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Epilepsy is a neurological disease characterised by recurrent seizures with complex aetiology. Temporal lobe epilepsy, the most common form in adults, can be acquired following brain insults including trauma, stroke, infection or sustained status epilepticus. The mechanisms that give rise to the formation and maintenance of hyperexcitable networks following acquired insults remain unknown, yet an extensive body of literature points towards persistent gene and epigenomic dysregulation as a potential mediator of this dysfunction. While much is known about the function of specific classes of epigenetic regulators (writers and erasers) in epilepsy, much less is known about the enzymes, which read the epigenome and modulate gene expression accordingly. Here, we explore the potential role for the epigenetic reader bromodomain and extra-terminal domain (BET) proteins in epilepsy. Using the intra-amygdala kainic acid model of temporal lobe epilepsy, we initially identified widespread dysregulation of important epigenetic regulators including EZH2 and REST as well as altered BRD4 expression in chronically epileptic mice. BRD4 activity was also notably affected by epilepsy-provoking insults as seen by elevated binding to and transcriptional regulation of the immediate early gene Fos. Despite influencing early aspects of epileptogenesis, blocking BET protein activity with JQ1 had no overt effects on epilepsy development in mice but did alter glial reactivity and influence gene expression patterns, promoting various neurotransmitter signalling mechanisms and inflammatory pathways in the hippocampus. Together, these results confirm that epigenetic reader activity is affected by epilepsy-provoking brain insults and that BET activity may exert cell-specific actions on inflammation in epilepsy.
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Claudin-5 is one of the most essential tight junction proteins at the blood-brain barrier. A single nucleotide polymorphism rs10314 is located in the 3'-untranslated region of claudin-5 and has been shown to be a risk factor for schizophrenia. Here, we show that the pumilio RNA-binding protein, pumilio-1, is responsible for rs10314-mediated claudin-5 regulation. The RNA sequence surrounding rs10314 is highly homologous to the canonical pumilio-binding sequence and claudin-5 mRNA with rs10314 produces 25% less protein due to its inability to bind to pumilio-1. Pumilio-1 formed cytosolic granules under stress conditions and claudin-5 mRNA appeared to preferentially accumulate in these granules. Added to this, we observed granular pumilio-1 in endothelial cells in human brain tissues from patients with psychiatric disorders or epilepsy with increased/accumulated claudin-5 mRNA levels, suggesting translational claudin-5 suppression may occur in a brain-region specific manner. These findings identify a key regulator of claudin-5 translational processing and how its dysregulation may be associated with neurological and neuropsychiatric disorders.
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Barrera Hematoencefálica , Claudina-5 , Proteínas de Unión al ARN , Humanos , Claudina-5/metabolismo , Claudina-5/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Barrera Hematoencefálica/metabolismo , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Animales , Biosíntesis de Proteínas/fisiología , Células Endoteliales/metabolismoRESUMEN
PURPOSE: The posterior orbit is a confined space, harbouring neurovascular structures, frequently distorted by tumours. Image-guided navigation (IGN) has the potential to allow accurate localisation of these lesions and structures, reducing collateral damage whilst achieving surgical objectives. METHODS: We assessed the feasibility, effectiveness and safety of using an electromagnetic IGN for posterior orbital tumour surgery via a comparative cohort study. Outcomes from cases performed with IGN were compared with a retrospective cohort of similar cases performed without IGN, presenting a descriptive and statistical comparative analysis. RESULTS: Both groups were similar in mean age, gender and tumour characteristics. IGN set-up and registration were consistently achieved without significant workflow disruption. In the IGN group, fewer lateral orbitotomies (6.7% IGN, 46% non-IGN), and more transcutaneous lid and transconjunctival incisions (93% IGN, 53% non-IGN) were performed (p = .009). The surgical objective was achieved in 100% of IGN cases, with no need for revision surgery (vs 23% revision surgery in non-IGN, p = .005). There was no statistically significant difference in surgical complications. CONCLUSION: The use of IGN was feasible and integrated into the orbital surgery workflow to achieve surgical objectives more consistently and allowed the use of minimal access approaches. Future multicentre comparative studies are needed to explore the potential of this technology further.
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OBJECTIVE: Preclinical validation study to assess the feasibility and accuracy of electromagnetic image-guided systems (EM-IGS) in orbital surgery using high-fidelity physical orbital anatomy simulators. METHODS: EM-IGS platform, clinical software, navigation instruments and reference system (StealthStation S8, Medtronic) were evaluated in a mock operating theatre at the Royal Victoria Eye and Ear Hospital, a tertiary academic hospital in Dublin, Ireland. Five high-resolution 3D-printed model skulls were created using CT scans of five anonymised patients with an orbital tumour that previously had a successful orbital biopsy or excision. The ability of ophthalmic surgeons to achieve satisfactory system registration in each model was assessed. Subsequently, navigational accuracy was recorded using defined anatomical landmarks as ground truth. Qualitative feedback on the system was also attained. RESULTS: Three independent surgeons participated in the study, one junior trainee, one fellow and one consultant. Across models, more senior participants were able to achieve a smaller system-generated registration error in a fewer number of attempts. When assessing navigational accuracy, submillimetre accuracy was achieved for the majority of points (16 landmarks per model, per participant). Qualitative surgeon feedback suggested acceptability of the technology, although interference from mobile phones near the operative field was noted. CONCLUSION: This study suggests the feasibility and accuracy of EM-IGS in a preclinical validation study for orbital surgery using patient specific 3D-printed skulls. This preclinical study provides the foundation for clinical studies to explore the safety and effectiveness of this technology.
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Cirugía Asistida por Computador , Humanos , Órbita/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Programas Informáticos , Fenómenos ElectromagnéticosRESUMEN
OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585× and 1360×, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes.
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Epilepsia Refractaria , Epilepsia del Lóbulo Temporal , Esclerosis del Hipocampo , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Epilepsia Refractaria/genética , Epilepsia Refractaria/etiología , Epilepsia Refractaria/patología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/patología , Filaminas/genética , Variación Genética , Esclerosis del Hipocampo/genética , Esclerosis del Hipocampo/patología , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/complicaciones , Malformaciones del Desarrollo Cortical/patologíaRESUMEN
The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating the degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. In this study, we show an age-dependent increase in polyubiquitination in the brains of wild-type mice, accompanied by an induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, the aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during ageing.
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OBJECTIVE: Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood. METHODS: Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma. RESULTS: Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day. SIGNIFICANCE: Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.
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Proteínas CLOCK , Epilepsia Refractaria , Epilepsia del Lóbulo Temporal , Melatonina , Proteínas de Unión al ARN , Estado Epiléptico , Animales , Humanos , Masculino , Ratones , Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo , Melatonina/sangre , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Convulsiones , Estado Epiléptico/inducido químicamente , Estado Epiléptico/genética , Factores de Transcripción/metabolismo , Proteínas CLOCK/genéticaRESUMEN
Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 µmol L-1 and 90% reduction at 3 µmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.
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MicroARNs , Encéfalo/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Oligonucleótidos , Oligonucleótidos AntisentidoRESUMEN
Blood-brain barrier (BBB) dysfunction is associated with worse epilepsy outcomes however the underlying molecular mechanisms of BBB dysfunction remain to be elucidated. Tight junction proteins are important regulators of BBB integrity and in particular, the tight junction protein claudin-5 is the most enriched in brain endothelial cells and regulates size-selectivity at the BBB. Additionally, disruption of claudin-5 expression has been implicated in numerous disorders including schizophrenia, depression and traumatic brain injury, yet its role in epilepsy has not been fully deciphered. Here we report that claudin-5 protein levels are significantly diminished in surgically resected brain tissue from patients with treatment-resistant epilepsy. Concomitantly, dynamic contrast-enhanced MRI in these patients showed widespread BBB disruption. We show that targeted disruption of claudin-5 in the hippocampus or genetic heterozygosity of claudin-5 in mice exacerbates kainic acid-induced seizures and BBB disruption. Additionally, inducible knockdown of claudin-5 in mice leads to spontaneous recurrent seizures, severe neuroinflammation, and mortality. Finally, we identify that RepSox, a regulator of claudin-5 expression, can prevent seizure activity in experimental epilepsy. Altogether, we propose that BBB stabilizing drugs could represent a new generation of agents to prevent seizure activity in epilepsy patients.
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Barrera Hematoencefálica , Células Endoteliales , Animales , Barrera Hematoencefálica/metabolismo , Claudina-5/genética , Claudina-5/metabolismo , Células Endoteliales/metabolismo , Humanos , Ratones , Convulsiones/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismoRESUMEN
Temporal lobe epilepsy is the most common and refractory form of epilepsy in adults. Gene expression within affected structures such as the hippocampus displays extensive dysregulation and is implicated as a central pathomechanism. Post-transcriptional mechanisms are increasingly recognized as determinants of the gene expression landscape, but key mechanisms remain unexplored. Here we show, for first time, that cytoplasmic mRNA polyadenylation, one of the post-transcriptional mechanisms regulating gene expression, undergoes widespread reorganization in temporal lobe epilepsy. In the hippocampus of mice subjected to status epilepticus and epilepsy, we report >25% of the transcriptome displays changes in their poly(A) tail length, with deadenylation disproportionately affecting genes previously associated with epilepsy. Suggesting cytoplasmic polyadenylation element binding proteins (CPEBs) being one of the main contributors to mRNA polyadenylation changes, transcripts targeted by CPEBs were particularly enriched among the gene pool undergoing poly(A) tail alterations during epilepsy. Transcripts bound by CPEB4 were over-represented among transcripts with poly(A) tail alterations and epilepsy-related genes and CPEB4 expression was found to be increased in mouse models of seizures and resected hippocampi from patients with drug-refractory temporal lobe epilepsy. Finally, supporting an adaptive function for CPEB4, deletion of Cpeb4 exacerbated seizure severity and neurodegeneration during status epilepticus and the development of epilepsy in mice. Together, these findings reveal an additional layer of gene expression regulation during epilepsy and point to novel targets for seizure control and disease-modification in epilepsy.
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Epilepsia del Lóbulo Temporal/metabolismo , Regulación de la Expresión Génica/fisiología , Poliadenilación/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Epilepsia del Lóbulo Temporal/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Transfer RNAs (tRNAs) are a major class of noncoding RNA. Stress-induced cleavage of tRNA is highly conserved and results in tRNA fragments. Here we find specific tRNA fragments in plasma are associated with epilepsy. Small RNA sequencing of plasma samples collected during video-EEG monitoring of focal epilepsy patients identified significant differences in three tRNA fragments (5', 5'AlaTGC, and 5'GluCTC) from controls. Levels of these tRNA fragments were higher in pre-seizure than post-seizure samples, suggesting they may serve as biomarkers of seizure risk in epilepsy patients. In vitro studies confirmed that production and extracellular release of tRNA fragments was lower after epileptiform-like activity in hippocampal neurons. We designed PCR-based assays to quantify tRNA fragments in a cohort of pre- and post-seizure plasma samples from focal epilepsy patients and healthy controls (n = 32/group). Receiver operating characteristic analysis indicated that tRNA fragments potently distinguished pre- from post-seizure patients (area under the curve of 0.8-0.95). Elevated tRNA fragments levels were not detected in patients with psychogenic non-epileptic seizures, and did not result from medication tapering. This study identifies a novel class of epilepsy biomarker and reveals the potential existence of prodromal molecular patterns in blood that could be used to predict seizure risk.
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Ácidos Nucleicos Libres de Células/sangre , Epilepsias Parciales/sangre , Epilepsias Parciales/fisiopatología , ARN de Transferencia/sangre , Adulto , Biomarcadores/sangre , Ácidos Nucleicos Libres de Células/genética , Electroencefalografía , Epilepsias Parciales/genética , Femenino , Humanos , Masculino , Reacción en Cadena de la Polimerasa , ARN de Transferencia/genéticaRESUMEN
Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons.SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE.
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OBJECTIVE: ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. METHODS: Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. RESULTS: Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus increased the transcription of the uracil-sensitive P2Y receptors P2ry2 , P2ry4 , and P2ry6 and decreased the transcription of the adenine-sensitive P2Y receptors P2ry1 , P2ry12 , P2ry13 . Protein levels of P2Y1 , P2Y2 , P2Y4 , and P2Y6 were increased after status epilepticus, whereas P2Y12 expression was decreased. In the chronic phase, P2ry1 , P2ry2 , and P2ry6 transcription and P2Y1 , P2Y2 , and P2Y12 protein levels were increased with no changes for the other P2Y receptors. In hippocampal samples from patients with temporal lobe epilepsy, P2Y1 and P2Y2 protein expression was increased, whereas P2Y13 levels were lower. Demonstrating a functional contribution of P2Y receptors to seizures, central injection of ADP exacerbated seizure severity, whereas treatment with UTP decreased seizure severity during status epilepticus in mice. SIGNIFICANCE: The present study is the first to establish the specific hippocampal expression profile and function of the P2Y receptor family after experimental status epilepticus and in human temporal lobe epilepsy and offers potential new targets for seizure control and disease modification.
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Epilepsia Refractaria/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Convulsiones/metabolismo , Animales , Western Blotting , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia del Lóbulo Temporal/metabolismo , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Estado Epiléptico/tratamiento farmacológicoRESUMEN
There is a need for diagnostic biomarkers of epilepsy and status epilepticus to support clinical examination, electroencephalography and neuroimaging. Extracellular microRNAs may be potentially ideal biomarkers since some are expressed uniquely within specific brain regions and cell types. Cerebrospinal fluid offers a source of microRNA biomarkers with the advantage of being in close contact with the target tissue and sites of pathology. Here we profiled microRNA levels in cerebrospinal fluid from patients with temporal lobe epilepsy or status epilepticus, and compared findings to matched controls. Differential expression of 20 microRNAs was detected between patient groups and controls. A validation phase included an expanded cohort and samples from patients with other neurological diseases. This identified lower levels of miR-19b in temporal lobe epilepsy compared to controls, status epilepticus and other neurological diseases. Levels of miR-451a were higher in status epilepticus compared to other groups whereas miR-21-5p differed in status epilepticus compared to temporal lobe epilepsy but not to other neurological diseases. Targets of these microRNAs include proteins regulating neuronal death, tissue remodelling, gliosis and inflammation. The present study indicates cerebrospinal fluid contains microRNAs that can support differential diagnosis of temporal lobe epilepsy and status epilepticus from other neurological and non-neurological diseases.
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Biomarcadores/líquido cefalorraquídeo , Epilepsia del Lóbulo Temporal/líquido cefalorraquídeo , Epilepsia del Lóbulo Temporal/genética , MicroARNs/líquido cefalorraquídeo , Estado Epiléptico/líquido cefalorraquídeo , Estado Epiléptico/genética , Adulto , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Epilepsia del Lóbulo Temporal/sangre , Exosomas/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Modelos Logísticos , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Análisis de Componente Principal , Transporte de ARN , Curva ROC , Reproducibilidad de los Resultados , Estado Epiléptico/sangreRESUMEN
Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.
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Regulación de la Expresión Génica , MicroARNs/metabolismo , Factores de Empalme de ARN/metabolismo , Sinapsis/fisiología , Animales , Biología Computacional , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Perfilación de la Expresión Génica , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neuropéptidos/metabolismo , Picrotoxina/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Proteoma/análisisRESUMEN
Current anti-epileptic drugs (AEDs) act on a limited set of neuronal targets, are ineffective in a third of patients with epilepsy, and do not show disease-modifying properties. MicroRNAs are small noncoding RNAs that regulate levels of proteins by post-transcriptional control of mRNA stability and translation. MicroRNA-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid (LNA), cholesterol-tagged antagomirs targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in mouse models of status epilepticus. Translation of these findings would benefit from evidence of efficacy in non-status epilepticus models and validation in another species. Here, we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre-treated with Ant-134 in the pentylenetetrazol model. Pre-treatment with Ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats, a toxin-free model of acquired epilepsy. Nevertheless, Ant-134 post-treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and Ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model. The potent anticonvulsant effects of Ant-134 in multiple models may encourage pre-clinical development of this approach to epilepsy therapy.
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The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis.
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Citoplasma/metabolismo , Epilepsia/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Helicasas/metabolismo , Análisis de Secuencia de ARN , Sinapsis/metabolismo , Transactivadores/metabolismo , Animales , Biología Computacional , Epilepsia del Lóbulo Temporal/genética , Hipocampo/patología , Humanos , Inmunoprecipitación , Masculino , Ratones Endogámicos C57BL , Unión Proteica , ARN Helicasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Estado Epiléptico/genética , Transactivadores/genéticaRESUMEN
UNLABELLED: Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1ß release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.
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Epilepsia del Lóbulo Temporal/complicaciones , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Gliosis/tratamiento farmacológico , Gliosis/etiología , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Convulsiones/tratamiento farmacológico , Convulsiones/etiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Adolescente , Adulto , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacología , Niacinamida/uso terapéutico , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Adulto JovenRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumour having a median survival of just 12-18 months following standard therapy protocols. Local recurrence, post-resection and adjuvant therapy occurs in most cases. NEW METHOD: U87MG-luc2-bearing GBM xenografts underwent 4.5mm craniectomy and tumour resection using microsurgical techniques. The cranial defect was repaired using a novel modified cranial window technique consisting of a circular microscope coverslip held in place with glue. RESULTS: Immediate post-operative bioluminescence imaging (BLI) revealed a gross total resection rate of 75%. At censor point 4 weeks post-resection, Kaplan-Meier survival analysis revealed 100% survival in the surgical group compared to 0% in the non-surgical cohort (p=0.01). No neurological defects or infections in the surgical group were observed. GBM recurrence was reliably imaged using facile non-invasive optical bioluminescence (BLI) imaging with recurrence observed at week 4. COMPARISON WITH EXISTING METHOD(S): For the first time, we have used a novel cranial defect repair method to extend and improve intracranial surgical resection methods for application in translational GBM rodent disease models. Combining BLI and the cranial window technique described herein facilitates non-invasive serial imaging follow-up. CONCLUSION: Within the current context we have developed a robust methodology for establishing a clinically relevant imageable GBM surgical resection model that appropriately mimics GBM recurrence post resection in patients.