RESUMEN
OBJECTIVE: Neurosteroids regulate neuronal excitability by potentiating γ-aminobutyric acid type-A receptors (GABARs). In animal models of temporal lobe epilepsy, the neurosteroid sensitivity of GABARs is diminished and GABAR subunit composition is altered. We tested whether similar changes occur in patients with epilepsy and if depolarization-induced increases in neuronal activity can replicate this effect. METHODS: We determined GABAR α4 subunit expression in cortical tissue resected from pediatric epilepsy patients. Modulation of human GABARs by allopregnanolone and Ro15-4513 was measured in Xenopus oocytes using whole-cell patch clamp. To extend the findings obtained using tissue from epilepsy patients, we evaluated GABAR expression and modulation by allopregnanolone and Ro15-4513 in cultured rat hippocampal neurons exposed to high extracellular potassium (HK) to increase neuronal activity. RESULTS: Expression of α4 subunits was increased in pediatric cortical epilepsy specimens encompassing multiple pathologies. The potentiation of GABA-evoked currents by the neurosteroid allopregnanolone was decreased in Xenopus oocytes expressing GABARs isolated from epilepsy patients. Furthermore, receptors isolated from epilepsy but not control tissue were sensitive to potentiation by Ro15-4513, indicating higher expression of α4 ßx γ2 subunit-containing receptors. Correspondingly, increasing the activity of cultured rat hippocampal neurons reduced allopregnanolone potentiation of miniature inhibitory postsynaptic currents (mIPSCs), increased modulation of tonic GABAR current by Ro15-4513, upregulated the surface expression of α4 and γ2 subunits, and increased the colocalization of α4 and γ2 subunit immunoreactivity. INTERPRETATION: These findings suggest that seizure activity-induced upregulation of α4 ßx γ2 subunit-containing GABARs could affect the anticonvulsant actions of neurosteroids.
Asunto(s)
Corteza Cerebral/metabolismo , Epilepsia Refractaria/metabolismo , Fenómenos Electrofisiológicos/fisiología , Agonistas de Receptores de GABA-A/farmacología , Neuronas/metabolismo , Neuroesteroides/metabolismo , Pregnanolona/farmacología , Receptores de GABA-A/metabolismo , Adolescente , Adulto , Animales , Azidas/farmacología , Benzodiazepinas/farmacología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Niño , Preescolar , Epilepsia Refractaria/cirugía , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Lactante , Masculino , Neuronas/efectos de los fármacos , Oocitos , Técnicas de Placa-Clamp , Ratas , Receptores de GABA-A/efectos de los fármacos , Xenopus , Adulto JovenRESUMEN
Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum.
Asunto(s)
Encéfalo/anomalías , Hemimegalencefalia/genética , Malformaciones del Desarrollo Cortical/genética , Megalencefalia/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética , Adolescente , Encéfalo/metabolismo , Niño , Preescolar , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Predisposición Genética a la Enfermedad/genética , Hemimegalencefalia/metabolismo , Hemimegalencefalia/patología , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Malformaciones del Desarrollo Cortical/metabolismo , Malformaciones del Desarrollo Cortical/patología , Megalencefalia/metabolismo , Megalencefalia/patología , Mutación , Neuroimagen , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
OBJECTIVE: A high incidence of structural brain abnormalities has been reported in individuals with pyridoxine-dependent epilepsy (PDE). PDE is caused by mutations in ALDH7A1, also known as antiquitin. How antiquitin dysfunction leads to cerebral dysgenesis is unknown. In this study, we analyzed tissue from a child with PDE as well as control human and murine brain to determine the normal distribution of antiquitin, its distribution in PDE, and associated brain malformations. METHODS: Formalin-fixed human brain sections were subjected to histopathology and fluorescence immunohistochemistry studies. Frozen brain tissue was utilized for measurement of PDE-associated metabolites and Western blot analysis. Comparative studies of antiquitin distribution were performed in developing mouse brain sections. RESULTS: Histologic analysis of PDE cortex revealed areas of abnormal radial neuronal organization consistent with type Ia focal cortical dysplasia. Heterotopic neurons were identified in subcortical white matter, as was cortical astrogliosis, hippocampal sclerosis, and status marmoratus of the basal ganglia. Highly elevated levels of lysine metabolites were present in postmortem PDE cortex. In control human and developing mouse brain, antiquitin immunofluorescence was identified in radial glia, mature astrocytes, ependyma, and choroid plexus epithelium, but not in neurons. In PDE cortex, antiquitin immunofluorescence was greatly attenuated with evidence of perinuclear accumulation in astrocytes. INTERPRETATION: Antiquitin is expressed within glial cells in the brain, and its dysfunction in PDE is associated with neuronal migration abnormalities and other structural brain defects. These malformations persist despite postnatal pyridoxine supplementation and likely contribute to neurodevelopmental impairments.
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Aldehído Deshidrogenasa/biosíntesis , Corteza Cerebral/metabolismo , Epilepsia/diagnóstico , Epilepsia/metabolismo , Neuroglía/metabolismo , Adolescente , Animales , Animales Recién Nacidos , Movimiento Celular/fisiología , Corteza Cerebral/química , Corteza Cerebral/patología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Ratones , Neuroglía/química , Neuroglía/patología , EmbarazoRESUMEN
Dravet Syndrome (DS) is an intractable genetic epilepsy caused by loss-of-function mutations in SCN1A, the gene encoding brain sodium channel Nav 1.1. DS is associated with increased frequency of sudden unexpected death in humans and in a mouse genetic model of this disease. Here we correlate the time course of declining expression of the murine embryonic sodium channel Nav 1.3 and the rise in expression of the adult sodium channel Nav 1.1 with susceptibility to epileptic seizures and increased incidence of sudden death in DS mice. Parallel studies with unaffected human brain tissue demonstrate similar decline in Nav 1.3 and increase in Nav 1.1 with age. In light of these results, we introduce the hypothesis that the natural loss Nav 1.3 channel expression in brain development, coupled with the failure of increase in functional Nav 1.1 channels in DS, defines a tipping point that leads to disinhibition of neural circuits, intractable seizures, co-morbidities, and premature death in this disease.
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Muerte Súbita , Epilepsias Mioclónicas/metabolismo , Regulación de la Expresión Génica , Canales de Sodio/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Humanos , Ratones , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Factores de TiempoRESUMEN
The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the beta(3)-alpha(5)-gamma(3) GABA(A) receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABA(A) receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of beta(3)/beta(2) and alpha(5)/alpha(1) subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage-clamp analysis of GABA(A) receptor currents was then performed. Studies of GABA(A) receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the alpha(1)-selective benzodiazepine-site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABA(A) receptor affinity and modulation by neurosteroids were unchanged. This shift in GABA(A) receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder.
Asunto(s)
Síndrome de Angelman/patología , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/fisiología , Subunidades de Proteína/metabolismo , Receptores de GABA/metabolismo , Adolescente , Adulto , Animales , Estudios de Casos y Controles , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Moduladores del GABA/farmacología , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Oocitos , Técnicas de Placa-Clamp/métodos , Fenobarbital/farmacología , Subunidades de Proteína/genética , Receptores de GABA/genética , Xenopus , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
PURPOSE: Expression of the protein subunits that make up the γ-aminobutyric acid (GABA)(A) receptor pentamer is known to change during postnatal brain development in animal models. In the present study, analysis of cortical GABA(A) subunit expression was performed in control human tissue obtained from infancy through adolescence, and was compared to that from similarly aged children with intractable focal epilepsy. METHODS: Twenty frozen pediatric control and 25 epileptic neocortical specimens were collected. The membrane fractions were isolated and subjected to quantitative western blot analysis. Subunit expression was correlated with clinical factors including age, pathology, and medication exposure. RESULTS: In control cortical samples, α1 and γ2 GABA(A) receptor subunits exhibited low expression in infancy, which increased over the first several years of life and then stabilized through adolescence. In contrast, α4 subunit expression was higher in infants than in older children. The level of the chloride transporter KCC2 increased markedly with age, whereas that of NKCC1 decreased. These patterns were absent in the children with epilepsy, both in those with focal cortical dysplasia and in those with cortical gliosis. Although there was marked variability in GABA(A) receptor subunit expression among the children with epilepsy, identifiable patterns of subunit expression were found in each individual child. DISCUSSION: Maturation of cortical GABA(A) receptor subunit expression continues over the first several years of postnatal human development. Intractable focal epilepsy in children is associated with disruption of this normal developmental pattern. These findings have significant implications for the treatment of children with medications that modulate GABA(A) receptor function.
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Corteza Cerebral , Epilepsia/patología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de GABA-A/metabolismo , Adolescente , Factores de Edad , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Niño , Preescolar , Epilepsia/complicaciones , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Malformaciones del Desarrollo Cortical/etiología , Malformaciones del Desarrollo Cortical/patología , Pediatría , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genética , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Miembro 2 de la Familia de Transportadores de Soluto 12 , Estadística como Asunto , Adulto JovenRESUMEN
In sympathetic neurons, it is well-established that the neurotransmitters, norepinephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary Ca(2+) sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by approximately 50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters.