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Hyperpolarization activated Cyclic Nucleotide (HCN) gated channels are crucial for various neurophysiological functions, including learning and sensory functions, and their dysfunction are responsible for brain disorders, such as epilepsy. To date, HCN2 variants have only been associated with mild epilepsy and recently, one monoallelic missense variant has been linked to developmental and epileptic encephalopathy. Here, we expand the phenotypic spectrum of HCN2- related disorders by describing twenty-one additional individuals from fifteen unrelated families carrying HCN2 variants. Seventeen individuals had developmental delay/intellectual disability (DD/ID), two had borderline DD/ID, and one had borderline DD. Ten individuals had epilepsy with DD/ID, with median age of onset of 10 months, and one had epilepsy with normal development. Molecular diagnosis identified thirteen different pathogenic HCN2 variants, including eleven missense variants affecting highly conserved amino acids, one frameshift variant, and one in-frame deletion. Seven variants were monoallelic of which five occurred de novo, one was not maternally inherited, one was inherited from a father with mild learning disabilities, and one was of unknown inheritance. The remaining six variants were biallelic, with four homozygous and two compound heterozygous variants. Functional studies using two-electrode voltage-clamp recordings in Xenopus laevis oocytes were performed on three monoallelic variants, p.(Arg324His), p.(Ala363Val), and p.(Met374Leu), and three biallelic variants, p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp). The p.(Arg324His) variant induced a strong increase of HCN2 conductance, while p.(Ala363Val) and p.(Met374Leu) displayed dominant negative effects, leading to a partial loss of HCN2 channel function. By confocal imaging, we found that the p.(Leu377His), p.(Pro493Leu) and p.(Gly587Asp) pathogenic variants impaired membrane trafficking, resulting in a complete loss of HCN2 elicited currents in Xenopus oocytes. Structural 3D-analysis in depolarized and hyperpolarized states of HCN2 channels, revealed that the pathogenic variants p.(His205Gln), p.(Ser409Leu), p.(Arg324Cys), p.(Asn369Ser) and p.(Gly460Asp) modify molecular interactions altering HCN2 function. Taken together, our data broadens the clinical spectrum associated with HCN2 variants, and disclose that HCN2 is involved in developmental encephalopathy with or without epilepsy.
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Pathogenic variants in HCN1 are an established cause of developmental and epileptic encephalopathy (DEE). To date, the stratification of patients with HCN1-DEE based on the biophysical consequence on channel function of a given variant has not been possible. Here, we analysed data from eleven patients carrying seven different de novo HCN1 pathogenic variants located in the transmembrane domains of the protein. All patients were diagnosed with severe disease including epilepsy and intellectual disability. The functional properties of the seven HCN1 pathogenic variants were assessed using two-electrode voltage-clamp recordings in Xenopus oocytes. All seven variants showed a significantly larger instantaneous current consistent with cation leak. The impact of each variant on other biophysical properties was variable, including changes in the half activation voltage and activation and deactivation kinetics. These data suggest that cation leak is an important pathogenic mechanism in HCN1-DEE. Furthermore, published mouse model and clinical case reports suggest that seizures are exacerbated by sodium channel blockers in patients with HCN1 variants that cause cation leak. Stratification of patients based on their 'cation leak' biophysical phenotype may therefore provide key information to guide clinical management of individuals with HCN1-DEE.
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Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.
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Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , AMP Cíclico/metabolismo , Antidepresivos/farmacologíaRESUMEN
Variants in HCN1 are associated with a range of epilepsy syndromes including developmental and epileptic encephalopathies. Here we describe a child harboring a novel de novo HCN1 variant, E246A, in a child with epilepsy and mild developmental delay. By parental report, the child had difficulty in discriminating between colors implicating a visual deficit. This interesting observation may relate to the high expression of HCN1 channels in rod and cone photoreceptors where they play an integral role in shaping the light response. Functional analysis of the HCN1 E246A variant revealed a right shift in the voltage dependence of activation and slowing of the rates of activation and deactivation. The changes in the biophysical properties are consistent with a gain-of-function supporting the role of HCN1 E246A in disease causation. This case suggests that visual function, including color discrimination, should be carefully monitored in patients with diseases due to HCN1 pathogenic variants.
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OBJECTIVE: To compare the frequency and impact on the channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-of-function KCNH2 variants as predictive biomarkers of SUDEP risk. METHODS: We searched for KCNH2 variants with a minor allele frequency of <5%. Functional analysis in Xenopus laevis oocytes was performed for all KCNH2 variants identified. RESULTS: KCNH2 variants were found in 11.1% (10/90) of SUDEP individuals compared to 6.0% (20/332) of epilepsy controls (p = 0.11). Loss-of-function KCNH2 variants, defined as causing >20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about threefold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (<1% allele frequency) associated with greater loss of function and an ~11-fold enrichment in the SUDEP cohort (nominal p = 0.03). In silico tools were unable to predict the impact of a variant on function highlighting the need for electrophysiological analysis. INTERPRETATION: These data show that loss-of-function KCNH2 variants are enriched in SUDEP patients when compared to an epilepsy population older than 50 years, suggesting that cardiac mechanisms contribute to SUDEP risk. We propose that genetic screening in combination with functional analysis can identify loss-of-function KCNH2 variants that could act as biomarkers of an individual's SUDEP risk.
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Canal de Potasio ERG1/genética , Epilepsia/epidemiología , Epilepsia/genética , Variación Genética/fisiología , Muerte Súbita e Inesperada en la Epilepsia/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores/metabolismo , Niño , Estudios de Cohortes , Canal de Potasio ERG1/metabolismo , Epilepsia/metabolismo , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Xenopus laevis , Adulto JovenRESUMEN
BACKGROUND AND PURPOSE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4) with distinct biophysical properties and functions within the brain. HCN4 channels activate slowly at robust hyperpolarizing potentials, making them more likely to be engaged during hyperexcitable neuronal network activity seen during seizures. HCN4 channels are also highly expressed in thalamic nuclei, a brain region implicated in seizure generalization. Here, we assessed the utility of targeting the HCN4 channel as an anti-seizure strategy using pharmacological and genetic approaches. EXPERIMENTAL APPROACH: The impact of reducing HCN4 channel function on seizure susceptibility and neuronal network excitability was studied using an HCN4 channel preferring blocker (EC18) and a conditional brain specific HCN4 knockout mouse model. KEY RESULTS: EC18 (10 mg·kg-1 ) and brain-specific HCN4 channel knockout reduced seizure susceptibility and proconvulsant-mediated cortical spiking recorded using electrocorticography, with minimal effects on other mouse behaviours. EC18 (10 µM) decreased neuronal network bursting in mouse cortical cultures. Importantly, EC18 was not protective against proconvulsant-mediated seizures in the conditional HCN4 channel knockout mouse and did not reduce bursting behaviour in AAV-HCN4 shRNA infected mouse cortical cultures. CONCLUSIONS AND IMPLICATIONS: These data suggest the HCN4 channel as a potential pharmacologically relevant target for anti-seizure drugs that is likely to have a low side-effect liability in the CNS.
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Canales Catiónicos Regulados por Nucleótidos Cíclicos , Preparaciones Farmacéuticas , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Ratones , Nucleótidos Cíclicos , Convulsiones/tratamiento farmacológicoRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I h , which is important for modulating neuron excitability. Four genes (HCN1-4) encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid in situ hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula (medial and lateral) and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of I h -mediated "sag" in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels.
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OBJECTIVE: To examine the genotype to phenotype connection in glucose transporter type 1 (GLUT1) deficiency and whether a simple functional assay can predict disease outcome from genetic sequence alone. METHODS: GLUT1 deficiency, due to mutations in SLC2A1, causes a wide range of epilepsies. One possible mechanism for this is variable impact of mutations on GLUT1 function. To test this, we measured glucose transport by GLUT1 variants identified in population controls and patients with mild to severe epilepsies. Controls were reference sequence from the NCBI and 4 population missense variants chosen from public reference control databases. Nine variants associated with epilepsies or movement disorders, with normal intellect in all individuals, formed the mild group. The severe group included 5 missense variants associated with classical GLUT1 encephalopathy. GLUT1 variants were expressed in Xenopus laevis oocytes, and glucose uptake was measured to determine kinetics (Vmax) and affinity (Km). RESULTS: Disease severity inversely correlated with rate of glucose transport between control (Vmax = 28 ± 5), mild (Vmax = 16 ± 3), and severe (Vmax = 3 ± 1) groups, respectively. Affinities of glucose binding in control (Km = 55 ± 18) and mild (Km = 43 ± 10) groups were not significantly different, whereas affinity was indeterminate in the severe group because of low transport rates. Simplified analysis of glucose transport at high concentration (100 mM) was equally effective at separating the groups. CONCLUSIONS: Disease severity can be partly explained by the extent of GLUT1 dysfunction. This simple Xenopus oocyte assay complements genetic and clinical assessments. In prenatal diagnosis, this simple oocyte glucose uptake assay could be useful because standard clinical assessments are not available.
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Genetic generalized epilepsy (GGE) is a common epilepsy syndrome that encompasses seizure disorders characterized by spike-and-wave discharges (SWDs). Pacemaker hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are considered integral to SWD genesis, making them an ideal gene candidate for GGE. We identified HCN2 missense variants from a large cohort of 585 GGE patients, recruited by the Epilepsy Phenome-Genome Project (EPGP), and performed functional analysis using two-electrode voltage clamp recordings from Xenopus oocytes. The p.S632W variant was identified in a patient with idiopathic photosensitive occipital epilepsy and segregated in the family. This variant was also independently identified in an unrelated patient with childhood absence seizures from a European cohort of 238 familial GGE cases. The p.V246M variant was identified in a patient with photo-sensitive GGE and his father diagnosed with juvenile myoclonic epilepsy. Functional studies revealed that both p.S632W and p.V246M had an identical functional impact including a depolarizing shift in the voltage dependence of activation that is consistent with a gain-of-function. In contrast, no biophysical changes resulted from the introduction of common population variants, p.E280K and p.A705T, and the p.R756C variant from EPGP that did not segregate with disease. Our data suggest that HCN2 variants can confer susceptibility to GGE via a gain-of-function mechanism.
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ADN Complementario/genética , Epilepsia Generalizada/genética , Epilepsia/genética , Mutación con Ganancia de Función/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Electrofisiología , Femenino , Humanos , Masculino , Modelos Biológicos , LinajeRESUMEN
Gabapentin (GBP) is widely used to treat epilepsy and neuropathic pain. There is evidence that GBP can act on hyperpolarization-activated cation (HCN) channel-mediated Ih in brain slice experiments. However, evidence showing that GBP directly modulates HCN channels is lacking. The effect of GBP was tested using two-electrode voltage clamp recordings from human HCN1, HCN2, and HCN4 channels expressed in Xenopus oocytes. Whole-cell recordings were also made from mouse spinal cord slices targeting either parvalbumin positive (PV+) or calretinin positive (CR+) inhibitory neurons. The effect of GBP on Ih was measured in each inhibitory neuron population. HCN4 expression was assessed in the spinal cord using immunohistochemistry. When applied to HCN4 channels, GBP (100 µM) caused a hyperpolarizing shift in the voltage of half activation (V1/2) thereby reducing the currents. Gabapentin had no impact on the V1/2 of HCN1 or HCN2 channels. There was a robust increase in the time to half activation for HCN4 channels with only a small increase noted for HCN1 channels. Gabapentin also caused a hyperpolarizing shift in the V1/2 of Ih measured from HCN4-expressing PV+ inhibitory neurons in the spinal dorsal horn. Gabapentin had minimal effect on Ih recorded from CR+ neurons. Consistent with this, immunohistochemical analysis revealed that the majority of CR+ inhibitory neurons do not express somatic HCN4 channels. In conclusion, GBP reduces HCN4 channel-mediated currents through a hyperpolarized shift in the V1/2. The HCN channel subtype selectivity of GBP provides a unique tool for investigating HCN4 channel function in the central nervous system. The HCN4 channel is a candidate molecular target for the acute analgesic and anticonvulsant actions of GBP.
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Objective: Genetic generalized epilepsy (GGE) encompasses seizure disorders characterized by spike-and-wave discharges (SWD) originating within thalamo-cortical circuits. Hyperpolarization-activated (HCN) and T-type Ca2+ channels are key modulators of rhythmic activity in these brain regions. Here, we screened HCN4 and CACNA1H genes for potentially contributory variants and provide their functional analysis. Methods: Targeted gene sequencing was performed in 20 unrelated familial cases with different subtypes of GGE, and the results confirmed in 230 ethnically matching controls. Selected variants in CACNA1H and HCN4 were functionally assessed in tsA201 cells and Xenopus laevis oocytes, respectively. Results: We discovered a novel CACNA1H (p.G1158S) variant in two affected members of a single family. One of them also carried an HCN4 (p.P1117L) variant inherited from the unaffected mother. In a separate family, an HCN4 variant (p.E153G) was identified in one of several affected members. Voltage-clamp analysis of CACNA1H (p.G1158S) revealed a small but significant gain-of-function, including increased current density and a depolarizing shift of steady-state inactivation. HCN4 p.P1117L and p.G153E both caused a hyperpolarizing shift in activation and reduced current amplitudes, resulting in a loss-of-function. Significance: Our results are consistent with a model suggesting cumulative contributions of subtle functional variations in ion channels to seizure susceptibility and GGE.
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Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.
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Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Convulsiones Febriles/genética , Convulsiones Febriles/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Animales , Estudios de Casos y Controles , Proteínas de Transporte de Catión/química , Línea Celular , Análisis Mutacional de ADN , Predisposición Genética a la Enfermedad , Humanos , Patrón de Herencia , Estimación de Kaplan-Meier , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Linaje , Ratas , Riesgo , Convulsiones Febriles/mortalidad , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The ß1 subunit of voltage-gated sodium channels, Nav ß1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel α subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Nav ß1 subneuronal and regional expression. Nav ß1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Nav ß1 expression at the AIS was detected throughout the brain, predominantly in the hippocampus, cortex, and cerebellum. Despite expression of Nav ß1 in both excitatory and inhibitory AIS, it displayed a marked and fine-grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Nav ß1 coexpressed with Nav 1.1, Nav 1.2, and Nav 1.6 subunits. The disruption of Nav ß1 AIS expression by a human epilepsy-causing C121W genetic mutation in Nav ß1 was also investigated using a mouse model. AIS expression of Nav ß1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Nav α subunit modulation by Nav ß1 contributes to the mechanism of epileptogenesis in these animals as well as in patients.
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Axones/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismo , Alquenos , Animales , Western Blotting , Encéfalo/patología , Modelos Animales de Enfermedad , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/patología , Humanos , Inmunohistoquímica , Interneuronas/citología , Interneuronas/metabolismo , Interneuronas/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Mutación , Piperidinas , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genéticaRESUMEN
The GABAAγ2(R43Q) mouse is an established model of absence epilepsy displaying spontaneous spike-and-wave discharges (SWD) and associated behavioral arrest. Absence epilepsy typically results from cortico-thalamic networks. Nevertheless, there is increasing evidence for changes in hippocampal metabolism and electrical behavior, consistent with a link between absence seizures and hippocampus-related co-morbidities. Hyperpolarization-activated-cyclic-nucleotide-gated (HCN) channels are known to be transcriptionally regulated in a number of seizure models. Here we investigate the expression and function of these channels in the hippocampus of the genetic epilepsy model. A reduction in HCN1, but not HCN2 transcript, was observed in GABAAγ2(R43Q) mice relative to their littermate controls. In contrast, no change in HCN1 transcript was noted at an age prior to seizure expression or in a SWD-free model in which the R43Q mutation has been crossed into a seizure-resistant genetic background. Whole-cell recordings from CA1 pyramidal neurons confirm a reduction in Ih in the GABAAγ2(R43Q) mouse. Further, a left-shift in half-activation of the Ih conductance-voltage relationship is consistent with a reduction in HCN1 with no change in HCN2 channel expression. Behavioral analysis using the Morris water maze indicates that GABAAγ2(R43Q) mice are unable to learn as effectively as their wildtype littermates suggesting a deficit in hippocampal-based learning. SWD-free mice harboring the R43Q mutation had no learning deficit. We conclude that SWDs reduce hippocampal HCN1 expression and function, and that the reduction associates with a spatial learning deficit.
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Epilepsia Tipo Ausencia/fisiopatología , Hipocampo/fisiopatología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Discapacidades para el Aprendizaje/fisiopatología , Canales de Potasio/metabolismo , Animales , Región CA1 Hipocampal/fisiopatología , Comorbilidad , Epilepsia Tipo Ausencia/epidemiología , Femenino , Humanos , Discapacidades para el Aprendizaje/epidemiología , Masculino , Aprendizaje por Laberinto/fisiología , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Mutación , Células Piramidales/fisiopatología , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Convulsiones/etiología , Convulsiones/fisiopatologíaRESUMEN
OBJECTIVE: To understand the molecular basis and differential penetrance of febrile seizures and absence seizures in patients with the γ2(R43Q) GABA receptor mutation. METHODS: Spike-and-wave discharges and thermal seizure susceptibility were measured in heterozygous GABA γ2 knock-out and GABA γ2(R43Q) knock-in mice models crossed to different mouse strains. RESULTS: By comparing the GABA γ2 knock-out with the GABA γ2(R43Q) knock-in mouse model we show that haploinsufficiency underlies the genesis of absence seizures but cannot account for the thermal seizure susceptibility. Additionally, while the expression of the absence seizure phenotype was very sensitive to mouse background genetics, the thermal seizure phenotype was not. CONCLUSIONS: Our results show that a single gene mutation can cause distinct seizure phenotypes through independent molecular mechanisms. A lack of effect of genetic background on thermal seizure susceptibility is consistent with the higher penetrance of febrile seizures compared to absence seizures seen in family members with the mutation. These mouse studies help to provide a conceptual framework within which clinical heterogeneity seen in genetic epilepsy can be explained.
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Epilepsia/genética , Mutación/genética , Receptores de GABA-A/deficiencia , Receptores de GABA-A/genética , Animales , Epilepsia/metabolismo , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones NoqueadosRESUMEN
OBJECTIVE: We examined whether glucose transporter 1 (GLUT1) deficiency causes common idiopathic generalized epilepsies (IGEs). METHODS: The IGEs are common, heritable epilepsies that usually follow complex inheritance; currently little is known about their genetic architecture. Previously considered rare, GLUT1 deficiency, due to mutations in SLC2A1, leads to failure of glucose transport across the blood-brain barrier and inadequate glucose for brain metabolism. GLUT1 deficiency was first associated with an encephalopathy and more recently found in rare dominant families with epilepsy and paroxysmal exertional dyskinesia (PED). Five hundred four probands with IGEs and 470 controls underwent SLC2A1 sequencing. Glucose transport was assayed following expression of SLC2A1 variants in Xenopus oocytes. All available relatives were phenotyped, and SLC2A1 was sequenced. RESULTS: Functionally validated mutations in SLC2A1 were present in 7 of 504 (1.4%) probands and 0 of 470 controls. PED, undiagnosed prior to study, occurred in 1 proband and 3 of 13 relatives with mutations. The IGEs in probands and relatives were indistinguishable from typical IGE. Three cases (0.6%) had mutations of large functional effect and showed autosomal dominant inheritance or were de novo. Four (0.8%) cases had a subtle functional effect; 2 showed possible dominant inheritance, and 2 did not. These alleles leading to subtle functional impairment may contribute to complex, polygenic inheritance of IGE. INTERPRETATION: SLC2A1 mutations contribute to approximately 1% of IGE both as a dominant gene and as a susceptibility allele in complex inheritance. Diagnosis of GLUT1 deficiency has important treatment (ketogenic diet) and genetic counseling implications. The mechanism of restricted glucose delivery differs from the current focus on IGEs as ion channel disorders.
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Errores Innatos del Metabolismo de los Carbohidratos/complicaciones , Errores Innatos del Metabolismo de los Carbohidratos/genética , Epilepsia Generalizada/etiología , Epilepsia Generalizada/genética , Transportador de Glucosa de Tipo 1/genética , Adulto , Anciano , Animales , Análisis Mutacional de ADN , Evolución Molecular , Femenino , Estudios de Seguimiento , Genotipo , Transportador de Glucosa de Tipo 1/deficiencia , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Monosacáridos/deficiencia , Proteínas de Transporte de Monosacáridos/genética , Mutación/genética , Fenotipo , Adulto JovenRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) can act as pacemakers in the brain making them strong candidates for driving aberrant hypersynchronous network activity seen in epilepsy. Transcriptional changes in HCN channels occur in several animal models of epilepsy. However, only recently have genetic studies demonstrated sequence variation in HCN1 and HCN2 genes associated with human epilepsy. These include a triple proline deletion in HCN2 that increases channel function and occurs more often in patients with febrile seizure syndromes. Other HCNx gene variants have been described in idiopathic generalized epilepsy although the functional consequence of these remains unclear. In this review we explore potential cellular and network mechanisms involving HCN channels in the genetic epilepsies. We suggest how new genetic sequencing technology, medium-throughput functional assays and the ability to develop syndrome-specific animal models will provide a more comprehensive understanding of how I(h) contributes to pathogenic mechanisms underlying human genetic epilepsy. We also discuss what is known about the pharmacological manipulation of HCN channels in the context of epilepsy and how this may help future efforts in developing HCN-channel-based therapy.
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Anticonvulsivantes/uso terapéutico , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Predisposición Genética a la Enfermedad , Canales Iónicos/metabolismo , Canales de Potasio/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Epilepsia/metabolismo , Epilepsia/fisiopatología , Variación Genética , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/genética , Canales de Potasio/genéticaRESUMEN
The stoned proteins, stoned A (STNA) and stoned B (STNB), are essential for normal vesicle trafficking in Drosophila melanogaster neurons, and deletion of the stoned locus is lethal. Although there is a growing body of research aimed at defining the roles of these proteins, particularly for STNB where homologues have now been identified in all multicellular species, their functions and mechanisms of action are not yet established. The two proteins are structurally unrelated, consistent with two distinct cellular functions. The evidence suggests a critical requirement for stoned proteins in recycling/regulation or specification of a competent synaptic vesicle pool. As stoned proteins may be specific to a particular pathway of endocytosis, studies of their function are likely to be valuable in distinguishing between the different mechanisms of membrane retrieval and their respective contributions to synaptic vesicle recycling, a subject of considerable scientific debate. In this review, we examine the published literature on stoned and comment on the available data, conclusions from these analyses and how they may relate to alternative models of vesicle cycling.
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Proteínas Portadoras/fisiología , Proteínas de Drosophila/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Endocitosis/fisiología , Exocitosis/fisiología , Humanos , Modelos Biológicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Changes in the conductance of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel that mediates Ih are proposed to contribute to increased network excitability. Synchronous neuronal burst activity is a good reflection of network excitability and can be generated in isolated hippocampal slice cultures by removing Mg2+ from the extracellular fluid. We demonstrate that Ih contributes to this activity by increasing both the frequency and duration of bursting events. Changes in HCN channel function are also implicated in altered seizure susceptibility. Short-term application of kainic acid (KA) is known to initiate long lasting changes in neuronal networks that result in seizures, and in slice cultures was found to alter HCN mRNA levels in an isoform and hippocampal sub-region specific manner. These changes correlate with the ability of each sub-region to develop synchronous burst activity following KA that we have previously reported. Specifically, a loss of synchronous activity in the CA3 correlated with an increase in HCN2 mRNA levels that normalized concomitantly with the restoration of CA3 burst activity 7 days post insult. In contrast, in CA1 an increase in synchronous burst duration correlated with a reduction in HCN2 mRNA levels and both changes were still evident for 7 days post insult. Lamotrigine, known to increase Ih, reversed the impact of KA on burst duration in CA1 at both time-points linking a transcriptional reduction in HCN2 function to increased burst duration.
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Potenciales de Acción/genética , Hipocampo/metabolismo , Canales Iónicos/genética , Red Nerviosa/metabolismo , Neurotoxinas/farmacología , ARN Mensajero/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/genética , Sincronización Cortical/efectos de los fármacos , Epilepsia/genética , Epilepsia/metabolismo , Epilepsia/fisiopatología , Antagonistas de Aminoácidos Excitadores/farmacología , Predisposición Genética a la Enfermedad/genética , Hipocampo/efectos de los fármacos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Ácido Kaínico/farmacología , Lamotrigina , Red Nerviosa/efectos de los fármacos , Técnicas de Cultivo de Órganos , Canales de Potasio , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Triazinas/farmacologíaRESUMEN
The novel dicistronic transcript encoded by the Drosophila melanogaster stoned gene was recognized as being unusual in that the protein encoded by the first open reading frame, stoned-A (STNA), contains no internal methionine residues in a protein of 93 kDa. The dicistronic nature of the stoned locus and the lack of methionine residues in STNA is conserved across dipteran species. A second methionine-free cistron, encoding Snapin, was identified in Drosophila and also found to be dicistronic, the second open reading frame (ORF) encoding a methyltransferase. We have replaced the methyltransferase cistron with green fluorescent protein (GFP) and used this dicistronic construct to show that the GFP cistron is translated in Drosophila S2 cells. The insertion of in-frame AUG codons into the snapin ORF attenuates the translation of GFP, and the level of attenuation correlates with the number of inserted AUGs. Increasing the efficiency of translation-initiation of the Snapin cistron also attenuates the translation of GFP. This indicates that failure to initiate translation at the first AUG allows ribosomes to scan through the Snapin ORF and to initiate translation of the second cistron, unless new AUG codons are inserted. These data are used to interpret the expression of the stoned locus and in particular, to explain the altered stoned protein levels in the stoned-temperature-sensitive mutant allele, which replaces a lysine with a methionine codon early in the first, stonedA, cistron.