RESUMEN
OBJECTIVE: γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are major causes of various epilepsy syndromes, including severe kinds such as Dravet syndrome. Although the GABAA receptor is a major target for antiseizure medications, treating GABAA receptor mutations with receptor channel modulators is ineffective. Here, we determined the effect of a novel treatment with 4-phenylbutyrate (PBA) in Gabrg2+/Q390X knockin mice associated with Dravet syndrome. METHODS: We used biochemistry in conjunction with differential tagging of the wild-type and the mutant alleles, live brain slice surface biotinylation, microsome isolation, patch-clamp whole-cell recordings, and video-monitoring synchronized electroencephalographic (EEG) recordings in Gabrg2+/Q390X mice to determine the effect of PBA in vitro with recombinant GABAA receptors and in vivo with knockin mice. RESULTS: We found that PBA reduced the mutant γ2(Q390X) subunit protein aggregates, enhanced the wild-type GABAA receptor subunits' trafficking, and increased the membrane expression of the wild-type receptors. PBA increased the current amplitude of GABA-evoked current in human embryonic kidney 293T cells and the neurons bearing the γ2(Q390X) subunit protein. PBA also proved to reduce endoplasmic reticulum (ER) stress caused by the mutant γ2(Q390X) subunit protein, as well as mitigating seizures and EEG abnormalities in the Gabrg2+/Q390X mice. SIGNIFICANCE: This research has unveiled a promising and innovative approach for treating epilepsy linked to GABAA receptor mutations through an unconventional antiseizure mechanism. Rather than directly modulating the affected mutant channel, PBA facilitates the folding and transportation of wild-type receptor subunits to the cell membrane and synapse. Combining these findings with our previous study, which demonstrated PBA's efficacy in restoring GABA transporter 1 (encoded by SLC6A1) function, we propose that PBA holds significant potential for a wide range of genetic epilepsies. Its ability to target shared molecular pathways involving mutant protein ER retention and impaired protein membrane trafficking suggests broad application in treating such conditions.
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Epilepsias Mioclónicas , Epilepsia , Fenilbutiratos , Ratones , Humanos , Animales , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA/metabolismo , Epilepsias Mioclónicas/tratamiento farmacológico , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/complicaciones , Convulsiones/complicaciones , Epilepsia/genética , Ácido gamma-Aminobutírico , Estrés del Retículo Endoplásmico/genéticaRESUMEN
A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.
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Retículo Endoplásmico , Epilepsias Mioclónicas , Proteolisis , Receptores de GABA-A , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Animales , Retículo Endoplásmico/metabolismo , Ratones , Humanos , Convulsiones Febriles/metabolismo , Convulsiones Febriles/genética , Degradación Asociada con el Retículo Endoplásmico , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Mutación , Células HEK293 , Chaperón BiP del Retículo Endoplásmico/metabolismoRESUMEN
OBJECTIVE: This research was devoted to estimating the outcomes of ginsenoside Rg1 on learning and memory ability and neuronal apoptosis in epileptic rats through ERK/CREB/BDNF pathway. METHODS: The epileptic rats induced by lithium chloride were stochastically separated into model subgroup, ginsenoside Rg1 different dose subgroups. The ginsenoside Rg1 subgroups were given 20, 30 and 40 mg/kg ginsenoside Rg1 by gavage individually. Another 6 normal rats were selected as the control subgroup. The seizures of each subgroup were estimated. Morris water maze was utilized for estimating the changes of cognitive function changes of rats. The injury and apoptosis of hippocampal neurons in each subgroup were detected by Nissl and TUNEL assays. HE staining was applied for the structural and pathomorphological changes of hippocampal neurons detection. The oxidative stress level in hippocampus of rats was estimated by ELISA. DCFH-DA probe was applied for the changes of reactive oxygen species (ROS) in brain tissue detection. The Bcl-2, Bax, ERK, p-ERK, CREB, p-CREB and BDNF levels in cerebral cortex were estimated by western blot, and PD98059, a blocker of ERK pathway, was used to intervene. RESULTS: In the control subgroup, Nissl bodies were abundant and evenly distributed, and cortical neurons were arranged neatly. In the model subgroup, the cytoplasmic staining of cortical neurons was insufficient and the arrangement of neurons was disordered. After treatment with ginsenoside Rg1, the morphology of neurons in the cerebral cortex was restored. The frequency of seizures, duration of seizures, Racine grade, escape latency, target quadrant residence time, MDA, TNF-α and ROS levels of cerebral cortex in the model subgroup boosted notablely versus the control subgroup. The frequency of crossing the original platform, the activity of SOD, the IL-10, p-ERK/ERK, p-CREB/CREB and BDNF levels in cerebral cortex were notablely lessened. The above-mentioned indexes in the ginsenoside Rg1 subgroup were notablely improved versus the model subgroup, and the three proteins levels in the PD98059 intervention subgroup were notablely lower. CONCLUSION: Ginsenoside Rg1 can improve cognitive dysfunction in epileptic rats, which may be concerned with ERK/CREB/BDNF pathway activation in cerebral cortex and lessening oxidative stress and inflammation.
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Factor Neurotrófico Derivado del Encéfalo , Ginsenósidos , Ratas , Animales , Sistema de Señalización de MAP Quinasas , Especies Reactivas de Oxígeno , Transducción de Señal , Ginsenósidos/farmacología , Hipocampo , Apoptosis , ConvulsionesRESUMEN
OBJECTIVE: Infantile spasms is an epileptic encephalopathy of childhood, and its pathophysiology is largely unknown. We generated a heterozygous knock-in mouse with the human infantile spasms-associated de novo mutation GABRB3 (c.A328G, p.N110D) to investigate its molecular mechanisms and to establish the Gabrb3+/N110D knock-in mouse as a model of infantile spasms syndrome. METHODS: We used electroencephalography (EEG) and video monitoring to characterize seizure types, and a suite of behavioral tests to identify neurological and behavioral impairment in Gabrb3+/N110D knock-in mice. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded from layer V/VI pyramidal neurons in somatosensory cortex, and extracellular multi-unit recordings from the ventral basal nucleus of the thalamus in a horizontal thalamocortical slice were used to assess spontaneous thalamocortical oscillations. RESULTS: The infantile spasms-associated human de novo mutation GABRB3 (c.A328G, p.N110D) caused epileptic spasms early in development and multiple seizure types in adult Gabrb3+/N110D knock-in mice. Signs of neurological impairment, anxiety, hyperactivity, social impairment, and deficits in spatial learning and memory were also observed. Gabrb3+/N110D mice had reduced cortical mIPSCs and increased duration of spontaneous oscillatory firing in the somatosensory thalamocortical circuit. SIGNIFICANCE: The Gabrb3+/N110D knock-in mouse has epileptic spasms, seizures, and other neurological impairments that are consistent with infantile spasms syndrome in patients. Multiple seizure types and abnormal behaviors indicative of neurological impairment both early and late in development suggest that Gabrb3+/N110D mice can be used to study the pathophysiology of infantile spasms. Reduced cortical inhibition and increased duration of thalamocortical oscillatory firing suggest perturbations in thalamocortical circuits.
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Espasmos Infantiles , Humanos , Ratones , Animales , Espasmos Infantiles/genética , Receptores de GABA-A/genética , Convulsiones , Células Piramidales , Electroencefalografía , Síndrome , EspasmoRESUMEN
Lennox-Gastaut Syndrome (LGS) is a developmental and epileptic encephalopathy (DEE) characterized by multiple seizure types, electroencephalogram (EEG) patterns, and cognitive decline. Its etiology has a prominent genetic component, including variants in GABRB3 that encodes the GABAA receptor (GABAAR) ß3 subunit. LGS has an unknown pathophysiology, and few animal models are available for studying LGS. The objective of this study was to evaluate Gabrb3+/N328D knock-in mice as a model for LGS. We generated a heterozygous knock-in mouse expressing Gabrb3 (c.A982G, p.N238D), a de novo mutation identified in a patient with LGS. We investigated Gabrb3+/N328D mice for features of LGS. In 2-4-month-old male and female C57BL/J6 wild-type and Gabrb3+/N328D mice, we investigated seizure severity using video-monitored EEG, cognitive impairment using a suite of behavioral tests, and profiled GABAAR subunit expression by Western blot. Gabrb3+/N328D mice showed spontaneous seizures and signs of cognitive impairment, including deficits in spatial learning, memory, and locomotion. Moreover, Gabrb3+/N328D mice showed reduced ß3 subunit expression in the cerebellum, hippocampus, and thalamus. This phenotype of epilepsy and neurological impairment resembles the LGS patient phenotype. We conclude that Gabrb3+/N328D mice provide a good model for investigating the pathophysiology and therapeutic intervention of LGS and DEEs.
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Epilepsia , Síndrome de Lennox-Gastaut , Masculino , Femenino , Ratones , Animales , Síndrome de Lennox-Gastaut/diagnóstico , Receptores de GABA-A/genética , Ratones Endogámicos C57BL , Epilepsia/genética , Convulsiones , Mutación , Electroencefalografía , Ácido gamma-Aminobutírico/genéticaRESUMEN
OBJECTIVE: Mutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. METHODS: We conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. RESULTS: We identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5-7 Hz spike-wave discharges and absence seizures. INTERPRETATION: SLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.
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Epilepsias Mioclónicas , Epilepsia Tipo Ausencia , Animales , Astrocitos/metabolismo , Epilepsias Mioclónicas/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Humanos , Ratones , Convulsiones/complicaciones , Convulsiones/genética , Ácido gamma-AminobutíricoRESUMEN
In recent years, many methods for the facile synthesis of pyridines and their derivatives have been developed. The [2 + 2 + 2] cycloaddition reaction of alkynes and nitriles catalyzed by transition metals has emerged as the most straightforward and efficient method to obtain pyridine derivatives. Recently, Earth-abundant cobalt has been employed as a versatile and economical catalyst for the synthesis of functionalized molecules, as compared to other transition metals. This review mainly focuses on the recent research and development of the Co-catalyzed intramolecular [2 + 2 + 2] cycloaddition of diynes-nitriles or intermolecular [2 + 2 + 2] cycloaddition reaction of alkynes or diynes with nitriles for the construction of chiral or achiral multi-substituted pyridines. Meanwhile, brief mechanistic insights are also discussed here to explain the observed regioselectivity.
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Nitrilos , Piridinas , Alquinos , Catálisis , Cobalto , Reacción de Cicloadición , DiinosRESUMEN
Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.
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Astrocitos/metabolismo , Epilepsia/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Trastornos del Neurodesarrollo/genética , Neuronas/metabolismo , Bases de Datos Factuales , Epilepsia/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Humanos , Trastornos del Neurodesarrollo/metabolismo , Transporte de Proteínas/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: Expression of the TAZ gene is closely related to the prognosis of glioma patients. We hoped to find long noncoding RNAs (lncRNAs) related to TAZ and a new target for glioma treatment. METHODS: TAZ-related genes were found by dual-luciferase reporter gene assay, and the correlation of each gene was analyzed by the Pearson method. Human glioma cell lines U87 MG and U251 and glioma rats were used for cytology assays, and the related genes were transfected. We conducted immunohistochemistry, RT-qPCR, Western blotting, CCK8 test, flow cytometry, transwell assays, clone formation analysis, and tumor weight measurements to verify the above relationship. RESULTS: We found that miR-125a-5p was closely related to the TAZ gene, and the lncRNA MIR4435-2HG was closely related to miR-125a-5p. Both MIR4435-2HG-OE and TAZ increased the expression of the TAZ gene, activated the Wnt signaling pathway, inhibited apoptosis, and promoted migration and proliferation in glioma cells. Besides, it also increased the tumor volume of gliomas in a rat model subcutaneously inoculated with glioma cells. We also found miR-125a-5p could block the effect of MIR4435-2HG-OE and TAZ. CONCLUSIONS: LncRNA MIR4435-2HG obstructs the functions of miR-125a-5p and promotes neuroglioma development by upregulating the TAZ gene.
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Neoplasias Encefálicas/patología , Glioma/patología , MicroARNs/genética , ARN Largo no Codificante/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/genética , Anciano , Animales , Apoptosis/genética , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Humanos , Masculino , Persona de Mediana Edad , Ratas Endogámicas F344 , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Regulación hacia Arriba , Vía de Señalización Wnt/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Neuroinflammation is a major theme in epilepsy, which has been characterized in acquired epilepsy but is poorly understood in genetic epilepsy. γ-Aminobutyric acid type A receptor subunit gene mutations are significant causes of epilepsy, and we have studied the pathophysiology directly resulting from defective receptor channels. Here, we determined the proinflammatory factors in a genetic mouse model, the Gabrg2+/Q390X knockin (KI). We have identified increased cytokines in multiple brain regions of the KI mouse throughout different developmental stages and propose that accumulation of the trafficking-deficient mutant protein may increase neuroinflammation, which would be a novel mechanism for genetic epilepsy. METHODS: We used enzyme-linked immunosorbent assay, immunoprecipitation, nuclei purification, immunoblot, immunohistochemistry, and confocal microscopy to characterize increased neuroinflammation and its potential causes in a Gabrg2+/Q390X KI mouse and a Gabrg2+/- knockout (KO) mouse, each associated with a different epilepsy syndrome with different severities. RESULTS: We found that proinflammatory cytokines such as tumor necrosis factor alpha, interleukin 1-beta (IL-1ß), and IL-6 were increased in the KI mice but not in the KO mice. A major underlying basis for the discrepancy in cytokine expression between the two mouse models is likely chronic mutant protein accumulation and endoplasmic reticulum (ER) stress. The presence of mutant protein dampened cytokine induction upon further cellular stimulation or external stress such as elevated temperature. Pharmacological induction of ER stress upregulated cytokine expression in the wild-type and KO but not in the KI mice. The increased cytokine expression was independent of seizure occurrence, because it was upregulated in both mice and cultured neurons. SIGNIFICANCE: Together, these data demonstrate a novel pathophysiology for genetic epilepsy, increased neuroinflammation, which is a common mechanism for acquired epilepsy. The findings thus provide the first link of neuroinflammation between genetic epilepsy associated with an ion channel gene mutation and acquired epilepsy.
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Citocinas/genética , Citocinas/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Epilepsia/genética , Epilepsia/metabolismo , Receptores de GABA-A/genética , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Epilepsia/patología , Femenino , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de GABA-A/deficienciaRESUMEN
We performed next generation sequencing on 1696 patients with epilepsy and intellectual disability using a gene panel with 480 epilepsy-related genes including all GABAA receptor subunit genes (GABRs), and we identified six de novo GABR mutations, two novel GABRA5 mutations (c.880G>T, p.V294F and c.1238C>T, p.S413F), two novel GABRA1 mutations (c.778C>T, p.P260S and c.887T>C, p.L296S/c.944G>T, p.W315L) and two known GABRA1 mutations (c.335G>A, p.R112Q and c.343A>G, p.N115D) in six patients with intractable early onset epileptic encephalopathy. The α5(V294F and S413F) and α1(P260S and L296S/W315L) subunit residue substitutions were all in transmembrane domains, while the α1(R112Q and N115R) subunit residue substitutions were in the N-terminal GABA binding domain. Using multidisciplinary approaches, we compared effects of mutant GABAA receptor α5 and α1 subunits on the properties of recombinant α5ß3γ2 and α1ß3γ2 GABAA receptors in both neuronal and non-neuronal cells and characterized their effects on receptor clustering, biogenesis and channel function. GABAA receptors containing mutant α5 and α1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Our study identified GABRA5 as a causative gene for early onset epileptic encephalopathy and expands the mutant GABRA1 phenotypic spectrum, supporting growing evidence that defects in GABAergic neurotransmission contribute to early onset epileptic encephalopathy phenotypes.
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Epilepsia/genética , Discapacidad Intelectual/genética , Receptores de GABA-A/genética , Sinapsis/genética , Niño , Preescolar , Epilepsia/complicaciones , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Discapacidad Intelectual/complicaciones , Masculino , Potenciales de la Membrana/fisiología , Potenciales Postsinápticos Miniatura/fisiología , Mutación , Cultivo Primario de Células , Receptores de GABA-A/biosíntesis , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Adulto Joven , Ácido gamma-Aminobutírico/metabolismoRESUMEN
GABRB3 is highly expressed early in the developing brain, and its encoded ß3 subunit is critical for GABAA receptor assembly and trafficking as well as stem cell differentiation in embryonic brain. To date, over 400 mutations or variants have been identified in GABRB3. Mutations in GABRB3 have been increasingly recognized as a major cause for severe paediatric epilepsy syndromes such as Lennox-Gastaut syndrome, Dravet syndrome and infantile spasms with intellectual disability as well as relatively mild epilepsy syndromes such as childhood absence epilepsy. There is no plausible molecular pathology for disease phenotypic heterogeneity. Here we used a very high-throughput flow cytometry assay to evaluate the impact of multiple human mutations in GABRB3 on receptor trafficking. In this study we found that surface expression of mutant ß3 subunits is variable. However, it was consistent that surface expression of partnering γ2 subunits was lower when co-expressed with mutant than with wild-type subunits. Because γ2 subunits are critical for synaptic GABAA receptor clustering, this provides an important clue for understanding the pathophysiology of GABRB3 mutations. To validate our findings further, we obtained an in-depth comparison of two novel mutations [GABRB3 (N328D) and GABRB3 (E357K)] associated with epilepsy with different severities of epilepsy phenotype. GABRB3 (N328D) is associated with the relatively severe Lennox-Gastaut syndrome, and GABRB3 (E357K) is associated with the relatively mild juvenile absence epilepsy syndrome. With functional characterizations in both heterologous cells and rodent cortical neurons by patch-clamp recordings, confocal microscopy and immunoblotting, we found that both the GABRB3 (N328D) and GABRB3 (E357K) mutations reduced total subunit expression in neurons but not in HEK293T cells. Both mutant subunits, however, were reduced on the cell surface and in synapses, but the Lennox-Gastaut syndrome mutant ß3 (N328D) subunit was more reduced than the juvenile absence epilepsy mutant ß3 (E357K) subunit. Interestingly, both mutant ß3 subunits impaired postsynaptic clustering of wild-type GABAA receptor γ2 subunits and prevented γ2 subunits from incorporating into GABAA receptors at synapses, although by different cellular mechanisms. Importantly, wild-type γ2 subunits were reduced and less clustered at inhibitory synapses in Gabrb3+/- knockout mice. This suggests that impaired receptor localization to synapses is a common pathophysiological mechanism for GABRB3 mutations, although the extent of impairment may be different among mutant subunits. The study thus identifies the novel mechanism of impaired targeting of receptors containing mutant ß3 subunits and provides critical insights into understanding how GABRB3 mutations produce severe epilepsy syndromes and epilepsy phenotypic heterogeneity.
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Epilepsia/genética , Receptores de GABA-A/genética , Animales , Encéfalo/embriología , Línea Celular , Membrana Celular/metabolismo , Niño , Preescolar , Análisis por Conglomerados , Epilepsia/metabolismo , Síndromes Epilépticos/genética , Femenino , Citometría de Flujo/métodos , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Mutación/genética , Técnicas de Placa-Clamp , Fenotipo , Subunidades de Proteína/genética , Transporte de Proteínas , Ratas , Receptores de GABA-A/metabolismoRESUMEN
OBJECTIVE: γ-Aminobutyric acid type A (GABAA ) receptor subunit gene mutations are significant causes of epilepsy, which are often accompanied by various neuropsychiatric comorbidities, but the underlying mechanisms are unclear. It has been suggested that the comorbidities are caused by seizures, as the comorbidities often present in severe epilepsy syndromes. However, findings from both humans and animal models argue against this conclusion. Mutations in the GABAA receptor γ2 subunit gene GABRG2 have been associated with anxiety alone or with severe epilepsy syndromes and comorbid anxiety, suggesting that a core molecular defect gives rise to the phenotypic spectrum. Here, we determined the pathophysiology of comorbid anxiety in GABRG2 loss-of-function epilepsy syndromes, identified the central nucleus of the amygdala (CeA) as a primary site for epilepsy comorbid anxiety, and demonstrated a potential rescue of comorbid anxiety via neuromodulation of CeA neurons. METHODS: We used brain slice recordings, subcellular fractionation with Western blot, immunohistochemistry, confocal microscopy, and a battery of behavior tests in combination with a chemogenetic approach to characterize anxiety and its underlying mechanisms in a Gabrg2+/Q390X knockin mouse and a Gabrg2+/- knockout mouse, each associated with a different epilepsy syndrome. RESULTS: We found that impaired GABAergic neurotransmission in CeA underlies anxiety in epilepsy, which is due to reduced GABAA receptor subunit expression resulting from the mutations. Impaired GABAA receptor expression reduced GABAergic neurotransmission in CeA, but not in basolateral amygdala. Activation or inactivation of inhibitory neurons using a chemogenetic approach in CeA alone modulated anxietylike behaviors. Similarly, pharmacological enhancement of GABAergic signaling via γ2 subunit-containing receptors relieved the anxiety. SIGNIFICANCE: Together, these data demonstrate the molecular basis for a comorbidity of epilepsy, anxiety, and suggest that impaired GABAA receptor function in CeA due to a loss-of-function mutation could at least contribute to anxiety. Modulation of CeA neurons could cause or suppress anxiety, suggesting a potential use of CeA neurons as therapeutic targets for treatment of anxiety in addition to traditional pharmacological approaches.
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Amígdala del Cerebelo/fisiopatología , Ansiedad/complicaciones , Ansiedad/genética , Epilepsia/complicaciones , Epilepsia/genética , Receptores de GABA-A/genética , Amígdala del Cerebelo/efectos de los fármacos , Animales , Ansiolíticos/farmacología , Ansiedad/tratamiento farmacológico , Conducta Animal , Comorbilidad , Electroencefalografía , Epilepsia/fisiopatología , Potenciales Postsinápticos Excitadores , Humanos , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Receptores de GABA-A/deficiencia , Transmisión Sináptica , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Genetic epilepsy is a common disorder with phenotypic variation, but the basis for the variation is unknown. Comparing the molecular pathophysiology of mutations in the same epilepsy gene may provide mechanistic insights into the phenotypic heterogeneity. GABRG2 is an established epilepsy gene, and mutations in it produce epilepsy syndromes with varying severities. The disease phenotype in some cases may be caused by simple loss of subunit function (functional haploinsufficiency), while others may be caused by loss-of-function plus dominant negative suppression and other cellular toxicity. Detailed molecular defects and the corresponding seizures and related comorbidities resulting from haploinsufficiency and dominant negative mutations, however, have not been compared. Here we compared two mouse models of GABRG2 loss-of-function mutations associated with epilepsy with different severities, Gabrg2+/Q390X knockin (KI) and Gabrg2+/- knockout (KO) mice. Heterozygous Gabrg2+/Q390X KI mice are associated with a severe epileptic encephalopathy due to a dominant negative effect of the mutation, while heterozygous Gabrg2+/- KO mice are associated with mild absence epilepsy due to simple haploinsufficiency. Unchanged at the transcriptional level, KI mice with severe epilepsy had neuronal accumulation of mutant γ2 subunits, reduced remaining functional wild-type subunits in dendrites and synapses, while KO mice with mild epilepsy had no intracellular accumulation of the mutant subunits and unaffected biogenesis of the remaining wild-type subunits. Consequently, KI mice with dominant negative mutations had much less wild-type receptor expression, more severe seizures and behavioural comorbidities than KO mice. This work provides insights into the pathophysiology of epilepsy syndrome heterogeneity and designing mechanism-based therapies.
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Conducta Animal , Epilepsia/genética , Genes Dominantes , Haploinsuficiencia , Mutación Missense , Receptores de GABA-A/genética , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Epilepsia/metabolismo , Epilepsia/fisiopatología , Humanos , Ratones , Ratones Noqueados , Receptores de GABA-A/metabolismoRESUMEN
Epileptic encephalopathies are a devastating group of severe childhood onset epilepsies with medication-resistant seizures and poor developmental outcomes. Many epileptic encephalopathies have a genetic aetiology and are often associated with de novo mutations in genes mediating synaptic transmission, including GABAA receptor subunit genes. Recently, we performed next generation sequencing on patients with a spectrum of epileptic encephalopathy phenotypes, and we identified five novel (A106T, I107T, P282S, R323W and F343L) and one known (R323Q) de novo GABRG2 pathogenic variants (mutations) in eight patients. To gain insight into the molecular basis for how these mutations contribute to epileptic encephalopathies, we compared the effects of the mutations on the properties of recombinant α1ß2γ2L GABAA receptors transiently expressed in HEK293T cells. Using a combination of patch clamp recording, immunoblotting, confocal imaging and structural modelling, we characterized the effects of these GABRG2 mutations on GABAA receptor biogenesis and channel function. Compared with wild-type α1ß2γ2L receptors, GABAA receptors containing a mutant γ2 subunit had reduced cell surface expression with altered subunit stoichiometry or decreased GABA-evoked whole-cell current amplitudes, but with different levels of reduction. While a causal role of these mutations cannot be established directly from these results, the functional analysis together with the genetic information suggests that these GABRG2 variants may be major contributors to the epileptic encephalopathy phenotypes. Our study further expands the GABRG2 phenotypic spectrum and supports growing evidence that defects in GABAergic neurotransmission participate in the pathogenesis of genetic epilepsies including epileptic encephalopathies.
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Epilepsia Refractaria/genética , Epilepsia Refractaria/fisiopatología , Epilepsia/genética , Epilepsia/fisiopatología , Receptores de GABA-A/genética , Niño , Preescolar , Fenómenos Electrofisiológicos , Exoma , Femenino , Células HEK293 , Humanos , Masculino , Mutación , Técnicas de Placa-Clamp , FenotipoRESUMEN
BACKGROUND: Early myoclonic encephalopathy (EME), a disease with a devastating prognosis, is characterised by neonatal onset of seizures and massive myoclonus accompanied by a continuous suppression-burst EEG pattern. Three genes are associated with EMEs that have metabolic features. Here, we report a pathogenic mutation of an ion channel as a cause of EME for the first time. METHODS: Sequencing was performed for 214 patients with epileptic seizures using a gene panel with 109 genes that are known or suspected to cause epileptic seizures. Functional assessments were demonstrated by using electrophysiological experiments and immunostaining for mutant γ-aminobutyric acid-A (GABAA) receptor subunits in HEK293T cells. RESULTS: We discovered a de novo heterozygous missense mutation (c.859A>C [p.Thr287Pro]) in the GABRB2-encoded ß2 subunit of the GABAA receptor in an infant with EME. No GABRB2 mutations were found in three other EME cases or in 166 patients with infantile spasms. GABAA receptors bearing the mutant ß2 subunit were poorly trafficked to the cell membrane and prevented γ2 subunits from trafficking to the cell surface. The peak amplitudes of currents from GABAA receptors containing only mutant ß2 subunits were smaller than that of those from receptors containing only wild-type ß2 subunits. The decrease in peak current amplitude (96.4% reduction) associated with the mutant GABAA receptor was greater than expected, based on the degree to which cell surface expression was reduced (66% reduction). CONCLUSION: This mutation has complex functional effects on GABAA receptors, including reduction of cell surface expression and attenuation of channel function, which would significantly perturb GABAergic inhibition in the brain.
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Síndrome de Opsoclonía-Mioclonía/genética , Receptores de GABA-A/genética , Convulsiones/genética , Espasmos Infantiles/genética , Encéfalo/diagnóstico por imagen , Encéfalo/fisiopatología , Cristalografía por Rayos X , Electroencefalografía , Células HEK293 , Humanos , Lactante , Masculino , Modelos Moleculares , Mutación Missense , Síndrome de Opsoclonía-Mioclonía/fisiopatología , Receptores de GABA-A/química , Convulsiones/fisiopatología , Espasmos Infantiles/fisiopatologíaRESUMEN
OBJECTIVE: The mutant γ-aminobutyric acid type A (GABAA ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABAA receptors, and affects trafficking of partnering α and ß subunits. Heterozygous Gabrg2+/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy. METHODS: We introduced the GABRG2 allele by crossing Gabrg2+/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2HA subunits, and compared GABAA receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording. RESULTS: Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and ß2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold. SIGNIFICANCE: Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits.
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Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/terapia , Mutación/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Convulsivantes/toxicidad , Estimulación Eléctrica , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Técnicas de Placa-Clamp , Pentilenotetrazol/toxicidad , Subunidades de Proteína/genética , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Corteza Somatosensorial/citología , Tálamo/citologíaRESUMEN
Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells, iGSCs) through expression of Oct4, Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells, iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/ß-catenin signalling and expression of CD133, CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents.
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Desdiferenciación Celular/genética , Proliferación Celular/genética , Reprogramación Celular/genética , Células Madre Neoplásicas/metabolismo , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Técnicas de Reprogramación Celular/métodos , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Mitógenos/farmacología , Células Madre Neoplásicas/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Temozolomida , beta Catenina/metabolismoRESUMEN
Genetic mutations in voltage-gated and ligand-gated ion channel genes have been identified in a small number of Mendelian families with genetic generalised epilepsies (GGEs). They are commonly associated with febrile seizures (FS), childhood absence epilepsy (CAE) and particularly with generalised or genetic epilepsy with febrile seizures plus (GEFS+). In clinical practice, despite efforts to categorise epilepsy and epilepsy families into syndromic diagnoses, many generalised epilepsies remain unclassified with a presumed genetic basis. During the systematic collection of epilepsy families, we assembled a cohort of families with evidence of GEFS+ and screened for variations in the γ2 subunit of the γ-aminobutyric acid (GABA) type A receptor gene (GABRG2). We detected a novel GABRG2(p.R136*) premature translation termination codon in one index-case from a two-generation nuclear family, presenting with an unclassified GGE, a borderline GEFS+ phenotype with learning difficulties and extended behavioural presentation. The GABRG2(p.R136*) mutation segregates with the febrile seizure component of this family's GGE and is absent in 190 healthy control samples. In vitro expression assays demonstrated that γ2(p.R136*) subunits were produced, but had reduced cell-surface and total expression. When γ2(p.R136*) subunits were co-expressed with α1 and ß2 subunits in HEK 293T cells, GABA-evoked currents were reduced. Furthermore, γ2(p.R136*) subunits were highly-expressed in intracellular aggregations surrounding the nucleus and endoplasmic reticulum (ER), suggesting compromised receptor trafficking. A novel GABRG2(p.R136*) mutation extends the spectrum of GABRG2 mutations identified in GEFS+ and GGE phenotypes, causes GABAA receptor dysfunction, and represents a putative epilepsy mechanism.