order cognitive function. These results suggest that disruption of physiologic KAR function precludes appropriate development of the nervous system.
RESUMEN
Kainate receptors are members of the glutamate receptor family that regulate synaptic function in the brain. They modulate synaptic transmission and the excitability of neurons; however, their contributions to neural circuits that underlie behavior are unclear. To understand the net impact of kainate receptor signaling, we generated knockout mice in which all five kainate receptor subunits were ablated (5ko). These mice displayed compulsive and perseverative behaviors, including over-grooming, as well as motor problems, indicative of alterations in striatal circuits. There were deficits in corticostriatal input to spiny projection neurons (SPNs) in the dorsal striatum and correlated reductions in spine density. The behavioral alterations were not present in mice only lacking the primary receptor subunit expressed in adult striatum (GluK2 KO), suggesting that signaling through multiple receptor types is required for proper striatal function. This demonstrates that alterations in striatal function dominate the behavioral phenotype in mice without kainate receptors.
Asunto(s)
Enfermedades Cerebelosas/genética , Enfermedades Cerebelosas/metabolismo , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/metabolismo , Animales , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2-/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth.SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2) is also expressed in DRG. We show here that it is a developmentally downregulated but dynamic component of KARs in these neurons, that it contributes to regulated neurite regrowth in adult neurons, and that it is increased in adult mice after nerve injury. Our data confirm Neto2 as a KAR auxiliary subunit and expand our knowledge of the molecular composition of KARs in nociceptive neurons, a key piece in understanding the mechanistic contribution of KAR signaling to pain-processing circuits.