RESUMEN
Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain.
Asunto(s)
Metilación de ADN/genética , Epigénesis Genética , Epilepsia del Lóbulo Temporal/patología , Hipocampo/patología , Adolescente , Adulto , Biología Computacional , Islas de CpG/fisiología , Epilepsia del Lóbulo Temporal/genética , Femenino , Regulación de la Expresión Génica , Hipocampo/metabolismo , Humanos , Inmunoprecipitación , Masculino , MicroARNs/metabolismo , Microdisección , Persona de Mediana Edad , Proyectos Piloto , Regiones Promotoras Genéticas , Esclerosis , Adulto JovenRESUMEN
When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.
Asunto(s)
Hipocampo/metabolismo , MicroARNs/metabolismo , Estado Epiléptico/prevención & control , Amígdala del Cerebelo/metabolismo , Animales , Antagomirs , Proteínas Argonautas/metabolismo , Regulación hacia Abajo , Agonistas de Aminoácidos Excitadores/toxicidad , Inyecciones Intralesiones , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/farmacología , Oligonucleótidos/farmacología , Regulación hacia ArribaRESUMEN
Exposure of the brain to brief, non-harmful seizures can activate protective mechanisms that temporarily generate a damage-refractory state. This process, termed epileptic tolerance, is associated with large-scale down-regulation of gene expression. Polycomb group (PcG) proteins are master controllers of gene silencing during development that are re-activated by injury to the brain. Here, we explored the transcriptional response of genes associated with polycomb repressive complex (PRC) 1 (Ring1A, Ring1B, and Bmi1) and PRC2 (Ezh1, Ezh2, and Suz12), as well as additional transcriptional regulators Sirt1, Yy1, and Yy2, in a mouse model of status epilepticus (SE). Findings were contrasted to changes after SE in mice previously given brief seizures to evoke tolerance. Real-time quantitative PCR showed SE prompted an early (1 h) increase in expression of several genes in PRC1 and PRC2 in the hippocampus, followed by down-regulation of many of the same genes at later times points (4, 8, and 24 h). Spatio-temporal differences were found among PRC2 genes in epileptic tolerance, including increased expression of Ezh2, Suz12, and Yy2 relative to the normal injury response to SE. In contrast, PRC1 complex genes including Ring 1B and Bmi1 displayed differential down-regulation in epileptic tolerance. The present study characterizes PcG gene expression following SE and shows prior seizure exposure produces select changes to PRC1 and PRC2 composition that may influence differential gene expression in epileptic tolerance.
RESUMEN
Emerging data support roles for microRNA (miRNA) in the pathogenesis of various neurologic disorders including epilepsy. MicroRNA-134 (miR-134) is enriched in dendrites of hippocampal neurons, where it negatively regulates spine volume. Recent work identified upregulation of miR-134 in experimental and human epilepsy. Targeting miR-134 in vivo using antagomirs had potent anticonvulsant effects against kainic acid-induced seizures and was associated with a reduction in dendritic spine number. In the present study, we measured dendritic spine volume in mice injected with miR-134-targeting antagomirs and tested effects of the antagomirs on status epilepticus triggered by the cholinergic agonist pilocarpine. Morphometric analysis of over 6,400 dendritic spines in Lucifer yellow-injected CA3 pyramidal neurons revealed increased spine volume in mice given antagomirs compared to controls that received a scrambled sequence. Treatment of mice with miR-134 antagomirs did not alter performance in a behavioral test (novel object location). Status epilepticus induced by pilocarpine was associated with upregulation of miR-134 within the hippocampus of mice. Pretreatment of mice with miR-134 antagomirs reduced the proportion of animals that developed status epilepticus following pilocarpine and increased animal survival. In antagomir-treated mice that did develop status epilepticus, seizure onset was delayed and total seizure power was reduced. These studies provide in vivo evidence that miR-134 regulates spine volume in the hippocampus and validation of the seizure-suppressive effects of miR-134 antagomirs in a model with a different triggering mechanism, indicating broad conservation of anticonvulsant effects.
Asunto(s)
Espinas Dendríticas/patología , Hipocampo/patología , MicroARNs/genética , Neuronas/patología , Estado Epiléptico , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Regulación de la Expresión Génica/efectos de los fármacos , Inyecciones Intraventriculares , Masculino , Ratones , MicroARNs/antagonistas & inhibidores , Oligonucleótidos/uso terapéutico , Pilocarpina/toxicidad , Memoria Espacial/efectos de los fármacos , Memoria Espacial/fisiología , Estadísticas no Paramétricas , Estado Epiléptico/inducido químicamente , Estado Epiléptico/patología , Estado Epiléptico/prevención & control , Factores de TiempoRESUMEN
14-3-3 proteins are ubiquitous molecular chaperones that are abundantly expressed in the brain where they regulate cell functions including metabolism, the cell cycle and apoptosis. Brain levels of several 14-3-3 isoforms are altered in diseases of the nervous system, including epilepsy. The 14-3-3 zeta (ζ) isoform has been linked to endoplasmic reticulum (ER) function in neurons, with reduced levels provoking ER stress and increasing vulnerability to excitotoxic injury. Here we report that transgenic overexpression of 14-3-3ζ in mice results in selective changes to the unfolded protein response pathway in the hippocampus, including down-regulation of glucose-regulated proteins 78 and 94, activating transcription factors 4 and 6, and Xbp1 splicing. No differences were found between wild-type mice and transgenic mice for levels of other 14-3-3 isoforms or various other 14-3-3 binding proteins. 14-3-3ζ overexpressing mice were potently protected against cell death caused by intracerebroventricular injection of the ER stressor tunicamycin. 14-3-3ζ overexpressing mice were also potently protected against neuronal death caused by prolonged seizures. These studies demonstrate that increased 14-3-3ζ levels protect against ER stress and seizure-damage despite down-regulation of the unfolded protein response. Delivery of 14-3-3ζ may protect against pathologic changes resulting from prolonged or repeated seizures or where injuries provoke ER stress.
Asunto(s)
Proteínas 14-3-3/genética , Retículo Endoplásmico/metabolismo , Hipocampo/metabolismo , Estado Epiléptico/metabolismo , Animales , Apoptosis , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Hipocampo/citología , Inmunohistoquímica , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Brief seizures (epileptic/seizure preconditioning) are capable of activating endogenous protective pathways in the brain which can temporarily generate a damage-refractory state against subsequent and otherwise harmful episodes of prolonged seizures (tolerance). Altered expression of microRNAs, a class of non-coding RNAs that function post-transcriptionally to regulate mRNA translation has recently been implicated in the molecular mechanism of epileptic tolerance. Here we characterized the effect of seizure preconditioning induced by low-dose systemic kainic acid on microRNA expression in the hippocampus of mice. Seizure preconditioning resulted in up-regulation of 25 mature microRNAs in the CA3 subfield of the mouse hippocampus, with the highest levels detected for miR-184. This finding was supported by real time PCR and in situ hybridization showing increased neuronal miR-184 levels and a reduction in protein levels of a miR-184 target. Inhibiting miR-184 expression in vivo resulted in the emergence of neuronal death after preconditioning seizures and increased seizure-induced neuronal death following status epilepticus in previously preconditioned animals, without altered electrographic seizure durations. The present study suggests miRNA up-regulation after preconditioning may contribute to development of epileptic tolerance and identifies miR-184 as a novel contributor to neuronal survival following both mild and severe seizures.
Asunto(s)
Hipocampo/metabolismo , MicroARNs/genética , Neuronas/patología , Convulsiones/genética , Animales , Western Blotting , Muerte Celular/genética , Convulsivantes/administración & dosificación , Electroencefalografía , Epilepsia/inducido químicamente , Epilepsia/genética , Epilepsia/metabolismo , Perfilación de la Expresión Génica , Hipocampo/patología , Hibridación in Situ , Ácido Kaínico/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Regulación hacia ArribaRESUMEN
Hippocampal sclerosis (HS) is a common pathological finding in patients with temporal lobe epilepsy (TLE) and is associated with altered expression of genes controlling neuronal excitability, glial function, neuroinflammation and cell death. MicroRNAs (miRNAs), a class of small non-coding RNAs, function as post-transcriptional regulators of gene expression and are critical for normal brain development and function. Production of mature miRNAs requires Dicer, an RNAase III, loss of which has been shown to cause neuronal and glial dysfunction, seizures, and neurodegeneration. Here we investigated miRNA biogenesis in hippocampal and neocortical resection specimens from pharmacoresistant TLE patients and autopsy controls. Western blot analysis revealed protein levels of Dicer were significantly lower in certain TLE patients with HS. Dicer levels were also reduced in the hippocampus of mice subject to experimentally-induced epilepsy. To determine if Dicer loss was associated with altered miRNA processing, we profiled levels of 380 mature miRNAs in control and TLE-HS samples. Expression of nearly 200 miRNAs was detected in control human hippocampus. In TLE-HS samples there was a large-scale reduction of miRNA expression, with 51% expressed at lower levels and a further 24% not detectable. Primary transcript (pri-miRNAs) expression levels for several tested miRNAs were not different between control and TLE-HS samples. These findings suggest loss of Dicer and failure of mature miRNA expression may be a feature of the pathophysiology of HS in patients with TLE.
Asunto(s)
ARN Helicasas DEAD-box/genética , Epilepsia del Lóbulo Temporal/genética , Hipocampo/patología , MicroARNs/genética , Ribonucleasa III/genética , Esclerosis/genética , Adulto , Animales , Biología Computacional , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Temporal lobe epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of neurological disorders. In experimental models of prolonged, injurious seizures (status epilepticus) and in human epilepsy, we found upregulation of miR-134, a brain-specific, activity-regulated miRNA that has been implicated in the control of dendritic spine morphology. Silencing of miR-134 expression in vivo using antagomirs reduced hippocampal CA3 pyramidal neuron dendrite spine density by 21% and rendered mice refractory to seizures and hippocampal injury caused by status epilepticus. Depletion of miR-134 after status epilepticus in mice reduced the later occurrence of spontaneous seizures by over 90% and mitigated the attendant pathological features of temporal lobe epilepsy. Thus, silencing miR-134 exerts prolonged seizure-suppressant and neuroprotective actions; determining whether these are anticonvulsant effects or are truly antiepileptogenic effects requires additional experimentation.
Asunto(s)
Silenciador del Gen , MicroARNs/genética , Fármacos Neuroprotectores/metabolismo , Convulsiones/genética , Convulsiones/prevención & control , Adulto , Animales , Región CA3 Hipocampal/efectos de los fármacos , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Muerte Celular/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/patología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/patología , Silenciador del Gen/efectos de los fármacos , Humanos , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Células Piramidales/efectos de los fármacos , Células Piramidales/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Estado Epiléptico/genética , Estado Epiléptico/patología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Crosslinking and the resultant changes in mechanical properties have been shown to influence cellular activity within collagen biomaterials. With this in mind, we sought to determine the effects of crosslinking on both the compressive modulus of collagen-glycosaminoglycan scaffolds and the activity of osteoblasts seeded within them. Dehydrothermal, 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde crosslinking treatments were first investigated for their effect on the compressive modulus of the scaffolds. After this, the most promising treatments were used to study the effects of crosslinking on cellular attachment, proliferation, and infiltration. Our experiments have demonstrated that a wide range of scaffold compressive moduli can be attained by varying the parameters of the crosslinking treatments. 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde treatments produced the stiffest scaffolds (fourfold increase when compared to dehydrothermal crosslinking). When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared to the other groups. Taken together, these results indicate that crosslinking can be used to produce collagen-glycosaminoglycan scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds.